Brain Development: Normal Processes and the Effects of Alcohol ...

BRAIN DEVELOPMEN T

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BRAIN DEVELOPMEN T 

NORMAL PROCESSES AND 

THE EFFECTS O F ALCOHOL 

AND NICOTINE 

EDITED BY 

Michael W. Miller 

OXPORD 

UNIVERSITY PRES S 

2006

OXPORD 

UNIVERSITY PRES S 

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Library of Congress Cataloging-in-Publication Data 

Brain development : normal processes and th e effect s o f alcohol an d nicotine / edited by Michael W. Miller. 

p. ; cm. 

Includes bibliographical references and index. 

ISBN-13 978-0-19-518313- 9 

ISBN 0-19-518313-4 

1. Developmenta l toxicology. 2 . Nenrotoxicology . 3 . Alcohol-Toxicology . 4 . Nicotin e —Toxicology. 

5. Alcoholis m i n pregnancy—Pathophysiology. 6 . Pregnan t women—Tobacco use —Physiological effect . 

[DNLM: 1 . Brain—dru g effects. 2 . Brain—growt h & development. 3 . Ethanol—pharmacology . 4 . Fetus—dru g effects . 

5. Neurons-dru g effects. 6 . Nicotin e - pharmacology. WL 300 B8125 2006] I . Miller , Michael W. 

RA1224.45.B73 2006 

616.8'6471-dc22 200501285 4 

135798642 

Printed in the Unite d States of America 

on acid-free paper

As such, the present volume is the product of decades 

of work by teams o f neuroscientists and developmen - 

tal biologists . I than k th e name d an d nameles s o f 

these dedicate d researcher s wh o hav e reache d m e 

through bot h ora l and/o r writte n communications . 

This volume builds on the foundation of research described 

i n a boo k publishe d 1 3 year s ago , Development 

of the Central Nervous System, Effects of Alcohol 

and Opiates (Miller , 1992). Since tha t book was published, 

our understanding of the mechanism s under - 

lying norma l brai n developmen t an d th e effect s o f 

ethanol neurotoxicit y o n neura l ontogen y ha s grown 

exponentially. Interes t i n th e clinica l proble m o f 

nicotine exposure has also increased. 

Personally, m y researc h ha s bee n supporte d b y a 

host of postdoctoral trainees, graduate and undergraduate 

students, and technicians . I am greatl y indebted 

to them fo r working toward our commo n goa l of un - 

derstanding th e intricacie s o f brai n development . 

Some o f my current students (Maria Bruns and Juli e 

Siegenthaler) an d collaborator s (Sandr a Mooney ) 

Acknowledgments 

contributed t o the writing of this book. My career has 

been consistentl y an d substantivel y supported b y the 

federal government , notably the Nationa l Institute of 

Alcohol Abus e an d Alcoholis m an d th e Departmen t 

of Veterans Affairs. Without the suppor t of these agen - 

cies I would not hav e been abl e t o study normal an d 

abnormal brain development, no r would I have been 

able t o mee t th e peopl e wh o wrot e chapter s fo r this 

volume. These people hav e brought considerabl e expertise 

and keen insights to their contributions of outstanding 

chapters . 

The dua l sculptors, nature and nurture, have bee n 

essential factor s in m y development as a scientist. M y 

fascination wit h science—ho w thing s wor k and ho w 

they fail—wa s fostere d b y m y parent s an d th e post - 

Sputnik America n culture . I t i s m y grea t hop e tha t 

children of all ages, boys and girls, retain their wonder 

as to why things are a s they are. This sense o f marvel 

should b e celebrate d an d hel d i n aw e by society. My 

candle was stoked by high schoo l an d college teachers 

(including Willia m Harri s and Willia m Tully , an d

vi ACKNOWLEDGMENT S 

Edward Hodgson an d Edwar d Maly, respectively), re- Finally , I would lik e to thank m y family , m y wif e 

search mentor s (includin g Pedro Pasi k and Ala n Pe - an d thre e children , fo r their continue d suppor t an d 

ters), and collaborators (notably Richard Nowakowski goo d humor . The y ar e m y sustenance , grounding , 

and Rober t Rhoades). They encouraged m e t o think an d reason for being, 

broadly and not to be confined by dogma o r methods.

List of Abbreviations i x 

Contributors xii i 

1. Model s o f Neurotoxicity Provide Unique 

Insight into Normal Developmen t 3 


Part I. Norma l Developmen t 

2. Cel l Proliferatio n 9 

Bernhard So ter 

Pradeep G . Bhide 

3. Neurona l Migration 2 7 

Huaiyu Hu 

4. Neurona l Differentiation: From Axons 

to Synapses 4 5 

C. David Mintz 

Iddii H. Bekirov 

Tonya R. Anderson 

Deanna L. Benson 

Contents 

vii 

5. Cel l Death 7 3 

Stevens K Rehen 

Jerold J.M. Chun 

6. Intracellula r Pathways of Neuronal 

Death 9 1 

Sandra M. Mooney 

George I. Henderson 

7. Developmenta l Disorder s and 

Evolutionary Expectations: Mechanism s 

of Resilience 10 4 

Barbara L. Finlay 

Jeremy C. Yost 

Desmond T . Cheung

viii CONTENT S 

Part IL Ethanol-Affecte d Developmen t 

8. Prenata l Alcohol Exposure and Huma n 

Development 12 3 

Claire D. Coles 

9. Influenc e of Alcohol on the Structur e of 

the Developing Human Brain 14 3 

Susanna L, Fryer 

Christie L. McGee 

Andrea D. Spadoni 

Edward P. Riley 

10. Prenatal Ethanol Exposure and Feta l 

Programming: Implications for Endocrine 

and Immune Developmen t an d 

Long-Term Healt h 15 3 

Joanna H, Sliwowska 

Xingqi Zhang 

Joanne Weinberg 

11. Earl y Exposure to Ethanol Affects th e 

Proliferation of Neuronal Precursor s 18 2 


12. Effect s o f Ethanol o n the Regulatio n of 

Cell Cycle in Neural Ste m Cell s 19 9 

W. Michael Zawada 

Mita Das 

13. Mechanism s of Ethanol-Induced 

Alterations in Neuronal Migration 21 6 

Julie A. Siegenthaler 


14. Effect s o f Ethanol on Mechanism s 

Regulating Neuronal Process 

Outgrowth 23 0 

Tara A. Lindsley 

15. Neurona l Survival Is Compromised b y 

Ethanol: Extracellular Mediators 24 5 


Maria B. Bruns 

Paula L. Hoffman 

16. Intracellular Events in Ethanol-Induce d 

Neuronal Death 26 7 


Michael W . Miller 


17. Neural Crest and Developmental Exposur e 

to Ethanol 27 9 

Susan M. Smith 

Katherine A. Debelak-Kragtorp 

18. Glial Targets of Developmental Exposur e 

to Ethanol 29 5 

Consuelo Guerri 

Gemma Rubert 

Maria Pasqual 

Part III. Nicotine-Affecte d Developmen t 

19. Tobacco use During Pregnancy : 

Epidemiology and Effect s o n Offspring 31 5 

Jennifer A. Willford 

Nancy L . Da y 

Marie D. Cornelius 

20. Prenata l Nicotine Exposur e and Animal 

Behavior 32 9 

Brenda M. Elliott 

Neil E. Grunberg 

21. Neurona l Receptors for Nicotine: Functiona l 

Diversity and Developmental Change s 34 1 

Huibert D. Mansvelder 

Loma W. Role 

22. Neura l Precursors as Preferential Targets 

for Dru g Abuse: Long Term Consequence s 

and Latent Susceptibilit y to Central 

Nervous System Disorders 36 3 

Kurt F. Hauser 

Nazira El-Hage 

Shreya Buch 

Gregory N. Barnes 

Henrietta S. Bada 

James R. Pauly 

23. Nicotini c Receptor Regulatio n of 

Developing Catecholamine System s 38 1 

Frances M. Leslie 

Layla Azam 

Kathy Goliardo 

Kathryn O'Leary 

Ryan Franke 

Shahrdad Lotfipour 

Index 39 9

5-HT serotoni n 

8-OH-DPAT 8-hydroxy-2-(di-n-propylamino)tetrali n 

ABCG2 ATP-bindin g cassette protein 

otBgTx a-bungarotoxi n 

ACh acetylcholin e 

AChE acetylcholinesteras e 

ACTH adrenocorticotropi c hormone 

ADH alcoho l dehydrogenase 

ADHD attentio n deficit hyperactivity disorder 

ADNF activity-dependen t neurotrophic factor 

ADNP activity-dependen t neuroprotective protein 

ADP adenosin e diphosphate 

ADX adrenalectom y 

AHP adul t hippocampal progenitor 

AIDS acquire d immunodeficiency syndrome 

AI F apoptosi s inducing factor 

List of Abbreviations 

ix 

ALC prenatall y exposed to alcohol, bot h with and 

without dysmorphic features 

AMPA a-amino-B-hydroxy-S-methyM-isoxazole - 

propionic acid 

AMPAR a-amino-3-hydroxy - 5-methyl-4-isoxazole 

propionic acid receptor 

AP-1 activatin g protein-1 

APAF apoptoti c protease activating factor 

ARIA acetylcholin e receptor-inducin g protei n 

ARND alcohol-relate d neurodevelopmenta l 

disorder 

ASD atria l septic defect 

ASR acousti c startle reflex 

Astn astrotacti n 

ATP adenosin e triphosphate 

AVP arginin e vasopressin 

BDNF brain-derive d neurotrophic facto r 

BEG bloo d ethano l concentratio n

x LIS T OF ABBREVIATION S 

β-E P β-endorphi n 

bFG F basi c fibroblast growth facto r (aka fibroblas t 

growth facto r 2 ) 

BH Bcl- 2 homolog y 

BMP bon e morphogeni c protei n 

BSID Bayle y Scales of Developmen t 

BrdU bromodeoxyuridin e 

[Ca 2+ 

] i intracellula r concentratio n o f Ca 2+ 

CAM cel l adhesio n molecul e 

cAMP cycli c adenosin e monophosphat e 

CAP cel l adhesio n protei n 

CBG corticosteron e bindin g globulin 

CC corpu s callosum 

CC K cholecystokini n 

CD13 3 cel l determinan t 13 3 (hematopoieti c ste m 

cell marker , aka promini n cdk cyclindependen 

t kinas e 

Cdk cyclin-dependen t kinase 

cGM P cycli c guanosine monophosphat e 

ChAT cholin e acetyltransferas e 

CKI cyclin-dependen t kinase inhibito r 

CN S centra l nervou s system 

CON A concanavali n A 

COR T corticosteron e 

CP cortica l plate 

CRE B cycli c AMP respons e elemen t bindin g protein 

CRH corticotropi n releasin g hormon e 

CRH-R 1 corticotropi n releasin g hormon e typ e 1 

recepto r 

CRM P collapsi n respons e mediato r protei n 

CT compute r tomograph y 

CVLT- C Californi a Verbal Learnin g Test- children' s 

version 

CYP2E1 cytochrom e P450 2E 1 

CytOx cytochrom e C oxidase 

DA dopamin e 

Dab disable d 

DC C delete d in colorecta l cance r 

DHβ E dihydro-β-erythroidin e 

DIABL O direc t IAP binding protein 

DI V day s in vitro 

DNA-PKc s DNA-dependen t protei n kinas e 

catalytic subunit 

DO I l-(2,5-dimethoxy-4-iodophenyl)-2 - 

aminopropan e hydrochloride 

DO R δ-opioid receptor s 

DR G dorsa l root ganglia 

DT I diffusio n tenso r imagin g 

EC 50 concentratio n effectin g a half maximal 

response 

ECM extracellula r matrix 

EG F epiderma l growth facto r 

EGF r epiderma l growth facto r recepto r 

EG L externa l granular layer of the cerebellu m 

EM electro n microscop y 

ERK extracellula r signal regulate d kinas e 

ES embryoni c stem 

ETS environmenta l tobacc o smok e 

FAD D Fas-associate d deat h domai n 

FAS feta l alcoho l syndrom e 

FASD feta l alcoho l spectru m disorder s 

FasL Fa s ligand 

FCM D Fukuyama-typ e congenita l muscular 

dystroph y 

FE E feta l ethanol-expose d 

FG F fibroblas t growth facto r 

FILI P filami n A-interactin g protei n 

fMRI functiona l magneti c resonanc e imagin g 

G gestationa l day 

GABA γ-aminobutyri c aci d 

Gadd45 γ growt h arres t and DN A damage-inducibl e 

GD F growt h differentiatio n facto r 

GD X gonadectom y 

GE ganglioni c eminenc e 

GF growt h fractio n 

GFA P glia l fibrillar y acidi c protei n 

GM C ganglio n mothe r cel l 

GnR H gonadotropin-releasin g hormone 

GR glucocorticoi d recepto r 

GR P glial-restricte d precursor 

GS H glutathion e 

GTPas e guanosin e 5'-triphosphatas e 

HG F hepatocyt e growth facto r 

hn heteronuclea r

HNE 4-hydroxynonena l 

hNSC huma n neura l stem cell 

HPA hypothalamic-pituitary-adrena l 

HPG hypothalamic-pituitary-gonada l 

IAP inhibitor y apoptotic protei n 

Ig immunoglobuli n 

IGF insulin-lik e growth factor 

IGF-lr insulin-lik e growth factor-1 receptor 

IGF-BP insulin-lik e growth factor binding protein 

IHZ intrahila r zone; or hippocampal field CA4 or 

the subgranular zone 

IL interleuki n 

IP intraperitonea l 

IP3 inosito l 1,4,5-triphosphat e 

IQ intelligenc e quotient 

ISEL i n situ end-labelin g 

IZ intermediat e zone 

KC1 potassiu m chlorid e 

Ki67 proliferation-associate d nuclear antige n 

KOR K-opioi d receptors 

Large like-acetylglucosaminyltransferas e 

LBW lo w birth weight 

LC locu s coeruleus 

LC-NE locu s coeruleus-noradrenergic (sympathetic 

adrenal medullary) 

LCN loca l circuit neuro n 

LF leavin g fraction 

LHRH luteinizin g hormone releasin g hormone 

LiglV ligasel V 

LPA lysophosphatidi c acid 

LPS lipopolysaccharid e 

LTP long-ter m potentiation 

LV laten t variable 

mAChR muscarini c acetylcholine recepto r 

MAP microtubule-associate d protei n 

MAPK mitogen-activate d protein kinase 

MDA malondialdehyd e 

MEB muscle-eye-brai n disease 

MHC majo r histocompatibility complex 

MHPCD Materna l Healt h Practice s and Child 

Development 

LIST OF ABBREVIATIONS x i 

MNTB media l nucleu s o f the trapezoid body 

MOR fl-opioi d receptor s 

MR mineralocorticoi d receptor 

MRI magneti c resonance imaging 

MZ margina l zon e 

NAc nucleu s accumbens 

nACliR nicotini c acetylcholine receptor 

NAD nicotinamide-adenin e dinucleotid e 

NAP asn-ala-pro-val-ser-ile-pro-gl n 

NBL neuroblasti c layer 

nCAM neura l cel l adhesion molecule 

NE norepinephrin e 

NGF nerv e growth factor 

NgCAM neuron-gli a cell adhesion molecul e 

NHEJ non-homologou s end-joinin g 

NMD neurona l migration disorder 

NMDA N-methyl-D-aspartat e 

NMDAR N-methyl-D-aspartat e recepto r 

NMJ neuromuscula r junction 

NO nitri c oxide 

NOND naturall y occurring neuronal deat h 

NPC neura l progenitor cel l 

Nr-CAM NgCAM-relate d cell adhesion molecul e 

NRT nicotin e replacemen t therap y 

NSC neura l stem cel l 

NT neurotrophi n 

NTS nucleus tractus solitarius 

NT-4 neurotrophi n 4 

OD ocula r dominance 

ODC ornithin e decarboxylase 

OPPS Ottaw a Prospectiv e Prenatal Stud y 

P postnata l day 

PA phosphatidi c aci d 

PACAP pituitar y adenylyl cyclase activating 

polypeptide 

PAFAHIb platelet-activating facto r acetylhydrolase 

isoform Ib , aka LISI 

PARP poly-adenosin e diphosphate ribos e polymerase 

PCD programme d cel l deat h 

PDGF platelet-derive d growth factor 

PEE prenata l ethanol exposure 

PET positro n emission tomograph y

xii LIS T OF ABBREVIATION S 

PEt phosphatidy l ethanol 

PF C prefronta l cortex 

PB К phosphatidylinosito l З'-phosphokinas e 

РК А protei n kinase A 

PKC protei n kinase С 

PLD phospholipas e 

PLS partia l least squares analysis 

PM pia l membran e 

PN projectio n neuro n 

PN S periphera l nervous system 

POMGnT l UDP-N-acetylglucosamin e protein-O - 

mannos e ßl ,2-N-acetylglucosaminyltransferase 

POMC pro-opiomelanocorti n 

POMT1 protein-O-mannos e ßl,2-Nacetylglucosaminyltransferase 

PPI prepuls e inhibitio n 

PSD post-synapti c densit y 

PSN principa l sensor y nucleus of the trigeminal 

nerve 

PSR phosphatidylserin e receptor 

РТЕ prenata l tobacc o exposur e 

PTV Piccolo-Bassoo n transport vesicle 

PVE pseudostratifie d ventricula r epitheliu m 

PVN paraventricula r nucleu s 

PWM pokewee d mitoge n 

Q probabilit y of cell cycl e exit 

RA retinoi c acid 

RAR retinoi c acid recepto r 

Rb retinoblastom a protei n 

RG radia l glia 

ROS reactiv e oxygen specie s 

SC subcutaneou s 

SES socioeconomi c statu s 

Shh soni c hedgeho g 

SHR P stres s hyporesponsiv e perio d 

SKY spectra l karyotypin g 

SID S sudde n infan t deat h syndrom e 

SIN- 1 3-morpholinosydnonimin e 

Smac secon d mitochondria-derive d activato r 

ofcaspase 

SMase sphingomyelinas e 

Smo Smoothene d 

sMRI structura l magnetic resonanc e imaging 

SN substantia l nigr a 

SNARE S solubl e n-ethylmaleimide-sensitiv e 

fusion protein-attachmen t protei n 

receptor s 

SNAP-2 5 synaptosom e associate d protein-2 5 

SNR P stres s nonresponsiv e period 

SOD Superoxid e dismutase 

SPEC T singl e photo n emissio n compute d 

tomograph y 

SPP secondar y proliferative populatio n 

SV synapti c vesicle 

SynCAM synapti c cell adhesio n molecul e 

SZ subventricula r zon e 

SZa anterio r subventricular zone 

T c tota l lengt h o f the cell cycl e 

TCR T cell receptor s 

TER T telomeras e reverse transcriptas e 

TG F transformin g growth facto r 

TGFßlr transformin g growt h facto r ßl receptor 

TGFßllr transformin g growt h factor ßll receptor 

TH tyrosin e hydroxylas e 

TNF tumo r necrosis facto r 

TRF telornerase-repeat-bindin g factor 

Trk tyrosin e kinas e recepto r 

TÚNEL termina l deoxynucleotidy l transferase 

mediated deoxyuridine nick en d labeling 

UCS unconditione d stimulu s 

VB ventrobasa l nucleus of the thalamus 

VBM voxel-base d morphometry 

VDCC voltage-dependen t Ca 24 " channel 

VIP vasoactiv e intestinal polypeptide 

VLM ventrolatera l medulla 

VSD ventra l septi c defec t 

VTA ventra l tegmental area 

VZ ventricula r zone 

VLDLr ver y low density lipoprotei n recepto r 

WCST Wisconsi n Card Sort Task 

WRAML Wid e Range Assessment of Memory and 

Learning 

WWS Walker-Warbur g syndrom e

TONYA R . ANDERSO N 

Mount Sinai School of Medicine 

New York NY 

LAYLA AZAM 

University of California College of Medicine 

Irvine CA 

HENRIETTA S. BADA 

University of Kentucky College of Medicine 

Lexington KY 

GREGORY N . BARNE S 


Lexington KY 

IDDIL H. BEKIROV 


New York NY 

DEANNA L. BENSO N 


New York NY 

Contributors 

xiii 

PRADEEP G . BHID E 

Massachusetts General Hospital 

Harvard Medical School 

Boston MA 

MARLA B . BRUN S 

SI/NY- Upstate Medical University 

Syracuse NY 

SHREYA BUCH 


Lexington KY 

DESMOND T . CHEUN G 

Cornell University 

Ithaca NY 

JEROLD J.M. CHUN 

Scripps Research Institute 

La Jolla CA 

CLAIRE D. COLE S 

Emory University School of Medicine 

Atlanta GA

xiv CONTRIBUTOR S 

MARIE D. CORNELIU S 

University of Pittsburgh School of Medicine 

Pittsburgh PA 

MITA DAS 

University of Colorado Health Sciences Center 

Denver CO 

NANCY L. DAY 


Pittsburgh PA 

KATHERINE A. DEBELAK-KRAGTOR P 

University of Wisconsin School of Medicine 

Madison WI 

NAZIRA EL-HAGE 


Lexington KY 

BRENDA M. ELLIOT T 

Uniformed Services University of the Health Sciences 

Bethesda MD 

BARBARA L. FINLA Y 


Ithaca NY 

RYAN FRANKE 


Irvine CA 

SUSANNA L. FRYE R 

San Diego State University 

San Diego CA 

KATHY GALLARD O 


Irvine CA 

CONSUELO GUERRI 

Centro de investigacion Principe Felipe 

Valencia, Spain 

NEIL E. GRUNBER G 

Uniformed Services University of the Health Sciences 

Bethesda MD 

KURT F . HAUSE R 


Lexington KY 

GEORGE I. HENDERSO N 

University of Texas Health Science Center 

San Antonio TX 

PAULA L. HOFFMA N 


Denver CO 

HUAIYU H u 

SUNY-Upstate Medical University 

Syracuse NY 

FRANCES M . LESLI E 


Irvine CA 

TARA A. LINDSLE Y 

Albany Medical School 

Albany NY 

SHAHRDAD LOTFIPOUR 


Irvine CA 

HUIBERT D . MANSVELDE R 

Vrije Universitat, 

Amsterdam, The Netherlands 

CHRISTIE L. McGE E 


San Diego CA 

MICHAEL W. MILLE R 

Department of Neuroscience and Physiology and 

Developmental Exposure Alcohol Research Center 

SUNY-Upstate Medical University 

Research Service 

Syracuse Veterans Affairs Medical Center 

Syracuse NY 

G. DAVI D MINTZ 


New York NY 

SANDRA M. MOONEY 


Syracuse NY 

KATHRYN O'LEARY 


Irvine CA 

MARIA PASCUA L 

Centro de investigacion Prìncipe Felipe 


JAMES R. PAUL Y 


Lexington KY

STEVENS K . REHEN 

Scripps Research Institute 

La Jolla CA 

EDWARD P. RILE Y 


San Diego CA 

LORNA W. ROL E 

Columbia University 

New York NY 

GEMMA RUBER T 

Centro de investigacion Principe Felipe 


JULIE A. SlEGENTHALE R 


Syracuse N Y 

JOANNA H. SLIWOWSK A 

University of British Columbia 

Vancouver BC 

SUSAN M. SMIT H 

University of Wisconsin School of Medicine 

Madison WI 

ANDREA D . SPADON I 


San Diego CA 

BERNHARD SUTE R 

Massachusetts General Hospital 

Harvard Medical School 

Boston MA 

JOANNE WEINBER G 


Vancouver BC 

CONTRIBUTORS x v 

JENNIFER A. WILLFORD 


Pittsburgh PA 

JEREMY C.YOST 


Ithaca NY 

XINGQI ZHAN G 


Vancouver BC 

W. MICHAE L ZAWADA 


Denver CO


BRAIN DEVELOPMEN T


Models of Neurotoxicity 

Provide Unique Insight into 

Normal Development 

Happy familie s ar e al l alike ; ever y un - 

happy family i s unhappy i n its own way. 

Anna Karenina, Leo Tolstoy 

The developin g nervous system is comprised o f complex 

set s o f elements tha t assemble int o a dynamic, 

interactive physical , chemical , an d electrica l mesh . 

In th e norma l animal , thi s assembl y proceeds i n a n 

orderly progression amounting t o the su m of additive 

(cell proliferation, migration, and neurit e outgrowth) 

and subtractiv e processes (pruning of axons and den - 

drites and neuronal death). Each o f these ontogenetic 

processes involve s the timely , intricat e choreograph y 

of molecular an d cellular players . That said, develop - 

ment does not follow a rigid sequence . 

The developin g brai n ha s considerable flexibility 

and adaptability . This adaptabilit y ca n b e overcom e 

by a multitud e o f challenges , an d th e variet y o f re - 

sponses ca n be as diverse as the challenges. Th e chal - 

lenges can be internal or external. Internal alterations 

1 


3 

include changes cascading from a spontaneous mutation 

i n the genome . Externa l challenges ca n be sub - 

tle (response to a stress) or profound (change s cause d 

by exposur e t o a toxin o r drug). Alterations induced 

by interna l an d externa l challenge s ca n b e superim - 

posed an d change s ma y be additive or synergistic. In 

any case , a n understandin g o f th e response s o f th e 

nervous syste m provide s uniqu e insigh t int o it s nor - 

mal developmen t an d it s plasticity . Doe s th e chal - 

lenged nervou s syste m consistentl y respon d i n th e 

same fashion , o r doe s i t respon d usin g a variet y o f 

strategies? 

Tolstoy's comment on his own work was that Anna 

Karenina did not solve a single problem; rather, it presented 

the m all, beautifully. Environmental exposur e 

to substance s suc h a s ethanol an d nicotin e present s 

situations o f compelling clinica l interest . Ethano l i s 

arguably the most fully studied teratogen . Unlike Tol - 

stoy's self-critique , studies o f ethano l toxicit y hav e 

not bee n restricte d t o phenomenologica l studies ; 

many mechanism s o f ethano l neurotoxicit y hav e

4 INTRODUCTIO N 

been pursued . Indeed , wor k o n ethano l provide s a 

model o f how t o stud y the negativ e effect s o f othe r 

substances. Moreover , study of the effect s o f ethanol 

provides a uniqu e insigh t int o norma l development . 

The approac h of this book is to look at three contrast - 

ing situations: normal development, an d the effect s of 

two different type s of challenges. Examinatio n o f th e 

normal and abnormal situations informs each of these 

situations. Thus, i t is only through geneti c an d envi - 

ronmental challenge s tha t a ful l appreciatio n o f th e 

complexity of brain development can be achieved. 

NORMAL DEVELOPMEN T 

Our understandin g o f norma l neura l developmen t 

has progressed by quanta during the las t dozen years . 

Not onl y hav e w e begu n t o understan d th e myria d 

genes tha t ar e expresse d i n th e developin g centra l 

nervous syste m (CNS) , bu t function s fo r th e tran - 

scripts and th e protein s have been ascribed . Two examples 

ar e rln an d dix, gene s tha t cod e fo r protein s 

important in stopping neuronal migration and in promoting 

the migration of neocortical loca l circuit neurons 

from th e ganglion eminence . 

The compellin g abnorma l organizatio n o f th e 

brain an d th e associate d dysfunctio n o f th e reeler 

mouse hav e fascinate d developmenta l neurobiolo - 

gists for more than five decades (e.g. , Falconer, 1951 ; 

Alter et al, 1968; Bruc k and Williams, 1970 ; Lambert 

de Rouvroit and Goffinet , 1998) . The invers e organi - 

zation o f the reele r mous e ha s provide d uniqu e in - 

sights int o mechanism s o f normal neurogenesi s an d 

the formatio n of neural networks. Yet it is only since 

1995 that the genetic basi s for this murine mutant has 

been discovered , the knockout of the gen e rln (D'Arcangelo 

e t al. , 1995 ; Hirotsun e e t al. , 1995 ; Ogaw a 

et al. , 1995) . Th e functio n o f th e gen e product , 

reelin, continues to be debated. 

The gen e dix i s a key player i n cortica l develop - 

ment (Marin and Rubenstein , 2003). It is critical for 

the generatio n o f loca l circui t neuron s i n th e gan - 

glionic eminence. A dearth of local circuit neurons is 

evident i n dix knockou t mice . Thus , a ne w doo r t o 

understanding th e complexit y o f cortica l neurono - 

genesis an d tangentia l pathway s o f neuronal migra - 

tion has been opened . 

The section on normal development describes our 

current understandin g o f cellula r an d molecula r 

events tha t defin e developmenta l phenomena . Fo r 

convenience, neural development has been divided 

into four phases : cel l proliferation , migration, differ - 

entiation, and deat h (Chapter s 2, 3 , 4, an d 5 and 6 , 

respectively). Althoug h thes e ar e discret e phases , 

they are highly integrated. That is, the number o f cycling 

cells ha s a direc t effec t o n th e wa y in whic h 

cells differentiate and on the incidence o f cell death. 

Thus, these chapter s address a single developmental 

phase whil e placin g ontogeneti c event s i n th e con - 

text o f the continuu m o f neural development. I n a 

provocative essa y (Chapter 7 ) at the en d o f this sec - 

tion, basic information on ontogeneti c event s is discussed 

i n term s o f mammalian evolution , an d thei r 

ramifications o n th e respons e of the nervou s system 

are addressed. 

ETHANOL-AFFECTED DEVELOPMEN T 

The concep t that ethano l affect s th e developmen t of 

the mammalia n (human ) brain came t o the for e i n 

1973 wit h th e publicatio n o f paper s b y Jone s an d 

Smith (Jone s an d Smith , 1973 ; Jone s e t al, 1973) . 

These were not the first papers to identity fetal alcoho l 

effects. Thoug h les s celebrated , report s b y Lemoin e 

and colleagues (1968) and Ulleland (1972) had already 

described feature s o f feta l alcoho l syndrom e (FAS) . 

Although it has been suggeste d that the first evidence 

linking alcoho l consumptio n durin g pregnanc y an d 

deleterious effect s o n offsprin g come s fro m Gree k 

and Roma n mytholog y an d fro m th e Bible , this no - 

tion was debunked by Abel (1984). 

To the best of my knowledge, the first documentation 

o f someone wit h FA S can b e attribute d t o th e 

artists Harvey Kurtzman in 195 4 and Norman Ming o 

in 1956 . The y dre w a perso n originall y known as 

Melvin Coznowsk i o r Me l Haney , late r know n a s 

Alfred E . Neuman (also spelled Newman). 1 Mr. Neuman 

ha s al l o f the classi c pathomnemonic craniofa - 

cial malformation s of FAS (thin uppe r lip, deficient, 

philtrum, narro w palpebrai fissures, flattened bridge 

of th e nose , an d lo w set ears) , alon g wit h acknowl - 

edged menta l dysfunctio n and probabl e intrauterine 

growth retardation (a s evidenced b y microencephaly). 

Although verificatio n o f prenata l exposur e i s un - 

known, such evidence i s not obligatory for a diagnosis 

of FAS (Stratton e t al., 1996) . As is typical of accomplished 

artists, Kurtzman and Ming o were perceptive 

observers and mad e associations that were as insightful 

a s thos e o f th e experience d pediatrician s wh o

described FA S more than a doze n year s later. Kurtzman 

and Mingo did not associate the craniofacial malformations 

an d brai n dysfunctio n wit h prenata l 

exposure t o alcohol, but the y di d link the diagnosti c 

features that characterize FAS. 

Ethanol can act through a number of mechanisms. 

This is not to say that ethanol is a "dirty drug". Indeed, 

evidence show s tha t i t affect s th e nervou s syste m i n 

very specifi c ways . Moreover , th e manne r i n whic h 

ethanol alter s developmen t instruct s u s abou t th e 

adaptability an d resilienc y of the developin g nervous 

system. Fro m a clinical perspective, a n understandin g 

of the consequence s o f early exposure to ethanol i s of 

paramount importance . Prenatal exposur e to alcoho l 

affects upward s o f 1 % of al l liv e births an d i t i s th e 

prime cause of mental retardation in the Unite d States 

(Sokol e t al., 2003). Furthermore, ou r appreciatio n o f 

ethanol-induced change s provide s a model o f and in - 

sights int o th e etiolog y o f numerou s neurodevelop - 

mental disorders, such as autism and schizophrenia. 

The firs t se t of chapters i n Par t II addresses global 

effects o f prenata l exposur e t o ethanol . Thes e in - 

clude th e prevalenc e o f alcohol-induce d deficit s 

among human s (Chapte r 8) , the structura l malfor - 

mations cause d b y prenata l exposur e t o ethano l 

(Chapter 9) , an d th e effect s o f ethanol o n nervous 

system-endocrine system interactions (Chapte r 10) . 

Following this discussio n i s a series of chapters tha t 

focus o n th e fou r ontogeneti c phases : cell prolifera - 

tion (Chapter s 1 1 and 12) , migration (Chapte r 13) , 

differentiation (Chapte r 14) , and death (Chapters 1 5 

and 16) . The las t two chapters o f this section exam - 

ine th e effect s o f ethano l o n tw o population s o f 

neural-related cells : neura l cres t cell s (Chapte r 17 ) 

andglia (Chapter 18) . 

NICOTINE-AFFECTED DEVELOPMENT 

The fina l sectio n o f the boo k examines the respons e 

of th e nervou s syste m t o a nicotin e challenge . 

Whereas ethano l i s acte d upo n b y endogenou s 

enzymes—for example , catalas e an d alcoho l 

dehydrogenase —it does not act at a specific receptor . 

In contrast , nicotin e allegedl y act s though a focused 

endogenous mechanis m a t receptor s fo r cholinergic 

neurotransmission. An understanding of the effect s of 

nicotine i s not onl y importan t fo r appreciatio n o f a 

basic scienc e issue , i t i s o f critica l clinica l impor - 

tance. A fetus is exposed to nicotine though increase d 

NEUROTOXICITY AND NORMAL DEVELOPMENT 5 

blood concentrations in the pregnant woman as nicotine 

freel y crosse s the placenta l "barrier," and infant s 

are exposed to nicotine through secondhand smoke. 

The clinica l problem o f fetal nicotin e exposur e is 

discussed i n Chapter 19 . Nicotine i s a rather ubiquitous 

substanc e — for thos e wh o smok e an d fo r thos e 

who ar e expose d t o secondhand smoke . Nicotin e exposure 

is often accompanied b y exposure to other substances 

suc h a s alcohol . Thus , clinica l studie s hav e 

fostered th e nee d t o conduc t well-controlle d anima l 

studies to understand the behaviora l consequences of 

exposure t o nicotin e pe r s e (Chapte r 20) . Althoug h 

nicotine i s thought to act through specific cholinergic 

receptors, it has broader effects through catecholaminergic 

and othe r systems. These issues are examined i n 

Chapters 2 1 and 22 , respectively. Chapter 2 3 explores 

the effect s o f nicotine on specific ontogenetic events . 

Separate studie s o f norma l developmen t an d o f 

development altere d b y ethanol o r nicotin e provide 

unique, thoug h limited , insigh t int o brai n develop - 

ment. Eac h line of inquiry opens windows of understanding 

tha t otherwis e migh t b e inaccessibl e fro m 

other vantag e points . B y consolidating th e informa - 

tion gathered from the three approaches, however, we 

can gain a fuller appreciatio n (a ) of the complexit y of 

neural development, an d (b ) of how i t can g o wrong 

during a disease process. 

Note 

Abbreviations 

CNS centra l nervous system 

FAS feta l alcohol syndrome 

1. I t wa s alleged that Harvey Kurtzma n an d Norman 

Mingo were not th e first to dra w a face resemblin g 

Alfred E . Neuman. Indeed , two lawsuits (base d on 

copyrights file d i n 191 4 an d 1936 ) atteste d tha t 

Kurtzman and Mingo were not the originators of the 

caricature no w referre d t o a s Alfred E . Neuman. It 

was used in advertisements for dental practices (see a 

copy of the Manitoba Free Press in 192 8 [http://www 

.imagehosting.us/index.php?action = show&ident = 

728813]) and a n imag e was even used by the Nazi s 

as part o f their anti-Semiti c campaig n i n th e 1930 s 

(Djerassi, 1988 ; see http://garfield.library.upenn.edu/ 

essaysM2pl61yl989.pdf). [n.b. The nam e Alfred E . 

Neuman an d th e imag e of the gap-toothe d person 

were first joined i n 1956. ] The principa l (winning ) 

defense was that earlier artists had themselves copied

6 INTRODUCTIO N 

a likenes s produced befor e 1914 , notably "Mickey 

Dugan, The Yello w Kid" drawn by Richard Felton 

Outcault in th e lat e nineteent h century , an d various 

other cartoons and advertisement s (see a copy 

of th e Winnipe g Tribun e i n 190 9 [http://www r 

.imagehosting.iis/index.phpFaction = show&ident = 

728816]). Interestingly , most o f the earlie r versions 

depict a normal upper lip, a distinc t philtrum, an d 

normal-appearing eyes. Therefore, it does appear that 

Kurtzman an d Ming o were the first to associate th e 

three characteristi c features o f FAS : facial dysrnor - 

phology, mental dysfunction, and microencephaly. 

References 

Abel E L (1984 ) Fetal Alcohol Syndrome and Fetal Alcohol 

Effects. Plenum , Ne w York. 

(1995) An update on incidence of FAS: FAS is not 

an equa l opportunit y birt h defect . Neurotoxico l 

Teratol 17:437-443 . 

Alter M, Lieb o J, Desnick SO , Stromme r B (1968) Th e 

behavior of the reele r neurologica l mutan t mouse . 

Neurology 18:289 . 

Bruck RM, Williams TH (1970 ) Light and electro n microscopic 

stud y of the hippocampa l regio n o f normal 

and reeler mice. J Anat 106:170-171. 

D'Arcangelo G, Mia o GG, Chen SC , Soare s HD, Morgan 

JI, Curran T (1995) A protein related to extracellular 

matri x proteins delete d i n th e mous e mutan t 

reeler. Nature 374:719-723. 

Djerassi C (1988 ) Th e ques t fo r Alfre d E . Neuman . 

Grand Street 8:167-174. 

Falconer D S (1951 ) Tw o new mutations , trembler an d 

reeler with neurological action in the house mouse. 

J Genet 50:192-201 . 

Hirotsune S, Takahara T, Sasak i N, Hiros e K, Yoshiki A, 

Ohashi T, Kusakab e M , Murakam i Y, Muramatsu 

M, Watanabe S , Nakao K, Katsuki M, Hayashizakiy 

(1995) Th e reeler gene encode s a protei n with a n 

EGF-like motif expresse d b y pioneer neurons . Na t 

Genet 10:77-83. 

Jones KL , Smit h D W (1973 ) Recognitio n o f th e feta l 

alcohol syndrom e i n earl y infancy . Lance t 2:999 - 

1001. 

Jones KL , Smit h DW , Ullelan d CN , Streissgut h A P 

(1973) Patter n o f malformatio n i n offsprin g o f 

chronic alcoholic mothers. Lancet 1:1267—1271 . 

Lambert de Rouvrait C, Goffinet AM (1998) The reele r 

mouse a s a model o f brain development. Ad v Anat 

EmbryolCellBioll50:l-106. 

Lemoine P , Haroussea u H , Borteyr u J-P , Menue t J- C 

(1968) Le s enfant s de parent s alcooliques : anom - 

alies observées à propos de 12 7 cas. Ouest Me d 21 : 

476-482. 

Marin O , Rubenstei n J L (2003 ) Cell migratio n i n th é 

forebrain. Annu Rev Neurosci 26:441-483. 

Ogawa M , Miyat a T , Nakajim a K , Yagyu K , Seik e M , 

Ikenaka K , Yamamoto H , Mikoshib a K (1995) Th e 

reeler gene-associated antigen on Cajal-Retzius neu - 

rons is a crucial molecule for laminar organization of 

cortical neurons. Neuro n 14:899-912 . 

Sokol RJ , Delaney-Black V, Nordstrom B (2003) Feta l 

alcohol spectru m disorder . JAM A 290:2996 - 

2999. 

Stratton K, Howe C, Battagli a F ( 1996) Fetal Alcohol Syndrome: 

Diagnosis, Epidemiology, Prevention and 

Treatment. National Academy Science Press , Washington, 

DC . 

Ulleland CN (1972 ) The offsprin g o f alcoholic mothers. 

Ann N Y Acad Sci 197:167-169.

I 

NORMAL DEVELOPMENT


Cell proliferatio n i s the earlies t step in the protracted 

process of development o f the mammalian brain, and 

the pac e o f cel l proliferatio n and th e numbe r an d 

types of cells produced ca n be modulated b y a variety 

of genetic an d environmenta l factors . The dynamic s 

of the proliferatio n o f precursor cells in th e develop - 

ing mammalian brai n ar e shape d b y various factors. 

This chapte r describe s th e spatiotempora l feature s 

of cell proliferatio n and effect s o f neurotransmitters, 

major constituent s o f th e chemica l environmen t o f 

the developin g brai n tha t modulat e th e proces s o f 

precursor cell proliferation. It focuses on three neurotransmitters 

that are among the mos t abundant one s 

in th e developin g brain : dopamine , y-aminobutyri c 

acid (GABA) , and glutamate . Th e goa l i s to presen t 

an overview of the organization and activit y of precursor 

cell populations and discuss the potential fo r modulation 

of precursor cell activity by neurotransmitters. 

The discussio n i s limited t o cell proliferation in tw o 

exemplary structure s i n th e centra l nervou s syste m 

(CNS): the cerebral cortex and neostriatum. 

2 

Cell Proliferatio n 

Bernhard Suter 

Pradeep G. Bhide 

9 

ORGANIZATION OF PRECURSOR 

CELL POPULATIONS I N 

THE EMBRYONIC TELENCEPHALON 

Cells of the nervou s system arise from precurso r cell s 

lining the inne r o r ventricular surface o f the neura l 

tube. The precurso r cell s are organized as a pseudostratified 

columnar epithelium. The epitheliu m is commonly 

referre d t o a s the pseudostmtified ventricular 

epithelium (PVE) , a s it borders th e ventricula r cavi - 

ties of the embryoni c nervous system (Boulder Committee, 

1970 ; Takahash i e t al. , 1993) . Th e regio n of 

the neura l tube o f the feta l nervou s system that con - 

tains th e PV E i s called th e ventricular zone (VZ) , a 

histologically define d regio n surroundin g the ventri - 

cles. Most cells in the PVE are bipolar and span virtually 

the entir e distanc e betwee n th e pia i (outer ) and 

the ventricular (inner) surfaces (Fig. 2-1A), especially 

during earl y neural tub e stages . A small numbe r o f 

cells ar e round an d contact only the ventricula r surface; 

most of these cells are mitotically active.

FIGURE 2- 1 Diagra m o f the architectoni c an d cytokineti c compartment s i n 

the developing mammalian brain . The ventricula r surface is to the lef t and th e 

piai surface to the right. A. Four cytoarchitectonic compartments can be recognized 

o n th e basi s o f cytologica l criteri a (Boulder Committee, 1970 ; Smart , 

1976): the ventricular zone (VZ, or the ependymal layer), subventricular zone 

(SZ, o r the subependyma l layer) , the intermediat e zon e (IZ , o r th e externa l 

sagittal stratum), and three differentiating fields—a subplate (SP), cortical plate 

(CP), and marginal zone (MZ). Cells in the VZ are organized a s a pseudostratified 

columna r epithelium , wherea s the S Z and SP/CP/M Z contain randoml y 

oriented, nonepithelial cells. The I Z consists predominantly of columnar arrays 

of bipolar cells and growin g axons (black lines). The border s of the S Z and I Z 

are ambiguous. The dashed lines indicate the approximate borders between th e 

different compartments . B . Precursor cells can be classifie d int o two groups on 

the basi s of their interkinetic nuclear migratory pattern: a pseudostratified ven - 

tricular epithelium (PVE ) and a secondary proliferati ve population (SPP) . PVE 

cells undergo interkineti c nuclea r migratio n (blac k arrows) such that nuclei of 

cells in M phase of the cell cycle are located a t the ventricular surface and th e 

nuclei o f cells i n S phase ar e farthes t fro m th e ventricula r surface. Nucle i of 

cells in Gl an d G2 phase s are at intermediate distances . SP P cells do not undergo 

interkinetic nuclear migration, and the nuclei of cells in the different cel l 

cycle phases are scattered throughout the VZ and I Z (soli d black arrows). PVE 

cells that leave the cell cycl e exit the VZ, migrate through th e S Z and IZ (broken, 

curved arrows), and settle in the CP/MZ. SPP cells that exit the cell cycl e 

also migrate to the CP/MZ. C. The spatial distribution of PVE, SPP , and CP/MZ 

cells i s illustrated to sho w the overla p among th e proliferativ e and postmitoti c 

populations. Th e PV E near the ventricular surface is a "pure" population; how - 

ever, the S-phas e zone (wher e nuclei of cells undergoing S phase are located) of 

the PV E overlap s with the SPP . The CP/M Z i s a population o f predominantly 

postmitotic cell s near the piai surface (right-hand side of the figure); however, in 

the SZ/IZ area, some postmitotic cell s mix with precursor cells of the SPP . Thus, 

the architectonic and cytokinetic compartments show considerable overlap in the 

developing nervous system. (Source: Modifie d from Bhide , 1996) . 

10

At late r stage s o f feta l development , variou s re - 

gions interpose betwee n th e VZ and the piai surface . 

Thus, additiona l architectoni c subdivision s and a n 

additional precurso r populatio n emerge . A n earl y 

population o f precursor cell s tha t emerge s i s calle d 

the secondary? proliferative population (SPP) . The ar - 

chitectonic subdivision in which the SPP comes to lie 

is called the subventricular zone (SZ) (Boulder Com - 

mittee, 1970) . Around the sam e time, a preplate ap - 

pears between th e S Z and th e pia i surfac e (Boulde r 

Committee, 1970 ; Marin-Padilla, 1983) . Shortly thereafter, 

th e preplat e i s split into a marginal zon e (MZ ) 

and subplat e b y the cortica l plat e (CP) . These thre e 

zones (the subplate, CP, and MZ) are fields that con - 

sist largely of postmitotic o r differentiatin g cell s pro - 

duced by the PVE and SPP precursors. In some parts 

of the nervou s system such as the cerebra l cortex, another 

architectoni c subdivisio n called th e intermediate 

zone (IZ ) develops . The I Z lie s between th e S Z 

and th e M Z an d contain s growin g axons, migrating 

postmitotic neurons, an d cell s in glial lineages. Note 

that the VZ, SZ, CP, and MZ are architectonic subdivisions 

o f the nervou s system whereas th e PV E an d 

SPP are cytokinetic subdivisions of the precursor populations. 

Th e architectoni c an d cytokineti c subdivisions 

overlap extensively, as shown in Figure 2-1C. 

The PV E i s a n interactiv e communit y o f cells , 

coupled t o on e anothe r vi a intercellula r junction s 

such as gap junctions (LoTurco and Kriegstein, 1991 ; 

Bittman e t al, 1997) . PV E cell s shar e biochemica l 

characteristics with radial glia (Hockfield and McKay, 

1985; Mille r an d Robertson , 1993 ; Malatest a e t al, 

2000, 2003; Noctor et al, 2001; Weissman et al, 2003). 

Until recently, radia l gli a wer e considere d onl y t o 

serve th e dua l function s of guiding newly generate d 

neurons t o thei r fina l destination s an d actin g a s a 

transitional stage in astrocytic ontogeny (Rakic , 1972; 

Voigt, 1989 ; Takahash i e t al. , 1990 ; Misso n e t al , 

1991; Miller and Robertson , 1993) . 

The PV E cell s underg o interkineti c nuclea r migration, 

a hallmar k o f pseudostratifie d epitheli a i n 

general (Sidma n e t al. , 1959 ; Fujita , 1960) . Thus , 

nuclei of the PVE cells are distributed within the VZ 

in accordance with the cel l cycle phase (Fig . 2-1B). 

Nuclei undergoing mitoses are located a t the ventricular 

border, whereas the nuclei in the DNA synthesis 

or S-phas e ar e i n abventricula r locations . Nucle i o f 

cells passing through Gl an d G2 are located at intermediate 

distances between th e tw o poles. The inter - 

kinetic nuclea r migrator y pattern distinguishe s the 

CELL PROLIFERATION 1 1 

PVE from th e SP P (His, 1887; Sauer, 1936 ; Boulder 

Committee, 1970 ; Takahashi e t al, 1993) . SPP cells 

are not organized into an epithelium an d d o not display 

interkineti c nuclea r migratio n (Raki c e t al. , 

1974). The SP P is a major component of the precursor 

cel l poo l i n som e part s o f th e nervou s system , 

such a s the neostriatum and the cerebra l cortex , and 

not i n othe r parts , suc h a s the hippocampu s o r th e 

hypothalamus. 

The PV E is a "pure" population o f precursor cell s 

in th e sens e tha t daughte r cell s produce d b y it an d 

that embark upon the path of differentiation int o neurons 

o r gli a exi t th e PV E rapidl y (Miller, 1999) . I n 

other words , a snapshot of the VZ, which house s th e 

PVE, reveal s proliferatin g precursor cell s an d virtu - 

ally n o differentiatin g neuron s o r gli a (Fig . 2-1C) . 

The SZ , b y contrast , whic h include s th e SPP , i s a 

mixture o f proliferating SPP cell s an d differentiating 

daughter cells generated fro m bot h th e PVE and th e 

SPP. These difference s i n the cellula r environment s 

may bestow unique physiological features on the PVE 

and SP P precursors and their differential response s to 

environmental substances such as ethanol (see Chapter 

11) . The intimat e cell-cell contact amon g precur - 

sor cell s i n th e V Z ma y facilitat e contact-mediate d 

signaling betwee n proliferatin g cells , whic h i s les s 

likely t o occu r i n th e SZ , althoug h contac t amon g 

precursor cell s an d postmitoti c cell s ma y b e mor e 

likely in the SZ . Close proximity of the SP P to growing 

axons in the I Z (Fig. 2-1A), in some part s of the 

nervous syste m suc h a s th e cerebra l wall , increase s 

the molecular diversity of the environment further . 

Neurons and glia arise from bot h the PV E and th e 

SPP. Contributions o f the PV E and the SP P to the total 

number s of cells i n different part s of the brai n vary 

significantly. Fo r example , i t has bee n suggeste d tha t 

the PV E and SP P generate neuron s primaril y in th e 

deep and superficial laminae of neocortex, respectively 

(Miller, 1989 ; Miller and Nowakowski, 1991) (Chapter 

11). In the neostriatum, the SP P appears to be the major 

contributor of neurons and glia, whereas in the hippocampus 

proper , th e PV E generate s al l cel l types . 

The PV E in the hippocampa l formatio n is the sourc e 

of pyramidal neurons of the hippocampus and granule 

neurons of the dentat e gyrus , early in the feta l perio d 

(Angevine, 1965 ; Stanfield an d Cowan , 1979 ; Altman 

and Bayer , 1990) . I n th e dentat e gyru s cells derive d 

from th e PV E earl y i n th e embryoni c period settle at 

the border between the granule cell layer and the hilar 

region an d continu e t o proliferate , formin g a secon d

12 NORMA L DEVELOPMENT 

and apparentl y permanen t precurso r poo l calle d th e 

subgranular, or intrahilar zone (IHZ) (Angevine, 1965 ; 

Stanfield an d Cowan , 1979 ; Altman and Bayer , 1990) . 

The IH Z is the sourc e o f the majorit y of granule neu - 

rons of the dentate gyru s later in development, including 

adult life (Angevine , 1965 ; Bayer , 1982 ; Camero n 

et al, 1993 ; Gag e et al, 1998 ; va n Praag et al, 2002 ; 

Kempermann et al., 2004). 

Although th e architectoni c an d cytokineti c subdivisions 

mentioned above appea r i n nearly all parts of 

the developin g telencephalon , considerable regiona l 

variation occur s i n term s o f the siz e o f the differen t 

FIGURE 2- 2 Regiona l difference s i n the siz e of the cytoarchitectoni c compart - 

ments i n th e developin g brain . A histologica l sectio n i n th e corona l plan e 

though th e hea d o f an 11-day-ol d mouse fetus is shown. The fetu s was exposed 

to th e S-phas e marke r bromodeoxyuridine (BrdU ) fo r 6 hours; Brd U was injected 

int o th e pregnan t da m an d th e fetu s wa s harvested 6 hours later . Th e 

section wa s processe d immunohistochemicall y t o revea l th e distributio n o f 

BrdU i n proliferatin g cells. BrdU-labele d cell s appear dar k and surroun d th e 

ventricular cavities of the brain (empty space at the center o f the micrograph). 

The compartment s o f the telencephalo n visibl e a t thi s corona l leve l ar e th e 

hippocampus (HIP) , cerebra l neocortica l wal l (CX) , latera l ganglioni c emi - 

nence (LGE) , and medial ganglionic eminence (MGE) . O n the left-hand side , 

the broken line marks the approximate boundar y between th e ventricular zon e 

(VZ) and the subventricular zone (SZ), and the solid line marks the boundary 

between th e S Z and the intermediat e zon e (IZ) . The broke n line i s drawn on 

the basi s of difference s i n th e packin g densit y and dispositio n o f the BrdU - 

labeled cell s (highe r densit y i n th e VZ , aligne d i n compariso n t o th e SZ , 

which has a lower density of BrdU-labeled cell s that are randomly distributed) . 

The soli d line is at the boundary between BrdU-labele d (precursor ) and unla - 

beled (nonproliferatin g or differentiating) cells . A relatively large VZ, SZ , and 

IZ area i s in the ganglioni c regio n (LG E an d MGE ) compare d t o that i n th e 

dorsal C X an d hippocampa l anlag e (HIP) , eve n a t thi s earl y stage o f brain 

development. Th e higher-magnificatio n image s (right ) sho w th e histolog - 

ical divisions of VZ, SZ, and PPZ in the different telencepahlic compartments . 

The primordi a of eyes (EYE) are also visible in this section. (Source: Modifie d 

from Shet h and Bhide, 1997 )

subdivisions and th e tim e o f emergence o f each sub - 

division i n the cours e of embryonic development. I n 

fact, these regiona l difference s distinguish th e precur - 

sors o f the differen t telencephali c compartments . I n 

the telencephalon of the developin g mous e brain, regional 

variation is evident as early as gestational da y 

(G) 1 1 (the day of conception i s designated a s GO). In 

coronal section s o f 11-day-ol d feta l mous e brain , a 

sizeable SZ can be seen i n the lateral ganglionic emi - 

nence (GE ) an d media l GE . Thes e specialize d seg - 

ments of the S Z contain precursor s of neurons i n th e 

basal ganglia and a source o f neocortical G AB A neu - 

rons (Fig . 2-2) . Th e murin e cerebra l cortex , hip - 

pocampus, an d hypothalami c region s do no t hav e a 

SZ at G11. In fact, an SZ does not develop i n the hippocampal 

and hypothalamic regions , although it does 

develop i n th e cerebra l corte x abou t 2- 3 day s later 

(Miller, 1999) . 

The V Z is the earlies t structure i n the developin g 

nervous system , thus i t contains th e earlies t forming 

precursor cel l populatio n (i.e. , the PVE) . Th e V Z is 

present i n al l part s o f the Gil telencephalo n (Fig . 

2-2). A distinct and sizeabl e MZ , consisting o f newly 

generated differentiatin g cells, is evident in the lateral 

and medial GEs by Gl 1, whereas the other regions of 

the telencephalon hav e a relatively smaller MZ at this 

age. Thus, the siz e and distributio n o f both proliferative 

and postmitotic subdivisions of the telencephalon 

show considerabl e variatio n i n th e feta l forebrain . 

The emergenc e o f heterogeneit y withi n th e telen - 

cepahlic anlage reflects influences of transcription factors 

(Puelles and Rubenstein , 1993 ; Rubenstein e t al., 

1998) and/o r neurotransmitter s (Ohtan i e t al. , 2003) 

that distribute in a regionally specific manner. The re - 

gional variation also foreshadows the structura l differ - 

ences tha t emerg e i n thes e region s a s developmen t 

proceeds. 

MOLECULAR REGULATION OF 

CELL PROLIFERATION 

Cell proliferatio n in th e nervou s syste m i s an intri - 

cately regulated process . The regulatio n occur s by the 

concerted action s of a variety of genetic an d environmental 

factor s a t the leve l of the cyclin g cells i n th e 

PVE an d SPP . Ther e ar e a t leas t thre e interrelated 

tiers of regulation: (1 ) the tota l numbe r o f cells pro - 

duced b y the PVE and SPP , (2) the emergence o f specific 

phenotype s amon g th e daughte r cells , an d (3 ) 


FIGURE 2- 3 Rol e played b y the cyclin-dependen t kinase 

(Cdk) inhibitor p27 Kl P 1 i n the regulatio n o f cell 

cycle a t th e Gl-to-S-phas e transition . Th e progres - 

sion of the cell cycle can be regulated at the transition 

from th e G 2 to M phase o r from th e Gl t o S phase . 

Genetic an d environmenta l factor s tha t modulat e 

progression o f cell cycl e i n th e developin g brai n ap - 

pear t o influence th e Gl-to-S-phase transition. Thi s 

transition i s regulate d b y Cdk s (Cdk2 , Cdk4 , an d 

Cdk6) an d their cyclin partners (cyclins A, E, and D) . 

Association o f Cdks an d thei r cycli n partners inacti - 

vates the retinoblastom a protein (Rb) by phosphorylation, 

which i n turn promote s th e transitio n fro m Gl 

to S phase b y releasing th e transcriptio n facto r E2F . 

p27 Kl P 1 inhibit s th e activit y of the Cdk-cycli n com - 

plexes an d therefor e inhibit s the transitio n fro m G l 

to S phase. Thus, genetic an d environmenta l factor s 

that alte r th e expressio n an d activit y of p27 Kl P 1 ca n 

modulate cel l proliferation in the developing brain. 

the temporal sequenc e of cell production an d specification 

tha t i s unique t o each regio n o f the brain . An 

understanding of the molecular mechanisms that regulate 

these processes is important not onl y for appreciating 

cytogenesi s durin g norma l developmen t bu t 

also for understanding (a ) the wa y in which cel l production 

ca n b e induce d i n specifi c region s o f th e 

adult brain to repair injury b y trauma o r disease an d 

(b) the mechanisms by which neoplasms in the CN S 

originate. 

Genetic an d environmenta l factor s regulat e cel l 

proliferation b y acting upo n th e molecula r machin - 

ery that regulates the cell cycle. This machinery con - 

sists of protein kinase s and thei r regulator y subunits , 

known a s cyclin-dependen t kinase s (Cdks ) an d cy - 

clins, respectively (Fig. 2-3). Essentially , Cdk activity


depends o n the synthesis and destruction o f cyclins as 

well as on the function o f Cdk-activating kinases and 

Cdk inhibitor s (LaBaer et al. , 1997 ; Morgan , 1997 ; 

Cheng et al, 1999 ; Sherr and Roberts, 1999 ; Murray, 

2004). Eac h Cd k ha s a preferre d cycli n partner(s) . 

For example, Cdk 2 associate s wit h cyclin s A and E , 

whereas Cdk 4 an d Cdk o associat e wit h D-typ e cy - 

clins. Association of Cdks wit h cyclin s results in th e 

regulation of the cel l cycle at two principal transition 

points: th e transition s fro m th e G l t o S phase, an d 

from G 2 t o M (Murra y and Hunt , 1993 ) (Fig . 2-3) . 

Cdk2, Cdk4 , an d Cdk 6 ar e principall y involved i n 

the regulation of the transition from Gl t o S, whereas 

Cdkl regulate s the G2- M transition . The transitio n 

from G l t o S phas e i s associate d wit h additiona l 

mechanisms o f regulatio n involvin g th e retinoblas - 

toma protei n (Rb ) and th e transcriptio n factor E2F . 

Association o f Cdk s wit h cyclin s inactivate s R b b y 

phosphorylation, which in turn result s in the releas e 

of E2F . E2 F promotes th e transitio n fro m G l t o S 

and DNA synthesis (Dyson, 1998 ; Frolov et al, 2001 ). 

Control ove r cell cycl e progressio n a t the Gl-to - 

S-phase transitio n i s exercise d b y Cd k inhibitors , 

which inhibi t th e activit y of th e Cdk-cycli n com - 

plexes (Ferguso n e t al., 2000) . Inhibitio n o f Cdk ac - 

tivity b y specifi c Cd k inhibitor s suc h a s p27 Ki P ] o r 

p21 Wafl suppresse s the entr y into the S phase (Zind y 

et al, 1997 , 1999 ; Delall e e t al., 1999 ; Siegenthale r 

and Miller, 2005) (Fig. 2-3). Muc h o f the regulation 

of cytogenesi s i n th e developin g brai n i s thought t o 

occur a t the Gl/S transitio n (Cavines s e t al, 1996 ; 

Ross, 1996) . Transcriptio n factors , growt h factors , 

hormones, an d neurotransmitter s ar e amon g th e 

most commo n regulator s o f cel l proliferatio n i n 

the developin g brain . They are believed t o act upo n 

the Gl/S transitio n poin t t o increase o r decrease th e 

output of neurons or glia (Caviness et al., 1996 ; Ross , 

1996). 

Much o f our understandin g of cell cycl e regula - 

tion derives from i n vitro studies. That said, the PV E 

and SP P cell s i n th e intac t mammalia n brai n diffe r 

from th e precursor cells maintained i n a Petri dish i n 

one importan t aspect. PV E an d SP P cell s in th e in - 

tact brain serve a histogenetic agenda, which warrants 

generation o f a specific number o f specific cell type s 

in a spatiotemporal gradient over a restricted period of 

time. Therefore, t o understand mechanisms that reg - 

ulate CNS histogenesi s we need to analyze cell prolif - 

eration i n population s o f PV E o r SP P cell s i n th e 

intact brain . A challeng e face d b y developmenta l 

neurobiologists i s to assimilat e mechanisti c insights , 

gained b y analysis of cell cycl e regulatory machinery 

from studie s of dissociated cells , into analyses of cel l 

cycle kinetic s an d cel l outpu t fro m population s o f 

PVE or SPP cells i n th e intac t brain . Th e followin g 

section describe s recent progress i n efforts along those 

lines. Complementary insigh t into these issue s is provided 

b y studyin g the proliferativ e response i n th e 

developing brai n followin g ethano l exposur e (se e 

Chapters 1 1 and 12) . Much of our knowledg e o f th e 

kinetics of PVE and SPP cell cycle in the intact brain 

is derive d fro m analyse s o f neocortica l PV E cells . 

Therefore, th e followin g section focuse s mainl y o n 

cell cycle kinetics of neocortical PVE . 

CELL CYCLE KINETICS 

AND DYNAMIC S OF 

CELL PROLIFERATION I N 

THE NEOCORTEX 

In an y tissue or organ system, the precursor cell popu - 

lation initiall y expands by producing large numbers of 

new precurso r cells . Th e precurso r cel l populatio n 

subsequently deplete s itsel f through (a ) the productio n 

of large numbers o f daughter cells , which exi t the cel l 

cycle, differentiate , an d acquir e matur e phenotypes , 

and (b ) th e deat h o f cyclin g cell s (se e Chapte r 5) . 

This type o f biphasic growth pattern ensure s the pro - 

duction o f a large numbe r o f cells withi n a specified 

time interval . Th e neocortica l PV E follow s thi s 

growth pattern. It undergoes two phases of growth: an 

initial phase of expansion during which mos t daugh - 

ter cell s retur n t o th e precurso r pool , an d a late r 

phase o f depletion whe n mos t o f the daughte r cell s 

differentiate int o neuron s o r glia (Smart, 1976 ; Cavi - 

ness e t al, 1995 ; Mille r an d Kuhn , 1995 ; Takahash i 

et al, 1996 , 1997) . Interestingly , during the phas e of 

expansion, th e PV E grow s in th e tangentia l dimen - 

sion, and thi s growth result s in expansion o f the PV E 

such tha t i t cover s th e underlyin g subcortica l struc - 

tures. There i s little expansio n o f the PV E i n th e ra - 

dial dimension ; growt h i n th e radia l dimensio n i s 

seemingly reserve d fo r late r developmenta l period s 

when newbor n neuron s an d gli a are release d fro m a 

depleting PV E (Miller, 1989) . 

The us e o f th e S-phas e marke r [ 3 H]thymidine 

(pH]dT) revolutionize d th e analysi s of neuroepithelial 

cel l cycl e kinetics by permitting identification of 

nuclei in S phase and tracking the nuclei labeled i n S

phase as they passed through G2 , M, and Gl phase s 

of the cel l cycl e (Sidman e t al., 1959 ; Angevine and 

Sidman, 1961 ; Fujit a an d Miyake , 1962 ; Sidman , 

1970). I t becam e possibl e t o estimat e cel l cycl e pa - 

rameters an d th e tim e o f generation o f different cel l 

types throug h cumulativ e o r puls e labelin g wit h 

[ 3 H]dT. Recen t developmen t o f nonradioactiv e 

S-phase marker s bromodeoxyuridin e (BrdU ) an d 

iododeoxyuridine further enhance d th e versatilit y of 

these method s (Mille r and Nowakowski , 1988, 1991 ; 

Takahashi e t al , 1992 ; Haye s an d Nowakowski , 

2000; Tarui et al., 2005). 

S-phase labeling methods hav e been exploited, often 

combinin g th e radioactiv e an d nonradioactiv e 

markers t o perfor m sophisticate d analyse s of cell cy - 

cle kinetic s an d cel l outpu t function s o f PV E an d 

SPP cells in the embryonic murine neocortex (Miller 

and Nowakowski , 1991; Cavines s e t al, 1995 ; Haye s 

and Nowakowski , 2000 ; Takahash i e t al , 2002 ; 

Siegenthaler and Miller , 2005 ; inter alia) . This work 

shows that the neocortical PVE cells in fetal mic e executed 

1 1 cell cycles throughout the cours e of generation 

of neocortical neuron s over the week from Gil 

to G17. During the 1 1 cell cycles, the length of the cell 

cycle increases (from abou t 8 to 1 8 hours) (Fig. 2-4) , 

the increas e bein g du e t o th e lengthenin g o f G l 

(from abou t 3 t o 1 2 hours). This pattern i s also de - 

tected i n ra t neocortica l proliferativ e zone s (Mille r 

and Kuhn, 1995). 

The probabilit y that a new daughter cel l exit s the 

cell cycl e (calle d Q ) increase d durin g th e perio d of 

cortical neuronogenesi s (Miller , 1993 , 1999 ; Mille r 

and Kuhn , 1995 ; Takahashi e t al, 1999) . I t was near 

zero fo r cel l cycl e 1 (o n Gil fo r the mouse ) an d 

nearly 100 % for cell cycle 1 1 (G16-17). Eac h cell cycle 

is associated with specific values of Q and cell cycle 

length (i.e. , the lengt h o f the Gl-phase) , the two 

parameters that change (increase) systematically over 

the cours e o f the 1 1 cel l cycle s (Fig . 2-4) . Th e Q 

value reache s th e halfwa y mar k during the sevent h 

and eight h cel l cycles , o n approximatel y G14 (Fig . 

2-4). Thi s probability value marks a significant milestone 

i n the lif e history of neocortical PVE , a s it indicates 

the switc h fro m th e expansiv e to the depletiv e 

phase of growth and fro m tangentia l to radial expansion 

o f the neocortex . Furthermore , i t mark s a significant 

mileston e i n neocortica l neuronogenesis : 

generation of neurons for the infragranular layers (layers 

V an d VI ) cease s an d tha t fo r the granula r layer 

(layer IV ) and supragranula r layers (layer s II and III) 


begins (Fig . 2-4) . Thus , quantitative estimates of the 

values o f Q a t each intege r cell cycl e hav e enable d 

the linkag e of Q to laminar destination of the daugh - 

ter cells. In other words, a novel concept emerges, i n 

which th e mechanism s regulatin g Q ar e linke d t o 

mechanisms regulatin g cel l fat e o r cel l clas s (Taru i 

et al, 2005). 

Neuronogenesis acros s th e cortica l mantl e doe s 

not procee d simultaneously ; i t abides by various spatiotemporal 

gradients (Smart, 1961; Smar t and McSh - 

erry, 1982 ; Miller , 1988a ; Baye r and Altman , 1991) . 

The transvers e gradients reflect th e gradient s in cel l 

cycle kinetics, especially the valu e of Q, i n the PV E 

(Caviness et al., 2000, 2003; Tarui et al, 2005). Thus, 

PVE cell s are distribute d along a spatia l continuu m 

of cell cycl e parameters and Q . PV E cell s located a t 

the rostrolatera l locatio n ar e mor e advance d tha n 

those a t the caudomedial location with respect to cell 

cycle length and values of Q. That is, rostrolateral precursor 

cell s hav e longe r cel l cycl e time s an d highe r 

values of Q than those of caudomedial precurso r cell s 

on any given day during the interval Gil t o G17. For 

example, a given location i n the neocortical PV E contains 

precursor cell s passin g through th e entir e range 

of cell cycle times and Q values over the interval Gil 

to G17. At any given time, th e neuroepithelium con - 

tains a range of cell cycl e times and Q values . Cells 

destined to multiple layer s in multiple neocortica l ar - 

eas aris e contemporaneousl y a t multipl e location s 

within the neuroepithelium. 

Apart fro m th e tangentia l neurogeneti c gradien t 

discussed above, neocortical neuronogenesis proceeds 

along a radial dimension, a characteristic common t o 

laminar structures throughout th e nervou s system. At 

any give n locatio n o f the neocortica l anlagen , neu - 

rons are generated i n an inside-out pattern (Angevin e 

and Sidman , 1961 ; Berr y et al , 1964 ; Berr y and 

Rogers, 1965 ; Caviness , 1982 ; Miller , 1985) . Deep - 

layer neuron s ar e generate d earlie r than th e superficial 

laye r neurons . Usin g a double S-phas e labelin g 

method, Takahashi an d colleague s (1999 ) correlate d 

the lamina r position of a neocortical neuron with its 

"birth hour. " Throug h th e us e o f this method , the y 

were able to correlate the time of generation and laminar 

destination of neocortical neurons with considerably 

higher spatiotempora l resolutio n tha n ha d bee n 

possible previously. 

A model correlatin g th e probabilit y of cell cycl e 

exit (which is equivalent to Q) with the probabilit y of 

the destinatio n o f a neuron i n a specifi c neocortica l

16 NORMA L DEVELOPMEN T 

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FIGURE 2- 4 Spatiotempora l gradient s o f cell proliferatio n and neurogenesi s i n th e cerebra l cortex . 

Neurogenesis in the cerebra l cortex progresses along tangential (A ) and radia l (B) gradients. A. Neo - 

cortical neurogenesi s begin s at the rostroventrolatera l regio n o n gestationa l da y (G) 1 1 in mice an d 

proceeds toward the caudodorsomedial regio n in a systematic gradient (curved arrow). This transverse 

neurogenetic gradien t translates into a corresponding gradient in cel l cycle length o f the precursor 

population an d the probability of cell cycle exi t (Q) for the daughte r cells . Thus, precursor cell s in 

the pseudostratifie d ventricula r epithelium (PVE ) i n the rostroventrolatera l region have longer cell 

cycle time and highe r values of Q compare d to their counterparts in the caudodorsomedial region. 

B. Neurogenesis als o proceeds alon g a radial gradient. Neurons of the deep layers are produced ear - 

lier than those in the superficial layers. Each neocortica l PVE cell in mice, on average, undergoes 1 1 

cell cycles over a 7-day period from Gil to G17. Cell cycle length increase s with each cell cycle and 

each cycle is associated with a unique value of Q. Thus, the cel l cycle length increase s from abou t 8 

to 1 8 hours over the cours e o f 1 1 cell cycles . The valu e of Q increase s from abou t 0. 1 t o 0.8 , ap - 

proaching the maximal value of 1.0, during this interval and reaches the halfway mark (0.5) betwee n 

cell cycle s 7 and 8 on G1 4 (broken line an d asterisk) . Each cel l cycl e produces cell s destine d fo r 

specific neocortical layer(s). Thus, cell cycle number, cell cycle length, Q, and layer destination are 

linked to one another during normal development. (Source: Base d on data of Takahashi et al., 1999 ; 

Tarili et al., 2005) 

layer wa s the n develope d b y Takahash i an d col - 

leagues (1999) . Theoretically , neuron s produce d a t 

any time point during the neurogenetic sequenc e ca n 

have som e probabilit y of occupying any neocortica l 

layer. I n reality , however , durin g norma l develop - 

ment neuron s generate d durin g cel l cycl e 1 hav e 

nearly a 100 % probabilit y of occupying laye r VI an d 

0% probability of occupying layer II—III, and neuron s 

generated durin g cel l cycl e 1 1 (th e fina l cel l cycle ) 

have a near 100 % probability of occupying layer II—III 

and 0 % probabilit y of occupyin g laye r VI . Sinc e a 

given cell cycle number is associated with given values

of Q, anothe r wa y of interpreting these finding s i s that 

the probability that a cell arising from a given cell cycle 

will occupy a specific laye r is tightly linked t o the values 

of Q. Lower Q values predict destination to deeper 

layers and higher values predict destination t o more superficial 

layers. What factors set the probability values? 

Overexpression of the cd k inhibitor p27 ki P* in th e 

PVE increases Q, and that the neurons produced during 

the cell cycles with the experimentally elevated Q 

values assum e superficia l lamina r position s tha t 

would be appropriate for the higher value of Q (Tarui 

et al. , 2005) . Therefore , Q an d lamina r destinatio n 

appear t o b e highl y correlated . Also , thes e finding s 

suggest that p27 kl P* expression and activit y play a role 

in setting the value s of Q. Recen t dat a sho w that an - 

other cdk inhibitor, p21, is also involved in cell cycle 

exit (Siegenthaler an d Miller , 2005) . Furthermore, a 

variety of genetic and environmenta l factors , such as 

transcription factors , growt h factors , an d neurotrans - 

mitters, influenc e intracellula r p27 ki P J an d p21 wofl 

content. Th e combine d action s o f these factor s ma y 

regulate Q and , therefore , neocortica l neurona l fat e 

during normal development . 

The discussio n above pertains directly to the regulation 

o f cel l proliferatio n i n th e neocortica l PVE . 

Neocortical neuron s aris e not only from the neocorti - 

cal PVE but also from the proliferative cells in the GE . 

Neocortical PV E i s the sourc e o f neocortica l (gluta - 

matergic) projection neurons (PNs), which constitut e 

70%-90% o f al l neocortica l neuron s (Peter s e t al , 

1985; Re n e t al, 1992 ; Beaulieu , 1993) . I n rodents , 

the majorit y o f GABAergi c loca l circui t neuron s 

(LCNs) o f th e cerebra l cortex , whic h constitut e 

10%-30% o f all neocortica l neurons , i s produced i n 

the media l an d cauda l GE s o f th e basa l forebrai n 

(Anderson et al, 1997a ; Tamamaki e t al, 1997 ; Lavdas 

et al, 1999 ; Powel l et al., 2001; Nery et al., 2002; 

Ang et al, 2003). These LCNs arrive in the cerebra l 

cortex via tangential migrator y pathways. In primates , 

however, i t appear s tha t mos t GABAergi c LCN s o f 

the cerebra l corte x derive from th e neocortica l PV E 

and only a minority come from the GÈ (Letinic et al., 

2002). Although neocortica l PN s and LCN s ar e produced 

i n different locations , both types of neurons located 

i n th e sam e neocortica l laye r appea r t o b e 

produced o n the same day, at least in rodents (Miller , 

1985, 1988b ; 1999 ; Fairé n e t al, 1986 ; Goba s an d 

Fairén 1988 ; Valcanis and Tan, 2003). 

Neocortical gli a ar e generate d i n th e SP P 

(Smart, 1961 ; Smar t an d LeBlond , 1961 ; L e Vine 


and Goldman, 1988a , 1988b ; Levison and Goldman , 

1993). One notabl e exception to this pattern is that of 

radial glia , which commonl y hav e cel l bodie s i n th e 

PVE, an d may undergo transformation into astrocytes 

near th e en d o f th e neuronogeneti c epoc h (Voigt , 

1989; Takahash i e t al , 1990 ; Misso n e t al , 1991 ; 

Miller an d Robertson , 1993) . A distinguishable SP P 

(SZ) appear s i n th e neocortica l proliferativ e regio n 

soon afte r th e first cortical neuron s are generated, b y 

G13 i n th e mous e (Miller , 1989 ; Takahash i e t al, 

1995). At this time, th e siz e of the SP P is


Bhide, 1997 ) (Fig . 2-2) . Therefore , th e SP P i s likely 

to b e a major sourc e o f neurons o f the neostriatum . 

This finding concurs with that in th e neocorte x (se e 

Chapter 11) . Studie s o n th e Distalless-relate d tran - 

scription factor s dlxl, dlx2, an d dlxS, whic h regulate 

cell proliferatio n and migratio n i n th e GE , confir m 

the origin of striatal projection neuron s from th e SP P 

of th e G E (Anderso n et al, 1997b ; Stenma n e t al. , 

2003). Furthermore , analyse s o f transgeni c mic e 

show that the radial glia in the PV E of the lateral and 

medial GE s giv e ris e t o nearl y all o f the gli a i n th e 

neostriatum and onl y to a subset of LCNs (parvalbumin 

and Er81-positive LCNs). 

Two studies using S-phase labeling methods com - 

parable t o thos e use d fo r th e neocortica l PV E esti - 

mated th e cel l cycl e parameters and cel l output fro m 

the PV E o f the latera l G E (Bhide , 1996 ; Shet h an d 

Bhide, 1997) . The cel l cycl e wa s longer an d th e values 

o f Q wer e greate r i n th e latera l G E o f mice o n 

Gil and G12 compared t o the corresponding parameters 

in the neocortica l PVE. Estimatio n of cell cycle 

kinetics of the PV E o f the latera l GE a t later stages of 

development i s complicated b y the fac t tha t durin g 

the late r developmenta l stage s thi s regio n acquire s 

migrating cell s produce d i n the adjacen t media l G E 

(Anderson et al., 2001). 

The strikin g structural and functiona l complexity 

of th e neostriatu m (Graybiel , 1990 ; Gerfen , 1992 ) 

has le d t o a number o f investigations of the tim e o f 

generation o f neostriata l neurons . Earl y studie s fo - 

cused o n the tim e of generation o f neurons destine d 

for th e patc h (striosome ) an d matri x compartments . 

These can be identified on the basis of distribution of 

opioid receptor s or neuropeptide conten t of the neu - 

rons. Neuron s destine d fo r th e patc h compartmen t 

are generated earlie r than thos e destine d for the ma - 

trix in rodents , carnivores , and primate s (Bran d an d 

Rakic, 1979 ; Graybie l an d Hickey , 1982 ; Fishel l an d 

van der Kooy, 1987 ; Fishel l et al, 1990; Son g and Harlan, 

1994). Other studies, which examined the time of 

generation o f neostriatal neurons regardles s o f loca - 

tion of a neuron within the patc h o r matrix compartments, 

found some overla p in the tim e o f generation 

of PNs an d LCN s (Bran d an d Rakic , 1979 ; Fernan - 

dez et al, 1979 ; Bayer , 1984). In rodents, striatal neu - 

rons containing different type s of neurotransmitters or 

neuropeptides ar e generate d a t distinc t period s o f 

neurogenesis (Semb a e t al. , 1988 ; Sadiko t an d Sas - 

seville, 1997) . Neostriata l neurogenesi s als o follow s 

spatiotemporal gradient s (Fernande z e t al. , 1979 ; 

Marchand and L a joie, 1986; Bayer and Altman, 1987 ) 

that are less systematic than th e transvers e and radial 

gradients definin g neocortica l neuronogenesis . I n 

fact, gradient s o f neuronogenesis ar e difficul t t o de - 

fine in nonlaminar structures suc h as the neostriatu m 

(Bayer and Altman, 1987) . 

NEUROTRANSMITTERS AND 

CELL PROLIFERATION 

Cell proliferatio n i s shape d b y a variet y of environ - 

mental factors such as growth factors and neurotrans - 

mitters. Growth factors, as the name indicates, tend to 

be growth-promotin g agent s and thei r influence s o n 

cell proliferatio n are no t unexpected . Som e growt h 

factors, such as transforming growth factor (3, however, 

have a negative effec t o n cell proliferation (see Chapter 

11) . Neurotransmitter s hav e well-characterize d 

functions a t the synaps e in the matur e brain. Neuro - 

transmitters and their receptors appear in the nervous 

system earl y i n th e feta l period , befor e synapti c 

contacts develop. Thus, it can be inferred that neurotransmitters 

influenc e developmental processes . Non - 

vesicular transpor t an d releas e o f neurotransmitter s 

(Démarque e t al. , 2002 ) and paracrin e mechanism s 

for neurotransmitter receptor activation (Owens et al., 

1999) hav e bee n propose d i n th e feta l brain . Suc h 

mechanisms ar e distinc t fro m thos e use d b y neuro - 

transmitters in the matur e brain, as are the composi - 

tion an d activit y o f neurotransmitte r receptors . Fo r 

example, GAB A receptors i n th e feta l brain diffe r i n 

their subunit composition, affinity , sensitivity , and signal 

transductio n mechanism s relativ e to their coun - 

terparts i n th e matur e brai n (Owen s and Kriegstein , 

2002). Thus, a role for neurotransmitters in th e feta l 

brain tha t ma y b e separat e fro m thei r rol e a t th e 

synapse i n th e matur e brai n appear s likel y (Lander, 

1988; Levit t et al, 1997 ; Nguye n et al, 2001; Owens 

and Kriegstein , 2002; Ohtani e t al, 2003). The influence 

of dopamine, GABA, and glutamate on cell proliferation 

an d neurogenesi s i n the neocorte x an d th e 

neostriatum is discussed below. 

Dopamine 

Dopamine appear s in the developing mouse brai n as 

early as G13 (Ohtani e t al., 2003). The cerebra l cor - 

tex an d th e neostriatu m expres s among th e highes t 

amounts o f dopamin e receptor s i n th e feta l brai n

(Sales e t al , 1989 ; Guennou n an d Bloch , 1993 ; 

Schambra e t al, 1994 ; Bakowsk a and Morrell, 1995 ; 

Jung and Bennett , 1996 ; Dia z e t al., 1997 ; Shearma n 

et al., 1997 ; Ohtani et al., 2003). Dopaminergic axons 

originating in the midbrain penetrat e th e VZ and S Z 

of the prefronta l cortex and latera l G E (precurso r of 

the neostriatum ) o f the mous e brai n a s early as G13, 

and growt h cone s of these axons are in close proxim - 

ity t o PV E an d SP P cell s i n thes e region s (Ohtan i 

et al., 2003; Popolo e t al., 2004) . 

Dopamine recepto r activatio n influences cell pro - 

liferation i n the latera l GE an d the dorsomedia l pre - 

frontal corte x (PFC) . I n th e latera l GE , Dl-lik e 

receptor activatio n reduce s entr y int o S (i.e. , an an - 

timitogenic effect ) an d D2-lik e receptor activatio n increases 

th e Gl/ S transitio n (i.e. , a mitogeni c effect ) 

(Zhang and Lidow, 2002; Ohtani et al, 2003 ; Popol o 

et al, 2004 ; Zhang et al, 2005) . Dopamine itself produces 

Dl-lik e effects , presumabl y becaus e o f th e 


preponderance o f Dl-lik e bindin g site s i n th e feta l 

brain (Ohtan i e t al, 2003) . Dopamine recepto r acti - 

vation influence s cel l proliferatio n i n th e neuroep - 

ithelium o f the PF C i n a manner comparabl e t o that 

in the lateral GE . 

Activating dopamine receptor s differentiall y affect s 

precursor population s i n th e V Z an d S Z (Fig . 2-5) . 

Precursor cell s i n th e V Z respon d predominantl y t o 

Dl-like receptor activation, whereas the precursors in 

the SZ respond to D2-like receptor activation (Ohtani 

et al, 2003 ; Popolo e t al, 2004) . VZ cells i n the lat - 

eral ganglionic region are more responsiv e to Dl-lik e 

receptor activatio n tha n thei r counterpart s i n th e 

PFC; a large r dos e o f th e Dl-lik e recepto r agonis t 

SKF 81297 is required to elicit a significant response 

from th e PF C (Popol o e t al , 2004) . Furthermore , 

D2-like recepto r activatio n did not produc e change s 

in th e proliferatio n o f precursor cel l i n the neocorti - 

cal VZ, although i t produced effect s i n the V Z of the 

FIGURE 2- 5 Schemati c representatio n o f distribution o f Dl-like an d D2-lik e 

receptors (R) . Dl-R and D2-R i n the ventricular an d subventricular zone s (V Z 

and SZ , respectively) of the telencepahli c neuroepithelium (A ) and th e effect s 

of dopamine recepto r activation on cel l proliferation (B) are shown. A. On th e 

basis o f dat a o n incorporatio n o f th e S-phas e marke r bromodeoxyuridin e 

(BrdU), precurso r cell s i n th e V Z appea r t o respon d t o Dl- R activatio n to a 

greater extent than D2- R activation, whereas the precursor cells in the S Z have 

an opposite respons e (Ohtan i e t al, 2003 ; Popolo e t al, 2004) . B. Activation of 

Dl-R decrease s th e Brd U labeling index , which i s a reflection o f a decrease i n 

the relative proportions of proliferating cells entering S phase. Activation of D2- 

R produces th e opposite effect (i.e. , increases BrdU labeling index) . Dopamine 

produces effect s simila r to thos e produce d b y Dl-R activation . (Source: Modi - 

fied from wor k by Popolo e t al, 2004) .


lateral ganglioni c region. Thus , significan t regiona l 

variation exist s in the responsivenes s of progenitors i n 

the neuroepithelia l domains of the dorsa l and ventral 

forebrain of the mouse fetus. This pattern likel y reflects 

variation i n th e relativ e abundance o f dopamine re - 

ceptor binding site s or differences i n signal transduction 

mechanisms . 

The Dl-lik e famil y o f dopamine receptor s i s coupled 

to G proteins, which activate adenylate cyclase and 

increase intracellular cyclic adenylate monophosphat e 

(cAMP) concentration s (Monsm a e t al. , 1990) . In - 

creased intracellula r cAM P conten t leads t o increases 

in the amounts of the Cdk inhibitor p27 Kl P ] , which in 

turn inhibit s entry int o S (Kat o et al. , 1994 ; L u an d 

DiCicco-Bloom, 1997) . Thus , th e antimitogeni c ef - 

fects o f Dl-like recepto r activatio n ma y b e mediate d 

via the cAMP-p27 Kl P 1 pathway. Other report s suggest 

that th e effect s o f Dl-lik e recepto r activatio n o n 

neocortical cel l proliferation is mediated vi a a cAMP - 

independent pathway involving Raf-1, a component o f 

the recepto r tyrosin e kinase-mitogen-activate d kinas e 

pathway (Zhang et al., 2005). Therefore, dopamin e re - 

ceptor activation may activate multiple second messen - 

ger systems in neuroepithelial cells . 

GABA 

GABA receptors appear early in the neuroepithelium 

and M Z o f the feta l brai n (Huntle y et al, 1988 ; Lo - 

Turco and Kriegstein, 1991 ; LoTurco e t al., 1995 ; Behar 

e t al., 1998 ; Hayda r et al., 2000). GABA receptors 

in th e feta l brai n ar e excitator y an d influenc e cell 

proliferation i n the VZ and SZ . Early studies indicate 

that GABA A receptor activatio n decreased entr y into 

S (antiproliferative ) i n neocortica l precurso r cell s 

(LoTurco e t al., 1995). These effects appear to be mediated 

by endogenously released GABA , as inhibition 

of the GABA A receptor s increase d Gl - to S entry in 

expiants o f neocortica l tissu e take n fro m ra t fetuse s 

(LoTurco e t al, 1995) . The effect s o f GABAA recep - 

tor inhibition were developmentally regulate d i n that 

the effect s wer e pronounce d a t G19 , bu t no t a t 

G14-G16 (LoTurc o e t al, 1995) . Presumably , thi s 

finding reflects maturational changes i n the activity of 

the receptors . Th e effect s o f GABA A recepto r activa - 

tion, lik e th e effect s o f dopamine recepto r activation , 

differ between the VZ and the SZ. GABAA receptor activation 

increase s Gl- t o S entr y i n th e V Z an d de - 

creases i t i n th e S Z (Hayda r e t al , 2000) . Th e 

intracellular signalin g cascad e tha t mediate s th e ef - 

fects o f GAB A recepto r activatio n o n th e cel l cycl e 

machinery is not fully understood. The effect s ma y be 

secondary t o suppressio n o f the actio n o f mitogenic 

factors such a s basic fibroblast growth factor (Antono - 

poulosetal, 1997) . 

Glutamate 

Glutamate recepto r activation influences the prolifera - 

tion o f precursor cell s i n th e cerebra l corte x and th e 

neostriatum. Activatio n o f bot h N-methyl-D-aspartat e 

(NMDA) and a-amino-3-hydroxy-5-methyl-4-isoxazol e 

propionate (AMPA)/kainat e receptors influence s Glto-S 

entry in the precursor cells . Early studies indicate d 

that activatio n of AMPA/kainate receptors b y endoge - 

nous glutamate decrease d entr y into S in the neocorti - 

cal V Z (LoTurc o e t al. , 1995 ; Hayda r e t al, 2000) . 

Activation o f AMPA/kainit e receptors , however , pro - 

duced th e opposit e effect s i n the S Z by increasing entry 

int o S (Hayda r e t al, 2000) . Although glutamat e 

receptor activatio n als o influence d proliferatio n o f 

neostriatal precursors , th e effect s wer e mediate d vi a 

the NMD A recepto r famil y rathe r tha n throug h th e 

AMPA/kainate receptor s (Lu k e t al. , 2003 ; Lu k an d 

Sadikot, 2004) . NMD A recepto r activatio n promote d 

entry into S among neostriata l precurso r cell s (Sadikot 

et al, 1998 ; Luk et al., 2003; Luk and Sadikot, 2004). 

CONCLUSIONS 

The discussio n above presents a n overvie w of the ef - 

fects of neurotransmitters on the proces s of cell prolif - 

eration. A summary of the effect s of dopamine, GABA , 

and glutamat e o n cel l proliferatio n in th e neocorte x 

and neostriatu m o f the developin g brain is presented 

in Table 2-1 . Thes e data sho w that imbalanc e i n the 

neurochemical environmen t o f the developin g brain 

can alte r th e proces s o f generatio n o f neuron s an d 

glia. Although th e direction of the effects may vary depending 

o n th e recepto r typ e activate d and th e pre - 

cursor population involve d (i.e., increase or decreas e 

in proliferation), it is reasonable t o assume tha t expo - 

sure o f proliferatin g precurso r cell s t o therapeuti c 

or illici t drug s tha t targe t an d produc e imbalance s 

in th e neurotransmitte r system s ca n produc e signifi - 

cant change s i n th e numbe r an d phenotyp e o f cells 

generated.

*Zhang and Lidow (2002); Popolo et al. (2004). 

f Popolo et al. (2004). 

'Ohtanietal. (2003). 

**Haydar et al. (2000); Owens and Kriegstei n (2002). 

M LoTurcoetal. (1995). 

-Sadikot et al. (1998); Luk et al. (2003); Luk and Sadiko t (2004). 

Abbreviations 

AMPA a-amino-34iydroxy-5-methyl-4-isoxazol e 

propionate 

BrdU Bromodeoxyuridin e 

e AMP cycli c adenylate monophosphat e 

Cdk cyclin-dependen t kinas e 

CNS centra i nervou s syste m 

CP cortica l plat e 


GABA y-aminobutyri c acid 

GÈ ganglioni c eminenc e 

[ 3 H]thymidine [ 

3 H]dT 

IHZ intrahila r zone 

IZ intermediat e zon e 

LCN loca l circuit neuro n 

MZ margina l zone 


PFC prefronta l cortex 

PN projectio n neuro n 

PVE pseudostratifie d ventricular epitheliu m 

Q probabilit y of cell cycle exit 

Rb retinoblastom a protei n 

SPP secondar y proliferative population 

SZ subventricula r zone 


References 


TABLE 2- 1 Summar y of effects o f dopamine, GABA, and glutamat e receptor activation on S-phas e labeling 

indices in neocortical and neostriatal precursor populations of the fetal brain 

Neocortex 

Neostriatum 

Precursor 

Population 

VZ 

sz 

VZ 

sz 

Dl 

Decrease* 

Decrease 1 

f f 

Decrease 

Decrease n 

Dopamine 

D2 

No effect 1 

Increase * 

Increase fî 

Increase u 

GABA 

Receptor Subtype 

GABAA 

Increase** 

Decrease ft 

Decrease** 

Not known 

Not known 

NMDA 

No effect - 

No effect * * 

1 f 

Increase 

No effec t 

Glutamate 

AMPA/Kainate 

Increase** 

Decrease**-"'** 

Decrease** 

No effect * * 

Noeffect u 

No effect 1 ' 

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During developmen t o f the mammalia n centra l ner - 

vous syste m (CNS) , neuron s ar e commonl y gener - 

ated at sites far from their final location (se e Chapters 

2, 11 , and 12) . They migrate to a n anlag e before differentiating 

int o thei r fina l morpholog y an d makin g 

synaptic connection s (se e Chapte r 4) . Disturbance s 

of neuronal migratio n underli e brai n disorder s suc h 

as menta l retardatio n an d epileps y (Dobyn s an d 

Truwit, 1995 ; Raymon d e t al. , 1995 ; Dobyn s e t al, 

1996). 

Neuronal migratio n i s widespread throughout th e 

developing periphera l nervou s system. For example, 

neurons usin g luteinizin g hormon e releasin g hor - 

mone (LHRH ) are generated in the developing olfactory 

placode , a transient structur e outsid e th e brain . 

They migrat e lon g distance s t o thei r fina l location s 

in th e media l basa l forebrai n an d hypothalamus . I n 

contrast, som e neuron s migrat e fro m th e CNS . A 

prime exampl e i s neural cres t cells . Thes e cell s ar e 

derived fro m th e dorsa l neural tub e an d giv e rise t o 

3 

Neuronal Migration 

Huaiyu Hu 

27 

the neurons an d glia of the peripheral nervou s system 

as well as non-neural cells , fo r example, facia l skele - 

ton, segment s o f the heart and great arteries, and pigment 

cells in the skin (see Chapter 17) . 

Most studie s o f neuronal migratio n hav e focuse d 

on neocortex. This is (a) because defect s in neuronal 

migration causing clinical disorders are commonly associated 

wit h corte x an d (b ) becaus e corte x ha s a 

highly defined structural an d functional organizatio n 

that derive s chiefly fro m a n orderl y neuronal migra - 

tion. Cortica l neurons ar e generated i n the neuroep - 

ithelium surrounding the lateral ventricles (Chapter s 

2, 11 , and 12) . The rule s defining neuronal migratio n 

appear to be similar among mammalian species. This 

present chapter focuses on rodents . 

Neuronal migratio n involve s fou r coordinate d 

steps outlined below. 

1. Th e proces s begins as cells pass through mitosis 

and permanentl y exi t from th e cel l cycl e (se e


Chapters 2 and 11) . I n th e rodent , thi s occur s 

during the secon d hal f of gestation (Angevin e 

and Sidman , 1961 ; Lun d an d Mustari , 1977 ; 

Gardette e t al, 1982; Miller, 1985) . 

2. Postmitoti c neuron s prepar e t o migrate . The y 

remain i n the proliferativ e zone s for up to 18 - 

26 hours, th e amoun t o f time increasin g wit h 

the age of the fetu s (Miller, 1999) . Durin g thi s 

time youn g neuron s exten d thei r leadin g pro - 

cesses whic h boas t pioneerin g filopodi a an d 

laniellipodia that explor e th e environment an d 

define th e directio n o f movemen t (Rakic , 

1972). They may move laterally within the proliferative 

zones (Noctor e t al., 2004). 

3. Th e neuron s actively move from th e prolifera - 

tive zone s throug h th e intermediat e zon e 

and ente r th e immatur e gra y matter. This ste p 

involves th e choreographe d movemen t o f 

the nucleus toward the leading process and th e 

retraction o f th e trailin g proces s (Hatte n an d 

Mason, 1990 ; Raki c et al, 1996) . Neuronal migration 

i n th e neocorte x o f mic e an d rat s i s 

completed b y the end of the first postnatal week 

(Berry and Rogers , 1965 ; Miller, 1999) . 

4. When a neuron reaches its final destination, its 

nucleus cease s th e forwar d movement . A t 

this site , th e neuro n differentiate s and elabo - 

rates a characteristic morphology (Miller, 1981 , 

1986). 

The migratio n o f neocortical neuron s can follow a 

radial o r tangential pat h (Rakic , 1990; Miller , 1992 ; 

Nadarajah an d Parnavelas , 2002) (Fig. 3-1) . Th e former 

describes a process whereby neurons migrate in a 

direction perpendicula r t o th e surfac e of the latera l 

Protein and Function 

Filamin A, actin binding protein 

Doublecortin, microtubule-associated protei n 

LISl/PAFAHIb, interacts with tubulin, dynein, 

NUDEL, mNUDE 

Reelin, ECM protei n 

POMT1, presumed glycosyltransferas e 

POMT2, presumed glycosyltransferase 

POMGnTl, glycosyltransferase 

Eukutin, presumed glycosyltransferase 

Large, presumed glycosyltransferas e 

FIGURE 3- 1 Multipl e migratio n route s o f cerebra l 

cortical neurons . Cerebra l cortica l projectio n neu - 

rons ar e generate d i n th e ventricula r zon e (VZ ) 

and subventricula r zon e (SZ ) o f the developin g ce - 

rebral wall . Th e migratio n rout e o f thes e neuron s 

is radia l (1) . Loca l circui t neuron s ar e generate d 

in th e media l ganglioni c eminenc e (GE) . The y 

undertake several migration routes (2 , 3, and 4) that 

involve tangentia l migratio n an d radia l migration . 

CP, cortical plate ; IZ, intermediate zone ; MZ, mar - 

ginal zone . 

ventricles (pathwa y 1) , wherea s th e latte r refer s t o 

neurons as they migrate along a pathway that parallels 

the surfac e o f th e latera l ventricl e (pathway s 2-4) . 

This chapter review s the basic principles of radial and 

tangential migration , molecula r event s underlyin g 

normal neurona l migration , an d som e molecula r de - 

fects tha t distur b neuronal migratio n to cause neuro - 

logical disorders (Table 3-1) . 

TABLE 3- 1 Genetic s of neuronal migration disorders in humans 

Gene 

FLNA 

Dcx 

PAFAHIB 

Rein 

POMT1 

POMT2 

POMGnTl 

FCMD 

Large 

Structural Phenotype 

Peri ventricular heterotopia 

Double cortcortex syndrome s 

Subcortical band heterotopi a 

Type I lissencephaly 

Lissencephaly 

Walker- Warburg syndrome Polymicrogyria 

Walker- Warburg syndrome Polymicrogyria 

Muscle-eye-brain disease Polymicrogyria 

Fukuyama congenital muscula r dystroph y 

Polymicrogyria 

MDC1D, Polymicrogyria

PATHWAYS O F MIGRATIO N 

Radial Migratio n 

Most cortica l neurons , notabl y projectio n neurons , 

are derive d from progenitor s i n th e ventricula r zon e 

(VZ) and subventricula r zone (SZ) . These zones are 

located alon g the interna l aspect o f the cerebra l wall. 

Most VZ an d S Z cell s migrat e t o corte x via a radial 

pathway. Radial migration appear s t o involv e at leas t 

two mechanisms. 

The firs t cohor t o f neurons tha t move s ou t o f the 

germinal area forms th e preplat e (Marin-Padilla , 1978 ; 

Allendorfer and Shatz, 1994). During the earliest stages 

of cortical development, neuron s take positions in th e 

preplate vi a somatic translocatio n (Britti s e t al. , 1995 ; 

Miyata e t al, 2001; Nadarajah e t al, 2001; Nadaraja h 

and Parnavelas , 2002). Each o f these neuron s send s a 

long process toward the piai surface as it leaves the VZ . 

Subsequently, a migrating neuron release s its ventricular 

attachments, but maintains its piai contacts. 

The second , an d large r cohor t of migrating neu - 

rons split s the preplat e int o tw o layers: th e marginal 

zone (MZ) and the subplate (SP) , the anlage of layers 

I and VIb, respectively. These newly arrived neurons 

take a position between th e MZ and SP and form the 

cortical plate (CP). The C P i s laid down by an insideout 

sequenc e i n which early-generate d neuron s tak e 

a positio n i n th e deepe r C P an d wave s o f later - 

generated neuron s migrat e t o positio n i n succeed - 

ingly superficia l position s (Angevin e an d Sidman , 

1961; Rakic , 1974 ; Miller , 1985 ; McConnell, 1995) . 

The CP differentiates int o layers Il-VIa. 

Neurons mov e int o th e C P migrat e radiall y via a 

different mod e o f migration tha n tha t use d b y neu - 

rons forming the preplate . Th e CP-destine d neuron s 

use radia l glia a s a physical substrate for their migration 

(Rakic, 1972,1990; Hatten, 1999) (Fig . 3-2). Dur - 

ing migration , eac h neuro n ha s a shor t leadin g 

process that i s not attache d t o the pia i surface and a 

trailing process that eventuall y lose s it s contact wit h 

the ventricular surface. Radial glial guidance provides 

a mechanism fo r later-generated neuron s to find positions 

beyond the postmigratory neurons, hence establishing 

the inside-out pattern characteristic of the CP. 

This migration is relatively fast. Apparently, regardless 

of when th e neuron s ar e generated, the y migrate at a 

rate of ~120|im per da y (Miller, 1999) . Simila r rates 

are eviden t i n th e radia l migratio n o f hippocampal 

neurons from th e VZ (Nowakowski and Rakic, 1981). 

NEURONAL MIGRATION 2 9 

FIGURE 3- 2 Radia l migration during development o f 

the cerebral cortex. Neurons migrate along the radial 

glia (RG ) to the cortica l plate (CP ) wher e later-born 

neurons migrat e pas t the early-bor n neurons . Reeli n 

(illustrated as dots) secreted by Cajal-Retzius neuron s 

(CR) i n th e margina l zon e (MZ ) i s required fo r th e 

inside-out patter n o f lamination . Withi n th e MZ , 

radial glia l endfee t (EF ) for m th e so-calle d glial - 

limiting membrane. Togethe r wit h some Cajal-Retzius 

cells, thes e endfee t appos e th e brai n surfac e base - 

ment membrane. SP, subplate. 

Radial gli a span th e ful l dept h o f the developin g 

cerebral wall. In the intermediat e zon e (IZ) , the radial 

glia l fiber s ar e groupe d i n fascicle s (Gressen s 

and Evrard, 1993). I n the mouse , these cell s are evident 

during the perio d o f neurogenesis and beyon d 

the cessatio n o f neurona l migratio n i n neocorte x 

(Miller and Robertson , 1993) . After neurona l migra - 

tion i s complete, radia l glia appea r t o transfor m to 

astrocytes (Schmeche l an d Rakic , 1979 ; Misso n 

et al , 1988 ; Voigt , 1989 ; Mille r an d Robertson , 

1993). Fo r many years, the identit y of radial glia has 

been considere d par t o f th e astrocyt e lineage . O n 

the basi s of recent data , however , i t appears tha t radial 

gli a ar e no t gli a —rather, the y ar e neura l ste m 

cells (Malatest a e t al. , 2000 ; Miyat a e t al. , 2001 ; 

Noctor e t al, 2001) . The y expres s markers for cytoskeletal 

protein s fo r immature (stem ) cells , fo r example, 

nesti n an d RC-2 , an d the y ca n generat e 

neurons and glia.


Tangential Migratio n 

Analyses of forebrain neuron s with retroviral markers 

show tha t a neurona l clon e derive d fro m a singl e 

founder i s dispersed far wider than woul d have heen 

predicted i f neurons onl y migrate d radiall y (Luski n 

et al, 1988; Walsh and Cepko, 1988) . Although most 

neurons ar e tightl y distribute d i n radia l column s 

of -200 |Llm, a notabl e numbe r ar e locate d a s muc h 

as 2 mm from this column. In these clones, cells tend 

to b e projectio n neuron s o r loca l circui t neuron s 

(LCNs) (Luski n et al., 1988 , 1993 ; Parnavelas et al, 

1991). Th e distributio n o f th e retrovirall y labele d 

cells is equally broad fo r clones o f projection and lo - 

cal circuit neurons. This dispersion principally results 

from the tangential movemen t o f cells within the proliferative 

zones before they ascend radially. 

On th e basi s of findings from trac t tracing studies, 

it appear s tha t neuron s generate d i n th e ganglioni c 

eminence (GE ) migrat e t o neocorte x (d e Carlo s 

et al., 1996) . Molecular an d anatomica l studie s show 

that thes e neuron s ar e LCN s (e.g. , Anderso n e t al. , 

1997; Powel l e t al , 2001 ; Marin an d Rubenstein , 

2003). These LCN s migrat e throug h th e SZ , IZ , or 

MZ (Fig. 3-1) (Tamanak i e t al, 1997 ; Lavdas et al , 

1999; Anderso n e t al , 2001 ; An g et al , 2003) . Accordingly, 

th e neuron s loo p aroun d th e latera l G È 

and cours e alon g th e VZ-SZ interface, in the I Z external 

t o th e SZ , o r throug h th e MZ . Afte r findin g 

their tangential position, the neurons associate with a 

radial gli a an d migrat e radiall y t o thei r appropriat e 

laminar residence. Thi s is supported b y data showin g 

that LCNs migrate to cortex via an inside-ou t patter n 

(Miller, 1985 , 1986, 1992; Fairén étal, 1986 ; Gobas 

and Fairén , 1988) . A n exceptio n i s a subpopulatio n 

of LCN s tha t expres s vasoactiv e intestina l polypep - 

tide (Miller , 1992) . Fo r thes e neurons , ther e i s n o 

birthdate- or lamina-specific pattern of the migration. 

The tangentia l pathway s ar e importan t becaus e 

many neocortica l LCN s migrat e vi a on e o f thes e 

pathways; dix knockout mice (mic e in which n o neurons 

ar e generate d i n th e GE ) hav e les s tha n onequarter 

o f th e LCN s eviden t i n wild-typ e mic e 

(Anderson e t al , 1997) . Thi s findin g concur s wit h 

those fro m retrovira l tracin g studie s showin g tha t 

clones commonly consist of either projection neurons 

or LCNs (Luski n et al, 1988 , 1993; Parnavelas et al , 

1991). Recen t evidenc e (An g et al , 2003 ; Nocto r 

et al, 2004 ) show s that LCN s derive d fro m th e G E 

that migrate at the VZ-SZ interface can dip into the 

VZ and re-ente r th e cell cycl e before they begin thei r 

radial migration . This observatio n contributes to th e 

finding that cells remain in the proliferative zones before 

the y commenc e thei r radia l migratio n (Miller , 

1999). Nevertheless , th e tangentia l migratio n is suffi - 

ciently fas t tha t LCN s generate d i n th e G E an d co - 

hort projectio n neuron s generate d concurrentl y i n 

the VZ and S Z migrate to cortex i n tandem (Miller , 

1985, 1986 , 1988, 1997). 

Neurons migratin g tangentiall y rel y o n divers e 

substrates. LHRH expressin g neurons migrat e on axons 

as they mov e t o their targe t (Schwanzel-Fukud a 

and Pfaff , 1990) . Othe r migratin g neuron s migrat e 

over "resident " cells . Thes e includ e olfactor y LCN s 

migrating throug h th e anterio r subventriciila r zon e 

(SZa) o n thei r wa y t o th e olfactor y bul b (Luskin , 

1993; Loi s et al.,1996). The substrat e for the tangen - 

tial migration of neurons from th e media l GE t o neocortex 

i s unknown; there i s no axona l or glial syste m 

for guidance . Thus , thes e neuron s presumabl y mi - 

grate ove r cell s i n th e variou s compartments o f th e 

developing cerebral wall (Anderson et al, 1997) . 

FACTORS REGULATIN G 

NEURONAL MIGRATION 

Soluble Stimulatin g Factor s 

Soluble factors can modulate radial migration. Migrating 

cortical neurons express TrkB, the high-affinit y receptor 

fo r brain-derived neurotrophi c factor (BDNF ) 

and neurotrophin- 4 (NT-4 ) (Beha r et al, 1997) . Thi s 

suggests a role of BDNF and NT-4 in their migration. 

Indeed, BDN F an d NT- 4 promote th e migratio n o f 

cortical neuron s in vitro. Consistent wit h culture stud - 

ies, BDNF or NT-4 infusion int o the latera l ventricle 

or applicatio n i n cultur e slice s induces heterotopias , 

which result from the overmigration of neurons (Brunstrom 

e t al, 1997) . Epiderma l growt h facto r recepto r 

(EGFr) an d it s ligands, includin g epiderma l growt h 

factor an d transformin g growth facto r (TGF ) a, ar e 

expressed i n the developin g cor f ex (Kornblu m e t al , 

1997). Mic e lackin g EGF r exhibi t mor e cell s i n th e 

proliferative zones , a finding suggesting inhibite d mi - 

gration, whereas high concentration s o f EGFr i n th e 

embryonic cortex enhance radia l migration (Threadgill 

et al, 1995 , Carie et al, 2001) . 

Tangential migratio n is stimulated by growth factors. 

One suc h ligand is hepatocyte growth factor (HGF). It

is expressed in th e teleneephalo n durin g LCN migra - 

tion from the medial G E t o the pallium (Powel l et al., 

2001). HG F increase s the numbe r o f cells migratin g 

away from th e subpallium , and neutralizatio n o f HG F 

by blocking antibodies inhibit s this migration. In addition, 

BDNF and NT-4 strongly stimulate the migration 

of LCNs that use y-aminobutyric acid (GABA) as a neurotransmitter 

(Polleux et al, 2002). 

TGFp promotes neuronal migration. TGFpl and 2 

are expressed by cells in the developing cortex; TGFpl, 

by neurons ; an d TGFp2 , b y radia l glia , particularly 

their inner segments (Miller, 2003). Elements in developing 

corte x als o expres s TGF p receptors—TGFpI r 

predominantly by radial glia and TGFpIIr by neurons. 

Thus, developin g corte x contains th e component s 

needed fo r TGFp to mediate neuron—gli a interaction s 

through bidirectional paracrine mechanism. Also, it appears 

that TGFp is involved in early stages of neuronal 

migration (stage s 1 , 2 , and 3) . These conclusion s are 

supported b y finding s fro m complementar y i n vitro 

studies. TGFpl inhibit s the proliferation of B104 neuroblastoma 

cell s (Lu o and Miller , 1999 ) an d culture d 

cortical neurons (Mille r and Luo, 2002a) . In addition, 

TGFpl move s VZ cells i n slices of cerebral corte x out 

of th e cyclin g populatio n an d accelerate s th e rat e o f 

neuronal migratio n b y 70% (Siegenthaler an d Miller , 

2005). Th e latte r effec t i s concentration-dependent . 

Migration i s maximall y potentiate d b y exposur e t o 

TGFpl a t a concentration of 10 ng/ml. In contrast, exposure 

to a higher o r lower concentration ha s little effect 

or it can even retard neuronal migration. 

Besides growt h factors , neurotransmitter s ar e in - 

volved i n modulatin g neurona l migration . GAB A is 

present i n th e developin g corte x (Rickman n e t al. , 

1977; Miller , 1986) . I t promote s cortica l neuron s t o 

migrate throug h GAB A receptors (Beha r et al. , 1996 , 

2001). It appears that GABA A/C receptors are involved 

in migratio n fro m th e proliferativ e zones t o th e I Z 

whereas GABA B receptor s ar e involve d i n migratio n 

from th e I Z t o th e CP . Anothe r neurotransrnitter , 

N-methyl-D-aspartate (NMDA), is also shown to modulate 

radial migration (Behar et al., 1999). Pharmacological 

blockad e o f NMD A recepto r activit y decrease s 

migration whereas enhancement o f NMDA recepto r 

activity increases the rate of cell movement. 

Cell Adhesion Molecules 

Interactions betwee n migratin g neuron s an d thei r 

substrates ar e mediate d b y cel l adhesio n molecules . 


Astrotactin (Astn ) 1 is a glycoprotein expressed by th e 

migrating neuron s (Fishel l an d Hatten, 1991 ; Zhen g 

et al., 1996) . Antibodies that block the functions of astrotactin 

abolis h th e adhesio n betwee n neuron s an d 

glia, thu s reducin g th e rat e o f migratio n (Stit t an d 

Hatten, 1990) . I n vivo , mice deficien t i n astrotacti n 

display slowed radial migration (Adams et al., 2002). 

Integrals are cell surface receptors that have been 

shown to regulate the migration of many cell types including 

neurons . The y mediat e essentia l cell-cel l 

and cell-extracellula r matri x interactions required for 

cell migration. Blocking of integrili oc ^ by specific antibodies 

reduce s th e rat e o f migration an d result s i n 

detachment of neurons from radia l glial fibers (Anton 

et al., 1999 ; Dulabo n et al, 2000). In mice deficient 

in integrin OC 3, some neurons appear to stop migrating 

prematurely an d fai l t o migrat e t o thei r appropriat e 

layer. Similarly , antibodie s agains t oc v integri n als o 

perturb neuron-glia interactions and may affect neu - 

ronal migration as well. 

The neural cell adhesion molecule (nCAM ) i s a 

member of the immunoglobuli n superfamil y of cel l 

adhesion molecule s though t t o pla y a rol e i n chai n 

migration. I t ha s a n unusua l carbohydrat e modifi - 

cation called polysialic acid, a linear polymer of sialic 

acid (Finn e e t al , 1983 ; Rutishauser , 1996) . I n 

nCAM knockou t mice , man y olfactory LCN precur - 

sors are retarded i n the SZ a and fai l t o migrate to the 

olfactory bul b (Tomasiewic z et al. , 1993 ; Grenie r 

et al. , 1994) . Interestingly , removal of the polysiali c 

acid moiet y of nCAM b y a specific enzyme i n wildtype 

animal s completel y mimicke d thi s knockou t 

phenotype (Ono e t al., 1994) . Cell migration in vitro 

is severel y abolishe d i n collage n culture s i n whic h 

polysialic acid is removed (Hu et al., 1996) . Thus, the 

carbohydrate modification of nCAM, polysialic acid , 

plays an importan t rol e i n the migratio n o f olfactory 

LCNs. 

TGFpl induce s nCA M an d integri n expression. 

Studies o f cortica l slice s sho w tha t thes e effect s ar e 

concentration-dependent; nCA M an d integri n ex - 

pression increas e wit h increase d TGFp l concen - 

tration (Siegenthale r an d Miller , 2004) . Likewise , 

TGFpl potentiate s nCA M expressio n b y culture d 

cortical astrocyte s (Lu o an d Miller , 1999) , neurons , 

and neuroblastom a cell s (Mille r an d Luo , 2002b) . 

In ligh t o f the effec t o f TGFpl o n th e rat e o f neu - 

ronal migratio n (se e above), i t appears that increase d 

nCAM an d integrin expression is facilitatory, but only 

to a certain point. I f the extracellula r space become s


too rich in cell adhesion proteins, neuronal migration 

is impeded , possibl y because th e microenvironmen t 

becomes too sticky. 

Extracellular Matrix Molecule Reelin 

Modern mous e genetic s hav e provide d som e muta - 

tions wit h neuronal migratio n disorder s (Tabl e 3-2) . 

The reeler mouse ha s received greate r attentio n fro m 

developmental neurobiologist s tha n ha s an y othe r 

mouse mutants . Thi s mous e exhibit s characteristi c 

lamination defect s of cerebral cortex , the hippocam - 

pus, and th e cerebellu m (Raki c and Caviness , 1995 ; 

Lambert de Rouvroit and Goffinet, 1998) . I n the mu - 

tants, neocortica l neuron s ar e generate d i n th e V Z 

and S Z as in the wild-typ e animals and initiall y their 

migration seems normal. A major differenc e i s that the 

preplate is never split into the M Z an d th e SP ; therefore, 

th e C P form s unde r the so-calle d superplate . As 

the migratin g neurons approac h th e CP , neuron s i n 

the reeler mic e fai l t o for m norma l architectoni c or - 

ganizations. Instea d o f forming b y a n inside-ou t pattern 

o f neuronogenesis , the birthdat e o f neuron s i n 

the mutant cortex is reversed. Early-generated neurons 

are locate d superficiall y and late-generate d neuron s 

are distributed in deep cortex. Remarkably, despite the 

Protein and Function 

Reelin, ECM protei n 

Astrotactin 1 , neuron-glia cell adhesion molecul e 

Integrin a^ subunit, cell surface receptor bind s to reelin 

positioning defects, neurons do make correct connections, 

although the axonal pathways are distorted. 

The clonin g o f th e defectiv e gen e relin ha s in - 

creased interes t in thi s mutatio n (D'Arcangel o e t al. , 

1995). Reelin is expressed by Cajal-Retzius neurons i n 

the M Z o f the developin g cerebral wal l (D'Arcangel o 

et al., 1997) . How reelin functions in neuronal migra - 

tion i s an are a o f intense research ; n o consensu s ha s 

been reached. Mos t results support the idea that reeli n 

controls laminatio n b y acting a s a sto p signa l fo r mi - 

grating neuron s (Dulabo n e t al. , 2000) . Accordingly , 

neurons migrate past the previously deposited neurons 

in the lower CP where reelin is not expressed. The mi - 

grating neurons proceed t o the superficia l edg e o f the 

CP borderin g the MZ where reelin i s expressed (Cur - 

ran and D'Arcangelo, 1998) . This finding implies that 

reelin tells the neurons to stop migrating. Some o f the 

migration defects characteristic of the reeler mice, such 

as th e lac k o f splitting o f th e preplate , i s rescued i n 

transgenic mic e i n which reeli n expressio n is targeted 

in the VZ and SZ. Thus, the role of reelin in developing 

cortex is more complicated than simply providing a 

migratory stop signal (Magdaleno et al., 2002). 

Reelin binds to two receptors expressed by migrating 

neurons , Apo E recepto r 2 and ver y low-density 

lipoprotein recepto r (VLDLr ) (D'Arcangel o e t al. , 

TABLE 3- 2 Mous e mutations that affect neuronal migration 

Integrin oc 6 subunit, cell surface receptor binds to laminin 

Integrin (3j , cell surface receptor interacts with reelin, 

laminin 

Laminin Y J subunit, ECM protei n 

Very low-density lipoprotei n receptor , reelin recepto r 

Low-density lipoprotein receptor-related protein, reeli n 

receptor also known as ApoE receptor 2 

Disabled homolog 1 , interacts with VLDLr/Lrp8 

Cyclin-dependent kinase 5, phosphorylates Dabi 

and NUDEL 

LISl/PAFAHIb, binds to dynein, microtubule 

Doublecortin, microtubule-associated protein 

Gene 

rein 

astn 

itga3 

itgao 

itgbl 

lame I 

vldlr 

Irp8 

dabl 

cdkS 

PAFAHIb 

dcx 

Structural Phenotype 

reeler mouse, inverted cortical layering , absence of 

preplate split 

Slowed radial migration 

Abnormal laminar positioning of projection neurons 

Basement membrane breac h 

Cortical layering perturbation 

Basement membrane breach 

Cortical layerin g perturbation 

Basement membrane breac h 

Cortical layering perturbation 

Reeler phenotype in Vldlr/LrpS doubl e knockout 

Reeler phenotype in Vldlr /Lrp8 doubl e knockout 

Reeler phenotype, mutated in yotari and scrambler 

Inverted cortical layering 

Null is lethal, hypomorphic mutations caus e 

migration defect 

No migration phenotyp e

1999; Hiesberger et al, 1999) . Mice deficient i n both 

of these receptor s exhibit phenotypes identical to that 

of the reeler mouse (Trommsdorff et al., 1999) . There 

may b e othe r receptor s involve d in transducin g the 

reelin signal . Reeli n als o binds to integri n oc 3pj an d 

cadherin-related neurona l receptor s (Senzak i e t al. , 

1999; Dulabo n e t al , 2000) . Apparently , reelin - 

integrin bindin g i s no t require d fo r reeli n functio n 

because integrin pj knockout mouse does not exhibit 

a ree/er-like phenotype. 

Downstream reelin-induced signalin g involves the 

phosphorylation o f Drosophila disable d homologu e 

(Dab) 1 (Howell et al., 1999). Mice with mutations in 

the gene s dabl, scrambler, and yotari exhibi t phenotypes 

similar to that of reeler mice. Dabi binds to th e 

intracellular domain s o f lipoprotein receptor s an d i s 

tyrosine phosphorylate d upo n ligand-recepto r bind - 

ing (Trommsdorff et al, 1998 ; Keshvar a et al., 2001). 

Surprisingly, reeli n ha s been show n t o posses s a serine 

protease activit y and can diges t extracellular matrix 

molecules (Quattrocchi et al, 2002). Whether its 

enzymatic activit y regulate s neurona l migratio n re - 

mains to be evaluated. 

The mous e knockou t of cyclin-dependent kinase 

(Cdk) 5 (a serine/threonine kinase ) or o f its activator 

protein, p35 , exhibit s a reeler-like cortica l migration 

phenotype (Gilmor e e t al. , 1998 ; Kwo n an d Tsai , 

1998). A major differenc e is that th e preplat e i s split 

into the MZ and the SP in these mutant mice. Thus, 

Cdk5 and p35 may function in a different pathwa y to 

control neurona l migratio n fro m reelin . Cdk 5 ca n 

phosphorylate Dabi independent o f reelin binding to 

its receptor s (Keshvara et al. , 2002) . There ma y b e 

crosstalk betwee n th e reeli n signalin g pathwa y an d 

the Cdk 5 pathway. 

MIGRATION DEFECTS 

X-linked Periventricular 

Heterotopias: Failure to 

Initiate Migration 

X-linked periventricula r heterotopia s ar e nodule s of 

neurons linin g the V Z o r SZ . Presumably there i s a 

failure o f neuron s t o migrat e ou t o f proliferat i ve 

zones. The geneti c defect in X-linked periventricular 

heterotopia i s mutations i n FLNA , an X-linke d gen e 

encoding filami n A (Fox et al., 1998) . Filami n A is a 

large actin-binding phosphoprotein with a molecular 


weight of 280 kDa. It is necessary for the locomotio n 

of several cell types and i s expressed by cells in all layers 

o f th e developin g cerebra l cortex . Hemizygou s 

males with null mutations die during the embryoni c 

period. Heterozygou s female s have epileps y that ca n 

be accompanie d b y othe r manifestation s suc h a s 

patent ductus arteriosus. I t i s believe d tha t rando m 

X-chromosome inactivatio n result s in inactivatio n o f 

FLNA expressio n in strande d neurons . Recently , affected 

male s with likely partial loss-of-function muta - 

tions in FLNA (e.g. , amino aci d 65 6 Leu t o Phe an d 

amino aci d 230 5 Tyr t o sto p codon ) hav e bee n re - 

ported (Shee n e t al., 2001; Moro e t al, 2002). Interestingly, 

male s wit h thes e mutation s hav e neuron s 

that either migrate normally or exhibit complete mi - 

gratory arrest . Thi s dichotom y suggest s tha t othe r 

functionally relate d gene s can compensate fo r filamin 

A deficiency . Indeed , a structurall y relate d gene , 

FLNB, i s als o expresse d i n th e developin g cerebra l 

cortical wall, and both proteins can form heterodimer s 

(Sheen et al., 2002). 

The mechanism throug h which filamin A regulates 

the initiatio n of migration is unclear. Likely it involves 

the abilit y o f filami n A t o cross-lin k F-acti n int o 

isotropie, orthogonal arrays (Stossel et al., 2001). Crosslinking 

of F-actin increase s the viscosit y and stiffnes s 

of actin an d ma y be involve d in th e initiatio n of migration. 

In the V Z and SZ , FLNA i s expressed by all 

cells —mitotic and postmitotic cells. If filamin A regulates 

the initiation of neuronal migration, why do only 

postmitotic neuron s migrat e ou t o f the V Z an d S Z 

when all cells express FLNA? A potential mechanism 

involves filamin A-interacting protei n (FILIP). FILIP 

is expressed in the VZ and SZ, but not in postmitoti c 

migrating neuron s (Nagan o et al. , 2002) . FILIP-fil - 

amin A binding induces the degradation of filamin A. 

Thus, the loss of FILIP expressio n in postmitotic neu - 

rons may enable filami n A to control the star t of migration. 

Double Cortex Syndrom e an d Type I 

Lissencephaly: Prematur e Cessatio n 

of Neuronal Migratio n 

Double cortex describe s a conditio n i n whic h a 

subcortical-band heterotopia form s i n the subcortical 

IZ, the anlag e o f the whit e matter . Mutations i n a n 

X-linked gene, doublecortin (dcx)y ar e a genetic caus e 

of the disorder (des Portes et al, 1998 ; Gleeso n e t al, 

1998). Doublecortin is a 40 kDa protein, expressed by


migrating an d man y differentiatin g neurons. I t i s a 

microtubule-associated protei n wit h n o know n ho - 

mology wit h othe r microtubule-associate d protein s 

(Francis et al., 1999; Gleeson e t al., 1999 ; Hores h et al., 

1999). Doublecorti n stimulate s microtubule polymer - 

ization b y binding t o 1 3 protofilament microtubule s 

(Moores e t al., 2004). It is principally localized i n th e 

leading processe s o f migratin g neuron s (Friocour t 

et al., 2003). Thus, doublecortin may promote move - 

ment by inducing microtubule polymerizatio n i n the 

leading processes of migrating neurons. 

Classical lissencephal y (type I ) i s a brai n malfor - 

mation characterize d b y absent o r reduce d gyratio n 

and a cortex that is thicker and has a rudimentary four 

layers. I t is associated with severe mental retardation , 

epilepsy, an d cerebra l palsy . Mos t case s ar e du e t o 

mutations o f lisi, encoding LIS I or platelet-activatin g 

factor acetylhydrolas e isofor m I b (PAFAHIb , a non - 

catalytic subunit o f the enzyme ) (Reine r e t al., 1993 ; 

Hattori et al., 1994). Besides being part of an enzyme , 

LISl/PAFAHIb ha s a nonenzymati c function . Th e 

homolog i n fungu s Aspergillus i s NudF, a n essentia l 

part of a signaling pathway that regulate s nuclea r mi - 

gration (Wynshaw-Bori s an d Gambello , 2001) . Evi - 

dence indicate s that LISl/PAFAHIb regulates nuclear 

movement b y an evolutionary-conserve d mechanis m 

similar to that of the fungus. It binds to and regulate s 

the function an d distributio n of dynein (mammalia n 

NudA homolog) , whic h function s a s a minu s end - 

directed microtubule-associate d moto r protein . Thus , 

LISl/PAFAHIb ma y be par t o f the protei n comple x 

that exert s force s o n th e microtubule s surroundin g 

the nucleu s i n migratin g neuron s an d pullin g th e 

nucleus into the leading process (Tanaka et al., 2004). 

A role for LISl/PAFAHIb in nuclea r translocatio n is 

also supported b y its binding t o two other dynein in - 

teracting proteins , NUDE L an d mNUD E (mam - 

malian Nud E homologs ) (Fen g e t al. , 2000 ; Lian g 

et al, 2004). NUDEL and mNUDE colocalize a t the 

centromere tha t migrate s ahea d o f the nucleus . Th e 

distance betwee n th e centromer e an d th e nucleu s 

is enlarge d i n LISl/PAFAHIb-deficien t neuron s 

(Tanaka e t al, 2004). Thus, LISl/PAFAHIb play s an 

important rol e i n nuclea r translocatio n durin g neu - 

ronal migration. 

Type II Lissencephaly: Overmigration 

Several form s o f congenita l muscula r dystrophies — 

Walker-Warburg syndrome (WWS) , muscle-eye-brain 

disease (MEB) , an d Fukuyama-typ e congenita l 

muscular dystroph y (FCMD ) —are associate d wit h 

cortical dysplasia . These are autosomal recessiv e dis - 

orders characterize d b y congenita l muscula r dystro - 

phy, ocula r abnormalities , an d cortica l dysplasi a 

(Santavuori e t al., 1989) . Magneti c resonanc e imag - 

ing of MEB patient s reveal s hydrocephalus associate d 

with polymicrogyra or pachygyra caused by ectopie lo - 

cation o f neura l tissue s i n th e leptomeninges , als o 

known a s cobbleston e complexe s o r typ e I I 

lissencephaly (Valann e et al. , 1994 ; Corman d e t al. , 

2001). Presumably , thes e abnormalitie s result whe n 

the neurons migrat e to o far, passing through th e MZ , 

the glial-limiting membrane, the piai basement mem - 

brane (PM), and the pia (Ross and Walsh, 2001). 

Each o f the congenita l muscula r dystrophie s ha s 

been associate d wit h mutatio n o f a spécifi e gene . 

In WWS , mutation s ar e i n th e gene s codin g fo r 

protein-O-mannosyl transferase s (POMT 1 an d 

POMT2) (Beltran-Valer o e t al, 2002 ; va n Reeuwij k 

et al. , 2005) . Th e defectiv e gen e fo r ME B map s t o 

chromosome lp32-3 4 (Corman d e t al. , 1999) , an d 

the mutated gen e is POMGnTl. Peopl e wit h FCMD 

have a retrotransposon insertio n int o the 3 ' untranslated 

regio n o f FCMD . Thi s insertio n result s i n a 

dramatic reductio n i n FCMD expression . 

The myodystroph y (myd) mous e ha s muscle , eye , 

and neurona l migratio n defects in the brain similar to 

those i n humans with congenital muscula r dystrophie s 

(Holzfeind e t al., 2002). This myd mouse ha s muscle , 

eye, an d brai n defect s associate d wit h overmigratio n 

(Holzfeind et al, 2002). In the myd mouse, the genetic 

defect i s a functional deletio n i n the Large gene (likeacetylglucosaminyltransferase) 

(Grewa l e t al. , 2001) . 

The my d mutation is now designated Large m y d . I n hu - 

mans, mutation s i n Large gene cause congenita l mus - 

cular dystroph y MDC1 D (Longman , e t al , 2003 ) 

Thus, four ne w genes, POMT1, POMGnTl, FCMD, 

and Large, have bee n associate d wit h congenital mus - 

cular dystrophies , an d eac h cause s simila r overmigra - 

tion defects when mutated . 

The functio n of POMGnTl ha s been describe d in 

vitro. POMGnTl encode s a n enzym e involve d i n 

O-mannosyl glycosylatio n o f protein s calle d UDP - 

N-acetylglucosamine: protein-O-mannos e p-l,2-N - 

acetylglucosaminyl transferas e (POMGnTl), Th e 

enzyme catalyze s the following reaction: 

UDP-GlcNAc + Man-R 

-> GlcNAc(31-2Man-R + UDP

where R is a protein and Ma n i s anchored t o Ser/Thr 

residues (Takahashi et al., 2001; Yoshida et al, 2001). 

The function s of POMTT, fukutin, an d Large , gen e 

products o f POMTI, FCMD , an d Large, hav e no t 

been determined . Bioinformatic s studies , however , 

show that eac h protei n ha s a glycosyltransferase-lik e 

domain (Aravin d an d Koonin , 1999 ; Jurad o e t al. , 

1999; Peyrar d e t al. , 1999 ; Beltran-Valer o e t al , 

2002). The implicatio n is that the dystrophies and migration 

defects result from abnormal protein glycosylation. 

How can protein glycosylatio n enzymes regulate 

neuronal migration? 

Conceivably, migration involves glycosylation substrate 

proteins. A candidate substrate is a-dystroglycan, 

a heavily glycosylated membrane protein . The mucin - 

like domain o f a-dystroglycan is heavily substituted b y 

O-linked mannosy l glycan s tha t contain th e linkag e 

catalyzed b y POMGnTl (Chib a e t al, 1997) . Th e 

O-mannosyl glycans may play important role s in me - 

diating a-dystroglyca n interaction s with lamini n (Ervasti 

and Campbell, 1993 ; Smalheiser, 1993), a major 

component o f th e basemen t membrane . Further , 

a-dystroglycan i n som e o f these disease s is underglycosylated 

and binding to laminin is reduced (Hayash i 

et aì., 2001; Holzfeind et al, 2002; Kano et al, 2002; 

Michele et al., 2002). These results suggest a connection 

between hypoglycosylatio n of a-dystroglycan an d 

neuronal migratio n defect s i n th e brain . A n impor - 

tant role for a-dystroglycan i n this process is also supported 

b y finding s o f neurona l migratio n defect s i n 

the brains of mice in which dystroglyean is conditionally 

knocked out (Michel e et al., 2002; Moore e t al., 

2002). 

Mechanisms o f overmigration may involve abrogation 

o f th e PM . Th e P M i s compose d mainl y o f 

laminin, collage n IV , nidogen, an d perlecan , al l o f 

which regulat e cel l proliferation , migration , an d differentiation 

b y interactin g wit h mainl y tw o cell sur - 

face receptors: integrilis and a-dystroglycan. Th e PM , 

to which radia l glia l endfee t ar e attached , i s locate d 

between th e pia mater an d the MZ . Electro n micro - 

scopic analyse s show breaches i n th e P M a t site s of 

ectopie neura l cluster s o f patient s wit h FCM D 

(Nakano e t al. , 1996 ; Ishi i e t al, 1997 ; Sait o e t al. , 

1999). Laminin i s reduced or abnormally distribute d 

in the brai n surfac e PM o f several mutant mice wit h 

overmigration, includin g Lmxl a (Dreher) mic e 

(Sekiguchi et al, 1994 ^ Costa et al, 2001 ) and mic e 

lacking rnyristolate d alanine-rich C kinas e substrat e 

(Blackshear e t al , 1997) , integri n a 6 (Georges - 


Labouesse et al, 1998) , an d integri n p] (Graus-Port a 

et al , 2001) . Interestingly , integrili (3 j nul l neuron s 

migrate to appropriate positions in the cerebral cortex 

in chimeri c mic e (Fassle r and Meyer , 1995) , imply - 

ing tha t overmigratio n i s not cause d b y a n intrinsi c 

defect of the migrating neurons but b y a defective environment 

i n thi s mutant . Whethe r breache s i n th e 

PM ar e th e caus e o r th e resul t o f overmigration re - 

mains to be clarified. 

DIRECTIONAL GUIDANCE OF 

NEURONAL MIGRATIO N BY 

DIFFUSABLE FACTORS 

LCNs o f th e olfactor y bul b (granul e cell s an d 

periglomerular cells ) ar e mainl y generate d postna - 

tally, during th e first 2 to 3 weeks after birt h (Altma n 

and Das , 1966 ; Hinds , 1968) , althoug h som e neu - 

ronogenesis continue s i n th e adul t (Corott o e t al , 

1993; Loi s an d Alvarey-Buvlla , 1994) . Retroviral - 

labeling studies demonstrate tha t mos t o f the LCN s 

are generate d i n th e S Z nea r th e anterio r forebrai n 

(SZa) an d migrat e t o th e olfactor y bulb throug h a n 

SZ pathway (Fig . 3-3 ) (Luskin , 1993 ; Zigova e t al , 

1996). Apparently , olfactory LCNs do not migrate o n 

radially oriented glia l processes, a s the orientatio n o f 

the radia l glia l fiber s i s orthogona l t o th e migratio n 

trajectory (Kish i e t al, 1990) . Further , th e migratio n 

pathway i n th e S Z i s also devoid o f axon projections 

(Kishi, 1987) . 

How d o olfactor y LC N migrate ? Immunohisto - 

chemical studie s o f polysialic acid an d Tu J 1 expression 

i n th e adul t S Z pathwa y sho w tha t migratin g 

cells tend to travel in chains or streams of cells (Rousselot 

et al, 1995 ; Doetsc h an d Alvarez-Buylla, 1996 ; 

Jankovski and Sotelo, 1996 ; Loi s et al, 1996 ; Doetsc h 

et al , 1997 ; Garcia-Verdug o e t al, 1998) . Althoug h 

these chain s of migrating cells cannot b e observe d in 

newborn animals because man y cells are migrating at 

the same time, such chains are often observed in cul - 

tures of SZ cell s plate d on collage n gel s (H u et al, 

1996) or Matrigel (Wichterle et al, 1997) . Therefore , 

chain migration of olfactory LCN precursor s seems to 

be mechanistically distinc t fro m radia l migration relying 

on radial glia. 

In vivo studies show that olfactory LCN precursor s 

migrate fro m th e SZ a to the olfactory bulb in a unidirectional 

manner (Luskin , 1993; Hu and Rutishauser, 

1996). Thi s findin g implie s a n activ e guidanc e


FIGURE 3- 3 Tangentia l migratio n o f olfactor y loca l 

circuit neuron s (LCNs) . A parasagitta l sectio n 

through th e middl e of the olfactor y bulb (OB) of the 

forebrain o f the mous e is shown. Olfactory LCNs ar e 

generated i n the anterior subventricular zone (SVZa ; 

shown betwee n arrows) . The y migrat e t o th e O B 

within the SVZa (show n in dotted areas) . After arriving 

in the bulb, they migrate i n a radial fashion to reach 

their fina l location s i n th e granul e cel l laye r an d 

glomerular layer . The septu m i s locate d media l t o 

this section . CC , corpu s callosum ; CP , caudoputa - 

men; CTX , cerebra l cortex ; fmi , forceps mino r cor - 

pus callosum. 

mechanism. Th e olfactor y bul b ma y secret e a 

chemoattractant(s) (Li u an d Rao , 2003) . Further - 

more, th e cauda l part s o f th e septu m an d th e 

choroid plexus, which lie behind the migratio n pathway, 

secret e a chernorepulsiv e factor(s ) (H u an d 

Rutishauser, 1996) . Th e chernorepulsiv e activity secreted 

from th e septum is a member of the Sli t family 

of protein s (Siiti , SlitZ , an d Slit3 ) (Hu , 1999 ; W u 

et al., 1999) . Slit , first discovered i n Drosophila, an d 

also found in mammals, regulates the growth of axons 

crossing a t th e midlin e (Ba-Charve t e t al. , 1999 ; L i 

et al. , 1999 ) b y interacting with roundabout (Robo) 

receptors (Kidd et al, 1998). Robo receptors are members 

of the immunoglobulin superfamily of transmembrane 

molecule s (Zalle n e t al. , 1998 ; Kid d e t al. , 

1999). Th e Slit/Rob o repulsive signal i s mediated i n 

part by the proteins enable d an d abelson, Rh o famil y 

guanidine triphospotase s (GTPases) , an d Slit-Rob o 

GTPase activatin g proteins (srGAPs) (Bashaw et al. , 

2000; Wong et al, 2001). 

Soluble factor s ma y als o b e involve d i n activ e 

guidance i n th e migratio n o f cortical neurons . Thi s 

conclusion i s supported b y studies of LCN migratio n 

from the GE to the cerebral cortex. In vitro, these migrating 

neurons are repelled b y the Sli t proteins that 

are expresse d b y th e G E (Zh u e t al. , 1999 ) an d at - 

tracted by substances released by the developing cortex 

(Marin e t al. , 2003 ; Wichterl e e t al , 2003) . Neuropilins 

are transmembrane receptor s that mediate th e 

repulsive guidance activitie s o f class 3 semaphorins. 

Neuropilin 1 and 2 are expresse d by the LCN s tha t 

migrate from th e GE t o cerebral cortex (Marin et al., 

2001). Neuropili n knockou t mic e hav e decrease d 

numbers of LCNs in neocortex. 

Successful navigatio n o f neurons involve s mor e 

than attraction and repulsion. While en route within 

the SZ a pathway , olfactor y LCN s d o no t deviat e 

from thei r rout e int o surroundin g regions (Luskin , 

1993). What confines the cell s to the SZ a pathway? 

The S Z migration pathway in adult mice is rich in astrocytes, 

tenascin, and chondroiti n sulfat e proteogly - 

can (Thomas et al., 1996). Thus, it is possible that the 

migrating cell s migh t hav e stronge r affinit y fo r th e 

SZ, becaus e th e S Z contain s differen t extracellula r 

matrix molecule s an d cel l type s fro m thos e i n sur - 

rounding regions. 

SUMMARY AND CONCLUSION S 

Neuronal migration is a complex phenomenon regu - 

lated b y man y factors . I n vitr o perturbatio n studies 

and molecula r studie s in bot h th e mous e an d hu - 

mans identify som e key molecules that are involved. 

Examples includ e factor s extrinsic an d intrinsi c to a 

migrating neuron . Thes e extrinsi c factors , suc h a s 

soluble an d extracellula r matri x molecule s (neu - 

rotrophins, reelin , an d Slit) , an d intrinsi c factors, 

such a s cytoskeletal proteins (filamin , doublecortin , 

and PAFAHIb) , work in concert. They likely interact 

with cel l surfac e receptor s an d signalin g pathways. 

Some o f th e mechanism s ar e jus t beginnin g t o 

emerge. I n addition , ther e ar e critica l interaction s 

between geneti c an d environmenta l o r epigeneti c 

factors. 

Astn astrotacti n 

Abbreviations 

BDNF brain-derive d neurotrophic factor 

Cdk cyc l in-dependent kinase 


Dab disable d 

EGFr epiderma l growth factor recepto r

FCMD Fukuyama-typ e congenital muscular dys - 

trophy 

FILIP filami n A-interactin g protein 


GÈ gangliom a eminence 

GTPase guanidin e triphosphotase 

HGF hepatocyt e growth facto r 

IZ intermediat e zone 

Large like-acetylglucosaminyltransferas e 

LCN local circuit neuron 

LHRH luteinizin g hormone releasing hormone 

MEB muscle-eye-brai n disease 


nCAM neura l cell adhesion molecule 


NT-4 neurotrophin- 4 

PAFAHIb platelet-activatin g facto r acetylhydro - 

lase isoform Ib , a.k.a. LISI 

PM pia i basement membrane 

POMGnTl UDP-N-acetylglucosamin e protein - 

O-mannose ßl,2-N-acetylglucos a 

minyltransferase 

POMT1 protein-O-mannosy l transferase 


SZa anterio r subventricular zone 

TGF transformin g growth facto r 


VLDLr ver y low-density lipoprotein receptor 

WWS Walker-Warbur g syndrome 

ACKNOWLEDGMENTS Th e autho r thank s Dr . Eri c 

Olson fo r critica l readin g o f th e manuscript . Result s 

from th e author' s laborator y wer e supporte d b y grant s 

NS038877 and HD04401 1 from th e Nationa l Institute s 

of Health. 

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Neuronal Differentiation : 

From Axons to Synapses 

A newly born neuro n generate s a singl e axo n an d a 

somatodendritic domain. As it matures, i t engages, by 

way of synapses, a select populatio n o f the 2 0 billion 

other differentiatin g neuron s i n th e huma n centra l 

nervous syste m (CNS) . Amazingly , the connection s 

that ultimately form are stereotyped and , for the mos t 

part, faithfull y recapitulate d acros s individuals. Thi s 

chapter outlines major event s in differentiation, high - 

lighting the key cellular events and molecular mecha - 

nisms governing the process . 

ACQUISITION O F 

NEURONAL POLARITY 

The neuroanatomis t Jan Purkinje was the first to note 

that ther e ar e tw o morphologicall y distinc t types of 

nerve fiber s i n th e adul t nervou s system, axons an d 

dendrites (Shepherd , 1991) . Axons traverse long distances, 

maintai n a constan t caliber , an d commonl y 

branch a t right angles. I n contrast , dendrites, which 

4 

C. David Mintz 

Iddii H. Bekirov 

Tonya R. Anderson 

Deanna L . Benson 

45 

are usually shorter, taper with distance from the soma 

and generall y branch a t acute angles. Earl y in devel - 

opment, neurite s ar e morphologicall y indistinguish - 

able from on e another. Over the course of maturation 

they assume the characteristics of axons and dendrites 

in a process known as polarization. 

Stages of Neurite Maturatio n 

Time-lapse imagin g of individual neurons growing in 

dissociated cultures reveals a stereotyped sequence of 

stages in neurite development, diagramme d in Figur e 

4-1 (Dott i e t al., 1988) . I n Stag e 1 , the neuro n exist s 

as a round soma with lamellipodia and filopodia , bu t 

without neurites . Immature neurites , whic h canno t 

be identified as either axons or dendrites, emerge during 

Stag e 2 . I n Stag e 3 , a singl e proces s undergoes 

rapid growth and becomes the axon. The shorte r processes 

develop into dendrites in Stage 4. During Stag e 

5, there i s further elaboration an d maturatio n o f dendrites 

and axon s as synapses form. Though it is not yet


FIGURE 4-1 Stage s of neurite development Th e Stag e 1 neuron lacks neurites 

entirely. In Stag e 2 , immature neurites emerg e tha t are capable o f becomin g 

either axon s or dendrites. When on e o f the immatur e neurites outgrows the 

others i t becomes th e axo n and th e neuro n reache s Stag e 3 . The remainin g 

immature processe s becom e dendrite s an d elaborat e an d branc h i n Stag e 4 . 

Synaptogenesis and spinogensi s occur i n Stag e 5 , which i s not show n in this 

diagram. 

proven definitively , it i s generally believe d tha t th e 

stages o f neurit e developmen t observe d i n cultur e 

also occu r i n situ . Nearly all data tha t addres s ques - 

tions about neuronal polarity are derived from experiments 

i n whic h neuron s ar e grow n i n dissociate d 

cultures where individual processes can be identified 

and followed over time. 

Polarization is Initiated b y 

Axon Specificatio n 

All early neurites o n a particular neuro n ar e capabl e 

of becoming axon s or dendrites . On e ultimatel y acquires 

axona l characteristic s an d th e remainde r as - 

sume a dendriti c fate . I n a Stag e 2 neuron , eac h 

process undergoe s cycle s o f extension an d retractio n 

until on e neurit e outgrow s th e other s b y a critica l 

length—lOjLim i n hippocampa l neurons—an d be - 

comes th e axo n (Gosli n an d Banker , 1989) . I f a 

recently formed axo n is transected to a length compa - 

rable to that of the other neurites, one of the other minor 

processe s typicall y becomes th e axo n an d th e 

stump of the original axon adopts dendritic character - 

istics (Dott i an d Banker , 1987) . Thus , ever y Stag e 

2 proces s ha s the potentia l t o become the axon , bu t 

the presenc e o f a specifie d axo n actively promotes a 

dendritic fat e for the remainin g neurites . This mode l 

is supporte d b y data showin g that dendriti c matura - 

tion ca n occu r onl y after axo n specification (Cacere s 

et al, 1991) . 

As one neurit e outgrows the other s by virtue of interactions 

wit h loca l environmenta l factors , i t give s 

rise t o a positiv e feedback loo p tha t create s tw o dis - 

tinct compartments (Crai g and Banker , 1994) . As development 

continues , som e protein s an d organelle s 

become segregate d int o on e o f th e tw o compart - 

ments, an d th e differen t complement s o f proteins in 

mature axon s an d dendrite s underli e th e functiona l 

and morphologica l differences betwee n the two types 

of neurites . I n recen t years, some progres s ha s bee n 

made in elucidating the molecular basis of axon specification 

an d subsequen t maturatio n events , bu t a 

complete mode l ha s not yet been formulated. 

Role of the Cytoskeleton in 

the Establishment of Polarity 

A considerabl e bod y o f evidenc e support s th e hy - 

pothesis that microtubules are involved in axon specification. 

Quantitativ e analyse s o f seria l electro n 

microscopy (EM ) reconstruction s o f minor processe s 

and axon s in Stag e 2 and Stag e 3 neurons revea l tha t

axon specification coincides with a dramatic increase 

in microtubule number (Y u and Baas, 1994). This increase 

raises the possibilit y that selective transport of 

microtubules t o a singl e neurit e produce s rapi d 

growth and subsequen t acquisitio n of axonal charac - 

teristics (Baas, 1999) . 

The efficienc y o f microtubule assembl y and th e 

stability of microtubules once formed are inextricably 

linked t o th e loca l compositio n o f mierotubule - 

associated proteins (MAPs). Data from culture d neu - 

rons indicat e tha t MAP s such a s Tau an d MAPlb , 

which are enriched i n axons, may also play a role i n 

axon specification (Caceres and Kosik , 1990 ; DiTell a 

et al, 1996). Mice deficient in both Tau and MAPlb, 

however, have only modest defect s in axo n development 

(Takei et al., 2000), a finding suggesting that the 

dependence on MAPs is exaggerated in culture or compensated 

for over the long term in situ. At the very least, 

MAPs stabilize microtubules, and this stabilization contributes 

t o rapi d microtubule-dependen t neurit e out - 

growth. Other proteins that regulate microtubules may 

play a more crucial role in axon specification. For example, 

collapsi n respons e mediator protein (CRMP ) 

2 i s enriched i n th e dista l regio n o f Stag e 3 axons 

where i t apparently facilitates microtubule assembl y 

(Fukata e t al., 2002a) . Supporting this possibility , a 

dominant negativ e CRMP- 2 lackin g th e tubuli n 

binding sit e prevent s axon specificatio n in culture d 

neurons (Inagak i e t al. , 2001) . Also , recen t studie s 

have demonstrated tha t glycogen synthas e kinase 3 $ 

and other signaling molecules directly upstream fro m 

CRMP-2 can influenc e axon formation (Jiang et al., 

2005; Yoshimura et al, 2005). It must be noted, however, 

that CRMP- 2 an d th e axona l MAPs are multifunctional 

proteins , makin g the precis e significance 

of microtubules in the establishmen t of neuronal polarity 

unclear. 

The acti n cytoskeleto n ha s bee n implicate d i n 

axon specification. Local applicatio n o f cytochalasin 

D, an actin-depolymerizing agent, to the growth cone 

of a single neurite o f a Stage 2 neuron increase s th e 

likelihood tha t thi s neurit e wil l becom e th e axon . 

Bath application of the sam e reagent results in an increase 

in neurons bearing multiple axons (Bradke and 

Dotti, 1999) . It should be noted, however, that a subsequent 

study argues that this effect i s actually the re - 

sult o f faste r growt h o f al l neurites , rathe r tha n 

aberrant polarization (Ruthel and Hollenbeck, 2000). 

In Aplysia growt h cones, actin depolymerization ca n 

trigger increase d microtubul e elongatio n (Forsche r 

NEURONAL DIFFERENTIATION: FROM AXONS TO SYNAPSE S 4 7 

and Smith, 1988). Hence, it is likely that axon specification 

depends on the coordinated regulation of both 

actin depolymerization and microtubule assembly. 

Signaling Pathways That 

Control Polarization 

The emergenc e of polarity requires coordination between 

Stage 2 neurites, suggesting the existence of intracellular 

signalin g pathways . Th e Rh o famil y o f 

small GTPases , whic h include s Rho , Rac , an d 

Cdc42, are candidates to control polarization, as they 

can regulat e bot h acti n an d microtubule s (Burridg e 

and Wennerberg , 2004 ; Gunderse n e t al. , 2004) . 

Broad-based inhibitio n o f th e entir e Rh o famil y i n 

Stage 2 neurons results in the emergence of multiple 

axons (Bradke and Dotti, 1999). Additional work supports 

the notio n that Rho, Rac, and Cdc42 ca n each 

play distinc t roles . Expressio n o f a Ra c dominant - 

negative mutant in Drosophila reduces axon outgrowth 

(Luo e t al. , 1994) , wherea s expression of a constitu - 

tively active Cdc42 mutant i n culture d hippocampa l 

neurons induce s multipl e axon s (Schwambor n an d 

Puschel, 2004) . In contrast, Rho can induce the col - 

lapse of the dista l ends o f neurites via phosphyorlyation 

of CRMP-2 (Arimur a et al, 2000). According to 

one model, the spee d of outgrowth of any given neurite 

i s determine d b y a balanc e betwee n retraction 

and collaps e promote d b y Rh o an d outgrowt h pro - 

moted by Rac and Cdc42 (Fukata et al, 2002b). This 

hypothesis, whic h ha s no t bee n full y validate d i n 

mammalian neurons, has the potential t o explain differential 

growth of axons and dendrites in terms of local 

Rho family activity. 

There ar e many parallels betwee n neurona l an d 

non-neuronal cel l polarizatio n (Rodriguez-Boula n 

and Powell , 1992) , an d ther e i s evidence that some 

signaling pathway s related t o polarizatio n ar e con - 

served amon g different cel l types. The Pa r complex, 

which consists of Par3, Parò, and atypica l phosphokinase 

C , control s th e establishmen t o f polarity i n a 

broad range of species and cel l types via regulation of 

actin and microtubule s (Wodarz , 2002). Recent data 

suggest that the Par complex may control axon specification 

upstream from th e Rho family. Par3 and Parò 

are polarize d to th e axon s o f hippocampal neurons, 

and overexpressio n of ectopie Pa r prevents polarization 

(Sh i e t al. , 2004) . Segregatio n o f Par comple x 

members to a single neurite, which can occur as early 

as Stag e 2 , i s precede d b y polarizatio n o f Ra p IB,


a smal l GTPas e tha t i s a Ra s famil y membe r 

(Schwamborn and Puschel, 2004). The dat a indicate 

that Ra p IB i s upstrea m o f bot h th e Pa r comple x 

and Cdc4 2 wit h respec t t o axo n specificatio n 

(Schwamborn an d Puschel , 2004) . I t is thought tha t 

extracellular factors, a s yet unidentified, might medi - 

ate th e effect s o f the loca l environmen t on polariza - 

tion b y acting upstrea m fro m intracellula r signaling 

molecules such as Rho, Par, and Rap IB. 

Polarized Distribution of 

Proteins an d Organelles 

The difference s i n morphology and function that distinguish 

axons and dendrites reflect different comple - 

ments o f protein s an d organelle s i n eac h typ e o f 

neurite. In the adult brain, certain proteins have a polarized 

distribution , meanin g tha t the y ar e concen - 

trated or even exclusively present in either dendrites or 

axons. This asymmetr y must be created durin g development 

b y compartment-specifi c traffickin g an d re - 

tention o f thes e proteins . Th e Stag e 2 neurit e tha t 

becomes the axo n in Stag e 3 typically receives more 

organelles, cytosoli c proteins, and Golgi-derive d vesi - 

cles than the processes that become dendrites (Bradke 

and Dotti , 1997) . Intac t trafficking i s required for polarization, 

as it is possible to prevent axon specification 

by blockin g th e traffickin g o f Golgi-derive d vesicle s 

with brefeldin A (Jareb and Banker, 1997). The polar - 

ized distribution of some proteins may either promote 

the emergin g difference s betwee n developin g axon s 

and dendrites or simply reflect thos e differences . Th e 

details o f how eac h polarize d protei n reache s it s appropriate 

destination in one compartment bu t not the 

other remain largely speculative. 

The polarizatio n of proteins ma y resul t from re - 

tention specifi c t o the appropriat e compartment. Experiments 

with optical tweezers, which allow traction 

force t o b e applie d t o specifi c proteins, sho w that a 

barrier t o th e diffusio n o f some axon-specifi c mem - 

brane protein s exist s a t th e axo n initia l segmen t 

(Winckler et al, 1999; Fâche et al, 2004). The barrier 

appears to be an accumulation of actin-tethered mem - 

brane proteins . Analysis of phospholipid diffusio n i n 

the membrane suggests that the diffusio n barrie r does 

not exis t prio r t o axo n specificatio n (Nakad a e t al. , 

2003), but it may play an important role in subsequent 

maturation. The exten t to which the diffusio n barrie r 

is a cause or consequent of the establishmen t of neuronal 

polarity remains unknown. 

NEURITE MOTILITY 

The neuroanatomis t Santiag o Ramon y Cajal (1894 ) 

observed tha t th e dista l exten t o f a growin g neurit e 

"end[s] in a spherical conical swelling . .. with a large 

number o f thick protrusions and lamella r processes, " 

which he speculated wer e like "an amoebic mas s that 

acts as a battering ram to spread the elements along its 

path." H e name d thi s morphologica l specializatio n 

the growth cone, and decade s of subsequent research 

have supporte d hi s inferenc e that growt h cone s ar e 

highly motile structures required for the elongation of 

both axon s and dendrites . Much i s known about th e 

structure and functio n of growth cones . The motilit y 

of growth cone s depend s o n acti n an d microtubule s 

and th e protein s tha t regulat e them . Additionally , 

growth cone s ar e th e sensor y organs for extracellular 

cues that guide axons to engage in the directed growth 

required for the establishmen t of correct connectivity. 

Though muc h wor k remains t o be done , a n under - 

standing o f ho w growt h cone s functio n i n neurit e 

growth is emerging. 

Growth Cone Structur e 

Although neuronal growth cones vary considerably in 

shape an d size , the y generall y share som e commo n 

structural features. Most investigators recognize thre e 

morphological zone s know n a s th e central , transi - 

tional, an d periphera l domains, which are indicate d 

in Figur e 4- 2 (Forsche r and Smith , 1988 ; Bridgman 

and Dailey , 1989) . Th e relativel y thick centra l do - 

main abut s th e neurit e an d i s enriched wit h micro - 

tubules. The transitional domain, which lies between 

the centra l an d periphera l domains , contain s mesh - 

work, arcs , and radia l ridge s o f F-actin. Th e periph - 

eral domai n consist s o f the lamellipodium , a wide, 

flat region, and filopodia, which ar e fine membrane 

protrusions tha t exten d distall y fro m th e lamel - 

lipodium. The periphera l domain i s greatly enriched 

with F-actin, organized a s meshwork and arc s in th e 

lamellipodia. Filopodia contai n bundles of filaments 

with th e barbe d ends pointing distally. Compared t o 

the centra l domain , th e periphera l domai n ha s a 

lower densit y o f microtubule s an d the y ar e notabl y 

more dynami c (Schaefe r e t al, , 2002) . The leadin g 

edge o f migrating fibroblasts was thought t o hav e a 

structure similar to that of the neuronal growth cone. 

Recent findings, however, show that the cytoskeletal 

configuration an d mechanic s of motility in these two

FIGURE 4- 2 Structur e o f the growt h cone . Th e mor - 

phological zone s o f a growth con e ar e show n usin g a 

differential interferenc e contras t micrograp h o f a dendritic 

growth cone from a cultured ra t neocortical neu - 

ron. Not e th e thic k centra l zon e (C) , separate d fro m 

the fla t periphera l zon e (P ) b y th e transitiona l zon e 

(T). Also , i n th e periphera l zone , th e lamellipodiu m 

and several filopodia are indicated by dashed lines. 

structures diffe r considerabl y (Strasse r e t al. , 2004) . 

Thus, the presen t discussio n of growth con e motilit y 

is confined to models from experiment s done o n neuronal 

growth cones . 

Growth Cone Motility 

The exac t relationship between filopodial, lamellipodial, 

an d neurit e motilit y i s no t completel y under - 

stood. Filopodi a sampl e th e environmen t fo r cues to 

determine th e directio n o f growth (O'Conno r e t al. , 

1990), and thus filopodial motility tends to reflect this 

scanning patter n rathe r tha n demonstratin g a tigh t 

link wit h neurit e growth . The movement s o f lamellipodia 

are better correlate d with neurite growt h than 

those o f filopodia , bu t lamellipodi a commonl y en - 

gage i n extensio n an d retractio n tha t d o no t directl y 

correlate with alterations i n the trajectory of the growing 

neurite. 

Growth con e motilit y requires dynamic regulation 

of th e cytoskeleton . Th e treadmil l model , whic h i s 

based on observations in filopodia, focuses on the rol e 

of acti n (Fig . 4-3 ) (Li n e t al, 1994) . Filament s o f 

actin are simultaneously polymerized at the dista l tips 

of filopodi a an d retracte d proximall y b y myosi n 


FIGURE 4- 3 Th e acti n treadmil l mode l o f motility. 

The tw o principle features of the treadmill model ar e 

the retrograd e flo w o f actin, whic h i s due t o myosi n 

motors, and the polymerization of actin at the leading 

edge o f the filopodi a (o n right) . I f polymerization i s 

faster tha t retrograd e flow , ther e i s forwar d move - 

ment; i f it is slower, there i s retraction. Note that actin 

is coupled t o the substrate by linker proteins that bind 

transmembrane CAMs . 

motors (Li n e t al., 1996) . I f the rat e of actin polymer - 

ization a t the leadin g edg e i s greater than th e rat e of 

actin retrograd e flow , th e filopodi a advances . I f polymerization 

is slower than retrograde flow, the filopodia 

retracts. If polymerization and retrograd e flow are balanced, 

ther e i s no ne t chang e i n th e positio n o f th e 

filopodia. The substrat e on which a neurite grows can 

interact wit h transmembran e adhesio n molecules , 

which ar e attache d t o actin indirectl y via linker pro - 

teins. Th e couplin g o f the substrat e t o th e acti n cy - 

toskeleton (a ) allow s fo r th e generatio n o f tensio n 

when force s are applied t o actin an d (b ) accounts for 

the influenc e o f substrat e o n growt h con e motilit y 

(Suter an d Forscher , 1998) . This mode l i s not com - 

plete, a s axon s treate d wit h acti n depolymerizin g 

agents stil l demonstrat e ne t growt h (Bentle y an d 

Toroian-Raymond, 1986) , an d applicatio n o f rnicro - 

tubule destabilizing reagents can prevent neurite out - 

growth (Bamburg et al., 1986) . It should be noted that 

current model s o f growt h con e motilit y ar e largel y 

based o n experiments in unpolarized Aplysia neuron s 

or in spinal or sensory neurons isolate d from frog s an d 

chicks. Som e mechanism s o f growt h con e motilit y 

may differ amon g species , cell types, and neurite type , 

but th e regulator y molecule s ar e likel y t o b e conserved. 

A large an d divers e group o f proteins tha t modu - 

late cytoskeleta l dynamic s throug h intracellula r 

signaling o r direc t actio n i s involve d i n regulatin g 

growth con e motility . Th e Rh o famil y o f proteins ,


which ca n influence both th e actin an d microtubule 

cytoskeleton, play s a n importan t rol e i n regulatin g 

growth con e motility . Th e contro l o f actin polymer - 

ization at the leadin g edge o f growth cones i s an area 

of activ e study . Member s o f th e Ena/Vas p family , 

which prevent barbed acti n capping , ca n regulate th e 

behavior o f filopodi a (Lebran d e t al. , 2004) . Othe r 

molecules tha t ma y pla y role s i n acti n dynamic s at 

the leadin g edg e includ e profilin , fascili , an d acti n 

depolymerizing factor/cofili n (Will s e t al. , 1999 ; 

Meberg an d Hamburg , 2000 ; Coha n e t al, 2001) . 

Further wor k i s require d t o construc t a cohesiv e 

model o f how thes e an d othe r protein s regulat e cy - 

toskeletal dynamics at the leading edge o f the growth 

cone. 

Directed Growth 

Growth cone s ar e generall y considered t o mediat e 

the respons e o f growing axons and dendrite s t o th e 

myriad of guidance cue s that direct the establishmen t 

of appropriate connectivity. It i s possible t o alter th e 

trajectory o f neurite growth by applying a gradient of 

a chemotropi c molecul e t o a growt h con e i n vitr o 

(Zheng e t al. , 1994) . I n fact , thi s assa y i s now com - 

monly use d t o tes t whether a protei n ca n serv e a s a 

guidance cue. Furthermore, many receptors for known 

guidance cues are expressed in the plasma membrane 

of growth cones. I t is believed that gradients of guidance 

cue s acros s the surfac e o f the growt h con e pro - 

duce asymmetri c effect s o n receptor s tha t mediat e 

growth cone steering (Goodman, 1996) . 

The respons e o f growt h cone s t o guidanc e cue s 

depends o n bot h th e acti n an d microtubul e compo - 

nents o f the cytoskeleto n and th e effector s tha t regu - 

late them downstrea m from guidance receptors . I t has 

been propose d that growth cone turning occurs when 

actin i s polymerize d asymmetricall y withi n th e 

growth cone and when there i s a subsequent increase 

in microtubul e entry into and polymerizatio n in th e 

actin ric h zone (Den t and Gertler, 2003) . Consistent 

with this model, disruptions of regulators of either the 

actin o r th e microtubul e cytoskeleto n ca n resul t i n 

defective axo n guidance. Example s include the actin 

interactors Arp2/3 and Ena/Vas p (Lanier et al., 1999 ; 

Lee e t al, 2004 ; Strasse r e t al, 2004 ) a s well a s the 

microtubule interactors , plu s end-trackin g protein s 

(Lanier et al, 1999 ; Lee et al, 2004). The precise significance 

of these molecules in directed growth, however, 

remains a subject of active investigation. 

Several molecule s tha t ca n signa l betwee n guid - 

ance receptor s an d th e cytoskeleto n i n growt h cone s 

have been identified. Rho family members, which can 

regulate both acti n and microtubules, have been im - 

plicated i n signalin g downstrea m fro m guidanc e 

receptors (Gua n an d Rao , 2003) . Also, relativ e con - 

centrations o f cycli c nucleotide s (cycli c adenosin e 

monophosphate [cAMP ] an d cycli c guanosin e 

monophosphate [cGMP] ) can modulate the respons e 

of growth cones to guidance cues . For instance, a gradient 

of brain-derived neurotrophi c facto r (BDNF), a 

factor tha t i s usuall y attractive fo r Xenopus growt h 

cones, becomes repulsive if the growth cone is treated 

with a cAM P inhibito r (Song e t al, 1997) . Anothe r 

potential signal downstream from guidanc e receptor s 

is Ca 2+ . Liv e imagin g o f growt h cone s i n cultur e 

shows that transient increases in Ca 2+ o n one sid e of 

the growt h con e correlat e wit h turnin g towar d th e 

contralateral side (Gomez e t al., 2001). Interestingly, 

larger Ca z+ transients can result in ipsilateral turning. 

This paradox i s explained b y data indicatin g that low 

concentrations o f Ca 2+ ac t primaril y throug h cal - 

cineurin phosphatase, whereas higher concentration s 

act vi a calcium-calmodulin-dependen t protei n ki - 

nase I I (We n e t al. , 2004) . The recen t findin g tha t 

Ca 2+ concentrations i n the growth cone can be mod - 

ulated by cAMP and cGMP control of calcium chan - 

nels suggests that Ca 2+ acts on growth cone guidanc e 

downstream fro m th e cycli c nucleotides (Nishiyama 

et al., 2003). Though data on interactions between receptors, 

signals , effectors , an d th e cytoskeleto n ar e 

emerging, muc h remain s to b e don e befor e a com - 

plete model o f how a specific guidance cu e result s in 

a change i n growth cone motilit y can be constructed . 

AXON GUIDANCE 

In th e lat e nineteent h century , Ramon y Cajal pro - 

posed tha t axon s coul d b e guide d chemotropically , 

but the ide a fel l ou t of fashion and was untested until 

it was revisited by Roger Sperry in th e 1940 s (Sperry, 

1963). I n a serie s of classic experiments, Sperr y severed 

a frog optic nerve, rotated the eye 180°, and then 

allowed connections t o form again. These frogs mad e 

180° error s i n their attempt s t o catch flies. In contra - 

diction to the popular view, including that of his thesis 

advisor , Sperr y conclude d tha t th e regenerate d 

axons di d no t innervat e ne w targe t region s t o com - 

pensate fo r th e ey e rotation , bu t projecte d t o thei r

original sites in the tectum. The outcome s o f this and 

other relate d experiment s le d Sperr y to propose tha t 

extending axon s ar e guide d b y "cytochemica l tags. " 

He further propose d that neural connectivity is specified 

with onl y a fe w such tag s or cue s distribute d in 

orthogonal gradient s (Sperry, 1963). Several of these 

cues have now been identified , validating these principles 

and indicatin g that axo n guidance i s best con - 

sidered in their context. 

Guidance cue s ca n b e secrete d fro m a restricted 

source to form long-range, diffusible gradient s that act 

as molecula r beacons , wherea s membrane-attache d 

cues ca n for m short-range , standin g gradient s (se e 

Directed Growth , above) . Concurrently , adhesio n 

molecules expresse d alon g th e surfac e o f axon s ca n 

interact wit h othe r axon s o r particula r substrate s to 

promote axo n fasciculation , extension , turning , o r 

stopping. Most cues have different function s at differ - 

ent stage s o f developmen t o r i n differen t pathways , 

and man y hav e multipl e receptor s an d co-receptor s 

that ca n mediat e differen t responses . Thi s hig h de - 

gree of complexity make s it difficult t o sort out which 

interactions ar e importan t fo r the generatio n o f particular 

connections, bu t i t supports Sperry's ide a tha t 

a smal l numbe r o f guidance cue s ca n generat e th e 

diverse response s require d fo r high-fidelit y network 

development. I n ligh t o f thi s diversity , exemplar y 

cases o f axo n guidanc e hav e been selecte d t o illus - 

trate how the challenges o f directed growth and specificity 

are met. 

Many individua l growt h cone s appea r t o expres s 

all o f the receptor s require d a t th e appropriat e tim e 

and place to respond t o the cues necessar y for their final 

targeting. Some pathways capitalize on the effort s 

of a fe w pioneer neurons . Pioneer s ar e bes t under - 

stood in invertebrates, where it is well established that 

they differentiat e an d mak e connection s earl y in de - 

velopment when distances are small. The connection s 

provide a pat h fo r axon s extendin g fro m later-bor n 

neurons. I n mammalian cerebra l cortex , th e develop - 

ing cortical plate is sandwiched between transient layers 

called the marginal zone and subplate that contai n 

the earliest born neurons (Marin-Padilla, 1971) . Populations 

of subplate neurons send axons to the thalamus 

before the layer V and VI neurons that form th e adult 

projections are even born (McConnell e t al., 1989) . In 

a very different way , Cajal-Retzius neurons in the marginal 

zone pioneer local intracortical connections and 

may be importan t for generating a columnar organi - 

zation of cortical networks (Sarnat and Flores-Sarnat , 


2002). I n thi s way , pioneer neuron s produc e frame - 

works that , fo r particula r networks , ar e critica l fo r 

specifying pathways. 

Midline Crossing in Vertebrate 

Ventral Spinal Cor d 

After closel y examining the appearanc e an d trajectories 

of Golgi-impregnated neurons in the spinal cord , 

Ramon y Cajal hypothesize d tha t group s o f dorsally 

disposed neuron s giv e rise to axons that ar e attracte d 

to th e ventra l midline . Th e proo f cam e nearl y 100 

years later , whe n dissecte d midlin e expiant s wer e 

demonstrated t o b e attractiv e to commissurall y pro - 

jecting axons (Tessier-Lavigne et al., 1988 ) (Fig . 4-4) . 

This system was used a s a bioassay to isolat e netrin-1 

and -2 , which ar e solubl e cue s tha t ac t a s attractive 

proteins for commissural axons. Subsequent work has 

demonstrated tha t netrin' s attractiv e action i s medi - 

ated b y binding t o it s receptor, deleted i n colorecta l 

cancer (DCC ) (Kenned y et al., 1994 ; Serafin i e t al, 

1994; Keino-Mas u et al., 1996 ; Fazel i et al, 1997) . Interestingly, 

mutant mice hypomorphi c for the netrin - 

1 allele or lacking DCC stil l show some commissural 

axon crossing (Serafini et al., 1996; Fazeli et al., 1997) . 

This finding indicates that netri n doe s no t ac t alone . 

Sonic hedgeho g (Shh) , a morphogen secreted b y the 

floor plate, i s better know n fo r it s role i n neura l pro - 

genitor specification (Charron e t al., 2003; Yoshikawa 

and Thomas, 2004). Shh can also attract or influence 

the directio n o f polarizatio n o f commissura l axon s 

through it s receptor smoothened (Smo). Assisting the 

actions o f both Shh and netrin, heterodimers of bone 

morphogenic protein 7 (BMP7) and growth differenti - 

ation facto r 7 (GDF7) secreted fro m dorsa l roof plat e 

cells repel the incipient commissural axons away from 

the dorsa l midlin e (Augsburge r et al. , 1999 ; Butle r 

and Dodd, 2003). 

Once attracted t o the floor plate, commissural axons 

cross the midlin e (Seege r e t al., 1993) . Other axons 

turn back and for m ipsilatera l longitudinal tracts. 

Crossing i s regulate d i n par t b y th e signalin g o f 

chemorepellants of the Sli t family which are secreted 

by midlin e gli a an d bin d Rob o famil y receptor s o n 

commissurally projectin g growt h cone s (Rothber g 

et al , 1988 , 1990 ; Eros e e t al. , 1999 ; Lon g e t al, 

2004). Unlike other Robo family members, th e recep - 

tor Rig- 1 appear s t o attenuat e Sli t sensitivity , since 

commissural axon s neve r cros s the midlin e i n Rig-1 - 

deficient mic e (Sabatie r e t al. , 2004) . I n wild-typ e


FIGURE 4- 4 Midlin e guidance . Schemati c diagram s o f developin g mam - 

malian spinal cord depict the trajectories of eommissurally projecting neurons 

relative to the roo f plate an d floor plate in cross-section (A) or molecular gradients 

i n longitudinal preparation through th e ventra l rnidline (B). Pre-crossing 

axons are repelled fro m dorsa l midline by BMP? in the roo f plate (no t shown) 

and attracte d ventrally by netrin an d Sh h gradient s emanatin g from th e floor 

plate (shade d circles). Post-crossing axons become sensitive to Slit - and Sema - 

phorin 3 B an d F-mediate d repulsio n an d d o no t retur n t o th e floo r plate . 

(Source: Modifie d from Williams et al, 2004) 

animals, Rig- 1 concentration s decreas e an d Robo- 1 

and - 2 amounts increas e soo n afte r crossing , whic h 

may serve to expe l axon s from th e midlin e (Sabatie r 

et al. , 2004) . Thi s expulsio n i s probabl y aide d b y 

Robo-Ts abilit y t o silenc e DCC-mediate d attractio n 

to netrin by binding directly to DCC i n the presenc e 

of Slit (Stein an d Tessier-Lavigne , 2001 ) or by growth 

cone desensitizatio n t o th e hig h concentration s o f 

netrin at the midline (Piper et al., 2005). Sema-3B, an 

additional solubl e repellan t secrete d b y th e floo r 

plate, prevent s axon s from returnin g t o th e midlin e 

via interactions with its receptor neuropilin- 2 (Zou et 

al., 2000) . Afte r crossing , th e positio n o f longitudi - 

nally runnin g tract s appear s t o b e regulate d b y th e 

midline Sli t gradien t interactin g wit h Robo- 1 o r -2 

(Rajagopalan et al. , 2000 ; Simpso n et al. , 2000) . 

Concurrently, Wnt-4 , actin g throug h it s recepto r 

frizzled3, an d Shh , actin g throug h hedgeho g inter - 

acting protein , hel p orien t axon s anteriorl y i n th e 

longitudinal axi s (Lyuksyutova et al., 2003; Bourikas 

er al., 2005) , but the interactio n betwee n thes e cue s 

has ye t t o b e investigated . Collectively , thes e dat a 

support th e idea s tha t certai n guidanc e cue s ca n 

trump th e other s an d sugges t that cues are processe d 

hierarchically rather than being integrated. 

Generating Retinotectal Maps 

Once axon s reach thei r target region, they terminat e 

in highly organized pattern s that reflect their points of 

origin. Th e projection s extending fro m retina l gan - 

glion cell s t o the tectu m i n fish and frog s an d t o th e 

superior colliculus in rodents has served as the canon - 

ical syste m fo r studying the developmen t o f pattern - 

ing. I n mos t mammals , a ligh t shown i n th e fa r lef t 

visual field projects on the ventronasal part of the lef t 

retina, whic h i n tur n project s contralaterall y t o th e 

caudomedial regio n o f the superio r colliculu s (Fig . 

4-5). I f a n experimente r systematicall y move s a 

recording electrode rostrall y through th e superior colliculus, 

th e stimulatin g ligh t sourc e need s t o b e 

moved i n a n orderl y progression fro m lef t t o righ t to 

activate th e neurons . Thus , th e superio r colliculu s 

contains topographic map s of the visual field. 

A larg e bod y o f dat a suppor t th e correlatio n o f 

ephrin gradient s i n the retin a an d superior colliculus

interactions (McLaughlin et al., 2003; van Horck et al., 

2004). EphA receptor protein kinases are expressed in 

high in temporal an d low in nasal retina. EphrinA ligands 

are expresse d mostl y in hig h cauda l t o low rostral 

gradient s in th e superio r colliculus. The ligand s 

guide the formation of retinotopic map s oriented i n a 

rostral-to-caudal progressio n by repulsive eyes . Mice 

having targeted disruption s in both ephrinA2 and A5 

genes sho w a nea r complet e los s of rostral-to-caudal 

patterning in the superior colliculus (Feldheim e t al., 

2000). Introducin g new gradient s b y overexpressing 

additional Eph A receptor s i n subpopulation s o f retinal 

ganglio n cell s results in the creatio n o f two maps 

along th e rostral-to-cauda l axis (Brow n et al. , 2000). 

Orthogonal gradient s o f EphB Z i n th e retin a an d 

ephrin-Bl an d 2 in the tectum labe l the dorsoventral 

axis (Braiste d et al. , 1997) . Member s o f the ephrin B 

subfamily appea r t o operate principall y via attractive 

rather tha n repulsiv e interaction s (Hindge s e t al. , 

FIGURE 4-5 Th e retinocollicula r map. Schematic diagram 

depict s orthogona l gradient s o f Eph s an d 

ephrins tha t mediat e retinotopi c mappin g togethe r 

with the trajectory of a single projection i n the roden t 

projection fro m retin a t o superio r colliculus . Ligh t 

from a point in the fa r left visual field strikes the nasal 

retina o f the lef t ey e (circle) in it s monocular region . 

Ganglion cell s i n thi s zon e projec t (dar k axon ) t o 

caudal-medial point s on the right superior colliculus 

(oval). Eph A tyrosin e kinase s bind an d ar e repelle d 

from high concentrations o f ephrinA ligands, whereas 

EphB tyrosin e kinases bind an d ar e attracted t o high 

concentrations of EphB ligand. 

NEURONAL DIFFERENTIATION : FROM AXONS TO SYNAPSE S 5 3 

2002; Mann e t al., 2002) . The mechanism s guiding 

retinal ganglio n cel l axon s appea r t o follo w Sperry' s 

chemoaffinity mode l of targeting by orthogonal stand - 

ing gradients (Sperry, 1963) . In a n importan t exten - 

sion o n thi s model, recen t wor k indicate s that at low 

concentrations, EphA-ephrin A interaction s ar e at - 

tractive fo r axon extension, wherea s high concentra - 

tions elici t a repulsiv e response . Thu s axon s wil l 

readily extend int o th e superio r colliculus until the y 

reach a point where positive and negativ e influences 

are balanced (Hanse n et al, 2004). 

It is noteworthy that in mice lacking ephrinA2 and 

A5, retinal ganglio n cel l axons still target the superio r 

colliculus, and thei r axonal arbors are evenly distributed 

within the entire space allotted (Feldhei m e t al., 

2000). The mechanis m determinin g this interaxonal 

spacing remains unknown. Furthermore, i n the sam e 

mice, ther e i s evidenc e i n th e clusterin g o f opti c 

nerve terminal s that som e nearest-neighbo r relation - 

ships, and thu s som e topographi c ordering , i s maintained 

over small distances. Such clusterin g has been 

proposed t o be th e resul t of correlated activit y (Feldheim 

et al, 2000) and supports th e ide a that activity 

plays a major rol e i n the prunin g and shapin g of the 

final map s tha t ar e generate d b y standing Ep h an d 

ephrin gradients. 

Cues Are Bifunctional 

Netrins ar e generall y attractiv e an d EphA-ephrin A 

interactions, repulsive, but nearl y all guidance cue s 

can generat e diametricall y opposed response s in particular 

circumstance s (Fig . 4-6) . Differen t response s 

are sometimes the function of different cel l types. For 

example, netrin-1, secreted b y the ventral floor plate, 

repels moto r axon s extending from th e trochlea r nu - 

cleus such that they exit the dorsal aspect of the brain 

at th e midbrain-hindbrai n border . Thi s stand s i n 

stark contras t to it s attraction for spinal commissural 

axons (Colamarino and Tessier-Lavigne, 1995) . More 

commonly, changes between attractio n and repulsion 

occur a t differen t stage s of outgrowth an d appea r t o 

be employe d withi n individua l growt h cones . Suc h 

dynamic signalin g appears t o b e require d fo r steady 

axon extensio n through transitio n zones, such a s the 

ventral midline, and to titrate growth cone responsiveness 

within a target t o achieve a smooth representa - 

tional map . 

Bifunctional response s resul t fro m differentia l 

activation o f downstrea m signalin g pathway s tha t


FIGURE 4-6 Guidanc e cues . Examples of long- and short-rang e axon guidance proteins 

used i n vertebrates. Molecules are depicted acros s from on e another as they would be in 

situ. Members of the ephrin family generate topographic maps , such as that found in th e 

retinotectal projection . Integrins , an d immunoglobuli n (Ig ) superfamil y member s o f 

the calcium-dependen t (classi c cadherins) an d calcium-independen t classe s (e.g. , Ng - 

CAM) mediat e short-rang e guidanc e throug h contact-dependen t mechanisms . Thre e 

major familie s o f long-range axo n guidance ar e indicate d here : netri n an d it s receptor, 

DCC; Sli t and it s receptor, Robo; and clas s 3 Semaphorins and it s co-receptor complex , 

neuropilins an d plexins . (Source: Modifie d fro m Benso n e t al. , 2001 , an d Y u an d 

Bargmann, 2001) 

ultimately regulat e acti n dynamic s (fo r review , se e 

Pasterkanip and Kolodkin , 2003). For instance, attractive 

growt h con e turnin g o f Xenopus spina l moto r 

growth cones to gradients of netrin-1 can be converte d 

to repulsio n upo n associatio n of the cytoplasmi c do - 

mains of the netrin DCC recepto r with a co-receptor, 

UNC5 (Hon g e t al, 1999) . Additionally , changing 

amounts o f cycli c nucleotide s ca n chang e growt h 

cone response s t o BDNF , netrin-1 , an d Sema3 D 

(Ming et al, 1997 ; Son g et al, 1998) , results suggesting 

that previous experience or environment ca n alter 

responsiveness. I n suppor t o f this possibilty, Xenopus 

retinal ganglion cell axons grown on laminin have decreased 

ratios of cAMP/cGMP relative to those grown 

on fibronectin, and are repulsed rather than attracte d 

by netri n (Hopke r e t al. , 1999) . Thes e example s

(a) suggest that the response to a cue can be modified 

by bot h outside-i n (recepto r t o signalin g pathway) 

and inside-ou t (signaling molecule to altered stat e of 

receptor responsiveness) mechanisms and (b) support 

the ide a that growt h cones hav e multipl e mean s by 

which to finely tune their responses . 

Adhesion and Guidance 

Growth cones explore their environment by regulating 

their attachment to other neurons, glia, or components 

of the extracellular matrix (ECM). Contact-dependent 

interactions betwee n cel l adhesio n molecule s 

(CAMs) o f th e immunoglobuli n superfamily , cad - 

herin superfamily , integrins, and th e EC M generat e 

traction sufficient fo r growth cone extension. Interactions 

between CAMs and ECMs that are inhibitory to 

growth creat e boundarie s (Sno w an d Letourneau , 

1992). It has long been suspecte d that standing gradients 

o f ECM s ma y als o b e abl e t o orien t o r stee r 

growth cones , bu t th e dat a directl y supportin g thi s 

role a s a guidanc e cu e ar e quit e recen t becaus e o f 

the difficult y o f generatin g continuous , substrate - 

bound gradient s (Adam s et al., 2005) . Cell-cell an d 

cell-substrate interaction s can promote axon bundling 

(fasciculation) an d defasciculation , thereb y makin g 

the process of guidance mor e efficient . O n th e othe r 

hand, there is little evidence indicating that selective 

fasciculation serve s as a critica l guidanc e cu e (Ben - 

son et al, 2001). 

Contact-dependent interaction s ca n sto p axo n 

extension when i t has reached it s final target region, 

preceding th e formatio n of terminal branches an d 

synapses. Axons usually terminate within small neural 

groups or a restricted number o f layers, greatly reducing 

th e numbe r o f potentia l individua l neural 

targets. Homophilic N-cadherin adhesion appears to 

prevent thalamocortica l an d retinocollicula r axon s 

from extendin g pas t thei r targe t layer s (Inou e an d 

Sanes, 1997 ; Poskanzer et al., 2003) and can apically 

restrict mossy-fibe r terminal field s i n hippocampu s 

(Bekirov e t al. , 2002) . Similarly , attractive interactions 

betwee n axonin-1/transien t axona l glycopro - 

tein 1 an d neuron-gli a CA M (Ng-CAM) , an d 

between Fll/contactin an d Ng-CAM-relate d CA M 

(Nr-CAM) contribut e t o lamina-specifi c termina - 

tions o f nociceptiv e an d proprioceptiv e fibers , re - 

spectively, in chick spinal cord (Perrin et al., 2001). 

Thus, CAM s appea r to encode laminar recognitio n 

cues, bu t the y ma y als o provid e a sto p signal , 


preventing axon s fro m extendin g further . Whethe r 

recognition an d stoppin g ar e mediate d concomi - 

tantly by a particular cue or combination of cues has 

yet to be investigated. 

Few experiments have addressed directly how adhesive 

cue s becom e integrate d wit h othe r guidanc e 

cues. Th e emergin g pictur e promise s a complicate d 

answer. The CAM L I can suppor t rapid axon extension, 

but in a cis complex with the receptor neuropilin- 

1, i t als o appear s t o b e require d fo r repulsio n o f 

cortical axons by the solubl e ligand Sema3 A (Castellani 

et al., 2000). Additionally, soluble LI (whic h can 

be generate d b y metalloproteas e cleavage ) can convert 

a repulsive Sema3A response to attraction (Castellani 

et al., 2000) in a manner reminiscent o f laminin's 

ability to convert an attractive netrin response to on e 

of repulsio n (se e Cue s Ar e Bifunctional , above ) 

(Hopkeretal., 1999) . 

Interactions betwee n substrat e and solubl e cue s 

may underli e ho w commissura l axon s ca n travers e 

the neutra l midline an d exten d laterally toward longitudinal 

tracts . Rob o activatio n by Sli t ca n elimi - 

nate N-cadherin-base d adhesio n tha t normall y 

promotes rapi d extensio n (Rhe e e t al. , 2002) . This 

activation correlates with the formation of a complex 

between Robo , N-cadherin, an d Abelson kinas e and 

is accompanie d b y phosphorylatio n o f p-catenin , a 

necessary cadherin binding partner. Participation in 

this complex, i n turn, occludes interactio n of N-cadherin 

wit h the acti n cytoskeleton . One ca n imagin e 

that rapi d N-cadherin-mediate d outgrowt h concur - 

rent with high Rig-1 expression would suffice t o permit 

progressio n throug h a n otherwis e neutra l 

midline t o th e poin t a t which Rob o concentrations 

increase, wher e axon s would disengage fro m N-cad - 

herin, turn , an d gro w longitudinally by a differen t 

mechanism. 

Thus, severa l attractiv e and repulsive , short - and 

long-ranged cue s ac t hierarchically and occasionally 

collaborate t o generat e a fina l stereotype d targetin g 

outcome. Som e cue s ac t similarl y throughou t th e 

rostral-caudal extent of the developing nervous system, 

but th e action s o f others appear to differ acros s CN S 

regions. Additionally, certain molecule s ar e co-opte d 

during multiple epochs of development for slightly different 

purposes. The temporal sensitivities of such cues 

are just beginning to be appreciated with the advent of 

conditional geneti c tool s an d improve d method s fo r 

transient transfections. These techniques ar e useful for 

determining th e virtuall y unknow n mechanisms tha t


stop axon extension or stimulate the generation of terminal 

arbors for synapse formation. 

SYNAPTOGENESIS 

Sherrington coine d th e ter m synapse i n 189 7 t o de - 

scribe the site s where axons contact th e dendrite s o r 

soma o f other neurons , i n keepin g wit h th e neuro n 

doctrine that nervous tissue comprises separat e cells . 

Although thi s doctrine also was espoused b y Ramon y 

Cajal? Kôlliker , His, Forél, and severa l other contem - 

poraries, Golgi and other s fervently supported a n opposing 

reticular theory that the nervous system was an 

anastomosing network (Shepherd, 1991) . Sinc e arguments 

fro m bot h side s were base d mostl y o n Golgi - 

impregnated sample s viewe d b y ligh t microscopy , 

unequivocal evidence supportin g the former doctrin e 

did not come until half a century later from a series of 

electrophysiological an d E M studies . Kuffle r (1942 ) 

and Fat t an d Kat z (1951 ) demonstrate d a t th e fro g 

neuromuscular junctio n tha t th e curren t underlyin g 

changes i n membran e potentia l a t th e postsynapti c 

muscle endplat e coul d no t deriv e directl y fro m th e 

presynaptic moto r axon, as the reticular theory would 

have predicted . The n studie s b y Palad e an d Pala y 

(1954) an d D e Roberti s an d Bennet t (1955 ) i n th e 

mid-1950s provided clear, high-resolution EM image s 

of a cleft separating pre- and postsynapti c membrane s 

at chemica l synapses . Ironically , soli d evidenc e 

supporting th e existenc e o f a n electrotoni c synaps e 

came shortly thereafter, with the identificatio n o f gap 

junctions, wher e Bennet t (1963 ) an d other s demon - 

strated tha t ioni c curren t ca n flow directly from on e 

neuron t o anothe r (Peter s e t al. , 1991 ; Shepherd , 

1991). 

Subsequent electrophysiological , anatomical, and 

biochemical findings continu e to challenge our col - 

lective notio n o f th e synaps e beyon d Sherrington' s 

general conceptualization . Thoug h characterizin g 

the matur e synapse in differentiate d neura l networks 

has occupied neurobiologist s fo r more than a century, 

more recen t wor k indicate s tha t earlie r stages o f assembly 

and differentiatio n ar e crucial for generating a 

normal network . In thi s vein, the las t sections o f this 

chapter describ e ho w axona l and dendriti c morpho - 

logies an d compositio n ar e modifie d t o assembl e 

synapses i n th e vertebrat e CNS . Sinc e maturatio n is 

generally assesse d b y th e exten t t o whic h adul t fea - 

tures are present, the following discussion of chemical 

and electrica l synapse s begin s wit h description s o f 

their mature form and composition . 

Structure an d Composition of 

Mature Chemical Synapse s 

Ultrastructural Features 

The matur e chemical synaps e is a polarized, adhesiv e 

junction formed most commonly between axon s and 

dendrites. In electro n micrograph s thi s synaps e 

(sometimes called a zonula adherens) i s characterized 

by rigidly parallel pre - and postsynapti c membranes , 

separated b y a clef t o f 1 2 to 2 0 nm, electron-dens e 

material withi n th e clef t and , i n most cases , thick , 

electron-dense materia l i n th e cytoso l subjacen t t o 

the postsynapti c membran e (Fig . 4-7) . Thi s synapse 

resembles othe r adhesive junctions , but can be distinguished 

b y th e presenc e o f synapti c vesicle s (SVs ) 

close to the junction i n the presynaptic cytoso l (Gray , 

1963). I n aldehyde-fixe d E M specimens , SV s ar e 

roughly 40-50 nm i n width, can appea r spherica l o r 

oblong (pleomorphic) , an d ca n hav e agranula r o r 

electron-dense core s (Grill o an d Palay , 1963 ; Val - 

divia, 1971) . A t mos t synapse s the y ar e foun d i n a 

readily releasable pool near the plasma membrane or 

in a more remove d reserv e pool. Vesicle s within thi s 

pool ca n b e foun d docke d withi n a gridded arra y of 

presynaptic cytomatri x component s or , i n photore - 

ceptors, along a presynaptic ribbon that runs orthogonal 

t o th e junctio n (Sjostrand , 1958 ; Gray , 1959a ; 

Bloom an d Aghajanian , 1966 , 1968) . Mitochondri a 

are ofte n foun d both pre - and postsynapticall y (Ibat a 

and Otsuka , 1968 ; Bodian , 1971) , an d a postsynapti c 

web of filaments is often subjacent to the postsynaptic 

density (PSD ) (D e Roberti s e t al, 1961 ; Gray , 1963 ; 

Peters an d Kaiserman-Abramof , 1969 ; Stewar d an d 

Levy, 1982 ; Space k and Harris , 1997) . 

Although dendrodendritic, dendrosomatic, and all 

other conceiveable pairing s do occur in the vertebrate 

CNS, th e larg e majorit y o f chemica l synapse s 

encountered ar e axosomati c o r axodendriti c (Peter s 

et al , 1991) . Axodendriti c synapses occur o n eithe r 

smooth dendriti c shaft s o r mushroom-shaped protru - 

sions calle d spines tha t stu d th e dendriti c arbor s 

of most glutamatergi c neuron s (Nimchinsk y e t al , 

2002). Spin e synapse s are typifie d b y a pronounced 

PSD, a relativel y wide clef t (nea r 2 0 nm), polyribo - 

somes i n clos e proximit y t o smoot h endoplasmi c 

reticulum, an d rounde d presynapti c vesicle s i n

FIGURE 4-7 Chemica l synapse . This electro n micro - 

graph (left ) an d schemati c diagra m (right ) o f un - 

stained tissu e section s take n fro m adul t ra t spina l 

cord depic t severa l ultrastructural features typical of 

mature type I, asymmetric synapses. Among these features, 

abundant presynaptic vesicles and a pronounced 

aldehyde-fixed tissues . By comparison, som e shaf t an d 

nearly all somal synapses have a much les s pronounced 

or absent PSD, a narrower cleft (near 12 nm), and con - 

tain both rounde d and oblong (pleomorphic ) vesicles. 

In Gray' s studie s o f cerebral cortex , h e terme d thes e 

synapses types I and II , respectively , althoug h Colon - 

nier's distinctio n o f asymmetrica l an d symmetrica l 

synapses based o n the appearanc e o f the PS D i s more 

widely used (Gray , 1959b; Colonnier, 1968). 

Molecular Constituents 

Morphological distinction s betwee n synapse s reflect 

functional differences : roun d SV s a t asymmetri c 

synapses usuall y release glutamate , an d pleomorphi c 

SVs a t symmetri c site s commonl y carr y inhibitor y 

neurotransmitters (Eccle s e t al , 1961 ; Uchizono , 

1965). Smal l dense-cor e SVs , i n contrast , generall y 

carry catecholaminergi c neuromodulators , an d larg e 

dense-core vesicle s (-100-20 0 nm) carr y neuropep - 

tides (D e Roberti s an d Pelligrin o d e Iraldi , 1961 ; 

Wolfe e t al. , 1962) . A s wit h mos t classificatio n 

schemes, thes e correlation s hav e notabl e exceptions . 

For instance , som e small , clea r SV s contai n neu - 

ropeptides suc h a s oxytoci n o r a combinatio n o f 


postsynaptic density (PSD) ar e the mos t reliabl e an d 

conspicuous. Als o visible are th e electron-dens e col - 

lection o f transmembrane an d matri x molecule s i n 

the synapti c cleft an d the filamentous web subjacent 

to th e PSD . Mitochondri a ar e presen t i n bot h th e 

presynaptic terminal and the dendritic spine. 

neurotransmitter an d neuropeptid e (Meiste r e t al. , 

1986), an d som e 8 0 nm dense-cor e vesicle s can carr y 

catecholamines. Additionally , another typ e o f 8 0 nm 

dense-core vesicl e i s though t t o delive r protein s re - 

quired a t nascen t synapse s (Ahrnar i e t al. , 2000 ; 

Bresler e t al., 2001 ; Shapir a e t al, 2003) . The latte r 

also has been called a Piccolo-Bassoon transport vesicle 

(PTV) for the proteins used to identify it during purification 

(Zhai , 2001) . 

Converging technica l approache s hav e bee n use d 

to identif y an d characteriz e a bev y o f synapti c con - 

stituents, suc h a s those involve d i n vesicl e traffickin g 

(Sudhof, 2004) . Synapsin s i n th e vesicl e membran e 

tether SV s to an acti n framewor k until they ar e phos - 

phorylated b y calcium-dependen t kinase s followin g 

Ca 2+ influ x (Bahle r an d Greengard , 1987 ; Petrucc i 

and Morrow , 1987) . Membrane-boun d Rab 3 appear s 

to modulat e transpor t o f thes e liberate d vesicle s t o 

active zone s (Leender s e t al. , 2001) . SV s doc k a t 

these site s throug h th e bindin g o f solubl e N - 

ethylmaleimide-sensitive fusio n protei n attachmen t 

protein receptor s (SNARES ) i n th e vesicl e mem - 

brane t o target s a t th e plasm a membrane . Amon g 

these interactions , th e comple x forme d by the vesicular 

SNARE S synaptobrevin/vesicle-associate d


membrane protei n wit h its target SNARES , synaptosomal 

associated protein (SNAP) 25 and syntaxin, was 

the first identified and is the most widely known (Sollner 

e t al. , 1993 ; Pevsne r e t al. , 1994) . Syntaxins , i n 

turn, ca n bind t o either Muncl S o r Muncl3 in th e 

presynaptic gri d to modulat e th e primin g event s for 

vesicle fusion (Hat a et al., 1993 ; Asher y et al, 2000 ; 

Misura et al, 2000). SNAP-25 binding by synaptotagmin 

i n th e vesicl e membran e appear s t o regulat e 

subsequent fusio n por e openin g durin g calcium - 

dependent exocytosi s (Jorgensen et al. , 1995 ; Sugit a 

and Sudhof, 2000; Littleton e t al, 2001). 

ECM protein s such as laminins, collagens, and proteoglycans 

are found i n and aroun d th e synapti c clef t 

(Dityatev an d Schachner , 2003) . As with othe r adhe - 

sive junctions, the density within the synaptic cleft con - 

tains CAMs such as Ng-CAM and N-cadherin (Chavi s 

and Westbrook , 2001; Phillips et al, 2001; Schuster et 

al., 2001; Guan an d Rao, 2003). Members o f receptorligand 

families such as Eph-ephrins and th e neurexinneuroligins 

ar e als o presen t (Scheiffel e e t al, 2000 ; 

Henderson e t al, 2001; Dean e t al, 2003; Grunwald 

et al. , 2004) . Transmembran e protease s suc h a s 

metalloprotease-disintegrins cleave the ectodomains of 

these transynapti c molecule s t o attenuat e signalin g 

(Tanaka e t al, 2000 ; Matsumoto-Miya i e t al. , 2003) . 

Other extracellula r proteases such as plasminogen activators 

can degrade components o f the ECM i n a manner 

suspecte d to permit morphological change s a t the 

synapse durin g development an d plasticit y (reviewed 

in Dityatev and Schachner, 2003 ; Berardi et al, 2004). 

In th e postsynapse , the PS D comprise s a number 

of signaling and scaffolding molecules for receptors in 

the plasm a membrane (Wals h and Kuruc, 1992 ; Walikonis 

et al, 2000; Kim and Sheng, 2004; Peng et al, 

2004; Phillips et al, 2004; Collins e t al, 2005). One 

quintessential componen t i s PSD-95 , a membe r o f 

the PSD/synapse-associate d protein/membrane - 

associated guanylate kinase family of scaffolding mol - 

ecules. I t is typically found at excitatory synapses an d 

directly bind s transmembran e component s suc h a s 

the N-methyl-D-aspartate receptor (NMDAR ) neuroligin, 

as well as molecules involve d in secondary signaling 

suc h a s neurona l nitri c oxid e synthetas e an d 

regulatory proteins such as synaptic guanidine triphos - 

photase activatin g protei n fo r Ra s (Iri e e t al. , 1997 ; 

Kim and Sheng , 2004) . Similarly, glutamate receptor - 

interacting protei n interact s with th e oc-amino - 

3-hydroxy-5-methyl-4-isoxazole propionat e recepto r 

(AMPAR) an d gephyri n wit h th e glycin e recepto r 

(Meyer e t al, 1995 ; Don g e t al, 1997 ; Lev i e t al. , 

2004). Th e cytoskeleta l protein s acti n an d spectri n 

concentrate a t both th e pré - and postsynapse . These 

proteins, alon g wit h tubulin , ar e though t t o under - 

lie th e characteristi c filamentou s appearanc e o f th e 

postsynaptic we b an d ma y contribut e t o change s i n 

postsynaptic recepto r compositio n durin g develop - 

ment and plasticity (Matus et al., 1980 ; Fische r et al., 

2000; Ki m an d Lisman , 2001 ; W u e t al, 2002 ; re - 

viewed in Moss and Smart, 2001; Luscher and Keller, 

2004; van Zundert e t al, 2004). 

Structural Correlates o f Synaps e 

Assembly and Maturatio n 

The pre - and postsynaptic elements of developing circuits 

aris e principally from interaction s betwee n ax - 

onal an d somatodendriti c filopodia . Axona l growt h 

cones ma y form termina l bouton s a s they d o a t th e 

neurornuscular junctio n (NMJ) , bu t predominantl y 

form e n passant ("i n passing") along the lengt h o f an 

axon (Bodian , 1968 ; Hind s an d Hinds , 1972 ; Skof f 

and Hamburger , 1974 ; Vaugh n et al, 1977 ; Vaugh n 

and Sims , 1978 ; Mason , 1982) . Coate d vesicle s and 

smooth endoplasmi c reticulu m i n branchin g axon s 

and coate d pit s near the postsynaptic densitie s o f nascent 

contacts ma y participate i n traffickin g o f synaptic 

components o r membrane recyclin g durin g early 

periods o f synaptogenesi s (reviewe d i n Vaughn , 

1989). Eighty-nanomete r granulate d PTV s als o have 

been foun d i n th e vicinit y of nascent synapse s (May 

and Biscoe, 1973 ; Ahmari et al, 2000). Another char - 

acteristic featur e o f man y immatur e synapse s i s a 

reversed asymmetry, from th e earl y presence of presynaptic 

vesicle s i n th e absenc e o f a pronounce d 

PSD (Haye s and Roberts , 1973 ; Vaughn e t al, 1977 ; 

Newman-Gage an d Westrum , 1984) . A s the synaps e 

differentiates, the PSD thickens, at least at type I sites, 

and presynapti c vesicle s steadil y increas e i n numbe r 

(Bloom an d Aghajanian , 1966 , 1968 ; Jones , 1983 ; 

Steward and Falk, 1986 ; Vaughn, 1989) . 

Mechanisms o f Synapse Initiatio n 

Spinal motor neuro n dendrite s arborize extensively as 

they ente r th e margina l layer , a zone occupie d pro - 

fusely b y axonal terminals (Vaughn et al., 1988) . Ob - 

servations suc h a s thi s on e an d th e branchin g o f 

dendritic filopodi a a t site s o f PSD-9 5 clusterin g 

suggest a "synaptotropic " branchin g o f dendriti c

arbors i n respons e t o presynapti c signal s (Vaugh n 

et al, 1988 ; Niel l e t al, 2004) . Axo n collaterals simi - 

larly form a t sites of synaptobrevin II clustering during 

initial contact, consistent with a role for retrograde signaling 

i n presynapti c differentiation. Signal s promot - 

ing early stages of synapse formation derive from bot h 

pre- an d postsynapti c sources , bu t th e sourc e an d 

identity of the signa l initiatin g thi s cascad e o f event s 

are still the subject o f intense investigation. Som e initial 

clues have come fro m a series of biochemical an d 

genetic studie s at the NMJ , whic h demonstrat e tha t 

motor neuron-derive d agri n promote s clusterin g o f 

acetylcholine receptor s a t site s o f innervation , 

whereas a n unidentifie d retrograd e myotube-derive d 

signal is required i n turn fo r normal differentiation of 

the presynapse (DeChiara et al., 1996 ; Gauta m et al., 

1996). 

This bidirectional signaling during NMJ differen - 

tiation i s recapitulated a t central synapses. Analogous 

to agri n a t th e NMJ , th e secrete d lecti n Nar p ca n 

cluster AMPA R an d promot e AMPAR-dependen t 

NMDAR clusterin g a t synapses i n cultures o f dissociated 

hippocampa l o r spinal neuron s (O'Brie n e t al, 

1999; Mi et al, 2004). Vesicles can be recruited to the 

presynapse either through th e binding of postsynaptic 

neuroligins t o presynapti c (3-neurexi n receptor s o r 

the presumabl y homophili c bindin g o f SynCA M 

(Scheiffele e t al, 2000 ; Biedere r e t al, 2002 ; Dea n 

et al. , 2003 ; Gra f e t al, 2004) . SynCA M signalin g 

also can promot e glutamatergi c transmission in non - 

neuronal cell s partnere d wit h neuron s i n cultur e 

(Biederer et al, 2002). Whether the los s of SynCAM 

blocks or attenuates synaps e induction remain s to be 

demonstrated; down-regulatio n o r blockad e o f neu - 

roligin isoform s attenuate s synaps e inductio n (Chi h 

et al., 2005). Additionally, blockade o f cadherin function 

o r ECM-integrin signaling soon afte r synaps e assembly 

compromise s nascen t o r matur e synaps e 

structure, respectivel y (Togash i e t al. , 2002 ; Bozdagi 

et al., 2004). Blocking of ECM-integrin signaling similarly 

compromise s matur e synaps e structur e (Chavi s 

and Westbrook , 2001 ; Schuste r e t al , 2001 ; Cha n 

et al., 2003). Together these findings support th e ide a 

that severa l recognition an d adhesio n factor s initiat e 

and maintain synapses coordinately. 

Maturation of Synapse Composition 

Naive site s of contact become competent for neuro - 

transmission throug h th e progressiv e transpor t an d 


stable recruitmen t o f synapti c components . N-cad - 

herin an d the presynaptic gri d components Bassoon , 

Piccolo, an d Rab3-interacting molecule appea r to be 

transported to presynaptic sites by PTVs (Wang et al., 

1997; Richte r e t al , 1999 ; Ahma d e t al. , 2000 ; 

Bresler et al, 2001; Phillips et al., 2001; Altrock et al, 

2003; Shapira et al, 2003; Jontes et al, 2004). Postsy - 

naptically, PSD-95 , NMDAR , an d AMPA R i n th è 

dendrite accumulate at the synaps e ove r a more pro - 

tracted time period covering a couple of hours. Timelapse 

imagin g o f dissociate d hippocampa l neuron s 

grown fo r 8-1 2 day s i n cultur e shows that PSD-9 5 

and NMDA R accumulat e a t synapse s gradually, implying 

tha t thes e component s ar e adde d piecewis e 

rather tha n i n large r increment s b y a fusion particl e 

(Friedman e t al, 2000 ; Bresle r e t al, 2001 , 2004) . 

Similar work in dissociated cortical neurons grown for 

2-4 day s suggests tha t NMDA R an d AMPAR can b e 

delivered in packets akin to presynaptic transport vesicles, 

potentiall y implicating distinct mechanisms for 

receptor deliver y at different stage s of development o r 

in different neuron types (Washbourne et al., 2002). 

Young synapse s exhibit heterogeneit y i n recepto r 

composition. Fo r instance , man y immatur e gluta - 

matergic site s contai n NMDA R cluster s bu t ar e 

thought t o lac k functiona l AMPAR. Ove r time , th e 

occurrence of these "silent " synapses diminishes with 

the insertio n o f AMPAR at thes e site s (Isaac , 2003). 

The NMDA R subuni t compositio n als o i s develop - 

mentally regulated , a s th e NR2 A subuni t replace s 

NR2B at synaptic sites (Sheng e t al, 1994) . Matura - 

tion of synapse receptor content and recepto r subunit 

composition i n thes e case s i s thought t o b e activitydependent 

(Ra o e t al. , 1998 ; Tova r an d Westbrook , 

1999; Mos s an d Smart , 2001 ; Christi e e t al, 2002 ; 

Anderson e t al, 2004; Luscher and Keller , 2004 ; van 

ZundertetaL, 2004). 

Dale originally postulated tha t individua l neurons 

release onl y one typ e of neurotransmitter to accoun t 

for differentia l muscl e responses to preganglionic an d 

postganglionic sympatheti c fibe r stimulation . Thi s 

principle has been reinterpreted a s counterexamples , 

primarily in developing systems , have come to light. 1 

Analogous t o th e aforementione d combinatio n o f 

neuropeptide- an d neurotransmitter-carryin g vesicl e 

populations in some adult terminals (De Robertis and 

Pellegrini d e Iraldi , 1961) , th e vesicula r glutamat e 

transporter-3 co-distributes wit h vesicular transporter s 

for glycine , y-aminobutyri c aci d (GABA) , acetyl - 

choline, an d monoamin e neurotransmitter s durin g


early brai n developmen t (Mclntir e e t al. , 1997 ; 

Chaudhry e t al, 1998 ; Takamor i e t al, 2000 ; Boul - 

land e t al. , 2004) . I n a simila r vein , glycin e an d 

GABA are co-release d i n individua l vesicle s b y affér - 

ents o f th e media l nucleu s o f th e trapezoi d bod y 

(MNTB) ont o th e latera l superio r olivar y nucleu s 

during a developmentally regulate d switc h i n neuro - 

transmitter (Nabekur a e t al. , 2004) . Mos t o f thes e 

afférents ar e concurrentl y glutamatergi c durin g th e 

first postnatal week , but ove r time th e incidenc e o f 

this glutamatergic phenotype among MNTB neurons 

diminishes (Gillespi e e t al. , 2005) . Likewise , several 

lines o f evidence sugges t tha t glutamatergi c dentat e 

gyrus granule cell s ca n synthesiz e an d releas e GAB A 

and tha t th e appearanc e o f this GABAergi c pheno - 

type is developmentally regulate d (Gutierrez , 2003) . 

Mechanisms of Synapse Maintenance 

and Disassembly 

AMuncl8-l nul l mutation abrogates vesicular neurotransmitter 

releas e (Verhage et al., 2000 ; Voets et al., 

2001). Despit e th e apparen t rol e fo r presynaptically 

derived signal s in earl y synaptogenesis , prenata l cytoarchitecture 

an d synapse ultrastructure in MunclS- 

1 mic e appea r surprisingl y normal . Bu t b y birth , 

massive neurodegeneratio n ha s ensue d (Verhag e 

et al., 2000), which suggests that presynaptic release is 

not require d for synaps e formation bu t i s important 

for maintenance. Thyroid hormone deficiencies similarly 

reduce dendritic spine number (Bicknell , 1998 ; 

Thompson an d Potter , 2000) . The augmentatio n o f 

spine numbe r b y estradic i an d termina l numbe r b y 

BDNF and glial-derive d factors is consistent with th e 

proposed role s fo r thes e factor s i n synaps e mainte - 

nance a s well (McEwen , 1999 ; Mauc h e t al, 2001 ; 

Ullianetal.,2001,2004). 

An exuberanc e o f initial contacts i s thought t o b e 

pruned several-fol d ove r the course of normal development 

(Knyiha r et al., 1978 ; Huttenlocher, 1979 ; Rakic 

et al. , 1986) , implicatin g mechanism s fo r selectiv e 

synapse maintenanc e an d elimination . Th e earl y 

F-actin dependenc e o f postsynaptic compositio n sug - 

gests tha t synapse-specifi c factor s interactin g wit h th e 

actin cytoskeleto n ca n promot e th e maintenanc e o f a 

subset o f sites i n th e vertebrat e CN S (Alliso n e t al, 

1998; Zhan g an d Benson , 2001) . Additiona l studie s 

suggest that stabilization of particular postsynaptic sites 

depends o n transmitter-evoke d recepto r activation , 

Ca 2+ release , o r loca l protei n synthesi s (Fre y an d 

Morris, 1997; Martin et al, 1997 ; Engertand Bonhoeffer, 

1999 ; Lohmann e t al., 2002; Hashimoto and Kano, 

2003; Tashiro e t al, 2003 ; Nagerl e t al, 2004) . Con - 

versely, depressed o r blocked activity leads to the elimi - 

nation o f affecte d spine s i n developin g hippocampa l 

slices (Nagerl et al, 2004 ; Zhou et al, 2004) . 

Notably, th e effect s o f activity on synapse numbe r 

in th e brai n appear t o be developmentall y restricte d 

(Fiala et al, 2002 ; Gmtzendler et al, 2002 ; Nagerl et 

al, 2004) . Thi s windo w fo r activity-dependen t 

changes i n synapse number, an d pruning in general, 

has bee n propose d t o underli e critica l period s o f 

experience-dependent plasticit y in the neocorte x an d 

cerebellum. Consisten t wit h thi s possibility , th e ef - 

fects of monocular deprivatio n o n ocular dominance 

(OD) i n cortex are attenuated whe n protei n synthesi s 

is blocke d durin g th e critica l period . Furthermore , 

degradation o f chondroiti n sulfat e proteoglycan s i n 

the perisynapti c ECM, whic h ar e thought t o inhibi t 

axonal sprouting , reactivate s thi s O D plasticit y i n 

adult mice (Berard i et al, 2004) . Additionally, blocking 

of NMDAR activatio n during a critical period in - 

creases climbing fiber innervation in the cerebellu m 

that correlate s wit h impaire d moto r coordinatio n 

(Kakizawa et al, 2000) . 

Cellular and Subcellula r 

Target Specificit y 

An axo n guided t o it s target lamin a mus t b e abl e t o 

identify it s appropriate target cell type (e.g., GABAergic 

interneuron vs. glutamatergic projectio n neuron ) 

and subcellula r domain (e.g. , distal vs. proximal dendrite). 

This final stage o f axon guidance i s thought to 

be mediate d b y the cell - and domain-specifi c expres - 

sion o f CAM s o r receptor-ligan d combination s wit h 

singular affinities . Fo r instance , GABAergi c basket - 

cell axon s are guided alon g cerebellar Purkinje axons 

by a n ankyrinG-base d gradien t o f neurofasci n (als o 

called neurofilament 168) to form axo-axonal synapses 

at th e axo n initia l segmen t (Jenkin s an d Bennett , 

2001; Ango et al, 2004) . This hypothesis of subcellular 

targeting , thoug h widel y accepted , i s otherwis e 

untested excep t wit h N-cadherin , whic h appear s no t 

to function in this capacity (Bozdagi et al, 2004) . 

A challenge fo r the developin g networ k i s how t o 

recruit o r selectivel y retai n synapti c component s a t 

specific sites of innervation. GluR6-containing kainate 

receptors ar e recruite d t o site s o f N-cadherin activa - 

tion (Cousse n e t al , 2002) . Similarly , th e EphB 2

eceptor directl y clusters NMDAR when activate d by 

its cognat e ephrinB Z ligan d i n youn g hippocampa l 

neurons (Dalva et al., 2000). Neurexin can induce independent 

postsynapti c clustering of GABA receptor, 

NMDAR, gephyrin , an d PSD-95 , a s wel l a s 

neuroligins-1 and -2. In turn, neuroligin-2 can cluster 

vesicles expressin g vesicula r transporter s fo r gluta - 

mate, vesicula r GAB A transporter , o r th e GABA - 

synthesizing enzyme glutamic acid decarboxylase 65 

(Graf et al, 2004; Chih et al, 2005). The abundance 

of synapti c factors wit h distinc t bindin g specificitie s 

and clusterin g activitie s suggest s that , i n combina - 

tion, these factor s not only specify synaps e placemen t 

but als o encode synapse composition durin g maturation 

and plasticity. 

Electrotonic Synapse s 

Whereas most of the literature on synapses focuses on 

the nature , modulation , an d assembl y o f chemica l 

synapses, there i s growing appreciation fo r the func - 

tional contributions o f electrical synapse s (also called 

gap junctions), Electrotoni c synapse s ar e forme d b y 

the abutment o f molecular hemichannels called connexons 

(Fig. 4-8). Th e coupled connexons , which are 

integral membran e proteins , produc e a hermeti c 


channel an d a synaptic ga p just 2 nm wid e (Goode - 

nough, 1976) . Gap junction s often form between th e 

same subcellula r domains , suc h as two dendrites or 

two somata (Peinado et al., 1993) . These synapses are 

commonly foun d in lower vertebrates like the fro g or 

chicken betwee n axon s an d dendrite s withi n th e 

spinal cor d an d som e brai n nuclei . I n mammals , 

these synapse s are widespread in th e retin a and als o 

are foun d i n th e brai n an d spina l cor d (Paternostro 

et al, 1995 ; Ras h et al, 1998 ; Venanc e e t al., 2000). 

Gap junction s ar e foun d wit h greate r frequenc y a t 

earlier developmental stages , and it has been hypoth - 

esized that strong electrical couplin g synchronizes activity 

an d stabilize s synapti c interaction s durin g 

development (Connor s e t al. , 1983 ; Peinad o e t al. , 

1993). "Mixed" synapses, which contain both electrotonic 

and chemica l elements , ma y serve this stabilizing 

rol e i n lowe r vertebrates and th e ra t spina l cord 

(Rash et al., 1998 , 2000) . 

Vertebrate connexon s ar e hexamer s o f connexi n 

isoforms (Goodenough , 1976 ; Segretai n an d Falk , 

2004). Connexin compositio n determine s channel gating 

properties and confers gap junctions with selective 

permeabilities for distinct ions and second messengers . 

Connexon compositio n i n th e nervou s system , a s i n 

other tissues, is cell-type specific an d developmentall y 

FIGURE 4-8 Ga p junction. Gap junction connexons purified from mouse liver 

and visualize d by transmission electro n microscopy (left ) an d X-ra y diffractio n 

(middle) form orderly lattices, indicative of their sixfold symmetry and composi - 

tion. A schematic representatio n o f gap junction s in th e plasm a membran e 

(right) depict s ho w the abutmen t o f connexon hemichannel s i n th e lipi d bilayer 

o f each synapti c membran e produce s a hermetic channe l an d a narrow 

synaptic gap. (Source; Image s modified from Caspa r and colleagues , 1977 , an d 

reprinted with permission from th e publisher.)


regulated (Bevan s et al, 1998 ; Suchyn a e t al, 1999 ; 

Bukauskas an d Verselis , 2004) . Thi s spatiotempora l 

control o f ga p junctio n gatin g an d permeabilit y i s 

likely to have consequences fo r nervous system assembly; 

however , the effec t o f connexin or gap junction 

deficiencies on synapse formation, targeting, or stability 

have yet to be examined. 

The mechanism s guiding spatial targeting of connexins 

or cell type-coupling specificity in the nervous 

system als o hav e ye t t o b e examined . Wor k i n non - 

neural tissues , however, suggests that molecules typi - 

cally associated with other types of junctions facilitate 

connexin targeting to sites in the plasm a membrane . 

For example , E-cadheri n overexpressio n ca n pro - 

mote normal concentrations o f connexin targetin g to 

the plasma membrane in skin papilloma cells and alter 

couplin g preference s i n transfecte d melanoma s 

(Prowse e t al. , 1997 ; Hs u e t al, 2000 ; Hernandez - 

Blazquez et al., 2001). Additionally, normal gap junction 

formatio n betwee n len s cell s i s disturbe d i n 

Nr-CAM-deficient mice an d b y antibodie s agains t 

N-cadherin (Frenzel and Johnson, 1996 ; Lustig et al., 

2001). The tigh t junctio n components zonula occludens 

1 an d som e claudin s als o associat e wit h ga p 

junctions with as yet undetermined functional conse - 

quences (Duffy e t al, 2002). 

CONCLUSIONS 

Neuroscientists hav e define d a serie s o f significant 

events o r stage s essentia l fo r norma l differentiatio n 

and integratio n of neurons into a functional network. 

Recent work links particular stages of differentiation 

with the function of particular molecules o r molecular 

families, and this work has provided the necessar y 

tools wit h whic h mechanism s ca n b e pursued . On e 

challenge fo r futur e experimenter s i s t o determin e 

how molecula r pathways of apparent overlappin g or 

converging functio n ar e parse d int o specifi c an d 

meaningful outcomes . 

Abbreviations 

AMPAR ot-amino-3-hydroxy-5-methyl-4 - 

isoxazole propionate recepto r 


BMP bon e morphogenic protein 

CAM cel l adhesion molecul e 

cAMP cycli c adenosine monophosphat e 

cGMP cycli c guanosine monophosphate 

CNS centra l nervou s system 

CRMP collapsi n response mediator protei n 

DCC delete d in colorectal cancer 


EM electro n microscopy 


GDF growt h differentiation factor 

MAP microtubule-associate d protein 

MNTB media l nucleus of the trapezoid body 

Ng-CAM neuron—gli a cell adhesion molecule 

NMDAR N-methyl-D-aspartat e recepto r 

NMJ neuromuscula r junction 

Nr-CAM NgCAM-relate d cell adhesion molecule 

OD ocula r dominanc e 

PSD postsynapti c density 

PTV Piccolo-Bassoo n transport vesicle 

Shh soni c hedgehog 

S mo smoot h ened 

SNARES solubl e n-ethylmaleimide-sensitiv e 

fusion protein-attachmen t protei n 

receptors 

SNAP-25 synaptosom e associated protein-25 

SV synapti c vesicle 

SynCAM synapti c cell adhesion molecul e 

ACKNOWLEDGMENTS Th e author s than k loan a 

Carcea, Elizabet h Kichula , and Cynthi a Kwong for their 

careful readin g and critica l comments on this chapter and 

Alice Elste for her image of a chemical synapse. The au - 

thors are supported by the Nationa l Institute s of Health 

(CDM, IHB , an d DLB) , Societ y fo r Neuroscienc e 

(TRA), and an Irma T. Hirschl Award (DLB). 

Equal contribution s for th e writin g o f thi s chapte r 

from Mint z (polarity and motility) , Bekiro v (guidance), 

and Anderson (synaptogenesis). 

Notes 

1. "Dale' s principle/ ' base d o n argument s that transmitter 

function o f a neuron is distinctive an d "un - 

changeable" (Dale, 1935), actually was first defined 

by Eccles (1957 ) t o state "that the sam e chemical 

transmitter is released from all the synaptic terminals

of a neurone. " Durin g th e perio d o f Dale' s Nobe l 

prize-winning research, onl y two neurotransmitters 

(acetylcholine and adrenaline) ha d been identified. 

A restatement of the principle by Eccles (1982), that 

"at al l th e axona l branche s o f a neuro n th e sam e 

transmitter substance or substances are liberated," is 

consistent wit h the later finding that neurotransmitter 

and neuropeptide can be released from th e same 

nerve terminal (D e Robertis and Pelligrin i de Irai di, 

1961) an d als o encompasses th e mor e recen t findings 

described i n this chapter's discussion. 

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Over the pas t several decades, the importanc e of cell 

death i n the formation of the nervou s system has become 

wel l established . Indeed , understandin g cel l 

death i n th e developin g brai n ha s been on e o f th e 

most exciting areas of research in neuroscience. Th e 

present chapter focuses on the mammalian fetal cerebral 

cortex as a paradigm of study. 

HISTORICAL RECOGNITION O F 

PROGRAMMED CELL DEATH 

AND APOPTOSIS 

Embryologists hav e lon g recognize d tha t regressiv e 

events play a major role in shaping organ development 

(Clarke an d Clarke , 1996) . Abou t S O years ago , th e 

term programmed cell death (PCD ) wa s coined . I t 

evolved from detaile d studie s of the cellula r degenera - 

tion durin g insec t metamorphosi s (Lockshi n an d 

Williams, 1965 ) and the morphogenesis o f avian limbs 

(Saunders, 1966) . PC D i s a geneticall y regulate d 

5 

Cell Death 

Stevens K. Rehen 

Jerold J.M. Chun 

73 

mechanism that plays a critical role in specifi c stages 

of development, sculptin g different organ s during the 

formation an d maturatio n o f tissue s (Elli s e t al. , 

1991). 

Apoptosis is the bes t known form o f PCD, charac - 

terized originally by morphological change s observe d 

by microscopy (Kerr et al., 1994). Apoptosis is characterized 

by nuclear size reduction, blebbing of the cel - 

lular membrane, chromatin condensation , and DNA 

fragmentation (Ker r et al, 1972 ; Wyllie et al, 1984) . 

In the absence of inflammation, the apoptotic corpses 

are usuall y eliminate d b y phagocytes (Arend s e t al, 

1990), whic h us e th e phosphatidylserin e recepto r 

(PSR) to identify and remove the dying cells (Zhuan g 

et al, 1998 , Wan g e t al, 2003) . Apoptosis is considered 

a counterpoin t t o cel l deat h b y necrosi s (Ker r 

et al. , 1972) . Necrosi s i s distinguishe d b y earl y 

swelling and ruptur e o f both intracellula r organelle s 

and the plasma membrane. 

Although th e definitio n o f apoptosi s i s based o n 

morphological criteri a and PC D i s characterized as


a physiologica l even t durin g development , bot h 

terms have been use d interchangeably since their inception 

(Torne i étal., 1994). This use reflects an early 

consensus amon g scientist s abou t th e programme d 

nature o f apoptosis, eve n whe n triggere d b y a non - 

physiological stimulus . It i s important t o emphasiz e 

that the often-neglected distinctio n between apoptosi s 

and PC D ha s somewhat muddied the field, delaying 

the appreciatio n o f other form s o f PCD tha t are also 

relevant to development of the nervous system, such as 

autophagy (Dunn, 1994 ; Xu e et al., 1999 ; Guimarae s 

et al, 2003). In this chapter, the term PC D refer s to 

all forms of cell death associate d with a developmen - 

tal process. 

GENETIC CONTRO L O F 

PROGRAMMED CELL DEATH 

The original concept of PCD suggests an event during 

normal developmen t tha t i s unde r geneti c control . 

Much o f what we know about thi s regulatio n arise s 

from th e pioneerin g work b y Sydne y Brenner, John 

Sulston, an d Rober t Horvitz , wh o wer e awarded th e 

2002 Nobel Priz e i n Physiolog y or Medicine fo r elucidating 

mechanisms of cell deat h i n th e nematod e 

Caenorhabditis elegans. 

During nematode development, the generation of 

its 109 0 somati c cell s i s followed by th e deat h an d 

elimination o f 13 1 specified cell s (Ellis and Horvitz , 

1986). Mos t of these eliminate d cell s ar e neural pre - 

cursors. I n a serie s of elegan t geneti c experiments , 

Horvitz an d colleague s define d the "centra l dogma " 

of PCD : tha t th e expressio n o f certain gene s (ced-3 

and ced-4 i n nematode ) i s require d fo r cel l death , 

whereas othe r gen e product s (ced-9) ca n bloc k th e 

phenomenon. 

In vertebrates, pro-death genes are the caspase family 

o f cystein e protease s an d th e apoptoti c protease - 

activating factor (APAF) 1, and anti-apoptotic protein s 

(including Bcl- 2 and Bcl-xL ) (se e Chapte r 6). The 

Bcl-2 family i s critically involved in th e regulatio n o f 

mammalian cell death and surviva l and include s not 

only anti-apoptoti c proteins , bu t als o pro-apoptoti c 

proteins actin g through bot h caspase-dependen t and 

caspase-independent pathway s (Korsmeyer , 1999) . 

This famil y i s divide d int o thre e majo r subgroup s 

based o n th e patter n o f Bcl- 2 homolog y (BH ) do - 

mains (Korsmeyer , 1999) . Anti-deat h Bcl- 2 famil y 

members, including Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 , 

contain fou r B H domains (termed BH1 , BH2, BH3, 

and BH4) . There are two pro-death subgroup s of the 

Bcl-2 family: the multidomain subgroup contains th e 

BH1 an d BH 2 domains and include s Bax, Bak, Bok, 

and Bcl-xS ; th e BH3-onl y subgroup include s Bini , 

Bid, Bcl-2-associated death protei n (Bad) , death pro - 

tein 5/hara-kiri , Puma, and Noxa . Bcl-2 family mem - 

bers ca n interac t wit h eac h othe r vi a thei r B H 

domains, and th e fina l fat e o f a cell ma y depend o n 

the intracellular balance between anti- and pro-death 

proteins (Akhtar et al, 2004). 

NEUROTROPHIC THEORY AND 

CLASSICAL CELL DEATH 

During th e 1940s-1950s , Vikto r Hamburger , Rit a 

Levi-Montalcini, and Stanle y Cohen conducted pio - 

neering studies on mechanisms controlling cell death 

that culminate d wit h th e identificatio n o f nerv e 

growth facto r (NGF ; Cohe n an d Levi-Montalcini , 

1957). I n 1986 , Levi-Montalcin i an d Cohe n wer e 

awarded th e Nobe l Priz e i n Physiolog y or Medicine 

for thei r importan t discover y of NGF an d it s role i n 

both neurit e outgrowt h an d cel l survival . Researc h 

on the function of NGF lea d to the neurotrophic theory, 

which postulate s tha t neurona l survival depends 

on competitio n fo r limite d amount s o f trophic fac - 

tor^) release d b y target s (Oppenheim , 1991) . Thi s 

early wor k ha s bee n th e impetu s fo r numerou s 

research group s investigatin g nove l neurotrophi c 

factors that regulate the surviva l of developing neural 

cells. Wit h th e clonin g o f the gen e fo r NGF (Scot t 

et al, 1983) , other related growth factors with similar 

amino aci d sequence s hav e bee n discovered . 

This famil y o f proteins , collectivel y referre d t o a s 

neurotrophins, consist s o f NG F an d brain-derive d 

neurotrophic facto r (BDNF ; Scot t e t al , 1983) , neu - 

rotrophin (NT)-3 (Ernfors et al, 1990), NT-4/5 (Berkemeier 

e t al , 1991 ; Hallbroo k e t al , 1991) , an d NT- 6 

and NT-7 (Gotz et al, 1994 ; Nilsson et al, 1998) . 

Two type s o f neurotrophi n receptor s hav e bee n 

identified o n the basi s of their pharmacological properties: 

a low-affinity recepto r calle d p75 , and a family 

of high-affinit y receptor s (trks ) tha t ar e tyrosin e pro - 

tein kinase s an d th e member s o f which bin d selec - 

tively to different neurotrophin s (Barbacid , 1994). In 

general, NGF activate s trkA, BDNF and NT-4/ 5 ar e 

ligands fo r trkB , an d NT- 3 activate s trkC , althoug h 

there i s som e degre e o f cross-tal k amon g them .

FIGURE 5- 1 Zone s in the developing cerebral cortex. The anatom y of the cerebral 

corte x change s a s development proceed s (increasin g age fro m lef t t o 

right). A. The earl y proliferative ag e consists of a pseudostratified epithelium . 

B, C. With development, th e cerebral wal l increases in width. In some cells, 

neurites processes stretch fro m th e ventricula r surface (blac k bar) to the pia i 

surface. C , D . With further development , mor e mature neurons (black triangular 

cells) begin to differentiate superficiall y in the cortical plate (CP), along 

with the ingrowt h and outgrowth of axons in the intermediat e zone (IZ ; lines 

parallel to the black bar). MZ, margina l zone; SZ , subventricular zone; VZ, 

ventricular zone. (Source: Adapted from Cowan , 1979 ) 

Activation o f tr k receptor s initiate s intracellula r sig - 

naling cascades tha t can produce rapid, long-term ef - 

fects o n neuro n growt h an d survival . Bindin g wit h 

p75 ca n promot e eithe r apoptosi s o r survival . I t ca n 

facilitate neurotrophi n signalin g throug h tr k recep - 

tors promotin g cel l survival . Interestingly , p7 5 i s 

structurally related to the tumor necrosis factor recep - 

tors tha t regulat e th e onse t o f cell deat h i n th e im - 

mune syste m (Majda n e t al , 1997) . Constitutiv e 

expression o f the intracellula r p75 domai n i n trans - 

genie mic e lead s t o cel l deat h i n severa l neurona l 

populations. Reader s wishin g t o lear n mor e abou t 

neurotrophic factor s and thei r receptor s ar e referre d 

to some o f the man y excellent recen t reviews on thi s 

topic (e.g., Huang and Reichardt, 2001 ; Dechant and 

Barde, 2002; Hempstead, 2002) . 

CELL DEATH 7 5 

DEVELOPING CEREBRA L CORTE X 

AS A MODEL FO R STUDYIN G 

MAMMALIAN NEURA L CELL DEAT H 

Neurons o f the mammalia n cerebra l corte x first arise 

during embryoni c developmen t fro m neuroproliferative 

zones that surround the cavities, or ventricles, that 

contain cerebra l spina l fluid . I n th e cerebra l corte x 

these proliferative regions are known as the ventricular 

zone (VZ) , which lines the ventricle, and th e subventricular 

zone (SZ) , whic h i s immediately suprajacen t 

to the VZ (Fig. 5-1). Developmental events in the VZ 

serve a s an archetypa l exampl e o f neurogenesis else - 

where in the neural tube, includin g the spinal cord . 

During neurogenesis , cell s underg o on e o f three 

general fates. They can continue to proliferate, become


postmitotie, or die. Those neurons that become postmitotic, 

a step referred to as the birth of a neuron, migrate 

ou t o f th e VZ , throug h a superficia l regio n 

termed the intermediate zone (IZ) , until they reach a 

position i n the superficia l aspec t o f the anlag e o f the 

cortical gra y matter known as the cortical plate (CP) . 

Once i n th e CP , neuron s differentiat e an d mak e 

synaptic connection s (Miller , 1988 ; Baye r an d Alt - 

man, 1991 ) o r di e (e.g. , Finla y an d Slattery , 1983 ; 

Miller, 1995) . 

Death of Post-Mitotic Cells 

Historically, on e reaso n th e contributio n o f PCD t o 

development ha s no t bee n well-recognize d i s tha t 

apoptotic bodie s ar e susceptibl e t o rapi d clearanc e 

and phagocytosi s in intac t tissue. Thus, it is difficul t 

to study the presenc e of cell death in static (fixed) tissue. 

A real-time approach could unequivocally determine 

the prevalence an d distribution of dying cells in 

situ, however , thi s metho d i s not currentl y practica l 

since e x viv o system s produc e artifactua l change s 

in cellular behavior and induce premature cell death. 

Approximately one-third or more of the post-mitotic 

neuronal populatio n undergoe s naturall y occurrin g 

PCD during development relate d to insufficient acces s 

to trophic signal s (e.g. , NG F an d BDNF ) fro m tar - 

gets an d afférent s (Oppenheim , 1991 ; Linden , 

1994). Th e amoun t o f neurona l deat h varie s wit h 

time, location , an d cel l typ e (e.g. , Miller, 1995) . I n 

cortex, fo r example , th e incidenc e o f deat h amon g 

different population s o f post-migrator y neurons ca n 

vary fro m 10 % to 60% . Neurons i n th e deepes t an d 

most superficia l corte x ar e mor e likel y t o di e tha n 

those i n mid-cortex. Moreover , local circuit neuron s 

within a particular layer are twice as likely to die relative 

to laminar-cohort projection neurons. 

PCD play s a crucia l par t i n th e developmenta l 

strategy of the cerebra l cortex. An overabundance of 

neurons i s generated t o create a transient scaffol d fo r 

the developing cortex and i s subsequently eliminate d 

as th e corte x matures . Durin g developmen t o f th e 

mammalian telencephalon , th e genesi s o f neuron s 

destined for the si x layers of the cerebra l cortex is preceded 

b y generation o f a population o f neurons tha t 

reside in the subplate (an anlage of the white matter ) 

and the marginal zone (the anlage of cortical layer I). 

These neuropeptide-containin g neuron s (Chu n 

et al, 1987 ; Al-Ghoul and Miller, 1989 ; Ghosh e t al, 

1990) ar e presen t i n larg e number s durin g 

development an d functio n i n early synaptic circuitry 

(Chun an d Shatz , 1989a ; 1989b) . A s the brai n ma - 

tures, their disappearance through neuronal death occurs 

in concert with the invasio n of the cortica l plat e 

by axona l projection s an d wit h th e eliminatio n o f 

synapses i n th e subplate . Thes e observation s sugges t 

that during development the subplate (a) is a transient 

synaptic neuropil (Chu n e t al., 1987) ; and (b ) serves 

as a guidepos t fo r ingrowin g thalamocortical axons 

subsequently eliminate d b y cell death . Thi s ide a o f 

sculpting the cortex through an initial overproductio n 

of neurons followed by paring back is in concert with 

the neurotrophic theory. 

Most studie s on PC D i n th e centra l nervou s system 

(CNS ) hav e focused o n th e period o f target in - 

nervation (se e below) , whe n differentiatin g neuron s 

are sendin g out axon s and makin g synaptic connec - 

tions with postmitotic neurons (Hamburger and Levi- 

Montalcini, 1949 ; William s and Smith , 1993 ; Miller, 

1995). An implicit assumption o f many of these studie s 

was that PC D playe d a minor rol e during embryoni c 

CNS development . Indeed , elaborat e mathematica l 

models have been constructed that assume there is no 

PCD durin g neuronogenetic phases of development 

(Cavinessetal., 1995) . 

Death of Proliferating Cells 

Death ca n affec t proliferatin g populations of cells, a 

phenomenon representin g a distinct for m an d phas e 

of cell death i n the developing CNS. Thi s populatio n 

is bes t represente d b y neura l progenito r an d ste m 

cells (NPCs). These cells, which have morphological 

and molecula r characteristic s o f radia l gli a (Nocto r 

et al., 2001), are located i n the VZ and SZ. They also 

share similarities with mitotic immune cell s such as T 

cells of the thymic cortex (Chun, 2001). Importantly, 

NPCs d o no t hav e synaptic contacts a t thi s stag e of 

development, and are still proliferative. 

Thymic T cells are notable a s a comparison popu - 

lation t o NPCs. Developin g T cell s underg o severa l 

distinct cell selection mechanism s (positive selection, 

negative selection , an d deat h b y neglect) tha t resul t 

in elimination of 97% of the developin g T-cell popu - 

lation (Egerto n e t al, 1990 ; Shortma n e t al. , 1990 ; 

Shortman an d Scollay , 1994) . Nevertheless , fo r de - 

cades controversy existed following the proposal of intrathymic 

cel l deat h (Metcalf , 1966) , becaus e o f th e 

absence o f histological evidence fo r intrathymic cell 

death (Post e an d Olsen , 1973) . I t wa s not unti l th e

development o f nove l hybridizatio n techniques col - 

lectively termed in situ end labeling, or ISEL, that intrathymic 

cel l deat h wa s convincingly demonstrated 

(Surh an d Sprent , 1994 ; Chun , 2001) . ISEL identi - 

fies breaks in double-stranded DN A produced during 

PCD b y attaching labele d nucleotide s t o fre e DN A 

ends. The labe l can then be visualized in tissue sections 

(Gavrieli et al., 1992 ; Abrams et al., 1993 ; Wijsman 

e t al., 1993 ; Wood e t al., 1993 ; Homm a e t al, 

1994; Whit e e t al, 1994) . I n sit u end-labelin g plu s 

(ISEL+) improves the sensitivit y of the original ISEL 

techniques t o visualize PCD i n th e developing ner - 

vous system (Blaschke et al, 1996 , 1998 ; Stale y et al, 

1997; Pompeiano e t al, 1998 , 2000 ; Zhu an d Chun, 

1998). Th e increase d sensitivity of ISEL+ ha s bee n 

validated i n severa l establishe d mammalia n model s 

of PCD . Fo r example , ISEL + reveal s normal PC D 

and quantitative increases in PCD withi n the thymic 

cortex following dexamethasone treatment of the thymus 

(Wyllie , 1980 ; Egerto n e t al , 1990 ; Shortma n 

et al 1990; Surh and Sprent, 1994) . Moreover, studies 

using ISEL+ i n the smal l intestinal villus, a model of 

PCD wher e the entir e population o f cells turns over 

every tw o t o fou r days , demonstrat e tha t apoptoti c 

DNA fragmentation can be identified in at least some 

dying cell s severa l day s befor e thei r actua l elimina - 

tion (Pompeiano et al, 1998) . 

The searc h fo r PCD i n th e developin g cerebral 

cortex wa s prompte d b y th e superficia l similarities 

between thymocyte development and cortical neurogenesis, 

particularly in ligh t of the lac k of histological 

evidenc e fo r cel l deat h i n th e thymus . Th e 

patterns o f labele d cell s reveale d b y ÏSEL + i n th e 

embryonic rodent brain suggest that PCD occur s on 

a large scale throughout the neuraxis (Blaschke et al, 

1998). 

As expecte d fro m previou s studie s o n target - 

dependent PCD , dyin g cells are present in the anticipated 

postmitoti c neurona l compartment s o f th e 

cerebral corte x (I Z an d CP). Strikingly , a larg e 

amount o f PCD i s also present i n the VZ . The tim e 

course of cell death i n mouse cortex varies across development 

and i s related to the progression of the cel l 

cycle (Blaschk e et al , 1996 , 1998 ; Thomaido u e t al , 

1997). Most PCD i s observed between gestational day 

(G) 1 2 and G18, when 25% to 70% of cells in the feta l 

cerebral corte x die (Fig . 5-2) . Pea k incidence o f cell 

death occur s abou t G14 . Prio r t o G1 0 an d i n th e 

adult, less than 1 % of cortical cells are dying. This pattern 

show s that ther e i s a substantia l amoun t o f cel l 


FIGURE 5- 2 Distributio n of dying cells in the embryonic 

mouse cortex. In situ end-labeling plus (ISEL+) 

from gestational day (G)10-G18 and in the adult cortex 

(A, C, E, G, I, and K), along with nuclear staining 

by 4,6-diamino-2-phenylindole of the sam e fiel d (B , 

D, F, H, J, and L). A, B. G10. Fro m this age through 

G14 (A-F) , th e neuroepitheliu m consist s o f a 

homogeneous-appearing proliferativ e layer (ppl). C, 

D. G12. E, F. G14. G , H. G16. Th e cerebral wall at 

G16 ha s stratifie d int o additiona l layer s (th e mar - 

ginal zon e [MZ] , cortica l plat e [CP] , intermediat e 

zone [IZ] , an d ventricular zone [VZ] ) tha t are also 

apparent at G18 (I , J). K, L. Adult (Ad) cortex. Th e 

ventricle (v) is at the lower edge of each photograph . 

Note th e spars e ISEL + labelin g a t G1 0 tha t in - 

creases through G14, and then declines with furthe r 

development. From G12 to G18, note the extensiv e 

labeling i n the proliferativ e zone s (ppl, VZ) a s well 

as in the postmitotic zone s (MZ , CP , and IZ) . Only 

rare dying cells are detected b y ISEL+ i n the adul t 

cortex. Thus cortical PCD i s developmentally regulated 

an d cease s by adulthood. Scal e bars = 50 mm. 

(Source: Modifie d fro m Blaschk e an d colleagues , 

1996) 

death withi n proliferatin g NPC populations . Thes e 

cells hav e no t ye t sen t ou t axon s t o synapti c targets, 

hence, classica l neurotrophi c theor y canno t accoun t 

for thi s earl y phase o f cell death . Thi s phenomeno n 

represents a novel form of PCD an d is discussed below.


FIGURE 5- 3 Examinatio n of caspase-9 and caspase-3 

knockout mice. Capase-9 +/ ~ (A) and caspas e 9~ /- (B) 

fetuses a t G16.5 , showin g th e large r an d mor - 

phologically abnorma l brai n i n th e knockout . Scal e 

bar= 1.0mm. C-F . Caspase- 3 mutant s hav e a n expanded 

proliferative zone. C, D. BrdU immunolabeling 

i n a sectio n throug h th e developin g cerebra l 

cortex of a hétérozygote an d mutan t embryo , respec - 

tively. E, E DAP I staining of the adjacent section i n a 

series fro m C an d D , respectively . I n th e caspase- 3 

knockout fetus , th e corte x i s distorte d int o fold s 

and th e latera l ventricle i s identifiable only as a thin 

slit in the center of the cortical mass. The thicknes s of 

the proliferativ e laye r relative to the corte x looks very 

much alike in the two embryos. Statistics showed that 

the percentage o f BrdU-labeled cells was only slightly 

increased i n th e -/ - fetuse s compare d t o th e +/ - 

fetuses, v , latera l ventricle . Scal e ba r = 500 Jim. 

(Source: A, B: adapted from Kuid a e t al., 1998 . C-F : 

adapted from Pompeian o e t al., 2000) 

Glial Death within 

the Developing Brain 

The rol e o f astrocytes or other gli a i n neurona l cel l 

death i s poorly understood. Interestingly, phagocytes 

of macrophage lineages such as microglia contribut e 

to the eliminatio n o f dead cell s i n the brain . Phagocytes, 

from mic e lackin g the gen e encodin g PS R are 

defective i n removin g apoptoti c cells . Thes e PCR - 

deficient mic e hav e developmenta l abnormalities , 

such as in the accumulation of dead cells in the lun g 

and brain . Som e o f these animal s also present a hyperplasic 

brai n phenotyp e resemblin g tha t o f mic e 

deficient i n the cel l death-associated gene s APAF-1, 

caspase-3, an d caspase- 9 (Fig . 5-3 ) suggestin g tha t 

phagocytes ma y also be involved in promoting apop - 

tosis (L i e t al. , 2003) . Actually , in th e developin g 

cerebellum, th e deat h o f Purkinje neuron s ca n b e 

promoted b y microglia. This findin g suggest s that a 

form o f engulfment-related cel l death ma y link PC D 

to th e clearanc e o f dea d cell s (Marin-Tev a e t al. , 

2004). 

DIFFERENT PHASES OF 

DEVELOPMENT, DIFFERENT 

MECHANISMS OF PROGRAMMED 

CELL DEATH 

Using an early molecular marker for post-mitotic neurons, 

Weine r an d Chu n (1997) , foun d tha t PC D i n 

neuroproliferative zones does not become pronounce d 

until NPC s begi n t o differentiat e int o postmitoti c 

cells. This correlation suggest s that the initial generation 

o f postmitotic neuron s ma y trigger the onse t of 

PCD i n a given proliferative zone. The natur e of this 

link; however, is currently unclear. 

Similar to the developing cerebral cortex, the retina 

of newborn mice and rat s is composed o f two strata 

containing cell s i n variou s stage s o f development . 

One laye r of differentiated cell s i s the ganglio n cel l 

layer an d th e othe r i s the neuroblasti c laye r (NBL). 

Like the cortica l VZ, the NB L is comprised o f NPCs 

(Alexiades and Cepko , 1997) . During differentiation, 

NPCs that are exiting the cell cycle, migrate from th e 

NBL to form other layers of postmitotic neurons (Linden 

et al, 1999) . 

PCD ca n be induced i n the NBL following inhibition 

o f protein synthesis . Interestingly, as the retin a 

matures, retinal cells become less sensitive to protei n 

synthesis inhibitors, and i n adult retinal tissue , thes e 

inhibitors have n o effec t o n cel l death (Rehe n et al, 

1996). Whe n BrdU , a marke r o f proliferatin g cells 

(Miller an d Nowakowski , 1988), i s used i n conjunction 

wit h protein synthesi s inhibitors, the vas t majority 

of cells undergoing PCD ar e not labeled with BrdU 

(Fig. 5.4) . This result suggests that the populatio n of

newly postmitotic neuron s is particularly sensitive to 

PCD throug h inhibitio n o f protein synthesis . Continuous 

synthesis of anti-apoptotic proteins appears to be 

important in the regulatio n o f PCD durin g neuronal 

migration an d differentiatio n i n th e retin a (Rehen s 

et al , 1999) . Consisten t wit h thi s hypothesis , mous e 

embryos deficient for Bcl-xL, an anti-apoptoti c mem - 

ber o f the Bcl- 2 family, exhibi t massiv e cell deat h of 

immature neurons in the cerebra l corte x (Motoyama 

et al, 1995). Bcl-xL is up-regulated i n early postmitotic 

neurons a s they migrat e fro m th e V Z an d thi s hig h 

level o f expression is maintained throug h adulthood . 

On th e othe r hand, Bcl-x L expressio n is low in NPCs 

(Motoyama et al, 1995). 

In summary, ISEL+ results indicate that PCD i s 

a commo n cel l fat e fo r NPC s throughou t th e 

neural tube. Studie s involving inhibition of protein 

FIGURE 5- 4 Identificatio n of newly postmitotic cell s 

sensitive to apoptosis induced by inhibition of protein 

synthesis. Immunolabeling for BrdU in the prolifera - 

tive zone o f a retinal expiant (arrows ) after inhibitio n 

of protein synthesis. Notice that the majorit y o f dying 

cells (arrowheads) , identifie d a s globula r apoptoti c 

bodies under differential interferenc e contrast, i s unlabeled 

wit h BrdU . Scal e ba r = 20 \im. (Source: 

Adapted from Rehe n e t al., 1999 ) 


synthesis i n th e developin g nervou s system and tar - 

geted deletio n o f the anti-apoptoti c gen e Bcl-x L suggest 

that during migration and initial differentiation, 

the PC D machiner y i s controlle d b y suppresso r 

proteins. 

PROLIFERATIVE CELL DEATH, DNA 

BREAKS AND END JOININ G 

Thymic PCD occur s during the selection o f functionally 

an d molecularl y distinc t thymocyte s (Sur h an d 

Sprent, 1994), based on somatic DNA rearrangements 

encoding T cell receptor s and immunoglobulins in T 

and B lymphocytes, respectively. This process, known 

as V(D)J recombination (Weaver and Alt, 1997) , i s responsible 

for the unparallele d degree of immunologicai 

diversit y tha t i s fundamenta l t o th e immun e 

response. A s describe d above , a paralle l exist s be - 

tween PCD i n the thymus and the feta l brain, where 

cell death takes place in conjunction with cell prolif - 

eration (Shortman et al, 1990 ; Chun, 2001). 

ISEL+ result s suggest that a form o f cell selectio n 

occurs i n th e neuroproliferativ e regions o f the brai n 

and that the occurrence of PCD i s associated with the 

maturation proces s (Blaschk e e t al. , 1996 , 1998) . I n 

the feta l brain , the pea k of cell deat h coincide s wit h 

the perio d whe n th e first neurons destine d t o com - 

prise th e matur e corte x ar e bein g generate d (Cavi - 

ness, 1982) . Thi s spik e i n PC D migh t allo w th e 

selection o f the first cortical neuron s havin g desired 

phenotypes which woul d serv e as a template fo r th e 

selection o f later-generate d cells . Thi s hypothetica l 

scenario leave s open th e questio n o f how cells migh t 

be mechanistically selected . 

Nearly a decade befor e geneti c recombinatio n was 

formally demonstrated in lymphocytes, Dreyer and colleagues 

(1967 ) postulate d tha t recombine d gene s en - 

coding cell-surface proteins might play a part in guiding 

the correct re-innervation of the goldfish tectum by retinal 

axons . These recombine d protein s would act a s a 

kind o f molecular cod e t o ensure that th e topographi - 

cally correc t retinotecta l mappin g i s achieved. Sinc e 

this idea was put forward, however, no gene undergoin g 

the requisit e recombination i n the nervou s system has 

been found . Thi s i s perhap s no t surprising , a s th e 

reagents that were instrumental in the discovery of lymphocyte 

V(D)J recombination—DNA sequences fro m 

a known candidate locus, and clonal cell lines in which 

the rearrangement even t takes place—do not currently 

exist for the nervous system.


An indirect approach to this issue is to ask whether 

genes involve d in immunologica l V(D) J recombina - 

tion ar e expresse d i n th e nervou s system . I n fact , a 

common featur e shared by cortical thymocytes (Turka 

et al, 1991 ) and VZ NPCs (Chun e t al., 1991 ) is expression 

o f a rang e o f gene s involve d i n th e initia l 

cleavage of recombined genes and th e rejoining of the 

free DN A end s t o complet e th e recombinatio n reac - 

tion. Par t o f th e initia l cleavag e reactio n i n lympho - 

cytes is driven by recombination-activating genes 1 and 

2 (RAG 1 an d RAG2) , which ar e involve d in V(D)J 

recombination (Schat z e t al, 1989) . Ragl - knockout 

mice, however , hav e n o obviou s brai n phenotyp e 

(Mombaerts et al, 1992) , and Rag2 is not expressed in 

the CNS. It is notable that broken DNA ends that exist 

in cells that, like those produced by recombinase cleavage, 

hav e blun t end s tha t ar e 5'-phosphorylated , al - 

though mos t of these ends appear to be predominantly 

associated with cell death (Staley et al, 1997). 

After cleavage , DN A en d joinin g take plac e b y 

means of several nonhomologous end-joining (NHEJ) 

proteins including Ku70, Ku80, DNA-dependent protein 

kinas e catalyti c subunit (DNA-PKcs) , XRCC4 , 

and DN A ligase IV (Lig IV). Through gene-targetin g 

in th e mouse , i t has been show n that XRCC4 or Lig 

IV deficiency causes growth defects, premature senescence, 

immun e respons e sensitivity , and inabilit y to 

support V(D)J recombination in primary murine cell s 

(Lieber, 1999). 

Strikingly, XRCC4 or Li g IV deficiency in mic e 

causes lat e embryoni c lethalit y accompanie d no t 

only by defective lymphogenesis but also by defective 

neurogenesis, manifested by extensive apoptotic deat h 

of newly generated postmitoti c neuronal cells (Gao et 

al, 1998) . I n the contex t o f Lig IV and XRCC4 defi - 

ciency, embryoni c lethalit y an d neurona l apoptosi s 

likely resul t fro m a p53-dependen t respons e t o unre - 

paired DN A damage (Fran k e t al, 2000 ; Ga o e t al. , 

2000). Ku7 0 and Ku8 0 deficiency results in dramati - 

cally increase d deat h o f developing neuron s i n mic e 

though DNA-PKcs deficiency does not lead to any neuronal 

death phenotype. The Ku-deficien t phenotype is 

FIGURE 5- 5 Massiv e cel l deat h o f newl y postmitoti c neuron s i n XRCC 4 

knockout mice. A-F. Hematoxyli n and eosi n stainin g of littermate XRCC4 +/ ~ 

and XRCC4~ / ~ brai n sections . Horizonta l brai n section s fro m G14. 5 

XRCC4 +/ - (A-C) and XRCC4- 7 - (D-F) mutants at a comparable leve l reveal 

severe acellularit y (cavitation , lon g arrow ) an d thinne r cortica l plat e (CP , 

small arrows) in the cerebra l hemispher e o f XRCC4~ / ~ brain. Higher magnifications 

sho w relativel y normal ventricula r zon e (VZ ) of th e mutan t corte x 

(A, B vs. D, E) , wherea s in th e intermediat e zone (IZ ) extendin g to th e C P 

there are numerous pyknotic nuclei (dense hernatoxylin stain) (E, F). CP, cortical 

plate; S, striatal analge or the ganglioni c eminence; v, ventricle. Original 

magnifications: A and D xlOO ; B , C, E , an d F , x400. (Source: Adapte d fro m 

Gao et al, 1998)

qualitatively similar to, but les s severe, than tha t associated 

with XRCC4 and Lig IV deficiency (Gu et al, 

2000). Th e lac k o f a neurona l deat h phenotyp e i n 

DNA-PKcs-deficient fetuses , alon g wit h th e milde r 

phenotype o f K u deficienc y (vs . XRCC4 - o r Li g 

IV-deficient fetuses) , is likely related t o relativ e low 

fidelity of DNA end-joining in the backgroun d strain 

of thes e mutants , show n b y a V(D)J recombinatio n 

end-joining assay. 

A intriguin g aspec t o f these studie s i s the consis - 

tency of abnormal cell death pattern s i n the developing 

cerebral cortex of mice deficien t in DN A ligase 

IV, XRCC4 (Barne s et al, 1998 ; Fran k e t al, 1998 ; 

Gao et al, 1998) , and Ku (Gu et al, 2000). Aberrant 

death i s not observe d a t the earlies t embryonic ages, 

and i t is not unti l th e perio d when mos t postmitotic 

neurons ar e generated tha t dea d cell s ar e clearly observed 

(Fig . 5-5) . Thi s genera l timin g i s seen i n al l 

examined region s o f the neura l tube , includin g th e 

spinal cord (Gao et al, 1998) . 

These studie s provide the first evidence that evolutionarily 

conserved gene s require d fo r completion o f 

V(D)J recombination in lymphocytes are required for 

normal neura l cel l surviva l i n th e developin g brain 

(Gao e t al, 1998 ; Chun , 1999 ; Chu n an d Schatz , 

1999). Several NHEJ proteins have roles in NPC sur - 

vival durin g the onse t o f neural differentiation . Th e 

synthesis o f thes e protein s coul d b e crucia l fo r th e 

normal behavior of newly postmitotic neurons during 

their migration from proliferativ e zones to final destinations 

in the CMS (Rehen e t al, 1996 , 1999) . 

The absenc e of proteins important for the process 

of immunologica l V(D) J recombinatio n promote s 

cell death i n newly postmitotic neurons. This finding 

suggests th e operatio n o f a nove l for m o f cell selec - 

tion that distinguishes between neurons that make advantageous 

DN A rearrangement s o r othe r genomi c 

alterations and those that do not. What kin d of DNA 

alterations could occu r i n NPCs within the develop - 

ing brain? 

ANEUPLOIDY, CELL SELECTION, 

AND PROLIFERATIV E CELL DEATH 

IN THE BRAI N 

As discussed above, there i s indirect evidence fo r somatic 

alteratio n o f th e genom e i n neuron s durin g 

neurogenesis base d o n a growin g list o f molecule s 

that functio n i n DN A recombination , repair , an d 


surveillance (Ga o e t al , 1998 , 2000 ; Chun , 1999 ; 

Chun an d Schatz , 1999 ; G u e t al , 2000 ; Le e e t al , 

2000; Rolig and McKinnon, 2000; Allen et al, 2001). 

Many o f these molecule s ar e als o implicate d i n th e 

genetic alteration s tha t ar e foun d i n cancers . On e 

prominent geneti c abnormalit y i n cance r cell s i s 

aneuloidy, a deviation from th e norma l diploid num - 

ber of chromosomes. Th e associatio n between cance r 

and neurodevelopmen t le d t o th e searc h fo r aneu - 

ploidy a s on e expressio n o f genomi c chang e i n 

neurons. 

Direct assessmen t o f chromosome s i n dividin g 

cells ca n b e performe d wit h spectra l karyotypin g 

(SKY) which uses fluorescent labels to uniquely identify 

eac h chromosome . Thi s technique reveal s that a 

large fractio n o f NPC s harbo r chromosoma l abnor - 

malities. Remarkably, one-third o f mouse NPCs have 

gained o r los t at leas t on e o f their 4 0 chromosome s 

(Fig 5-6 ; Rehe n e t al, 2001). NPCs isolated fro m th e 

VZ o f the feta l mous e cerebra l corte x ar e ofte n ob - 

served wit h on e o r mor e chromosome s displace d 

from the mitotic spindle. Such chromosome displace - 

ment suggest s tha t NPC s frequentl y mis-segregat e 

their chromosomes , whic h ma y represen t on e wa y 

that neurons can become aneuploi d during development 

(Yan g et al, 2003). 

Aneuploidy ha s als o bee n detecte d i n th e adul t 

cerebral cortex , usin g interphas e fluorescen t i n sit u 

hybridization (FISH ) t o visualize individual chromosomes. 

In adult male cerebral cortex , FISH fo r X and 

Y chromosomes show s that approximately 1 % of neurons 

gained or lost a sex chromosome (Fi g 5-7) . Thi s 

implies that a t least som e fractio n o f aneuploid cell s 

that ar e produce d durin g neurogenesi s surviv e t o 

adulthood. Theoretically , th e overal l percentag e o f 

aneuploid neuron s i n th e adul t coul d b e muc h 

higher than 1% , assuming the rat e of gain/loss is similar 

fo r al l chromosome s (Kausha l e t al , 2003) , al - 

though furthe r studie s ar e require d t o addres s thi s 

issue. 

Although some aneuploid cells survive, Aneuploid 

cells may account for a significant proportion of PC D 

during neurogenesis. Cell deat h i s likely a commo n 

fate fo r aneuploid cells . I n cancers , man y cell s tha t 

are thought to be dying have an altered genome. Further, 

the high amount s of PCD in neuroproliferativ e 

zones supports the ide a of a selection proces s against 

undesirable geneti c abnormalities . A s a first approximation, 

aneuploid y appear s t o b e generate d i n a 

stochastic fashion . On e ca n speculat e tha t rando m


FIGURE 5-6 Aneuploi d neural progenitor cell s (NPCs ) 

in the embryonic mouse brain. A. Spectral karyotyping 

(SKY) fro m a representativ e aneuploi d embryoni c 

NPC. Spectra l (left) an d invers e DAPI (center) images 

of the chromosom e sprea d are shown, along with th e 

karyotype table (right) . Not e that each differen t chro - 

mosome ha s a uniqu e spectra l colo r (indicate d b y 

shades of gray). Euploid chromosome numbe r i n mic e 

aneuploidy impart s diversity and complexit y t o neu - 

ronal populations , an d tha t aneuploi d NPC s ar e 

selected o n the basi s of their genomic contents , with 

one probable fate being cell death. The pervasiveness 

of adul t neura l aneuploid y may indicat e a selective 

advantage acquire d b y som e aneuploi d neurons , a s 

has bee n note d i n certai n aneuploi d cance r cell s 

(Lengauer e t al , 1997) . Thus , variatio n in chromo - 

some numbe r can b e considere d a new mechanis m 

for generating cellular diversity in the CNS . 

The main conclusion from these data is that aneuploid 

cells are found in the developing and adult ner - 

vous system, demonstrating tha t brain cells can diffe r 

at th e leve l o f thei r genome . Whethe r DN A re - 

arrangements are als o present i n thes e cell s remains 

an ope n question . Thes e somati c change s i n th e 

DNA of a cell could affec t a significant portion of the 

genome, alterin g hundred s o f gen e copie s whic h 

would hav e important consequences fo r the physiol - 

ogy of that cell. The globa l effect o f this process could 

is 40. The sprea d has an extr a copy of chromosome 2 , 

but ha s only one cop y of chromosomes 1 5 and 1 7 (39, 

XY, +2, -15, -17). B . Graphs show the percentage of 

loss (left ) an d gai n (right) of specific chromosome s i n 

NPCs. Bar s are coded o n th e basi s of spectral color of 

each chromosome (excep t for sex chromosomes). Spe - 

cific chromosome s ar e los t a t rate s o f 1.6%—8.4 % o f 

cells analyzed and are gained at rates of less than 2%. 

result in a mature brai n that i s a unique genetic mosaic, 

characterize d b y a euploi d populatio n inter - 

mixed with a diverse aneuploid population of neurons 

(Rehen e t al , 2001 ; Kausha l e t al , 2003 ; Yang et al , 

2003). 

LYSOPHOSPHATIDIC ACID, 

CELL CYCLE EXIT, DECREASED 

PROGRAMMED CELL DEATH, AND 

INCREASES IN BRAIN DIMENSION S 

A variety of extracellular factors hav e been show n to 

influence NPC s an d youn g neuron s o f th e cortex . 

Neurotransmitters (LoTurco et al., 1995 ) and peptid e 

factors (Drag o e t al. , 1991 ; Ghos h an d Greenberg , 

1995; Temple and Qian , 1995 ) affect neurogeni c parameters 

o f developin g cortica l cells , althoug h i t i s 

generally unclea r ho w thes e signal s influenc e th e 

growth and morpholog y of the intac t cerebral cortex.

FIGURE 5- 7 Aneuploi d cell s i n th e adul t corte x are 

neurons. A . Th e presenc e o f bot h microtuble - 

associated protein (MAP ) 2-positive (large arrow) an d 

MAPZ-negative (smal l arrows ) cells i n a 1 0 Jim sec - 

tion throug h adul t cerebra l corte x demonstrate s th e 

specificity o f MAP 2 labelin g a s a neurona l marker . 

Nuclei wer e counterstained wit h DAPI . B-G . Cell s 

in 1 0 ^Lm sections through the adult male cortex were 

hybridized wit h X and Y chromosome paints . C , E , 

G. Cell s i n these same 1 0 \im sections were then im - 

munostained for MAP2. B. The cel l o n th e lef t con - 

tains bot h a n X and a Y chromosome (note d b y a n 

asterisk), whereas the cel l on the righ t has an extra Y 

chromosome. C. The cel l with two Y chromosomes is 

MAP2-positive. D, E. A cell in motor cortex contains 

an extr a X chromosom e (D ) an d i s MAP2-positiv e 

(E). F, G. A cell i n motor corte x contains a n extr a Y 

chromosome (F ) an d i s MAP2-positiv e (G) . Scal e 

bars =10 Jim (D , F ) an d 5. 0 Jim (H , J) . (Source: 

Adapted from Rehe n e t al., 2001) 


Some trophi c factor s suc h a s fibroblast growth facto r 

2 (FGF-2 ) an d NT- 3 ar e als o known to regulate th e 

transition fro m dividin g NPCs to terminally differen - 

tiated neuron s in the CNS , (Ghos h an d Greenberg, 

1995). Tha t said , factor s abl e t o modulat e PC D 

within the VZ are less well understood . 

A lipid molecule, lysophosphatidic acid (LPA), has 

been implicate d i n th e regulatio n o f neurogeneti c 

processes, including proliferative cell death. Man y of 

the effect s o f LPA are mediated by G protein-coupled 

receptors tha t ar e member s o f the lysophospholipi d 

receptor gen e famil y (Fukushim a e t al., 2001 ; Chun 

et al, 2002 ; Anlike r an d Chun , 2004 ; Ishi i e t al, 

2004). A role for LPA signaling in nervous system development 

wa s initially suggested b y the discover y of 

the firs t lysophospholipi d receptor , LPA 1? whic h 

shows enriche d expressio n in th e V Z (Hech t e t al. , 

1996). Subsequen t studie s hav e identifie d LPA 2 ex - 

pression i n postmitotic cell s o f the embryoni c cortex 

(Fukushima et al, 2002; McGiffert e t al., 2002) and 

LPA3 expressio n withi n th e earl y postnata l brai n 

(Contos e t al. , 2000) . LPA , i s als o presen t i n th e 

brain (Das and Hajra, 1989 ; Sugiur a et al, 1999 ) an d 

can b e produce d b y postmitoti c neuron s an d 

Schwann cell s i n cultur e (Weine r an d Chun , 1999 ; 

Fukushima e t al.,2000). 

In a n e x vivo brain culture system , LPA exposure 

rapidly alters the organizatio n of the developing cere - 

bral corte x (Kingsbur y et al. , 2003) . Afte r jus t 1 7 

hours, LPA-treated hemispheres displa y striking cortical 

fold s an d a widenin g o f the cerebra l wall , com - 

pared t o untreate d opposit e hemisphere s obtaine d 

from th e sam e animal s (Fig . 5-8 ) (Kingsbur y et al. , 

2003, 2004) . This expansio n o f cortica l thickness is 

due t o a n increas e i n cell s within bot h proliferative 

and postmitoti c region s withou t a correspondin g 

change i n cell density. Whereas LP A is a mitogen for 

many cell types in culture (Moolenaar , 1995 ; Moolenaar 

e t al. , 1997) , LP A exposure appears to decrease 

proliferation i n a n e x vivo brain culture syste m in favor 

of terminal mitosi s (Kingsbury et al., 2003). In addition, 

LPA exposure in this ex vivo system promotes 

the surviva l o f VZ cells . Therefore , th e increas e i n 

cortical thicknes s followin g LP A treatmen t i s du e 

to (1 ) the increase d surviva l of many NPCs , whic h 

enlarges the proliferating pool, and (2 ) the inductio n 

of termina l mitosis , which increase s the numbe r o f 

postmitotic neurons. Most importantly from a mechanistic 

standpoint , neithe r foldin g no r increase s i n


FIGUR E 5- 8 Effec t o f lysophosphatidi c aci d (LPA ) on 

cortica l development . Intac t cerebra l hemisphere s ex - 

posed t o (LPA ) e x viv o exhibi t cortica l foldin g an d 

widenin g of the cerebra l wall compare d t o contro l hemi - 

spheres fro m th e sam e animal . Whole-moun t view s (A) 

and sagitta l section s (B ) fro m gestationa l da y (G ) 1 4 

hemisphere s sho w dramati c cortica l foldin g followin g 

cultur e with LPA, compared t o contro l medium . D, dorsal; 

R , rostral ; Ctx , cortex ; GE , ganglioni c eminence . 

Scale ba r = 0.50 mm . C . G1 4 cortice s labele d with th e 

nuclea r stain DAP I show increase d thicknes s following 

cortica l thickness , mitosis , an d cel l surviva l ar e ob - 

served i n LPA 1 /LPA 2 recepto r nul l mic e (Kingsbur y et 

al., 2003) , result s demonstratin g tha t th e effect s o f 

LPA in an ex vivo system ar e recepto r mediated . 

The rapi d increas e i n cortica l siz e followin g LPA 

exposure i s consisten t wit h studie s showin g tha t res - 

cue o f V Z cel l death—likel y multipl e round s o f 

death , given th e ~2-hou r clearanc e tim e estimate d for 

dying cell s i n th e corte x (Thomaido u e t al. , 1997 ) — 

can hav e majo r consequence s fo r cortica l growt h 

(Kuid a e t al. , 1996 ; Pompeian o e t al. , 2000) . More - 

over, th e increase d presenc e o f postmitoti c neuron s 

(β-tubulin-III-labele d cells ) in th e VZ followin g LPA 

treatmen t ma y represen t prematur e differentiatio n of 

NPC s tha t contribut e to increase d VZ thickness . Th e 

switch t o a postmitoti c statu s coul d trigge r th e pro - 

ductio n o f suppressor protein s (i.e. , Bcl-x L) able t o abrogate 

PC D in cells migratin g from VZ t o CP. 

LPA signalin g ca n hav e majo r effect s o n cell s 

within th e embryoni c cerebra l corte x that are distinct 

LPA-treatment . CP , cortica l plate ; IZ , intermediat e 

zone; SZ , subventricula r zone ; VZ , ventricula r zone . 

Scale bar=100μm . LP A promote s termina l mitosi s 

throug h a n increas e i n M-phas e cell s immunolabele d 

with anti-phosphohiston e H 3 antibod y (D) , a decreas e 

in S-phas e cell s (dat a no t shown) , an d a n increas e i n 

differentiatin g neuron s immunolabele d wit h anti-β - 

tubulin II I antibody (E) . LP A decreases th e numbe r o f 

dying cells immunolabele d for anti-activ e caspase-3 antibody 

(F) . (Source: Modifie d fro m Kingsbur y e t al , 

2004) 

from cel l proliferation . Interestingly , th e foldin g pat - 

tern observe d i n brain s treate d wit h LP A are superfi - 

cially reminiscen t o f thos e observe d i n hyperplasti c 

brains from knockou t mice fo r caspase s (Kuid a et al., 

1996; Rot h e t al., 2000) or PS R (L i et al., 2003). De - 

spite thes e similarities , LPA-mediate d cytoarchitec - 

toni c change s diffe r fro m thos e of caspase deletio n i n 

several aspects . Disruptio n o f cel l deat h b y caspas e 

deletio n result s in reduce d NP C deat h an d increase d 

VZ siz e withou t a marke d increas e i n C P thicknes s 

(Kuid a e t al., 1996 ; Hayda r et al., 1999b ; Pompeian o 

et al. , 2000). Augmente d cel l cycl e re-entr y by over - 

expression o f β-cateni n produce s fold s vi a th e tan - 

gential expansio n o f cortica l surfac e are a withou t 

increase s in cortica l width (Chen n an d Walsh, 2002 ; 

Zechne r e t al. , 2003) . B y comparison , receptor - 

mediate d LP A signaling increase s cortica l thicknes s 

by alterin g bot h proliferativ e and postmitoti c popula - 

tions, an d furthe r produce s regularl y arrange d corti - 

cal folds .

Future studie s that involv e the inhibitio n o f specific 

G-protein s an d downstrea m effector s i n cortical 

progenitors should provid e insight int o how receptor - 

mediated LP A signalin g promote s termina l mitosi s 

and cel l surviva l i n th e developin g cerebra l cortex . 

Such studie s may also reveal links between cell death 

and suppresso r proteins , includin g thos e relate d t o 

V(D)]/NHEJ an d Bcl- 2 family molecules, fo r the survival 

an d prope r functio n of newly postmitotic neu - 

rons within the developing brain. 

CONCLUSIONS 

Tissue homeostasi s i s currentl y viewed a s a carefu l 

balance betwee n proliferatio n and cell death . Th e 

number, compositio n an d distributio n o f cells within 

both adul t an d developin g organs , includin g th e 

CNS, critically depend o n this balance. The dramatic 

increase i n cerebra l cortica l siz e acros s mammalia n 

evolution is paralleled by increased cell numbers and 

an expansio n of cortical surface are a due t o change s 

in th e regulatio n o f proliferation, differentiation and 

PCD withi n th e feta l brai n (Raki c an d Caviness , 

1995; Caviness et al, 1995 ; Kuda et al 1996 ; Ceccon i 

et al, 1998 ; Yoshida et al, 1998 ; Hayda r et al, 1999b ; 

Pompeiano e t al, 2000; Roth e t al, 2000; Chenn and 

Walsh, 2003 ; Li et al, 2003). The presen t chapte r focused 

o n th e rol e o f PC D i n shapin g th e develop - 

ment of the cerebral cortex . 

Collectively, th e result s discusse d i n th e presen t 

chapter indicate that in the developing CNS, PC D is 

a significant cell fate for proliferating NPCs, and thi s 

PCD coul d b e responsibl e fo r a selectio n proces s 

based o n alteration s o f DNA , particularl y involving 

chromosomal aneuploidy . During migration and initial 

differentiation, cell death i s prevented o r delayed 

by th e expressio n o f suppresso r proteins, includin g 

members o f th e Bcl- 2 famil y an d th e V(D)J/NHEJ 

factors, thus allowing these newly postmitotic cell s to 

find their final positions and differentiate. After reach - 

ing thei r fina l destination , neural cell s start to com - 

pete fo r limite d trophi c facto r suppor t t o surviv e 

another roun d o f PCD. A newly identified group of 

factors tha t affec t NPC s ar e th e lysophospholipid s — 

small lipi d signal s actin g throug h cognat e G pro - 

tein-coupled receptors . LP A signalin g result s i n 

decreased cel l deat h an d induce d cel l cycl e exit , 

which can be considered new influences on cerebral 

cortical growth and anatomy. Future studies will better 


define th e mechanism s o f selectio n an d cel l deat h 

during nervous system development. 

Abbreviations 

APAF apoptoti c protease-activating factor 


BH bcl- 2 homolog y 



CP cortica l plat e 

DNA-PKcs DNA-dependen t protei n kinas e cat - 

alytic subunit 

FGF fibroblas t growt h factor 


ISEL i n situ end-labeling 


Lig I V ligas e IV 

LPA lysophosphatidi c acid 

MAP microtuble-associate d protei n 

NBL neuroblasti c layer 


NHEJ nonhomologou s end-joining 

NPC neura l progenitor cell 


PCD programme d cel l death 

PSR phosphatidylserin e receptor 

SKY spectra l karyotyping 


trk tyrosin e protein kinase 


ACKNOWEDGMENTS W e ar e gratefu l t o Am y Yang , 

Marcy Kingsbury, Brigitte Anliker, Christine Paczkowski, 

and Suzanne Peterson for the critical reading of this manuscript. 

This work was supported by the National Institute 

of Mental Health, National Institut e for, and Pe w Latin 

American Program in Biomedical Sciences. 

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Cell death i s critical to normal development i n multiple 

orga n systems . For example, it is required during 

the formatio n of digits and durin g the generatio n of 

the middl e ea r spac e an d th e vagina l opening (e.g. , 

Saunders, 1966 ; Rodrigue z et al. , 1997 ; Robert s an d 

Miller, 1998) . Likewise, in the central nervous system 

(CNS) o f the norma l developin g mammal , a n esti - 

mated 20%-80 % mor e neuron s than th e final num - 

ber ar e generate d befor e th e surplu s i s eliminate d 

(Miller, 1995 ; Sastry and Rao , 2000). This loss of neurons 

varies by brain structure an d b y specific neuro n 

populations. 

In som e brai n structure s there i s a wholesale loss 

of transien t neurona l populations . Suc h cullin g o f 

populations i s required for proper developmen t t o ensue. 

Two exemplary populations ar e subplate neuron s 

(e.g., Kostovic and Rakic, 1980 ; Chun et al., 1987 ; Al- 

Ghoul and Miller, 1989) , and Cajal-Retziu s neuron s 

(e.g., Dere r an d Derer , 1990 ; Meye r e t al., 1998 ) i n 

neocortex (Marin-Padilla , 1998 ; Supe r e t al. , 1998 ; 

Sarnat an d Flores-Sarnat , 2002) . I t i s believe d tha t 

6 

Intracellular Pathway s 

of Neuronal Death 

Sandra M. Moone y 


91 

these populations die after they serve their role in cortical 

development . Th e subplat e neuron s provid e a 

temporary targe t populatio n fo r thalamocortica l ax - 

ons that arriv e i n the subplat e befor e the neuron s of 

their target layer (cortical layer IV) have migrated t o 

their final position (Ghosh e t al., 1990) . Cajal-Retzius 

neurons expres s reelin, which i s vital for normal mi - 

gration of cortical neurons (Lambert de Rouvroit and 

Goffinet, 1998 ; Rice and Curran, 2001). This association 

is indicated b y the disrupted cortical organization 

that occur s i n reeler mic e (Lamber t d e Rouvroi t and 

Goffinet, 1998 ) an d whe n Cajal-Retziu s neurons are 

prematurely los t (Frotscher , 1998 ; Hartman n e t al. , 

1999; Saftigetal, 1999) . 

Target-dependent neurona l deat h ma y b e th e 

most important proces s controlling neurona l deat h in 

the CNS . Throug h thi s mechanis m th e nervou s system 

sculpts itself , matching th e number o f neurons in 

communicating part s o f a syste m fo r optimal outpu t 

and maximizin g efficien t function . Neurona l deat h 

occurs i f (a ) ther e i s a lac k o f input/innervation ,


(b) axon s reac h th e wron g plac e withi n th e targe t 

area, (c ) axons project t o the wron g target , (d ) ther e 

are simply too many axons terminating within the target, 

or (e) no target exists. One exampl e o f the latter is 

the damage d trigeminal-somatosensor y system ; tran - 

section o f the infraorbita l nerv e causes a los s of neurons 

i n th e trigemina l brai n ste m nucle i an d th e 

ventrobasal nucleu s o f th e thalamu s (Klei n e t al. , 

1988; Mille r e t al., 1991 ; Wait e e t al. , 1992 ; Miller , 

1999; Sugimot o et al., 1999 ; Baldi et al., 2000). 

It is important to note that programmed neurona l 

death i s a phenomenon mainl y confined to developing 

invertebrates . As the ter m implies , neuron s ca n 

die a s a consequence o f a genetic program. The bes t 

examples are provided by Caenorhabditis elegans (see 

Apoptosis, below). Thus, programmed cell death must 

be distinguishe d fro m naturall y occurrin g neurona l 

death, whic h principall y represents target-dependen t 

neuronal death . 

Many developin g neuron s di e soon afte r the y ar e 

generated (se e Chapter 5) , perhaps a s a consequence 

of an error in DNA replication. Thi s adaptive mechanism 

allows an organism t o protect itsel f from basin g 

its circuitr y on a cadr e o f neurons wit h mutate d o r 

damaged DNA . 

EXPERIMENTAL MODEL SYSTEMS 

This chapte r consider s in vitro and i n viv o studies of 

neuronal death. Eac h experimenta l approac h ha s its 

own advantages and limitations. A major advantage of 

in vitr o studie s is that a homogeneou s populatio n o f 

cells i s addressed . Thi s settin g i s crucia l t o studie s 

of the induction of neuronal death by xenobiotics, because 

cells within the brain differ widel y in their susceptibility 

(Watts et al., 2005). Additionally, the entir e 

population is likely to respond t o a manipulation i n a 

similar manne r an d develo p synchronously . Thi s 

means that small changes in transcript and protein expression 

o r post-translational modification s ar e mor e 

likely to be detectable. Disadvantage s t o this method 

are tha t cultur e condition s ca n influenc e th e path - 

way^) activated , an d th e model ma y not b e physio - 

logically relevant . I n contrast , i n vivo studie s us e a 

highly heterogenous syste m in which ther e ar e mul - 

tiple cel l type s i n divers e developmenta l stage s a t 

a give n time . Thi s tempora l asynchron y make s i t 

harder to differentiate transcrip t and protei n change s 

from a background of ontogenetic noise . In addition, 

changes tha t ar e see n ma y be difficul t o r impossibl e 

to interpret . O f course , th e majo r advantag e t o thi s 

method i s that it is the more physiologically and clini - 

cally relevant model . 

Direct evidenc e o f neurona l deat h i s difficult t o 

amass because th e degenerativ e process ca n be rapid 

and the debris can be removed quickly (Reddien and 

Horvitz, 2004) . Thus, cells can expres s death-related 

proteins (e.g. , activ e caspas e 3 an d bax ) for a lon g 

time befor e the y di e (e.g. , Chen g an d Zochodne , 

2003), and no t al l cells rely on caspas e as an effecto r 

of cell deat h (se e Caspase-Independen t Cel l Death , 

below). Anothe r widel y used metho d o f identifyin g 

dying cell s i s termina l deoxynucleotidy l transferas e 

mediated deoxyuridin e nic k end labelin g (TUNEL) , 

in which free , adenylate d ends of degenerating DN A 

are labeled . Unfortunately , TUNE L ca n generat e 

false positive results; in at least one mode l syste m the 

amount o f TUNEL exceeds cel l los s (Gordo n e t al., 

2002). 

The onl y incontrovertible proo f of neuronal deat h 

is documentation o f a decrease i n neuronal number s 

in a longitudinal, population-base d study . Final neu - 

ronal numbers ar e a function of three developmenta l 

events: generation, migration , and death. A study performed 

at a single time-point in a mature animal cannot 

determin e th e relativ e contribution s o f thes e 

developmental events . A s a resul t o f th e potentia l 

of generatin g fals e positiv e results , a mathematica l 

method wa s devised to determine the number s of dying 

neuron s (Miller , 2003). The grea t advantag e of 

this approac h i s that th e estimat e o f cell deat h de - 

pends on the documented cel l numbers and the proliferative 

activity of the population . 

MODES O F CELL DEAT H 

The mode s o f cell deat h includ e apoptosis , necrosis , 

and autophag y (e.g. , Gree n an d Reed, 1998 ; Lock - 

shin an d Zakeri , 2004) . Apoptosi s i s a highl y con - 

trolled mechanism , durin g whic h specifi c transcrip t 

and protein change s occur . Apoptosis has been calle d 

cellular suicide , a s it only affects th e cel l undergoin g 

it. I n contrast , durin g necrosis , cell s an d thei r or - 

ganelles swel l and burst , releasing enzymes int o th e 

local environment . A resul t o f thi s typ e o f deat h i s 

damage to neighboring cells, thus causing a cluster of 

dying cells. Autophagy involves lysosymal engulfmen t 

of damaged cell s o r cell fragments . In man y model s

of disease or injury, dyin g cell neighbor s ma y exhibit 

different mode s o f cell deat h an d a n individua l cel l 

may exhibit features of different modes . 

One importan t poin t t o conside r i n cel l deat h i s 

that the lif e o f a critically damaged cel l ma y be tem - 

porarily maintained, but i t will likely die. That is, the 

blocking of one mode of cell death ma y merely cause 

a cell to shift to another mode of cell death. Thus, the 

outcome i s that the cel l still dies, but i t may do so by a 

different sequenc e o f event s (e.g. , D'Mell o e t al, , 

2000; Denecker et al, 2001; Zhou et al., 2005). Alternatively, 

blocking of cell death may allow cell survival, 

which is not necessarily a good thing. Fo r example, in 

mice deficien t i n apoptosi s protease-activatin g facto r 

(APAF) 1 , caspase 3 , o r caspas e 9 , ther e i s a lac k o f 

cell deat h i n the neura l tube , and thi s prevent s normal 

closur e o f th e tub e (Kuid a e t al. , 1996 , 1998 ; 

Cecconi e t al, 1998 ; Hake m e t al, 1998 ; Yoshid a 

et al, 1998 ; Honarpou r et al, 2000). It should als o be 

considered that enhanced cel l deat h i s not th e etiol - 

ogy of a pathological state, rather, neuronal death is a 

process or end poin t associated with a given disease. 

Apoptosis 

The ter m apoptosis wa s coined t o describe a series of 

morphological step s throug h whic h dyin g cells pass 

(Kerr e t al. , 1972) . With furthe r study , apoptosis has 

become associate d with a metabolically active process 

(e.g., Wyllie, 1997 ; Putch a an d Johnson , 2004) . Accordingly, 

chromosoma l DN A i s cleaved , th e chro - 

matin condenses , an d th e nucleu s i s broke n int o 

small pieces . Eventuall y the cel l shrink s and breaks 

into small , membrane-boun d apoptoti c bodie s tha t 

are engulfe d b y neighborin g cell s o r macrophages . 

This mechanism ensures that proteolytic enzymes are 

contained withi n membrane-boun d entitie s and tha t 

the apoptoti c cel l die s withou t causin g deat h o f 

neighboring cells. 

Much o f the initia l fundamental information on 

the pathway s of apoptosi s cam e fro m studie s o f th e 

nematode C. elegans (e.g., Horvitz et al., 1983 ; Hent - 

gartner an d Horvitz , 1994 ; Reddie n an d Horvitz , 

2004). O f the 109 0 cell s produced durin g the devel - 

opment of C. elegans, 13 1 die. Products o f two genes 

are essentia l for this death: Ced 3 and Ced 4 (Horvitz 

et al., 1983 ; Ellis and Horvitz , 1986). Los s or inactivation 

o f eithe r o f these gene s prevent s cel l death . I n 

the 95 9 cells that do not die , the produc t o f another 

gene, Ced9 , bind s t o Ced4 , an d prevent s i t fro m 

INTRACELLULAR PATHWAY S OF NEURONAL DEATH 9 3 

activating Ced3. Mammalian homolog s of these critical 

genes hav e been identified. Ced3 is homologou s 

with th e caspas e family , Ced 4 wit h APAF-1 , an d 

Ced9 wit h th e Be l family . Althoug h th e gene s an d 

gene product s involve d i n apoptosi s are highl y con - 

served acros s species , th e mammalia n pathway s are 

considerably mor e comple x an d convolute d tha n 

those identifie d in C . elegans. For example, differen t 

pathways of apoptosis ar e invoke d in cerebellar granule 

cell s dependin g o n whethe r th e cell s ar e pre- or 

postmigratory (Lossi et al., 2004). 

As state d above , us e o f th e ter m apoptosis ha s 

changed ove r the years . The definitio n ha s been re - 

worked fro m a se t o f morphologica l feature s t o in - 

clude molecula r an d biochemica l features . Putch a 

and Johnson (2004) suggest that apoptosis b e reserved 

for a caspase-dependen t cel l deat h tha t cause s th e 

morphological features described by Kerr et al. (1972; 

see above) . They conten d tha t caspase-independen t 

cell deat h i s nonapoptotic , despit e acknowledgin g 

that i t would b e possibl e t o see apoptotic morpholg y 

without caspas e activation . Man y researcher s us e 

apoptosis an d programmed cell death interchange - 

ably, bu t thi s i s inappropriat e becaus e a program 

connotes geneti c determination , a s i n th e cas e o f 

C. elegans. In mammalian systems , this type of genetic 

deat h i s rare , an d mos t apoptoti c deat h result s 

from environmenta l factors . I n thi s case , apoptoti c 

death i s best considere d a s naturall y occurring cel l 

death. I n this chapter, apoptosis include s morpholgi - 

cal and biochemical events . 

Major Component s of 

Apoptotic Pathways 

Bel Family 

Bel protein s play pivotal roles i n cel l death . I n contrast 

to C. elegans, which only expresses a single gene, 

Ced9, many organisms have multiple member s of this 

family. Ther e ar e tw o functionall y distinc t group s 

within th e Be l family : thos e tha t induc e deat h an d 

those that protect against death. Bel proteins can also 

be subdivide d structurally: there ar e three subgroup s 

defined b y th e numbe r o r typ e o f Bcl- 2 homolog y 

(BH) domains. Anti-apoptotic members o f the family , 

including Bcl-2 , Bcl-x L, Bcl-w , Mcl-1 , an d Al/Bfl-1 , 

contain fou r B H motifs : BH1 , BH2, BH3 , an d BH4 . 

Pro-apoptotic member s ma y b e i n on e o f tw o sub - 

groups: (1 ) those tha t contai n multipl e B H domain s


but lac k BH4-e.g., Bax , Bcl-x s, Bak , and Bok/Mtd , 

or (2) the BH3-only subgroup that includes Bid , Bini/ 

Bod, Bad, Bmf, Bik/Nbk, Blk, Noxa, Puma/Bbc3, an d 

Hrk/DP5. 

Bel family members form dimers . Homodimers of 

two anti-apoptotic members , o r heterodimers formed 

by one pro-apoptotic an d one anti-apoptotic member , 

for example , Bcl-2-Bax , ar e pro-survival . I n contrast , 

homodimers o f pro-apoptoti c members , o r het - 

erodimers o f tw o pro-apoptoti c members , resul t i n 

death. The BH 3 domain appears to be the prime suspect 

fo r inducin g apoptosis . Protein s wit h th e BH 3 

domain have two functions: (1 ) to bind to and inactivate 

the survival-inducing Bel proteins, and (2 ) to activate 

th e pro-apoptoti c Be l protein s wit h multipl e B H 

domains, such a s bax and ba k (Desagher e t al, 1999 ; 

Wei et al, 2000, 2001; Degterev et al, 2001). According 

to on e mode l o f apoptosis , Ba k i s a trans-membran e 

protein in the outer mitochondrial membran e an d Bax 

is i n th e cytoplas m (Lucken-Ardjomand e an d Marti - 

nou, 2005) . An apoptoti c signa l cause s a conforma - 

tional change in both proteins that results in exposure 

of their N terminal s an d translocatio n o f Bax to th e 

mitochondrial oute r membrane . Onc e i n the mem - 

brane, Bak and Bax form oligomers that induce mem - 

brane permeabilit y an d thu s allo w movemen t o f 

molecules suc h a s cytochrorne C , HtrA2/Omi , an d 

Smac/DIABLO. 

Caspases 

Caspases ar e a group of structurally related enzyme s 

in mammals. Genes fo r caspases have homology with 

the C. elegans gene Ced3 (e.g., Jiang and Wang, 2004; 

Shiozaki an d Shi , 2004) . Lik e th e gen e produc t o f 

Ced3, caspase s are importan t fo r cell death . I n con - 

trast to Ced3, however, caspases are no t require d for 

death (se e below) . Caspases ar e proteases ; a t leas t 1 4 

caspases have been identified. They have cysteine s i n 

their activ e site s an d cleav e thei r targe t protei n adja - 

cent to an asparti c aci d residue . Caspase s ar e synthe - 

sized a s inactiv e isoform s an d ar e cleave d int o larg e 

and small subunits. Active caspases are tetramers com - 

posed of two large and two small subunits. 

Caspases ar e groupe d int o thre e subfamilies : 

interleukin-lp-converting enzyme-lik e caspases , ini - 

tiator caspases (e.g., caspase 8 or caspase 9), and effec - 

tor caspase s (e.g. , caspas e 3 ) (Shi , 2002) . Initiato r 

caspases can cleav e themselves and effecto r caspase s 

are cleave d b y othe r enzymes , includin g initiato r 

caspases. Activatin g initiato r caspase s ca n induc e a 

caspase cascade i n which effecto r caspase s are cleave d 

and the n othe r apoptosis-relate d downstrea m mole - 

cules are activated. Caspase s ca n cleave more than 40 

target proteins ; o f particula r importanc e ar e a n in - 

hibitor of DNases, nuclea r laminins, and cytoskeletal 

proteins. Inactivatio n o f DNas e inhibitor s allow s 

DNases to cut DNA into fragments that are multiples 

of 180-200 bp. The resul t of this serial snipping is the 

stereotypical DNA ladder on a Southern blot that is associated 

with apoptosis. Cleavage o f nuclear laminins 

results i n nuclea r fragmentation . Similarly, cleavage 

of cytoskeleta l element s result s i n membran e bleb - 

bing and, ultimately, cell fragmentation. 

The importanc e o f caspase s i n norma l develop - 

ment i s reflected in mice that lack effective caspas e 3 

or 9 . Bot h mutant s hav e craniofacia l malformations 

and abnorma l brai n morpholog y (Kuid a et al., 1996 , 

1998; Ceccon i e t al , 1998 ; Hake m e t al , 1998) . 

Specifically, these animals exhibit enlarged prolifera - 

tive zones , heterotopi c cluster s o f neurons , an d in - 

creased number s o f neurons. Interestingly , caspas e 3 

deficiency ca n b e offse t b y deficienc y i n th e anti - 

apoptotic gen e Bcl-x L (Rot h e t al. , 2000) , implyin g 

that Bcl-x L can ac t through a caspase-3-independent 

mechanism. 

Apoptosis Protease Activating Factor 1 

APAF-1, homologous to Ced4, exists in the cytoplasm 

as an inactiv e protein (Ferrar o et al, 2003 ; Shiozaki 

and Shi, 2004). Interaction with cytochrome C in the 

presence of adenosine triphosphate (ATP) results in a 

conformational chang e i n APAF- 1 that allow s seven 

molecules o f APAF-1 to assemble an d for m th e cen - 

tral portion o f the apoptosome . I n addition, th e con - 

formational chang e expose s the caspas e recruitmen t 

domain, which bind s procaspase 9 . In the absence of 

an inhibitory apoptotic protein (IAP ) such as X-linked 

IAP (XIAP) , caspas e 9 i s activate d vi a a n unknow n 

mechanism. Activ e caspase 9 recruits and activates effector 

caspases 3 and 7 . 

APAF-1 knockou t mic e ar e embryoletha l (Cec - 

coni e t al, 1998) . The y have craniofacia l an d CN S 

malformations tha t appear to result from insufficien t 

cell deat h durin g development . Interestingly , their 

limbs ar e differentiall y affected . O n gestationa l da y 

(G) 15.5 , the limbs are less mature those than in wildtype 

animals, as evidenced by the persistence of interdigital 

webbing . B y G16.5 , however , thei r limb s

appear normal; the webbing is lost after undergoin g a 

caspase-independent form o f cell death . This findin g 

implies tha t som e tissu e ca n overcom e th e los s o f 

APAF-1, but the CM S cannot, an d that deat h vi a the 

caspase-independent pathwa y takes longer. 

Cytochrome C 

Cytochrome C i s normally found betwee n th e inne r 

and oute r mitochondria l membrane s and i s used for 

transporting electrons between th e Cytochrome B-C1 

and cytochrom e oxidas e complexe s i n th e electro n 

transport chain. When the mitochondrial permeability 

transition pore i s compromised, substances i n th e 

intramembrane space , includin g cytochrom e C , are 

allowed t o move into the cytoplasm . Once in the cytoplasm, 

cytochrome C interact s with APAF-1, ATP, 

and caspase 9 to form the apoptosome. 

Poly-Adenosine Diphosphate 

Ribose Polymemse 

Poly-adenosine diphosphate (ADP ) ribose polymerase 

(PARP) 1 is found i n the nucleu s an d mitochondria . 

It can b e activated i n response to DNA damage o r to 

oxidative stress (e.g., Hong et al., 2004; Nguewa et al., 

2005). Followin g DNA damage , PARP- 1 becomes ac - 

tive an d synthesize s poly(ADP-ribose ) an d catalyze s 

the addition of ADP-ribose to the DNA (e.g., Bouchard 

et al, 2003) . Followin g poly(ADP-ribosyl)ation, DNA 

repair enzyme s ca n b e recruite d an d activated . Gas - 

pases 3 and 7 cleave PAR P in it s nuclear localization 

sequence, resulting in an inactive form of the enzyme 

that canno t ad d ADP-ribose, thus , th e DN A canno t 

undergo repair . 

During th e synthesi s o f poly(ADP-ribose) , 

nicotinamide-adenine dinucleotid e (NAD ) i s cleaved 

into ADP-ribos e an d nicotinamide . T o restor e NA D 

levels (and thus the NAD/NAD H ratio) , the cel l con - 

verts nicotinamid e t o NA D b y mean s o f tw o mole - 

cules o f ATP , thu s depletin g th e energ y pool . 

Following severe DNA damage, the loss of cellular energy 

is likely responsible for a necrotic cel l death afte r 

excessive DNA damage (Szab o and Dawson, 1998 ; H a 

and Snyder , 1999 ; Fuerte s et al, 2003). Interestingly , 

the los s of energy may be necessary , but i s unlikely to 

be sufficient, for cell death (Wan g et al, 2004). 

Mitochondrial PARP- 1 may be involve d i n apop - 

tosis via release of apoptosis inducing factor (AIF ) (Yu 

et al , 2002 ; D u e t al , 2003) . Genomi c damag e 

INTRACELLULAR PATHWAY S OF NEURONA L DEATH 9 5 

causes poly(ADP-ribosylation) in the nucleus, resultin g 

in cellular NAD depletion, activatio n of mitochondrial 

PARP, AIF release, and subsequent cell death. 

Apoptosis Inducing Factor 

In normal , undamage d cells , AI F i s importan t fo r 

protection agains t oxidativ e stres s (Klei n et al. , 2002 ; 

Lindholm et al, 2004; Vahsen et al, 2004). Following 

PARP activation, AIF i s released fro m th e mitochon - 

dria into the cytosol (see Poly-Adenosine Diphosphate 

Polymerase, above). AIF translocates into the nucleu s 

where i t i s involve d i n condensatio n o f chromati n 

and DN A fragmentatio n (Hon g e t al, 2004) . Th e 

PARP-dependent releas e o f AIF i s a caspase-indepen - 

dent mechanism o f cell deat h (se e Caspase-indepen - 

dent Cel l Death , below) . I n addition , caspase s ca n 

cause release of AIF, as can changes in mitochondria l 

membrane permeability , fo r example , b y Ba x an d 

Bak. Bcl-2 can interfer e with AIF-mediated apoptosi s 

by blocking th e cellula r redistributio n o f AIF that is 

necessary for death (Susi n et al., 1996 , 1999 ; Dauga s 

et al, 2000). 

Apoptosis-Related Receptors 

Ligands (e.g. , tumor necrosi s factor [TNF]-a , Apo2/ 

TRAIL, FasL , an d possibl y growth factors ) tha t inter - 

act with receptors o f the TN F famil y activat e the extrinsic 

apoptoti c pathway . TNF-oc binds to the TN F 

receptor, Apo2/TRAIL binds t o TNF-related apopto - 

sis inducin g ligand-recepto r 1 (TRAIL-R1)/death re - 

ceptor (DR) 4 and TRAIL-R2/DR5, and FasL binds to 

Fas(CD95,APO-l). 

Effectors o f Apoptosis 

p53 

p53 i s a multifunctiona l housekeepin g protei n in - 

volved i n cel l cycl e regulation , recognitio n o f DN A 

damage and consequent repair , and cell death (Mille r 

et al, 2000 ; Morrison e t al, 2002) . Although i t is a 

transcription factor , i t als o respond s t o genotoxi c 

stress (e.g . Bertran d e t al., 2004) . p5 3 activation ca n 

halt the cel l cycle and initiat e DNA repair or apoptosis 

(Harms et al, 2004). 

p53 usually has a short half-life (Ki m et al, 2004) . 

When stabilized, for example, through phosphorylation 

on serin e residues , p53 accumulates i n th e nucleus ,


where i t ha s transcriptiona l activities . p53-regulate d 

genes critica l for normal brain development includ e 

cell cycl e proteins p21 and murine double minut e 2 , 

the DN A repai r protei n growt h arres t an d DN A 

damage-inducible gene 45 , and th e apoptosis-related 

proteins insulin-lik e growth facto r binding protei n 3 

and Ba x (Levine , 1997) . I n addition , p5 3 regulate s 

the Fa s receptor (Somasundaram, 2000) and th e hormone 

polyadenylat e cyclase-activatin g polypeptid e 

receptor 1 (e.g., Cian i e t al, 1999 ; Johnson , 2001) 

(see Chapters 1 5 and 16) . 

p53 can play a direct role in cell death by interacting 

wit h th e Be l famil y o f protein s (Chipu k e t al. , 

2003, 2004 ; Mihar a e t al , 2003) , APAF-1 , an d 

apoptosis-related receptors . Thes e interaction s ar e 

pro-apoptotic. p5 3 down-regulate s pro-survival Bcl-2 

and Bcl-x , and up-regulate s Bax, Bid, and APAF-1 . It 

also up-regulate s TRAIL-R2/DR5 , Fas , an d FasL . I n 

addition, p53 represses c-Fos (Kle y et al, 1992) , thus 

potentially interferin g with activato r protei n 1 and , 

hence, transcription. 

Oxidative Stress 

Oxygen, thoug h centra l t o life , ca n b e toxic . I n it s 

ground state, it possesses two unpaired electron s with 

parallel spin states. This setting makes a two-electron 

reduction kineticall y unlikely ; however , sequentia l 

one-electron reduction s can occur, hence generating 

oxygen fre e radicals , reactive oxyge n specie s (ROS) . 

In th e biologica l setting , th e initia l one-electron ex - 

change generate s th e superoxid e anio n radical . Th e 

protonated two-electro n reductio n produce s H 2O2 

(the protonated for m o f the peroxide ion), with the final 

protonate d four-electro n produc t bein g water . 

The oxyge n radicals resulting from thi s process generate 

a plethor a o f reactiv e specie s wit h othe r mole - 

cules, suc h a s nitroge n an d iron , al l o f which ca n 

produce a pro-oxidan t environment i n cells , termed 

oxidative stress. 

The centra l role s for ROS in apoptosis, both as initiators 

an d a s signalin g event s withi n th e apoptoti c 

process, ar e wel l documente d (Curti n e t al. , 2002 ; 

Fleury e t al , 2002 ; Polste r an d Fisku m 2004) . Although 

the specific mechanism s by which ROS elicit 

and/or maintai n apoptosi s remai n undefined , thes e 

compounds hav e a n effec t o n a variet y o f cellula r 

components: proteins , DN A base s and sugars , polysaccharides, 

an d lipids . On e ROS-relate d pathwa y 

that has been connecte d t o ethanol-mediated neuro n 

apoptosis i s th e productio n o f pro-apoptoti c prod - 

ucts o f lipi d peroxidatio n withi n mitochondria (Ra - 

machandran e t al , 2001 , 2003) . RO S reac t wit h 

unsaturated fatt y acids , initiatin g a self-perpetuatin g 

peroxidation o f membrane lipid s (Kappus , 1985) . In 

addition t o direc t damag e t o biomembranes , thi s 

ubiquitous proces s generate s highl y reactive aldehydes, 

th e mos t studie d an d th e mos t toxi c bein g 4 - 

hydroxynonenal (HNE ) (Esterbaue r e t al , 1990 ; 

Uchida e t al, 1993) . Importantly , HN E formatio n 

generates apoptotic death o f neurons, and it s production, 

secondar y t o oxidativ e stress , ha s bee n com - 

pellingly linke d t o neuro n deat h i n a variet y o f 

neurodegenerative diseases (Zarkovic, 2003). 

APOPTOTIC PATHWAY S 

Caspase-Dependent: Intrinsic vs. 

Extrinsic Pathways of Apoptosi s 

The pathway s of apoptosis can be divided into intrinsic 

an d extrinsi c on th e basi s of activation site (Budi - 

hardjo e t al , 1999 ; Shiozak i an d Shi , 2004) . Th e 

intrinsic, mitochondria-associate d pathwa y depend s 

on Bel proteins. The extrinsi c pathway is mediated by 

activation o f an apoptosis-relate d receptor. Th e path - 

ways ar e no t trul y independent , a s ther e i s muc h 

cross-talk, and both can activate caspas e 3 (Fig. 6-1) . 

In addition, activation of the extrinsic pathway can activate 

a feedback loop to the intrinsic pathway. 

Intrinsic Pathways 

The fundamenta l featur e o f the intrinsi c pathwa y i s 

the releas e o f pro-apoptoti c compound s fro m mito - 

chondria. Thi s pathwa y i s activated b y intracellula r 

stress signal s includin g bu t no t limite d t o oxidativ e 

stress or DNA damage. Thus, major effector s ma y be 

ROSandp53. 

Mitochondrial permeabilit y i s affected b y the Be l 

family o f proteins . Ba x homodimer s allo w io n flu x 

through th e mitochondrial membrane , an d this some - 

how allow s movemen t o f cytochrom e C , possibl y 

through enlargement of the voltage-dependent anio n 

channels (Banerje e an d Ghosh , 2004 ) o r by opening 

the mitochondria l permeabilit y transition por e (e.g. , 

Marzo et al., 1998) . Breach of the membrane cause s a 

loss of membrane potentia l and release of cytochrome c, 

AIF, HtrA2/Omi, and/or second mitochondria-derive d

activator of caspase/direct IAP binding protein, which 

has a low isoelectric poin t (Smac/DIABLO) . I n nor - 

mal cells , thes e substance s ar e sequestere d betwee n 

the inne r an d oute r mitochondria l membranes . Re - 

lease int o th e cytoplas m initiate s apoptoti c pathway s 

(Fig. 6-1). It should be note d tha t permeabilization of 

the mitochondria l membran e doe s no t necessaril y allow 

dumping of all intermembrane substances. Instead, 

there i s som e selectivity . For example , applicatio n o f 

INTRACELLULAR PATHWAY S OF NEURONA L DEAT H 9 7 

n-methyl-D-aspartate to cultured primar y mouse corti - 

cal neurons can cause release of AIF and cytochrome c 

(Wang et al., 2004). In contrast , oc-amino-3-hydroxy-5 - 

methyl-4-isoxazoleproprionic aci d ca n induc e th e re - 

lease of cytochrome c, but no t AIF. 

In response to DNA damage or ROS, p53 accumulates 

in the mitochondria (e.g., Marchenko et al., 2000; 

Erster e t al. , 2004) , where i t interacts with Bel-famil y 

proteins i n a transcriptional-independen t manner . 

FIGURE 6-1 Pathway s underlying neural cell death. Three cell death pathways 

tracing the flow of information from externa l effects t o nuclear response are depicted: 

intrinsic and extrinsi c caspase-dependent pathways and a caspase-inde - 

pendent pathway . Variou s player s involve d i n thes e pathway s ar e shown . 

Positive an d negativ e effector s ar e depicte d b y line s endin g i n arrow s an d 

blunted tips , respectively. FADD, Fas-associate d deat h domain ; FLIP , FADD - 

like interleukin-1-converting enzyme inhibitory protein for other abbreviations 

see Abbreviations list. See text for details.


This interaction explain s the rapi d response of a cel l 

to a death signal . P53 alters expression of Bel famil y 

members i n a pro-apoptotic manner, i.e. , expression 

of Ba x is increased an d concurrentl y Bcl- 2 i s down - 

regulated. I n addition , p53 up-regulate s APAF- 1 expression. 

Release d cytochrom e C form s a comple x 

with seven APAF-1 molecules, an d i n the presenc e of 

ATP, caspase- 9 i s recruite d int o thi s complex , th e 

apoptosome, an d activated . Active caspase 9 cleaves 

and activate s effecto r caspase s 3 , 6 , and/o r 7 . Not e 

that at this point, the intrinsi c and extrinsi c pathways 

merge a s caspase s 8 an d 10 , activated b y receptor s 

(see below), cleave and activate effector caspases . 

Extrinsic Pathways 

The extrinsi c pathway is activate d b y factor s outsid e 

the cell membrane via ligand-receptor binding (Muzio 

et al, 1996 ; Almasan and Ashkenazi, 2003; Choi and 

Benveniste, 2004). Binding of the ligand to the recep - 

tor causes recruitment of cytoplasmic element s suc h 

as Fas-associate d deat h domai n an d TN F receptor - 

associated deat h domai n tha t for m th e death - 

inducing signalin g complex. Thi s proces s result s in 

recruitment and activatio n of initiator caspases 8 and 

10 through proteolysis . Caspases 8 and 1 0 then acti - 

vate effector caspase s 3, 6, and 7 , and ca n als o cleave 

a membe r o f the Be l famil y o f proteins, Bid. At this 

point, th e intrinsi c pathwa y also come s int o pla y as 

Bid translocates to the mitochondrio n an d cause s insertion 

o f Bax-Bax homodimers into the mitochondr - 

ial membrane, thu s allowing release o f cytochrome c 

and th e resultin g downstrea m events , an d effecto r 

caspase activatio n (L i e t al. , 1998 ; Lu o e t al. , 1998 ; 

Wei et al., 2001; Scorrano an d Korsmeyer, 2003). 

Substrates of Caspases 

Substrates o f effecto r caspase s ma y numbe r i n th e 

hundreds (e.g., Earnshaw et al., 1999; Nicholson et al., 

1999; Fischer et al., 2003; Fuentes-Prior and Salvesen, 

2004). Thre e ar e o f particular interest—Bid, PARP , 

and DFF40/CAD . Activated , i.e. , cleaved , Bi d 

translocates t o th e mitochondria , wher e i t interact s 

with othe r pro-apoptoti c member s o f the Be l family , 

specifically Ba x an d Bak , thu s causin g membran e 

permeability, and leakage of cytochrome C (L i et al., 

1998; Lu o et al, 1998 ; Wei et al, 2001). Cleavage of 

PARP b y caspase s 3 an d 7 inactivate s the DN A re - 

pair mechanism (Lazebnik et al., 1994) . Activation of 

DFF40/CAD by caspase 3 results in enzymatic cleavage 

of double-stranded DNA, the hallmark characteristic 

o f fragmente d DNA . Thi s structur e ca n b e 

visualized on a n agaros e gel as a ladder o r can b e labeled 

in vivo by means of TUNEL. 

Caspase-Independent Cell Death 

There i s some debat e as to whether caspase-indepen - 

dent cel l deat h i s a for m o f apoptosi s (Chipu k an d 

Green, 2004 ; Putch a an d Johnson , 2004) , however, 

pathways o f caspase-independen t cel l deat h ar e fre - 

quently activated concomitant with caspase-dependent 

pathways (Ramachandra n e t al. , 2003) . Caspase - 

independent cell death i s initiated by the sam e effec - 

tors a s thos e i n typica l apoptosis—for example , p5 3 

and ROS (Hill et al, 2000; Godefroy et al, 2004), but 

the morphological outcom e differs. Caspase-indepen - 

dent deat h doe s no t resul t in DN A fragmentation or 

cell blebbing , no r d o th e mitochondri a los e mem - 

brane potential. In addition, the caspase-independen t 

pathway may be initiate d by overactivation of PARP-1 

(Hong et al, 2004). 

As wit h caspase-dependen t pathways , the releas e 

of mitochondrial intermembrane substances AIF, endonuclease 

G , HtrA2/Omi , and/o r Smac/DIABL O 

appears t o b e pivota l fo r caspase-independent deat h 

(Susin e t al., 1999 ; Crega n e t al, 2002 ; Hong e t al , 

2004; Yakovle v an d Faden , 2004) . Releas e o f AI F 

from the mitochondria results in activation of DNases 

that cause DNA fragmentation. Endonuclease G i s a 

mitochondrial nucleas e necessar y fo r replicatio n o f 

mitochondrial DN A (Cot e an d Ruiz-Carrillo , 1993) . 

If released into the cytoplasm , it translocates into th e 

nucleus wher e i t causes internucleosoma l DN A frag - 

mentation (L i et al , 2001) . HtrA2/Om i an d Smac / 

DIABLO als o appear to induce caspase-independen t 

death, althoug h th e exac t mechanis m i s unknow n 

(Yakovlev and Faden , 2004). 

CONCLUSIONS AN D SUMMARY 

Misleadingly referred t o a s a regressiv e process, neuronal 

deat h i s essentia l fo r norma l CN S develop - 

ment. Withou t it , th e nervou s syste m ca n becom e 

overpopulated wit h poorl y integrate d neuron s an d 

circuitry that produces functional deficits. This observation 

i s supported b y studies of knockout mice defi - 

cient i n caspase 3 or 9 (Oppenheim e t al, 2001) . In

these animals , th e amoun t o f neuronal deat h i s reduced 

and learning an d memory is compromised. 

Although neurona l deat h ma y b e describe d a s 

apoptotic o r necrotic , dyin g cell s commonl y exhibi t 

features o f both (Eva n an d Littlewood , 1998 ; Gree n 

and Reed, 1998 ; Lockshi n and Zakeri, 2004). More attention 

ha s been directe d t o understanding apoptosis . 

The pathway s activated durin g apoptotic degeneratio n 

are comple x an d ofte n appea r t o b e conflicting . The y 

include so-calle d intrinsi c an d extrinsi c systems , bot h 

of whic h culminat e i n th e activatio n o f caspas e 3 . 

Some cells , however , di e throug h a caspase-independent 

mechanism. The curren t and future understanding 

o f neuronal death ha s been an d wil l be garnere d 

from studie s of the action s o f toxins (e.g. , ethano l an d 

neurotransmitter blockers), radiation , stress , an d othe r 

challenges. Suc h studie s provid e valuabl e insigh t int o 

the interpla y betwee n the variou s modes of cell deat h 

and th e pathway s underlyin g thes e degenerativ e 

events. 

Abbreviations 

ADP adenosin e diphosphate 

AIF apoptosi s inducing factor 

APAF apoptosi s protease-activatin g factor 

ATP adenosin e triphosphat e 

BH Bcl- 2 homology 

CNS centra l nervou s syste m 

DIABLO direc t IAP binding protein 


IAP inhibitor y apoptoti c protein 

NAD nicotinamide-adenin e dinucleotide 

PARP poly-adenosin e diphosphat e ribos e poly - 

merase 

ROS reactiv e oxyge n specie s 

SMAC secon d mitochondrial-derive d activato r 

of caspase 

TNF tumo r necrosis factor 

TUNEL termina l deoxynucleotidy l transferas e 

mediated deoxyuridine nick end labeling 

ACKNOWLEDGMENTS Wor k o n thi s chapter was supported 

b y th e Nationa l Institut e of Alcohol Abus e an d 

Alcoholism. 

INTRACELLULAR PATHWAY S O F NEURONAL DEAT H 9 9 

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7 

Developmental Disorders and 

Evolutionary Expectations: 

Mechanisms of Resilience 

Every livin g organism ca n trac e it s lineage bac k t o 

the unicellular organisms that first populated Earth . 

We are thus th e descendant s o f creatures wh o hav e 

not onl y survived but hav e successfull y reproduce d 

in the fac e of gross atmospheric shifts, blasts of ionizing 

radiation, the impact s of comets, ice ages, global 

warming, earthquake s an d hurricanes , plague s o f 

any number o f viral an d bacteria l forms, prédation, 

starvation and drought , th e toxin s of plants and ani - 

mals, parasites, competing tribes, jealou s fathers, indifferent 

mothers , an d everyda y accident s o f al l 

kinds. Any survivor of this wildly improbable lineage 

is mad e of toug h stuff . The poin t of the presen t 

chapter i s to tur n th e usua l discussio n o f develop - 

mental vulnerability on its head and, rather than discuss 

the effect s o f an "environmenta l insult(s) " on a 

fragile infant , examin e th e desig n feature s o f th e 

tough stuf f o f whic h w e ar e made . W e argu e tha t 

only in this evolutionary context wil l the disorder s of 

development tha t sometime s d o emerg e mak e 

mechanistic sense. 

Barbara L. Finlay 

Jeremy C. Yost 

Desmond T . Cheung 

104 

EVOLUTIONARY DRIVES 

Catastrophe 

Conservation o f th e buildin g block s o f th e embry o 

across the invertebrat e an d vertebrat e lineage s and th e 

resilience of that embryo have been part of a general rethinking 

of the characte r of the evolutionar y process to 

which our ancestors wer e subjected, along with greate r 

awareness of the effect s o f periodic globa l catastrophe s 

on th e natur e of the genom e (Gerhar t and Kirschner, 

1997). Struggle for success among individuals in an evolutionary 

landscape of potential niche s and their associated 

fitness peaks, success defined by better adaptation 

resulting i n greate r reproduction , i s the essentia l Dar - 

winian view . The kind s of adaptations highlighte d b y 

this proces s ar e both increasingl y subtl e organis m an d 

environment fits and enhancement s importan t in sexual 

selection, a central aspect of evolutionary change. 

Catastrophe, loca l o r global , provide s a forc e i n 

which all organisms, across radiations and species, ar e

subjected t o a sever e filter ; succes s arise s fro m sur - 

vival i n suddenl y change d environment s (Alvare z 

et al., 1980 ; Albritton, 1989) . Variation in gene trans - 

mission during catastrophe ca n aris e from individua l 

competition a s well, but whole radiations may be lost. 

The best-know n example , o f course, i s the extinctio n 

of the dinosaur s and surviva l o f mammals i n the lat e 

Cretaceous period . Selectio n wil l favo r th e robus t 

and adaptabl e organism, able t o weather disaster, to 

relocate or create its favored nich e o r get by in a ne w 

one, an d t o reproduc e a s muc h a s possible . Ou r 

genomes carr y th e histor y o f bot h type s o f change . 

Moreover, th e natur e o f geneti c chang e appear s to 

make i t likel y tha t thi s histor y is no t overwritte n in 

such a way that it is inaccessible, but rather added to, 

as we will elaborate later. 

Evolvability 

"Evolvability" i s a related , bu t distinc t ide a tha t car - 

ries impor t fo r how a genom e ca n respon d t o chal - 

lenge. This idea has been used in two distinct ways in 

the allie d field s o f evolutionary biology an d artificia l 

intelligence an d robotic s b y mean s o f genetic algo - 

rithms. The first, and somewha t stricter , sense of this 

concept aros e from th e hypothesi s an d late r demonstration 

(in bacteria) that in times of extreme stress and 

crisis, "deliberate " additiona l variation and mutatio n 

would arise as an adaptiv e mechanism, suppressed in 

favored environment s (Gerhart an d Kirschner , 1997; 

Radman e t al. , 1999) . Th e second , mor e general , 

sense is the observation that some types of genomic or 

informational structure s more readily produce usable 

adaptive changes and the potential for evolution than 

others (Lipso n an d Pollack , 2000 ; Nolf i an d Flore - 

ano, 2002 ; Baum, 2004) . Everythin g else equal , th e 

"evolvable" organism is more likel y to be wit h us today. 

Both the strict and the general sense of evolvability 

will be important in understanding the response of 

organisms to developmental challenge . 

Adaptive Response O r Pathology? 

The thir d conceptual thread t o be woven into the argument 

for evolutionar y chang e is the recastin g of 

some state s usually viewed as illness or patholog y as 

adaptations b y th e emergin g fiel d o f evolutionary 

medicine (Ness e an d Williams , 1996) . A vim s co - 

opting ou r genom e fo r it s ow n purpose s i s a ba d 

thing. Consequently , a n immun e respons e disables 

DEVELOPMENTAL DISORDERS AND EVOLUTIONARY EXPECTATIONS 10 5 

the viru s a t the cellula r leve l (antibodies) , make s its 

environment inhospitabl e (fever) , an d expel s i t (al l 

the variou s miseries of flu). Each o f these symptom s 

can be viewed as adaptive responses, no t as pathology. 

Of course , no t ever y response t o disease o r trauma is 

adaptation, an d th e urg e t o tell such "just-so " stories 

must be strictly policed. For example, exsanguination 

when a n arter y i s severed migh t cleans e th e woun d 

and quickl y lessen metabolic load, but obviousl y it is 

not a n adaptiv e response. Conceptua l clarit y in thi s 

domain get s particularl y muddled whe n organism s 

are i n clos e competition . Fo r example , i t would ap - 

pear that the enterprising rhinovirus has co-opted th e 

expulsion component o f the immun e respons e for its 

own adaptiv e purpose o f spreading itself around th e 

environment—whose adaptation is the sneeze? 

Evolutionary medicine become s particularl y relevant 

to the developmenta l respons e to shortages an d 

pathogens when the normal, tactical nature of the response 

o f the adul t organis m t o challenges i s considered 

an d applie d t o developmen t (Bateso n e t al. , 

2004; Gluckma n and Hanson , 2004) . The bes t and 

most elaborate d exampl e i n physiology is the balanc - 

ing o f requirements fo r short- and long-ter m surviva l 

in th e hypothalamic-pituitary-adrena l axi s i n re - 

sponse to acute and chroni c environmental stressor s 

(Sapolsky et al., 1986) . In order to survive an immediate 

threat, energy is mobilized by metabolic change s 

that, if they persist long-term, will damage the animal. 

Alterations i n nervou s system s an d behavio r i n re - 

sponse t o earl y environmenta l challenges , particu - 

larly commonl y encountere d ones , migh t als o b e 

tactical adaptations and shoul d no t automaticall y be 

construed a s pathology. Fo r example , perhap s i t i s a 

good bet to alter interest in food compare d t o general 

exploration i n perpetuit y i f earl y experienc e show s 

food to be in unusually short supply (Smart and Dob - 

bing, 1977 ; Levitsky , 1979 ; Bateson e t al , 2004 ; 

Gluckman and Hanson, 2004) . 

Environmental Expectatio n 

The fourt h thread i s environmental expectation . This 

is usually raised in the contex t of predictable environ - 

ments an d a genome predispose d t o lear n (Gree - 

nough an d Black , 1992) : i f informatio n abou t th e 

future structur e of visual informatio n in th e worl d is 

well predicted by present structure, and ther e i s time 

to learn it, why waste genome specifyin g it? Considering 

on e o f the classi c case s o f ethology, i f mother i s


the first large, moving thing any duck likely to survive 

sees, then learning the qualities of the first large, moving 

object i s an efficien t wa y to get the information . 

On th e othe r hand , th e physiologica l armamentar - 

ium availabl e indicate s tha t w e als o expec t trauma , 

infection, an d poo r nutritio n o f al l kinds . Ther e i s 

no reaso n to assum e tha t the brai n is not similarl y 

prepared. Recen t compariso n o f the genom e o f th e 

chimpanzee an d of the human, showing rather more 

changes i n gene s involve d i n foo d metabolis m an d 

immune respons e tha n i n th e brain-relate d genes , 

has underline d tha t geneti c respons e t o often - 

encountered challenges may be fairly rapid, and "diet 

and pathogen s ar e dominan t selectiv e force s fo r al l 

species" (Olso n an d Varki , 2004) . Relevan t t o thi s 

chapter i s the cultura l history of alcohol. A s alcohol 

has served as a common sourc e of nutrition and foo d 

preservation i n Wester n Europe , thos e population s 

(compared to Asian ones) have become equippe d t o 

metabolize alcohol, an d ther e i s some evidenc e tha t 

the incidenc e o f alcoholis m fall s th e longe r fer - 

mented product s hav e been use d i n a particular cul - 

ture (Goedd e e t al , 1992 ; Whitfield , 1997) . T o 

understand a developmental respons e t o an environ - 

mental challenge , i t will make a great dea l o f differ - 

ence whethe r th e challeng e i s "expected, " suc h a s 

alcohol, o r unprecedented—for example , a designe r 

drug such as thalidomide that may mimic the form of 

an early regulatory gene. 

A Warning and a Road Map 

In the followin g section , we first examine a few central 

cases of the developmenta l consequences of alcohol 

an d othe r substanc e abuse , an d tur n th e usua l 

style o f presentation upsid e down to show the outra - 

geous situations in which the typical embryo can survive. 

A stron g cavea t i s necessar y here: a t n o poin t 

should a demonstration that th e "typical " infant ca n 

be born without defect—for example , following a persistent 

bath o f alcohol—be mistaken as an argumen t 

endorsing alcoholis m i n pregnan t mothers . A sub - 

stantial percentag e o f infants exposed t o ethano l d o 

have defects , a probabilit y n o prospectiv e paren t 

should ignore . No r ar e we in an y way attempting t o 

minimize the value of locating and ameliorating thos e 

defects cause d b y developmental insults described i n 

many of the other chapters of this volume. Rather, our 

point is that if we fail to appreciate the robus t nature of 

the developin g embry o an d consistentl y mislabe l 

deviations as pathology instead o f adaptations (or just 

deviations), w e will be i n a n explanator y real m tha t 

makes n o sens e i f we try to understan d th e patholo - 

gies of development tha t can and do occur. 

Next, we will turn to some general desig n feature s 

of developmen t tha t ar e stabl e i n th e fac e o f chal - 

lenge, and conside r som e particula r cases of nervous 

system development tha t show those principles. Over - 

all, the guiding principal in this discussion is one tha t 

is true for all life sciences but i s often ignored in med - 

icine: "nothing i n biology makes sense except in th e 

light of evolution." (Dobzhansky, 1973) 

CONTEXTUALIZING THE 

DEVELOPMENTAL EFFECT S 

OF ALCOHOL, NICOTINE, 

AND THALIDOMIDE 

Alcohol 

The huma n bod y has a variety of mechanisms fo r the 

screening an d disposa l o f toxins. Alcohol i s no excep - 

tion. The presence o f alcohol i n our evolutionary and 

developmental environmen t ha s le d t o specifi c en - 

zymes responsibl e fo r it s metabolism: alcoho l dehy - 

drogenase an d acetaldehyde dehydrogenase . Ethano l 

is naturally encountered i n fermentation in fruit, an d 

also is a direct result of fermentation of starches in th e 

gut. I n man y human cultures , alcoho l i s sought no t 

only for the obviou s psychoactive consequence s bu t 

also becaus e i t serve s a usefu l rol e a s a preservative 

and secondarily as a source of calories (Goedde e t al., 

1992; Whitfield, 1997 ; Dudley , 2000, 2002). Alcohol 

can thus be considered not simply a completely alien 

disruptive element but also a normal part of the developmental 

environment . This i s not t o downplay the 

dangers o f ethanol t o a developin g organis m bu t t o 

place it in the contex t of a regulating system. We not e 

in passin g tha t thi s evolutionar y histor y ma y hav e 

a significant impact o n the relevanc e o f animal mod - 

els to human development , wher e frugivores and omnivores 

migh t b e considerabl y bette r models tha n 

carnivores. 

With th e assumptio n o f the robustnes s o f the de - 

velopmental syste m i n mind , w e tur n t o a recen t 

study fro m Martinez-Fria s and colleague s (2004 ) o n 

the relationshi p between alcoho l consumptio n dur - 

ing pregnancy and congenital anomalies , paying specific 

attentio n t o thos e anomalie s indicativ e of feta l

alcohol spectru m disorder s (FASD) . Fro m a corpu s 

of dat a collecte d ove r 2 4 years , Martinez-Fria s an d 

colleagues dre w a grou p o f 4705 malforme d infant s 

and a comparable group of 4329 normal infants whose 

mothers reporte d alcoho l consumptio n durin g preg - 

nancy. These infant s wer e examine d i n th e firs t 3 

days of lif e for a variet y of congenita l anomalies , including 

variou s nervou s syste m defect s an d a se t o f 

craniofacial anomalies. These facial anomalies, includ - 

ing a hypoplastic nose , are indicativ e o f several cognitive 

impairment s (Swille n e t al. , 1999 ; Donna i an d 

Karmiloff-Smith, 2000) . In particular , they are typical 

of FAS D and , i n conjunctio n wit h materna l alcoho l 

consumption, ar e considered reliabl e predictor s of the 

condition (Astle y and Clarren, 1997 ; Coles et al, 2000; 

Streissguth and O'Malley, 2000; Sood et al, 2001). 

Subjects can be grouped by maternal alcohol con - 

sumption int o five categories, ranging from Grou p 1 , 

who experience d low , sporadi c exposur e throug h a 

range of daily doses, to Group 5 , who were exposed to 

over 92 g of absolute alcoho l per day throughout ges - 

tation, includin g severa l glasse s o f distille d spirits . 

Group 5 also included th e offsprin g of those who self - 

identified a s alcoholics. An odds rati o was calculate d 

for eac h birt h defect t o indicat e the effec t o f alcohol 

consumption o n the ris k of developing that defect. 

To begin wit h the worst-cas e scenario, we should 

expect infant s o f Grou p 5 mother s t o sho w sever e 

deficits. I n adults this level of alcohol consumption i s 

associated wit h anoxia, the pathologica l deficienc y of 

oxygen, as well as renal failure an d sever e nutritional 

deficiencies. As a result, it would be expected that this 

level of exposure would cause a variety of deficits i n a 

developing organism . Amon g 8 7 infant s expose d t o 

this amount o f ethanol, 6 7 had a t least on e physica l 

defect. Thus, only 23% were normal at birth. Lookin g 

to th e nervou s system , however , 79 % o f the infant s 

showed no central nervou s system (CNS) defect s under 

standard infant neurological examinations. Given 

a level of alcohol exposur e known for its damaging effects 

on the physiology of adults, this is remarkable resilience. 

Becaus e th e neurologica l componen t of the 

suite of FASD-associated symptom s is not consistentl y 

detectable a t thi s age , facia l phenotype s alon e ar e 

used as a proxy for FASD (Astle y and Clarren , 1997) ; 

fully 60% of the infant s in Group 5 had a normal facial 

phenotype. Although 40% damage i s a tragic statistic, 

even i n this extreme cas e o f shockingly irresponsibl e 

alcohol consumption , mor e tha n one-hal f o f the in - 

fants show evidence o f being quit e normal . 


Normality dominate s th e res t o f th e groups . I n 

Group 4 (the second-highes t group), th e offsprin g of 

mothers wh o consumed betwee n 5 6 and 88 g ethanol 

per day, 95% of the infant s showed normal facial phe - 

notypes. According to the odds ratio, children of these 

mothers wer e twic e a s likel y t o develo p th e typica l 

FAS characteristics, but th e differenc e wa s not statistically 

significant . Onl y Grou p 3 , with lo w dail y con - 

sumption, showe d a statistically significant relationship 

between alcoho l consumptio n an d thi s phenotype . 

At this consumption level , 97% of the infant s showe d 

the norma l phenotype . I n th e sporadi c alcohol con - 

sumption group s ( 1 an d 2) , no t onl y wer e FASD - 

predictive anomalie s presen t i n onl y 3 % and 2 % of 

the infants , respectively , but the odds ratio s suggeste d 

that alcohol consumptio n decrease d th e risk of anomalies, 

although thes e difference s wer e not statistically 

significant. 

An important consideration i n this study, and gen - 

erally i n th e field , i s tha t o f socioeconomi c factors . 

Children of mothers of lower socioeconomic statu s are 

at greater risk , for a variety of reasons. These mother s 

are mor e likel y to be expose d t o other ris k factors , in - 

cluding lead (Malcoe et al., 2002; Morello-Frosch e t al., 

2002), chemica l solvent s (Cordie r e t al. , 1992) , an d 

illegal drug s (Hans , 1999) . Thes e ris k factor s ar e als o 

associated with cognitive impairments. Hence, it is difficult 

to separate alcohol fro m other potential causativ e 

factors. Additionally , eve n give n equivalen t alcoho l 

and dru g exposure , childre n o f higher socioeconomi c 

status ar e mor e likel y to b e normal . A substantial re - 

view o f neurobehaviora l studie s o f alcohol-expose d 

children (Mattson and Riley, 1998) confirms the prevalence 

o f low scores on cognitiv e tests in groups of low 

socioeconomic statu s (SES), but also includes startlin g 

examples of children who perform at normal levels and 

even above on cognitive tests (Streissgut h e t al, 1980 ; 

Fried an d Watkinson , 1988 , 1990 ; Frie d e t al., 1992) . 

Indeed, th e ter m low-risk group i s used synonymously 

with higher socioeconomic statu s in these studies. Notably, 

given low to moderate consumptio n and a high 

level of maternal education , intelligenc e quotien t an d 

other cognitive scores fall well within the normal range , 

and occasionally with mean scores a full standard deviation 

above normal. Although thes e score s were lower 

than thos e achieved by children in the same socioeconomic 

leve l without fetal alcoho l exposure , i t is clear 

that multipl e factor s i n th e developmenta l environ - 

ment beyond alcoho l consumptio n ar e at work in chil - 

dren who show substantial deficits .


Further, studie s o f th e effect s o f exposur e t o lo w 

(Forrest et al, 1991 ; Greene et al., 1991 ; Frie d e t al, 

1992) or even moderate (Man , 1980) amounts o f alcohol 

fail to find a cognitive deficit. Although thes e stud - 

ies focu s o n exposur e t o lowe r amount s o f ethanol , 

they ar e significan t i n ligh t o f conventional wisdo m 

that an y an d al l alcoho l consumptio n durin g preg - 

nancy is an unacceptable risk . Rather than being a silver 

bulle t capabl e o f derailing normal neurologica l 

development, alcoho l exposur e a t lo w level s has ef - 

fects that are hard to demonstrate, which makes sense 

in term s o f the long-ter m presenc e o f alcohol i n ou r 

ancestry. Even at pathologically high levels it does no t 

have 100 % penetrance . 

Nicotine 

Nicotine i s certainl y les s commo n tha n alcoho l i n 

our evolutionary history. Indeed, consumption o f any 

significant amoun t o f nicotin e i s a recen t develop - 

ment i n human history . The suit e of developmental 

effects cause d by smoking (the most typical way of using 

nicotine ) i s complex , involvin g respiratio n an d 

oxygen availabilit y directly, periphera l vasoactiv e ef - 

fects, and action o n central neurotransmitte r systems. 

Still, a numbe r o f it s effect s fal l withi n a well - 

developed evolutionar y context i n development, tha t 

of reduced resourc e availability. Although w e canno t 

review this context and it s effects full y here , i t is well 

established tha t th e developmenta l progra m show s 

significant flexibility with regard to nutrient availability. 

Within limits , growth ca n b e scale d bac k o r de - 

layed i n time s o f poor nutrition , favorin g the brai n 

whose generatio n i s largely prenatal, an d caugh t u p 

when condition s ar e favorable (Lucas and Campbell, 

2000). 

Tobacco exposur e restricts the availability of nutrients 

t o th e developin g fetu s (Lamber s an d Clark , 

1996). Carbo n monoxid e from tobacc o smok e binds 

with hemoglobin , th e oxygen-carryin g molecule i n 

blood, t o form a stable carboxyhemoglobin, limiting 

oxygen transport in the mother and thereby depriving 

the fetu s of oxygen a s well. Nicotine increase s hear t 

rate an d cause s vasoconstriction , increasin g bloo d 

pressure i n the mother , reducin g uterin e bloo d flow, 

and therefor e decreasin g a fetus's acces s t o al l nutri - 

ents. Hypoxia , malnutrition , an d th e ensuin g meta - 

bolic challenge s forc e th e fetu s t o mak e d o wit h 

inadequate resources. As such, it is not surprising that 

the mos t common effec t o f nicotine exposur e i s low 

birth weight (Ellard et al, 1996 ; Lamber s and Clark, 

1996). Lo w birth weigh t i s independently associate d 

with cognitiv e deficit s (Chaudhari e t al. , 2004 ; Corbett 

and Drewett, 2004; Viggedal e t al, 2004), regard - 

less o f th e causativ e agent . Indeed , a s a systemati c 

problem, i t is generally associated wit h growth deficits 

throughout th e body , includin g ostéopathi e effect s 

(Nelson et al., 1999), yielding a generally underdeveloped 

organism . A s such, i t i s difficul t t o determin e 

whether an y cognitiv e deficit s ca n b e directl y attributed 

t o nicotine or tobacco or if they secondarily stem 

from anoxi a and generally decreased resources. 

The direc t effect s o f nicotine o n neurologica l de - 

velopment ar e controversial, an d i n a way that is reminiscent 

o f th e comple x interaction s o f postnata l 

environment wit h prenatal malnutrition . A variety of 

studies indicat e n o cognitiv e deficit s i n infant s an d 

young children (Streissgut h et al., 1980 ; Forrest et al., 

1991), an d ther e i s some evidenc e o f a transitive effect, 

with scores on the Bayley Scales of Developmen t 

(BSID) that ar e low at 1 2 months comin g u p t o nor - 

mal level s b y 2 4 month s (Frie d an d Watkinson , 

1988). Ofte n test s tha t d o indicat e a n effec t i n chil - 

dren of heavy smokers fail to separate it from postna - 

tal exposur e (Richardso n e t al. , 1995) , whic h ha s 

more reliabl y been associate d wit h decrease d score s 

on cognitiv e test s (DiFranz a e t al. , 2004) . Eve n so , 

this effect o n BSID tes t scores is typically small when 

compared t o other factors , includin g number o f toys 

in th e household , SES , numbe r o f infan t illnesses , 

and even the identity of the examiner. Essentially, the 

data indicat e that on e woul d b e bette r of f spending 

the cigarett e money o n toys, as a deficiency of toys in 

the child' s hom e i s a mor e reliabl e predicto r o f decreased 

cognitiv e score s tha n nicotin e an d tobacc o 

exposure. 

Embryological Silver Bullets 

In contras t to our relativel y robust developmental de - 

fense agains t alcohol, for which we have evolved specific 

mechanism s dedicate d t o it s detoxification and 

metabolism, an d agains t nicotine, whic h ma y be subsumed 

unde r mechanism s alread y available to buffe r 

resource limitations, there stands a new class of potential 

teratogens : designe r drugs . These drug s are ofte n 

evolutionarily novel , base d o n molecule s neve r en - 

countered b y ou r ancestor s i n th e natura l environ - 

ment. O r the y ca n b e simila r to o r eve n base d o n 

molecules naturall y produced b y th e huma n body ,

potentially bypassing protective mechanisms, to ma - 

nipulate the developmental process directly. 

Though hardly typical of modern entrie s int o th e 

category, thalidomide i s certainly the mos t infamou s 

example o f a designer drug. Introduced i n 195 7 as a 

sedative an d anti-nause a agent , i t was considered s o 

safe a s to be regularl y prescribed to combat morning 

sickness an d insomni a i n pregnan t women . With - 

drawn fro m th e marke t i n 1961 , i n les s than 4 years 

thalidomide ha d cause d a worldwid e epidemic o f 

birth defects . Whil e n o accurat e censu s wa s eve r 

taken, it is estimated that between 10,00 0 and 20,00 0 

babies wer e born disable d a s a resul t o f thalidomid e 

(Knightley e t al. , 1979 ; se e http://www.thalidomid e 

.ca, http://www.marchofdimes.com/professional/681 _ 

1172.asp). Completely unknown is the rat e of embryonic 

rejectio n i n th e fac e o f thi s drug . Thu s i t i s 

difficult t o estimate the penetration o f the teratogeni c 

effects o f thalidomide. 

The method s o f action responsibl e for the terato - 

genic effect s o f thalidomide are stil l under investigation 

nearl y 5 0 year s afte r it s introduction . Indeed , 

study has increased drasticall y in recent year s as new 

applications fo r th e dru g hav e bee n foun d i n treat - 

ments fo r leprosy , lupus, rheumatoi d arthritis , an d 

other disorders. As a result, at least 30 different mech - 

anisms have been propose d for the teratogeni c effect s 

(Stephens an d Fillmore , 2000). Some hav e been discredited, 

other s are contradictory, whereas others can 

coexist handily. 

An affinity fo r DNA sequences rich in guanine has 

been established , suc h a s thos e i n th e GC-box , a 

hexanucleotide promote r i n th e huma n genom e 

(GGGCGG). Thalidomid e ca n inser t int o suc h se - 

quences, interferin g wit h thei r functio n (Jonsson , 

1972). Approximately 9% of the huma n genome uses 

G-rich promoter s to the exclusio n of other commo n 

promoters such as TATA and CCAA T boxe s (Bûcher, 

1990). Additionally , thalidomide ha s been linke d t o 

the fibroblast growth factor (FGF ) famil y of transcription 

factors . Wolper t (1976 , 1999 ) champion s th e 

idea tha t thalidomid e inhibits FGF-8, an initiato r of 

sonic hedgehog (shh). Stephen s (1988 ) implicate d i t 

in th e insulin-lik e growt h facto r (IGF ) I/FGF-2 / 

angiogenesis pathway . A follow-u p t o thi s wor k 

(Stephens and Fillmore, 2000) details the interrupted 

pathway and it s promoter sequences, effectively tying 

together th e leadin g theories o n thalidomid e embry - 

opathy to make a strong case for intersecting vulnera - 

bilities in certain systems. 


Within th e thalidomide-affecte d population , cer - 

tain trend s i n th e abnormalitie s are relevan t t o ou r 

discussion. Som e defect s are much more commo n i n 

children tha t survive d to birth. Lim b outgrowt h defi - 

ciencies are far and awa y the mos t common, presen t 

in approximatel y 90% of the group . Ea r an d ey e defects 

ar e th e secon d mos t commo n pathologies , af - 

fecting approximatel y 60% of the grou p (Yan g et al. , 

1977; Quibell, 1981 ; Lenz, 1985). 

In th e developin g limb , th e IGF-I/FGF-2/angi o 

genesis pathwa y i s necessar y fo r norma l outgrowth . 

Of 1 0 genes in the pathway, nine rely on GC-box pro - 

moters lacking TATA or CCAAT promoter sequences . 

This pathwa y i s particularl y vulnerabl e t o interca - 

lation b y thalidomide . A simila r situatio n exist s fo r 

the vascula r endothelial growth factor-integrin path - 

way, whic h ha s bee n implicate d i n deformitie s o f 

the ear . Regardles s o f which o r ho w man y o f these 

mechanisms ar e eventuall y implicate d i n develop - 

mental disorders , it i s notable that th e explanations , 

as a class , directly implicate developmenta l contro l 

mechanisms. 

FEATURES OF EVOLUTION 

AND DEVELOPMEN T THAT MAXIMIZE 

STABILITY 

The prio r example s sho w tha t w e have a phenomenon 

t o account for developmental stability of most infants 

i n th e fac e o f what would appea r t o b e sever e 

challenges, particularl y i n th e cas e o f "expected " 

challenges. Mos t of the other chapters i n this volum e 

give accounts o f abnormality; here we make some argument 

for forces for normality. 

A list of the feature s o f the genom e an d develop - 

ment that produce "evolvability " and the lis t of those 

that produc e stabl e solution s t o developmental chal - 

lenges ar e quit e similar . Bot h problem s hav e th e 

defining featur e o f producin g a functiona l conse - 

quence i n the even t of a deviation. In the cas e of evolution, 

the deviation to be assimilated comes from th e 

genome; i n th e developmenta l case s w e ar e dis - 

cussing here, the deviation is introduced from the environment. 

The principa l difference i s that evolution 

lacks the tactical nature of some developmental solutions. 

Evolutio n ha s n o plan s for the future , an d i n 

the genom e an d lif e historie s o f curren t organism s 

we see the direc t evidenc e o f what i s evolvable. De - 

velopment, however , mos t certainl y ha s intentio n


and tactics , a s they hav e bee n selecte d an d writte n 

into th e genome . No developmenta l progra m ha s as 

its end point a perfect, nonreproductive infant. Trade - 

offs an d fault s ca n b e gambled o n to produce a n im - 

perfect, reproductive adult. 

Duplicate and Vary 

An overal l quantitativ e feature of evolution i s that a s 

organisms becom e mor e complex , fro m prokaryote s 

to eukaryote s an d t o plant s an d animals , the y hav e 

more DN A an d mor e gene s (Lync h an d Conery , 

2003). That an increase in complexity should be partnered 

wit h an increase i n genes a t first seems reason - 

able on it s face, unti l the amoun t o f conservatism in 

basic metaboli c pathway s is confronted an d th e ac - 

tual histor y of genetic chang e i s investigated (Szath - 

mâry e t al., 2001) . I t i s not clea r tha t increasin g th e 

number o f gen e product s pe r s e shoul d necessarily 

lead t o fancie r animal s —if Shakespear e ha d ha d a 

few mor e letter s o f th e alphabe t t o wor k with , fe w 

would argue that the plays would thereby b e deeper. 

The historica l record of the genom e a s viewed in 

current animals does not show linear increases of genetic 

materia l consisten t with the notio n o f progressive 

additio n o f complexity . Rather , duplication s 

occur many times over, gene duplication followed by 

variation o f one o f the duplicate d genes, bot h a t th e 

level o f the individua l gene an d a t th e leve l o f th e 

whole genome , a s wel l a s a t variou s intermediat e 

steps (Lynch and Conery , 2000) . Below are a few examples. 

The trichomati c color vision of primates has 

arisen three separate times, by duplication of the "yel - 

low" opsin followed by one o r two minor amino aci d 

changes i n on e o f th e gene s producin g a sligh t 

change i n wavelengt h selectivit y (Jacobs, 1998) . A 

whole-genome duplicatio n occur s a t th e chordate - 

vertebrate boundary , implicate d particularl y i n th e 

subsequent elaboration of the cranium , jaw, and fore - 

brain (Northcutt, 1996). 

The regulator y gene s controllin g bod y plan , 

which ar e conserve d acros s vertebrate s an d inverte - 

brates, sho w evidenc e o f multipl e replication s i n 

evolutionary histor y (Gerhart an d Kirschner , 1997) . 

The us e o f this strategy is clear—one gen e ca n con - 

tinue it s essential roles , while a second ca n be fre e t o 

vary, assum e ne w functions , or b e produce d i n different 

contexts . Th e abilit y t o duplicat e an d var y 

"semantic" aspects of the genome—tha t is, aspects of 

the genome that relat e t o meaningful component s of 

the organism s —is a featur e tha t allow s evolvability , 

and has been employed t o advantage in "genetic algorithms" 

i n compute r scienc e (Lipso n an d Pollack , 

2000; Nolfi an d Floreano , 2002; Baum, 2004). 

Duplicate-and-vary b y it s natur e make s develop - 

ment responsiv e t o local geneti c accident . On e frus - 

trating aspect o f early genetic wor k (to geneticists) is 

that whe n i t becam e possibl e to "knock out " singl e 

genes (i n the cas e of nonlethal omissions) , the usua l 

effect wa s often nil, largely because of the massiv e re - 

dundancy o f gene copies . I t shoul d b e emphasize d 

that extr a o r slightl y altere d gen e copie s di d no t 

evolve i n anticipatio n o f futur e accident . Onc e i n 

place, however, creature s wit h the duplication s hav e 

had a developmenta l edge . Man y othe r version s of 

the duplicate-and-var y strateg y can b e see n i n addi - 

tion t o gene duplication an d variation . For example, 

the segmentai structure o f the vertebrate an d inverte - 

brate brai n an d bod y pla n i s anothe r versio n o f 

duplicate-and-vary, at a higher level of complexity. 

Large neura l structures , suc h a s th e corte x an d 

cerebellum, ma y als o b e case s o f duplicate-and-var y 

(or multiply-and-vary), regardless of whether the uni t 

duplicated i s a cel l assembly , a "cortica l column, " 

a region , o r a cortica l area . Cortica l area s hav e th e 

analogous strategi c feature s o f conserve d structur e 

throughout, suc h that cortical areas may be functionally 

substituted for each other , a s shown from myriad 

examples o f plasticity either earl y or lat e i n life , bu t 

particular cortica l areas, such a s visual or somatosensory 

cortex, appear to have functionally relevant local 

features "wire d in " (reviewe d in Kingsbur y an d Fin - 

lay, 2001 ; Pallas , 2001) . Finally , i t i s possibl e tha t 

some o f th e peculia r feature s o f ou r bilatera l bod y 

(and brain) plan migh t be placed in this context. Th e 

exact caloric requirement s o f each bod y part appea r 

to b e ver y tightly regulated . Fo r example , th e brai n 

size and gu t lengt h (tw o metabolically expensiv e or - 

gans) co-vary negatively with each other very precisely 

in primates with the result of keeping basal metabolic 

rate unchange d (Aiell o and Wheeler , 1995) . Ye t the 

amount of each organ we possess seems to be far from 

the bar e minimum required, or even the amount required 

fo r everyday function. Jared Diamon d calcu - 

lates from variou s successful surgeries in humans that 

we ca n ge t b y with on e kidney , half o f one lung , a 

third of a liver, half the cerebral cortex, and so on (Diamond, 

1994) . I t ha s no w bee n acknowledge d tha t 

creative use of brain lateralization, in the duplicate-and - 

vary version, can be found throughout th e invertebrat e

and vertebrat e lineage , i n frui t fly , fish , birds , an d 

many arthropods, and , of course, ourselve s (Vallorti - 

gara and Rogers , 2005) . Humans d o quite peculiarl y 

well with early, full-hemispheric deletions. 

Convergent Redundancy 

Frank duplication an d variatio n of identifiable devel - 

opmental mechanism s see m t o hav e occurre d nu - 

merous times , a s can b e see n i n th e man y isoform s 

and othe r variant s o f cel l recognitio n molecules , 

signaling molecules , an d trophi c molecules . A n absolute 

hallmar k of development, however , is redundancy 

o f a differen t type , wher e multipl e distinc t 

mechanisms ar e al l directe d towar d th e sam e outcome. 

Th e retinotecta l system , i n whic h th e twodimensional 

layou t o f cells i n th e retin a i s faithfull y 

transferred t o the two-dimensiona l surface o f the tecturn 

for the organization of eye and body movements, 

has served as a "model system " for the investigatio n of 

axon guidance, synaptogenesis, and topographic ma p 

formation sinc e Roge r Sperry first investigated i t half 

a centur y ag o (Sperry , 1963) . Thi s mode l syste m 

provides th e cleares t instanc e o f mechanisti c re - 

dundancy. Eac h propose d mechanis m ha s ha d it s 

researcher-champion, unti l th e realizatio n dawne d 

that evolution had taken no vow of mechanistic parsimony. 

There ar e a numbe r o f logica l possibilitie s o f 

mapping an arra y A-B-C-D ont o a secon d arra y 

A*-B*-C*-D*, eac h o f which alon e ca n solv e th e 

developmental puzzle . A could recogniz e A*; B recognize 

B*, and s o on, which is called "chemoaffinity, " 

Sperry's first hypothesis (Sperry, 1963). Alternative to 

attraction, A could b e repelled b y D*, and less by C* 

(Bonhoeffer and Huf , 1992) . Or, A might stic k to B 

better than to C, preserving intrinsic order in the fiber 

array, and then have some rule about how to position 

itself relative to A*-D* (Fraser , 1980) . Or , tempora l 

order i n developmen t migh t b e exploited , havin g A 

and A* mature first, then B and B* , and s o on (Reese, 

1996). Or , the element s i n both array s could b e produced 

i n excess, connected at random, an d error s removed 

b y some secon d rule , a s cell deat h i s a majo r 

component of brain development (Oppenheim, 1991). 

Or, th e ma p coul d self-organiz e by a Hebbia n rule , 

using the featur e that th e contras t level o f neighboring 

elements in the visual world is more highly correlated 

tha n distan t element s (Schmidt , 1985 ; Wong, 

1999). The answe r is, of course, that every single on e 


of these logically separate mechanisms cooperate s t o 

produce orderl y maps. During development, i t is possible 

to disable one or more of these mechanisms alto - 

gether, yet still preserve topographic map order, much 

as in gene knockout experiments, as residual mechanisms 

rescue the map. 

These two separate forms of redundancy, duplicateand-vary 

and convergen t redundancy , contribut e to a 

massively parallel , semimodula r architectur e o f de - 

velopmental mechanism s livin g beneath th e surface 

of a n organis m maturin g ove r time . Startin g fro m 

scratch t o buil d a comple x organism , one migh t b e 

predisposed t o a sequential , assembly-lin e mecha - 

nism, producing one part at a time, and connecting it 

up, bu t th e relativel y greater vulnerabilit y of such a 

system to loss of any component is obvious. It is interesting 

to observe the present-day parallel evolutio n of 

manufacturing systems from th e strictly serial Ford assembly 

line to current, multiple-component "just-intime" 

manufacturing systems. The fac t that evolution 

must tinke r with existin g systems rather tha n desig n 

from scratc h i s ofte n give n a s a n argumen t fo r th e 

unique, redundan t natur e o f biological construction , 

but i t is possible evolution ha s com e upo n solution s 

for efficien t biologica l constructio n tha t w e ma y no t 

yet be able to recognize. 

Catastrophe Curve s 

It will not do to make a large fraction of an organism : 

it is a waste of energy for the parents and certainl y no 

good fo r the offspring . Restated , partia l solutions ar e 

no goo d i n development—i f uncorrectabl e defect s 

are detected, n o more energy should be spent on the 

embryo to compound th e metabolic loss . For this reason, 

bailin g ou t i s perhap s th e mos t commo n re - 

sponse t o developmenta l defect . Fo r man y species , 

particularly "reselected" animals who are specialized 

for rapi d reproduction i n challengin g environments , 

embryos ma y b e resorbe d unti l lat e developmenta l 

stages i n respons e t o very small change s i n environmental 

stress , or the fetuses may be aborted an d cannibalized, 

a s any researche r attemptin g t o stud y th e 

etiology of developmental disorder s in rodents knows. 

Even fo r primates, wh o spen d muc h energ y o n th e 

growth and car e of one o r two infants, a large proportion 

o f conceptions ar e aborte d an d resorbe d in th e 

first trimester; th e commo n figur e give n i n medica l 

advisories about pregnancy is "up to 50%," although i t 

is har d t o locat e th e empirica l basi s o f thi s claim .


Even i f the numbe r i s much lower , the recognitio n 

of developmental disorde r and terminatio n o f development, 

particularl y i n earl y stages , ar e a s muc h 

an importan t face t o f developmen t a s constructiv e 

mechanisms. 

If a commitment t o the offsprin g i s unavoidable — 

for example , if it is the onl y chance a t reproduction , 

or a life-threatenin g metaboli c commitmen t ha s al - 

ready bee n made—the n ever y subsequent choic e t o 

preserve the offsprin g should be made a s is physically 

possible. Particularl y in shortag e situations , clearly 

strategic choices ar e made—for example , t o preserve 

the brai n of the infant , which i s generated onl y early 

in development, ove r and against the musculoskeletal 

system, whos e growt h ca n b e delayed, o r to favor th e 

infant's nutritiona l need s ove r th e calori c require - 

ments o f the mothe r (Marty n et al., 1996 ; Luca s an d 

Campbell, 2000) . One "difficulty " i n research on developmental 

disorders, which may not be entirely obvious 

to those working in well-established paradigms, 

is to be able to find just those regime s that lie on th e 

cusp betwee n th e plateau s o f embryoni c rejectio n 

and death, an d full normality . 

Analogous strategi c similaritie s can be seen in various 

developmenta l mechanism s i n th e nervou s sys - 

tem, fo r example , a certai n numbe r o f neurons , a 

certain convergenc e rati o between neurons , or a feature 

of physiology. Central mechanism s ca n gir d th e 

system against environmental stressors , but a t a critical 

value, this defense will suddenly and catastrophi - 

cally collapse. For example, if the number o f neurons 

in th e corte x is progressively depleted b y the mitoti c 

inhibitor methylazoxymethanol during early develop - 

ment, the thalamus projecting to that cortex will show 

no los s of neurons unti l mor e tha n 70%-80 % o f its 

target i s gone, an d the n collaps e itsel f (Wo o e t al. , 

1996). Simila r event s ma y b e a t pla y with th e anti - 

mitogen ethanol. Prenatal exposur e to ethanol causes 

a

Neurogenesis and the Process 

of Cell Type Specification 

Neurogenesis comprise s th e productio n o f neuron s 

and gli a tha t wil l reside i n a structure; this i s analogous 

to organogenesis, which is the production of cells 

that wil l constitut e a n organ . Thus, neurogenesis includes 

the proliferatio n o f neural precursors, the fat e 

decision, th e migration , differentiation , an d some - 

times deat h o f th e cells . Mos t neurona l precursor 

cells are generated in the proliferativ e zones that line 

the inne r surface o f the neura l tube (se e Chapters 2, 

11, and 12) . In thes e zones , precursor cells undergo 

mitosis, wit h "symmetric" divisions, producing more 

precursors. Eventually, some precursors begin "asymmetric" 

divisions , with one daughte r cel l exitin g th e 

cell cycl e and the proliferativ e zone, and then begin - 

ning the transformatio n into particular types of neurons 

o r gli a (Cavines s e t al. , 1995 ; Ohnum a an d 

Harris, 2003). The origina l size of the progenitor pool 

combined wit h th e duratio n o f neurogenesis o f any 

given par t o f the brai n predict s it s size (Finla y an d 

Darlington, 1995 ; Finlay et al, 2001). 

Not every precursor cel l i n the nervou s system differentiates. 

In some regions of the brain, stem cells remain 

undifferentiate d throughou t th e lif e o f th e 

animal, and recent work suggests that these cell s can 

be presse d int o service to effec t repair s for damaged 

neural circuit s even i n the adul t brain (e.g. , Alvarez- 

Buylla and Garcia-Verdugo, 2002; Gage, 2002; Picard- 

Riera e t al. , 2004) . That is , neuronal ste m cell s may 

provide a substrat e fo r compensator y plasticit y i n 

some systems of the brains of adults. 

We concentrat e her e o n mechanism s tha t con - 

trol th e assignmen t o f cel l identity , with particular 

attention pai d to how the proces s may regulate itself 

if disturbance s occur . Researcher s firs t frame d th e 

question o f cell specificatio n in term s o f where cel l 

specification occurs . Conceivably , thi s decision i s (a) 

intrinsic, suc h tha t specifi c precurso r population s 

give ris e t o particula r subpopulation s o f cell s only ; 

(b) determine d b y a cloc k producin g a certai n cel l 

type after a required number of precursor cell subdivisions 

or with regard to some externally specified stage; 

or (c) extrinsic, defined b y the externa l milieu of the 

precursor cells , i n eithe r th e proliferativ e zone s o r 

their eventual destination. The answe r that ma y vary 

from on e brai n regio n t o anothe r i s likely th e usua l 

developmental answer: all of the above. We give a few 

specific examples , an d conside r th e implication s of 


hybrid mechanism s fo r producing norma l structure s 

in response t o challenge. 

In vivo and i n vitro experiments demonstrat e tha t 

the order of neurogenesis and cell type are correlated . 

For example, i n th e vertebrat e nervou s system, mos t 

glia ar e produce d afte r th e completio n o f neurona l 

generation (Ohnuma and Harris, 2003). In the retina, 

the si x types of cells are produced i n order, although 

in al l cases , th e productio n o f cel l type s overlap s 

(Policy e t al, 1989) . Evidenc e suggest s that a com - 

bination of clock-like initiation of specification o f particular 

cel l type s an d feedbac k processes tha t asses s 

how many cells of a class have been produced . On e 

of these, p27 Ki P 1 , a cel l cycl e inhibitor, gradually increases 

in progenitors until a critical level is reached, 

whereupon cell s designate d a s oligodendrocytes exit 

the cel l cycle and differentiat e (Freeman , 2000 ; Dyer 

and Cepko , 2001 ; Ohnuma and Harris, 2003). Thus, 

the gradua l accrual o f p27 Kl P 1 i s a negativ e feedbac k 

mechanism tha t tells precursor cells when to start differentiating 

into glia as opposed to neurons . 

Injury i s capabl e o f resettin g th e neurogeneti c 

mechanism. I n frog s an d fishes, for example, Mulle r 

glia ca n re-ente r th e cel l cycl e i n respons e t o retinal 

injury, an d ca n produc e progen y tha t differentiat e 

into neurons (Reh and Levine, 1998) . In mature glia, 

p2yKipi j s expresse d a t high levels . Following retinal 

injury, however , p27 Klpl i s down-regulate d i n cells 

that re-ente r th e cel l cycle , allowing for the genera - 

tion o f ne w neurons . Regulatio n o f specificatio n of 

cell classe s has als o been show n t o occu r a t a mor e 

detailed level—fo r example , i f the retin a is depleted 

of a particular class of amacrine cells while retinogenesis 

is ongoing, the subsequent production of that cell 

type is up-regulated (Reh, 1987) . 

Negative feedback control of the rat e of histogenesis 

provides flexibility and robustnes s in th e develop - 

ing organis m relativ e t o mor e rigi d developmenta l 

schemes tha t migh t plausibl y hav e evolved . Fo r in - 

stance, suppos e tha t onl y gli a are produced o n a set 

day following the onset of neuronogenesis, or that glia 

are produce d onl y afte r a certain numbe r o f cell cy - 

cles wit h n o feedbac k regulation . I n eithe r o f these 

cases, if anything interferes with cel l productio n at a 

certain tim e point , a n essentia l cel l grou p migh t 

never be generated. On the other hand, if progression 

through cel l classe s is regulated only by feedback, in 

the cas e o f lo w resourc e availability , neurogenesis 

could stal l at an earl y point an d neve r produce late - 

generated cel l groups . Intrinsi c clocks , overlapping


distributions of cell production, an d feedbac k regulation 

of the amoun t of each cel l typ e in concer t together 

virtually guarantee that some members of each 

cell clas s are generated , an d thei r norma l ratio s defended. 

Cell Migratio n 

The migratio n of developing neurons to their adult positions 

in the brain is a critical step in the developmen t 

of the nervou s system. Several distinc t developmenta l 

mechanisms, such a s radial an d tangentia l migration 

and cellular adhesion molecules, are thought to govern 

this proces s (Rosenwei g et al, 1999 ; Corbi n e t al, 

2001). Thi s i s a proces s that ca n g o wrong, and "ec - 

topia," cel l group s lying elsewher e than thei r normal 

terminal site, is one o f the principal morphological deviations 

associate d wit h retardatio n (Evrar d e t al. , 

1989a, 1989b) . Even i f migration abnormalitie s resul t 

in misplaced cells, however, all is not lost. 

One o f the mos t striking demonstrations o f developmental 

robustnes s comes fro m on e mode l syste m 

of perturbed cell migration, in reeler mice. Reelin is a 

protein secreted by several neuronal populations during 

development, and i s vital for allowing neurons to 

complete migratio n an d adop t thei r ultimat e posi - 

tions i n a number o f laminar structures in th e CN S 

(Rakic and Caviness , 1995 ; Ric e and Curran , 2001) . 

Mice lackin g reeli n (reeler mice , so named fo r thei r 

staggering gait , whic h result s primaril y from a cere - 

bellar anomaly ) show abnormal pattern s o f cerebra l 

cortical lamination. The arrangemen t of the six layers 

of the cerebra l cortex is inverted, so that th e earliest - 

generated cells form th e most superficial layer s of the 

cortex and late-generate d neurons tend t o be distributed 

i n dee p cortex . Despite thi s perturbation, affer - 

ent projections to the visual, somatosensory, olfactory, 

and moto r cortice s fin d thei r correc t targe t cells . I n 

addition, th e overal l organizatio n o f majo r system s 

and the physiological response s of individual neurons 

in reeler mic e ar e comparable t o those in the norma l 

brain. Reeler mice have other anatomical abnormalities 

relative to wild-type mice. These include change s 

in synaptic density, distribution, and topology that are 

present in a number of brain structures including the 

hippocampus, piriform cortex , and cerebellum . Nev - 

ertheless, althoug h morpholog y i s grossly abnormal, 

function i s spared. This result suggests caution i n any 

assumption tha t disturbe d morpholog y ha s negativ e 

functional consequences . 

Reeler mic e provid e a n additiona l demonstratio n 

of the robustnes s of the "duplicate-and-vary " strategy. 

Only mic e homozygou s fo r the mutan t reelin allele 

show the neuronal abnormalities listed above. Studies 

of mice heterozygous for reelin have neuronal and be - 

havioral phenotype s virtuall y indistinguishabl e fro m 

those of wild-type mice (Salinger et al., 2003). 

Maturation o f Cell Types during 

Later Development 

The maturatio n o f neuronal morpholog y provides an 

example o f convergen t redundancy . I n vitr o experi - 

ments sho w tha t granul e an d Purkinj e cell s isolate d 

from thei r normal connections gro w in a typical manner 

(Sei l et al, 1974) . This finding implies that signals 

intrinsic to cells can regulate aspects of neuronal differ - 

entiation. The neura l environment of developing cells, 

however, also influences cell morphology and connectivity. 

For instance, som e spinal cells are directed t o become 

moto r neuron s unde r th e influenc e o f cell s 

ventral to the notochor d (Roelin k et al., 1994) . I f a second 

lengt h o f notochord i s inserted abov e th e spina l 

cord, cells begin to differentiate a s motor neurons on either 

side of the notochord. Cell-cell interactions of this 

sort, in which cells influence the fate s o f adjacent precursor 

cells, are referred t o as induction, and have been 

documented extensivel y in the vertebrate brain (Rosenweig 

et al., 1999). The abilit y of extrinsic cell-cell interactions 

to regulate cel l fate is not restricted to precursor 

cells. Cortical transplan t studie s consistentl y sho w that 

sensory and motor cortex cells grafted ont o distant cortical 

region s ca n assum e morphological , connectional , 

and functional properties appropriate to that region. For 

instance, fetal visua l cortex of rats transplanted into rat 

neonatal somatosensor y cortex develops the barrel-like 

whisker representation s characteristi c o f primar y so - 

matosensory cortex (Schlaggar and O'Leary, 1991), and 

the expressio n o f the limbi c system-associate d mem - 

brane protei n tha t mark s specific functional region s of 

the cerebra l corte x can be regulated by environmental 

stimuli (Levitt et al, 1997) . 

Patterned sensor y activity can alter cell fate. Cross - 

modal rewirin g o f retina l axon s int o ferre t auditor y 

thalamus provides visually patterned activity to the au - 

ditory cortex (Pallas et al., 1999). Primary auditory cortex 

provided with earl y visual input resemble s visual 

cortex topographicall y (Ro e e t al. , 1990) , physiologi - 

cally (Roe et al., 1992) , and perceptually (vo n Melchner 

e t al. , 2000) . Neuron s i n thi s altere d auditor y

cortex no t onl y assum e th e functiona l propertie s an d 

arrangement o f neurons i n th e visua l corte x but als o 

are capabl e o f mediatin g visuall y guide d behavio r 

(von Melchner et al, 2000). 

Hence, intrinsi c factors suc h a s gen e expressio n 

and extrinsi c factor s suc h a s induction , neuron - 

neuron interactions , and patterne d activit y are all capable 

of regulating cell fate at various (and sometimes 

overlapping) point s i n development . No t al l of thes e 

mechanisms ar e necessaril y invoke d o r eve n neces - 

sary during the production o f any given neuron . 

Postnatal Development and 

Critical Period s 

Critical periods ar e tim e window s i n postnata l de - 

velopment durin g whic h neuron s an d circuitr y ar e 

particularly receptive to acquiring certain kinds of information 

critica l fo r normal developmen t (Hensch , 

2004). Critica l period s hav e bee n extensivel y doc - 

umented fo r sensor y systems , moto r systems , an d 

multimodal function s suc h a s imprinting , birdson g 

learning, soun d localization , an d huma n languag e 

learning. How are critical periods useful fo r ensuring 

normal development, when i t seems that the unlucky 

absence of a particular experience at a particular time 

might permanently derail normal development? Th e 

answer i s that the critical-perio d onset , duration, and 

termination are not regulated simpl y by age, but rathe r 

by experience. I f appropriate neura l activatio n i s not 

provided at all, then developing circuit s often remai n 

in a waiting state until such input is available. For instance, 

the segregation of ocular dominance column s 

depends o n visual experience. If all visual experience 

is denied, the representatio n of the tw o eyes does no t 

begin it s segregation an d th e specia l neurotransmit - 

ters an d receptor s that ar e responsibl e fo r this structural 

chang e ar e hel d i n their initia l state (Kirkwoo d 

et al., 1995) . Within certai n constraints , whe n experi - 

ence i s reinstated, anatomical , pharmacological , an d 

physiological event s then progress as they would have 

independent of the ag e of the animals. A similar phenomenon 

ha s als o been observe d i n bird s that learn 

their songs from tutors—fo r the unfortunate nestlings 

born to o late i n th e seaso n t o hear an y of the spring 

songs that establish territory, the critical period is held 

over unti l nex t spring , whe n singin g begin s agai n 

(Doupe an d Kuhl , 1999) . Critica l period s hav e vul - 

nerabilities. If activation i s only partial, i t may initiate 

the progressio n o f the critica l period , stabilizin g a n 


abnormal state , as has been seen i n the production of 

ocular dominanc e columns : i f both eye s ar e closed , 

the critica l period i s delayed. O n th e othe r hand , if 

just on e ey e i s closed , th e critica l period proceeds , 

dedicating al l resource s t o th e ope n ey e (Kat z an d 

Shatz, 1996) . 

ETHANOL AND THE DEVELOPING 

NERVOUS SYSTEM: 

EVOLUTIONARY CONSIDERATIONS 

Here we look at a few of the cases in which the effect s of 

ethanol o n developin g system s have been investigated 

and consider them in the context of self-regulation. The 

effects o f ethanol o n th e nervou s system—at any stage 

of development—are known to be numerou s an d varied 

(se e Chapter s 11-18) . Fo r instance , ethano l ca n 

both inhibi t an d stimulat e neuro n proliferation , de - 

pending o n th e physica l locatio n o f the cells , an d th e 

concentration an d eve n timin g (da y or night) o f expo - 

sure (Luo and Miller, 1998; see Chapter 11) . 

Given tha t the effect s o f ethanol o n th e develop - 

ing nervous system are numerous, complex, and con - 

tingent upo n man y factors , ca n w e b e sur e tha t al l 

these effect s ar e malignant? To be sure, some ar e un - 

ambiguously deleterious . Ethano l ca n induc e neu - 

ronal deat h i n vitro and i n vivo, oxidative stress, an d 

excitotoxicity (Chapter s 1 5 an d 16) ; interfer e wit h 

glucose transpor t an d uptake; an d reduce th e expres - 

sion an d adhesiv e propertie s o f cell adhesio n mole - 

cules (Chapter 13) . Other effects of ethanol, however, 

may no t necessaril y be maladaptive . Fo r instance, i n 

addition t o causin g neurona l deat h i n developin g 

cerebellum (L i et al. , 2002 ) and corte x (Jacob s an d 

Miller, 2001) , ethano l ca n slo w th e productio n o f 

developing cerebella r (L i e t al. , 2002 ) an d cortica l 

(Miller an d Nowakowski , 1991 ; Miller , 2003 ) neu - 

rons, and it can increase th e time during which post - 

mitotic cortica l neuron s remai n i n th e proliferativ e 

zone before commencin g thei r migratio n whil e als o 

decreasing th e rat e o f neurona l migratio n (Miller , 

1993). Give n tha t developmen t i s dynamically regulated 

an d sensitiv e t o environmenta l cues , i t i s no t 

implausible that these delays might be an adaptive response 

to a hostile developmenta l environmen t (hig h 

concentrations o f ethanol) i n whic h cel l productio n 

and migratio n ar e retarded unti l a more favorable environment 

becomes available. This interpretation gen - 

erates testable predictions . I f cell-cycle an d migratio n


delays are a respons e t o a hostile environment , then 

we should expec t t o se e a restoration o f these activi - 

ties to normal (o r even above-normal ) rate s once th e 

normal developmenta l environmen t i s restored . I n 

practice, we might expec t t o see resumption o f neu - 

ron proliferation and migration after transient exposur e 

to ethanol (afte r the restoration of normal cellular environment). 

Certainly , researc h ha s demonstrate d tha t 

the nervous system can and does recover from ethanol - 

related developmenta l insult s (Ander s and Persaud , 

1980; Riley, 1990 ; Popova, 1997 ) but the precise mechanisms 

mediatin g recover y are poorl y characterized. 

We suggest that some of the observed ethanol-induced 

changes in the developing nervous system may not be 

disorders, but remnant s o f processes tha t hav e medi - 

ated recovery. 

SUMMARY AND CONCLUSIONS 

Development ca n go wrong. One nee d onl y consider 

the appearanc e o f a poorly placed o r underfertilize d 

garden plan t compare d t o it s well-nourished siblings 

to counteract th e ros y glow that this chapter, wit h its 

relentless underscorin g o f mechanism s designe d fo r 

developmental stability , may have produced . Never - 

theless, ther e ar e man y reason s fo r taking a vie w of 

pathology that begins with evolution, rather than with 

human developmen t i n isolation. 

First, althoug h du e cautio n shoul d b e employe d 

in advisin g pregnan t mothers , communicatin g th e 

wild improbabilit y o f gros s developmenta l disorder s 

from tin y infractions in alcoho l consumptio n durin g 

pregnancy would relieve unnecessary anxiety. 

Second, animal "models " should b e examine d fo r 

their appropriateness t o understanding huma n pathol - 

ogy. Vertebrate nervous systems are quite conservativ e 

in structure; curren t wor k comparing th e genomes of 

different specie s suggest s that their defensive packaging 

is quite different, depending on such factors as "r" 

and "k " selection an d the dietary and pathogen expo - 

sure i n th e evolutionar y history of the species . Th e 

kind of modeling of development we do must change 

and is , in fact, in the proces s of change—from th e assumption 

of linear dose-response curve s to pathogens 

and teratogens , t o multicomponen t chaoti c system s 

in whic h "normality " i s the stronges t attractor, o r i n 

which severa l attractors, each representin g a developmentally 

stable strategy, may exist. 

Finally, we need to establish bette r causa l links in 

developmental model s tha t g o fro m fundamenta l 

physiology to morphology t o behavior, i n order to discriminate 

pathologica l chang e fro m compensator y 

change and from simple deviation. Evolutionar y mod - 

els ca n provid e scaffoldin g fo r suc h understandin g 

that pathology-based researc h can never provide . 

Abbreviations 

BSID Bayle y Scales of Developmen t 


FASD feta l alcohol spectru m disorde r 


IGF insulin-lik e growth factor 

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II 

ETHANOL-AFFECTED 

DEVELOPMENT


8 

Prenatal Alcohol Exposure 

and Human Development 

Although a relationshi p betwee n materna l drinkin g 

and reproductiv e problem s had bee n suspecte d lon g 

ago (Warner and Rosett , 1975 ; Abel, 1984) , it was the 

description o f fetal alcoho l syndrom e (FAS ) in 197 3 

(Jones and Smith, 1973 ; Jones et al, 1973 ) that initi - 

ated comprehensiv e studie s of the effect s o f prenatal 

alcohol exposur e on human development . Researc h 

over the las t 3 0 years ha s confirmed the origina l observations 

that affected childre n sho w growth retardation; 

birth defects , particularly a characteristic facia l 

dysmorphia; and behaviora l deficits indicative of central 

nervous system (CNS) damage (Fig . 8.1). Animal 

models as well as epidemiological and clinical studies 

in humans hav e provided evidence o f the effect s o n 

both physica l an d behaviora l characteristic s o f ex - 

posed offspring , bu t th e CN S effect s an d thei r per - 

sonal and social consequences have received the most 

study in human samples . There have been a number 

of comprehensive reviews of the result s of prenatal alcohol 

exposure on development, particularl y in earl y 

childhood (e.g. , Coles , 1992 , Stratto n e t al. , 1996 ; 

Claire D. Coles 

123 

Streissguth, 1997 ; Mattso n an d Riley , 1998 ; U S De - 

partment o f Health and Huma n Services , 2000). Recently, 

publication s i n thi s are a hav e increase d s o 

much that it is now impossible to review the entire literature 

in a single chapter. For that reason, this chapter 

refer s briefl y t o earlier findings and deal s in mor e 

detail with recent studies. 

A decad e ago , th e followin g area s wer e amon g 

those tha t seeme d t o hav e th e mos t researc h poten - 

tial: threshold effects , neuroimaging , long-term development 

i n affected individuals , specific versus global 

effects, an d wha t i s now calle d "translation " amon g 

the different discipline s studying this problem (Coles , 

1992). These areas have remained active. In addition, 

several other issues have come to the fore. With better 

diagnosis and identificatio n of affected individual s by 

professionals and increasin g demand fo r services and 

resources by families, th e nee d fo r improving education 

an d remediatio n o f affecte d individual s has be - 

come evident . Also , a s clinica l an d longitudina l 

research cohort s hav e matured , differen t area s o f

124 ETHANOL-AFFECTE D DEVELOPMENT 

FIGURE 8- 1 Rang e o f effects o f prenatal alcohol exposure o n th e centra l ner - 

vous system. 

behavior hav e becom e salient . Fo r instance , socia l 

and adaptiv e function s hav e bee n identifie d a s im - 

paired i n individual s with FA S (Kell y e t al. , 2001) , 

and patient s fro m clinica l sample s ar e reporte d t o 

have a hig h frequenc y o f lega l an d menta l healt h 

problems (Streissgut h et al, 1996 ; Fam y et al, 1998 , 

Fast an d Conroy , 2004) . Thes e issue s are discusse d 

below, in addition to recent researc h o n neurodevel - 

opmental outcomes in affected individuals. 

METHODOLOGICAL CONSIDERATIONS 

Despite 3 0 year s o f study , ther e remai n significan t 

disagreements i n the literatur e and among th e pub - 

lic and man y professionals about the effect s o f prenatal 

alcohol exposure . Some of these discrepancies can 

be accounte d fo r b y difference s i n methodologie s 

used to study this phenomenon. Before undertaking a 

review of the researc h literatur e i n thi s area, i t is important 

t o understan d ho w methodolog y affect s out - 

comes an d t o acknowledg e tha t thes e issue s are no t 

specific to this field. 

It i s obviousl y impossible , ethicall y an d practi - 

cally, to carry out tru e experimental studie s of the effects 

o f prenatal alcohol exposur e on human growth , 

behavior, and cognition. For those aspects of development 

tha t cannot b e modele d i n animal studies , research 

mus t b e don e i n eithe r clinica l sample s o f 

already affected individual s or i n cohort s of offsprin g 

of wome n wh o drin k durin g pregnancy . Eithe r ap - 

proach ha s rea l value and rea l difficulties . A s experimental 

contro l o f the "independent " variable i s no t 

possible, suc h studie s mus t b e correlationa l an d de - 

scriptive. In addition, it is usually not possible to con - 

trol completely the confounders or effect modifier s in 

such situations . When conductin g studie s with sam - 

ples o f individual s drawn fro m clinica l settings , re - 

searchers mus t tak e int o accoun t systemati c biase s 

and confounders . Childre n diagnose d wit h FA S or 

other effect s o f prenatal alcohol exposur e come to the 

attention o f professionals because o f problems i n be - 

havior or development. Thus, it is inappropriate to use 

such sample s to evaluate the relationshi p between alcohol 

exposure and these particular outcomes. Nevertheless, 

stud y o f suc h childre n ma y b e valuabl e fo r 

other research purposes . Also, many clinically identified 

alcohol-affecte d childre n hav e negativ e earl y 

caregiving histories resulting from materna l substanc e 

abuse, out-of-hom e placement , and othe r caregiving 

failures, and researc h wit h suc h group s must include 

similar contrast group s to avoid confusing the effect s 

of pre- and postnata l environment s on the outcome s 

of interest. 

In addition to being very expensive and time con - 

suming, exposur e samples also have methodological 

limitations. Wome n wh o drin k durin g pregnancy 

usually also smoke tobacco and ma y use other drugs 

(Day e t al , 1993 ) (se e Chapte r 19) , introducin g 

other confounders into the research. They may have

a genetic predispositio n to alcoholism and associated 

mental health problems (Zucke r et al., 1995) , have a 

family histor y characterized by substance us e an d it s 

social correlates, and be selected fro m a low socioeconomic 

status (SES) environment, a s such women are 

more likely to be availabl e for recruitment int o exposure 

studies. Another limitation o f the use of exposure 

samples i s tha t mos t sampl e population s ar e com - 

posed o f wome n drinkin g i n th e lo w t o moderat e 

range (e.g. , Day et al, 1993 ; Jacobso n e t al, 1998) . 

Although i t is valuable to have information about th e 

effects o f lower amounts of ethanol exposure. The rel - 

ative absence o f exposure studies of infants of heavier 

drinkers i s due i n som e par t t o th e difficult y i n re - 

cruiting such women . 

For these reasons , when tryin g to understand th e 

effect o f alcohol exposur e on offsprin g development , 

it i s important to take into consideration th e charac - 

teristics of the researc h sample an d th e resultan t implications. 

An y singl e stud y ma y no t b e sufficien t 

to understan d thes e teratogeni c effects , wherea s th e 

body of literature as a whole can provide considerable 

insight. 

THRESHOLDS AN D 

DOSE-RESPONSE EFFECTS OF 

PRENATAL ALCOHOL EXPOSUR E 

There is a great deal o f interest i n the questio n o f the 

"dose" o f alcoho l necessar y t o caus e negativ e out - 

comes. Fo r public health reasons, it would be valuable 

to know if there i s a "safe" level of prenatal exposure. 

This is a complicated question . Some researchers posit 

that the construc t of threshold i s not usefu l becaus e it 

oversimplifies the rea l relationship between the teratogen 

an d th e multipl e outcome s tha t ar e measure d 

(Sampson et al., 2000). Sampson and colleagues argue 

in their review that there are no meaningful differences 

in behavior, standard scores, academic outcomes, an d 

behavior betwee n thos e individual s labeled a s having 

FAS and thos e characterized as having alcohol-related 

neurodevelopment disorder s (ARND) . The y believ e 

that behavioral outcome doe s not vary with variation in 

physical teratogeni c effects . I n addition , thes e re - 

searchers present evidence from thei r large prospectiv e 

exposure stud y suggestin g tha t th e relationshi p be - 

tween prenatal exposur e and neurodevelopmental outcomes 

i s monotonie, o r without meaningfu l threshol d 

PRENATAL ALCOHOL EXPOSURE AND HUMAN DEVELOPMEN T 12 5 

effects "whe n dos e an d behaviora l effect s ar e quanti - 

fied appropriately" (p. 421). 

Other investigator s have identifie d threshol d ef - 

fects in their samples. Jacobson and colleagues (1998) 

found tha t aspects of infant cognition sho w threshol d 

effects. A t 2 6 month s i n thei r cohort , psychomoto r 

functioning an d aspect s o f fin e moto r functionin g 

are affecte d onl y in th e offsprin g o f heavier drinker s 

(Kaplin-Estrin e t al, 1999) , althoug h non e o f these 

children exhibi t physica l dysmorphia . I n anothe r 

sample, Da y and colleague s (1991 , 1999 , 2002 ) show 

that growt h i s affecte d i n a dose—respons e fashion . 

Growth i s more affecte d i n individual s whose moth - 

ers drank most heavily (>1 drink/day); they average 14 

pounds les s tha n controls . Simila r relationship s ar e 

evident fo r cognitio n an d achievement . Whe n thi s 

cohort was 6 years old, a threshold o f exposure to on e 

drink dail y i s foun d fo r academi c achievemen t i n 

reading and spelling, whereas effects o n mathematic s 

skills are linear (Goldschmidt et al., 1996) . 

In addition, as discussed below, the morphologica l 

outcomes characteristi c of prenatal alcoho l exposur e 

appear t o b e affecte d i n a dose-respons e manne r 

(Lynch e t al. , 2004) . All of these report s sugges t that 

heavier drinkin g i s associate d wit h greate r physica l 

and neurodevelopmental effects . The exten t to which 

results can b e described a s monotonie or reflective of 

"thresholds" in alcoho l us e may depend o n th e typ e 

of sample s recruite d an d th e amoun t o f exposure i n 

the sampl e a s wel l a s b y th e method s use d i n th e 

study and in the data analysis. 

PHYSICAL EFFECTS OF PRENATAL 

ALCOHOL EXPOSUR E 

Facial Dysmorphi a 

The facia l dysmorphi a associate d wit h heav y expo - 

sure is one o f the three diagnostic features used to define 

FAS. These features include midface hypoplasia 

(anteverted nares) , shor t palpebra i fissures, and flattened 

o r indistinc t philtrtim, usually associated with 

a thi n o r flattene d uppe r vermillio n border . Othe r 

minor anomalie s als o ar e commonl y foun d i n af - 

fected children , including epicanthal folds, low nasal 

bridge, ea r anomalies (lo w set and rotated) , and mi - 

crognathia. The craniofacia l anomalie s ar e believe d 

to be the resul t of disturbances o f cell migration dur -


ing organogenesi s (U S Departmen t o f Healt h an d 

Human Services , 2000; see Chapter 17) . In practice, 

assessment of these anomalies is complicated b y variance 

associate d wit h ag e an d ethnicity . Whe n af - 

fected individual s are followe d ove r childhoo d int o 

early adulthood, som e o f the individua l features tha t 

are salien t durin g infanc y and earl y childhoo d be - 

come harde r t o identify , particularl y afte r pubert y 

(Streissguth, 1997) . 

Several different "systems " of diagnosis are use d i n 

clinical diagnosi s an d researc h o n FAS . Astle y an d 

Clarren (2001 ) have propose d a syste m tha t focuse s 

on "sentine l features, " whic h the y hav e identifie d 

through analysi s of a large sample o f children wh o applied 

for diagnostic services. In this large clinical sample, 

severa l facia l feature s hav e bee n foun d t o b e 

associated with the diagnosi s at each stag e of development. 

Thes e feature s ar e (1 ) a n absen t o r indistinc t 

philthrum, (2 ) a thinne d uppe r vermillion , an d 

(3) shortene d palpebra i fissures . I n thei r four-digi t 

code metho d for diagnosis, these facia l features constitute 

on e dimensio n necessar y fo r diagnosi s (Astley , 

2004). Othe r dimension s ar e growt h (les s than thir d 

percentile), materna l alcoho l use , an d evidenc e o f 

CNS involvement. 

The Collaborativ e Initiativ e o n Feta l Alcoho l 

Spectrum Disorders , sponsored b y the Nationa l Insti - 

tute o n Alcoholis m an d Alcoho l Abuse , ha s devel - 

oped a Dysmorphia Core Physica l Exam that i s used 

in international studies to diagnosis participants of all 

ages and ethnicities (Ma y et al, 2000; Hoyme e t al, 

2005). Thi s method , develope d b y Jone s an d col - 

leagues (1973) , i s based o n th e origina l description s 

of FAS and i s a modification o f the criteri a offered b y 

the Institute of Medicine (Stratto n et al, 1996) . As in 

other systems , th e chil d i s measured an d examine d 

for physica l an d facia l anomalies , a s well a s height , 

weight, head circumference , and heart and neurolog - 

ical problems . The n a determinatio n i s made a s t o 

whether th e chil d ca n b e rate d a s (1 ) havin g FAS , 

(2) not having FAS, or (3) deferred. The classification 

"deferred" indicate s tha t som e characteristic s ar e 

present bu t tha t the diagnosi s cannot b e mad e with - 

out mor e information (e.g., neurodevelopmental tes t 

results). Thi s methodolog y ha s bee n use d i n Russi a 

and Sout h Afric a as well as the Unite d State s (Hoym e 

et al, 2005). 

Coles an d colleague s (Fernhoff e t al., 1980 ; Black - 

ston e t al , 2004 ; Lync h e t al. , 2004 ) hav e use d a 

slightly differen t methodology . I n a longitudinall y 

followed exposur e sample report , a dose-response pattern 

between alcohol exposur e and physical characteristics 

can be identified both in the neonatal perio d and 

later i n development. Th e sam e dysmorphi c features, 

however, ar e no t alway s salien t ove r development . 

Rather, it is the total "dysmorphia score" that is reliable 

from infanc y through middle adolescence. Mor e longitudinal 

research is needed t o determine th e continuity 

of physica l characteristics , particularl y facia l anom - 

alies, to provide guidelines for making the diagnosis in 

older childre n and adults . This kind of research i s important 

als o because there may be ethnic differences in 

facial characteristics that can affect diagnosis. Finally, it 

is necessar y t o us e a longitudina l approac h becaus e 

use o f clinical sample s in a cohort desig n exaggerates 

the salienc e o f feature s tha t ar e expecte d t o b e par t 

of th e criteri a an d ignore s individuals whose features 

are not consistent with these expectations . 

Effect of Alcohol Exposur e 

on Growt h 

Growth retardatio n is useful fo r the identificatio n of 

FAS during infanc y an d th e preschoo l period , but i t 

is les s consistentl y eviden t i n olde r childre n an d 

youth (Streissguth , 1997 ; Cole s et al, 2002). It is difficult 

t o evaluat e thi s facto r i n clinica l sample s 

because th e argumen t i s necessaril y circular . Thi s 

criterion i s use d i n makin g a clinica l diagnosis , 

hence suc h individual s ar e necessaril y growt h re - 

tarded. Expose d individual s who presen t fo r diagno - 

sis do no t receiv e i t i f they ar e no t growt h retarded . 

Long-term follow-u p o f childre n identifie d earl y i n 

life i s necessary to confirm persistent growth deficits . 

For thi s reason , exposur e studie s provid e a bette r 

method fo r evaluatio n o f thi s outcome . Generally , 

children expose d t o alcohol appea r to be statistically 

smaller tha n control s o r tha n nationa l norm s (Da y 

et al, 1999 , 2002) . Ofte n mean s ar e within norma l 

ranges, however , s o althoug h thi s characteristi c i s 

statistically "real, " i t ma y o r ma y no t b e o f clinical 

significance, and i t is not necessaril y helpful in iden - 

tification o f specifi c individual s if prenatal alcoho l 

exposure i s no t known . I t i s no t ye t establishe d 

whether lowe r birt h weight , height , an d hea d cir - 

cumference associate d wit h prenata l alcoho l expo - 

sure (and, perhaps, associate d polydrug use) are ris k 

factors for other conditions occurring later in life .

NEURODEVELOPMENTAL EFFECT S 

OF PRENATAL 

ALCOHOL EXPOSURE 

Effects on Brain Structure : 

Information from 

Neuroimaging studies 

The followin g sectio n briefl y review s neuroimaging 

studies of the effect s o f prenatal alcoho l exposur e o n 

brain structure. Brain morphology could be described 

as a "physica l effect " o f prenata l exposure , a s well ; 

a fulle r discussio n o f thi s subjec t i s provide d i n 

Chapter 9 . 

Both human studie s of behavior and animal models 

leave no doubt that alcohol i s a teratogen that has 

toxic effects o n the brain. Until recently, it was impossible 

t o evaluat e directl y it s effec t o n huma n brai n 

structure and functioning, but i n the last decade, im - 

aging studie s o f alcohol-affecte d patient s hav e bee n 

conducted. Severa l researc h group s usin g clinica l 

samples of patients with fetal alcoho l spectrum disor - 

ders (FASD ) hav e reporte d effect s o n th e brai n i n 

older childre n an d adults . Mos t o f thes e publishe d 

studies have use d structura l magneti c resonanc e im - 

aging (sMRI) and hav e focused on morphology . Sev - 

eral studie s (Bookstei n e t al , 2001 , 2002 ; Sowel l 

et al , 200lb ) hav e correlate d structura l outcome s 

with neurobehavioral data. Although functiona l magnetic 

resonanc e imagin g (fMRI ) i s being develope d 

as a researc h too l i n thi s area , n o studie s have been 

published a s of this writing. 

In sMRI studies , microcephaly ha s been the mos t 

consistent finding, with FAS patients having a general 

reduction i n brain volume compare d t o that o f con - 

trast groups (Riley et al, 1995 ; Archibald et al, 2001; 

Sowell e t al, 2001a , 2001b ; Bhatar a e t al, 2002) . 

More specifically , relativ e reductions in white matte r 

have been noted the corpus callosum and the parietal 

lobe (Riikone n e t al, 1999 ; Archibal d e t al , 2001 ; 

Sowell et al, 200lb ) in patients with FAS. The corpu s 

callosum ha s bee n a focus o f interest becaus e i t i s a 

midline structure and i s readily identifiable. Agenesis 

and hypoplasi a o f this structur e hav e bee n reporte d 

repeatedly, particularly in extremely dysmorphic individuals 

(Rile y e t al , 1995 ; Johnso n e t al , 1996 ; 

Swayze e t al , 1997 ; Riikone n e t al , 1999 ; Sowel l 

et al , 2001b ; Bhatar a e t al, 2002) . Sowel l an d col - 

leagues (20 0 lb) reporte d reduction s an d displace - 

PRENATAL ALCOHOL EXPOSURE AND HUMAN DEVELOPMENT 12 7 

ments i n th e anterio r region o f the corpu s callosu m 

as wel l a s i n th e splenium , whic h correlate d wit h 

deficits i n verba l learning an d memor y i n th e sam e 

patients. Bookstei n an d colleague s (2001 , 2002 ) re - 

ported tha t there i s greater variability in the shap e of 

the corpu s callosu m amon g alcohol-affecte d patients 

and tha t thicke r o r thinner structure s ar e relate d t o 

specific pattern s o f neurodevelopmenta l deficits : a 

thicker corpu s callosum i s associated wit h deficit s i n 

executive functioning, whereas a thinner one accom - 

panies motor deficits . 

The cerebellu m ca n be affected b y prenatal expo - 

sure t o ethanol . I t i s smaller i n childre n wit h FA S 

(Riikonen e t al, 1999 ; Autti-Ram o et al, 2002) , particularly 

in the anterio r vermis (lobule s I-V) (Sowell 

et al, 1996) . These findings are supported b y animal 

studies (e.g. , Goodlet t an d Lundahl , 1996) . Man y 

studies report other specific deficits in CNS morphol - 

ogy, although non e as consistently as those o f the cor - 

pus callosum an d the cerebellum . 

Studies o n th e structura l effec t o f prenatal expo - 

sure on the brain indicate that significant morphological 

change s ca n b e found . I t i s not clear , however , 

that a specific area of deficit is present in all cases. This 

finding suggest s tha t th e teratogeni c effec t i s mor e 

general. Severa l candidates fo r further researc h hav e 

emerged fro m thes e data , but eve n th e corpu s callo - 

sum, which has been implicate d frequently , does no t 

show specific macrostructural patterns related to alcohol 

exposure across all studies. For instance, althoug h 

callosal agenesis has been reported (Riley et al, 1995 ; 

Johnson et al, 1996 ; Bhatara et al, 2002) , other stud - 

ies fin d thinning o f these area s or posterior displace - 

ment (Sowell et al, 200lb) , and yet other studies find 

macrostructural anomalies only in a minority of individuals 

with FAS (Autti-Ramo et al, 2002) . This situation 

i s further complicate d b y a study in nonhuman 

primates showin g that repeated , episodi c exposure t o 

ethanol during pregnancy can result in an increase in 

the siz e o f the corpu s callosum , especiall y th e ante - 

rior segmen t (Mille r e t al , 1999) . Thes e outcome s 

suggest that, particularly in individuals whose alcoho l 

effects ar e subtle, gross anatomical studie s may not be 

observable. 

A complementary imagin g technique t o sMR I i s 

diffusion tenso r imagin g (DTI) , whic h allow s th e 

measurement o f whit e matte r integrity . I n FA S pa - 

tients, sMR I studie s frequentl y repor t effect s o n in - 

tegrity o f white matte r tract s (Johnso n e t al , 1996 ;


Swayze e t al, 1997 ; Clar k e t al, 2000 ; Archibal d 

et al, 2001; Sowell et al, 2001b; Bhatara et al, 2002). 

Through DTI , M a and colleague s (2005 ) found that 

fractional anistrop y in the gen u an d spleniu m o f the 

corpus callosum of alcohol-affected youn g adults was 

significantly lower than tha t of age-matched controls . 

This suggest s a n associatio n o f teratogenic exposur e 

and white matter integrity. 

Although i n a n earl y stag e o f exploration , neu - 

roimaging procedure s hol d promis e fo r understand - 

ing th e teratogeni c effect s o f prenata l exposure . 

When fMR I studie s ar e publishe d an d functiona l 

outcomes ar e correlate d wit h behaviora l outcomes , 

knowledge o f structure-function-behavio r relation - 

ships in these patients will be greatly advanced. 

Motor and Neuromotor Effects 

Alcohol effect s o n psychomoto r functio n hav e bee n 

observed regularl y i n infant s an d toddler s (Cole s 

et al, 1985; Coles, 1993 ; Jacobson et al, 1993 ; see discussion 

of Jacobson e t al., 1988 , above) . Problems i n 

motor functioning in older children are less well doc - 

umented bu t ar e reporte d (Aronso n e t al. , 1985 ; 

Kyllerman et al, 1985 ; Janzen et al., 1995) . Descrip - 

tive studie s o f childre n wit h FA S consistentl y not e 

deficits in motor skills and coordination (Mattso n and 

Riley, 1998) . Among quasi-experimenta l research de - 

signs with children diagnose d with FAS are deficits i n 

visual-motor integratio n (Conry , 1990 ; Cole s e t al. , 

1991; Mattso n e t al, 1993 ) an d fine-moto r strengt h 

and coordinatio n (Bar r e t al. , 1990 ; Conry , 1990) . 

Kyllerman and colleagues (1985 ) found tha t children 

(mean ag e 70 months) born to women wh o abuse alcohol 

ha d lowe r motor developmen t score s an d di d 

more poorl y on test s of motor coordinatio n tha n di d 

those i n a comparison grou p who were not prenatally 

exposed. Children wit h FAS also show deficits i n balance 

(Marcus , 1987 ; Roebuc k e t al, 1998a , 1998b) , 

increased clumsines s (Steinhause n e t al. , 1982) , ab - 

normal gait (Marcus, 1987 ; Conn-Blower , 1991 ) an d 

tremors (Aronso n e t al, 1985 ; Marcus , 1987) . Roe - 

buck an d colleague s (Roebuc k et al, 1998a , 1998b ) 

suggest tha t damag e t o th e cerebellu m fro m heav y 

prenatal alcohol exposure may interfere with efficien t 

use o f visual an d somatosensor y syste m cues . Usin g 

electromyography, thes e researcher s conclude d tha t 

this defici t wa s th e resul t o f damag e t o th e CN S 

rather tha n t o the periphera l nervou s system control - 

ling muscles or vestibular system. 

Peripheral systems are affected b y prenatal exposure 

to alcohol . Avari a an d colleague s (2003 ) examine d 

nerve conductio n i n median , ulnar , peroneal , an d 

tibial nerve s i n newborn s wh o wer e re-examine d a s 

toddlers at 1 2 and 1 4 months. Ulna r and tibial motor 

nerve velocitie s ar e slowe r an d dista l amplitude s 

are lower in ethanol-exposed children . The observe d 

changes differe d fro m thos e i n alcoholi c periphera l 

neuropathy o f adult s an d reflecte d bot h myeli n in - 

volvement (reduced velocity) and axonal damage (de - 

creased amplitude). In contrast, assessment of sensory 

nerve conduction doe s no t identif y significan t differ - 

ences betwee n alcohol-expose d infant s an d thos e i n 

the contro l group . No clinical correlates are noted i n 

either group of infants. 

Although researc h i n thi s are a i s limited , thes e 

studies and persisten t clinical reports suggest that further 

exploratio n o f effect s o n moto r an d sensory / 

motor developmen t i s warranted. Deficits in these areas 

hav e a significan t effec t o n earl y life, potentiall y 

affecting physical , cognitive, and socia l development . 

In addition , earl y motor deficit s ar e ofte n correlate d 

with specifi c learnin g an d academi c problem s a t 

school ag e and later. 

Global and Specific Effects o n 

Neurocognitive Functioning 

Global Cognitive Effects 

Global intellectua l deficit s ar e the primar y neurodevelopmental 

outcom e associate d wit h prenata l alco - 

hol exposure . Thi s i s consisten t wit h th e result s o f 

other earl y neurological insult s that, contrar y t o th e 

adult pattern , ten d t o b e mor e globa l tha n specific . 

Deficits i n genera l intellectua l functio n (tha t is , 

scores on intelligence or other ability tests) have been 

observed i n sampl e population s compose d o f chil - 

dren diagnose d wit h FA S and i n thos e draw n fro m 

longitudinal studies of heavy gestational alcohol expo - 

sure. Children meetin g the diagnostic criteria for FAS 

show a relatively broad range of intellectual outcome s 

with sampl e estimate s rangin g fro m sever e intellec - 

tual deficienc y to averag e levels of functioning (e.g. , 

IQs range fro m 2 0 to 120 ; Mattson an d Riley , 1998 ; 

and fro m 1 6 to 105 ; Streissgut h e t al, 1978) . Mea n 

performance on standardized measures of intellectual 

function typicall y hav e bee n i n th e borderlin e t o 

mildly deficien t rang e (IQ s rang e fro m 6 5 t o 75 ) 

(Stratton e t al. , 1996) . I n larg e retrospectiv e an d

prospective studies , hal f o f th e childre n wit h FA S 

meet criteri a for mental retardation, an IQ < 70 on a 

standardized measur e o f intelligenc e (Abel , 1998) . 

There has also been discussio n of the effec t o f prenatal 

exposur e to low or moderate levels of alcohol an d 

most researc h studie s hav e reporte d tha t i n suc h co - 

horts there is a decrement o f several IQ points (4 to 6) 

attributable t o th e exposur e (e.g. , Streissgut h e t al. , 

1993). 

Specific Cognitive Effects 

In additio n t o moto r problems , function s that hav e 

been examined an d appear to be affecte d b y prenatal 

alcohol exposur e includ e specifi c deficits in visual - 

spatial processing, arousal and regulation of attention, 

w ; orking memory, planning an d organizationa l skills, 

mathematical achievement , an d behaviora l regulation. 

In reading the literature, it is important to examine 

th e exten t t o whic h globa l deficit s hav e bee n 

taken into consideration i n the interpretation o f "specific" 

deficit s i n outcome . Individual s showing mental 

retardatio n o r borderline intellectua l functionin g 

(IQ


the regulatio n o f attentio n i s o f grea t importance , 

as master y of arousal and attentio n i s a prerequisite 

for learnin g an d socia l developmen t (MacKintosh , 

1975) and thus has the potential to mediate a number 

of long-ter m neurodevelopmenta l outcomes . Man y 

children with FAS have difficulties wit h regulation of 

arousal; thi s proble m ma y b e associate d wit h thei r 

ADHD diagnosis (Streissguth et al., 1986, 1995 ; Nanson 

an d Hiscock , 1990 ; Kopera-Fry e e t al , 1997 ; 

Oesterheld an d Wilson, 1997 ) o r other alteration s i n 

attention (Streissgut h et al, 1984; Cole s et al, 1997) . 

Investigations int o th e effec t o f prenatal alcoho l 

exposure o n attentiona l regulatio n tha t hav e use d 

prospective longitudina l design s hav e produce d in - 

consistent results . Streissguth an d colleague s (1984 ) 

found tha t prenata l alcoho l exposur e resulte d i n 

more errors and slower reaction times on a task of vigilance 

and sustaine d attention i n preschoo l childre n 

with moderat e alcoho l exposure . In the same cohort , 

observations o f children' s behavio r b y traine d ob - 

servers an d rating s b y parent s sugges t tha t expose d 

children ar e les s attentive, less compliant, an d mor e 

fidgety (Landesman-Dwyer and Ragozin, 1981). These 

results hav e no t alway s bee n confirme d b y othe r 

prospective studie s using exposure samples (i.e., Boyd 

et al, 1991; Coles étal, 1997) . Brown and colleagues 

(1991) als o sho w an associatio n betwee n gestationa l 

alcohol exposur e and regulation of attention, but they 

attribute their results to the effect s o f the postnatal environment. 

A four-factor, multidimensiona l mode l o f 

attentional regulatio n (Mirsky et al, 1991 ) wa s used 

to asses s 7-year-old s prenatall y expose d t o alcoho l 

(Coles et al., 1997). These children differ significantl y 

from a contras t grou p o f childre n diagnose d wit h 

ADHD i n thei r abilit y t o attend . Alcohol-affecte d 

children hav e mor e difficult y encodin g informatio n 

and in flexibility in problem solving, but they are similar 

t o control s i n thei r abilitie s to focu s an d sustain 

attention. Th e childre n diagnose d wit h ADHD have 

a contrasting pattern. 

Hypothesizing that early difference i n arousal levels 

note d i n alcohol-expose d infant s (Cole s e t al. , 

1985) might be associated with later problems i n self - 

regulation and attention, Kable and Coles (2004 ) further 

examine d th e relationshi p o f alcoho l exposur e 

and informatio n processing i n a differen t sampl e o f 

high-risk, low-birth-weigh t infant s wh o ha d bee n ex - 

posed t o moderate amount s of alcohol. Behaviorally , 

at 6 and 1 2 months, these infants show small but significant 

developmenta l difference s i n menta l an d 

psychomotor development a s well as in behavior regulation 

(Coles et al., 2000). When they were 6 months 

old, th e infants ' attentio n wa s evaluated b y us e o f a 

habituation paradigm with cardiac orienting response 

as outcome. Infant s with mor e alcoho l exposur e re - 

spond more slowl y than "low-risk " controls to presentation 

o f bot h visua l an d auditor y stimul i an d ar e 

rated as significantly higher i n arousal level. This difference 

i n information-processin g speed i s consistent 

with earlier work (Jacobson et al., 1994 ) showin g that 

fixation duration (lookin g time) i s longer i n alcohol - 

exposed infants . The implicatio n i s that alcohol i s related 

to the speed o f information processing. 

The relationshi p betwee n attentio n an d arousa l 

regulation i s suggestive. The abilit y to focus an d sus - 

tain attentio n depend s o n th e abilit y t o moderat e 

arousal to allow information to be processed efficientl y 

(Ruff an d Rothbart , 1996) . I t ma y b e tha t alcohol - 

exposed infant s cannot modulat e arousa l effectivel y 

and ar e therefore less efficient i n processing environmental 

information . Findings amon g adolescents and 

adults o f inefficiencies i n informatio n processin g i n 

the visual modality (e.g., Coles et al., 2002) or in both 

auditory and visua l modalities (Conno r e t al., 1999 ) 

may be influence d by arousal level as well. This suggestion 

is supported by findings from a study of the ef - 

fects of maternal substance abus e on attention. Sues s 

and colleague s (1994 ) foun d tha t prenata l exposur e 

to alcohol, but no t opiates , influenced attention an d 

psychophysiology (cardiac functioning and vagai tone) 

at school age . How this compromise i s related to de - 

velopmentally later and more complex cognitive functions 

and attention and behavioral regulation has not 

yet been investigated . 

Executive Function Skills. Executiv e function i s a 

construct referred t o as, "perhaps the mos t appealing , 

yet least understood, aspect of cognition and metacognition" 

(Borkowski and Burke , 1996 , p . 235) . Executive 

function involve s higher-order cognitive processes 

that range from attentiona l regulation , working memory 

skills , planning an d organizationa l thinking an d 

problem solvin g (Lyon, 1996; Morris, 1996) . From an 

information-processing prospective , attention , mem - 

ory, and executiv e functio n ma y be interrelate d pro - 

cesses that affect cognitiv e functioning and the ability 

to carr y out goal-oriente d behavior s (Borkowski an d 

Burke, 1996). 

Both clinica l description s o f childre n wit h FA S 

and longitudinal exposure studies implicate ethanol i n

compromising executiv e function. Investigators have 

approached thi s construc t fro m severa l theoretica l 

positions. Som e investigator s aggregate measure s t o 

obtain a n estimat e o f executive function (e.g. , Samp - 

son et al, 1997 ; Connor et al, 2000), whereas other s 

evaluate specific area s thought t o represent aspects of 

executive functio n (e.g. , Kern s et al, 1997 ; Mattso n 

et al, 1999 ; Schonfel d e t al, 2001) . It is difficult t o 

compare studie s becaus e th e construc t pe r s e is no t 

well defined. Therefore, th e followin g review will examine 

the effec t o f prenatal alcoho l exposur e o n various 

component s tha t ar e usuall y understoo d t o b e 

part of executive functioning. 

Memory an d Metamemory . Memor y i s a broa d 

field o f study, and onl y some aspect s o f memory ar e 

considered to be part of executive function. Childre n 

with FAS exhibit deficits in memory skills, but the im - 

pairments are not always stable and their extent seems 

to be influence d b y modality of the tas k used an d b y 

the specifi c cognitive demands of tasks (Mattson an d 

Roebuck, 2002) . For instance , a n interactio n o f age 

and tas k characteristic s ma y determin e whethe r 

effects ar e shown . Usin g th e McCarth y Scale s o f 

Children's Abilitie s (McCarthy , 1972) , Janze n an d 

colleagues (1995 ) do not find difference s i n memory 

function betwee n youn g childre n diagnose d wit h 

FAS and a contrast group selected fro m day-car e settings. 

This finding contrasts with a study by Coles and 

colleagues (2004) , wh o use d a differen t test . The y 

find that children in the same age range do have spe - 

cific memory deficits. 

The effec t o f presentation modalit y (i.e. , visualauditory) 

on memory has been investigated. Learnin g 

and recal l o n task s presented visuall y o r i n narrative 

form w ; ere compared (Platzma n et al., 2001). Adolescents 

with FA S have poorer recall of visual elements 

than control s and age mates in special education pro - 

grams, wherea s long-ter m recal l o f auditoril y pre - 

sented item s i s not impaired . Short-ter m memor y for 

auditorily presented item s (i.e., random string s of digits; 

Wechsler Intelligenc e Scal e for Children-Revised, 

Digit Span) is also similar to that of contrast groups. In 

a different sampl e o f clinically referred children, age s 

3 to 9 years old, wit h a diagnosis o f FASD, a similar 

auditory tas k an d a visual/spatia l analo g wer e contrasted. 

Relativ e deficit s for spatial spa n ar e evident , 

whereas performance on th e digi t span task is consistent 

wit h overal l abilit y leve l (Cole s e t al , 2004) . 

These findings suggest specific deficits in memory with 


language-related or auditorily presented materials relatively 

spared . Thi s patter n o f performanc e i s als o 

noted b y Mattson and Roebuck (2002) among schoolaged 

an d adolescen t childre n diagnose d wit h FA S or 

having ha d heav y prenata l exposure . I n thi s group , 

memory performance is compared t o that o f normal 

controls o n fiv e standardize d measure s o f learnin g 

and memory: a verbal learning task (California Verbal 

Learning Test-Children' s Versio n [CVLT-C] ; Deli s 

et al., 1987) , the Biber Figure Learning Test, and three 

subtests from th e Wid e Rang e Assessment o f Memory 

and Learnin g (WRAML ; Sheslow an d Adams , 1990) . 

These subtest s ar e Verbal Learning , Visua l Learning , 

and Soun d Symbol . The constructio n o f the CVLT- C 

is important for understanding the result s of these studies. 

This test i s a verbal learning tas k with orall y pre - 

sented lists of words in several categories (i.e., clothing, 

fruits, an d toys). Due t o the lis t structure, a n effectiv e 

memory strategy (grouping by semantic category) can 

be used to aid learning and recall . 

Through th e use of tests of verbal and visual memory, 

it has been show n that alcohol-affected childre n 

learn fewe r item s (bot h word s an d figures ) overal l 

than control s (Mattso n an d Roebuck , 2002) . After a 

standard dela y period , however , th e percentag e re - 

called (o f the materia l that ha d bee n learned ) i s not 

different betwee n th e group s for verbal material. O n 

the nonverba l material , th e alcohol-affecte d grou p 

show 7 relativ e deficits in delaye d recall . Thus , verbal 

memory i s les s affecte d tha n nonverba l memory . 

Data fro m thes e studie s hav e been reanalyze d t o examine 

the effec t o f stimulus differences i n two of the 

verbal memory tasks (Roebuck-Spencer an d Mattson , 

2004). Accordingly, children with FASD demonstrat e 

semantic clusterin g on th e CVLT-C , indicatin g tha t 

they are able to take advantage of the list characteris - 

tics t o ai d recall . The y perfor m bette r o n thi s tas k 

than o n th e WRAM L verba l memor y task , whic h 

does no t offe r th e sam e opportunit y t o us e th e "im - 

plicit" memor y strateg y inherent i n the stimulu s material. 

O n th e basi s o f thes e findings , th e author s 

argue tha t previousl y observe d difference s in verba l 

and nonverba l memory ma y result from us e of more 

effective memor y strategie s o n verba l learning tasks . 

Other work (i.e., Platzma n e t al, 2001; Coles et al., 

2004, se e discussion above) does not suppor t this explanation 

becaus e visuall y and auditoriall y presente d 

materials are equivalent in these studies. 

Various studie s repor t tha t childre n with FAS D 

have memory deficits on verbal learning tasks (Mattson


et al, 1996; Kerns étal, 1997 ; Schonfel d étal, 2001; 

Willford e t al, 2004). A longitudinal follow-u p study 

of 14-year-olds shows that verbal-auditory rathe r than 

visuospatial memor y i s affected wit h learning ; short - 

and long-ter m memor y deficits on a paired associate 

task are detected (Willfor d et al, 2004) . I n a sampl e 

of adolescents an d youn g adult s classifie d a s having 

an "averag e IQ " o r "belo w averag e IQ, " Kern s an d 

colleagues (1997 ) show that individuals with FAS perform 

belo w expectation o n the CVLT-C. The greates t 

difference i s not o n initia l learning trials, but o n th e 

fifth trial. These results suggest that affected individu - 

als are not able to use semantic memory strategies appropriately 

t o maintain a comparable learnin g slop e 

over repeated trials . That is, the individual s with FAS 

do not use the categorie s inheren t i n the lists to organize 

and facilitate their recal l an d thei r response s do 

not sho w the clusterin g o f semantically related item s 

that is usually found i f effective memor y strategies are 

used. This findin g contrast s t o a lack of difference i n 

primacy effec t an d seria l clustering scores , which re - 

lies less on strategy use. 

Mattson an d Riley (1999) contras t intentional an d 

incidental memor y b y measuring recal l o f information 

(a ) whe n childre n wit h FA S ar e awar e o f th e 

memory tas k involved and (b ) when the y learn infor - 

mation usin g effective memor y strategies provided by 

the investigator s without bein g awar e of the nee d t o 

learn the information in expectation o f recall. The in - 

vestigators conclude tha t incidental memor y is not affected 

b y prenatal alcohol exposure. 

Coles an d colleague s (1997 ) gav e a n alcohol - 

exposed an d a contras t grou p o f 7-year-olds a paire d 

associate tas k requirin g tha t th e childre n continu e 

with the list-learnin g process until the y achiev e mas - 

tery. At both immediat e an d delaye d recall , alcohol - 

affected childre n ar e no t differen t fro m control s with 

this methodology . Th e numbe r o f repetition s re - 

quired t o achiev e lis t mastery , however , i s signifi - 

cantly higher for alcohol-affected children . 

Cumulatively, the abov e findings suggest that one 

aspect o f th e memor y defici t observe d i n alcohol - 

affected individual s involves employing strategies. That 

is, they may not be exhibiting effective "metarnemory " 

(an important aspect of executive functioning) in tha t 

they ma y hav e mor e difficult y i n selectin g an d em - 

ploying effective learnin g strategies and the y may not 

be aware of the level of effort required t o achieve mas - 

tery on a particular task. It may also be tha t becaus e 

alcohol-affected individual s process informatio n mor e 

slowly, learning time is spent less efficiently an d mor e 

trials are required. Whether these deficit s are uniqu e 

to individuals with FAS D o r are characteristic of slow 

learners in general i s not clear and should b e the sub - 

ject of further study . 

An aspect of memory that is considered a factor in 

executive processin g i s active working memory. This 

process involve s menta l manipulatio n o f element s 

stored briefl y i n short-term memor y and has been referred 

to as a "mental scratc h pad " (Ashcraft , 1995) . It 

requires that informatio n be store d an d processe d i n 

meaningful "units " at a given moment in time. Man y 

of the cognitiv e task s comprising neurodevelopnien - 

tal tes t batterie s includ e us e o f active working mem - 

ory because higher-order processing or problem solving 

requires tha t severa l unit s o f informatio n b e main - 

tained simultaneously . 

In alcohol-affected individuals , problems i n active 

working memor y hav e bee n identifie d earl y i n in - 

fancy. Usin g a visua l expectanc y paradig m (Hait h 

et al. , 1988 ; Jacobson e t al. , 1992 ) as a measur e o f 

working memory, Was s and Hait h (1999 ) show that 3month-old 

infants exposed to alcohol prenatally have 

more difficult y tha n nonexpose d control s i n main - 

taining an d manipulatin g three item s of information 

simultaneously, a result implying a reduction i n working 

memory capacity . On th e same task, these infant s 

have difficultie s shiftin g fro m on e rul e t o anothe r 

(that is , learning to anticipate wher e item s woul d b e 

presented next ) an d wit h masterin g comple x spatia l 

sequences. Kodituwakk u an d colleague s (1995 ) use d 

several neurodevelopmental tasks tapping activ e working 

memory in older individuals. They conclude that a 

problem i n managing goal s i n working memory i s the 

mechanism underlyin g much o f the cognitiv e impairment 

see n i n childre n wit h FAS . I n practica l terms , 

such a defici t wil l affec t dail y functionin g an d academic 

performanc e a s well a s results of neuropsychological 

tests. For instance, children diagnosed with FAS 

consistently sho w impairment s i n doing mathematics , 

as demonstrate d b y performanc e o n th e arithmeti c 

subtests from the Wechsler series (e.g., Wechsler, 1991) 

of intelligence tests . These tests rel y heavily on activ e 

working memor y skill s by requiring the manipulatio n 

of number s an d arithmeti c operation s i n short-ter m 

memory to generate correct answers . 

Planning an d Organization . Peopl e wit h FAS D 

are described a s having impairments in planning, organization, 

an d problem-solvin g aspect s o f executiv e

functioning (Streissguth , 1997) . Tw o task s that have 

been use d to evaluate functioning in this area are progressive 

planning tasks (e.g., Tower of London; Shal - 

lice, 1982) and tasks assessing learning and reversa l or 

shifts i n discriminativ e stimuli (i.e., Wisconsin Car d 

Sort Task [WCST] ; Heato n e t al, 1993) . Bot h kinds 

of tasks were originally used t o measure th e effect s o f 

frontal lobe damag e i n adult s (Shallice , 1982) . Pro - 

gressive planning task s require tha t th e perso n bein g 

tested replicat e a visuall y presente d desig n b y rearranging 

circular disks or balls that are placed o n vertical 

pegs. There are rules restraining the numbe r an d 

type o f moves permitted . Th e tas k require s tha t th e 

subject retai n these rule s a s well a s the sequenc e o f 

moves in working memory to correctly solve the prob - 

lem. Initially , th e tas k i s simpl e an d require s littl e 

planning, bu t a s task complexity increases , the indi - 

vidual must be able to visualize a multistep sequence 

of moves to complete th e tas k successfully . 

Hie WCST and similar tasks require the individual 

to identif y o r sor t according to specifi c characteristics 

of the stimul i (i.e., color, number, o r shape) presente d 

to them, with feedback provided to facilitate learning. 

For th e initia l se t o f trials , th e correc t dimensio n 

may be color, so that all responses that match i n color 

are rewarded . Once th e correc t dimensio n i s reliably 

learned, a differen t dimension become s "correct " s o 

that, fo r instance, numbe r become s th e correc t solution 

and i s rewarded. This task requires cognitive flexibility, 

becaus e th e individua l must inhibi t previously 

learned response s an d shif t set s or rule s learned fro m 

previous trials. 

Using both kinds of tasks, children wit h FAS have 

been foun d to perform more poorly than control s and 

to mak e perseveratio n error s (repetition o f response s 

that ar e incorrec t o r no t reinforced ) (Kodituwakk u 

et al, 1995 ; Cole s e t al. , 1997 ; Kern s et al, 1997) . 

These findings suggest that they have difficulty incor - 

porating environmental feedback to correct a response. 

Children wit h FA S typicall y hav e mor e difficult y 

learning th e shifts , particularl y reversa l shifts , tha n 

would b e expecte d (Kodituwakk u et al., 1995 ; Cole s 

et al, 1997 ; Kerns et al., 1997) , a pattern implying difficulties 

wit h inhibitio n o f learne d response s o r a n 

inattentiveness to new information. 

Kodituwakku and colleague s (2001 ) used anothe r 

paradigm t o discriminat e emotion-lade n learnin g 

from conceptua l se t shiftin g (th e reversa l shif t task s 

discussed above ) to further document neurodevelopmental 

impairment s associate d wit h FAS . These au - 


thors expecte d performanc e of alcohol-exposed indi - 

viduals to be impaire d i n tasks that involve d an emo - 

tional component. I n this study, performance on th e 

WCST wa s contrasted wit h tha t o n a differen t tas k 

that assessed visual-discrimination reversal learning as 

well a s extinctio n o f reward-respons e association s 

(Rolls et al., 1994) . This second tas k was assumed t o 

measure emotion-relate d learnin g becaus e a reward 

was provided rather than the feedback provided in the 

WCST. Afte r controllin g fo r conceptua l se t shiftin g 

(performance on the WCST) and for general intellec - 

tual abilities , school-age , alcohol-expose d childre n 

perform mor e poorl y tha n th e referenc e sampl e o n 

emotion-related learning; they achieve fewer reversals 

and sho w mor e variabilit y in extinction . I n addition , 

there i s a significant relationship between thes e mea - 

sures of emotion-related learnin g and conceptua l shift - 

ing and parent-reported behavio r problems. Th e ability 

to regulate arousal may have affected performanc e on 

this learning task, although thi s issue i s not addresse d 

in this studv. 

ACADEMIC ACHIEVEMENT 

Given th e neurocognitiv e problem s identifie d i n exposed 

individuals , i t i s no t surprisin g tha t academi c 

achievement i s frequentl y affected . Learnin g prob - 

lems, schoo l failure , repeatin g grades , an d droppin g 

out of school have all been cite d as negative outcome s 

associated wit h FA S in clinica l sample s (Streissgut h 

et al, 1996 ; Autti-Ramo, 2000). It is important to bear 

in min d tha t fo r alcohol-expose d a s well a s for othe r 

children, academi c achievemen t i s determine d b y 

complex interaction s betwee n th e child's neurodevelopmental 

status, environmental supports for academic 

success, and emotional stability. 

Overall academi c achievemen t i s predicted b y the 

individual's genera l intellectua l abilit y an d b y SES . 

Academic problem s consisten t with globa l abilit y 

deficits ar e typically seen. There ma y also be specifi c 

areas of academic weaknes s for the child wit h FAS beyond 

those associate d with cognitive deficit an d social 

disadvantage. Researc h suggest s that , relativ e t o gen - 

eral intellectua l ability , alcohol-exposed childre n an d 

adolescents hav e specific learning problems with mathematics 

(Spoh r an d Steinhausen , 1984 ; Streissgut h 

et al., 1994) . Deficit s in math achievemen t associate d 

with prenata l alcoho l exposur e hav e bee n identifie d 

regularly i n bot h longitudina l an d clinica l studie s


(Streissguth e t al, 1994 ; Kodituwakk u e t al, 1995 ; 

Goldschmidt e t al , 1996 ; Mattso n e t al , 1996) . 

Streissguth an d colleague s (1993 ) repor t tha t arith - 

metic disabilitie s are related t o prenatal alcoho l expo - 

sure i n thei r longitudina l cohort , particularl y when 

there wa s "massed" o r heavy drinking. These investigators 

also note that their findings in the longitudina l 

cohort ar e consisten t wit h observation s fro m thei r 

older, clinical sample of patients diagnosed wit h FAS. 

Other longitudinal sample s have also found evidenc e 

of specific deficit s in thi s area . Coles and colleague s 

(Coles e t al, 1991 , 1997 ; Howel l e t al, 2006 ) not e 

specific deficit s i n mat h functionin g i n a cohor t o f 

African-American schoo l childre n eve n whe n globa l 

delays ar e controlled . I n thi s population , thes e prob - 

lems appear to be related to deficits in visuospatial and 

visual-motor functioning . Jacobson (1999 ) note prob - 

lems in math i n a Detroit longitudina l cohor t a s well 

and attribut e these difficultie s t o problems with working 

memory and executive functioning skills. 

In a sample population includin g both inner-cit y 

and suburba n 4V2-year-ol d childre n wit h lo w birt h 

weight, specific deficits in preacademic mathematica l 

skills are identifie d among alcoho l expose d childre n 

(Kable e t al, 1999) . Highe r score s o n a cumulative 

risk inde x related t o alcohol an d othe r dru g us e ar e 

significantly relate d t o poore r performanc e o n th e 

Test of Early Mathematics Ability, 2nd Edition (Gins - 

burg an d Baroody , 1990) , o n a numbe r o f specifi c 

preacademic skills , including cardinality , constancy , 

counting, an d visua l recognitio n o f numbers. Apparently, 

problem s wit h workin g memory , visua l per - 

ception, an d executiv e functionin g underli e thes e 

functional problems . Adult s wit h FA S als o showe d 

mathematics-related difficulties , includin g computa - 

tion, an d i n solving problems requirin g estimation o f 

magnitude (Kopera-Fry e et al., 1996) . Thus , there i s 

considerable evidenc e tha t thi s academi c are a i s a 

problem i n affecte d individuals , due, n o doubt , t o 

deficits i n cognitiv e processe s tha t suppor t mat h 

achievement. Som e candidat e processes are those areas 

discusse d abov e i n thi s chapter—i.e. , attention , 

executive functioning, and workin g memory as well 

as visuospatial skills (Geary, 1993 ; Ashcraft, 1995) . 

SOCIAL AND EMOTIONA L FUNCTIONING 

Problems wit h behavior and emotiona l regulatio n are 

the most frequent reason for clinical referral of children 

suspected o f fetal alcoho l exposur e (e.g. , Steinhause n 

et al, 1993;Janzenetal, 1995; Kopera-Frye et al, 1997 ; 

Oesterheld an d Wilson , 1997 ; Roebuc k e t al, 1999 ; 

Mattson an d Riley , 2000 ; Kodituwakk u et al, 2001) . 

Social an d emotiona l problem s ar e so commonly re - 

ported b y parents an d i n clinica l studie s i t has been 

suggested that "people wit h FAS appear to differ fro m 

the mentall y retarde d populatio n becaus e o f addi - 

tional problem s i n th e socia l domain " (Kell y e t al. , 

2001, p . 143) . In a survey of a clinical population of 

individuals with FAS and ARND, Streissgut h and col - 

leagues (1996 ) repor t a hig h frequenc y o f menta l 

health, legal , and social problems among the second - 

ary disabilities in this group. The vas t majority (94% ) 

of individual s ar e reporte d t o hav e menta l healt h 

problems o f some kind , with the mos t commo n psychiatric 

problem s bein g ADH D an d depression . 

These reports ar e certainly a concern, but i t may be 

too early to interpret thes e outcome s a s the direc t results 

o f the teratogeni c exposure , particularl y a s thi s 

survey did no t includ e a compariso n grou p an d was 

drawn fro m a clinicall y referre d populatio n tha t 

should be expected t o have such problems . 

In interpretin g report s fro m clinica l sample s o r 

from sample s tha t ar e self-referre d (e.g. , O'Conno r 

et al., 2002), some othe r factors that might influenc e 

behavior shoul d b e take n int o account . Man y chil - 

dren wit h FAS D experienc e environmenta l factor s 

known t o contribut e t o behavioral, social, an d emo - 

tional problems, an d it is disingenuous to ignore thes e 

issues t o attribut e problem s t o prenata l exposure . 

Children diagnose d wit h FA S often com e from low - 

income or socially disadvantaged families and usually 

have limited acces s to educational o r other resources . 

Caregiving disruption s are the nor m rathe r than th e 

exception i n children referre d to clinical settings, an d 

many children are in foster car e following a history of 

neglect and abuse . 

To examine the frequency of behavior concerns in 

a clinica l sampl e o f alcohol-affected children , Cole s 

and Kabl e (unpublishe d results ) evaluate d 28 7 pa - 

tients (mea n age , 4.5 years) referred for evaluation t o 

a FAS specialty clinic. Of the 10 9 children diagnose d 

with FAS , 54.7 % wer e describe d a s "to o active, " 

30.5% had difficultie s wit h tantrums, and 41.1% were 

described a s having problems wit h aggression. Whe n 

these childre n ar e compared t o those referre d t o th e 

same clini c wh o have n o evidenc e o f dysmorphia o r 

growth problems an d t o those wit h no histor y of prenatal 

alcoho l exposure , th e frequenc y o f th e sam e

problems i s 53.1% , 25.0%, an d 43.8% , respectively . 

Upon furthe r examinatio n o f the clini c sampl e an d 

dividing it into four groups (FAS, ARND, nondysmorphic 

FASD , an d unexposed) , n o significan t relationship 

betwee n diagnosti c categor y an d behaviora l o r 

emotional outcome s i s evident . Thes e finding s sug - 

gest that problems with emotional an d behavioral reg - 

ulation, commo n i n children wit h FAS , is associated 

as much wit h clinica l referra l statu s or advers e early 

experiences as with prenatal exposure. 

The relationshi p betwee n prenata l alcoho l expo - 

sure and conduct disorder and delinquency in adolescence 

i s an area of interest. Some researchers and FAS 

advocates have suggested tha t prenatal alcohol expo - 

sure i s associated wit h delinquen t o r criminal behavior 

during adolescence (e.g. , Streissguth e t al., 1996 ; 

Fast e t al. , 1999 ; Fas t an d Conry , 2004) . These con - 

clusions ar e ofte n based o n studie s o f adolescents al - 

ready referre d t o clinic s fo r treatmen t o r i n th e 

juvenile justic e system. In a surve y o f secondary disabilities, 

Streissgut h an d colleague s (1996 ) describ e 

that caregiver s report tha t a majorit y o f yout h an d 

young adults with FASD are involved in "delinquent" 

or problematic behaviors. Of 25 3 patients 1 2 years of 

age o r older , 60 % have disruption s i n schoo l experi - 

ence (e.g., suspensions), 50 % have demonstrated "in - 

appropriate" sexua l behavior , an d 30% , alcohol an d 

drug problems. Fas t and colleagues (1999 ) als o report 

an associatio n between prenata l exposur e and crimi - 

nal behavior . In a sample of 287 adjudicated adolescents 

remande d fo r inpatien t forensi c psychiatri c 

assessment, 23.3% were found to have some prenatal 

alcohol-related diagnosis. 

In studie s of the relationshi p of prenatal exposure 

and behavio r i n les s high-risk groups , th e result s ar e 

more mixed . The Achenbac h (1991 ) Child Behavior 

Checklist an d th e teache r version , th e Teache r Report 

Form, hav e often been used to measure behavio r 

problems. Elevate d score s o n proble m indice s hav e 

been reported in some studies (i.e., Olson e t al., 1997 ; 

Mattson and Riley, 2000) but not others (Steinhausen 

et al. , 1993) . Eve n whe n score s fo r childre n wit h 

FASD ar e statisticall y highe r tha n thos e o f age - 

matched contro l groups , th e score s d o no t alway s 

reach clinicall y significan t levels (e.g. , Brow n e t al, 

1991). Lync h an d colleague s (2003 ) specificall y examine 

the question o f delinquency an d conduct problems 

i n alcohol-expose d adolescent s compare d t o 

contrast group s fro m th e sam e populatio n o f low - 

income, urba n youth . The y sho w with a sampl e o f 


248 children, mea n ag e o f 1 5 years, male s ar e mor e 

likely than females to report delinquent behavior, but 

there i s no relationshi p between prenatal alcoho l exposure 

and outcomes. Delinquen t behavio r is related 

to parenting factor s an d environmenta l stress . In th e 

same grou p o f youn g people , examinatio n o f aca - 

demic an d schoo l record s indicate s n o highe r inci - 

dence of conduct problems, attendanc e problems , o r 

suspensions i n alcohol-expose d yout h (Howel l e t al., 

2006). 

Methodological factor s mus t be considere d when 

assessing th e result s o f research . Interpretatio n o f 

reports fro m clinica l studie s i s particularly speculative 

i n th e are a o f behavio r an d social/emotiona l 

functioning. Investigator s mus t b e ver y carefu l t o 

identify appropriat e contras t group s as well as the ef - 

fects o f confoundin g an d mediatin g factor s whe n 

attempting t o understan d observe d outcomes . Fo r 

the mos t part , thes e methodologica l restraint s have 

not been observe d in this area of study, and fo r these 

reasons, i t i s to o earl y t o dra w conclusion s abou t 

the effec t o f prenatal exposur e o n thi s are a o f func - 

tioning. 

CURRENT ISSUES AND SUGGESTIONS 

FOR FUTURE RESEARC H 

Despite greatl y increased understandin g o f the con - 

sequences o f prenatal exposure , some issue s are stil l 

being debated. These areas of controversy and explo - 

ration sugges t the directio n fo r researc h durin g th e 

next decade. 

Description of Characteristic s 

Over Time 

Although som e sampl e population s an d a number o f 

exposure studie s hav e bee n followe d longitudinally , 

there remains limited informatio n about the trajectories 

o f development resultin g from prenata l alcoho l 

exposure amon g thos e wit h eithe r FA S o r ARND . 

Even objective issues such a s facial dysmorphi a have 

not been studie d in the sam e individuals over time to 

evaluate th e salienc y of features at different point s i n 

development. Outcome s from longitudina l cohort s in 

which repeate d wave s of data have been collected o n 

the sam e individual s (Streissgut h e t al. , 1993 ; Da y 

et al, 2002) have often been reporte d by time period , 

as i f thes e wer e cross-sectiona l cohorts , instea d o f


examining them ove r time to evaluate developmental 

trajectories. Sinc e these database s do exist , investigators 

hav e th e opportunit y t o investigat e th e longi - 

tudinal aspect s o f developmen t i n alcohol-affecte d 

children and , when the y have done so , to greatly increase 

understanding in this area. 

Behavior and Conduct Disorder s 

A largely unresolved questio n i s that of the relation - 

ship betwee n prenata l exposur e an d late r behaviora l 

and emotiona l problems . Man y o f these problems a s 

well a s psychiatri c disorder s (Fam y e t al. , 1998 ; 

O'Connor et al., 2002) have been identified i n clini - 

cally referred samples. I t is likely that the nex t decad e 

will produc e a numbe r o f studies o n thes e relation - 

ships an d furthe r examinations of the effec t o f expo - 

sure o n th e ris k fo r lega l problem s an d substanc e 

abuse. I f studies are carried out wit h the appropriat e 

methodologies and control for the multiple confounding 

factors associate d with maternal substanc e abuse , 

the contributio n o f prenata l exposur e t o suc h out - 

comes may be better understood. 

Behavioral Phenotypes 

In th e rea l world , i t i s often difficul t t o ascrib e ob - 

served outcome s t o maternal alcoho l use . There has 

been a good dea l o f interest i n identifyin g a "behav - 

ioral phenotype" that characterizes peopl e wh o are affected 

b y prenatal exposure . Suc h a constellatio n o f 

behavioral outcome s i s believed t o characterize bot h 

those wit h th e ful l FA S and thos e wh o d o no t hav e 

identifying facia l features . Clinician s woul d lik e t o 

have a checklist of such behaviors that can be used by 

teachers, parents, and othe r observer s to discriminate 

affected fro m unaffecte d children and t o facilitate referral. 

The searc h for such a phenotype does not take 

into account th e multipl e factors tha t affec t develop - 

ment an d th e man y etiologie s tha t ca n hav e a final 

common pathwa y in behavioral terms. 

Intervention an d Standards o f Care 

The mos t neglecte d are a o f researc h o n th e behav - 

ioral effect s o f alcohol i s on method s fo r intervention 

to improve outcomes fo r affected individuals . Parents 

and caregiver s o f affecte d childre n stat e tha t thei r 

most urgen t nee d i s appropriat e interventions . I n 

1996, a report b y the Institut e of Medicine (Stratto n 

et al, 1996 ) recommende d tha t studie s b e don e i n 

this area an d tha t standards of care fo r affected indi - 

viduals be written. To date, n o reports of intervention 

studies i n huma n sample s hav e bee n published , al - 

though ther e are currently five such studies being car - 

ried ou t unde r th e aegi s o f the Center s fo r Diseas e 

Control an d Prevention . N o standard s of care hav e 

been propose d b y any agenc y o r professional organi - 

zation. Althoug h i t is apparently difficul t t o carry ou t 

work in this area, it seems likel y that as more information 

i s accumulated abou t cognitiv e an d behaviora l 

consequences of prenatal exposure , interventio n an d 

treatment wil l be a focus of research i n the future. 

Abbreviations 

ADHD attentio n deficit , hyperactivity disorder 

ARND alcohol-relate d neurodevelopmenta l dis - 

order 


CVLT-C Californi a Verba l Learnin g Test - 

children's version 

DTI diffusio n tenso r imagin g 

FAS feta l alcohol syndrom e 

FASD feta l alcohol spectrum disorders 

fMRI functiona l magnetic resonance imagin g 

SES socioeconomi c status 

sMRI structura l magnetic resonanc e imaging 

WCST Wisconsi n Car d Sort Task 

WRAML Wid e Rang e Assessmen t o f Memor y 

and Learnin g 

ACKNOWLEDGMENTS Th e autho r would lik e t o ac - 

knowledge th e continuin g contributions of th e facult y 

and staf f o f the Materna l Substance Abuse and Chil d 

Development Laboratory, Department of Psychiatry and 

Behavioral Sciences , Emory University School of Medicine, 

an d thos e o f th e researc h participant s an d thei r 

families wh o make research on these problems possible. 

Some o f th e wor k referre d t o i n thi s chapte r was supported 

by awards to the autho r from th e followin g agencies: 

th e Georgi a Departmen t o f Huma n Resources 

(DHR), Maternal Substance Abuse Prevention Program, 

the National Institute of Alcohol Abuse and Alcoholism, 

and the Centers for Disease Control and Prevention.

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Influence of Alcohol on Structur e 

of the Developing Human Brain 

A diagnosis of fetal alcohol syndrome (FAS) requires a 

combination o f three clusters o f symptoms: (1 ) a distinct 

craniofacia l appearance, (2 ) growt h deficiency , 

and (3 ) centra l nervou s syste m (CNS ) dysfunction . 

Characteristic FAS-relate d craniofacia l dysmorphol - 

ogy includes short palpebrai fissures (eye openings), a 

smooth philtrum (the area above the upper lip), a flat 

nasal bridge an d mid-face , and thinnes s o f the uppe r 

lip. Th e growt h deficienc y related t o FA S manifests 

either prenatall y and/or postnatally, and ca n necessi - 

tate neonatal car e for failure to thrive. The thir d clas s 

of symptoms , CN S dysfunctio n an d accompanyin g 

structural brain changes , ar e the subjec t of the pres - 

ent chapter. The effect s o f prenatal alcoho l exposur e 

on cognitio n an d behavio r ar e variabl e an d wide - 

reaching. Commo n difficultie s includ e attentio n 

deficits; decrement s i n genera l intelligenc e quotien t 

(IQ); behavioral problems; visual , auditory, and per - 

ceptual disturbances ; fin e an d gros s motor problems ; 

and learnin g disabilitie s (Streissgut h an d Connor , 

2001). Indeed , th e syndrom e ha s bee n cite d a s th e 

9 

Susanna L. Fryer 

Christie L. McGee 

Andrea D. Spadon i 

Edward P. Riley 

143 

leading preventable cause of mental retardatio n (Pulsifer, 

1996) . 

The stud y o f brai n structur e i n alcohol-expose d 

individuals offers a means to understand the etiologi - 

cal relationship s tha t underli e th e neurobehaviora l 

abnormalities observed i n thes e individuals . By examining 

relationships between brain structure and behavior, 

we gain insight s int o th e mechanism s underlyin g 

alcohol teratogenicity. Before delving into a discussion 

of brain structural changes arisin g from feta l alcoho l 

exposure, i t is important t o ad d a not e o n terminol - 

ogy. In recognitio n of the wide-rangin g effect s of 

prenatal alcohol exposure, the term fetal alcohol spectrum 

disorders (FASD ) ha s been adopte d t o describ e 

the entir e rang e o f outcomes fro m prenata l alcoho l 

exposure (Bertran d e t al. , 2004) . Thes e effect s var y 

from th e pronounced facial , growth, and CNS abnor - 

malities traditionally ascribed to FAS , to subtl e neu - 

robehavioral, growth , o r physica l deficit s tha t ma y 

also occu r a s a resul t o f suc h exposure . Usin g thi s 

classification, FA S corresponds to dysmorphic FAS D


(i.e., al l thre e o f the diagnosti c criteri a ar e met , in - 

cluding th e combinatio n o f spécifi e dysmophi a re - 

quired t o mee t FA S criteria). Therefore, individual s 

with a history of prenatal alcohol exposur e and deficits 

thought to be related t o the alcohol exposure , but without 

th e necessar y criteria for an FA S diagnosis, would 

be considere d a s nondysmorphic FASD . Th e termi - 

nology use d throughou t th e presen t chapter refers to 

alcohol-exposed individual s as having FAS when they 

meet th e thre e majo r criteri a for the diagnosis , an d 

nondysmorphic FAS D when the y do not . I t is important 

to note that although there is a general consensu s 

on th e wide-rangin g an d variabl e effect s o f feta l 

alcohol exposure , diagnostic an d classificatio n terminology 

remains controversial . Thus, honing the diag - 

nostic criteri a tha t describ e feta l alcoho l effect s i s 

currently a priority for the field (Riley et al., 2003). 

AUTOPSY STUDIES 

Dysmorphic Fetal Alcohol 

Spectrum Disorde r 

Autopsy, which literall y means, "t o see for oneself," in - 

volves examinatio n t o determin e th e exac t caus e o f 

death. Autopsies are useful i n the stud y of brain disease 

processes. Postmorte m examinatio n permit s a mor e 

direct study of neuroanatomy than i n vivo techniques. 

Examination of expired tissue, however, presents a confound 

whe n attemptin g t o generaliz e th e finding s t o 

live cases. In other words, autopsies allow for a detailed 

description no t possibl e with les s invasive techniques, 

but the data they provide are not necessarily representative 

o f living cohorts. Historically , autopsy cas e report - 

ing wa s one o f th e initia l method s use d t o examin e 

teratogenic effects o f alcohol on brain structure. In th e 

autopsy case s o f children wit h FAS , death usuall y occurred 

because of major CNS o r cardiac dysfunction. 

The handfu l o f subsequent autops y report s sho w 

that developing brain s sufficiently expose d t o alcoho l 

have a host of structural abnormalities. These include 

gross microcephaly , cellula r disorganization , an d 

anomalies of specific brain structures such a s the cor - 

pus callosum an d cerebellum . Th e first autops y of a 

FAS case wa s o f an infan t wh o die d a t 5 days of ag e 

(Jones, 1975) . The infant' s brain exhibite d enlarge d 

lateral ventricles and agenesis (absence ) of the corpu s 

callosum. In addition, neuroglial heterotopias, a type 

of microdysplasia in which abnormal neural and glia l 

tissue cover parts of the brain surface, were described. 

Notably, suc h microdysplasia s ar e though t t o resul t 

from aberran t cortical neural migration and ar e com - 

mon i n autops y case s o f prenatal alcoho l exposure . 

For instance , i n a n autops y repor t o f five cases, het - 

erotopias wer e note d i n eac h case , althoug h th e 

amount o f abnormal tissu e was variable among indi - 

viduals (Wisniewsk i e t al. , 1983) . Anothe r autops y 

study that examine d brain s from fetuses , infants, an d 

one chil d corroborate d th e presenc e o f microdys - 

plasias and commente d on the diversit y of malformations 

observed (Peiffe r e t al., 1979) . Furthermore, th e 

authors suspecte d tha t dosage , i n additio n t o tim e 

course o f exposure, likely influences the degre e an d 

nature of structural damag e t o the developing brain. 

Recent neuropathologica l studie s associat e FA S 

with specific types of complex cerebral malformations 

(Coulter et al., 1993) . Evaluation o f a 2-month-old infant 

expose d t o a bing e patter n o f alcoho l exposur e 

during th e firs t trimeste r o f pregnancy wa s the firs t 

observation associatin g feta l alcoho l exposur e wit h 

midline cerebral dysgenesis. Due t o elements of septooptodysplasia, 

midlin e cerebra l dysgenesi s i s a typ e 

of midlin e malformatio n characterize d b y a lac k o f 

the septum pellucidum, optic nerv e damage , an d endocrine 

abnormalities . I n additio n t o thi s midlin e 

damage, th e cas e showe d genera l microcephal y an d 

cerebellar Purkinj e neuro n disruption . Specifically , 

the cerebellar neurons were unusually positioned and 

had abnormal dendritic structure. 

Non-Dysmorphic Feta l 

Alcohol Spectrum Disorde r 

An earl y case stud y that examine d th e brain s o f four 

neonates include d case s o f bot h dysmorphi c an d 

nondysmorphic FAS D (Clarre n et al., 1978) . Disrup - 

tions t o brain structure wer e note d i n addition t o microcephaly, 

includin g heterotopia s an d histologica l 

structural aberration s associated wit h error s i n neu - 

ronal an d glia l migration . Th e degre e o f structura l 

damage t o the alcohol-expose d brain s prompted th e 

authors to conclude "tha t problems of brain morpho - 

genesis can occur as the predominant effec t o f ethanol 

exposure in utero" (p. 67). Brain alterations may more 

directly indicat e damag e fro m alcohol-relate d effect s 

than facia l dysmorphia. 

As th e consensu s o f initia l autops y report s indi - 

cating extensiv e and diffus e damag e throughou t th e 

alcohol-exposed brai n mounted , investigator s bega n

to speculat e tha t n o particula r patter n o f behavioral 

or intellectual functioning is characteristic of individuals 

with FAS (Clarren, 1986) . Yet, neuropsychological 

studies suggest a syndrome-specific pattern o f cognitive 

and behavioral deficits associated with fetal alcohol exposure 

(Mattso n an d Riley , 1998) . Th e subject s that 

comprise autops y studie s likely represen t th e mos t severe 

cases of prenatal alcohol exposure, i.e. , those with 

damage incompatibl e with life . Thus , finding s fro m 

autopsy studies may not be representative of the major - 

ity of individuals with fetal alcohol effects. This is especially 

true in the case of FAS, because mos t of the brain 

damage cause d by fetal alcoho l exposur e doe s not preclude 

viability (Clarren, 1986) . 

IMAGING STUDIE S 

Medical imagin g technologie s detec t inheren t differ - 

ences i n biological tissu e density (e.g., gray brain matter 

vs. white brain matter vs. bone), displayin g them a s 

images with contrast differences. Imagin g technologie s 

offer th e abilit y to examine structural brain damage i n 

vivo an d includ e larger , more representativ e sample s 

than the descriptions of brain structure provided by autopsy. 

Give n tha t CNS deficit s ar e a hallmark feature 

of FASD, brain matter quantification through imaging 

studies provide s a crucia l insigh t int o specifyin g ho w 

normative brain-behavio r relationship s migh t b e af - 

fected by alcohol exposure. In contrast to autopsy, magnetic 

resonanc e imagin g (MRI ) an d othe r i n viv o 

techniques provid e information, albeit indirectly, of living 

tissue. Moreover, a s there i s no inheren t selectio n 

bias, the findings of these studies are likely more repre - 

sentative of the populatio n o f interest. 

Structural brai n imag e analyse s revea l tha t chil - 

dren an d adolescent s prenatall y expose d t o alcohol, 

with o r without dysmorphi c facial features, hav e pat - 

terns of brain structure malformations consistent with 

the neuropsychologica l an d behaviora l effect s foun d 

in thi s population . Mos t o f the existin g researc h o n 

neurostructural change s associate d wit h prenata l alcohol 

exposur e relies on structural MRI techniques . 

Total Brain Volume and Shape 

Early MR I Studies 

Most MRI studies of individuals prenatally exposed to 

alcohol hav e focuse d o n measure s of brain volume . 

ALCOHOL AN D THE DEVELOPIN G HUMA N BRAIN 14 5 

Reliably, quantitativ e volumetri c analyse s have con - 

firmed the overal l reductions i n size of the brai n an d 

the cerebra l vaul t (Swayz e e t al. , 1997 ; Archibal d 

et al, 2001 ; Autti-Ramo e t al, 2002) . Analytic tech - 

niques examining regional variations in brain size and 

shape have suggested tha t fetal alcoho l exposur e pro - 

duces a pattern o f differential brai n damage. Specifi - 

cally, th e corpu s callosum , cerebella r vermis , basa l 

ganglia, and parietal regions may show particular sensitivity 

to the effect s o f prenatal alcohol . A discussion 

of findings from specifi c studies follows. 

A series o f imaging studies from a group o f collaborators 

in San Diego has focused on images collected 

from a sample o f children an d adolescent s prenatall y 

exposed t o alcohol , bot h wit h an d withou t dysmor - 

phic feature s (ALC ) (Archibal d e t al., 2001). Analysis 

of total brain volume show s that there are lobar differ - 

ences between AL C and contro l groups . After statisti - 

cally controllin g fo r overal l brai n reduction s i n th e 

ALC group, the parieta l lobe is disproportionately reduced, 

suggestin g tha t thi s regio n i s particularly vulnerable 

t o alcoho l exposur e durin g development . 

Such data parallel findings in the mature rat, in which 

parietal corte x i s reduced b y one-third followin g prenatal 

exposure to ethanol (Mille r and Potempa, 1990 ; 

Mooney an d Napper , 2005) , but occipita l corte x is 

unaffected. 

The regiona l tissue composition i s affected b y prenatal 

exposure to ethanol. Ra w volume reductions are 

evident for both gra y and whit e matte r when FA S individuals 

are compared with controls. Yet , when over - 

all reduction s i n brai n volum e wer e statisticall y 

accounted for, only white matte r reduction s reache d 

statistical significance . Thus, global whit e matte r hy - 

poplasia appear s t o b e mor e sever e tha n globa l gra y 

matter hypoplasi a i n brain s o f individuals with FAS . 

In addition, exploratio n o f proportional tissu e composition 

i n eac h lob e reveale d tha t parieta l gra y an d 

white matte r volume s ar e disproportionately reduce d 

in individual s with FA S relative to controls , wherea s 

occipital lob e whit e matte r i s proportionally larger in 

FAS subjects. These finding s sugges t relative sparing 

of white matter in this region. 

Voxel-Based Morphometry 

Whole-brain voxel-base d morphometry (VBM ) aims 

to localize cortica l abnormalitie s by examining eac h 

voxel, o r point, o n th e brai n image . Thi s methodol - 

ogy avoid s th e nee d t o defin e boundarie s o n eac h


image, as is often necessar y with standard volumetric 

brain imagin g methods . Thus , VB M allow s for th e 

study of brain regions without clear gyral or structural 

boundaries. As voxel intensity values are considered to 

be a proxy for tissue density, in VBM, statistica l parametric 

map s depictin g average d tissu e densitie s are 

created fo r both gray and white matter. 

Results from a VBM analysis of children an d ado - 

lescents with prenatal alcohol exposure (Sowell et al., 

200 lb) complemen t earlie r volumetri c finding s o n 

the same sampl e (Archibal d et al, 2001). The VBM 

study reveal s prominen t abnormalitie s i n th e peri - 

Sylvian cortice s o f th e lef t tempora l an d parieta l 

lobes. Specifically , alcohol-exposed subject s have excess 

gra y matte r densit y and decrease d whit e matte r 

density i n thes e regions , compare d t o age-matche d 

controls. 

Shape Analysis 

Shape analysi s determines th e siz e o f th e brai n b y 

measuring th e distanc e fro m th e cente r t o variou s 

landmark point s o n th e brai n surface . Accordingly, 

the averag e brai n surfac e exten t i s smalle r fo r th e 

alcohol-exposed subject s tha n fo r control s (Sowel l 

et al. , 2002a) . This resul t i s consistent wit h myria d 

studies showin g tha t prenata l exposur e t o alcoho l 

causes microcephaly . Furthe r examinatio n reveal s 

prominent regiona l pattern s i n alcohol-induce d siz e 

and shape differences. Specifically , large group differ - 

ences ar e evident in the peri-Sylvia n and parieta l regions, 

with characteristi c narrowin g observed i n th e 

alcohol-exposed subject s and increase s in gray matter 

density. I n addition , portions o f the anterio r an d or - 

bital fronta l cortex , particularl y i n th e lef t hemi - 

sphere, ar e affected i n ALC subjects. These pattern s 

confirm tha t alcohol-induce d abnormalitie s ar e spe - 

cific rathe r tha n reflectin g diffuse damag e o r globa l 

microencephaly. 

The surfaced-base d analyti c method s use d b y 

Sowell and colleagues (2002a) allow researchers to locate 

relatively small regions of gray matter volume in - 

creases masked by reductions in gray matter. In othe r 

words, gros s volumetri c studie s hav e misse d subtl e 

anomalies b y taking the tota l brai n volume int o ac - 

count. Regiona l shap e abnormalitie s ma y occu r i n 

alcohol-exposed subjects because of abnormal myelin 

deposition tha t (a ) prevent s norma l growt h an d 

(b) leads to cortical thinning in select areas. Evidence 

of fronta l lob e abnormalitie s ar e consisten t wit h 

findings from th e cognitiv e an d behaviora l literature 

citing deficit s i n executiv e function s suc h a s plan - 

ning, cognitive flexibility, and response inhibition following 

prenatal exposur e t o alcohol (Mattso n e t al, 

1999; Connor et al, 2000; Kodituwakku et al, 2001). 

Brain shape has been explore d with surface-based 

image analysis techniques designe d to match cortica l 

anatomy betwee n hemisphere s base d o n delineate d 

landmarks (Sowel l et al, 2002b). From thi s stud y of 

differences i n brain shape and gray matter density, the 

authors not e tha t th e lef t hemispher e appear s t o b e 

more affecte d tha n th e righ t hemisphere an d specu - 

late tha t AL C subject s migh t hav e altere d hemi - 

spheric symmetr y compare d t o th e patter n see n i n 

typically developing subjects. Specifically, gray matter 

asymmetry i s more prominen t i n th e tempora l lob e 

for nonexpose d controls , wit h th e righ t hemispher e 

having more gray matter tha n th e left . This asymmetry 

i s significantl y reduce d i n alcohol-expose d sub - 

jects. The suggestio n o f altered brain asymmetry as a 

consequence of prenatal alcohol exposur e is provocative, 

although furthe r studie s are neede d befor e this 

claim ca n b e substantiate d an d it s implication s fo r 

possible behavioral effects understood . 

Taken together , dat a fro m th e Sa n Dieg o grou p 

show tha t asid e fro m globa l reduction s i n brai n 

volume, parieta l region s ar e disproportionatel y af - 

fected an d whit e matte r i s more affecte d tha n gra y 

matter. 

Cerebellum 

Several MRI studies describe cerebellar hypoplasia in 

individuals prenatall y expose d t o alcoho l wit h an d 

without FA S (Mattson e t al, 1996 ; Riikone n e t al, 

1999; Archibald et al, 2001; Autti-Ramo et al, 2002). 

Both cerebellar gray and white matter are reduced i n 

alcohol-exposed individual s (Archibald et al, 2001) . 

Reductions ar e particularl y evident i n vermal corte x 

of earlier maturing lobules (Sowel l et al., 1996) . Th e 

methodology use d t o generat e thes e result s i s as follows. 

For each subject, the midsagitta l section i s identified 

an d the vermis is divided into three regions : the 

anterior vermis (lobules I-V), lobules VI and VII, and 

lobules VIII-X. Alcohol-induced difference s ar e only 

evident i n th e anterio r vermis , wher e th e alcohol - 

exposed children showed significant reductions. This 

pattern of cerebellar vermal dysmorphology is distinct 

from th e pattern s found in children wit h other disorders, 

such a s autism, William syndrome , an d Dow n

syndrome (Raz et al, 1995 ; Courchesne e t al, 2001; 

Schmittetal.,2001). 

Animal model s o f feta l alcoho l exposur e consis - 

tently sho w abnormalitie s o f th e cerebella r vermi s 

and hemispheres . Variou s investigators have exam - 

ined th e effect s o f episodic ethano l exposur e during 

the firs t postnata l wee k (se e Goodlet t e t al. , 1990 ; 

Light e t al. , 2002) . Followin g suc h exposure , th e 

number of Purkinje neurons is reduced. As in human 

studies, the greates t differences ar e apparent in earlymaturing 

region s o f th e cerebella r vermis . Thus , 

ethanol-induced change s i n th e anima l studie s no t 

only concu r wit h th e finding s i n human s bu t als o 

show that th e Purkinj e cel l i s a key target of ethanol 

toxicity. 

Corpus Callosum 

The corpu s callosu m i s a bundle o f fibers that con - 

nects th e tw o cerebral hemispheres . I t develops during 

the secon d trimester of gestation. Autopsy reports 

document agenesi s o f thi s structur e i n infant s ex - 

posed t o alcohol i n utero. Partial or complete agene - 

sis i s describe d i n severa l imagin g studie s (Rile y 

et al, 1995 ; Johnson et al, 1996 ; Swayz e et al, 1997 ; 

Riikonen e t al, 1999 ; Clar k e t al, 2000) , and i t is 

suggested tha t prenatal alcoho l exposure is a leadin g 

cause of this condition (Jere t et al., 1986) . Mos t individuals 

expose d t o alcoho l prenatall y d o no t hav e 

such sever e alterations. Careful evaluation indicates 

that alcohol induce s subtle yet significant changes in 

the siz e an d shap e o f this structure . Studie s o n th e 

morphology of the corpus callosum reveal that anomalies 

within specific cortica l regions often correspon d 

to particular callosal subregions through whic h thos e 

regions send white matter fibers (Barkovich and Norman, 

1988) . 

Authors of the first study to systematically evaluate 

the corpus callosum divid e a midsagittal are a sectio n 

of th e callosu m int o fiv e subregion s (Rile y e t al. , 

1995). Afte r excludin g tw o subject s with complet e 

agenesis and controllin g for overall brain size, group 

differences emerge i n three callosa l subsections. Th e 

alcohol-exposed grou p display s disproportionate are a 

reductions i n th e gen u (mos t anterior section) an d 

two areas that correspond to the splenium (mos t posterior 

region). These data are consistent wit h a study 

by Autti-Ramo and colleague s (2002) , who show that 

the spleniu m o f corpus callosum i s reduced i n area, 

length, an d diamete r in children prenatally exposed 

ALCOHOL AND THE DEVELOPIN G HUMAN BRAI N 14 7 

to alcohol . Strikin g differences ar e eviden t o n map s 

charting th e averag e corpus callosu m displacemen t 

in alcohol-expose d subject s (Sowel l e t al. , ZOOla) . 

Specifically, the posterior portion o f the corpus callo - 

sum i s the sectio n mos t significantly displaced , lying 

more inferio r an d anterio r i n th e alcohol-expose d 

subjects. Thi s alcohol-induce d chang e i n callosa l 

placement correlate s wit h performanc e o n a verbal 

learning task . Indeed , displacemen t i s a bette r pre - 

dictor o f performance than testin g o f verbal IQ. I n 

other words , individual s wit h greate r callosa l dis - 

placement exhibi t increase d verba l learnin g impair - 

ments. 

A series of studies on a large sample of adolescents 

and adults exposed to alcohol prenatally evaluates the 

corpus callosu m b y mean s o f landmar k strategie s 

(Bookstein et al, 2001, 2002a , 2002b). This work develops 

usefu l discriminatio n protocols t o distinguish 

ALC fro m contro l subjects , a goal distinc t fro m tha t 

of othe r imag e analysi s research , whic h analyze s 

mean grou p difference s fo r th e purpos e o f descrip - 

tion. Variabilit y in th e corpor a callos a i s greater i n 

adolescents (Bookstei n e t al. , 2002a ) an d adult s 

(Bookstein et al, 2001) that wer e prenatally expose d 

to alcohol tha n i n age-matched controls . The author s 

suggest tha t hypervariatio n i n callosa l shap e withi n 

the AL C grou p i s a functio n o f th e timin g an d 

amounts o f exposure during th e developmen t o f this 

structure. Thus, differences i n the manner of the feta l 

alcohol exposur e may explain the numerou s patterns 

deviating from norma l callosa l variation . In addition , 

the dysmorphi c an d nondysmorphi c FAS D group s 

are indistinguishabl e wit h regar d t o callosa l hyper - 

variance. This findin g i s consistent with researc h i n 

the behaviora l domai n (Mattso n an d Riley , 1998 ; 

Connor et al., 2000 ) and speak s to the utilit y of augmenting 

the us e of facial features associated with FAS 

with MRI findings as markers of alcohol-related brain 

damage. 

Two callosa l summar y score s an d fou r land - 

mark point s ca n b e use d t o creat e a classification 

algorithm tha t discriminate s th e sampl e (alcohol - 

exposed vs . nonexposed) wit h hig h sensitivit y (10 0 

out o f 11 7 subjects ) an d specificit y (4 9 ou t o f 6 0 

subjects) (Bookstei n et al. , 2002a) . This type o f discrimination 

protoco l ma y b e especiall y usefu l a s a 

diagnostic too l fo r th e ag e rang e studie d a s man y 

conventional sign s used i n th e diagnosi s o f youn g 

children becom e les s apparent by adolescence an d 

adulthood.


A subsampl e fro m th e Seattl e studie s wa s use d 

to examin e th e relationshi p betwee n th e alcohol - 

induced callosa l hypervarianc e an d decrease d neu - 

ropsychological performance (Bookstein et al., 2002b). 

The relatio n o f callosal shap e an d neuropsychologi - 

cal performanc e wa s analyze d usin g partia l least - 

squares (PLS) analysis. Briefly, a PLS analysi s applies 

traditional multiple regression methods to latent variables 

(LVs), which are variables created fro m a factor 

analytic procedure to combine and summarize multiple 

measure s o f a construct . Th e dat a reductio n capabilities 

o f PL S ca n b e use d t o comba t bot h th e 

complexity brough t abou t b y larg e number s o f out - 

come variables and the statistical problems (e.g., multicollinearity) 

associate d wit h multiple , indirec t 

measurement o f complex constructs. On th e basi s of 

this analysis , exces s shap e variatio n correlates with 

two different profiles of cognitive deficit that are unrelated 

t o IQ o r the dysmophic/nondysmoprhi c grou p 

distinction. A relatively thick callosa l trac t i s associated 

with deficits in executive function, whereas a relatively 

thin callosum is related to motor deficits . 

The effec t o f ethanol o n the corpu s callosum has 

been examine d in nonhuman primates (Miller et al., 

1999). MRI studies and analyses of postmortem tissue 

show tha t th e callosu m i s large r i n som e ethanol - 

exposed animals, the most affected segmen t being the 

anterior par t (includin g the rostrum) . This segmen t 

interconnects th e fronta l cortice s and i s involved in 

executive function . Moreover, th e numbe r o f axons 

in th e anterio r callosu m i s increase d i n ethanol - 

exposed monkeys . I t i s importan t t o not e (a ) tha t 

these ethanol-inducec l change s ar e dose-dependen t 

and (b) that they are evident in monkeys with dysmorphic 

and nondysmorphic FASD. 

Basal Gangli a 

MRI studies show that the volume of the basal ganglia 

is reduced i n individual s prenatally expose d t o alco - 

hol (Mattson et al, 1996) . Although both the caudat e 

and the lenticular nuclei are reduced in volume, only 

the caudat e i s reduced afte r brai n size i s taken int o 

account. A larger study that further delineates subcortical 

structure s (Archibal d et al., 2001 ) describe s sig - 

nificant difference s i n childre n wit h FAS . N o 

differences ar e eviden t i n childre n wit h nondysmorphic 

FASD . Th e latte r data concu r wit h findings of 

a MR I analysi s o f th e basa l gangli a showin g n o 

abnormalities i n childre n wit h dysmorphi c an d 

nondysmorphic FASD (Autti-Ramo et al., 2002). 

Hippocampus 

The hippocampu s i s associated wit h short-term mem - 

ory an d learning . Accordin g t o neuropsychologica l 

studies of children with FASD, it appears that the hip - 

pocampus is damaged by prenatal alcohol exposure. In 

a recent study, evaluating spatial learning and memory 

in childre n wit h feta l alcoho l exposur e (Hamilto n 

et al. , 2003) , a virtual Morri s maz e tas k based o n ap - 

proaches routinely used in animal studies was used as a 

measure o f hippocampal functio n (Sutherlan d e t al. , 

2001; Johnso n an d Goodlett , 2002) . Alcohol-exposed 

children have impaired place learning relative to controls 

bu t ar e equall y proficien t durin g th e cue - 

navigation phase . Thu s th e result s o f th e huma n 

studies, like those with animals, implicate tha t ethano l 

interferes with hippocampal-mediated place learning . 

Imaging studie s sho w hippocampa l damag e i n 

alcohol-exposed subjects , althoug h no t al l studie s 

confirm thi s finding . I n a smal l sampl e o f Finnis h 

adolescents prenatally exposed to alcohol, some chil - 

dren hav e hippocampa l abnormalitie s includin g hy - 

poplasia an d regiona l thinnin g (Autti-Ram o e t al , 

2002). Moreover , i t appear s that th e hippocamp i i n 

these individuals is asymmetrical; specifically, the right 

hippocampus are significantly larger than the lef t hippocampus 

(Riikonen et al., 1999) . No lateralization is 

evident in controls. In contrast, another study describes 

relative sparing of the alcohol-exposed hippocampu s i n 

an otherwise hypoplastic brain (Archibald et al., 2001). 

Attempts to replicate existing MRI studies are neede d 

to clarify such conflicting findings. Particularly, longitudinal 

studie s will assis t i n assessin g developmental 

trends; i t i s difficul t t o meaningfull y compare cross - 

sectional sample s that differ o n crucial variables such 

as age. 

Optic Nerv e 

Eye abnormalities are frequently documented i n individuals 

with FAS . Optic nerve hypoplasia i s the mos t 

frequent for m o f ocula r dysmorpholog y associate d 

with prenata l alcoho l exposur e (Stròmlan d an d 

Pinazo-Durân, 2002) . Ten o f 1 1 childre n diagnose d 

with FA S showed evidenc e o f optic nerv e hypoplasia 

when evaluated with MRI, ophthamological examinations, 

an d electroretinogra m (ERG ) (Hu g e t al. , 

2000). In addition to the structural damage to the optic 

nerve , visual acuit y was decreased i n al l but on e 

subject. Report s o f the frequenc y o f vision problem s 

in FA S vary ; ove r hal f o f th e childre n studie d i n

a Swedis h sample ha d visual impairment, an d >10 % 

showed sever e acuity problems (Strômland , 1985 , in 

Strômland an d Pinazo-Durân , 2002) . This findin g 

indicates tha t prenata l alcoho l exposur e ha s detri - 

mental consequence s o n th e developin g visua l system, 

a conclusion corroborate d b y ERG results . Ten 

of 1 1 subject s i n th e stud y b y Hu g an d colleague s 

(2000) ha d abnorma l ER G results , attributabl e t o a 

lack o f sufficient retina l sensitivity . Data fro m oph - 

thalmological studie s o f FA S subject s sugges t tha t 

ocular deficits should be considered amon g the con - 

stellation o f presentin g symptom s o f a n alcohol - 

exposed individual . Thus, a n ey e examinatio n ma y 

be o f diagnosti c us e i n identifyin g individuals wit h 

prenatal alcoho l exposur e (Strômlan d an d Pinazo - 

Durân, 2002). 

OTHER IMAGING APPROACHES 

Ultrasonography 

In ultrasonography, sound wave s are recorded t o produce 

image s of internal organs and body tissues. This 

imaging modality 7 has been use d to examine the con - 

sequences o f prenatal alcoho l exposur e o n th e feta l 

development o f the fronta l cortex (Was s et al., 2001). 

The sampl e consist s o f 16 7 women , almos t hal f o f 

whom consume d varyin g amounts o f alcohol whil e 

pregnant. Result s from thi s stud y represen t th e spec - 

trum of fetal alcohol exposure effects; the prospectively 

identified sampl e relie s o n neithe r spontaneousl y 

aborted fetuse s nor severel y affected childre n identi - 

fied throug h retrospectiv e methods , scenario s tha t 

overrepresent case s o f heav y exposure . Th e fronta l 

lobe measuremen t wa s operationalized a s the linear 

distance fro m th e posterio r cavu m septu m pellu - 

cidum to the inner surface of the calvarium. It should 

be note d tha t this measurement i s logically less com - 

prehensive and precise than area or volume measure - 

ments o f brai n structure . Regressio n analyse s wer e 

used t o determine whic h o f several variable s studie d 

were predictiv e of i n viv o feta l fronta l lob e size . I n 

general, alcohol exposur e i s a significant predictor of 

reduced fronta l lob e size . Interestingly , other brai n 

measurements taken fro m th e ultrasonographs , suc h 

as th e distanc e betwee n th e posterio r thalamu s an d 

the inner calvarium, are less sensitive to alcohol exposure 

than th e frontal cortex . 

There is an interaction between the effect of alcohol 

exposure on the developing frontal corte x and maternal 

ALCOHOL AND THE DEVELOPIN G HUMAN BRAI N 14 9 

age (Was s e t al. , 2001) . Withi n th e alcohol-expose d 

women, a maternal age of greater than 30 years old increases 

the ris k of a fetus having a significantly smaller 

frontal lobe . Thi s findin g i s consistent wit h previou s 

research identifyin g advance d materna l ag e a s a ris k 

factor for having a child born with FAS (May and Gos - 

sage, 2001) . Thus , i t appears tha t th e deleteriou s ef - 

fects o f alcoho l exposur e o n th e developin g fronta l 

cortex are exacerbated with increased materna l age. 

Emission Computed Tomography 

Emission compute d tomograph y (CT ) technologies , 

such a s positron emissio n tomograph y (PET ) an d single 

photon emissio n computed tomograph y (SPECT), 

can be used t o measure cerebra l metabolism . Accord - 

ingly, a radiotracer is injected int o a subject's body an d 

images o f a nonsedate d individual , wh o migh t b e 

asked to perform a behavioral task, are taken. The im - 

ages are reconstructed t o appreciate the activity within 

nuclei o f the brain . Fo r the purpose s o f the researc h 

discussed i n th e presen t chapter , measurement s ob - 

tained in this way are a proxy for brain function in that 

increased metaboli c rate s within a brain regio n indi - 

cate more neural activity in that region. 

The effec t o f prenatal alcoho l exposur e o n brai n 

function wa s assesse d vi a PE T analysi s i n 1 9 youn g 

adults diagnose d wit h FA S (Clark et al, 2000). Glu - 

cose metabolis m i n subcortica l areas , includin g th e 

thalamic an d caudat e nuclei , i s decreased . I n th e 

most severe situations, ethanol causes gross structural 

deficits, wherea s othe r case s sho w subtl e effect s tha t 

vary b y region o r cel l type . These change s ar e remi - 

niscent o f ethanol-induced reductions i n glucose uti - 

lization amon g specifi c subcortical structure s i n th e 

rat brai n (Vinga n e t al. , 1986 ; Mille r an d Dow - 

Edwards, 1993) . Thus , fetal alcoho l exposur e cause s 

a continuum o f damage to the developing brain. 

SPECT has been used to evaluate the brain function 

o f 1 1 children (mea n age , 8. 6 years ) wit h FA S 

(Riikonen e t al., 1999) . Thes e children exhibi t mod - 

erate decrease s i n cerebra l bloo d flo w i n th e lef t 

parieto-occipital region . Thi s i s consisten t wit h th e 

abnormalities identified by structural MRI studies. Altered 

functio n withi n thi s regio n ma y b e relate d t o 

difficulties tha t childre n wit h FA S have wit h arith - 

metic an d speech . Additionally , th e sampl e o f chil - 

dren wit h FA S show s a n asymmetrica l patter n o f 

frontal lob e perfusion, or the patter n i n which th e radiotracer 

i s incorporate d b y th e tissue . Specifically, 

the righ t frontal region showed sligh t hyperperfusio n


compared to the pattern i n the lef t region. This differ - 

ence ma y relate t o th e attentio n deficit s commonl y 

seen in chidren wit h FAS ? although thi s findin g con - 

trasts wit h th e hypoperfusio n i n th e lef t fronta l an d 

prefrontal lobe s of children wit h attention defici t hy - 

peractivity disorde r (Sie g e t al. , 1995 ; Ame n an d 

Carmichael, 1997 ; Langlebe n e t al., 2001) . Notably, 

structural imagin g i n th e childre n wit h FA S showed 

that the brains of 6 of the 1 1 subjects ha d gros s struc - 

tural abnormalities , includin g cortica l atroph y an d 

anomalies of the corpus callosum and cerebellum . 

A recen t SPEC T stud y evaluate d cerebra l bloo d 

flow i n thre e individual s diagnosed wit h FA S (on e 

child, on e adolescent, an d one adult) (Bhatara et al., 

2002). All three case s no t only have structural abnor - 

malities but als o reduce d cerebra l bloo d flow in th e 

temporal cortex . Simila r to th e finding s o f Riikonen 

and colleagues (1999), the case study report noted lef t 

hemisphere hyperfusion , which is consistent with lef t 

hemisphere dysfunction. 


Teratogenic exposur e t o alcoho l (1 ) cause s perma - 

nent change s i n brain structure and (2 ) differentiall y 

affects specifi c components and region s o f the brain . 

These structura l change s likel y underli e deficit s i n 

the cognitiv e ability and adaptiv e functioning o f exposed 

individuals . Structura l brai n deficit s reveale d 

by quantitative image analyses of alcohol-exposed in - 

dividuals includ e reductions i n overall brain volume 

and alteration s i n brai n shape , density , an d bilatera l 

symmetry. Additionally, some structures are especially 

vulnerable t o th e teratogeni c effect s o f alcohol, no - 

tably the cerebellum , corpu s callosum , an d th e vari - 

ous structures that make up the basal ganglia. 

From a neurobehavioral poin t o f view, fetal alco - 

hol effects compris e a continuum, resulting in a range 

of devastatin g behaviora l an d cognitiv e disabilities . 

Targeted researc h definin g the degre e o f insult t o a 

structure an d th e continuu m o f neurobehaviora l effects 

will be of great clinical utility. Given that individuals 

with histories of heavy prenatal alcoho l exposur e 

face long-lastin g deficits affectin g multipl e aspect s o f 

cognitive and behavioral functioning, identification of 

brain areas likely associated wit h such deficit s will improve 

diagnostic and treatment strategies. 

Multidisciplinary research i s needed t o fill in th e 

gaps in our current knowledge and open new light on 

how structura l deficit s i n th e alcohol-expose d brai n 

relate t o neurobehaviora l outcome . Accordingly , a 

recent meetin g o f internationa l collaborator s high - 

lighted th e critica l importanc e o f research focusin g 

on the relationship between brain structure and function 

i n FASD (Rile y et al., 2003). In particular, func - 

tional imaging studies will provide an in vivo account 

of ho w prenata l alcoho l exposur e affect s neura l re - 

source allocation , o n a cognitive task-by-tas k basis. I t 

is possibl e tha t futur e researc h wil l sho w tha t a 

pathognomonic FAS D brai n doe s no t exist . Regardless 

of the wide-rangin g neura l complication s tha t fetal 

alcoho l effect s ma y exhibit , th e keyston e o f 

effective interventio n rests on explicatin g how behav - 

ioral sequelae resul t from teratogenic alteration o f the 

brain. 

Abbreviations 

ALC prenatall y expose d t o alcohol , bot h wit h 

and without dysmorphic features 


CT compute d tomograph y 


FASD feta l alcohol spectru m disorder s 

IQ intelligenc e quotien t 

LV laten t variable 

MRI magneti c resonance imaging 

PET positro n emission tomography 

PLS partia l least squares analysis 

SPECT singl e photo n emissio n compute d to - 

mography 

VBM voxel-base d morphometry 

ACKNOWLEDGMENTS Thi s wor k wa s supporte d b y 

the Nationa l Institutes of Heat h (AA01352 5 an d AA01 

0417). 

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10 

Prenatal Ethanol Exposure 

and Fetal Programming: 

Implications for Endocrine 

and Immune Development 

and Long-Term Health 

Ethanol use and abuse result in clinical abnormalities 

of endocrin e functio n an d neuroendocrin e regula - 

tion (Morgan, 1982 ; Adler, 1992). Direct and indirect 

effects o f ethanol o n man y hormone systems , including 

th e adrenal , gonadal, and thyroi d axes, as well as 

on aldosterone , growt h hormone , parathyroi d hor - 

mone, calcitonin , insulin , and glucagon , hav e been 

reported. Secondar y complications suc h a s liver disease, 

malnutrition , and othe r medica l condition s often 

presen t i n alcoholic s ma y i n an d o f themselves 

have endocrine consequences an d can potentially exacerbate 

the adverse effects o f ethanol. 

Whether ethanol-induce d endocrin e imbalance s 

contribute t o th e etiolog y o f fetal alcoho l spectru m 

disorders (FASD) is unknown, but i t is certainly a possibility 

(Anderson , 1981) . Th e effect s o f ethanol o n 

interactions betwee n th e pregnan t femal e an d fetu s 

are comple x (Rudee n an d Taylor , 1992 ; Weinberg , 

1993a, 1993b , 1994) , and bot h direc t and indirec t effects 

o f ethanol o n feta l developmen t occur . Ethano l 

readily crosse s th e placenta , thu s directl y affectin g 

Joanna H. Sliwowska 

Xingqi Zhang 

Joanne Weinberg 

153 

developing fetal cell s and tissues , including those re - 

lated t o endocrin e function . I n addition , ethanol - 

induced change s i n endocrin e functio n ca n disrup t 

the hormona l interaction s betwee n th e pregnan t female 

and feta l systems , altering the normal hormon e 

balance an d indirectl y affecting th e developmen t of 

fetal metabolic , physiological , an d endocrin e func - 

tions. Ethanol-induce d change s i n metaboli c and/o r 

endocrine function can also affect a female's ability to 

maintain a successful pregnancy , resulting in miscarriage 

or , i f the fetu s i s carried to term, possible con - 

genital defects. Of particular relevance to the present 

Chapter i s the notio n tha t disturbances of the recip - 

rocal interconnection s betwee n (a ) th e pregnan t 

female an d fetu s o r (b ) th e materna l an d neona - 

tal hypothalamic-pituitary-adrena l (HPA ) axes, ma y 

provide a common pathway by which perinatal exposure 

t o differen t agent s results in feta l programmin g 

(Angelucci e t al. , 1985) . Fetal o r early programming 

refers to the concept that early environmental or nongenetic 

factors , includin g pre- o r perinatal exposur e


to drugs or other toxic agents, ca n permanentl y organize 

o r imprint physiological and behaviora l systems 

and increas e vulnerabilit y t o illnesse s o r disorder s 

later i n lif e (Matthews , 2000 , 2002 ; Bakke r e t al , 

2001; Welberg and Seckl , 2001). 

The presen t Chapte r focuse s o n th e advers e ef - 

fects of prenatal ethanol exposur e on neuroendocrin e 

and immun e function , wit h particula r emphasi s o n 

the concept of fetal programming and the HP A axis, a 

key playe r i n th e stres s response . Th e HP A axi s i s 

highly susceptibl e t o programmin g durin g feta l an d 

neonatal developmen t (Matthews , 2000 , 2002 ; Wel - 

berg an d Seckl , 2001) . Earl y environmental experi - 

ences, includin g exposure to ethanol, ca n reprogra m 

the HP A axi s suc h tha t HP A ton e i s increase d 

throughout life . Thi s chapte r present s dat a demon - 

strating tha t gestationa l ethano l exposur e increase s 

HPA activity in both th e pregnant female and th e off - 

spring. Evidenc e suggestin g that increase d exposur e 

to endogenou s glucocorticoid s ove r th e lifespa n ca n 

alter behavioral and physiological responsiveness and 

predispose th e organis m t o developmen t o f certai n 

diseases late r i n lif e i s also described . Alteration s i n 

immune function may be one of the consequence s o f 

fetal HP A programming. Th e chapte r discusse s stud - 

ies demonstratin g tha t ethano l i s an immunoterato - 

genic agen t an d tha t programmin g o f HP A activity 

may mediat e som e o f the advers e effect s o f prenatal 

ethanol exposur e o n immun e competenc e i n late r 

life. 

THE CONCEPT O F STRESS 

The ter m stress, used in the biological sense, was popularized 

by Hans Selye , who proposed tha t stres s can 

be understoo d withi n th e contex t o f th e "genera l 

adaptation syndrome. " Thi s concep t gre w ou t o f 

Selye's observation s that a wid e variet y of physically 

noxious stimuli , suc h a s col d o r hea t exposure , sur - 

gery, muscular exercise , bacteria, toxins, or X-irradiation, 

resulted in essentially the same triad of symptoms: 

(1) adrena l cortica l enlargement , (2 ) thymic involu - 

tion o r atrophy, and (3 ) gastrointestinal ulcers (Selye , 

1936, 1950) . 

The respons e tria d hold s despit e th e fac t tha t a 

highly specific adaptive response exist s for any one o f 

these agents by itself. These symptoms are considere d 

a nonspecific adaptive response o f the bod y to a physical 

stressor and par t of the "alar m reaction, " th e first 

stage o f the genera l adaptatio n syndrome . This stag e 

is characterized b y activation of the HP A system, an d 

the correspondin g change s i n immune functio n an d 

gastric ulcération are thought to be mediated t o som e 

extent b y this HPA activation. If the stressfu l stimulu s 

persists, the organism enters the second, or resistance, 

phase o f th e genera l adaptatio n syndrome , durin g 

which adrenocortica l activatio n i s maintained. Afte r 

repeated o r constan t exposur e t o th e stresso r the or - 

ganism enter s th e thir d phase , whe n th e capacit y of 

the adrena l corte x t o synthesize , store , an d secret e 

glucocorticoids i s exceeded (Selye , 1946) . Althoug h 

not all aspects of the general adaptation syndrome are 

supported b y subsequent experimenta l evidence , thi s 

concept has provided a powerful context fo r stress research 

fo r many years. 

The concep t of nonspecificity (Sely e 1936 , 1950 ) 

has raise d a critica l question : throug h wha t mecha - 

nisms coul d s o man y divers e agent s transmi t th e 

common "message " of stress ? Sely e speculate d tha t 

there mus t b e som e physiologica l "firs t mediator " o f 

stress, i.e. , som e nonspecifi c chemica l o r byproduc t 

of biological reaction s that produced thes e symptom s 

indicative o f stres s (Mason , 1975) . Th e searc h fo r 

physiological firs t mediator s wa s largel y unproduc - 

tive. By the 1960s , stres s researchers became increas - 

ingly awar e tha t (a ) psychologica l an d socia l factor s 

had effect s simila r to and perhap s eve n mor e poten t 

than thos e o f physical stressors and (b ) that th e HP A 

axis wa s particularly sensitive t o thes e psychologica l 

stimuli (Mason , 1968) . Thi s growin g understandin g 

of th e rol e o f psychologica l variable s le d Maso n 

(1975) t o sugges t tha t th e unrecognize d firs t media - 

tors i n man y o f Selye' s experiment s ma y hav e bee n 

the substrat e i n the central nervou s system (CNS) in - 

volved in emotional arousal . That HPA activation frequently 

occurred durin g novel or aversive stimulation 

is not surprising because th e HP A axis is an excellen t 

indicator o f arousa l (Henness y an d Levine , 1979) . 

This understanding altere d th e concep t o f nonspeci - 

ficity, at least in terms of the HP A response. Instea d of 

viewing hormona l response s a s bein g elicite d b y a 

great diversity of stimuli, they coul d b e viewed as being 

elicited largel y by the emotiona l arousa l or activation 

commo n t o mos t i f no t al l nove l an d aversiv e 

situations. 

A concept o f stress emphasizing the "emergency " 

function o f the sympatheti c nervou s system an d th e 

adrenal medull a an d thei r role s i n maintainin g in - 

ternal homeostasi s come s fro m th e wor k of Cannon

FIGURE 10- 1 Schemati c representatio n of circuitry of 

the stress system. The tw o major system s central to the 

stress respons e ar e th e hypothalamic-pituitary-adrena l 

(HPA) axis and the locus coeruleus noradrenergic sympathetic 

adrenal medullary (LC-NE) system , which interact 

to maintain homeostasis . Th e HP A axis consists 

of a cascade o f responses. Corticotropin-releasing hor - 

mone (CRH) , secrete d b y th e hypothalamus , stimu - 

lates th e releas e o f adrenocorticotropi c hormon e 

(ACTH) fro m th e anterio r pituitary , which i n tur n 

stimulates release of the glucocorticoid hormones fro m 

the adrena l cortex . Th e glucocorticoid s fee d bac k a t 

multiple levels of the axi s to inhibit activity. In the LC - 

NE system , norepinephrine (NE) , release d primarily 

from sympatheti c nerv e terminals , an d epinephrine , 

released primaril y from th e adrena l medulla , activat e 

sympathetic responses. Homeostasis is restored by activation 

of the parasympathetic system. The LC-N E system 

allow s the organis m t o reac t rapidly , while th e 

HPA hormones act over a longer time frame. There are 

intimate reciproca l interactions between th e HP A axis 

and th e LC-N E system , a s well a s reciproca l neura l 

connections betwee n th e tw o systems. CRH an d N E 

stimulate each other, and the two systems are regulated 

by similar neurotransmitters and b y mesocortical an d 

mesolimbic influences . Glucocorticoid s ar e though t 

to restrain both system s to prevent the consequence s 

of prolonge d o r excessiv e activation . Finally , activit y 

and sensitivit y of both system s are modulated by stress 

and circadia n influences . AVP, arginine vasopressin; 

BZD, benzodiazepine ; GABA , y-aminobutyri c acid ; 

NPY, neuropeptid e Y ; POMC, proopiomelanocortin ; 

ETHANOL EFFECTS ON ENDOCRINE AND IMMUNE FUNCTION 15 5 

and colleague s (Canno n 1914 , 1929 ; Canno n an d 

de la Paz, 1911) . Accordingly, homeostasi s i s viewed 

as th e operatio n o f coordinate d physiologica l pro - 

cesses that maintain the steady state of the organism. 

These authors also recognize th e importanc e o f psychological 

ove r physica l stimul i i n elicitin g a stres s 

response. 

In moder n stres s research , i t i s no w widel y ac - 

cepted tha t the respons e to stress is mediated b y both 

the HP A axis and th e locu s coeruleu s noradrenergi c 

sympathetic adrena l medullar y system (referre d t o as 

the LC-NE system) and that these system s interact to 

maintain homeostasi s (Fig . 10-1) . Th e LC-N E system 

i s involved i n th e "fight-or-flight " respons e an d 

enables th e organis m t o reac t rapidly . Tw o majo r 

players i n thi s rapi d respons e ar e norepinephrin e 

(NE), secreted from sympathetic nerve terminals, and 

epinephrine, release d fro m th e adrena l medulla . I n 

contrast, th e HP A axis acts ove r a longer tim e fram e 

and help s orchestrat e th e respons e an d adaptatio n of 

the bod y to the stresso r through variou s physiological 

and metabolic changes . 

The ter m stress ha s prevaile d ove r decade s be - 

cause i t attempt s t o addres s a basi c principl e o f na - 

ture: (1) the maintenance o f balance, equilibrium , or 

harmony i n th e fac e o f disturbin g stimul i an d (2 ) 

the counteracting , re-establishin g response s tha t re - 

establish homeostasi s (Chrouso s e t al. , 1988) . Man y 

definitions an d meaning s hav e been an d stil l are ascribed 

t o the term stress, largely because the term has 

been use d to refer t o the disturbin g stimuli, the stat e 

of disturbe d equilibrium , and/o r th e result s o f th e 

counteracting responses . Thi s chapte r use s th e fol - 

lowing definition s (Johnso n e t al. , 1992 ; Mille r an d 

O'Callaghan, 2002). Stress is a state of threatened in - 

ternal balance or homeostasis. The threatenin g or disturbing 

force s ar e define d a s stressors. Thes e ca n 

range from rea l threats to survival (e.g., immune chal - 

lenges or physical stressors) to perceived threats (e.g., 

psychological o r socia l stressors) . The counteractin g 

forces activate d t o neutraliz e th e effect s o f a stressor 

are adaptive responses, which ca n b e both behaviora l 

and physica l o r physiological , an d serv e t o re - 

establish homeostasis. 

SP, substanc e P . Activatio n i s represente d b y soli d 

lines, inhibitio n b y dashe d lines . (Source: Reprinte d 

from Chrousos (1998) with permission fro m Ne w York 

Academy of Sciences)


In recen t years , the concept s o f allostasis and al - 

lostatic loa d hav e been added t o the stres s literature 

and hav e extende d ou r thinkin g about homeostasi s 

(McEwen, 1998 ; McEwen an d Wingfield, 2003). In 

this framework , th e ter m homeostasis refer s t o th e 

stability o f physiologica l system s tha t ar e essentia l 

for lif e an d applie s strictl y to a limite d numbe r o f 

systems, suc h a s pH , bod y temperature , glucos e 

contents, an d oxyge n tension , tha t ar e maintaine d 

within narrow ranges optimal for current life condi - 

tions. I n contrast , th e ter m allostasis mean s th e 

achieving o f stability through chang e an d refer s t o 

systems suc h a s the HP A axis, catecholamines , an d 

cytokines that maintai n homeostati c system s in bal - 

ance. Thes e system s ar e typicall y no t require d fo r 

immediate surviva l an d hav e much broade r bound - 

aries than thos e o f homeostatic systems . Further, a s 

environments o r lif e circumstance s change , th e se t 

points or boundaries of these allostati c systems may 

also change. 

Allostatic system s enabl e u s t o respon d t o ou r 

physical state s —for example , awake , asleep , exer - 

cising, etc . —and t o cop e wit h challenge s suc h a s 

noise, crowding, isolation, extremes of temperature, 

danger, an d infectio n (McEwen , 1998) . Th e bod y 

responds t o challenge s b y turning o n allostati c responses 

(mos t commonly , fo r example , activatio n 

of th e HP A axi s and/o r LC-N E system ) that initi - 

ate comple x adaptiv e response s an d the n shuttin g 

off these response s when the threat is past. The ter m 

allostatic state refer s t o altere d an d sustaine d ac - 

tivities of the primar y response mediators , th e HP A 

and catecholamin e hormones , tha t integrat e phys - 

iological an d behaviora l response s t o challenge . 

Allostatic state s ca n b e sustaine d fo r limite d peri - 

ods, however, if inactivation i s inefficient o r th e or - 

ganism i s exposed repeatedl y to intense challenges . 

The cumulativ e resul t o f an allostati c stat e i s allo - 

static load , whic h ca n hav e pathophysiologi c con - 

sequences. 

The HP A Axis and p-Endorphin Syste m 

The HP A axi s include s a numbe r o f brai n areas , 

such a s the paraventricular nucleus o f the hypothal - 

amus (PVN), the anterior pituitary, and adrenal cortex, 

that act together t o produce a hormonal cascad e 

in respons e t o stressor s (Fig . 10.1) . Corticotropin - 

releasing hormone (CRH ) an d arginin e vasopressin 

(AVP) are synthesized in the parvocellular PVN an d 

released int o th e media n eminence , reachin g th e 

anterior pituitary via the hypophysia l portal system. 

CRH an d AV P act synergisticall y at the anterio r pituitary 

t o stimulat e th e synthesi s o f proopiome - 

lanocortin (POMC) , a large precursor hormone tha t 

when cleave d result s i n th e subsequen t releas e o f 

adrenocorticotropic hormon e (ACTH ) an d th e en - 

dogenous opioi d p-endorphi n (p-EP ) (Guillemi n 

et al, 1977 ; Gillies et al., 1982) . ACTH then act s at 

the adrena l corte x t o stimulat e th e synthesi s an d 

release o f glucocorticoids —cortisol i n human s an d 

corticosterone (CORT ) i n rats . In addition to a variety 

of important physiologica l and metaboli c func - 

tions, th e glucocorticoid s hav e a negativ e feedbac k 

action, causin g inhibitio n o f HP A activit y b y act - 

ing a t th e anterio r pituitary , th e PVN , an d othe r 

brain regions , particularl y th e hippocampu s an d 

prefrontal cortex . During norma l endocrine adapta - 

tion t o stressors , CRH, ACTH , an d glucocorticoi d 

concentrations ar e kep t withi n level s tha t promot e 

health. Whe n copin g fails , a n imbalanc e develop s 

between driv e an d feedbac k mechanisms , result - 

ing i n th e releas e o f too muc h o r too littl e o f these 

hormones an d increase d vulnerabilit y t o diseas e 

(Chrousosetal, 1988) . 

The Concept of Fetal Programmin g 

Both epidemiologica l dat a an d experimenta l stud - 

ies sugges t tha t environmenta l factor s operatin g 

early in life markedly affect developin g systems , per - 

manently alterin g structur e an d functio n through - 

out lif e (Fig . 10-2) . Althoug h th e ide a tha t earl y 

experiences ca n hav e long-ter m effect s i s not new , 

it i s reall y onl y i n th e pas t decad e o r s o tha t th e 

concept o f fetal o r early programming ha s emerge d 

as a ke y concep t i n development . Thi s concep t 

arises fro m a larg e bod y o f dat a showin g tha t lo w 

birth weigh t an d othe r indice s o f feta l growt h ar e 

associated wit h increase d biologica l ris k fo r coro - 

nary hear t disease , hypertension , an d typ e I I dia - 

betes o r impaire d glucos e toleranc e i n adul t life . 

Adult lifestyl e factor s suc h a s smoking , ethano l 

consumption, an d exercis e appea r t o b e additiv e 

to earl y lif e influences , thu s th e earl y lif e effect s 

may hav e distinc t role s an d causes . Thes e find - 

ings hav e le d t o th e hypothesi s tha t commo n 

adult disease s migh t originat e durin g feta l devel -

opment, i.e. , th e "feta l origin s o f adul t disease " 

hypothesis. 

The biologica l significanc e of physiological an d 

behavioral programmin g i s not known . I t has been 

suggested tha t environmenta l factor s actin g o n th e 

pregnant femal e and fetu s ca n alte r the se t point or 

responsiveness of physiological systems and thus prepare 

th e organis m fo r the environmen t int o whic h 

it will be born and develop . If , however, this process 

is initiate d b y advers e factor s durin g pregnanc y 

(e.g., placenta l insufficienc y o r prenata l ethano l 

exposure) o r i f th e environmenta l circumstance s 

later i n lif e diffe r fro m wha t wa s anticipated, the n 

the physiologica l adaptation s migh t alon e resul t i n 

maladaptive response s an d ultimatel y predispos e 

the organis m t o diseas e (Welber g an d Seckl , 2001 ; 

Matthews, 2002). 

The underlyin g processe s tha t lin k earl y growth 

restriction with these long-term healt h consequence s 

are no t full y understood . I t i s generall y accepte d 

that lo w birt h weigh t pe r s e i s unlikel y t o caus e 


these increased risks for disease. Rather, birth weight 

likely serves as a marke r fo r th e effect s o f earl y lif e 

events, and commo n factors probabl y underlie bot h 

the intrauterine growth retardation and altered physiological 

responsivenes s (Welber g an d Seckl , 2001) . 

The resettin g of key hormonal system s by early environmental 

event s ma y be on e mechanis m linkin g 

early lif e experience s wit h long-ter m healt h conse - 

quences. Studie s have identified the HP A axis as one 

of th e ke y system s likel y involve d (Fig . 10-2) . Th e 

HPA axis is highly susceptible to programming during 

development (Phillip s e t al. , 1998 , 2000 ; Matthews , 

2000, 2002 ) an d studie s demonstrat e stron g correla - 

tions betwee n birt h weight , plasm a cortiso l concen - 

trations, an d th e developmen t o f hypertensio n an d 

type I I diabetes. Thus, i t has been suggeste d tha t intrauterine 

programming o f the HP A axis i s a mechanism 

underlyin g th e observe d association s betwee n 

low birth weight and increase d ris k for disease. In order 

t o understan d feta l programmin g mor e fully , 

it is important to describe how the HP A axis develops. 

FIGURE 10- 2 Route s b y whic h th e feta l an d earl y neonata l environmen t 

can progra m adul t hypothalamic-pituitary-adrena l (HPA ) functio n an d 

behavior. Th e developin g linibi c system , hypothalamus , an d pituitar y synthesize 

hig h level s of corticosteroid receptors and ar e sensitive t o glucocorti - 

coids (GCs) . Early exposure to GCs alter s the developmen t an d subsequen t 

activity an d functio n o f both th e limbi c syste m an d th e HP A axis . I n th e 

periphery, th e overal l effec t o f programmin g durin g developmen t i s al - 

tered exposur e t o endogenou s GC s throughou t life . Increase d exposur e ha s 

long-term effect s o n behavior , cognition , learning , an d memory , an d pre - 

disposes th e individua l t o neurological , metabolic , an d immun e disease s 

and disorder s i n late r life . Conversely , i f programming reduces exposur e t o 

GCs ove r th e lifespan , thi s migh t ac t t o protec t agains t thes e pathologica l 

changes. (Source: Reprinte d fro m Matthew s (2002 ) wit h permissio n fro m 

Elsevier)


DEVELOPMENT OF THE HP A AXIS 

The HPA Axis in 

Human Developmen t 

As a result of the difficult y o f sampling an d measurin g 

ACTH, few data exis t on regulatio n o f ACTH secre - 

tion i n the human fetus . By 9 weeks of gestation, th e 

human feta l pituitar y contain s measurabl e corti - 

cotropic activit y and the content and concentration o f 

ACTH increase s wit h gestationa l ag e (Kasti n e t al. , 

1968; Pavlov a et al, 1968) . B y 12 weeks of gestation, 

ACTH is detectable b y radioimmunoassay in huma n 

placenta. ACT H concentratio n i s hig h prio r t o 3 4 

weeks an d the n fall s significantl y i n lat e gestatio n 

(Winters et al, 1974) . Infusio n o f ACTH to the preg - 

nant female at term does not affect the concentration s 

of plasma ACTH in the fetus, providing evidence that 

this hormone doe s no t cros s the placent a (Miyakaw a 

et al, 1976) . 

In contras t t o ACTH , concentration s o f cortiso l 

are hig h i n th e bloo d o f the pregnan t woma n an d 

there i s transplacental transfer o f cortisol (Pasqualini 

et al., 1970) . Feta l exposur e t o cortisol i s minimized , 

however, because cortisol is rapidly inactivated by the 

placental enzym e 1 1 p-hydroxysteroid dehydrogenas e 

type 2 (Seckl , 2004a , 2004b) . Abou t 80 % o f cortisol 

derived fro m th e pregnan t femal e i s converte d t o 

CORT (Beitin s e t al. , 1973 ; Murph y e t al, 1974) . 

The remainin g fractio n make s a n importan t contri - 

bution t o feta l plasm a cortisol . Childre n expose d t o 

dexamethasone ( a syntheti c glucocorticoid ) durin g 

early pregnancy becaus e o f a risk of congenital adre - 

nal hyperplasia show increased emotionality, unsociability, 

avoidance, and behavioral problems (Trautma n 

et al., 1995) . Th e rol e of prenatal glucocorticoid s i n 

animal model s o f FAS D i s discussed i n th e sectio n 

Prenatal Ethano l Exposur e Alters th e Immun e Sys - 

tem o f Offspring (below). 

The HPA Axis in Rodent s 

Studies on rats show that CORT secretion i n respons e 

to stressor s i s ver y hig h durin g th e lat e feta l perio d 

(Milkovic and Milkovic , 1963) . I n contrast, beginnin g 

on abou t postnatal da y (P) 2 and continuin g int o th e 

second wee k of life, the HPA response i s markedly suppressed 

in rat pups. The pioneerin g work of Schapiro 

(1962) identifie s thi s developmenta l stag e an d call s 

it th e "stres s nonresponsiv e period " (SNRP) . Late r 

studies sho w tha t HP A responsivenes s i s not actuall y 

abolished durin g thi s perio d bu t simpl y suppressed , 

and thi s perio d ha s bee n rename d th e "stres s hypo - 

responsive period " (SHRP) . Th e SHR P i s character - 

ized b y blunte d adrena l COR T secretion , despit e 

normal pituitary ACTH response s to a variety of stressors. 

Hypothalamic CRH content is decreased t o about 

20% o f that found i n 21-day-ol d animals, and ther e is 

also a decrease i n functionally active glucocorticoid re - 

ceptors t o about 20% of those found i n adults (Levine 

et al, 1967; Meane y et al, 1985a, 1985b ; Walker et al, 

1986a, 1991 ; Witek-Janusek, 1988) . This blunted basal 

and stimulate d COR T secretion i s believed t o be du e 

in par t t o a decreas e i n adrena l sensitivit y to ACTH 

(Levine et al., 1967 ) an d increas e in sensitivity to HPA 

feedback (Walke r e t al, 1986b) . Th e lo w circulating 

glucocorticoid concentration s durin g th e SHR P ap - 

pear to have a protective functio n and are essential for 

normal brain and behaviora l development (Rosenfel d 

et al, 1992 ; Walke r e t al, 2002) . Fo r example , rat s 

treated wit h glucocorticoid s durin g th e firs t wee k o f 

life hav e reduce d brai n weights , alteration s i n neu - 

rons (e.g. , reduced number s of dendritic spines), glia, 

and myelin , an d behaviora l change s (Sapolsk y an d 

Meaney, 1986) . Similarly, CORT treatment during the 

first postnata l wee k result s i n lowere d adul t basa l 

CORT concentrations (Turne r an d Taylor, 1976 ) an d 

neonatal treatmen t wit h ACTH o n P7-P9 and PI 7- 

P19 suppresses th e COR T circadian rhyth m i n adul t 

rats (Taylo r e t al. , 1976) . Th e presenc e o f th e da m 

appears t o be critica l fo r maintaining th e SHRP , an d 

separation fro m th e mothe r "releases " th e pu p HP A 

axis from inhibition . I n 10-day-ol d pups, fo r example , 

maternal separatio n (removin g activ e sensory stimula - 

tion, food , an d passiv e contact ) enhance d basa l an d 

stimulated ACTH and CORT secretion afte r exposur e 

to ethe r vapor s an d insulin-induce d hypoglycemi a 

(Walker, 1995) . Thu s i t appear s tha t th e firs t 2 post - 

natal weeks represent a critical period correspondin g to 

specific stages in the normal development o f hypothalamic 

an d forebrai n structure s tha t modulat e ACT H 

and COR T secretio n a s wel l a s CN S structure s in - 

volved in circadian regulation. 

By weaning age (P21-P25), the plasma ACTH and 

CORT responses t o stressors appear to be at essentially 

adult concentrations. Measures of CRH hav e shown a 

similar developmenta l pattern , with adul t responsive - 

ness bein g reache d b y weanin g ag e (Hiroshig e an d 

Sato, 1970) . I n contrast , negativ e feedbac k mecha - 

nisms continu e t o increas e i n effectivenes s betwee n

weaning age and adulthood . Whe n expose d t o ethe r 

or shock, for example, the plasm a CORT response of 

the weanling shows a later peak and delayed return to 

resting concentration s compared with th e patter n i n 

adulthood (Goldman et al, 1973). 

In summary, in the rat, the postnatal period is critical 

for the developmen t and integratio n of the CN S 

and peripheral nervous system mechanisms necessary 

to maintain homeostasis. I n contrast , many of these 

systems reach a greater degree of organization at birth 

in guine a pigs , sheep, nonhuma n primates , and hu - 

mans (Weaver et al., 2004). 

GESTATIONAL ETHANOL 

EXPOSURE AND HP A ACTIVITY 

Human Studies 

Compared t o the fairl y larg e literature on the effect s 

of ethanol consumption on the HP A axis of adults following 

acut e o r chroni c ethano l intake , onl y a few 

human studie s have examined the effect s o f drinking 

during pregnanc y o n th e HP A axi s o f th e develop - 

ing child . I n cas e studie s b y Roo t an d colleague s 

(1975), plasma cortisol concentrations are within normal 

rang e i n childre n wit h FASD . B y contrast, dat a 

from Jacobson and colleague s (1999 ) show that heavy 7 

drinking during pregnancy and a t conception ar e associated 

with higher basal and post-stress (blood draw) 

cortisol concentrations i n 13-month-ol d infants. Basal 

cortisol concentrations are also higher in 2-month-old 

infants expose d i n uter o t o ethano l o r cigarette s 

(Ramsay et al., 1996) . Animal studies strongly support 

this finding that ethano l exposur e i n utero ca n hav e 

major effect s o n developmen t an d functio n o f th e 

offspring HP A axis. Not surprisingly , HPA activity in 

the pregnan t femal e i s also altered b y ethano l consumption. 

Animal Studie s 

Effects of Ethanol Consumption on 

HPA Activity of the Pregnant Female 

Data fro m roden t model s indicat e tha t gestationa l 

ethanol consumption increases adrenal weights, basal 

CORT concentrations , th e COR T stres s response , 

and the CORT stress increment in the pregnant dam 

(Weinberg and Bezio , 1987) . This occurs a s early as 


gestational da y (G) 11 , persists throughout gestation , 

may increase as gestation progresses, and occurs even 

with low concentrations o f ethanol i n the die t (Weinberg 

and Bezio , 1987 ; Weinber g and Gallo , 1982) . 

Importantly, thi s activatio n appears to b e relate d t o 

ethanol rather tha n t o nutritional factor s a s maternal 

nutritional status has no major impact on these effect s 

(Weinberg and Bezio , 1987) . The stimulator y effect s 

of ethanol o n basal hormone concentration s and th e 

corticoid response to stressors also can extend throug h 

parturition, eve n whe n ethano l administratio n i s 

discontinued prio r t o parturitio n (Weinberg , 1989) . 

Thus regula r consumption o f high dose s o f ethano l 

during pregnancy not only raises the set point of HPA 

function i n th e pregnan t femal e b y increasin g both 

basal an d stres s concentrations of CORT but als o results 

in HPA hyperresponsiveness to stressors. The ad - 

ditional finding that binding capacity of corticosterone 

binding globuli n (CBG ) i n ethanol-consumin g fe - 

males is similar to or less than that in control females, 

both durin g pregnanc y an d a t parturition , indicate s 

that the elevated CORT concentrations are functionally 

important an d support s the conclusio n tha t pre - 

natal ethanol exposur e results in both hypersécrétio n 

and hyperresponsivenes s of the HP A axi s (Weinber g 

and Bezio, 1987 ; Weinberg, 1989) . 

The pregnan t femal e an d th e fetu s constitut e a n 

interrelated functiona l unit. Hence, ethanol-induced 

alterations i n HP A activit y i n th e pregnan t femal e 

have implications for fetal HPA development. COR T 

crosses the placenta , a t least to some extent (Eguchi, 

1969), resultin g i n suppressio n o f endogenou s feta l 

HPA activity. At the same time, ethanol ca n cros s the 

placenta an d directl y activate the feta l HP A axis. Together 

thes e influences may have permanent organi - 

zational effect s o n neura l structure s tha t regulat e 

HPA activit y throughout lif e (Levin e an d Mullins , 

1966). Whether th e increased plasma CORT concentration 

i n pregnant female s plays a role i n mediatin g 

HPA hyperresponsivenes s i n feta l ethanol-expose d 

(FEE) offsprin g i s as yet unresolved . Adrenalectom y 

(ADX) o f the pregnan t dam ha s n o effec t o n th e in - 

creased CORT responses to restraint stress in FEE female 

offsprin g (Slon e an d Redei , 2002) . Similarly , 

CORT treatment o f ADX dams does no t mimi c th e 

effect o f prenatal ethanol exposure on HPA activity of 

offspring (Le e and Rivier , 1992 ) On th e basis of these 

data, i t appear s tha t th e increase d stres s responsive - 

ness of FEE offsprin g i s not mediated primarily by increased 

COR T concentration s i n pregnan t females .


In contrast , ADX of the pregnan t female can reverse 

the effect s o f prenatal ethanol exposur e o n pituitar y 

POMC mRNA concentrations in FE E offsprin g (Re - 

dei e t al. , 1993) . Furthermore , Trit t an d colleague s 

(1993) have suggested that removal of the adrenal cortex, 

but no t the medulla, in the pregnant female prevents 

th e growth-retardin g effect s o f prenatal ethano l 

and ma y reverse the delay in postpartum weight gain 

in FEE offspring . Thu s the advers e effects o f ethanol 

on birth weight are mediated at least partially through 

the adrenal gland. Further studies must be done to resolve 

the rol e of corticosteroids derive d from th e preg - 

nant female in mediating the adverse effects o f ethanol 

on their offspring. I t is likely, however, that a comple x 

interaction betwee n direc t an d indirec t effect s o f 

ethanol mediates the adverse consequences of ethanol 

consumption durin g pregnancy on fetal developmen t 

and programming of fetal HP A activity. 

Effects of Prenatal Ethanol Exposure 

on HPA Activity in Offspring 

The Preweanin g Period. Th e comple x interactio n 

of direct and indirec t effect s o f ethanol i s apparent i n 

the offsprin g followin g parturition. FE E fetuse s exhibit 

decrease d COR T concentration s compare d t o 

control fetuses on G19 (Revsko y et al., 1997) . I n con - 

trast, a t birth , FE E neonate s hav e elevate d plasm a 

and brai n contents of CORT, decrease d CB G bind - 

ing capacity, elevated plasma and pituitary concentrations 

of p-EP, and reduced pituitary concentrations of 

p-EP (Kakihana et al, 1980 ; Taylor et al, 1983; Weinberg 

et al., 1986; Angelogianni and Gianoulakis, 1989; 

Weinberg, 1989). Throughout the preweaning period, 

FEE offsprin g exhibi t blunte d HP A an d p-E P re - 

sponses t o a wide range o f stressors, including ether , 

novelty, saline injection, and col d stress (Taylor et al., 

1986; Weinber g e t al, 1986 ; Angelogiann i an d Gi - 

anoulakis, 1989 ; Weinberg , 1989) . In addition, prenatal 

ethanol exposur e alters the ontogeneti c expressio n 

of mRNAs fo r CRH an d POMC , delaying and exag - 

gerating the rise in CRH expression in female (but not 

male) pups, and suppresses POMC mRNA concentrations 

i n mal e (bu t no t female ) pup s throughou t th e 

preweaning period (Aird et al, 1997) . The implicatio n 

is that sexually dimorphic effects o f prenatal ethanol exposure 

on these two important glucocorticoid-regulated 

genes contribute to both the immediate and the longterm 

effect s o f prenatal ethano l o n stres s responsive - 

ness of the offspring . 

The significanc e of the reduced hormonal responsiveness 

described i n FEE pup s during early development 

remain s to be determined . Evidenc e indicate s 

that i n additio n t o th e reduce d adrenocortica l re - 

sponses to stressors , plasma CBG bindin g capacity is 

also reduced in FEE compare d to that in controls, at 

least durin g th e firs t wee k o f lif e (Weinberg , 1989) . 

Thus it is possible that although th e CORT stress response 

i s reduced i n FEE pups , the ratio of bound-tofree 

steroid s ma y no t b e altered . Th e reductio n i n 

CBG binding capacity may represent a compensatory 

response t o maintai n norma l neuroendocrin e func - 

tion i n FE E offspring . Thi s possibility remains to be 

tested. 

Long-Term Effect s o f Prenata l Ethano l Exposure . 

Importantly, th e reduce d HP A an d p-E P respon - 

siveness i n FE E ra t pup s earl y in lif e i s a transien t 

phenomenon. Following weaning, FEE rat s are typically 

hyperresponsive to stressors and t o drugs such as 

ethanol and morphine (Taylor et al., 1988 ; Weinberg, 

1993a, 1993b ; Ki m e t al, 1996 ; Le e e t al, 1990 , 

2000). Simila r result s hav e bee n describe d i n non - 

human primate s (Schneide r e t al. , 2004) . I n rhesu s 

monkeys, prenatal exposur e to moderate amount s of 

ethanol induce d highe r plasm a ACTH an d margin - 

ally highe r plasm a cortiso l response s t o th e stres s of 

maternal separation. 

An interesting findin g fro m roden t model s i s that 

the sexua l dimorphis m i n th e effect s o f prenata l 

ethanol exposur e see n durin g th e ontogen y o f th e 

HPA axis (Aird et al, 1997 ) extend s int o adulthood . 

Sex differences i n response s of male an d femal e offspring 

compare d t o thos e o f their contro l counter - 

parts are often observed an d ma y vary depending o n 

the natur e o f the stresso r and th e tim e cours e an d 

hormonal en d poin t measure d (Weinberg , 1988 , 

1992a, 1995 ; Halas z e t al, 1993 ; Le e an d Rivier , 

1996). Fo r example , i n adulthood , FE E male s an d 

females exhibi t increased CORT , ACTH, and/o r p - 

EP response s to stressors , such a s repeated restraint , 

footshock, an d immun e challenge s (Taylo r e t al. , 

1988; Weinberg, 1993b ; Kim et al, 1996 , 1999b ; Lee 

and Rivier , 1996 ; Weinber g e t al, 1996 ; Le e et al., 

2000). Bot h male s an d female s sho w increase d ex - 

pression o f mRNA s for immediat e earl y genes an d 

CRH followin g stressors (Lee e t al., 2000) as well as 

deficits i n habituatio n t o repeate d restrain t (Wein - 

berg e t al, 1996) . I n contrast , i n respons e t o pro - 

longed restrain t o r col d stress , HP A hyperactivity is

seen primarily in FEE male s (Weinberg , 1992a ; Kim 

et al., 1999a) , wherea s in respons e t o acute restraint 

or acut e ethano l o r morphin e challenge , increase d 

CORT and ACTH concentrations occur primarily in 

FEE female s (Taylo r e t al, 1982 , 1988 ; Weinber g 

et al. , 1986 ; Weinberg , 1988) . Similarly , altered re - 

sponses i n a consummator y tas k (Weinberg , 1988 ) 

and t o predictable an d unpredictabl e restrain t stress 

(Weinberg, 1992b ) sugges t deficit s i n th e abilit y of 

FEE female s but not males to use or respond to environmental 

cues. 

Possible Mechanisms Mediating 

Ethanol-Induced HPA Hyperresponsiveness 

The mechanism s underlyin g HP A hyperrespon - 

siveness i n FE E offsprin g ar e unknow n a t presen t 

but coul d reflec t change s at all levels of the axis . Increased 

HP A activit y coul d resul t fro m increase d 

secretion o f secretagogues (e.g., POMC, CRH, an d 

AVP), increase d pituitar y and/o r adrena l respon - 

siveness t o thes e secretagogues , increase d driv e t o 

the hypothalamus , deficits in feedback regulation of 

HPA activity , and/o r alteration s i n centra l neuro - 

transmitters regulatin g th e HP A axis . Indeed , i t i s 

likely tha t multipl e mechanism s pla y a role . Fur - 

thermore, the finding that prenatal ethano l exposur e 

differentially alter s HP A responsivenes s i n male s 

and females compared t o that in their control counterparts 

suggest s that th e gonada l hormone s o r al - 

tered adrenal-gonadal interaction s play a significant 

role i n mediatin g prenatal ethano l effect s o n HP A 

activity. This chapter focuses on four possible mechanisms 

considered importan t i n mediatin g HP A hyperresponsiveness 

in FEE animals : (1 ) alterations in 

HPA drive, (2 ) alterations i n HP A feedback regula - 

tion, (3 ) altered neurotransmitter regulation of HPA 

activity, an d (4 ) possibl e interaction s betwee n th e 

HPA an d hypothalamic-pituitary-gonada l (HPG ) 

Altered HPA Drive and/or Feedback 

A numbe r o f studie s sho w tha t stimulator y inputs o r 

drive to the PVN of the hypothalamus are enhanced by 

prenatal ethano l exposure . Weanling FE E mal e an d 

female pup s exhibi t increased basal concentrations of 

CRH mRN A in the PVN (Lee et al., 1990) , and adul t 

FEE male s hav e increase d basa l concentration s o f 

both hypothalamic CRH mRNA and pituitary POMC 


mRNA (Redei et al., 1993 ) compare d t o those of controls. 

Not all studies, however, show these alterations in 

central HP A regulation o f basal activity. Kim and col - 

leagues (1996) reported no differences i n basal CRH or 

AVP mRN A concentration s amon g adul t FE E an d 

control animals. Lee and colleagues (2000) showed no 

differences i n basa l CRH heteronuclea r (hn ) RNA or 

in basal CRH and AVP median eminence protein con - 

centrations i n FEE rat s compared t o controls. Impor - 

tantly, however, the latter investigators also showed that 

in respons e to both footshoc k and lipopolysaccharid e 

(LPS; an endotoxin used to mimic infection or inflammation) 

challenge, FEE animal s exhibit enhanced hypothalamic 

neurona l activit y compare d t o tha t i n 

controls, reflecte d i n increased mRN A concentration s 

of two immediate earl y genes (c-fos an d NGFI-B) , as well 

as significantly increased CRH hnRN A content. Thus, 

although ther e is some controversy in the literatur e as 

to whethe r HP A regulatio n i s altere d i n FE E ani - 

mals under basal conditions, the dat a of Lee an d col - 

leagues (2000 ) provide the first evidence o f a selective 

stress- and cytokine-induce d increas e i n activity of hypothalamic 

CR H neuron s in FEE animals , indicating 

a potentia l hypothalami c mechanis m throug h whic h 

ethanol up-regulates the HPA axis. 

Weinberg an d colleague s (Glava s e t al. , 2001 ; 

Zhang e t al. , 2005b ) undertoo k studie s t o resolv e 

conflicting finding s o n whethe r FE E animal s sho w 

changes in centra l HPA regulatio n unde r basa l or 

nonstressed conditions . Steady-stat e HP A functio n 

and th e rol e of CORT in mediating changes i n HPA 

regulation were examined i n ADX rats with or without 

COR T replacement . I n additio n t o hormona l 

concentrations, thes e investigator s assesse d th e con - 

centrations o f mRNA for (a) hypothalamic CR H an d 

AVP a s measures o f hypothalamic activity , (b) CR H 

type 1 receptor (CRH-R ^ an d POM C a s indices of 

pituitary activity, and (c ) hippocampal mineralocorticoid 

receptor (MR) and glucocorticoid receptor (GR) 

mRNA concentrations a s measures of feedback regulation. 

As expected , followin g ADX, animal s exhibi t a n 

increase in basal plasma ACTH concentrations com - 

pared t o thos e i n sham-operate d animal s (Glava s 

et al, 2001, Zhang et al., 2005b). Importantly, ACTH 

elevations are greater in FEE male s but no t females, 

compared t o thos e i n thei r contro l counterparts . 

These dat a sugges t tha t a CORT-independent alter - 

ation i n HP A regulation i s unmasked b y remova l o f 

the COR T feedbac k signal , a t leas t i n males . ADX


also reveals significant alterations in gen e expressio n 

in FEE animals . Followin g ADX, hypothalamie CRH 

mRNA concentrations ar e significantly higher i n FE E 

males an d female s than i n their contro l counterparts , 

despite the finding that plasma ACTH concentration s 

are elevated only in FEE males . FEE male s also have 

blunted AVP mRNA responses to ADX and lower pituitary 

CRH-R j mRN A expression overall compared t o 

controls, but there are no significant differences amon g 

groups in pituitary POMC mRNA levels. In addition, 

CORT replacemen t i s les s effectiv e i n normalizin g 

ADX-induced alteration s in FEE tha n in control ani - 

mals at several levels of the HP A axis, including hip - 

pocampal M R mRN A concentrations i n bot h male s 

and females , hippocampa l G R an d hypothalami e 

AVP mRNA in males, and anterior pituitary CRH-R! 

mRNA i n females . The alteration s i n M R an d G R 

mRNA concentrations are particularly noteworthy, as 

previous studie s have found no ethanol-induce d differences 

i n M R an d G R recepto r concentration s o r 

binding affinity i n the hippocampu s an d othe r brain 

regions amon g FE E an d contro l animal s (Weinber g 

and Petersen , 1991 ; Ki m et al, 1999c) . Thes e dat a 

are th e firs t t o provid e direc t evidenc e fo r possibl e 

deficits i n sensitivit y t o COR T regulatio n o f HP A 

feedback i n FEE animals . They suggest an alteration 

in th e balanc e betwee n HP A drive and COR T feedback 

and exten d previous work supporting alterations 

in feedbac k sensitivity in th e intermediat e (Osbor n 

et al, 1996) , bu t not the fas t (Hofman n et al, 1999 ) 

feedback time domain. 

An altere d balanc e betwee n HP A drive an d feed - 

back in FEE compare d t o control animals is also suggested 

b y dat a o n HP A responsivenes s followin g 

glucocorticoid recepto r blockade (Zhan g et al, 2005). 

Adult female offspring fro m FE E an d contro l groups 

were injected with the MR antagonist spironolactone, 

the G R antagonis t RU38486 , o r vehicle . Followin g 

collection o f basal blood samples, rats were subjected 

to a 1 hr restraint stress, and additiona l samples were 

collected durin g and followin g stress. Both M R an d 

GR blockades increase d basa l ACTH concentration s 

in FE E bu t no t control animals , compare d t o vehicle 

injection . Furthermore , M R blockad e increase d 

ACTH concentration s i n contro l bu t no t FE E fe - 

males during restraint, whereas GR blockade increased 

ACTH concentration s i n FE E female s durin g re - 

straint an d i n contro l female s durin g restrain t an d 

recovery, compared t o their vehicle-injected counterparts. 

This differential patter n of responses under both 

basal an d stres s condition s suggest s tha t prenata l 

ethanol exposure enhance s HP A drive and alter s sen - 

sitivity to feedback regulatio n b y MR and GR , possi - 

bly reflecting an altere d balanc e betwee n HP A drive 

and feedback. 

In summary, ethanol-exposed animals exhibit HPA 

dysregulation unde r basa l conditions , eve n i n th e 

face o f similar basal hormone levels , and difference s 

are furthe r unmaske d followin g perturbation s of th e 

system by stress, ADX, or receptor blockade. Dysregulation 

occur s a t multipl e sites , includin g th e hip - 

pocampus, hypothalamus, an d pituitary , and reflect s 

changes both in HPA drive and CORT feedback reg - 

ulation and/or in the balance between driv e and feedback. 

Althoug h FE E animal s initiat e compensator y 

mechanisms to maintain normal basal hormone con - 

centrations under mos t circumstances, perturbations 

of the syste m reveal that tonic or basal HPA tone in - 

creases an d likel y play s a ke y role i n raisin g the se t 

point o f responsiveness following stress. The altere d 

CRH-Rj mRN A responses and the lac k of differences 

in POM C mRN A concentration s i n FE E animal s 

suggest compensator y mechanisms , probabl y a t th e 

level o f th e pituitary , suc h tha t th e enhance d ac - 

tivation i n th e hypothalamu s doe s no t translat e di - 

rectly int o enhance d pituitar y activity. This ma y b e 

protective fo r FE E animals , minimizin g enhance d 

basal pituitary activity in th e fac e o f enhanced hypo - 

thalamie drive. Finally, prenatal ethanol exposure has 

sexually dimorphi c effect s o n HP A regulation. Thi s 

finding implies a role for the gonadal steroids or possibly 

an alteration i n adrenal-gonadal interactions me - 

diating thes e effect s o f ethanol o n HP A activity an d 

regulation. 

Ethanol Effects on Neurotransmitters 

Regulating HPA Activity 

Fetal ethanol exposur e may alter HPA responsiveness 

through effect s o n centra l neurotransmitte r system s 

that regulat e HP A function. Data indicat e that CR H 

release i s stimulated b y acetylocholine an d epineph - 

rine in a dose-dependent manner, by low doses of NE, 

and b y serotonin (5-HT) , an d i s inhibited b y |3-EP, yaminobutyric 

acid (GABA), dynorphin, and substanc e 

P (Assenmache r e t al, 1987 ; Plotsky , 1987) . Prenata l 

ethanol exposur e alter s th e developmen t o f centra l 

neurotransmitter system s in offsprin g (Druse , 1992) . 

Of particular relevance to this review are data demon - 

strating long-term change s i n NE, 5-HT , nitri c oxide

(NO), glutamate, and GAB A in FEE rat s in brain regions 

critical for HPA regulation. 

Norepinephrine. Ethanol-induce d change s i n 

steady-state concentration s o f NE appea r to var y b y 

brain regio n a s wel l a s th e timin g o f th e exposure . 

Basal concentrations of hippocampal NE were not altered 

b y prenata l ethano l exposur e (Rudee n an d 

Weinberg, 1993), whereas exposure through both prenatal 

and earl y postnatal development (equivalen t of 

all thre e trimesters ) increase d hippocampa l N E 

content in males and females in adulthood (Tra n and 

Kelly, 1999) . Whethe r prenata l ethanol exposur e 

alters hypothalamic NE concentration s i s not ye t resolved. 

N o difference s i n hypothalami c N E concen - 

trations were detected betwee n FE E an d control s of 

either se x (Rudee n an d Weinberg , 1993) . O n th e 

other hand , Deterin g an d colleague s (1980 , 1981 ) 

showed that hypothalamic NE concentrations ar e decreased 

i n bot h youn g an d adul t rat s expose d t o 

ethanol eithe r pre - o r postnatally . Uniqu e t o th e 

three-trimester mode l (Tra n an d Kelly , 1999) , however, 

ethanol-induce d increase s i n basa l hypothala - 

mic NE are found in females, but not males. 

Exposure to stressor s differentiall y alter s NE con - 

centrations i n FE E compare d t o contro l animals . 

Ethanol exposur e increase s hippocampa l N E i n 

females (Kelly , 1996b ) an d decrease s cortica l an d 

hypothalamic N E concentrations , bu t i t increase s 

hippocampal NE conten t in both males and female s 

(Rudeen an d Weinberg , 1993) . Thes e dat a sugges t 

that the NEergic system in the hippocampus and hypothalamus 

i s particularly vulnerable to ethanol an d 

that alteration s in N E regulatio n i n these area s may 

contribute to the ethanol-induce d alterations in HPA 

activity observed in FEE animals . 

Serotonin. Th e effect s o f prenata l ethano l expo - 

sure o n th e 5-HTergi c syste m have received special 

attention becaus e 5-H T als o acts a s a neurotrophic 

factor an d earl y insult t o thi s syste m ma y affec t it s 

own development as well as that of other neurotransmitter 

systems . Prenatal ethano l exposur e results in 

decreased concentrations of 5-H T or it s metabolites 

during feta l lif e an d i n weanlin g animal s (Drus e 

et al , 1991 ; Rathbu n an d Druse , 1985) . Impor - 

tantly, prenata l administratio n of buspiron e o r ip - 

sapirone, partia l 5-HT 1A agonists , i n conjunctio n 

with ethanol , ameliorate d som e o f thes e deficit s 

(Tajuddin an d Druse , 1993) . O n th e othe r hand , 


postnatal ethano l exposur e increase d hypothalami c 

5-HT content s i n adul t rat s o f both sexes , with females 

showin g greater overal l concentration s tha n 

males (Kelly, 1996a). Exposure during pre- and postnatal 

life di d not differentiall y alte r hypothalamic 5- 

HT concentration s (Tra n an d Kelly , 1999) . Feta l 

ethanol exposure also increased the number of binding 

sites for the serotonin transporter in some areas of 

the developin g ra t brai n bu t decrease d transporte r 

binding site numbers in other areas. This finding indicates 

that ethano l exposur e i n utero permanentl y alters 

5-HT transmission in discrete brain regions (Zafar 

et al., 2000). In addition, Sari and Zhou (2004) show 

that prenatal ethanol exposure results in long-lasting 

5-HT deficit s i n mic e an d propos e tha t ethano l o r 

its metabolite s caus e los s o f serotonergi c neuron s 

through apoptosis. 

Animals prenatally exposed to ethanol exhibit physiological 

an d behaviora l abnormalitie s consisten t 

with altered 5-HT function, including lack of response 

inhibition an d increase d anxiet y an d aggression . 

FEE animal s have altered hypothermi e response s to 

8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) , 

a 5-HT 1A receptor agonist, and FEE females , but no t 

males, show an increase d rate o f "wet dog shakes" to 

l-(2, 5-dimethoxy-4-iodophenyl)-2-aminopropan e hydrochloride 

(DOI), a ^-HT^c recepto r agonist. These 

effects ar e consisten t wit h ethanol-induce d alter - 

ations i n 5-H T recepto r functio n (Hofman n e t al. , 

2002). Furthermore , FE E female s exhibi t blunte d 

ACTH responses to 8-OH-DPAT bu t increased ACTH 

responses t o DO I compare d t o controls , which suggests 

an altered interaction between the HPA axis and 

the 5-HTergi c syste m i n FE E animal s (Hofman n 

et al, 2001). 

Nitric Oxide . Alteration s in NO concentration s induced 

by prenatal ethano l exposur e may play a role 

in mediatin g HP A hyperresponsiveness i n FE E ani - 

mals. NO i s an intercellula r gaseous signaling molecule 

that has multiple biological functions, including 

regulation o f vascula r tone , modulatio n o f neuro - 

transmission, involvemen t i n cell—cel l communica - 

tion and killing of pathogens in nonspecific immune 

responses (Lama s e t al. , 1998 ; Moncada , 1997a , 

1997b). NO i s generated fro m L-arginin e by NO syn - 

thase (NOS) , a n enzym e foun d i n magnocellula r 

neurons o f the PV N an d t o som e exten t als o i n th e 

parvocellular divisio n (Le e an d Rivier , 1993 ; Siau d 

et al, 1994 ; Hatakeyama et al., 1996) . NO acts within


the hypothalamu s to activate the HP A axis, with specific 

stimulator y effect s exerte d o n CRH-producin g 

neurons (Rivier, 1999). 

Ethanol-induced brai n damag e i s closel y re - 

lated t o glutamate-induce d exitotoxicity , mediate d 

through N-methyl-D-aspartat e (NMDA ) receptors . 

Chronic ethano l exposur e increase s NMD A re - 

ceptors and Ca 2+ channel activity , event s associated 

with seizure s during alcoho l withdrawa l (Lancaster , 

1992). It has also been postulated that chronic prenatal 

ethano l exposur e change s glutamate-NMD A 

receptor-NOS signal transduction in the developing 

guinea pi g brain (Kimura et al., 2000). Kimur a an d 

coworkers (Kimur a e t al. , 1996 , 1999 ; Kimur a an d 

Brien, 1998 ) reporte d decrease d feta l hippocampa l 

NOS activit y and , consequently , decrease d forma - 

tion of NO, bu t thos e changes are not accompanied 

by a los s o f NOS-containin g neuron s i n th e hip - 

pocampal CA I o r CA3 areas or changes in localization 

o f hippocampal NO S protei n i n th e near-ter m 

guinea pig . The critica l period for prenatal ethanol - 

induced los s o f hippocampa l CA I neuron s i n th e 

guinea pig brain appears to be between G62 and PI2 

(McGoey et al, 2003). Glutamate an d NMDA binding 

site s decrease i n th e hippocampu s o f near-term 

fetal guinea pigs chronically exposed to ethanol (Abdollah 

an d Brien , 1995) . Thus it has been propose d 

that chronic prenatal exposure to ethanol suppresses 

the function of the glutamate-NMDA receptor-NO 

signal transductio n syste m i n th e developin g hip - 

pocampus. Furthermore , i t has been postulated tha t 

persistent suppressio n o f thi s signalin g syste m dis - 

rupts or delays normal neuronal development, which 

temporally precede s th e los s o f hippocampa l CA I 

pyramidal cells in postnatal lif e (Gibso n e t al., 2000; 

Kimura et al, 2000; McGoey et al, 2003). In view of 

the importan t role of the hippocampus in HPA feedback, 

suc h alteration s coul d underli e th e ethanol - 

induced change s i n HP A feedback regulatio n tha t 

have been observed. 

Changes i n N O synthesi s in FE E animal s may 

mediate th e altere d HP A responses t o immun e sig - 

nals reporte d i n FE E rat s compare d t o thos e i n 

controls. Gottesfel d (1998 ) repor t increased NO formation 

i n respons e t o LP S i n FE E rats . Further - 

more, blockade of NO synthesis prevents the blunted 

sympathetic respons e to LPS or interleukin (IL)- l i n 

FEE rat s but no t i n contro l rat s (Gottesfeld, 1998) . 

Similarly, changes i n hypothalamic responsivenes s to 

NO ma y mediate the increased HPA responsiveness of 

FEE animal s to stressors . Intracerebroventricula r in - 

jection o f the N O dono r 3-morpholinosydnonimin e 

(SIN)-l increase s the ACTH respons e and th e con - 

centration o f mRN A fo r th e immediat e earl y gen e 

NGFI-B in FEE male s (Lee et al., 2003). Moreover, 

blockade o f NO formatio n abolishes difference s be - 

tween FE E an d contro l animal s in respons e t o IL- 1 

(Rivier, 1995) . In normal (no ethanol exposure ) rats, 

SIN-1 als o up-regulate s CRH , AVP , an d CRH-R L 

gene expressio n in the PVN . Th e functiona l impor - 

tance o f the increase d concentration s o f CRH an d 

AVP transcripts is evidenced b y immunoneutraliza - 

tion o f endogenous CR H an d AVP , which abolishe s 

or blunts, respectively, the ACTH respons e to SIN-1 

(Lee e t al. , 1999) . I t woul d b e fascinatin g to stud y 

these relationship s in animal s prenatally exposed t o 

ethanol. 

Altered HPA-HPG Interactions 

There ar e bidirectiona l interaction s betwee n th e 

stress syste m and the HPG axi s (Fig . 10-3) . Hor - 

mones o f th e HP A axi s hav e inhibitor y effects o n 

HPG activity . In turn, the gonadal steroids feed back 

to activat e (estradiol) or inhibi t (testosterone) HPA 

activity. Recen t studie s hav e investigate d a possibl e 

role o f ethanol-induce d alteration s i n HPA-HP G 

interactions i n mediatin g th e sexuall y dimorphi c 

effects o f ethano l o n mal e an d femal e offspring . 

Studies hav e examine d th e effect s o f gonadectom y 

(GDX), wit h o r without hormon e replacement , o n 

concentrations o f the HP A hormones in bot h sexe s 

under basal conditions and following stress. Preliminary 

data show that FEE male s have higher plasma 

ACTH concentration s tha n control s followin g re - 

straint stress , an d tha t GD X eliminate s th e dif - 

ferences amon g group s (La n e t al. , 2004) . Thus , 

HPA hyperresponsiveness i n FE E male s appear s t o 

be mediated , a t leas t i n part , b y ethanol-induce d 

changes i n testosteron e regulatio n o r in HP A sensitivity 

to testosterone. That is, the suppressiv e effect s 

of testosterone and/or the stimulatory effects o f GDX 

on stress-induced ACTH release are altered i n FE E 

compared t o contro l males . I n contrast , th e rol e of 

the gonada l hormone s i n HP A regulation i n FE E 

females i s mor e complex . Preliminar y dat a (Ya - 

mashita et al., 2004) show that overall, FEE females 

are les s responsiv e tha n control s t o th e effect s o f 

estradiol o n bot h reproductiv e an d nonreproduc - 

tive measures. Furthermore, FEE female s appear to

FIGURE 10- 3 Interaction s betwee n th e stres s system 

and reproductiv e axis . Th e stres s syste m consist s o f 

the hypothalamic-pituitary-adrena l (HPA ) axi s an d 

the locu s coeruleu s noradrenergi c sympatheti c ad - 

renal medullar y (LC-NE ) syste m (describe d i n de - 

tail i n Fig . 10.1) . Th e reproductiv e axi s consist s o f 

gonadotropin-releasing hormon e (GnRH ) neuron s 

which stimulat e secretio n o f luteinizin g hormon e 

(LH) an d follicl e stimulatin g hormone (FSH ) fro m 

the pituitary , which i n turn stimulate secretion of estrogens 

(mainl y estradiol ) an d androgen s (mainl y 

testosterone) fro m th e gonads . The reproductiv e axi s 

is stimulated by the LC-NE system, primarily through 

oc-noradrenergic (oc rNE) receptors , and i s inhibite d 

at al l level s by component s o f the HP A axis. Activa - 

tion is represented by solid lines, inhibition by dashed 

lines. ACTH , adrenocorticotropi c hormone ; CRH , 

corticotropin releasin g hormone ; E ?, estradiol ; T , 

testosterone. (Source: Reprinte d fro m Strataki s an d 

Chrousos (1995 ) wit h permissio n fro m Ne w Yor k 

Academy of Sciences) 

have decreased tissue responsiveness to estradiol and 

altered HP G sensitivit y to acute stress . Further stud - 

ies ar e neede d t o elucidat e th e effect s o f prenata l 

ethanol exposur e o n interaction s betwee n th e HPA 

and HPG axes . 


PRENATAL ETHANOL EXPOSURE ALTERS 

THE IMMUN E SYSTE M 

OF THE OFFSPRING 

Impairments in immune competence o f children with 

FAS have been demonstrate d broadl y in bot h innat e 

and adaptive immunity. Innate immunit y is not majo r 

histocompatibility complex (MHC ) restricted . I t provides 

a firs t lin e o f defens e agains t man y commo n 

pathogens throug h phagocyte s such a s monocytes , 

macrophages, polymorphonuclear leukocytes, natural 

killer cells , an d solubl e mediator s suc h a s comple - 

ment an d C-reactiv e protein . Adaptiv e immunit y is 

MHC restricte d and classifie d a s cellular or humoral, 

mediated b y T o r B lymphocytes , respectively . Im - 

mune response s agains t microorganisms ar e mediated 

by multipl e cel l types , an d interaction s betwee n th e 

innate and adaptiv e immune system s are necessary to 

launch a n effective immun e response. An understanding 

of the timeline an d events in normal developmen t 

of the immune system, a complicated an d multisystem 

process, is important to an understanding of the effec t 

of ethanol o n the developing immune system. 

Normal Timing of the Ontogeny 

of the Immune Syste m 

Although developmen t o f the immun e syste m take s 

place throughou t life , th e basi c framework of immunity 

develop s befor e birth . Th e misconceptio n tha t 

the newbor n is immunologically naive has been cor - 

rected with data demonstrating that neonatal T and B 

cells ar e matured to a stag e that the y ca n moun t a n 

antigen-specific immun e respons e t o antigen s en - 

countered i n utero , suc h a s the parasit e Ascaris an d 

tetanus toxoi d vaccinatio n (Gil l e t al. , 1983 ; Kin g 

étal, 1998) . 

Hematopoietic ste m cell s for m an d develo p i n 

the yolk sack until 4 weeks of gestation, migrate to the 

liver 3 to 4 weeks later, and then further migrate to the 

thymus and spleen. Ste m cell s are in the bone marrow 

at 11-1 2 week s of gestation (Migliacci o e t al, 1986 ; 

Holt and Jones, 2000). Susceptibility of early lymphohematopoiesis 

an d cel l migratio n t o environmenta l 

influences ca n occu r a s early as 7-10 week s postcon - 

ception (West , 2002) . As early a s 1 4 weeks o f gesta - 

tion, differentiate d T and B cells can be identified in 

fetal bloo d (Goldblatt , 1998 ) an d splee n (Peakma n 

et al., 1992). The splee n i s considered completely im - 

munocompetent b y 1 8 week s o f gestation . A t thi s


time, splenic T cells have adult levels of expression of 

CD3, CD4 , an d CDS and are able to respond to mitogens 

(Ka y et al., 1970) , an d spleni c accessor y cells 

are full y functiona l i n deliverin g co-stimulatory signals 

(Hol t an d Jones , 2000). Fetuse s stil l hav e fewe r 

memory T cells than adults do at this time (Hol t and 

Jones, 2000). Yolk sack-derived pro- B cells develop in 

the liver and acquire surface immunoglobulin (Ig ) M, 

IgD, and CD2 0 expression by 10-13 weeks of gestation 

(Hofma n et al., 1984) . B cells ar e detectabl e i n 

lymph node s fro m 16-1 7 weeks, in splee n a t 16-2 1 

weeks, an d ar e abundant i n bon e marro w at 16-2 0 

weeks of gestation (West , 2002). 

The structur e of the thymus develops as a result of 

epitheliomesenchymal interactions. Endoderm of the 

third pharyngeal pouch differentiate s int o thymic epithelium. 

Neural crest cells then migrat e through th e 

branchial arche s an d becom e th e mesenchym e tha t 

will form the layers around the epithelial primordium 

of the thymus and the connective tissue framework. T 

cell progenitor s arriv e in th e thymu s fro m th e live r 

during the ninth wee k of gestation, an d th e structur e 

of th e thymu s differentiate s int o a corte x an d a 

medulla at 10-1 2 week s (West, 2002). Prothymocytes 

express CD 7 i n th e feta l live r at 7 week s and CD 3 

at 8- 9 week s of gestation, whe n thes e cell s migrat e 

to the thymus (Holt and Jones, 2000). Gene rearrangement 

start s a t aroun d wee k 11 , an d expressio n o f 

the T cel l receptor s (TCR) , CD4 , an d CD 8 fol - 

lows. Thymi c "education " o f immature CD4 + /CD8 + 

(double-positive) T cell s take s plac e i n th e secon d 

trimester b y positiv e selection , wit h th e abilit y o f 

TCR t o bin d t o polymorphi c part s o f nonagonis t 

MHC-encoded molecules on nonlymphoid cells, and 

by negativ e selectio n of TCR, wit h hig h affinit y to 

self-antigens t o avoi d autoimmunit y (vo n Boehme r 

et al, 2003). Export of mature CD4+ or CD8+ (single 

positive) cell s begin s afte r wee k 13 , and a rapi d expansion 

o f the T cel l poo l happen s durin g 14-1 6 

weeks (Kay et al., 1970 ; Berr y et al, 1992) . At 13-14 

weeks, thymocytes acquire proliferative ability to most 

mitogens. During the second trimester , susceptibility 

to environmenta l factor s cause s disruptio n o f th e 

thymic educatio n processe s an d cel l proliferation , 

leading to a defective T cell repertoire. 

As roden t gestatio n (~ 3 weeks ) i s substantially 

shorter tha n tha t o f humans (~4 0 weeks) , immun e 

system developmen t o f newbor n ra t an d mous e 

pups occurs at a time equivalent to the end of the sec - 

ond trimeste r i n human s (Zaja c an d Abel , 1992) . 

Therefore, studie s i n roden t model s o f prenata l 

ethanol exposur e have focused primarily on the early 

events in ontogeny of the immune system, i.e., during 

the late gestational period and early postnatal life . 

Impaired Immunit y following Prenatal 

Exposure to Ethanol 

Children prenatall y expose d t o alcoho l hav e a n in - 

creased incidenc e o f bacteria l infections , suc h a s 

meningitis, pneumonia , recurren t otiti s media , gas - 

troenteritis, sepsis , urinar y tract infections , an d fre - 

quent uppe r respirator y trac t infection s (Johnso n 

et al, 1981 ; Churc h an d Gerkin, 1988) . These children 

als o have lower cell count s o f eosinophils and 

neutrophils, decrease d circulatin g E-rosette-formin g 

lymphocytes, reduce d mitogen-stimulate d prolifera - 

tive response s b y periphera l bloo d leukocytes , an d 

hypo-y-globulinemia (Johnson et al., 1981). 

Research usin g animal model s t o investigat e im - 

mune function of FEE offsprin g has (a) substantiated 

the clinica l evidenc e o f impaire d immunit y associ - 

ated wit h FASD and (b ) greatly increased ou r under - 

standing of these immune deficits in terms of both th e 

spectrum of effects i n differen t orga n systems and th e 

mechanisms mediatin g th e immunoteratogeni c ef - 

fects o f ethanol . Deficit s i n innat e immunit y hav e 

typically no t bee n observe d i n anima l studies . Fo r 

example, on e larg e stud y o n nonhuma n primate s 

(Macaco, nemestrina) show s that i n uter o ethano l ex - 

posure doe s not result in significant differences i n total 

numbers of white blood cells, leukocyte subsets, or 

monocyte phagocyti c activit y compare d t o tha t i n 

control subjects (Grossmann et al., 1993). In contrast, 

marked deficit s in adaptiv e immunity involving both 

cell-mediated an d humora l immunit y ar e eviden t 

in FE E animals . Furthermore , a s describe d i n th e 

discussion below , a marke d sexua l dimorphis m i n 

ethanol effect s ha s been observed , wit h the majorit y 

of deficits occurring in male offspring . 

Deficits i n Adaptive Immunit y 

Inborn errors of immunocompetent cells in children 

with FASD result in immunodefiency disorders or increased 

susceptibilit y to infections. Recurrent opportunistic 

infection and infectio n caused b y ubiquitous 

microorganisms, such a s bacteria, viruses , and fungi , 

typically occu r wit h deficit s in cell-mediate d immu - 

nity, wherea s deficit s i n B cells , immunoglobulins ,

complement, an d phagocyte s usuall y lead t o infec - 

tion by encapsulated and pyrogeni c bacteria, such as 

Haemophilus influenzae, Streptococcus pneumoniae, 

and Staphylococcus aureus. I n childre n wit h FAS D 

both types of recurrent infections have been reported, 

suggesting tha t bot h T an d B cell-mediated immu - 

nity are compromised (Johnso n et al., 1981) . 

Work with animal models confirms the findings in 

human subjects . For example, th e immun e respons e 

of FEE neonate s t o the intestina l parasit e Trichinella 

spiralis indicate s a diminished capacit y to respond t o 

the pathogen , demonstrate d b y a n increase d intes - 

tinal wor m coun t (Steve n e t al. , 1992 ; Seeli g e t al. , 

1996). The abnormalitie s involve depressed T an d B 

cell-mediated response s suc h a s lowe r seru m IL- 2 

and tumor necrosis factor (TNF ) contents , an d lower 

IgM an d Ig G antibod y titers. Interestingly, the detri - 

mental effect s o f ethano l appea r t o increas e acros s 

generations. Tha t is , FEE pup s (secon d generation) 

mothered b y FE E adul t offsprin g (firs t generation) 

who themselves consumed ethanol during pregnancy 

show reduce d proliferativ e response s t o T . spiralis 

antigen and stimulation with concanavalin A (Con A), 

lower titer s o f serum Ig M an d Ig G anti-T . spiralis, 

and lower percentages of T cells and cytotoxic T cells, 

compared t o th e first-generatio n FE E an d pair-fe d 

groups (Seelig et al., 1999) . The nex t two sections review 

r studie s tha t describ e specifi c deficit s i n eithe r 

cell-mediated o r humoral immunity. 

Deficits in Cell-Mediated Immunity 

Prenatal ethano l exposur e alters development o f the 

thymus i n rat s an d mice . Delaye d thymi c ontogen y 

(Ewald and Walden, 1988) , decreased tota l number s 

of thymocytes, and diminishe d mitogen-induce d cel l 

proliferation hav e been reported i n near-term fetuses 

(Ewald an d Frost , 1987) . Decrease d thymu s weight, 

size, and cell counts have also been observe d at birth 

(Redei et al., 1989) . These changes persisted through 

the preweanin g perio d an d eve n int o adolescenc e 

(Ewald an d Frost , 1987 ; Ewald , 1989 ; Giberso n an d 

Blakley, 1994 ; Taylo r e t al , 1999a) , althoug h on e 

study o f mic e foun d tha t tota l thymocyt e number s 

could retur n t o contro l level s as early as P6 (Ewald, 

1989). Similarly , mitogen-induce d proliferativ e re - 

sponses of thymic cells was suppressed in FEE male s 

at weaning (Redei et al, 1989) , but may be increased 

in adolescen t (44-day-old ) rat s (Chiappell i e t al , 

1992; Wong et al., 1992) . This increase i n thymocyte 


proliferation durin g adolescence doe s no t appea r to 

be mediate d b y change s i n numbe r o f GR s o n th e 

thymocytes; furthe r studie s ar e neede d t o elucidat e 

the mechanism s underlyin g these varyin g effect s o f 

ethanol. 

The advers e effects o f ethanol on thymic develop - 

ment have been confirme d by in vitr o studies usin g 

organotypic cultures . Tota l cel l number s an d per - 

centages of immature fetal thymocytes (CD4+/CD8+) 

decrease i n a concentration-responsiv e manne r i n 

ethanol-treated orga n culture s (Bra y e t al , 1993) . 

This decrease is a consequence o f accelerated apopto - 

sis, which then result s in a n increase d percentag e of 

more mature thymocytes expressing CD4 + /CD8~ and 

Y/8 TC R (Ewal d an d Walden , 1988 ; Ewal d e t al , 

1991; Bray et al., 1993 ; Ewald and Shao , 1993) . Similar 

outcome s ar e observe d i n 20 - to- 40-day-old FE E 

animals. That is , thymic cel l count s an d tota l num - 

bers of CD4 + and CD8 + cells are decreased throughout 

thi s period , an d immatur e CD8 + /TCR + an d 

CD8+/CD4SRC+ thymocyte s ar e reduce d b y P3 5 

(Taylor e t al. , 1999a) , finding s suggestin g tha t pre - 

natal ethano l treatment alters the late r stage s of thymocyte 

maturation , suc h a s afte r double-positiv e 

(CD4 + /CD8 + ) thymocytes acquire TCR expression. 

Prenatal ethano l exposur e ha s long-ter m advers e 

effects o n the immune system that last well into adulthood. 

FE E animal s hav e decrease d number s o f 

Thyl.2 + , CD4 + , CD8 + , and IgG + splenocytes (Ewald 

and Huang, 1990 ; Giberson and Blakley, 1994; Giber - 

son e t al. , 1997) . Decrease d percentage s o f Thyl.2 + 

splenocytes ar e also evident i n pups born t o mother s 

consuming ethano l durin g pregnanc y an d lactatio n 

or during lactation alone , indicatin g direct effect s of 

ethanol o n postnatal development o f the immune system 

(Giberso n an d Blakley , 1994) . Similarly , roden t 

and primat e studie s indicat e tha t spleni c lympho - 

cytes from FEE male s have decreased proliferative responses 

to mitogens from adolescenc e throug h youn g 

adulthood (Norma n et al, 1989 ; Rede i e t al, 1989 ; 

Gottesfeld e t al, 1990 ; Weinber g and Jerrells , 1991 ; 

Grossmann et al, 1993 ; Jerrells and Weinberg, 1998 ) 

and that the response normalizes by 8 months of age 

(Gottesfeld an d Ullrich, 1995 ; Norman et al, 1991) . 

Furthermore, deficit s no t onl y i n th e respons e o f 

freshly isolated splenic T cells but also in the response 

of T blas t cell s (obtaine d followin g treatment wit h 

Con A ) to IL- 2 or furthe r Co n A stimulation (Got - 

tesfeld e t al, 1990 ; Norma n e t al, 1991 ; Weinber g 

and Jerrells , 1991; Jerrell s and Weinberg , 1998 ) hav e


been observed . I n contrast , th e change s in mitogen - 

induced proliferation of thymocytes observed in FE E 

animals normalized i n young adulthood (Chiappell i 

et al, 1992). 

Deficits in Humoral Immunity 

Humoral immunit y is les s affecte d b y feta l ethano l 

exposure than is cell-mediated immunity. Prepubertal 

FEE rat s d o no t diffe r fro m control s i n seru m im - 

munoglobulin conten t afte r primar y and secondar y 

immunization (Gottesfel d an d Ullrich , 1995) . Simi - 

lar result s ar e eviden t i n nonhuma n primate s afte r 

immunization wit h tetanus toxi n (Grossman n e t al. , 

1993). On the other hand, deficits in humoral immu - 

nity do occur . Abnormal development o f B cell lineages 

in murine hernatopoietic organs, including bone 

marrow, spleen , an d liver , occu r fro m feta l age s 

through adolescence . The tota l number of splenic B 

cells an d thei r proliferativ e respons e t o LP S i s de - 

creased a t birt h an d throughou t th e preweanin g 

period (Wolcott et al, 1995). In addition, B cell maturation 

in fetal live r is delayed (Biber et al., 1998 ) an d 

the numbe r o f B220-positive hernatopoietic cell s i n 

liver is decreased i n rat fetuses at the en d o f gestation 

(Robinson and Seelig, 2002). At birth, the numbers of 

both immature (IgM + /IgD~) and mature (IgM + /IgD + ) 

B cells in spleen and bone marrow are decreased, but 

most recove r to norma l levels by 3 to 4 week s afte r 

birth, excep t fo r pre- B cell s (B220 + /IgM~) i n bon e 

marrow, which remain at low levels through 5 weeks 

of life (Moscatell o et al, 1999) . 

Ethanol Exposure an d Vulnerability 

to Stress-Induced Suppression of 

Immune Function 

The nervous , endocrine, an d immun e system s exist 

within a comple x regulatory network in whic h bidirectional 

communication occurs via shared receptors 

and hormona l mediator s t o maintai n homeostasi s 

(Fig. 10-4) . Th e CM S ca n modulat e bot h th e en - 

docrine and immune system s through releas e of neurotransmitters 

an d throug h direc t innervatio n o f 

organs. Feedbac k t o th e CN S occur s throug h hor - 

mones an d cytokines , many of which ar e simila r or 

even identical in structure. Immune signals can activate 

the HP A axis and th e glucocorticoid s play a major 

rol e in the stress-induce d suppression of immune 

and inflammatory reactions. 

FIGURE 10- 4 Interaction s betwee n th e stres s system 

and immune system. The long-ter m stress response is 

maintained vi a secretio n o f stres s hormone s (corti - 

cotropin releasin g hormon e [CRH] , adrenocorti - 

cotropic hormon e [ACTH] , an d glucocorticoids) . 

The locu s coeruleu s noradrengergi c (LC-NE ) sys - 

tem participate s i n th e effect s o f stres s o n th e im - 

mune respons e through bot h its interaction with th e 

hypothalamic-pituitary-adrenal (HPA ) axi s an d it s 

ability t o transmi t neural signal s from th e peripher y 

to th e immun e system . Activatio n o f th e HP A axi s 

occurs durin g th e stres s o f infection , inflammation , 

or autoimmun e processes , a s wel l a s accidenta l o r 

operative trauma . Durin g thi s activation , cytokines, 

neuropeptides, platelet-activatin g facto r (PAF)/prosta - 

noids are secrete d an d caus e stimulatio n o f the HP A 

axis. The glucocorticoids , en d hormone s o f the HP A 

axis, play a major role in the stress-induced suppression 

of immune an d inflammator y reactions. Activation is 

represented b y solid lines, inhibition b y dashed lines . 

(Source: Reprinted from Strataki s and Chrousos (1995) 

with permission from Ne w York Academy of Sciences) 

Chronic stress in adulthood provide s a challenge to 

the immune system that differentially affect s FE E an d 

control animals. Similar to the data demonstrating that 

basal hormone concentration s are typically normal in 

FEE animals , specific deficits in the immun e syste m

may no t b e observe d under nonstresse d conditions , 

and become apparent only when FEE animal s are exposed 

to stressors. In addition , sexually dimorphic effects 

of prenatal ethanol exposure may be observed, 

In FE E males , exposur e t o chroni c intermitten t 

stressors selectivel y down-regulates th e number s o f 

thymic and periphera l blood CD43 + cells as well as 

peripheral bloo d CD4 + T cells , an d marginall y decreases 

the numbe r o f peripheral bloo d MH C Clas s 

II Ia + (antige n presenting ) cells . I n contrast , CD4 3 

antigen expressio n o n periphera l bloo d T cell s i s 

selectively up-regulate d (Giberso n an d Weinberg , 

1995). Stres s doe s no t differentiall y alte r thes e im - 

mune measure s i n FE E females . The findin g tha t 

FEE males exhibit greater stress-induced immune abnormalities 

tha n FE E females , despit e showin g less 

adrenal hypertrophy , suggest s tha t male s ar e mor e 

sensitive t o smal l change s i n glucocorticoi d con - 

centrations tha n females . Role s fo r estroge n a s a n 

immunoprotective hormon e an d testosteron e a s a n 

immunosuppresive hormone i n these sexually dimorphic 

immune responses remain to be determined. 

One o f the first demonstrations tha t stress differentially 

alters immune functio n in FE E an d contro l females 

is the finding of an interaction between prenata l 

ethanol an d chroni c col d stres s i n femal e offsprin g 

(Giberson et al., 1997) . After 1 day of cold stress, FEE 

females exhibi t increase d lymphocyt e proliferatio n in 

response t o pokewee d mitoge n (PWM ; a T cell - 

dependent B cell mitogen) an d Con A challenge. No 

ethanol-induced difference s i n immun e responsive - 

ness ar e detecte d amon g males . I n contrast , FE E 

males expose d t o 1 or 3 days of col d stres s have in - 

creased basa l COR T concentration s compare d t o 

those o f FEE male s not expose d t o cold. These findings 

ar e consisten t wit h dat a fro m Halas z an d col - 

leagues (1993) , wh o foun d tha t th e challeng e o f 

chronic ethano l exposur e i n adulthood selectivel y increased 

Co n A-induce d lymphocyt e proliferatio n in 

FEE females but not in males. These data have important 

implication s for understandin g th e mechanism s 

underlying immune deficits induced by prenatal exposure 

to ethanol, because defective interactions between 

T an d B cells would significantly hinder th e develop - 

ment of a normal immune response . 

Cytokines and the HPA Axis 

Prenatal ethano l exposur e blunt s th e LPS-induce d 

febrile respons e i n mal e rat s (Taylo r e t al. , 1999b , 


2002a). Thi s effec t o f ethano l ma y b e mediate d b y 

a decrease d respons e o f centra l thermoregulator y 

systems to IL-1(3 , which coul d b e mediate d b y a decreased 

hypothalami c IL-1( 3 respons e t o LP S ad - 

ministration, possibl y throug h a n impaire d releas e 

of endogenous pyrogens (Ylikorkala e t al. , 1988 ; Yirmiya 

et al, 1993 , 1996) . Interestingly , both ADX and 

sham surger y i n th e pregnan t femal e abrogat e th e 

effect o f ethanol o n th e febril e respons e o f female 

offspring t o IL-1(3 , bu t onl y AD X ha s a n effec t o n 

male offspring . Thi s observatio n implies tha t mater - 

nal adrena l mediator s play an importan t rol e i n th e 

blunted febrile response of FEE male s and that nonadrenal 

mediator s participat e in modulatio n of ther - 

moregulatory system s in FE E female s (Taylo r et al. , 

2002a, 2002b). 

Parallel to blunted hormonal responses to stressors 

observed i n FEE animal s during th e preweaning period, 

FE E preweanling s exhibi t reduce d ACTH , (3- 

EP, an d TNF-o c response s t o immun e challenge s 

(IL-1(3, LPS) , Thi s reductio n persist s int o adoles - 

cence i n FEE male s but not in females (Lee and Rivier, 

1993 , 1996 ; Chiappell i e t al., 1997 ; Ki m et al, 

1999b). A n altered abilit y of IL-1 to stimulat e secre - 

tion o f POMC-relate d peptide s ma y underli e thi s 

reduced responsiveness (Lee and Rivier, 1993). Interestingly, 

ovariectom y prior to puberty eliminates th e 

difference i n ACTH response between FE E an d con - 

trol females (Lee and Rivier, 1996), a finding suggesting 

tha t ethano l an d femal e se x hormones regulat e 

HPA activity through a common pathway. 

In contras t to the preweaning period, ACTH an d 

CORT responses t o immune signal s such a s LPS or 

IL-1 (3 increase i n FE E animal s b y adulthoo d (Ki m 

et al. , 1999b) . Thi s respons e parallel s thei r HP A 

hyperresponsiveness to stressors. The finding that prenatal 

ethano l exposur e doe s no t alte r cytokin e re - 

sponses to immune challenge s suggest s that cytokine s 

probably d o no t mediat e th e ethanol-induce d in - 

crease in HPA responsiveness to immune signals . O n 

the other hand, FE E male s exhibit increased plasma 

concentrations o f pro-inflammatory cytokine s to LP S 

challenge followin g repeate d restrain t stres s (Eske s 

and Weinberg , 2003 ; Eske s e t al , 2004) . Wherea s 

CORT response s t o LP S ar e comparabl e amon g 

groups, FE E animal s hav e greate r an d mor e sus - 

tained elevation s o f IL-1(3 , TNF-ot , an d IL- 6 tha n 

those i n controls. These data support and extend pre - 

vious studie s suggestin g tha t although FEE animal s 

may no t diffe r i n cytokin e response s unde r basa l o r


nonstressed conditions, they may in fact be more vulnerable 

t o th e advers e effect s o f stres s o n immun e 

function (Eske s et al., 2004). 

Mechanisms Underlyin g 

the Effects of Ethanol 

on the Developing Immune Syste m 

Direct and Indirect Effects 

of Ethanol on the Fetus 

Ethanol ca n directl y disrupt the developmen t o f the 

fetal thymus. The highl y specified microenvironment 

of the thymu s provided by epithelial an d mesenchy - 

mal cell s i s pivotal i n attractin g immature lymphoi d 

precursors to enable them to be selected and differen - 

tiated int o matur e T cells . Exces s ethano l exposur e 

during th e developmen t o f the thymu s inhibit s th e 

ontogeny o f the thymi c epitheliu m an d disrupt s th e 

microenvironment fo r maturation o f T cells , whic h 

leads to impaired T cell immunity (Bockman, 1997) . 

These data are interesting in light of the share d clinical 

characteristics o f FASD and DiGeorg e syndrome 

(Ammann et al., 1982) . The latte r is a congenital im - 

mune deficienc y syndrome involvin g mainly T cell s 

and cause d by an abnormality in the developmen t of 

neural crest-derive d component s o f the thymu s and 

parathyroid gland s (Kirb y and Bockman , 1984) . Tar - 

geted effect s o f ethanol o n neural crest are describe d 

in Chapter 17 . 

Selective effect s o f ethanol o n thymi c CR H an d 

POMC gene expression in male fetuses have been reported. 

Significan t increase s i n thymi c CR H an d a 

decrease i n thymi c POM C gen e expressio n hav e 

been observe d on G1 9 (Revsko y et al., 1997) . Thes e 

changes d o no t appea r t o be relate d t o COR T con - 

centrations in the fetus or pregnant female, but possibly 

ar e induce d b y th e feta l testosteron e surge . 

Similarly, the ontogen y of GR sites per thymocyte i n 

FEE animal s in the first 2 months o f life differ s fro m 

that in control animal s (Chiappelli et al., 1992) . This 

indicates a rol e for th e glucocorticoi d hormone s i n 

the differentia l thymi c developmen t describe d i n 

FEE and control animals. 

Altered IL-2/IL-2 receptor interaction s ma y play a 

role in the developmen t of altered immune function 

in FEE animal s (Weinberg and Jerrells, 1991; Taylor 

et al., 1993 ; Chang et al, 1994). For example, despite 

showing a reduced proliferative response to mitogens, 

FEE animal s have norma l amount s o f IL-2 produc - 

tion, IL- 2 recepto r expressio n an d distribution , cal - 

cium influ x i n T cells , an d bindin g o f IL- 2 t o it s 

receptor. Th e internalizatio n and/or utilization of IL- 

2 by lymphoblasts, however, is reduced, an d th e half - 

time fo r dissociatio n o f IL- 2 fro m it s recepto r i s 

increased i n T cell s fro m FE E animals . Therefore , 

impaired intracellula r signalin g events , mediate d b y 

IL-2/IL-2R interactions , ma y underli e th e immun e 

deficits observe d i n FE E animals . In contrast , direct 

treatment o f lymphocyte s wit h acetaldehyde-seru m 

protein, whic h form s i n viv o a s a metabolit e o f 

ethanol consumption , result s i n decrease d IL- 2 pro - 

duction bu t no t IL- 2 recepto r expressio n (Brau n 

et al. , 1995) . This result suggests that, a t least under 

some conditions , decrease d IL- 2 productio n con - 

tributes to impaired proliferative responses. 

Altered neurotransmitte r regulatio n o f immun e 

function ma y play a role in altered immun e function 

of FE E animals . Prenata l exposur e t o ethano l de - 

creases concentration s o f NE an d (3-adrenoreceptor s 

in the lymphoid organs and diminishes synaptic transmission 

i n the splee n an d thymus , but no t the hear t 

(Gottesfeld e t al. , 1990) . Altere d NEergi c synapti c 

transmission, including a higher rat e of NE turnove r 

leading to reduced NE concentratio n and reduce d (3adrenoreceptor 

density , could affect immune capacit y 

in terms of NE-mediated IL-2 secretion an d cytotoxi c 

T cell responses . 

Immunity at the 

Fetoplacental Interface 

Immunity a t th e fetal-placenta l interfac e i s biase d 

toward humoral immunity; cell-mediated immunity is 

suppressed t o preven t feta l rejection . Cell-mediate d 

immunity i s skewed towar d Th 2 response s an d pro - 

duction o f cytokine s suc h a s IL-3 , IL-4 , an d IL-5 , 

rather tha n towar d Thl response s and production of 

cytokines such as IL-2 and IFN-y (Lin et al, 1993). It 

has been suggested that progesterone an d CD4 + regulatory 

T cell s are the key factors enabling the fetus to 

evade immune rejection by the pregnant female, thus 

allowing th e femal e t o maintai n pregnancy . Proges - 

terone suppresse s cytotoxi c activit y o f lymphocyte s 

from pregnan t wome n vi a progesteron e receptor s 

(Szekeres-Bartho e t al, 1990) . A timely increas e i n 

CD4 + /CD25 + regulator y T cells , systematicall y and 

locally at the interface between the fetus and pregnant

female, play s a n importan t rol e i n maintainin g 

tolerance vi a suppressio n o f a n allogenei c respons e 

directed agains t th e fetu s (Szekeres-Barth o e t al. , 

1990; Aluvihar e e t al. , 2004) . I n uter o exposur e t o 

ethanol coul d alte r th e balanc e betwee n regulator y 

T cells and T effecto r cells , and thus contributes to 

the increase d incidenc e o f spontaneou s abortion s 

and premature births. This conclusion i s supported 

by observations of elevated cor d bloo d Ig E concentrations 

i n ethanol-expose d infants , indicatin g in - 

creased activit y o f Th2-typ e response s (Somerse t 

et al. , 2004) . Long-ter m alteration s i n CD4 + regu - 

latory T cells in FEE male s may play a role in mediating 

deficit s i n T cel l functio n (Zhan g e t al. , 

2005a). Dexamethasone-induced apoptosi s an d res - 

cue by IL-2 of CD4+/CD25+ T regulator y cells fro m 

FEE animal s an d control s hav e bee n examined . 

CD4+/CD25+ regulator y T cell s fro m FE E male s 

were more resistant to dexamethasone-induced apoptosis 

i n th e presenc e o f IL-2, resulting i n increase d 

survival o f T regulator y cells . A s T regulator y cell s 

are suppressive in nature, th e increase d number s of 

surviving T regulator y cell s coul d pla y a rol e i n 

ethanol-induced immun e deficits. 

Neuroendocrine-Immune Interactions 

The CN S i s critical fo r th e developmen t an d mat - 

uration o f th e feta l immun e syste m throug h bot h 

sympathetic activit y an d neuroendocrin e mediators , 

such as growth hormone an d the HPA and HPG hor - 

mones (Fig . 10.4) . Th e sympatheti c nervou s system 

innervates lymphoid organs, and lymphocytes express 

receptors fo r CRH , ACTH , cortisol , NE , an d epi - 

nephrine. Th e glucocorticoi d hormone s ca n exer t 

profound influences on T cell function throug h their 

interaction with GRs on T cells, which modulate trafficking 

an d homing , proliferation , activation , an d 

apoptosis (Gonzal o e t al , 1994 ; Dhabha r e t al , 

1996). Fo r example, transgeni c mic e wit h decrease d 

GR binding capacity but normal basal concentration s 

of ACTH an d COR T sho w a partia l blockag e o f T 

cell differentiatio n and decrease d apoptosi s in the fetal 

period but no t in adult life (Sacedo n e t al.7 1999) . 

In addition, glucocorticoids cause a shift from Thl to 

Th2 responses and a change from a pro-inflammatory 

cytokine patter n (e.g. , IL-1 an d TNF-ot ) t o a n anti - 

inflammatory cytokin e pattern (e.g. , IL-1 0 and IL-4 ) 

(DeRijk e t al. , 1997 ; Elenko v an d Chrousos , 1999) . 


Growth hormon e increase s IFN- y productio n an d 

MHC clas s I an d I I expressio n b y thymic epithelia l 

cells, macrophages , dendriti c cells , fibroblasts , an d 

extracellular matri x (Savin o an d Dardenne , 2000) . 

These results suggest that growth hormone facilitate s 

MHC-mediated influence s o n thymocyt e differentia - 

tion. Thymic epithelia l cells and lymphocyte s in rat s 

express estroge n an d androge n receptor s fro m G1 6 

(Tanriverdi et al., 2003) and thus are influenced by circulating 

sex steroid hormones. Furthermore, adminis - 

tration o f gonadotropin releasin g hormon e (GnRH ) 

restores fetal thyrnic and liver-derived T cell proliferative 

response s afte r surgica l ablation o f the forebrain 

or the entir e brai n includin g th e hypothalamu s an d 

pituitary in 18-day-ol d fetuses (Zakharova et al., 2000). 

Thus, GnRH appear s to be involve d in regulation of 

T cel l developmen t eve n during prenatal ontogene - 

sis. These examples show how closely development of 

the thymus is regulated by the neuroendocrine system. 

Cytokines secreted by immune cells, such a s IL-1, 

IL-2, IL-4, an d IL-6 , influence the functio n of hypothalamic 

neurosecretor y an d thermoregulator y neu - 

rons and pituitary cells (Cunningham an d De Souza, 

1993; Rivier , 1994; Zalcma n e t al., 1994 ; Dun n an d 

Wang, 1995) , resultin g in activation of the HP A axis 

and inducing "sickness behavior" (Watkins and Maier, 

2000; Dantzer, 2001). IL-1, IL-6, and TNF-oc are also 

produced i n th e hypothalamu s b y microgli a an d 

macrophages (Hetie r et al., 1988 ; Sebir e et al, 1993 ) 

and thus can directly influence neuroendocrine func - 

tion. For example, IL-1 stimulates the release of CRH 

and AV P from th e hypothalamu s (Sud a e t al, 1990 ; 

Chover-Gonzalez e t al. , 1994) , an d IL-1 , IL-6 , an d 

TNF-oc stimulate ACT H secretio n fro m th e anterio r 

pituitary (Kehre r e t al. , 1988 ; Shar p e t al , 1989 ; 

Lyson and McCann, 1991). 

Given th e intimat e interaction s between th e neu - 

roendocrine an d immun e system s (Fig. 10.4 ) durin g 

development and in adulthood , feta l ethanol-relate d 

developmental change s i n neuroendocrin e activit y 

could affec t immun e competency . I n turn , feta l 

ethanol-induced change s in cytokine secretion by immune 

cell s can affec t neuroendocrin e activity . Therefore, 

th e altere d HP A response s t o immun e signal s 

(Lee and Rivier, 1993 ) and differential vulnerability to 

stress-induced immun e suppressio n (Giberso n an d 

Weinberg, 1995; Giberson et al, 1997) shown by FEE 

rats compared to that of controls could be mediated by 

the altered neuroendocrine-immune interactions.


PRENATAL ETHANOL EXPOSURE 

REPROGRAMS FETA L HP A 

AND IMMUNE FUNCTIO N 

The route s by which the feta l an d earl y neonatal en - 

vironment ca n progra m adult HPA function and be - 

havior are describe d eloquentl y b y Matthews (2002) 

(Fig. 10.2 ) and Welberg and Seckl (2001). The developing 

limbic system, primarily the hippocampus, hypothalamus, 

an d anterio r pituitary , synthesize hig h 

amounts o f GRs and ar e highly sensitive to glucocorticoids. 

Exposure to high concentration s o f glucocorticoids 

durin g earl y lif e ca n alte r th e developmen t 

and subsequen t functio n o f both th e limbi c syste m 

and the HPA axis. The limbi c system, particularly the 

hippocampus, regulates HPA activity, and in turn, endogenous 

glucocorticoid s modif y numerou s limbi c 

system functions. Mechanisms underlyin g these mu - 

tual effects involv e modification of developing neuro - 

transmitter systems , thei r transporte r mechanism s 

in the brain stem, the development o f GR expression 

in th e hippocampus , an d th e developmen t an d re - 

sponsiveness o f the hypothalami c PVN, whic h regu - 

lates glucocorticoid secretion. Prenatal events that can 

program HP A function includ e stres s durin g preg - 

nancy, exposure to synthetic glucocorticoids, and nutrient 

restriction . Postnata l event s tha t ca n progra m 

HPA function includ e earl y handling, alteration s i n 

maternal behavior, and exposure to exogenous glucocorticoids 

and infection. 

The overal l effec t o f earl y developmenta l pro - 

gramming i s altered exposur e t o endogenou s gluco - 

corticoids throughout life (Liu et al., 2001; Matthews, 

2002). This altered exposur e in turn modifie s behavior, 

cognition , learning, memory, and emotion , an d 

predisposes th e individua l t o cardiovascular , metabolic, 

and immune disorders. Although environmental 

factors pla y an importan t rol e i n feta l programming , 

it i s likely tha t perinata l environmental an d geneti c 

factors mutually influence each othe r in determinin g 

HPA activity and behavio r later i n life. Moreover , although 

th e effect s o f programmin g ar e ofte n long - 

lasting, postnatal and later environmental events can 

modulate the effect s o f prenatal programming. 

What is the mechanism underlying fetal or neonatal 

programmin g b y earl y lif e experiences ? Earl y 

experiences resul t i n long-ter m consequence s fro m 

the behavioral to the molecular level through epige - 

netic processes (Weaver et al., 2004). In the model of 

Weaver and colleagues, naturally occurring variations 

in materna l behavio r ar e associate d wit h the devel - 

opment o f individual differences i n behaviora l an d 

HPA responses to stressors . Thus, offsprin g of moth - 

ers showing high level s o f maternal behavio r (larg e 

amounts of licking, grooming, and arched-back nurs - 

ing o f their young ) are les s fearful an d sho w better - 

modulated HP A responses t o stressors than offsprin g 

of mothers showin g low levels of maternal behavior . 

This outcome i s not due t o a change in the underlying 

geneti c profile ; i f pup s ar e cross-fostere d fro m 

low- t o high-materna l behavio r mother s a t birth , 

the offsprin g profil e i s associated wit h th e adoptiv e 

rather tha n th e biologica l mother . Therefore , varia - 

tions in maternal behavior can serve as a mechanism 

for nongenomi c transmissio n o f individua l differ - 

ences i n stres s reactivity across generations. At least 

two majo r epigenomi c mechanism s ar e though t t o 

be involved : demethylatio n o f on e ke y sit e i n th e 

NGFI-A bindin g sequenc e o f th e firs t exo n o f th e 

GR gene, and increase d acetylatio n o f the histone s 

surrounding th e G R gen e (Weave r e t al. , 2004) . 

These two alterations result in increase d expression 

of GRs an d increase d access of transcription factors 

to GRs , thu s increasin g HP A feedback regulatio n 

and alterin g th e physiologica l an d behaviora l pro - 

files of the offspring . 

In our model, prenatal ethanol exposure is an en - 

vironmental variable that causes long-term alterations 

in th e physiologica l and behaviora l profile o f the offspring 

(e.g. , Weinber g an d Jerrells , 1991 ; Giberso n 

and Weinberg , 1995 ; Ki m et al, 1996 ; Glava s et al , 

2001; Hofmann et al, 2002 ; Lan et al, 2004 ; Zhang 

et al, 2005b) . We hypothesize tha t prenata l ethano l 

exposure reprogram s th e feta l HP A axis, resultin g i n 

long-term alteration s in HP A regulation and respon - 

siveness under basal and stress conditions and increasing 

HPA tone throughout life. In view of the evidenc e 

(Phillips e t al , 1998 , 2000 ) tha t feta l programmin g 

of the HPA axis, at least in part, underlies the connection 

betwee n th e earl y environment an d adul t stressrelated 

an d behaviora l disorder s i n humans , furthe r 

studies are essential to elucidate the mechanisms underlying 

prenatal ethano l effect s o n programmin g of 

HPA activity. 


The presen t chapte r discusse s the advers e effects o f 

prenatal exposur e to ethanol o n neuroendocrine an d

immune functio n i n th e offspring , wit h particula r 

emphasis o n th e HP A axi s an d th e concep t o f feta l 

programming. Ethanol-induce d disturbance s o f th e 

reciprocal interconnections betwee n th e pregnant female 

an d th e fetu s ma y provide a common pathway 

by which perinatal exposure to different agent s results 

in feta l programming . Th e feta l HP A axi s i s reprogrammed 

by ethanol exposure such that HPA tone is 

increased throughou t life . HP A activit y is increased 

and HP A regulation i s altered unde r bot h basa l an d 

stress conditions . Increase d exposur e t o endogenou s 

glucocorticoids over the lifespa n ca n alte r behavioral 

and physiological responsiveness and increase vulnerability 

t o illnesse s late r i n life . Deficit s i n immun e 

competence ma y b e on e o f th e long-ter m conse - 

quences of fetal HP A programming. Prenatal ethanol 

exposure compromise s offsprin g immun e functio n 

and increases vulnerability to the immunosuppressive 

effects o f stress. 

It is now essential t o explore possible epigenomic 

mechanisms mediatin g altere d physiologica l and be - 

havioral functio n followin g prenata l ethano l expo - 

sure. Th e fac t tha t ethano l cause s alteration s i n th e 

methylation stat e withi n cell s support s the possibil - 

ity tha t mechanism s simila r t o thos e describe d b y 

Weaver an d colleague s (2004 ) mediat e th e change s 

induced b y ethano l exposur e i n utero . Dat a fro m 

such investigation s will have important implications 

for th e developmen t o f therapeutic intervention s focused 

o n reversin g the long-ter m advers e effect s o f 

prenatal alcohol exposure . 

Abbreviations 

ACTH adrenocorticotropi c hormon e 

ADX adrenalectom y 

AVP arginin e vasopressin 

J3-EP p-endorphi n 

CBG corticosterone-bindin g globulin 


CON A concanavali n A 

CORT corticosteron e 

CRH corticotropin-releasin g hormone 

CRH-R! corticotropin-releasin g hormone type 1 

receptor 

DO! l-(2 , 5-dimethoxy-4-iodophenyl)-2-amino - 

propane hydrochloride 


8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) - 

tetralin 


FEE feta l ethanol-exposed 

S-HT serotoni n 



GDX gonadectom y 

GnRH gonadotropin-releasin g hormone 

GR glucocorticoi d receptor 

hn heteronuclea r 


HPG hypothalamic-pituitary-gonada l 

Ig immunoglobuli n 

IL interleuki n 

LC-NE locu s coeruleu s noradrenergi c (sympa - 

thetic adrenal medullary) 

LPS lipopolysaccharid e 

MHC majo r histocompatibility complex 

MR mineralocorticoi d receptor 



NO nitri c oxide 

NOS nitri c oxide synthase 


POMC pro-opiomelanocorti n 

PVN paraventricula r nucleus 

PWM pokewee d mitogen 

SHRP stres s hyporesponsive period 

SIN-1 3-morpholinosydnonimin e 

SNRP stres s nonresponsive period 

TCR T cell receptors 

TNF tumo r necrosis factor 

ACKNOWLEDGMENTS Th e researc h from ou r laboratory 

tha t i s reporte d in thi s revie w wa s supporte d by 

grants fro m th e Nationa l Institut e o f Alcoho l Abus e 

and Alcoholis m and fro m th e Universit y o f British Co - 

lumbia Huma n Earl y Learnin g Partnershi p to J.W . 

J.H.S. was supported by a Bluma Tischler postdoctoral 

fellowship.


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11 

Early Exposure t o Ethanol 

Affects the Proliferation o f 

Neuronal Precursor s 

The numbe r o f neurons constituting the brai n is a direct 

reflection o f the number of cells produced during 

early development . Th e presen t chapte r explore s th e 

effects o f ethano l o n spatiotempora l pattern s an d 

systems regulatin g cell proliferation . During develop - 

ment mor e neuron s ar e generate d tha n th e numbe r 

comprising th e matur e brain . A "correction " occur s 

through the natural death of many neurons (see Chapter 

5) . Thi s proces s i s als o affecte d b y exposur e t o 

ethanol. Th e consequen t effect s ar e addresse d i n 

Chapters 15 , 16 , and 17 . 

SPATIOTEMPORAL PATTERNS 

The proliferatio n of neuronal population s occur s i n 

defined site s ove r restricte d period s o f time . Thes e 

spatiotemporal pattern s are affected b y early exposure 

to ethanol. Variou s structures involved have been examined, 

includin g segment s o f th e telencephalo n 


182 

(the neocorte x an d hippocampa l formation ) an d 

brain stem (cerebellum and pons). 

Telencephalic Structures 

Neocortex 

Patterns o f Neurona l Generation . Neocortica l 

neurons ar e largel y generated ove r a protracted, bu t 

defined prenata l period. It has also been argue d tha t 

neocortical neuron s continu e t o b e generate d a t a 

low rate in the matur e cortex (see Gould an d Gross , 

2002; Rakic, 2002). For the purpose s o f this review , 

any potential contribution fro m adul t neuronogene - 

sis in neocortex can be considered to be too low as to 

have a n impac t o n neurona l number . A possibl e 

model o f postnatal neurona l generatio n ma y be th e 

proliferative activit y in the intrahilar zone (IHZ ; also 

known a s hippocampa l fiel d CA4 ) o f hippocampa l 

formation wherein the generation of granule neurons

continues throughout lif e (se e Hippocampal Forma - 

tion, below). 

In th e rat , afte r productio n of the pallia i preplate 

cortical neuronogenesis begins on gestational day (G) 

11 and ends on G21 (Bruckner et al, 1976; Lund and 

Mustari, 1977 ; Miller , 1985 , 1988 ; Al-Ghou l an d 

Miller, 1989) . Thi s proces s i s orderly and follow s a 

well-defined inside-ou t sequence . Accordingly , th e 

first neurons generate d becom e laye r Vi a neurons ; 

later generations of neurons ar e destined for progressively 

more superficial laminae. 

Ethanol exposur e alter s th e numbe r an d se - 

quence o f neuronogenesi s (Miller , 1986 , 1988) , 

delaying and extending the period of neuronal generation 

b y 1 day each (Fig . 11-1) . O n eac h day , the 

number of neurons with a particular time of origin is 

reduced. The exceptio n to this pattern is the substantial 

increas e in neurona l generatio n a s cortical neu - 

ronogenesis i s winding down (after G19) . The earl y 

depression i n neurona l generatio n i s followed by a 

late surg e i n term s o f as neuronal density ; on G2 0 

and succeedin g days, neuronal generation is signifi - 

cantly greate r i n th e cortice s o f ethanol-treated rat s 

than i n controls. Interpretation of these data leads to 

four conclusions. 

The first is that the delaye d onset of cortical neuronal 

generation suggests an ethanol-related retarding 

effect o n the acquisitio n of critical numbers of proliferating 

cells required to initiate neuronal generation. 

That is, the population of proliferating neural precursors 

has to reach a threshold size before neuronal precursors 

begin to pass through their final cell divisio n 

and th e youn g neurons begi n to leav e the prolifera - 

tive population . Thi s patter n i s consisten t wit h th e 

concept of founder cells (see Chapter 2). 

The secon d conclusio n i s that ethano l ha s com - 

plex effects o n cell proliferation. Initially, the prolifer - 

ation of neuronal precursor s is depressed an d late r it 

is promoted. This conclusion is supported by various 

studies discusse d belo w (se e Site s o f Proliferation, 

below). 

The thir d observation , fro m th e comple x time - 

dependent effect s o f ethanol o n th e numbe r o f neurons 

being generated, i s that the lat e surge may be an 

effort o f the proliferative population to compensate for 

the earl y depression . Indeed, th e densit y of neurons 

generated ove r the entir e period of neuronogenesis is 

unaffected b y ethanol exposure. This is an interestin g 

finding in that the total number of cortical neurons, at 

ETHANOL AND PROLIFERATION OF NEURONAL PRECURSORS 18 3 

FIGURE 11- 1 Neurona l generatio n i n moto r cortex . 

These dark-fiel d micrograph s sho w tha t i n contro l 

rats, neurons generated on gestational day (G) 1 6 and 

on G20 are distributed in deep and superficial cortex, 

respectively. In ethanol-treated rats, neurons born on 

G16 ar e als o distribute d i n dee p cortex , althoug h 

their number s are fewe r an d the y ten d t o b e mor e 

deeply disposed. Neurons generated o n G20 that are 

properly distribute d i n superficia l cortex ar e als o of 

fewer number . Man y late-generate d neurons , how - 

ever, ar e ectopicall y distribute d i n dee p cortex . 

(Source: Image s taken, with permission, from Mille r 

(1986), Science 233:1308-1311.) 

least in somatosensory cortex, is significantly lower in 

ethanol-treated rats (Miller and Potempa, 1990) . The 

implication i s that ethanol doe s not affec t th e num - 

ber o f neuron s produce d fro m a n ontogeneti c col - 

umn; rather, it reduces the numbe r of columns. An


ontogenetic column i s a hypothetical uni t of developing 

cortex that includes the proliferative cells and the 

radial array of the progen y (e.g. , Rakic, 1978, 2000). 

This conclusion i s supported b y data showing that the 

density o f radia l glia l fiber s (th e guides commonl y 

used t o coordinate neurona l migration ) is unaffected 

by prenatal exposur e t o ethanol (Mille r and Robert - 

son, 1993). 

Finally, th e inside-ou t sequence i s largely maintained 

until the end of cortical neurona l generation . 

At this time , man y neuron s destine d fo r superficial 

cortex en d u p i n deep corte x because o f a defect in 

neuronal migratio n (se e Chapter 13) . The effect s o f 

ethanol o n neuronal generation may be best appreciated 

b y tracing the ontogen y of select populations of 

cortical neurons , fo r example, corticospinal (Miller , 

1987) an d callosa l projectio n (Miller , 1997 ) neu - 

rons. In general, they are distributed in laminae that 

are normal for their phenotype, with the notabl e exception 

o f the late-generated callosa l neurons. Many 

of thes e neuron s resid e i n infragranula r laminae . 

Thus, in addition to the conclusions described above, 

it appears that (a) ethanol doe s not affect the connectional 

phenotype of a particular neuron (regardles s of 

a migratio n defect) and (b ) the decisio n about neuronal 

lineag e occur s a t the tim e o f neuronal origin . 

Similar cortica l disorganizatio n o f neurona l pheno - 

types occur s i n cortice s in whic h cell s i n th e proliferative 

zone s ar e heterochronicall y transplante d 

(McConnell, 1988) . 

Sites o f Proliferation . Th e proliferatio n o f neuronal 

precursor s occur s i n specifi c site s generall y 

located proxima l t o th e ventricula r syste m (se e 

Chapter 2) . For neocortex, cell proliferation occurs in 

three sites (Fig. 11-2). The ventricula r zone (VZ) is a 

pseudostratified epitheliu m linin g th e latera l ventri - 

cles (Sauer , 1936 ; Watterso n e t al., 1956 ; Saue r an d 

Chittenden, 1959 ; Jacobson, 1991) . The subventricu - 

lar zone (SZ ) is a stratified epitheliu m externa l to the 

neocortical V Z (Ramo n y Cajal , 1909-1911 ; Rakic 

et al, 1974 ; Shoukimas and Hinds, 1978) . Th e ganglionic 

eminenc e (GE ) generates som e loca l circui t 

neurons (Tamamaki et al, 1997 ; Zhu et al, 1999 ; Anderson 

étal, 2001; Wichterle étal, 2001). 

The tw o primary neocortical proliferatio n zone s 

are th e V Z an d SZ , an d ther e i s some controvers y 

about the contribution of the VZ and S Z in the generation 

o f neocortica l neurons . Som e investigator s 

FIGURE 11- 2 Effect s o f ethanol o n the thre e sources 

of neocortica l neurons . Neuron s tha t populat e th e 

cortical plat e (CP ) are derive d from th e ventricular 

zone (VZ) , subventricular zone (SZ) , and ganglioni c 

eminence (GE) . In norma l rats , V Z an d S Z cell s 

principally giv e rise to infra - an d supragranula r neurons, 

respectively (solid lines). The G E generate s local 

circui t neuron s tha t ar e distribute d throughou t 

neocortex. Followin g prenata l exposur e t o ethanol , 

many late-generate d neuron s (originatin g largely in 

the SZ ) complet e thei r migration s i n dee p corte x 

(dashed line) . IZ, intermediat e zone ; MZ , marginal 

argue that the V Z i s the sit e o f neuronal origi n an d 

that th e S Z generate s gli a an d glia l precursor s 

(Gomes et al, 2003 ; Samuelson et al, 2003) . In contrast, 

other s argu e tha t al l neuron s ar e derive d fro m 

the S Z and that the VZ is merely a self-replacing population 

(Doetsc h e t al , 1997 , 1999; Alvarez-Buylla 

and Garcia-Verdugo , 2002 ; Marten s e t al , 2000 , 

2002). Other studies support a different mode l o f cortical 

development, that both th e VZ and S Z give rise 

to cortica l neuron s (Miller , 1992 ; Luo an d Miller , 

1998). 

Studies on th e consequence s o f prenatal exposure 

to moderate amounts of ethanol (sufficien t to produce

peak blood ethano l concentrations o f 15 0 mg/dl) provide 

unique insight into the controversy over the con - 

tribution o f th e V Z an d S Z t o th e populatio n o f 

neocortical neurons. Dail y neuronal production dur - 

ing th e firs t hal f o f the perio d o f neuronongenesis is 

depressed b y prenata l exposur e t o ethano l (Miller , 

1986, 1988) . Durin g thi s time, th e V Z i s the promi - 

nent neocortica l proliferativ e zone . Change s i n cell 

proliferation withi n the V Z paralle l the earl y reduc - 

tions i n neurona l generation . Prenata l exposur e t o 

ethanol reduce s th e tota l numbe r o f cell s an d th e 

number of cycling cells in the VZ (Miller, 1989) an d 

it increases the time cells need t o transit through th e 

cell cycle (Miller and Nowkowski, 1991). 

The ethanol-induce d late surge in neuronal generation 

occur s when the S Z becomes th e predominant 

proliferative zone . The S Z i s affected b y ethanol i n a 

way opposit e tha t o f th e VZ . Th e tota l numbe r o f 

cells i n th e S Z an d th e numbe r o f cycling cells ar e 

increased b y ethano l (Miller , 1989 ; Mille r an d 

Nowakowski, 1991) . 

Taken together , th e correlativ e dat a o n th e birt h 

dates o f cortica l neuron s an d characteristic s o f V Z 

and S Z cells show that both the VZ and S Z produc e 

neurons. Thi s thesi s wa s tested i n a n experimen t i n 

which fetuses were exposed t o ethanol for only 4 days 

during th e perio d o f (a ) S Z prominenc e (betwee n 

G18 an d G21) , (b ) VZ prominenc e (betwee n G1 2 

and G15) , o r (c ) ste m cel l productio n (betwee n G 6 

and G9) (Miller, 1996a). SZ cell proliferation and the 

production o f late-generate d neuron s ar e onl y af - 

fected whe n th e ethano l exposur e occur s betwee n 

G18 an d G2 1 (Fig . 11-3) . Moreover, this late exposure 

t o ethano l increase s both cel l proliferatio n an d 

production. VZ cell proliferation and early-generated 

neurons, b y contrast , ar e maximall y affected b y exposure 

t o ethano l tha t occur s betwee n G1 2 an d 

Gl 5, i.e., when the VZ is most prominent. This exposure 

to ethanol inhibit s VZ cel l proliferatio n an d re - 

duces th e numbe r o f neuron s generate d o n G15 . 

Exposure to ethanol between G6 and G9 does not affect 

th e proliferatio n o f cell s i n eithe r proliferativ e 

zone o r th e numbe r o f early- or late-generate d neu - 

rons. Furthermore , base d o n timing , i t appear s tha t 

the VZ gives rise to neurons distributed in infragranu - 

lar corte x an d th e S Z generate s supragranula r 

neurons. This conclusion i s supported b y the tracin g 

of derivative s of cell s tha t expres s transcripts fo r th e 

genes Svetl (Tarabyki n e t al. , 2001 ) an d eux (Niet o 

ETHANOL AND PROLIFERATION OF NEURONA L PRECURSORS 18 5 

et al. , 2004 ) fro m th e S Z t o th e supragranula r laminae. 

Radial Glia. Radia l glia, thought to serve as guides 

for neurona l migratio n (Rakic , 1971 , 1978) , ar e 

among th e first cortical cell s t o be generated . Tradi - 

tionally, radial glia have been considered transitiona l 

astrocytes (e.g. , Schmeche l an d Rakic , 1979 ; L u 

et al, 1980 ; Voigt, 1989 ; d e Lima e t al, 1997) , how - 

ever, mor e recen t evidenc e show s that som e radia l 

glia ca n differentiat e int o neuron s (Malatest a e t al. , 

2000; Hartfuss et al, 2001; Levers et al, 2001; Noctor 

et al , 2001 , 2002 ; Alvarez-Buyll a an d Garcia - 

Verdugo, 2002; Gôtz e t al., 2002; Sanai et al, 2004). 

This dual lineage is consistent with glial expression of 

nestin, a cytoskeleta l protein associate d with neural 

stem cell s (Hockfiel d an d McKay , 1985 ; Miller an d 

Robertson, 1993) . Although n o studie s have directly 

assessed th e effect s o f ethanol o n th e proliferatio n of 

radial glia , it appears that th e periodicit y of the net - 

work o f radia l glia l fiber s i s unaffecte d b y ethano l 

(Miller an d Robertson , 1993 ; Vallè s e t al , 1996 ; 

Minana e t al., 2000). This finding implies that these 

cells proliferat e t o maintai n a consisten t densit y 

within the expandin g telencephalic vesicle. A discussion 

o f the effect s o f ethanol o n radia l glia and thei r 

role in neuronal migratio n is provided in Chapter 3. 

Hippocampal Formation 

Neurons in the hippoeampal formation are generated 

over a protracted period of time: i n the rat , from G1 5 

to adulthoo d (Altman , 1962 ; Angevine , 1965 ; Sch - 

lessinger e t al , 1978 ; Bayer , 1980 ; Miller , 1995a) . 

Most neuron s i n th e thre e hippoeampa l fields , 

CA1-CA3, and th e dentat e gyru s are generated pre - 

natally. The sit e of origin of these neurons is the VZ . 

A notable exception is that most (-85%) granule neurons 

i n th e dentat e gyru s ar e generate d postnatally . 

Postnatal granule cell generation occurs in the IHZ. 

Hippocampal cel l number s ar e substantiall y affected 

by ethanol exposure. These effects ar e spatially 

and temporally defined. Total DN A and protein con - 

tent i n hippoeampa l formatio n i s lowe r i n animal s 

exposed to ethanol prenatally (Miller, 1996b). I n contrast, 

postnatal exposur e increases both DNA and protein 

content. Anatomical studies have provided mor e 

detailed informatio n o n thes e ethanol-induce d 

effects. Animal s expose d t o ethano l prenatall y have


FIGURE 11- 3 Time-dependen t effects o f ethanol on the tw o neocortical prolif - 

erative zones. A . Exposure to ethanol durin g three prenata l tim e period s was 

examined: durin g the perio d o f neocortical ste m cel l generatio n (gestationa l 

day [G ] 6 to G9), prominenc e o f the ventricula r zone (VZ ) (G12-G15) , and 

prominence o f the subventricula r zone (SZ ) (G18-G21). B. Exposure during 

the period o f stem cel l generation doe s not affect th e proportio n o f cells prolif - 

erating o n G1 5 an d G2 1 (whe n th e V Z an d S Z predominate, respectively) . 

Later exposures, by contrast, hav e specific effects o n the zone most active during 

the tim e o f exposure, i.e., the V Z between G1 2 an d G1 5 and th e S Z between 

G1 8 an d G21 . (Source: Image s taken , wit h permission , fro m Mille r 

(1996a), Alcohol Clin Ex p Res 20:139-143.) 

fewer neurons in the pyramidal cell layer of CAI an d 

granule cel l laye r of the dentat e gyru s than d o con - 

trols (Miller, 1995a) . Interestingly, whereas postnatal 

exposure generall y has littl e effec t o n th e numbe r 

of these neurons, it can increase the numbe r of granule 

neuron s i n th e dentat e gyru s (Miller , 1995b ; 

Pawlak et al., 2002). This effect o n granule neurons is 

concentration-dependent Moderat e amount s o f 

ethanol ar e stimulator y and hig h concentration s of 

ethanol are depressive. 

The ethanol-induced changes in neuronal number 

directly reflect alterations in neuronal generation . Fo r 

example, the generation of dentate granule neurons is 

reduced b y prenatal ethano l exposur e an d promote d 

by postnatal exposure . Thus, ethanol affect s develop - 

ing hippocampal formation in the sam e manner tha t 

it affect s th e generatio n o f neocortica l neurons . As 

postnatal generation of granule neurons occurs in th e 

IHZ, i t appears that ethano l affect s al l telencephali c 

auxiliary (derived) proliferative zones similarly. 

Brain Ste m 

The effec t o f chronic gestationa l exposur e t o ethano l 

on th e principa l sensor y nucleu s o f th e trigemina l 

nerve (PSN) and cerebellum has been examined. The 

PSN i s a simple structure. All of its neurons are derived 

from th e pontine VZ. Prenatal exposure to ethanol reduces 

the numbe r of neuons i n the PS N (Mille r and 

Muller, 1989 ; Miller , 1995b) . This reductio n i s partially 

(two-thirds ) due t o decreased cell generation an d 

partially (one-third ) to exacerbatio n of mechanisms of 

naturally occurring neuronal death (Miller, 1999a). The 

reduction in cell generation appears to be equivalent for 

neurons and gli a and neurona l generation i s delayed by 

about 1 day (Miller and Muller, 1989) . 

The cerebella r corte x is also vulnerable to ethano l 

toxicity. A gross indication that ethanol affects cel l generation 

in the cerebellu m i s the effec t o f ethanol on its 

size (Bauer-Moffett and Altman, 1975; Kornguth et al., 

1979; Dia z an d Samson , 1980 ; Pierc e an d West ,

1986; Bonthiu s and West, 1988) . Exposur e to moder - 

ate amounts of ethanol (bloo d ethanol concentration s 

of-150 mg/dl) causes reductions (15%-20%) in cerebellar 

siz e similar to tha t eviden t fo r cerebral corte x 

(Miller, 1996b) . Followin g exposur e to high amount s 

(sufficient t o produc e bloo d ethano l concentration s 

of >250 mg/dl), the size of the cerebellum i s more adversely 

affected . 

Cerebellar neuron s ar e generate d i n tw o site s — 

the VZ and a derived proliferative zone, th e externa l 

granular layer (EGL). Attention ha s been directed toward 

the EGL because i t is most prominent in the rat 

postnatally an d i t give s birth t o a populou s neuro n 

type i n th e centra l nervou s system (CNS) , th e cere - 

bellar granul e neuron . Postnata l exposur e t o hig h 

amounts o f ethano l prolong s th e existenc e o f th e 

EGL an d delay s its depletion (Bauer-Moffet t an d Altman, 

1977 ; Korngut h e t al. , 1979) . Moreover , cel l 

proliferation i n the EG L i s reduced. These data suggest 

that the EG L i s different fro m othe r derive d proliferative 

zone s i n tha t it s proliferative activity is no t 

stimulated by ethanol. Th e EG L differ s fro m th e S Z 

and IHZ insofar as it is the sol e source of granule neurons, 

whereas the S Z and IH Z produce neuron s that 

can be generated i n the VZ. Nonetheless, thi s conclu - 

sion remain s unconfirme d unti l dose-respons e dat a 

analysis of the EG L i s performed. 

CELL CYCLE KINETICS 

In vivo and In Situ Studies 

As cells proliferate the y pass through a defined set of 

steps, cumulativel y known a s the cell cycle. The cel l 

cycle has four phases: Gl, S, G2, and M. During th e 

Gl phas e th e cell s eithe r prepar e fo r anothe r pass 

through the cell cycle or exit from the cell cycle. Durings, 

cells replicate their nuclear DNA . G 2 i s a short 

period whe n preparatio n fo r mitosi s take s place , 

which occur s durin g M . Dat a o n th e cel l cycl e ar e 

best appreciated for VZ cells. The somat a of VZ cells 

move rhythmically as the cell s pass through th e fou r 

phases (Sauer , 1936 ; Watterso n e t al. , 1956 ; Saue r 

and Chittenden , 1959 ; Atla s an d Bond , 1965) . 

Through thi s process , calle d interkinetic nuclear migration, 

mitotic cell s tha t ar e a t th e ventricula r surface 

reconstitute and move their nuclei to positions in 

the external third of the VZ where they replicate their 

DNA during S. 


Ethanol doe s no t affec t th e patter n o f interkinetic 

nuclear migration in the VZ per se (Kennedy and Elliott, 

1985; Miller, 1989). On the other hand, ethanol 

does affec t th e tim e a VZ cel l take s to pass throug h 

the cell cycle. For example, in the neocortical V Z of a 

21-day-old rat fetus (Miller and Nowakowski, 1991) or 

a slic e o f corte x explante d fro m a 17-day-ol d fetu s 

(Siegenthaler an d Miller , 2005a) , the tota l length of 

the cel l cycl e (T c) i s increased 26%-29% . Thi s in - 

crease ha s a direct negativ e effec t o n th e numbe r o f 

cells produce d i n th e VZ. An increase i n th e Tc is 

also eviden t i n th e EG L o f the cerebellu m (Borge s 

and Lewis , 1983) . Ethanol-induce d increase s in Tc 

primarily result from a lengthening o f Gl. Thes e effects 

o f ethanol ar e no t th e sam e fo r all proliferative 

populations. Fo r example, th e Tc of the neocortica l 

SZ i s not affecte d b y moderate exposure s to ethano l 

(Miller and Nowkowski, 1991). 

In addition to cell cycle kinetics, the production of 

new cells is defined by three othe r features of proliferating 

populations: th e growt h fraction (GF), th e pro - 

portion of cells that exit the cell cycle, and the leaving 

fraction (LF) . The G F i s the proportio n o f total cel l 

population tha t i s actively cycling. Overall , th e G F 

for th e cerebra l wal l extends ove r th e perio d o f neo - 

cortical neuronongenesi s (Mille r an d Kuhn , 1995 ; 

Takahashi e t al, 1995 , 1996) . Althoug h ther e i s no 

ethanol-induced differenc e i n th e G F a t th e begin - 

ning of this period, as neuronal generation proceeds a 

significant differenc e takes place . Ethano l cause s a n 

increase in the GF. The effec t of ethanol on the overall 

GF i s largely due t o ethanol-induced increase s in 

the SZ . Both the specifi c G F fo r the S Z (Miller and 

Nowkowski, 1991 ) and th e numbe r o f cells in the S Z 

(Miller, 1989 ) ar e increase d b y ethanol. Th e G F for 

the VZ is either unaffecte d by ethanol in vivo (Miller 

and Nowkowski , 1991) or subtly, but significantly , reduced 

i n th e V Z o f an organotypi c slice preparation 

(Siegenthaler an d Miller , 2005a) . The effec t o f th e 

increases i n GF i n the S Z is accentuated wit h time as 

the S Z become s th e majo r neocortica l proliferativ e 

zone. 

After cells pass through M, the daughter cells either 

re-enter the cycle or exit the cell cycle. The numbe r of 

cells tha t exi t th e cel l cycl e ca n b e determine d b y 

tracing the proportio n o f cells that have incorporate d 

a DN A precursor during S relative to the tota l popu - 

lation o f cycling cells, for example, cells that express 

Ki-67 (Chen n an d Walsh , 2002 , 2003 ; Siegenthale r 

and Miller , 2005a ; 2005b) . Accordingly , a n i n sit u


study show s tha t ethano l increase s th e numbe r o f 

cells that exit the cell cycl e (Siegenthaler an d Miller, 

2005a). 

A third featur e contributin g to neurona l produc - 

tion i s the LF . Thi s differ s fro m th e exi t fraction i n 

that i t encompasses cel l cycl e exi t and th e proces s of 

moving fro m th e proliferativ e zones . Th e L F rise s 

steadily over the period of neuronal generation (Miller, 

1993, 1999b ; Takahashi et al, 1996) . Ethanol does not 

affect th e L F during this period (Miller , 1993) . Inasmuch 

a s ethano l doe s affec t th e numbe r o f exitin g 

cells, thi s lac k o f increas e i n th e L F implie s tha t 

ethanol induce s postmitoti c cell s t o remai n i n th e 

proliferative zones for a longer time before commenc - 

ing their migration. 

Various cell cycle-relate d proteins ar e affecte d b y 

ethanol: th e expressio n o f cyclin A (a regulator o f S 

and G2), cycli n Dl, and cdk2 (proteins that regulate 

the progressio n of cells from G l t o S ) i s reduced b y 

ethanol exposur e (Li et al, 2001, 2002; Siegenthale r 

and Miller , 2005a) . Cerebellar expressio n o f cycli n 

D2, cdk4, and cdk6 (regulators of Gl) ar e unaffected 

by ethanol (L i et al, 2002). By contrast, p27 ( a protein 

involve d in movin g cells from G l ou t o f the cycle) 

i s depresse d b y ethano l i n th e V Z o f cortica l 

expiants (Siegenthale r an d Miller , 2005a ) and i n developing 

rat cerebellum (L i et al., 2002). Thus, the effects 

of ethanol on cell cycle-related protein s not only 

corroborate, bu t provid e a mechanis m fo r ethanol - 

induced increases in the Tc and cell cycle exit and a 

decrease i n the GF. 

Cell Culture Studies 

Studies i n cel l cultur e model s replicat e th e i n viv o 

data and provide systems in which to explore cellular 

and molecula r mechanism s underlyin g th e ethanol - 

induced changes . Variou s cell s hav e bee n use d a s 

models o f proliferatin g neura l precursors—primar y 

cultures of neurons (Jacobs and Miller , 2001; Miller, 

2003a) an d astrocyte s (Kenned y and Mukerji , 1985 ; 

Guerri e t al. , 1990 ; Snyde r e t al, 1992 ; Kòtte r an d 

Klein, 1999 ; Lu o an d Miller , 1999a ; Cost a an d 

Guizzetti, 2002 ; Mille r an d Luo , 2002a ) an d neu - 

ronal (Lu o an d Miller , 1997a , 1999b ; Mille r an d 

Luo, 2002b ) o r glia l (Wazir i e t al , 1981; Isenber g 

et al. , 1992 ; Resnicof f et al, 1994 , 1996b ; Lu o an d 

Miller, 1996 ) cance r cel l lines . There i s consensus 

that ethanol increase s the time these neural cells take 

to double the size of their populations. 

Doubling tim e i s a comple x yardstic k reflectin g 

the su m o f simpler parameters. On e o f these parame - 

ters is an increase in the Tc, and most of this increase 

is from a lengthened Gl (Guerr i et al., 1990 ; Luo an d 

Miller, 1997a ; Jacobs and Miller , 2001). Remarkably, 

the effect s o f ethanol o n th e proliferatio n o f dissoci - 

ated neuron s an d B10 4 neuroblastom a cell s strongl y 

parallel thos e o f V Z cell s i n viv o (cf . Mille r an d 

Nowakowski, 1991 ; Lu o an d Miller , 1997a ; Jacob s 

and Miller, 2001). Another factor is the GF. Alteration 

in the GF has an invers e effect on doubling time. For 

example, a s i n th e cas e o f B10 4 cells , a n ethanol - 

induced decrease in the numbe r of cycling cells con - 

tributes t o a n increas e i n doublin g tim e (Lu o an d 

Miller, 1997a) . A third facto r definin g th e doublin g 

time i s the incidenc e o f cell death . Deat h normall y 

occurs amon g proliferatin g population s (Blaschk e 

et al., 1996; Thomaidou et al, 1997; Jacobs and Miller, 

2000) (see Chapter 5) . Ethanol affects th e balance between 

cell proliferation and death (Jacobs and Miller, 

2001; Miller , 2003a) . For example , ethano l cause s a 

steady decreas e i n th e numbe r o f primar y culture d 

cortical neurons . Mos t (two-thirds ) of the decreas e is 

due to compromised cell proliferation, whereas the remainder 

i s from ethanol-induce d cel l death. Ethano l 

affects neurona l generatio n an d deat h i n th e imma - 

ture PSN in a similar manner (Miller , 1999a). 

LIGAND-MEDIATED SYSTEM S 

Many factors may contribute t o the differentia l effec t 

of ethano l o n specifi c population s o f proliferating 

cells. A major playe r is the myria d of growth factor s 

that tightly regulate cell proliferation. This section focuses 

o n a smal l numbe r o f growth factor s tha t ha s 

been studie d i n relatio n t o th e effect s o f ethanol . 

These factor s includ e platelet-derive d growt h facto r 

(PDGF), basi c fibroblas t growt h facto r (bFG F o r 

FGF-2), insulin-lik e growth factor (IGF)- l an d IGF - 

2, an d transformin g growth facto r (TGF ) pi . The y 

can b e classifie d as mitogeni c (FGF-2 , IGF-1 , an d 

PDGF) or anti-mitogenic (TGFp). 

Mitogenic Factors 

Platelet-Derived Growth Factor 

The cyclin g activit y o f neura l cell s i s potentl y pro - 

moted by PDGF. It is noteworthy that PDGF receptors

are expresse d i n the S Z of normal rodent s (Sasahara 

et al, 1991 ; Yeh et al, 1991) . Th e growth-promoting 

effect o f PDGF has also been described for astrocytes 

(Luo an d Miller , 1999a ) an d B10 4 neuroblastom a 

cells (Luo and Miller, 1997a) . 

PDGF is active as a dimer, forme d as a combina - 

tion o f th e tw o isoforms , PDGF- A an d PDGF-B . 

PDGF-BB i s the mos t potent ligand , doubling or trebling 

the growt h of B104 cells an d astrocytes , respec - 

tively, over 3 days (Luo and Miller , 1997a , 1999a) . In 

contrast, PDGF-A A onl y increase s cel l growt h b y 

-50% (B104 neuroblastoma cells ) or -70% (astrocytes) 

over the sam e period. Although the tota l length o f the 

cell cycl e i s reduced, mos t o f the growt h result s fro m 

the recruitment o f cells into the cyclin g population— 

i.e., throug h a n increas e i n th e GF . Thi s patter n i s 

reminiscent o f th e respons e o f S Z cell s t o ethano l 

(Miller an d Nowakowski , 1991 ; Mille r an d Kuhn , 

1995). 

The PDGF-mediate d increase in cell proliferation 

is mediated throug h tw o intracellular signal transduction 

cascades : the ras-raf pathwa y and th e protein kinase 

C (PKC ) pathway (Fig . 11-4 ) (Lu o and Miller , 

1999a). Th e ultimat e effect s o f PDGF ar e th e tran - 

sient (phasic ) activation o f mitogen-activated protei n 

kinase and extracellular signal regulated kinase (ERK) 

and the transcription of proliferation-associated genes . 

PDGF-mediated growt h i s compromise d b y 

ethanol, th e growt h amon g B10 4 cell s (Lu o an d 

Miller, 1997a) and astrocytes (Luo and Miller, 1999a) 

being reduced in a concentration-dependent manner . 

Interestingly, the effect s o f ethanol o n the tw o ligand 

isoforms are cell type-specific. The actio n of PDGF- 

BB on B10 4 cells i s twice as sensitive to ethanol a s it 

is to PDGF-AA (Lu o and Miller , 1997a) , an d th e in - 

verse situatio n i s eviden t fo r neocortica l astrocyte s 

(Luo and Miller, 1999a) . This may mean tha t PDGF 

differentially affect s cel l lineage s i n vivo . Moreover , 

the variatio n i n effect s ma y underlie th e differentia l 

activity o f SZ cells , whic h i n th e ra t ar e largel y neuronogenetic 

prenatally and gliogenetic postnatally. 

The effec t o f ethanol o n ligand-mediate d activity 

apparently reflects ethanol-induced changes in recep - 

tor expression and activation. Two receptors can bind 

PDGF ligands : PDGF-a r an d PDGF-pr . I n B10 4 

cells, ethanol target s the PDGF-ar, which ultimately 

leads to a change in ERK activation (Luo and Miller, 

1999a). Ethano l doe s not stop ERK phosphorylation; 

rather, i t induces a sustained ER K activation, which 

in turn can lead to changes in gene transcription. 


Fibroblast Growth Factor-2 

FGF-2 i s an importan t regulator o f cell proliferatio n 

in th e V Z an d derive d zone s o f developing rodents . 

An immunohistochemical study shows that FGF-2 receptors 

ar e predominantl y expresse d i n V Z durin g 

normal, cortical development (Weis e et al., 1993) . SZ 

cells in the neonatal rat brain are also FGF-2 responsive 

(Bartlet t et al, 1994 ; Gritt i et al, 1999 ; Wagne r 

et al, 1999 ; Decke r e t al. , 2002) . Fo r example , in - 

traperitoneal injection of FGF-2 stimulates the prolif - 

eration o f neuronal precursor s in the S Z (Lachapell e 

et al. , 2002) . Similarly , intraventricula r administra - 

tion of FGF-2 expands the population of SZ neuronal 

progenitors (Kuhn et al., 1997 ) an d increase s mitoti c 

activity in the SZ and IH Z in adul t rat s (Wagne r 

et al, 1999) . 

FIGURE 11- 4 Signa l transductio n triggere d b y 

ethanol-sensitive growt h factors . Cel l proliferatio n is 

regulated b y platelet-derive d growt h facto r (PDGF ) 

and transforming growth factor (TGF) pi . PDGF acts 

as a dime r o f A o r B isoforms . Thes e growt h factor s 

bind t o specifi c trans-membran e receptor s tha t au - 

tophosphorylate. Thi s proces s trigger s a cascad e o f 

phosphorylation event s throug h variou s intermedi - 

aries. Interestingly , although PDG F and TGFpl in - 

duce opposit e effect s (mitogeni c an d anti-mitogeni c 

actions, respectively) , they both stimulat e extracellular 

signal-related kinase (ERK). The circle d plus and 

minus sign s identif y th e action s o f ethanol . Briefly , 

ethanol ha s a specific effec t o n a single PDGF receptor 

isofor m (PDGF-ar ) and , throug h bot h pro - an d 

antimitogenic factors , ethanol stimulates ER K activ - 

ity. This in turn can alter nuclear gene transcription. 

MEK, methylethylketone ; nCAM , nerv e cel l adhe - 

sion molecule; PKC, protein kinase C.


Dissociated cultures provide model systems to illuminate 

th e effect s o f FGF-2 on proliferating populations. 

Cell cycle kinetics among B104 cells is affected 

by FGF-2 (Luo and Miller, 1997a) . The Tc and GF 

are equally affected b y FGF-2. These data are consistent 

with the mixe d expression of FGF-2 response by 

VZ and SZ cells. Other preparation s that presumably 

model V Z and S Z cells impl y that FGF- 2 promote s 

the proliferation of VZ and SZ derivatives (Martens et 

al., 2000, 2002). 

The effect s o f ethano l o n FGF-2-mediate d cel l 

proliferation ar e not well understood. Studie s explor - 

ing thi s interactio n hav e examine d C 6 gliom a cell s 

(Luo an d Miller , 1996 ; Mille r and Luo , 2002a ) and 

telencephalic neuroepithelial cell s (Ma et al., 2003). 

They sho w tha t ethano l eliminate s th e FGF-2 - 

promoted increas e i n cell proliferatio n such tha t th e 

effects o f FGF-2 and ethanol nullif y each other . 

The depressiv e effect s o f ethano l ar e mediate d 

through th e FGF- 2 receptor , fl g (Lu o an d Miller , 

1996b, 1997b) . ER K phosphorylation, a downstream 

event of flg activation, increases i n cortica l astrocyte s 

(Smith an d Navratilova , 2003 ) an d ste m cell s (M a 

et al. , 2003 ) treate d with ethanol . Althoug h ethano l 

does not alter protein expressio n of FGF-2 receptors, 

ERK activity remains elevated, implying that ethano l 

affects recepto r phosphorylation . 

Insulin-like Growth Factor-1 

Prenatal exposure to ethanol inhibition of intrauterine 

and early postnatal growth correlates with the effec t of 

ethanol o n the IGF system (Breese et al, 1993 ; Sing h 

et al., 1994) . Ethanol halves plasma IGF-1 content i n 

the pregnant dam (Singh et al., 1994; Breese and Son - 

ntag, 1995 ) and in the fetal brain (Singh et al, 1996) . 

Data o n the effect s o f prenatal exposur e to ethanol o n 

IGF-2 are mixed. One repor t states that the amount of 

IGF-2 is doubled (Brees e and Sontag , 1995) , whereas 

others sho w tha t IGF- 2 expressio n i s halve d (Sing h 

et al., 1994 , 1996) . Likewise , the effec t o f ethanol o n 

binding protei n (IGF-BP ) i s als o mixed . Som e evi - 

dence show s tha t IGF-B P expressio n i s reduce d 

(Breese an d Sontag , 1995) , an d othe r studie s sho w 

that it is increased (Singh et al, 1994, 1996) . Although 

the reason s for these disparat e data ar e unclear , i t is 

agreed tha t (1 ) ethanol affect s circulatin g plasma an d 

fetal expressio n of IGF-2 an d IGF-B P an d (2 ) ethanol 

causes opposing effects o n the expressio n of these two 

proteins. The reduction in IGF-1 persist s in the young 

offspring (u p t o 3 weeks old), but i t abates by 40 day s 

of ag e (Brees e e t al, 1993) . I n contrast , IGF- 2 an d 

IGF-BP ar e unaffected in preweanling rats. 

The effec t o f IGF-1 ha s been studie d in C6 gliom a 

cells (e.g. , Resnicof f e t al , 1994 , 1996a , 1996b) . 

These cells have the advantage of being able to proliferate 

i n a serum-free medium an d i n th e absenc e o f 

any priming growth factors (e.g., Isenberg et al., 1992 ; 

Resnicoff e t al, 1994 ; Lu o and Miller, 1996) . IGF- 1 

is a potent mitoge n fo r proliferating C6 cell s (Resni - 

coff e t al., 1994 , 1996a , 1996b) . Thi s effec t i s at least 

partially growt h factor-specific , a s proliferatio n o f 

these cell s i s unaffected b y PDGF (Resnicof f et al. , 

1994). It is worth noting that this differential respons e 

to growth factors may be a quality of the cell s becaus e 

primary cultur e astrocyte s and B10 4 neuroblastoma 

cells are highly regulated b y PDGF (Luo and Miller , 

1997a, 1999a ) (se e Platelet-Derive d Growt h Factor , 

above). 

Ethanol inhibit s th e proliferatio n o f C 6 cell s 

(Waziri e t al, 1981 ; Isenber g e t al, 1992 ; Resnicof f 

et al, 1994 , 1996a , 1996b ; Luo and Miller, 1996) , and 

it has been suggested that this inhibition i s mediated by 

ethanol-induced disruption s of IGF-1 recepto r (IGF - 

lr; Resnicof f e t al , 1994 , 1996a , 1996b) . Ethano l 

blocks the mitogeni c effec t o f the ligan d (IGF-1 ) an d 

interferes with receptor (IGF- 1 r) activation. Autophosphorylation 

o f the IGF- 1 r is inhibited b y low concentrations 

o f ethanol . Also , ethano l inhibit s th e 

anti-apoptotic effect s o f IGF-1 (Gu i étal, 1997) . 

Anti-mitogenie Facto r 

TGFp ligand s ar e intracortica l factor s tha t tur n off 

neural cel l growt h (Camero n e t al. , 1998 ; Bôttne r 

et al, 2000). Exogenous TGFpl inhibit s the growth 

of variou s cells , includin g C 6 cells , astrocyte s (Ro - 

zovsky e t al, 1998 ; Ric h e t al. , 1999 ; Rob e e t al, 

2000; Mille r an d Luo , 2002a) , B104 neuroblastoma 

cells (Luo and Miller, 1999b) , and neural progenitor s 

(Miller an d Luo , 2002b) . TGFp l doe s thi s b y removing 

cell s fro m th e proliferativ e population, i.e. , 

reducing th e G F (Siegenthale r an d Miller , 2005b) . 

Interestingly, it does not affect cel l cycle kinetics. 

Transforming Growth Factor- 

Mediated Signal Transduction 

The effec t o f TGFpl o n cel l proliferatio n is medi - 

ated throug h ER K activatio n (Fig . 11-4) . TGFp l

induces sustaine d (>9 0 min ) activatio n (Lu o an d 

Miller, 1999b) , a n anti-intuitive result in that ERK is 

generally associated with mitogenic activity. A recent 

study of proliferating cells i n organotypi c cultures of 

fetal cortex shows that cycling cells in the VZ respond 

to TGFpl (Siegenthale r an d Miller , ZOOSb) . As with 

the dissociated cells, TGFpl inhibits VZ cell proliferation 

in situ by increasing the exit of cells from the cycling 

populatio n an d concomitantl y decreasin g 

the GF. 

Combined treatmen t o f cortica l slice s wit h 

ethanol and TGFp 1 affects the expressio n of cell cycle-related 

proteins (Siegenthale r an d Miller, 20 0 5b) . 

For example , tota l cycli n D l expressio n i s reduce d 

in th e VZ, an d becaus e ethano l an d TGFpl d o no t 

affect th e frequenc y o f cyclin Dl-positive cells , i t ap - 

pears tha t th e amoun t o f cycli n D l pe r cel l i s reduced. 

A simila r decremen t i n p2 7 expressio n ha s 

been detected . A s for p21, co-treatment with ethano l 

and TGFp l eliminate d th e increas e i n p2 1 expression 

promoted b y TGFpl alone. Thus, three proteins 

involved in the transition o f cells from Gl (t o either S 

or GO ) ar e depresse d b y combine d exposur e t o 

ethanol and TGFpl. It appears that ethanol interferes 

with TGFpl regulation o f cell cycle activity. 

In Vivo Transforming Growth 

Factor Systems 

Various immunohistochemica l analyse s hav e show n 

that th e developin g nervou s system contains a n en - 

dogenous TGFp system (Flanders et al., 1991 ; Pelto n 

et al, 1991 ; Miller , 2003b) . TGF p ligand s ar e expressed 

i n th e V Z an d S Z o f th e cerebra l vesicl e 

in vivo (Fig . 11-5) . TGFpl i s expressed i n th e V Z 

pre- an d postnatall y and TGFp2 i s expressed during 

the first postnata l week . Potentiall y mor e importan t 

than ligan d expressio n i s the distributio n o f the tw o 

chief TGF P receptors , TGFpI r an d TGFpIIr . Th e 

TGFpIr is expressed in both neocortica l proliferativ e 

zones throughou t life , wherea s th e TGFpII r i s only 

expressed i n th e VZ . Moreover , bot h TGFp 2 an d 

TGFpIr are expressed by radial glia. 

Prenatal exposur e to ethano l affect s endogenou s 

TGFP syste m expressio n i n viv o (Miller , 2003b) . 

TGFpl expression falls in immature and mature rats, 

whereas TGFp 2 expressio n rise s onl y i n perinates . 

Changes i n recepto r expressio n are mos t eviden t for 

the TGFpIr ; it s expression i s significantly highe r i n 

ethanol-treated rats, regardless o f age. These ethanol - 


FIGURE 11- 5 Transformin g growth facto r (TGF ) P 

ligands an d receptor s i n th e developin g ventricula r 

and subventricula r zones . Pair s of images depict th e 

distribution o f immunolabeling for TGFpl (to p left) , 

TGFp2 (to p right), TGFpIr (botto m left) , an d TGF - 

pIIr (bottom right ) in control (Ct) and ethanol-treate d 

(Et) perinates . Ethano l markedl y affect s ligan d an d 

TGFpIIr expressio n in the subventricula r zone (SZ) . 

Open and solid arrows identify immunolabeling i n the 

ventricular zone (VZ ) and SZ, respectively. Curved arrows 

labe l radia l gli a i n th e intermediate zon e (IZ) . 

Scale bars =100 jlm. (Source; Images taken, with permission, 

fro m Mille r (2003b) , J Com p Neuro l 

460:410-424.) 

induced change s ar e associate d wit h alteration s i n 

the distributio n o f cell s tha t ar e TGFpl , TGFp2 , 

TGFpIr, an d TGFpII r immunoreactive . Th e mos t 

marked change s ar e increase s i n S Z expressio n o f 

TGFp ligands an d especiall y receptors . Thi s finding 

may underli e th e increas e i n gliosi s that occur s fol - 

lowing early postnatal ethanol exposure (Fletcher an d 

Shain, 1993 ; Goodlett et al, 1993) . 

Lineage Specificity 

Studies o f th e effect s o f exogenou s TGFp l impl y 

that the endogenous TGFp system in the developin g 

and/or adul t brain i s affected b y ethanol. In general ,


ethanol block s TGFpl inhibition , however , thi s effect 

is lineage-dependent Fo r astrocytes and C6 cells , 

the combine d effec t o f ethanol an d TGFp l i s competitive 

(Mille r and Luo , 2002a), that is , in the pres - 

ence o f ethano l an d TGFpl , th e proliferatio n o f 

either of these cell types is the sam e as that occurring 

in untreate d cultures . I n contrast , th e individua l inhibitory 

effects o f ethanol an d TGFpl i n B10 4 cells 

(Luo and Miller , 1999b ) an d cell s determined to be 

neurons (Mille r and Luo, 2002b) are additive. Treatment 

o f B10 4 cell s wit h a n agen t tha t block s ER K 

activity largely eliminates the inhibition of [ 3 H]thymidine 

incorporation . Thi s resul t implie s tha t bot h 

ethanol and TGFpl-mediated inhibition of neuronal 

progenitors is transduced through ERK. 

Interestingly, th e Tc for in situ V Z cell s treated 

with TGFpl an d ethano l i s the sam e a s the Tc for 

untreated preparations. The sam e effect i s evident for 

the GF: TGFpl and ethanol cancel the inhibitory action 

o f th e othe r anti-mitogen . Thus , V Z cell s i n 

organotypic cultures behave the same way as cultured 

cortical astrocytes . This findin g i s particularly interesting 

in light of data showing that radial glia, at least 

in cortex, are stem cells that can give rise to neurons or 

astrocytes (see Radial Glia, above). This phenomeno n 

begs th e questio n o f ho w astrocyte s are identifie d — 

i.e., is the commonl y use d marker , glia l fibrillary 

acidic protein, astrocyte-specific (se e Chapters 1 3 and 

18)? Equally , if no t mor e important , i s th e findin g 

that after a cell i s determined as becoming a neuron, 

even though i t may retain mitotic ability (Jacobs and 

Miller, 2000), its reaction to its environment, such as 

combined TGFp l an d ethanol , ha s changed . Th e 

implications o f thi s shif t i n responsivit y ma y fram e 

our understandin g of stem cell s and progenitor s an d 

their future utilit y in transplantation. 

Interactions amon g Growth Factors 

Growth factor s d o no t ac t i n vivo i n isolation . They 

are multiply expressed, and cell s express multiple re - 

ceptors. Th e effect s o f PDGF, FGF-2 , IGF-1 , an d 

TGFpl ar e affecte d b y the mediu m i n whic h th e 

cells ar e raised . The actio n o f each facto r i s amplified, 

or onl y expressed, when th e mediu m contain s 

fetal seru m (Lu o an d Miller , 1997a , 1999b ; Miller 

and Luo , 2002a) . (Seru m i s ric h i n a plethor a o f 

growth factors.) A specific exampl e of an interactio n 

among growt h factor s i s tha t betwee n FGF- 2 an d 

TGFpl (Mille r an d Luo , 2002a) . I n culture s o f 

neocortical astrocyte s and C 6 cells , these two factors 

effectively cance l ou t the actio n o f each other . Like - 

wise, ethanol inhibits the primary action of each factor. 

Th e amoun t o f cell growt h tha t occur s amon g 

cells treate d wit h FGF-2 and TGFpl, with or without 

ethanol, is the sam e as that evident in untreate d 

cultures. 

The multipl e effect s o f ethano l and/o r growt h 

factors o n cel l proliferatio n appea r t o depen d o n a 

single gatekeeper , ERK . Wit h ou r curren t under - 

standing, however , it is difficult to deciphe r the action 

o f this transduction funnel. Ethano l ca n inhibit 

cell proliferatio n by altering ERK activit y (Lu o an d 

Miller, 1999a , 1999b) . Furthermore , bot h mitogeni c 

(e.g., PDGF and IGF-1) an d antimitogenic (TGFpl) 

growth factors activate ERK. It seems parsimonious to 

conclude tha t ERK is not a simple switch that is either 

o n o r off ; ERK i s activate d b y a mitoge n an d 

turned off by an anti-mitogen. Instead, ERK i s a complex 

switch that ca n b e activate d b y substances with 

opposing effects , an d th e characte r o f that activatio n 

(acute or sustained) defines th e effec t o n subsequen t 

transcription. 

The effect s o f ethanol ar e not ubiquitous, they are 

quite specific. For example, ethanol affects th e expression 

and activity of the PDGF-oer, but not the PDGFpr. 

Potentially , thi s differentia l effec t o f ethano l 

depends on the distribution of the receptors. Could it 

be tha t on e recepto r i s associate d with a lipi d raft , 

whereas another is not? 

WHAT CONTROLS CELL NUMBER? 

Ethanol studie s sugges t that , a t leas t i n som e telen - 

cephalic structures, there is a dynamic interaction between 

th e tw o proliferativ e zones . A t lo w an d 

moderate exposure s t o ethanol , cel l proliferatio n in 

the V Z i s reduced, bu t proliferatio n i n th e S Z an d 

IHZ i s increased (Miller , 1989 , 1995a) . Hence , th e 

implication is that there is a set point, an implicit goal 

for cel l number, and that the stimulated proliferation 

in the S Z and IH Z i s compensating for the earl y depression 

of VZ proliferation. 

The numbe r o f neurons i n a defined cylinder of 

neocortex i s unaffecte d b y moderat e ethano l expo - 

sure (Miller and Potempa, 1990) . Nevertheless, cortex 

is smaller (Miller, 1987 , 1996b) . This decrease i n size 

likely results from th e ethanol-induce d elimination of 

ontogenetic columns . Presumably, this alteration i s a

esult of the goa l of the developin g nervous system to 

maintain th e integrit y of ontogenetic columns , even 

if th e numbe r o f column s i s reduced. Exposure s t o 

high concentration s of ethanol overwhel m the com - 

pensatory response. 

The change s i n th e telencephalo n contras t with 

those i n region s o f the brai n derive d from onl y th e 

VZ. In the absence of a derived proliferative zone, no 

compensatory respons e i s possible an d reductio n i n 

cell number occurs regardless of ethanol concentra - 

tion. Th e depressio n o f proliferative activit y i n th e 

VZ ca n occu r throug h tw o nonmutuall y exclusiv e 

mechanisms. Ethanol ca n inhibit the action of mitogenie 

growt h factor s and/o r ethano l potentiate s 

the actio n o f anti-mitogeni c factors . Empirica l evidence 

fro m cel l cultur e studie s support s both o f 

these theses. 

Derived proliferatin g populations, fo r example , 

the SZ, are affected b y ethanol in a complex, bimodal 

fashion. This provides a mechanism for the S Z to off - 

set primary damage to the VZ in an attempt to generate 

a structure with "proper" cell density. The abilit y 

of the S Z to compensate fo r reductions in VZ prolif - 

eration ma y resul t from th e differentia l sensitivit y of 

the tw o cell populations to ethanol. As such, it would 

reflect differentia l responsivit y t o particula r growth 

factors. Key support for this conclusion i s that the two 

zones respon d t o differen t combination s o f growth 

factors and they express different receptors. By pulling 

together al l o f the dat a o n th e effect s o f ethanol o n 

growth facto r activity , w e postulat e tha t ethano l af - 

fects S Z cells in defined ways. 

Take th e exampl e of cell proliferatio n i n th e SZ . 

The S Z contain s receptor s fo r PDG F an d TGFp . 

Ethanol block s PDG F bindin g wit h a n ED 50 o f 

100-200 mg/dl, dependin g o n th e recepto r isofor m 

(Luo an d Miller , 1999a) . Thi s result s i n a reduce d 

mitogenic effec t o f PDGF . Ethano l interfere s 

with TGFp-induce d inhibitio n wit h a n ED 50 o f 


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12 

Effects of Ethanol on the Regulatio n 

of Cell Cycle in Neural Stem Cells 

The effect s o f early exposure to ethanol on the pro - 

liferation o f neurona l precursor s ar e describe d i n 

Chapter 11 . Th e presen t chapte r discusse s the ef - 

fects o f ethanol on the cel l cycle with the particular 

reference t o neural ste m cell s (NSCs ) i n the devel - 

oping brain . Relativel y littl e i s know n abou t th e 

mechanism b y which ethano l affect s th e cel l cycl e 

machinery i n NSC s becaus e mos t studie s focu s 

on the effect s o f ethanol on other cel l types. Understanding 

ho w ethano l affect s th e cel l cycl e o f 

NSCs migh t be valuable in the contex t of treatment 

of ethanol-induce d developmenta l defects , suc h 

as those observed in fetal alcohol spectrum disorders 

(FASD). Befor e delvin g into th e effect s o f ethano l 

on th e progressio n o f th e cel l cycle , NSC s an d 

the majo r molecula r component s o f cel l cycl e ar e 

defined. 

W. Michael Zawada 

Mita Das 

199 

DEFINITION O F NEURAL STEM CELLS 

Origin of Neural Stem Cells 

Stem cell s make the developmen t an d maintenanc e 

of mammalia n tissue s an d henc e organ s possible. 

Embryonic stem (ES) cells reside in the inner mass of 

a blastocyst, are pluripotent, and are capable of generating 

cells o f all three primar y germ lineages (endo - 

derm, mesoderm , an d ectoderm. ) A subdivisio n of 

the ectoderm , neuroectoderm , give s rise to th e cen - 

tral nervous system (CNS). In the developing CNS, a 

subset of stem cells known as NSCs shape s the struc - 

tural and functional layout of the brain. 

Neural stem cells ar e define d a s cells with a high 

proliferative capacit y tha t allow s fo r self-renew 7 al, 

retention o f multilineag e potentia l (multipotency) ,


and clonality , which permit s a single NS C t o gener - 

ate progeny with the sam e multi-lineag e potential as 

its own. In contrast, neuronal precursors are probably 

best defined a s a type of stem cel l that is oligopotent, 

that is, capable o f generating onl y a few selected type s 

of cells , i n thi s cas e neurons . Althoug h adul t brai n 

NSCs are less abundant an d generall y characterize d 

by a reduce d proliferativ e potential (se e Cel l Cycl e 

of Ste m Cells) , the y maintai n th e homeostasi s o f 

cell pool s wit h hig h turnove r rates , suc h a s granule 

cell neuron s o f th e hippocampu s an d granul e an d 

periglomerular interneurons of the olfactory bulb. 

Characteristics of Neural Stem Cells 

During development o f the CNS, tw o distinct populations 

o f NSCs exist . Th e firs t populatio n consist s o f 

relatively homogenous populatio n tha t participate s i n 

neural tub e formation . Thes e cell s ar e primaril y located 

in the ventricular zone (VZ) and lack expression 

of markers typical of differentiated neura l cells . Their 

proliferation an d surviva l are controlled by transmissible 

protein s suc h a s basi c fibroblas t growt h facto r 

(bFGF or FGF-2) (Kilpatrick and Harriett, 1995). 

Mitotically activ e cell s derive d fro m th e V Z con - 

tribute t o derive d proliferative zones suc h a s the sub - 

ventricular zone (SZ; also known as the subependymal 

zone) i n th e cas e o f th e telencephalon . Thi s zon e 

grows as the VZ diminishes in thickness (Miller, 1989) . 

With th e ris e of the SZ , a second identifiabl e population 

o f NSC s appear s (Reynold s an d Weiss , 1992 , 

1996). These stem cells proliferate in response to both 

bFGF an d epiderma l growt h facto r (Vescov i e t al. , 

1993). Thus, during the secon d hal f o f telencephali c 

generation, tw o populations o f NSCs exist, a principal 

population i n th e V Z an d a growin g populatio n i n 

the SZ. 

Despite differences i n the VZ and SZ, both popu - 

lations shar e similaritie s in protei n expression . Cell s 

in thes e zone s expres s nestin , ATP-bindin g cassett e 

protein (ABCG2) , telomeras e revers e transcriptas e 

(TERT), telomerase-repeat-bindin g facto r (TRF ) 1 , 

and TRF 2 (Klappe r e t al, 2001) . Moreover , telom - 

erase activit y is detectable i n bot h th e V Z and SZ . A 

fuller description o f telomerase i s provided i n the sec - 

tions Stem Cells , and Telomerase . 

In addition t o the VZ and SZ , NSCs, some radial 

glia, and cell s expressing glial fibrillary acidic protein 

(GFAP) hav e bee n postulate d t o b e ste m cell s 

(Doetsch e t al. , 1997 ; Garcia-Verdug o e t al. , 1998 ; 

Hartfuss e t al. , 2001 ; Alvarez-Buyll a an d Garcia - 

Verdugo, 2002) . I t shoul d b e note d tha t expressin g 

GFAP doe s no t necessaril y mean tha t a cell i s an astrocyte. 

Rather, it means that a cell expresses an intermediate 

filamen t als o expresse d b y astrocytes . Fo r 

example, radia l gli a ca n expres s GFAP , bu t the y d o 

not appea r t o be astrocytes . Instead , the y hav e trait s 

associated wit h NSCs, such a s the abilit y to generat e 

neurons an d glia . Consequently , th e effect s o f 

ethanol exposur e o n GFAP-positiv e cell s an d radia l 

glia during development also have wide-ranging consequences 

fo r NSCs (see Chapters 1 3 and 18) . 

In adul t rodents, four distinc t cell type s have been 

identified in the SZ (Doetsch e t al., 1997). They can be 

discriminated on the basis of the length of their cell cycle 

and ultrastructural characteristics. These are (1) proliferating 

migratory neuroblasts (type A cells), (2) slowly 

dividing astrocyte s (typ e B cells) , (3 ) rapidl y dividing 

progenitors (typ e C cells) , and (4 ) ependyma (typ e D 

cells) tha t lin e th e ventricula r wall. Whether ependy - 

mal cells themselves are stem cells is contested (Chias - 

son e t al., 1999 ; Johansson et al., 1999 ; Spassky et al., 

2005). Furthermore , parenchyma l cell s positiv e fo r 

neural cel l adhesio n molecul e an d transdifferentiate d 

cells are candidates for NSCs (Pevny and Rao, 2003). 

Symmetric and Asymmetric Cell 

Division among Neural Stem Cells 

Like other type s of stem cells , NSC s undergo eithe r 

symmetric o r asymmetri c cel l division s (Zhong , 

2003). During a symmetric cel l divisio n both daugh - 

ter cell s shar e th e sam e fate . Accordingly , both cell s 

remain in the proliferative population o r both exit the 

cell cycle . I n contrast , a n asymmetri c divisio n pro - 

duces two cells with different fates . For example, on e 

remains a stem cel l an d th e othe r assumes a defined 

phenotype such as that of a neuronal progenitor. How 

is this fate determination possible? 

Although mitoti c cel l divisio n produce s tw o 

daughter cell s containin g identica l geneti c material , 

the epigeneti c molecule s withi n th e cytoplas m ca n 

be distribute d unequall y betwee n th e tw o daughte r 

cells. Such differentia l distributio n of cytoplasrnic determinants 

i s postulate d t o affec t cel l fat e decision s 

(Zhong, 2003) . Asymmetric cel l divisio n is indeed a 

fundamental attribut e o f ste m cells , becaus e fo r a 

stem cell both to self-renew and to produce differenti - 

ated progeny , the cel l mus t have an abilit y to divide 

asymmetrically. A specifi c exampl e o f asymmetri c

cell divisio n come s fro m th e Drosophila CN S i n 

which a neuroblas t give s ris e t o a smalle r daughter 

cell know n a s the ganglio n mothe r cel l (GMC) . A 

GMC i n turn produces two neurons or glial/neuronal 

siblings (Doe et al., 1998), 

Among severa l component protein s tha t partici - 

pate i n asymmetri c divisions in Drosophila, prosper o 

and Numb are retained in the basal region of a dividing 

neuroblast at metaphase and preferentiall y segregate 

int o th e ganglio n mothe r cel l (Knoblic h e t al. , 

1995). Although tracing cell lineage trees is currently 

impossible in vertebrates in vivo, it is feasible to trace 

the progen y of single cells labeled retrovirally . Other 

methodologies fo r tracing stem cell s in vivo , such a s 

transplantation o f labeled cells , ar e discusse d belo w 

in the sectio n Us e of Neural Ster n Cell Transplanta - 

tion to Examine the Effect s o f Ethanol on Cell Regulation 

In Vivo . A discussion on the asymmetri c cel l 

division i s particularly relevant here , becaus e asym - 

metric distributio n o f cytoplasmi c determinant s i s 

cell-cycle dependent . Althoug h i t i s no t know n 

whether ethanol ca n disrupt this distribution, the possibility 

exists because the asymmetric cell division occurs 

early in the proces s of fate determination . Thus, 

even small changes during this period might result in 

substantial abnormalities later in development. 

Markers of NSC Populations 

No discussio n o f NSC s i s complet e withou t a n at - 

tempt t o define the m b y means o f unique ste m cel l 

markers. Although n o absolut e marke r for NSCs exists, 

th e presenc e o f a hematopoieti c ste m cel l 

marker, C D (cel l determinant ) 13 3 (known as prominin 

i n rodents) , i s require d fo r th e clonogeni c 

NSCs that self-rene w and retai n niultilineage differ - 

entiation propertie s (Uchid a e t al. , 2000) . Suc h 

clonogenic NSC s ca n b e derive d fro m huma n feta l 

brain tissue s by fluorescence-activate d cel l sortin g for 

cells that are GDI33% CD34-, CD45~, and CD23- 710 . 

These cells appear t o be the onl y human feta l brain 

cells that display clonogenic characteristics , retain the 

ability t o differentiat e int o bot h neuron s an d glia , 

and participat e i n a neurogenetic progra m following 

transplantation int o the roden t CN S (Uchid a et al. , 

2000; Tamaki et al., 2002). 

Evidence of the importanc e o f CD 133 as an indi - 

cator of "sternness" (i.e., their proliferative an d differ - 

entiative potential ) wa s discovered whe n testin g th e 

cancer ste m cel l hypothesis . As few as 10 0 CD133 + 

EFFECT OF ETHANOL ON NEURAL STEM CELLS 20 1 

cells isolate d fro m huma n brai n tumor s giv e rise to 

phenotypically identica l tumor s whe n transplante d 

into brain s o f immunodeficien t mic e (Sing h e t al. , 

2004). By contrast, th e CD133 " cells fai l t o produc e 

tumors in these transplants, even when engrafted as a 

bolus of 10 5 cells. These findings do not imply that all 

NSCs are tumorigenic, but that stem cells introduce d 

into the brai n ectopically and/o r at an inappropriat e 

time hav e a capacity for tumor generation . Althoug h 

GDI33 labelin g ha s been use d mostl y for detectio n 

of human NSCs , expression of other potentially useful 

marker s fo r NSC s suc h a s Sox-1 , Sox-2 , nestin , 

musashi, Hu , neuralstemnin , Le X (trisaccharid e 3 - 

frucosyl-N-acetyllactosamine)/stage-specific embryoni c 

antigen 1 , an d ABCG 2 ha s bee n use d t o identif y 

NSCs i n a variet y of specie s (Capel a an d Temple , 

2002; Pevny and Rao , 2003). 

CELL CYCLE 

The mos t fundamental property of living organisms is 

their ability to reproduce themselves. This property is 

based upon th e feature that cell s are able t o duplicate 

by a process known as the cel l or mitotic cycle. Mammalian 

brai n developmen t depend s o n a precis e sequence 

o f cell division s controlled b y the cel l cycle . 

Sculpting of the CNS an d other organs in the embryo 

is greatly affected b y the kinetic s o f the cel l cycl e an d 

the qualit y o f chromosoma l replicatio n tha t occur s 

during this process. Alterations in the amounts of necessary 

trophic factors (e.g., glucose and growth factors), 

mutations in the genes that control the cell cycle (e.g., 

cyclins and cyclin-dependent kinases [Cdks]), and various 

biophysica l stresse s (e.g. , excessiv e hypoxi a an d 

acidemia) occur naturally. These alterations ca n affec t 

the cell cycle and result in stunted or abnormal development, 

an d they cannot be easily controlled. Cel l cycle 

regulation , an d hence , developmen t ca n b e als o 

affected b y unnatural causes such as excessive ethanol 

consumption. Befor e discussin g the specifi c effects o f 

ethanol o n the cel l cycle , it is important to review the 

components o f cel l cycl e machiner y an d th e differ - 

ences between the cell cycles of somatic and ES cells. 

Cell Cycle of Somatic Cells 

As mentione d above , th e cel l cycl e i s the mean s b y 

which organisms reproduce themselves. In virtually all 

cells, the cell cycle is composed o f four discrete phases :


(1) the DNA synthesis phase (the S phase), (2) the cel l 

division phase (the M phase), and tw o gap phases, (3 ) 

the Gl phas e between M and S and (4) the G2 phase 

between S and M (Fig. 12-1). During Gl, the cell prepares 

for DNA replication that results in duplication of 

the chromosome s durin g S . The secon d ga p period , 

G2, is the post-DNA synthetic phase during which the 

cell prepares for mitosis. The ter m cell cycle checkpoint 

refers to a mechanism b y which the cel l actively halts 

the progression of the cell cycle until it can ensure that 

an earlier process, such as DNA replication o r mitosis, 

is complete. Suc h cell cycle checkpoints are mediated 

by p53 at the end of Gl an d during G2 (Tabl e 12-1) . 

P53 is also known as a tumor suppressor and whe n inactivated 

permits oncogenes to promote transformation 

and tumorigenesis (Lowe et al., 2004). 

Cyclins and their partners, the Cdks, constitute th e 

basis o f the molecula r mechanis m tha t control s cel l 

cycle regulation (Fig. 12-1) (Murray , 2004). Cdks are 

serine/threonine protei n kinase s that require binding 

of a cycli n t o becom e activated . Mammalia n cell s 

contain multipl e Cdk s that ar e activated b y multiple 

cyclins. Much o f the difference betwee n the behavior 

of different cyclins reflects their location and timing of 

expression rather than their direct effects o n substrate 

specificity. Th e activit y of Cdks i s highly regulated b y 

a number of mechanisms: (1 ) the level of their respec - 

tive partne r proteins, the cyclins , (2) the amount s of 

inhibitory proteins of the Cip/Kip (e.g., p21, p27, and 

p57) and Ink4 (e.g., pi5, p!6, p!8, and p!9) familie s 

(Cdk inhibitors or CKIs), and (3 ) inhibitory and stim - 

ulatory phosphorylatio n o f variou s Cd k residue s 

(Table 12-1 ) (Cheng , 2004 ; Siegenthale r an d Miller , 

2005). High concentrations of cyclins therefore generally 

stimulate cell-cycl e progressio n and proliferation 

through activatio n of Cdks, whereas high amounts of 

CKIs antagonize these processes. 

Cyclins ar e activatin g subunits tha t interac t wit h 

specific Cdk s t o regulat e their activit y and substrat e 

specificity. I n mammals , thes e complexe s include : 

(1) the D-type cyclins (cyclins Dl, D2, and D3) that 

activate Cdk4 and Cdk6 to execute critical regulatory 

events in Gl; (2 ) the E - and A-type cyclins that activate 

Cdk2 to affect event s in S including DNA replication; 

and (3 ) the A-typ e cyclins and B-typ e cyclins 

that activate Cdkl t o direct structural and regulatory 

events in M (Fig . 12-1) . Inactivation o f Cdkl i n late 

M contributes to progression into G L 

In somatic cells , movement throug h G l an d int o 

S is driven by the activ e forms o f the cycli n Dl, D2 , 

or D 3 complexin g with Cdk4 or Cdk6. This triggers 

an intricat e cascad e o f events. Formatio n o f a cyclin 

D-Cdk comple x induce s th e phosphorylatio n o f 

retinoblastoma (Rb) protein (Fig . 12-2 ) (Classo n an d 

Harlow, 2002). Before a cell prepares to enter a ne w 

cell cycl e durin g the Gl/S transition, a critical tran - 

scription factor , E 2F-1 i s inactive and boun d t o Rb . 

Once the new cell cycle begins, Rb is phosphorylated 

and E 2F-1 i s partially release d fro m R b inhibition . 

This activates cell cycle regulators including cyclin A 

and cycli n E tha t comple x wit h Cdk 2 an d Cdc25 A 

phosphatase. Cdc25 A phosphatas e remove s phos - 

phates tha t inhibi t Cdk 2 an d th e resultan t cyclin 

E/Cdk2 comple x further phosphorylate s Rb . The re - 

sult is a complete release of E2F and the transcription 

of a series of genes essential for progression into S and 

DNA synthesis . Parallelin g thi s activit y is the direc t 

contribution of the c-myc pathway to the Gl/S transi - 

tion b y elevatin g the transcriptio n fo r cycli n E an d 

Cdc25A(Fig. 12-2). 

In additio n t o th e positiv e regulation b y cyclins, 

Cdks ar e regulate d (1 ) by multipl e phosphorylatio n 

and dephosphorylation events and (2) by several CKIs 

that physicall y associat e wit h th e cyclin-Cd k com - 

plexes to inhibit their activities and promote cell cycle 

arrest or delay. Since phosphorylatio n i s fundamental 

to the regulation of cell cycle progression, a large subset 

ofDrosophila protei n kinases (kinome) studied for 

their necessity in the normal cell cycle was examined 

by silencin g genes wit h RNA-mediate d interferenc e 

(Bettencourt-Dias e t al. , 2004) . O f 22 8 protei n ki - 

nases that are down-regulated by this silencing, downregulation 

o f 8 0 o f the m ca n induc e cel l cycl e 

dysfunction. Man y o f thes e enzyme s wer e already 

known to phosphorylate microtubules , actin , and associated 

proteins, but as a consequence o f these studies, 

th e protei n kinase s hav e bee n assigne d ne w 

functions i n cel l cycle . Thes e finding s underscor e 

the complexity of cell cycle regulation and the multi - 

tude o f potentia l target s tha t ethano l migh t affec t 

during development. 

Cell cycle progression is regulated by growth factors 

(see Chapter 11) , the extracellular matrix, and cell-cel l 

contacts. Upo n deprivatio n o f cells fro m a n essentia l 

component (nutrient s o r growt h factors ) th e cell s 

become quiescen t an d ente r th e so-calle d GO-phase . 

This exit from th e cell cycle can be reversible in astrocytes 

(L i e t al. , 1996 ; MacFarlan e an d Sontheimer , 

2000) or irreversible, as evident in many types of postmitotic 

neurons (Zawada et al., 1996) . In mammalian

SOMATIC 

CELLS 

ES 

CELLS 

FIGURE 12- 1 Cel l cycle o f embryonic ste m (ES ) and somatic cells . Note tha t 

ES cell s hav e greatly shortened ga p phases, exhibi t constant expressio n o f cyclins 

A, E, and D , and lac k p53 checkpoints tha t allow for rapid cell division . 

bFGF, basic fibrobals t growt h factor ; IGF, instilin-lik e growt h factor ; PDGF , 

platelet-derived growth factor; TGFpl, transforming growth factor (31 . 

203


TABLE 12- 1 Endogenou s cel l cycl e inhibitor s slow down cel l cycl e at differen t stage s by acting on various 

cyclin-CDK complexes . 

Cell Cycle Stage 

Gl 

Gl/S 

S 

G2/M 

Ink4 Family 

Cyclin-Cdk Complexes plS pi 6 pl8 

cyclinD-Cdk4/6 + + + 

cyclinE-Cdk2 - 

cyclinA-Cdk2 - 

cyclinB-Cdk2 - 

cells, th e cel l cycl e machiner y tha t determine s 

whether cell s continue proliferatin g or ceas e dividing 

and differentiate appear s to operate mainly in the Gl. 

Cell Cycle of Stem Cells 

Embryonic Cells 

In stem cells, there i s a distinct cell cycle control tha t 

operates i n orde r t o maintai n thei r sternness . Th e 

need for higher rate s of proliferation i s met b y minimizing 

the length o f time that ES cells spend i n G l 

and G 2 (Fig . 12-1) . Mouse ES cells have a defective 

Rb pathwa y an d a nonresponsiv e p5 3 pathwa y 

p!9 p2l 

+ + 

- + 

+ 

+ 

Cip /Kip Family 

P27 

+1- 

+ 

- 

- 

pS7 

+/- 

+ 

+ 

- 

pS3 

(Savatier and Malashicheva, 2004). Furthermore, ES 

cells have constitutive phosphatidyl-inositol-3-kinase 

activity an d ectopi c cycli n E/Cdk 2 kinas e activity , 

features ofte n eviden t i n tumor s (Sherr , 1996) . Th e 

uncoupling o f G l fro m th e influenc e o f extrinsi c 

stimuli migh t explai n i n par t the rapi d proliferation 

rate o f E S cells . Anothe r metho d throug h whic h 

stem cells ar e abl e t o maintai n a hig h rat e o f self - 

renewal i s reduction i n expressio n o f CKIs, particu - 

larly p2 1 an d p2 7 (Chen g an d Scadden , 2004 ; 

Siegenthaler an d Miller , 2005) . I t i s likely tha t th e 

distinct cell cycle profile i n stem cells is mediated either 

b y distinct upstrea m intracellula r mediator s or 

by uniqu e combinatoria l relationship s o f commo n 

FIGURE 12- 2 Majo r regulators of cell cycl e initiatio n in Gl . Se e tex t for details. 

Cdk, cyclin-dependent kinase , RB, retinoblastoma. 

— 

+ 

- 

+

iochemical mediators that limit the intensit y of signals 

to exit the cell cycle. 

Stem cell s are not only key building blocks in normal 

developmen t bu t the y are als o candidates as the 

cell o f origin for cancer becaus e o f their pre-existing 

capacity o f self-renewa l an d unlimite d replicatio n 

(Beachy et al., 2004) . Cancer mos t often emerge s i n 

those tissues undergoing constant renewal by the proliferation 

o f ste m cells . B y retaining o r re-acquiring 

stem cell behavior, malignant cells may be able to recreate 

a tumor during metastasis or after therapeuti c 

elimination o f th e bul k o f th e tumo r populatio n 

(Beachy et al., 2004). 

Neural Stem Cells 

During normal development o f the CNS, th e division 

rates o f NSC s withi n th e germina l zone s var y wit h 

brain regio n an d wit h time (se e Chapter s 2 and 11) . 

The principl e of the cel l cycl e o f ES cell s (described 

above) composed solely of S and M no longer applies. 

By contrast, as the developmen t o f CNS progresses , a 

multitude a cel l cycl e "types " emerge s (Fig . 12-1) . 

These cell cycle s differ i n length, whic h i s most com - 

monly determined by the period of time that the cell is 

in Gl and/or G2. 

Studies of murine cortical neurogenesis detail th e 

cell cycle over the 6-da y long process (Caviness et al., 

1999). Founde r cell s an d thei r progen y underg o 1 1 

cell cycles. The proportio n of the cells becoming post - 

mitotic increases with eac h successiv e cel l cycl e an d 

the length o f the cycl e increases due to a gradual prolongation 

of Gl. Gl i s characterized by two main enzymatic 

activities : Cdk4 i n mid-Gl an d Cdk 2 i n late 

Gl jus t prio r t o initiatio n o f S (Fig . 12-2) . A s described 

abov e i n Origi n o f Neura l Ste m Cells , th e 

SZ cells ca n b e subdivide d int o slowl y dividin g 

GFAP-positive (typ e B) cells, rapidly dividing transitamplifying 

progenitors (type C cells), and proliferating 

migratory neuroblast s (typ e A cells) . On e propose d 

model of neurogenic program defines th e flowin g lineage 

progression : typ e B t o typ e C t o typ e A cell s 

(DoetschetaL, 1997) . 

The CK I p27 is an inhibito r of Cdk2 that can pro - 

long th e entr y o f a proliferatin g cel l int o S . Durin g 

neurogenesis, loss of p27 has no effec t o n the numbe r 

of stem cells (type B cells), but selectively increases the 

number o f transit-amplifying type C cells , whil e con - 

comitantly reducing the number o f type A neuroblasts 

(Doetsch e t al., 2002). By contrast, NSC s in transgenic 


mice over-expressing p27 have longer Gl (Mitsuhash i 

et al., 2001). The lengt h o f cell cycle (Tc) in germinal 

zones is under temporal and spatial control. Fo r example, 

i n th e mouse , divisio n rate s ca n b e a s rapi d a s 

7-10 hr i n early gestation an d u p to 1 8 hr long at the 

end o f cortica l neuronogenesi s (Takahash i e t al. , 

1995). Likewise, the lengt h o f the cel l cycl e increases 

in rat cortex from 1 1 to 1 7 hr across the perio d of neuronogenesis 

(Miller and Kuhn, 1995). Cell cycle varies 

by location, too. The Tc for the rat neocortical VZ on 

G21 i s 1 9 hr an d 1 6 hr fo r S Z cell s (Mille r an d 

Nowakowski, 1991) . In the adul t brain, the frequency 

of cel l proliferatio n can b e a s infrequen t as day s o r 

months (Garcia-Verdug o et al., 1998 ; Morshea d et al., 

1998; Doetsch e t al., 1999) . In addition to the apparent 

differences i n cell cycle lengths, the details of molecular 

controls dictatin g characteristics o f various cell cy - 

cles of NSCs are speculative. 

EFFECTS O F ETHANOL ON CEL L CYCLE 

Somatic Cell s 

Reports on the effect s o f ethanol o n cell cycl e regulatory 

molecule s sugges t tha t th e ethanol-mediate d 

effects o n ke y regulator s o f cel l cycl e ar e tissue - 

specific an d cell type dependent. Fo r example, in human 

lymphocytes , ethanol-induce d inhibitio n o f 

mitosis i s mediate d throug h G2/ M blockad e 

(Ahluwalia e t al. , 1995) . I n huma n epithelia l cells , 

ethanol delays cell cycle progression by up-regulating 

the expressio n of transcripts for p21 ( a key CKI) an d 

inhibiting Cdk 2 kinas e activit y in G l (Gu o e t al. , 

1997). I n epithelia l cells , thes e ethanol-mediate d ef - 

fects on the cell cycl e are independent o f the cellula r 

status of a checkpoint mediator , p53. Other studies in 

a variet y o f tissues corroborate th e finding s tha t spe - 

cific CKI s serve as targets o f ethanol actions . Fo r example, 

a n increas e i n p2 l gen e expressio n upo n 

ethanol infusio n ha s bee n reporte d i n th e isolate d 

perfused hear t (Jankala et al., 2001). Hepatocyte proliferation 

afte r partia l hepatectom y i s als o inhibite d 

by ethano l throug h amplificatio n of Gl checkpoin t 

mechanism involvin g induction o f p21 gen e an d in - 

hibition of cyclin D (Koteis h et al., 2002). Yet the effects 

o f ethano l migh t no t influenc e onl y a singl e 

phase o f the cel l cycle. Transient blockade of cell cycle 

a t G2/M an d preventio n o f the cel l fro m exitin g 

the cycl e by ethanol exposur e hav e been shown i n a


fibroblastic cel l lin e derive d fro m activel y dividing 

mouse connective tissu e (Mikami etal., 1997) . 

Ethanol intak e by pregnant rat s inhibits growth of 

the intestina l epitheli a (Pere s e t al. , 2004) . Ethano l 

substantially attenuates cryp t cell proliferatio n in bot h 

the jejunu m an d ileu m an d prolong s cel l cycl e tim e 

among crypt cells. In ethanol-fe d rabbits , neo-intimal 

hyperplasia i n coronar y arteries following balloo n an - 

gioplasty is diminished (Merrit t et al., 1997) . Similarly , 

local deliver y o f ethano l b y balloo n catheter s i n 

balloon-injured coronar y arterie s inhibit s intima l hy - 

perplasia (Liu et al., 1997). In cultured vascular smooth 

muscle cells , ethanol inhibit s the progressio n o f cells 

from the Gl to S by modulating the expression/activity 

of key regulatory molecules of the cycl e (Sayeed et al., 

2002). I n particular , ethano l induce s th e expressio n 

of p21, inhibit s Cdk 2 activity , and halt s th e induc - 

tion o f cycli n A , resultin g i n a decreas e o f hyper - 

phosphorylation of Rb and failure to initiate a new cell 

cycle. Cumulatively , report s o n ethanol-mediate d ef - 

fects on the cell cycle of somatic cells suggest that p21, 

the ke y negative regulator of mammalian cell cycle, is 

an important target of ethanol. 

Despite a n overwhelmin g number o f reports sup - 

porting th e anti-proliferativ e rol e o f ethanol, instea d 

of perturbin g th e cel l cycl e progression , it s action s 

can actually induce the cell cycle in certain cell types 

under particula r conditions. I n suc h cases , reduce d 

expression o f cell cycl e inhibitor s upon exposur e t o 

ethanol accelerate s progressio n from th e Gl t o S. Indeed, 

ethanol-mediate d inhibitio n o f pi8, pi9, and 

p21 expression, increased R b phosphorylation and increased 

proliferation have been reported i n squamous 

cell carcinoma cell lines of the head and neck (Hager 

et al., 2001; Kornfehl e t al., 2002). Increased hepato - 

cyte proliferation in rats after chronic ethanol feedin g 

has als o bee n demonstrate d b y severa l investigator s 

(Chung e t al., 2001). Immortalized NSCs , grown under 

proliferation-encouragin g conditions , als o re - 

spond t o ethano l b y increasin g th e rat e o f thei r 

division (Morgan et al., 2003) (described in more detail 

below). Likewise, following prenata l exposur e to 

moderate amount s o f ethanol, S Z cel l proliferatio n 

increased (Mille r and Nowakowski , 1991) becaus e of 

recruitment o f cells int o th e cyclin g population, no t 

because o f a decrease i n the Tc or the lengt h o f the 

Gl. Taken together , thes e findings indicate tha t th e 

effects o f ethanol on cell cycle progression are highly 

variable and depen d o n the molecular , cellular, and 

system contexts under which they occur . 

Stem Cells 

Neural Stem Cells 

Mechanisms o f neuronogenesis during developmen t 

(Miller, 1986 ) an d adulthoo d (Herrer a e t al. , 2003 ) 

are affecte d b y ethano l (se e Chapter s 1 1 an d 13) . 

Much of the followin g discussion is based on findings 

from adul t NSCs, as studies examining molecular de - 

tails of cell cycle modulation b y ethanol durin g brain 

development ar e few . Nonetheless, approache s an d 

findings presented her e should b e useful i n studying 

effects o f ethanol o n th e characteristic s o f cell cycl e 

in the course of neurogenesis. 

Both bing e (Nixo n and Crews , 2002) and chroni c 

(Miller, 1995; Rice etal., 2004; He etal, 2005) ethanol 

consumption reduc e the postnatal proliferation of neural 

progenito r cell s i n th e S Z o f the latera l ventricl e 

and th e intrahila r zone (IHZ ; also known as the sub - 

granular zone) o f the dentat e gyrus. Adult rats receiving 

liquid diet containing ethanol (6.5 % v/v) for 3 days 

reduced thei r neura l progenito r proliferatio n i n th e 

IHZ a s evidenced b y a decrease i n the number of cells 

that incorporat e bromodeoxyuridin e (BrdU ) (Ric e 

et al., 2004). In contrast, animal s receiving an ethano l 

diet for 1 0 or 3 0 days n o longe r displa y reduced pro - 

genitor proliferation , indicating activation o f a com - 

pensatory respons e t o th e actio n o f ethanol . A t least 

during the early postnatal period, the effect s o f ethanol 

are concentration-dependen t (Miller , 1995) ; hig h 

doses inhibi t cel l proliferatio n an d lowe r dose s ar e 

stimulatory. During feta l development , ethano l migh t 

also stimulate proliferatio n of neural progenitor s a s illustrated 

by enhanced proliferation of cerebral cortical 

neuroepithelial precursor s upon exposur e t o alcoho l 

(Miller an d Nowakowski , 1991 ; Miller , 1995 ; Santil - 

lano e t al. , 2005) . Treatment wit h ethano l fo r 4 days 

increases th e size , variation , an d numbe r o f cortica l 

precursor neurospheres, induces S , and increase s pro - 

gression through G2/ M (Santillano et al., 2005). 

Analysis of the effect s o f ethanol o n postnata l neu - 

rogenesis shoul d b e conducte d i n term s o f the fina l 

numbers of cells generated an d retained. This measure 

is affected b y not only the rat e of cell proliferation, but 

also by the rat e o f cell deat h (Miller , 2003). The im - 

portance o f this approac h ha s been documente d b y 

studies reportin g that ethano l actuall y increases cel l 

proliferation an d concomitantly increases cell death as 

determined wit h terminal deoxynucleotide transferasemediated 

deoxyuridin e triphosphate nick-en d labelin g

(TUNEL) (Pawlak et al., 2002; Miller, 2003). TUNEL 

identifies nucleosome-sized products of DNA degradation. 

These findings underscore the complexit y of the 

effects o f ethanol o n cel l cycl e and cel l survival . On e 

study explores the effect s of a 24 hr exposure to ethanol 

on C17.2 cells, a line of NSCs derived from a postnatal 

mouse cerebellu m (Morga n et al. , 2003) . Exposure 

to ethano l double d th e numbe r o f surviving NSCs 

when grown under proliferation-favoring culture conditions 

(10% serum/uncoated culture plate). In contrast, 

ethanol reduces the number of NSCs grown under dif - 

ferentiating condition s (1.0 % serum/poly-L-lysine - 

coated cultur e plate) . A s thi s NS C lin e ha s bee n 

immortalized with v-myc, it is conceivable that the in - 

crease i n proliferatio n of C17. 2 durin g exposur e t o 

ethanol i s related to the effect s o f ethanol o n the activity 

of the oncogene, v-myc. Oncogenes ar e known activators 

of telomerase, which in turn maintains telomere 

length necessar y for unabated proliferatio n (see Ste m 

Cells, and Telomerase, below). 

Mechanisms Underlying Cell Cycle 

Despite the abundance o f literature describing the effects 

o f ethano l o n neura l progenito r proliferatio n 

during neurogenesis , onl y a fe w studies have examined 

th e molecula r mechanism s mediatin g thes e 

changes. One o f the few models used to date is that of 

cerebellar neurogenesis . Cerebella r granul e neuron s 

represent a majo r neurona l typ e i n th e cerebellu m 

and aris e fro m th e proliferatin g externa l germina l 

layer durin g the first 2-3 postnata l week s i n th e ra t 

(Miale an d Sidman , 1961 ; Altman, 1972) . Cycli n A 

and Cdk2 , tw o positive regulator s o f the cel l cycle , 

exert contro l ove r neurogenesi s i n th e developin g 

cerebellum (L i et al., 2002) . Cerebellar granule progenitors 

express predominantly Cdk 2 (Courtne y an d 

Coffey, 1999 ; L i e t al. , 2001) . L i an d colleague s 

(2002) repor t that i n cerebella r granul e progenitor s 

derived from 3-day-ol d rat pups, ethanol exposure decreases 

th e expressio n o f Cdk 2 an d cycli n A . This 

ethanol-mediated reduction o f Cdk2/cyclin A expression 

may induce cell cycle arrest in neuronal precursors 

by increasing the duration of Gl and S. 

P27, a negative regulator of the cell cycle, plays an 

important role during neurogenesis of the developing 

cerebellum. I n th e neonata l cerebellum , p2 7 i s severely 

down-regulated after early postnatal exposure to 

ethanol (L i et al., 2002). P27 induces cell cycle arrest 

in neuronal precursors and accelerate s differentiatio n 


of neurons (Perez-Juste and Aranda, 1999; Farah et al., 

2000; Miyazaw a et al. , 2000) . Repressio n o f p27 b y 

ethanol exposur e may reactivate postmitotic neurons 

and evok e apoptotic cel l death . Collectively , report s 

on the effect s o f ethanol o n neurogenesis suggest that 

p27, Cdk2 , an d cycli n A ar e primar y targets o f th e 

ethanol in the developing cerebellum. Unfortunately, 

such detailed analysis of cell cycle proteins during prenatal 

exposure to ethanol is unavailable. 

Different neurona l population s fro m divers e locations 

in the developing brain exhibit different degrees 

of susceptibilit y t o ethano l toxicit y (Maie r e t al. , 

1999). Ethanol als o inhibits carbachol-induced DN A 

synthesis o f astrogli a (Guizzett i e t al. , 2003) . Mus - 

carinic agonist-drive n progression from th e G l t o S 

also ca n b e affecte d b y ethano l i n thes e cell s 

(Guizzetti an d Costa , 1996 ; Guizzett i e t al. , 2003). 

Taken together , thes e report s on ethanol-induce d ef - 

fects o n th e cel l cycl e o f different neurona l an d as - 

troglial population s sugges t tha t S an d G l ar e th e 

primary targets of ethanol-induced effects . 

Not onl y ca n cel l cycl e pathway s be studie d i n 

vitro and i n vivo (see Use of Neural Stem Cell Transplantation 

to Examine the Effect s o f Ethanol on Cell 

Cycle Regulation In Vivo), but gene array technology 

makes i t possibl e t o scree n ste m cell s fo r ethanol - 

niediated gene expressio n patterns in an effor t t o discover 

ne w ethanol-responsiv e genes . A repor t b y 

Treadwell and Sing h (2004), who used microarrays of 

adult mous e brai n gen e expressio n followin g acut e 

ethanol exposure , suggests that a significan t proportion 

of ethanol-responsive gene s have roles in cell cycle 

transitio n an d arrest . Th e genes , p2 l an d th e 

growth arres t an d DN A damage-inducibl e gen e 

(Gadd45r), ar e up-regulate d i n respons e t o ethanol . 

P21 an d Gadd45 r ar e implicate d i n bot h p53 - 

mediated an d p53-independen t inductio n o f G l 

growth arres t i n respons e t o ethano l an d oxidativ e 

stress (Guo et al., 1997; Kitamur a et al., 1999). 

The molecula r mechanism s o f ethanol-induce d 

effects o n cel l cycl e involv e ethanol-responsive ele - 

ments o n promoter s o f target genes . Ethano l alter s 

the expressio n an d DN A bindin g activit y of various 

transcription factors , suc h a s activatin g protei n 1 

(AP-1), nuclear factor K-B , and earl y growth response 

gene (Beckman n e t al. , 1997 ; Ceber s e t al. , 1999 ; 

Fried e t al, , 2001 ; Acquaah-Mensa h e t al. , 2002) . 

These transcription factors regulat e the transcriptio n 

of genes encoding cyclins , Cdks, and CKI s (Sylvester 

et al., 1998 ; Beier et al., 2000; Milde-Langosch et al.,


2000; Passegu e an d Wagner, 2000 ; Henniga n an d 

Stambrook, 2001) . AP- 1 bind s t o th e cAMP - 

responsive element in the promoter of cyclin A gene 

(Sylvester e t al. , 1998 ; Beie r e t al. , 2000 ) an d en - 

hances transcription of cyclin A. Suppression of AP-1 

activity b y prenata l ethano l exposur e (Acquaah - 

Mensah e t al., 2002) inhibits the transcriptio n of cyclin 

A in developin g cerebellum . Give n th e critica l 

role of Cdks and CKI s in neurogenesis, disruption of 

the Cdk/CKI system may be the mechanism underlying 

ethanol-induce d damag e o f man y developin g 

CNS nuclei, including the cerebellum . 

Telomerase 

A potential opportunit y for ethanol t o influence stem 

cell proliferatio n i s t o affec t th e chromosoma l ter - 

mini, the telomeres. In all vertebrates, telomeres con - 

sist o f non-coding G-rich DN A repeat s (TTAGGG) . 

These specialized chromati n structures are necessary 

for successfu l cel l divisio n an d ar e shortene d wit h 

each roun d o f DN A replication . The y ca n als o b e 

preserved b y elongatio n mediate d b y a n enzym e 

called telomerase. I n norma l huma n somati c cells , 

consecutive round s o f telomer e shortenin g induc e 

replicative senescence, particularl y in the absenc e o f 

telomerase. Los s o f TTAGG G sequence s lead s t o 

telomere dysfunctio n characterize d b y end-to-en d 

chromosome fusio n tha t result s in genomic instability. 

Telomere s ar e protecte d b y a collectio n o f pro - 

teins that, i n additio n to telomerase, include several 

other protein s regulatin g telomer e length , includin g 

protection o f telomeres 1 (Lei et al., 2004) and other s 

(Bekaert et al., 2004). Telomerase activity requires (1) 

the reverse transcriptase TERT and (2) an RNA component 

tha t add s TTAGG G repeat s t o th e end s o f 

chromosomes to prevent chromosomal shortening . 

Telomerase activity in the developing rodent brain 

is high on gestational day (G) 1 3 but declines by G18 , 

remains low until postnata l da y (P) 3 , and finally becomes 

undetectabl e b y P1 0 (Klappe r e t al. , 2001) . 

These observations are consistent with a role of telomerase 

in proliferation o f NSCs. Interestingly, accelerated 

shortenin g o f telomere s i n mic e deficien t i n 

telomerase abrogate s proliferation of adult NSCs isolated 

fro m th e SZ , however, it does no t hav e that effect 

in the NSCs isolated from th e feta l brain despite 

severe telomere erosion resulting in chromosomal ab - 

normalities and nuclear accumulation of p53 (Ferro n 

et al. , 2004) . Thi s observatio n parallel s th e findin g 

that mouse E S cells lack p53-dependent checkpoint s 

(Savatier an d Malashicheva , 2004) . Whethe r feta l 

NSCs a t variou s stages o f development shar e inde - 

pendence i s not clear , bu t variou s "types" o f cell cycles 

diffe r i n lengt h an d differin g expressio n pattern s 

of cell cycl e regulator y proteins hav e been identified 

both in somatic cells and ES cells (Fig. 12-1) . 

At least some NSCs display cell cycl e characteris - 

tics like those of somatic cells. This is evident in adult 

NSCs, whic h ca n hav e a prolonge d G O (Takahashi 

et al. , 1995 ; Garcia-Verdug o e t al. , 1998 ; Morshea d 

et al., 1998; Doetsc h etal., 1999) and those dependent 

on the p53 checkpoints (Meletis et al., 2005). In neurospheres 

derive d fro m th e S Z o f p53-null mice , th e 

number of BrdU-positive cells is increased as much as 

20%, whereas p2 1 content is reduced (Meleti s e t al., 

2005). Concomitan t wit h thi s increase d capacit y for 

self-renewal i s a reduction i n cell death, evidence d by 

reduced caspase 3 activity and annexin V staining. 

When examining neurogenesis, it is important no t 

to presume tha t the proteins involve d share the sam e 

function a t all stages of development. Suc h is the cas e 

for caspas e 3, a pro-apoptotic protease , i n late r stages 

of brain organogenesi s an d adulthood . Caspas e 3 is 

necessary for normal growth of morula and blastocys t 

embryos, a s it s inhibitio n stall s th e growt h o f suc h 

embryos (Zwaka etal., 2005). NSCs undergoing telomere 

shortening, invariabl y enter an irreversible growth 

arrest, an d eventuall y senesce . Developmen t o f perpetual 

NS C line s fo r studies o n huma n feta l NSC s 

has bee n successfull y attempte d usin g transfectio n 

with a proto-oncogene, v-myc (Vill a e t al, 2004). I n 

such immortalize d NSCs , v-myc maintain s hig h 

telomerase activit y and allow s culturing of NSCs for 

up to 4 years with preservation of short, but stable and 

homogenous telomeres . 

Ethanol affect s th e expression of a proto-oncogene , 

c-myc, but it fails to change the amounts of p53 in the 

skeletal muscl e o f rat s expose d t o chroni c ethano l 

(Nakahara et al., 2003). Prenatal exposur e to ethanol 

increases gestational expression of p53 in the cerebra l 

cortex (Kuh n an d Miller , 1998) . Thi s finding parallels 

th e effect s o f ethanol o n S Z cells , bu t whethe r 

ethanol alter s th e expressio n an d activit y o f onco - 

genes and p53 in NSCs remains to be examined. 

Ionic and Metabolic Homeostasis 

Calcium an d magnesiu m ar e bot h believe d t o pla y 

an importan t rol e i n cel l proliferatio n (Wol f an d

Cittadini, 1999 ; Ziesch e e t al. , 2004) . Ethano l ca n 

cause a decrease i n cytosolie-free Ca 2+ (Zhang e t al., 

1992) and a rapid depletion of intracellular Mg 2+ in 

vascular smooth muscl e cell s (Altur a et al., 1995) . In 

addition, ethano l ca n adversel y affect mitochondria l 

cytochrome oxidas e (Kennedy, 1998 ) an d AT P synthesis 

(Dev i e t al. , 1994) . Therefore , th e effect s o f 

ethanol o n io n distributio n and/o r cellula r bioener - 

getic metabolis m should no t b e overlooke d as possible 

modulators of cell cycle regulatory molecules an d 

cellular proliferation. 

USE O F NEURAL STEM CELL 

TRANSPLANTATION TO EXAMINE 

THE EFFECTS O F ETHANOL ON 

CELL CYCLE REGULATION IN VIVO 

Labeling Neura l Ste m Cells 

for In Vivo Studie s 

Cultured cell s are much more amenable t o the study 

of cel l cycl e regulators than ar e cell s i n situ . Therefore, 

mos t studie s hav e examine d NSC s tha t hav e 

been derive d fro m th e CN S an d experimentall y 

manipulated i n culture . Bot h th e remova l o f cell s 

from their natural surrounding in the brain and artifi - 

cial culture condition s contribut e t o a degree o f uncertainty 

a s t o whethe r observation s fro m tissu e 

culture truly reflect the natural responses of stem cell s 

in th e intac t brain. This issue can onl y be addressed 

through examination of NSCs i n live animals. Many 

novel technique s ar e currentl y availabl e t o stud y 

NSCs i n situ. 

The initia l ste p o f any suc h stud y is to develo p a 

method fo r identifying the cell s of interest i n vivo . A 

review o f these method s i s beyond th e scop e o f this 

chapter. Nonetheless, one approac h i s to use genetic 

techniques tha t allo w creatio n o f transgeni c mic e 

with expression of marker genes under NSC promot - 

ers such a s nestin o r CD 133. Viral vectors delivering 

a labe l suc h a s green fluorescen t protei n o r LacZ (3galactosidase, 

or the exhaustivel y used BrdU-labeling 

can b e use d a s well (Zawada et al. , 1998 ; Ouredni k 

et al. , 2001) . Another approac h i s transplantation o f 

NSCs int o the brain , following thei r ex vivo labeling 

and/or geneti c modification . Potentia l advantage s of 

this approach includ e th e ability to control th e num - 

ber o f cell s t o b e studied , thei r placement , an d th e 

ability to follow their movement and differentiation i n 

EFFECT OF ETHANO L ON NEURAL STEM CELLS 20 9 

vivo. I n suc h labeled , transplante d ste m cell s an d 

their progeny, one can assess the effect s o f ethanol in - 

take o n cel l cycl e regulator y proteins . A suitabl e 

model o f neurogenesis tha t i s as close a s possible t o 

human neurogenesi s (i.e. , transplantation o f human 

NSCs into a pregnant primate that i s available for examination 

o f effects o f ethanol o n cel l cycl e of stem 

cells) has been develope d (Ouredni k e t al., 2001) (for 

details see section below). 

Transplanted Huma n Neural Ste m Cells 

Participate in Brain Development 

It has been hypothesize d that multiple bonafide ste m 

cell pool s exis t an d represen t descendants o f a com - 

mon NS C (Ouredni k e t al. , 2001) . Thes e cell s 

emerge, by design, in the contex t of a single developmental 

proces s o f allocation an d segregatio n during 

the earliest stages of eerebrogenesis and then establish 

a lifelong reservoir, presumably for use in homeostasis 

and repair . This phenomeno n coul d represen t a developmental 

strateg y i n whic h plasticit y i s "pro - 

grammed" into the CN S a t the singl e cell leve l fro m 

the earlies t stage s o f embryogenesis . Thi s progra m 

depends on both progenito r proliferation and differen - 

tiation. 

A clon e o f NSCs o f human derivatio n (hNSCs ) 

was grafte d int o developin g brain s o f feta l bonne t 

monkeys (Macaca radiatd), a n Ol d Worl d monke y 

species (a s close a s feasible experimentally to the hu - 

man condition ) (Ouredni k e t al., 2001) . Unilaterally 

injected hNSC s distribut e themselve s evenl y 

throughout bot h cerebral hemispheres an d a t all levels 

of the neural axis, settling in diverse widespread regions 

of the telencephalon , principally at the fronta l 

and frontoparietal levels. Although individua l hNSCs 

are clonally related, they appear to segregate into two 

subpopulations. 

Cells in subpopulatio n 1 traverse great distance s 

through th e developing cerebrum b y migrating from 

the periventricula r germinal zones alon g host radial 

glia to terminate a t developmentally an d temporall y 

appropriate cortica l lamina e an d differentiat e ac - 

cordingly int o severa l neuronal an d glia l cel l types . 

Those hNSCs tha t migrated to the mor e distan t superficial 

neurogenic lamina become neurons , identified 

b y dua l immunoreactivit y t o antibodie s agains t 

neuron-specific nuclea r protein , calbindin, and neurofilament, 

intermixe d seamlessly with the monkey' s 

own neurons .


FIGURE 12- 3 Transplante d neura l ste m cell s (NSC) . 

Neural ste m cell s ca n b e grow n i n vitro and success - 

fully transplanted int o mammalian brain for studies examining 

thei r survival , proliferation , differentiation , 

and migration. A. C17.2 murine NSCs cultured in low 

serum differentiate int o astrocytes (large GFAP-positive 

cells), whereas some remain undifferentiated an d positive 

fo r nestin (fou r cell s abov e th e asterisks) . B. En - 

largement o f the tw o nestin-positive C17. 2 ste m cell s 

from pane l A . C. Te n day s after transplantatio n int o 

striatum an d nucleu s accumben s o f adul t C57BL/ 6 

mice, C17. 2 NSC s surviv e an d expres s activ e p - 

galactosidase as evidenced by staining this 40 jam-thick 

coronal sectio n fo r 5-bromo-4-chloro-3-indoly l p - 

d-galactopyranoside (X-gal) . D. Enlargemen t o f X-galpositive 

cell s i n boxe d are a i n pane l C . E . Fou r 

markers of various stages of cell cycle. Shown here are: 

Most hNSC-derived neurons are found i n superfi - 

cial cortica l layer s II/III (which , at the tim e o f transplant, 

profite d fro m a n intensiv e suppl y o f newl y 

formed neurons ) (Frant z an d McConnell , 1996) . 

hNSC-derived cells , that stop and integrate within the 

cortical layer s IV-VI, differentiat e appropriatel y int o 

glia, a s identified b y immunoreactivity t o GFA P (fo r 

astrocytes) o r t o 2 / ,3 / -cyclic-nucleotide phosphodi - 

esterase (fo r oligodendrocytes). Gli a o f dono r origi n 

are als o observe d i n the margina l zone (MZ ; laye r I) 

and in subcortical regions. Som e donor cell s also con - 

tribute to the population o f radial glia. 

Cells i n subpopulatio n 2 are small, non-processbearing, 

undifferentiate d BrdU-positiv e cell s dis - 

persed throughou t th e S Z as single cell s o r as small 

clusters intermingle d wit h th e germina l cell s o f th e 

host. When double-stained for cell type-specifi c antigens, 

thes e cell s expres s vimenti n (a n immatur e 

progenitor/stem cel l marker) , but ar e negativ e fo r all 

other marker s of differentiation . A smal l numbe r o f 

subpopulation 2 cells , however , ar e presen t withi n 

the striatum an d cortex, intermixed with the differen - 

tiated cell s appropriate to the specific lamina. 

The transplantatio n experiment answers a number 

of important questions concerning NSCs an d provides 

a model fo r studying individual NSCs during neurogenesis. 

(1) The dat a provid e a plausible explanatio n for 

the generatio n of multiple, disparate stem cell populations 

as part of a unitary strategy of NSC allocation . (2) 

The clona l progen y o f a given NSC segregat e t o yield 

differentiated cell s for organogenesis (e.g. , subpopula - 

tion 1) , whereas others (e.g. , subpopulation 2) are de - 

posited i n germinal zones (e.g. , the SZ ) as a reservoir. 

(3) Grafte d hNSCs can integrat e int o th e morphoge - 

netic program of the developing primate host brain. 

Examination o f Cell Cycle 

Machinery I n Vivo 

Although the mode l of neurogenesis described abov e 

is instructive, it is very labor intensive and costly . Simpler 

model s hav e bee n developed . A prototypica l 

NSC-line, C17.2 , ha s bee n derive d fro m postnata l 

murine cerebellum . Thes e cell s ar e nestin-positiv e 

and differentiat e int o neurons, astrocytes (Fig. 12-3) , 

and oligodendrocyte s i n culture . C17. 2 cell s ar e 

proliferating cel l nuclea r antige n (PCNA ) that label s 

the entir e cel l cycle , Ki-6 7 (nearl y entire cycle) , bro - 

modeoxyuridine (BrdU) (a marker for DNA synthesis), 

and phosphorylated histone H 3 (pHisH3, for G2/M).

highly engraftable i n the brain and ca n be identified 

by their expression of (3-galactosidase (Fig. 12-3) . Immunocytochemical 

determinatio n o f th e particula r 

phase i n whic h NSC s ar e presen t i s als o possible . 

Four commo n cel l cycl e markers , proliferatin g cell 

nuclear antigen that labels the entire cell cycle, Ki-67 

(nearly entir e cycle) , Brd U (DN A synthesis) , an d 

phosphorylated histone H3 (pHisH3, for G2/M), have 

been commonl y use d (Fig . 12-3) . I n on e elegan t 

study, these markers were used to determine that morphine 

induces premature mitosi s in proliferating cells 

in the adult mouse SZ (Mandyam et al., 2004). Commercially 

availabl e antibodie s agains t cyclins , Cdks , 

and CKI s ar e usefu l fo r exploratio n o f expression of 

cell cycle mediators in even greater detail. 


Prenatal exposur e t o ethano l ca n lea d t o FASD . 

Ethanol in the developing brain not only triggers cell 

death, bu t als o negativel y affect s cel l division . Sinc e 

the length o f the cel l cycle differs fo r ES cells, NSCs , 

and somati c cells , the effec t o f ethanol o n cel l cycl e 

progression varies in these cell populations and i s developmentally 

controlled . Take n together , ethano l 

generally reduces proliferatio n of NSCs during brain 

development b y affecting th e expressio n of Cdks, cyclins, 

and CKIs such a s p21 and p27. Although thes e 

complex responses are cell-typ e dependent , an d spa - 

tially and temporarily controlled, the net result generally 

i s prolongation o f ga p phase s o f th e cel l cycle , 

particularly Gl . Othe r potentia l cel l cycle-alterin g 

targets o f ethano l i n NSC s migh t b e telomerases , 

transcription factors , and protein s regulatin g cellula r 

bioenergetics. With the adven t of novel technologies, 

many ne w cell cycl e regulator s are being discovered 

and wil l become importan t player s in futur e studie s 

examining how ethanol affect s NS C proliferatio n and 

differentiation i n vivo. 

Abbreviations 

ABCC2 ATP-bindin g cassette protein 

AP-1 activatin g protein-1 

bFGF basi c fibroblast growth factor (als o known 

as FGF-2 ) 


CD 13 3 cel l determinan t 

Cdk cyclin-dependen t kinase 


CKI cyclin-dependen t kinase inhibitor 


ES embryoni c stem 



Gadd45 growt h arres t an d DN A damag e in - 

ducible 

GFAP glia l fibrillary acidic protein 

GMC ganglio n mother cel l 

hNSC huma n neural stem cell 


Ki-67 proliferation-associate d nuclear antigen 


NSC neura l stem cel l 


Rb retinoblastom a 


Tc tota l length o f the cell cycl e 

TERT telomeras e revers e transcriptase 

TRF telomerase-repeat-bindin g factor 

TUNEL termina l deoxynucleotid e transferase - 

mediated deoxyuridin e triphosphat e 

nick-end labeling 


ACKNOWLEDGMENTS Th e author s thank Andy Poczobutt 

for figure design and th e Nationa l Institute o f Alcohol 

Abuse an d Alcoholis m an d th e Integrativ e 

Neuroscience Initiative on Alcoholism for support of our 

research. 

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13 

Mechanisms of Ethanol-Induced 

Alterations i n Neuronal Migratio n 

Neuronal migration i s the proces s b y which postmi - 

totic neurons translocate fro m thei r birthplace in proliferative 

zone s t o th e appropriat e target structur e 

where th e neuron s integrate int o th e emergin g net - 

work. I n th e cerebra l cortex , fo r example , neuron s 

generated i n one o f two germinal zone s adjacent to a 

lateral ventricle , th e ventricula r an d subventricula r 

zones (se e Chapters 2 and 11) , migrate in an orderl y 

fashion to a defined laminar residence (Angevine and 

Sidman, 1961 ; Berr y and Rogers , 1965 ; Rakic , 1972 , 

1974;Rakicetal., 1994) . 

The inabilit y of neurons to properly migrate is one 

cause of neurological disorders. The cerebra l cortex is 

the brain region most frequently described as exhibiting 

malformation s designated a s neuronal migratio n 

disorders (NMDs). Tw o likely reasons for this are (1 ) 

its immense siz e and the large number o f constituen t 

neurons and (2 ) its highly laminated structure. These 

features mak e morphologica l disruption s more read - 

ily identifiable. 

Julie A. Siegenthale r 


216 

Structural Malformations i n 

Human Brain 

Two type s o f cortica l malformation s are eviden t i n 

humans wit h NMDs : lissencephal y o r heterotopias . 

Both malformation s are ofte n associate d wit h debili - 

tating cognitiv e defect s i n individual s who are men - 

tally retarde d and/o r epilepti c (Anderrnann , 2000 ; 

Pilzetal.,2002). 

The ter m lissencephaly, meanin g "smoot h brain, " 

is derived from the eve n appearance o f the surfac e of 

the brai n cause d b y a reductio n (pachygyria ) or absence 

(agyria ) of cortical gyri . Othe r defect s associ - 

ated wit h lissencephal y includ e increase d cortica l 

thickness an d reduce d o r poorl y define d cortica l 

lamination (Crome , 1956 ; Friede, 1989) . 

Heterotopia refer s to a cluster of neurons that is abnormally 

distributed relativ e to neurons with a com - 

mon phenotype , suc h a s birthdat e o r connectivity . 

Heterotopias ca n b e detecte d a t four sites . Cluster s

of ectopic neuron s may be distributed withi n the cor - 

tical gray (Miller, 1986 ; Anto n e t al., 1999) . Heterotopias 

can als o be distributed in the subcortica l white 

matter. A common exampl e i s a subcortical band het - 

erotopia, a thick strip of ectopic cells beneath the cor - 

tex. A ban d heterotopi a i s ofte n associate d wit h 

lissencephaly (Chevassus-au-Loui s and Represa, 1999 ; 

Kato and Dobyns, 2003) . Heterotopias are also found 

outside th e corte x beyond th e ventricula r (Eksiogl u 

et al, 1996 ) o r pia l surfac e (William s e t al, 1984 ; 

Saito et al., 1999 ; Taked a e t al, 2003). 

Ethanol-Induced Malformation s 

Neuronal migratio n disorder s in humans hav e gene - 

tic and/o r epigeneti c roots . Mutation s i n gene s tha t 

transcribe protein s regulatin g neurona l migratio n 

lead t o a spectru m o f defects , includin g cortica l 

lissencephaly an d heterotopias (Mochid a an d Walsh, 

2004). Exposur e t o drug s o f abuse , i n particula r 

ethanol, can lea d t o NMDs. Feta l brain s exposed to 

ethanol i n uter o ma y exhibi t a diminutio n o f gyr i 

and sulc i an d disruption s i n cortica l laminatio n 

(Fig. 13-1) (Clarre n e t al. , 1978 ; Wisniewsk i et al. , 

1983; Konovalov et al, 1997 ; Roebuc k e t al, 1998) . 

Morphological defect s cause d b y gestational ethano l 

exposure likely contribute t o the cognitive defects and 

mental retardatio n associate d with feta l alcoho l syn - 

drome (FAS) . 

Several brai n region s i n anima l model s o f FAS 

display morphologica l defects , includin g hetero - 

topias an d structura l disorganizatio n (Fig . 13-2A) , 

similar t o those reporte d i n humans. These region s 

include the cerebra l corte x (Miller , 1986 ; Kotkoski e 

and Norton , 1988 ; Komats u e t al., 2001 ; Moone y e t 

al, 2004) , striatu m (Heato n e t al, 1996) , midbrai n 

(Sari et al., 2001; Zhou e t al, 2001), and cerebellu m 

(Kornguth e t al , 1979 ; Borge s an d Lewis , 1983 ; 

Quesada e t al, 1990a , 1990b) . Th e similaritie s between 

human s an d laborator y animal s establis h th e 

utility o f animal model s fo r examinin g th e effec t o f 

ethanol o n neurona l migration . Rigorou s measure s 

of all aspects of cell migration are required to evaluate 

the effec t o f ethanol o n neuronal migration . Du e 

to the obviou s limitation s o f human studies , anima l 

models o f FAS have bee n use d (a ) t o further docu - 

ment the phenomenon of ethanol-induced defects in 

neuronal migratio n an d (b ) t o stud y underlyin g 

mechanisms. 

ETHANOL AFFECTS NEURONAL MIGRATION 21 7 

FIGURE 13- 1 Brai n malformations in humans exposed 

to ethanol in utero. The gyra l pattern of the normal human 

infant brain A. contrasts with the smoother cortical 

surface i n the brain of an infant with FAS B. The brain 

is marke d b y it s small size , which i s likely caused b y 

ethanol-induced defect s in both neurona l proliferatio n 

and cel l death. The brai n also exhibits a paucity of gyri, 

and these gyri tend to be broad. These features are signs 

of lissencephaly . Moreover , th e brai n i s covere d b y 

sheets of heterotopic cells over the surfac e o f the brain. 

The lissencephal y and heterotopias are consistent with 

defects i n neurona l migration . C . Ethano l exposur e 

can also induce heterotopias t o form i n other regions of 

the brain, includin g th e brain stem. Arrow s depict th e 

breach i n pia l surfac e throug h whic h th e migratin g 

neurons presumabl y streamed t o form th e heterotopia . 

D. Heterotopia s ar e als o collection s o f cells tha t pre - 

sumably terminate thei r migrations in inappropriate locations. 

Suc h neuron s are ofte n foun d in the cerebra l 

cortex. Cells with large (arrow ) and smal l (arrowhead) 

somata form a heterotopia within a portion o f layer I, a 

region of the cortex that is normally cell-sparse. (Source: 

Images provided courtesy of Sterling Clarren.) 

MIGRATION DEFECTS IN ANIMAL 

MODELS OF FETAL ALCOHOL SYNDROME 

A method commonl y use d t o stud y neurona l migra - 

tion i s mappin g o f spatiotempora l change s o f cell s 

with [ 3 H]thymidine autoradiography . Throug h thi s


method, proliferating cells can be permanently radio - 

labeled an d the migratio n o f tagged cell s traced fro m 

their "birth " through th e completio n o f their migra - 

tion. Durin g norma l development , mos t neuron s la - 

beled with pHjthymidin e earl y i n corticogenesi s 

eventually ar e distribute d i n dee p layers , wherea s 

neurons labeled late r in development occup y superficial 

layer s (Angevin e and Sidman , 1961 ; Berr y an d 

Rogers, 1965 ; Rakic , 1974 ; Mille r an d Nowakowski , 

1988). Thus, cortical layer s are formed in an orderly 

inside-out manner , during which migratin g neurons 

move past those that have already completed cel l migration. 

Migration ca n b e divide d int o three phases . Th e 

first is the onset of migration, which occurs in the proliferative 

zones . Second , youn g neurons actively migrate 

throug h th e intermediat e zone (IZ ; the anläg e 

of the white matter) to the superficia l limit of the cortical 

plate, the predecessor of the cortical gray matter. 

The fina l ste p i s the terminatio n of neuronal migra - 

tion in the developing structure . 

Although th e basi c inside-ou t developmen t o f the 

cortex i s largely preserved i n roden t model s o f FAS, 

each phas e o f migratio n i s affected b y ethanol . Th e 

birth of cells that will occupy analogous site s in cortex 

is 1 to 2 days later in ethanol-treate d rat s than i n con - 

trols (se e Fig. 11-1 , Chapte r 11 ) (Miller , 1986 , 1987 , 

1993). Par t o f this delay (as much a s 3 1 hr) i s due t o 

postmitotic cell s lingerin g longer i n th e proliferativ e 

zones befor e initiatin g thei r migrations . Thi s likel y 

contributes to the increase in size of the subventricular 

zone, which contains many young neurons beginning 

their migrations (Miller, 1989). On the other hand, the 

number o f neuron s beginnin g migratio n a t an y on e 

time, the leaving fraction, i s not affected b y ethanol. 

Ethanol als o reduce s th e rat e o f migratio n 

throughout development (Fig. 13-2B ) (Miller , 1993). 

For example, in the cortice s of 17-day-old fetuses, the 

mean rat e o f migratio n i s 138u,m/day , wherea s i n 

ethanol-treated fetuse s th e mea n rat e i s 8 2 jilm/day. 

Studies usin g slic e culture s o f corte x sho w tha t 

ethanol ha s a simila r effec t i n vitr o (Siegenthale r 

and Miller, 2004) and the rate of migration is reduced 

in a concentration-dependen t manne r (Hira i e t al. , 

1999b). The findin g tha t the rat e of migration i n situ 

is th e sam e a s tha t i n vivo suggest s tha t neurona l 

migration i s regulated b y local microenvironment — 

i.e., that extracortica l signal s (monoamines) ma y not 

be critical regulators. 

G13 G17 

TIME OF ORIGIN 

G20 

FIGURE 13- 2 Anima l model s o f feta l alcoho l syn - 

drome (FAS) . A. Malformations incidental to prenatal 

alcoho l exposure , includin g cortica l heterotopia s 

(box indicates area magnified in B), can be replicated 

in anima l model s o f FAS. B. Thi s particula r hetero - 

topia appears to be caused by cells streaming through 

layer 1 at multiple sites (arrows), forming a layer of ectopic 

cell s outsid e th e cortex . C . Th e rat e o f migration 

throughou t corticogenesi s i s particularl y 

vulnerable to ethanol. Pulse-chas e experiment s using 

[ 3 H] thymidin e sho w tha t ethano l cause s significant 

decreases i n th e rat e o f migration o f cells generate d 

on gestational da y (6) 13 , G17, and G20 . The migra - 

tion o f cell s generate d o n G1 7 i s mos t effecte d b y 

ethanol. On Gl 3 and G17, the cells that begin migration 

first (outermost 10 ) and the total migrating population 

(mea n population ) ar e similarl y slowe d b y 

ethanol. I n contrast , migratio n o f th e outermos t 1 0 

cells generate d o n G2 0 i s les s sensitiv e t o alcoho l 

than tha t o f the tota l population . (Source: Image s A 

and B provided courtesy of William Shoemaker and 

Gordon Sherman. ) 

During late r stage s o f cortica l development , 

ethanol cause s migratin g neuron s t o finis h thei r 

migration i n inappropriat e locations . Fo r instance , 

neurons i n the corte x o f mature, norma l rat s that are 

generated o n gestationa l da y (G) 2 0 are destine d fo r

layer II/III. Followin g prenata l exposur e t o ethanol , 

however, most of these neuron s eithe r terminat e thei r 

migrations i n deep laminae (layer s IV, V, and VI ) o r 

they overmigrat e int o laye r I (Miller , 1986 , 1993). 

Other studie s sho w tha t ethano l ca n caus e th e production 

o f suprapial heterotopias, o r warts (Kotkoskie 

and Norton , 1988 ; Komatsu e t al , 2001 ; Mooney 

et al., 2004) . The implicatio n i s that ethanol disrupts 

mechanisms involved in stopping neuronal migration. 

MIGRATION MACHINERY: PROTEIN 

TARGETS OF ETHANOL TOXICIT Y 

The thre e phases of neuronal migration are regulated 

by a number o f different protei n complexe s an d signaling 

pathways that work in concert t o move young 

neurons fro m th e proliferativ e zone s t o their targets. 

Each phase of neuronal migration is vulnerable to the 

effects o f ethanol . Therefore , protein s tha t regulat e 

migration, includin g cel l adhesio n protein s (CAPs) , 

cytoskeletal proteins, extracellula r matrix (ECM) proteins, 

and growt h factors , ar e likely candidates a s targets 

of ethanol toxicity . 

Cell Adhesion Proteins 

The migratio n of most cortica l neuron s begin s soo n 

after thei r birth in a zone proximal to the latera l ventricle 

(see Chapter 3) . The neuro n migrates out of the 

germinal are a usin g radial fibers that spa n th e widt h 

of the cerebral wall and remains apposed t o the guiding 

fiber throughou t its migratory tre k (Rakic , 1971, 

1972; Edmondso n an d Hatten , 1987) . Appropriat e 

adherence to the glia l guide is regulated by cell adhe - 

sion receptor s tha t lin k th e neura l cytoskeleto n t o 

CAPs, component s o f th e ECM , an d membrane - 

bound moitiés of the glia l fiber. 

Cell Adhesion Proteins Regulate Neuronal 

Migration in Normal Animals 

Adhesion protein s from bot h th e cel l adhesio n mole - 

cule (CAM ) and integri n families are particularly important 

fo r neuronal migratio n (Ron n e t al. , 1998; 

Clegg et al, 2003; Schmid and Anton, 2003). Neural 

CAM (nCAM ) and L I mediat e neurona l migration 

through homophilic binding and consequen t activation 

o f intracellula r signalin g cascade s centra l t o 

ETHANOL AFFECTS NEURONA L MIGRATION 21 9 

cytoskeleton reorganization (Panicker et al., 2003). Integrin 

receptors bind integrin s and ECM component s 

(e.g., laminin , fibronectin , an d glycoproteins) . Inte - 

grin bindin g trigger s cytoskeleta l change s throug h a 

cascade of phosphorylation events (Juliano, 2002). 

Up-regulating expression of CAPs i s an importan t 

part of initiating and propellin g neurona l migration. 

CAPs ar e richl y expresse d in region s of the cerebra l 

wall, th e IZ , an d th e cortica l plat e (CP) , containin g 

migrating neurons (Edmondson e t al., 1988 ; Seki and 

Arai, 1991 ; Anton et al, 1999 ; Siegenthaler an d Miller, 

2004). Disruptio n o f normal cel l adhesio n slow s th e 

progression o f glial-guided migration an d ca n lea d t o 

detachment o f neurons fro m th e glia l fibe r (Chuon g 

etal, 1987 ; Antonetal, 1996, 1999). 

Ethanol-lnduced Changes in 

Cell Adhesion Proteins 

Ethanol exposur e reduce s th e rat e o f migration an d 

causes neurons t o be marooned i n ectopic cortical locales 

(Miller , 1986 , 1987 ; Mille r an d Robertson , 

1993). A likel y mechanis m underlyin g ethanol - 

induced defect s i n neurona l migratio n i s disrupte d 

cell adhesion . Fo r example, ethano l inhibit s LI-LI 

mediated cel l adhesion by physically blocking LI-LI 

homophilic bindin g (Charnes s e t al , 1994 ; Ramanathan 

et al, 1996 ; Wilkemeyer et al, 1999 , 2000, 

2002a, 2002b) . Ethano l treatmen t als o inhibit s Li - 

mediated neurit e outgrowt h b y culture d cerebella r 

granule cells ; however , ethanol apparentl y doe s no t 

affect LI-LI binding per se (Bearer et al, 1999) . The 

implications o f this findin g ar e that ethano l ca n disrupt 

LI-mediated cell adhesio n throug h mechanism s 

other than the LI protein-protein interaction. 

Unlike LI , th e effect s o f ethanol o n nCA M ap - 

pear to be at the leve l of protein expression. Ethanol 

treatment increases nCAM protei n expression in primary 

cortical neurons and i n slices from feta l corte x 

(Fig. 13-3A ) (Mille r an d Luo , 2002a ; Siegenthale r 

and Miller , 2004) . Specifically , ethanol-induce d 

increases i n nCAM expression ar e noted on the surface 

o f migratin g neuron s i n cortica l expiant s (Fig. 

13-3B, 3C ) (Hira i e t al , 1999b ; Siegenthale r an d 

Miller, 2004) . Th e expressio n and post-translationa l 

processing o f th e thre e isoform s o f nCAM i n th e 

postnatal brain are differentiall y affecte d b y prenatal 

ethanol exposure , however, there ar e n o significan t 

changes i n nCA M niRN A expressio n with ethanol ,


FIGURE 13- 3 Expressio n o f cel l adhesio n protein s i s effecte d b y ethanol . 

A. Expression o f three isoform s (120 , 140 , an d ISOkD a isoforms ) o f neural 

cell adhesio n molecul e (nCAM ) i s increase d i n primar y cortica l culture s 

(top) and organotypic slice cultures (180 kDa only) (bottom). B. Cells i n th e 

intermediate zone (IZ ) fro m untreate d organotypi c slices immunolabel with 

nCAM antibody. nCAM expression is highest proximal to the cell membrane 

and appear s to localize to patches (arrows). C. Patche s o f nCAM immunore - 

activity appear to be larger, denser, and mor e frequent i n IZ cells exposed to 

ethanol. Ethano l exposur e also increases the expressio n of integrin subunits 

a?, oc v, and ßj i n organotypic cortical cultures D. Larg e increases in nCA M 

and integri n receptors, like those observed with ethanol exposure , may cause 

a migrating cell to adhere too tightly to the surrounding environ and thus slow 

motility. (Source: From Siegenthaler an d Miller, 2004, with permission). 

suggesting a defec t i n nCA M translatio n (Minan a 

et al., 2000). 

Ethanol-induced changes i n integri n expressio n 

and function are only beginning to be investigated. A 

compelling reason to explore integrins is the similarities 

between cortica l migratio n defects i n mic e lack - 

ing specifi c integri n subunit s an d anima l model s o f 

FAS. I n mic e lackin g integrin oc 3 an d i n animal s exposed 

prenatally to ethanol, cortical neurons destine d 

for superficia l layer s li e i n ectopic , deep-laye r posi - 

tions (Miller, 1986 , 1993 ; Anton etal, 1999). Neuralspecific 

integri n ß } knockou t mic e als o displa y 

abnormal neuronal migration . Ectopi c cell s are common 

i n the margina l zone and th e pia l surface i s disrupted, 

analogou s t o th e heterotopia s see n wit h 

prenatal ethanol exposur e (Graus-Porta etal., 2001). 

Despite th e similarities between anima l models of 

FAS and integri n knockou t animals , i n vitro studies 

indicate that ethanol potentiate s integri n protein con - 

tent (Fig . 13-3D ) (Siegenthale r an d Miller , 2004) . 

Interestingly, thi s findin g i s similar to th e effec t ob - 

served wit h nCA M expressio n (Hira i e t al. , 1999b ; 

Luo and Miller, 1999 ; Miller and Luo, 2002a; Siegen - 

thaler an d Miller , 2004) . Ethano l increase s th e ex - 

pression of integrin subunits OC 3, otv, and ß j in cortica l 

expiants (Siegenthaler and Miller, 2004). In culture d 

neurophils (Bautista , 1995 ) an d hepatocyte s (Schaf - 

fert et al., 2001), integrin oCj , oc 5, and ß! are increase d 

following treatmen t wit h ethanol , settin g u p th e 

possibility tha t to o muc h integrin , lik e to o muc h 

nCAM, is detrimental t o neuronal migration . Indeed , 

neurons tha t overexpres s integri n receptor s hav e impaired 

migratio n (Palece k e t al. , 1997) . Presumably , 

excess CA P cause s increase d cel l adhesio n an d pre - 

vents migratin g neuron s fro m breakin g adhesions a t 

the rea r o f the cell . Essentially , the adhesio n i s too 

sticky. 

Alterations i n integri n expressio n ma y als o con - 

tribute t o ethanol-induce d defect s i n th e radia l glia l 

scaffold. Radia l glial fibers traverse the migration path - 

way in the developin g corte x and provid e a guide for 

neurons durin g their migratio n t o their ultimat e resi - 

dence. Afte r neurona l migratio n i s complete , radia l 

glia transform into stellate astrocyte s (Schmechel an d 

Rakic, 1979 ; Voigt , 1989 ; Culican et al, 1990 ; Mille r 

and Robertson , 1993) . Ethano l exposur e i n uter o 

leads t o th e prematur e differentiatio n o f radia l gli a 

into astrocytes in both th e corte x (Miller and Robert - 

son, 1993 ; Miller , 2003) and cerebellum (Shett y and 

Phillips, 1992) . Neuron s activel y migrating o n thes e

transforming radia l glia ar e stranded i n deep cortica l 

layers, unable to complete thei r migration. This finding 

i s further supporte d b y [ 3 H]thymidine studies in 

which neuron s intende d fo r superficia l layer s ar e 

found i n dee p corte x followin g ethano l exposur e 

(Miller, 1986 , 1993) . Incidentally, absence of integrin 

OC3 also causes premature transformation of radial glial 

(Anton e t al, 1999 ) an d attachmen t o f radia l glia l 

endfeet i s disrupte d i n integrin-ß } knockou t mic e 

(Graus-Porta e t al., 2001). Thus, ethanol-induced disruptions 

i n integri n function o r expressio n may pre - 

cede the premature withdrawal of radial glia. 

Disruptions in Ca 2+ channel flux during neuronal 

migration ma y contribute to the ethanol-induce d reduction 

in the rat e of migration. Ca 2+ currents in migrating 

neurons are mediated by N-methyl-D-aspartate 

(NMDA) subtype of glutamate receptors . Cel l migration 

decrease s followin g treatment wit h NMD A an - 

tagonists (Komur o an d Rakic , 1992 , 1993 ; Hira i e t 

al., 1999a) . Ethanol affect s bot h th e expressio n an d 

function o f NMDA receptors. Chronic ethano l expo - 

sure up-regulates specific subunit s of the NMD A re - 

ceptor i n feta l cortica l neuron s (Kumari , 2001) . I n 

contrast, acut e treatmen t wit h ethano l block s 

NMDA-dependent Ca 2+ current s i n culture d hip - 

pocampal neuron s an d slice s (Lovinger et al. , 1989 , 

1990). Ca 2+ currents are particularly important at the 

trailing edg e o f migratin g cells . Los s o f integrin - 

dependent cel l adhesio n a t th e rea r edg e o f motil e 

cells depend s o n increase s i n intracellula r Ca 2+ 

(Marks an d Maxfield , 1990 ; Lawso n an d Maxfield , 

1995). Thus , ethanol-dependen t change s i n norma l 

Ca 2 + currents might slow neuronal migration by preventing 

normal separation at the rear edge of the cell. 

Extracellular Matrix 

The pia l membran e (PM ) (o r pial basemen t mem - 

brane) is an ECM-rich region. The EC M contribute s 

to the role of the PM in the normal developing cortex 

as a barrier (chemical and physical ) around developing 

neural tissu e and a n attachmen t poin t fo r radial 

glia (Siever s et al, 1994) . Breache s in the P M allow 

neurons to migrate beyond the pia l surface an d for m 

heterotopias (Marin-Padilla, 1996) . 

Prenatal ethano l exposur e ca n induc e formation 

of heterotopia s i n human s expose d prenatall y t o 

ethanol (Clarre n e t al., 1978) . Roden t model s o f prenatal 

ethanol exposure also show heterotopias i n both 

the cerebellu m an d corte x (Kotkoski e an d Norton , 


FIGURE 13- 4 Ethanol-induce d heterotopia s ar e associated 

wit h disruption s i n th e margina l zon e (MZ ) o f 

organotypic cortica l cultures . A . Severa l cel l som a oc - 

cupy a breach (ope n arrows) in calretinin-positive fibers 

within th e MZ . Th e cell s appea r t o b e streamin g 

through th e break and contributing t o the heterotopia , 

or "wart, " formin g outsid e th e cerebra l wall . Cajal - 

Retzius cel l bodie s (close d arro w wit h line ) an d pro - 

cesses (closed arrows) that occupy the MZ immunolabel 

with calretini n antibodie s (close d arrows). CP , cortica l 

plate. B. Breaches in the MZ (* ) contain nestin-positive 

radial glial fibers (arrows). These fibers normally terminate 

a t the pia l membrane (PM ) adjacen t to the MZ . 

Ethanol-induced disruption s i n th e M Z and/o r P M 

likely allow the fibers to course inappropriately into the 

heterotopia. (Source: Fro m Moone y e t al. , 2004 , with 

permission).


1988; Komatsu et al, 2001; Sakata-Haga et al, 2001; 

Mooney et al., 2004). In rodents, breaches in the glial 

limitans an d margina l zone , a superficia l cortica l 

region adjacen t t o th e PM , ar e ofte n foun d a t th e 

base o f ethanol-induce d heterotopia s (Fig . 13-4 ) 

(Komatsu e t al , 2001 ; Sakata-Hag a e t al , 2001 ; 

Mooney e t al, 2004). 

Despite repeated report s of heterotopias associate d 

with prenata l ethano l exposure , a clea r link betwee n 

defects i n th e EC M constituent s o f th e P M an d 

ethanol has not been made . Nevertheless, the appear - 

ance o f heterotopia s i n animal s i n whic h specifi c 

ECM protein s ar e dysfunctiona l strongl y indicate s 

that these proteins are disrupted by ethanol. Deletio n 

or dysfunction of PM protein s lamini n (Halfte r et al., 

2002), perlecan (Costel l e t al., 1999) , and chondroitin 

sulfate proteoglyeans (Blackshear et al, 1997 ) leads to 

overmigration o f neuron s an d formatio n o f ectopia s 

reminiscent of ethanol-induced heterotopias . Integrin 

receptors bind t o and organiz e EC M proteins . Mice 

lacking the integrin a6 or integrin ßj feature defects in 

the cortica l pia l membran e tha t lea d t o th e appear - 

ance o f ectopic cel l bodie s (Georges-Labouesse e t al, 

1998; Graus-Porta et al, 2001). As noted above, ethanol 

does affect th e expressio n of integrin subunits , includ - 

ing ßj (Siegenthale r an d Miller , 2004) . Conceivably , 

ethanol targetin g of these integrins is key to the gener - 

ation o f heterotopia s and , b y deduction , thes e inte - 

grins are key to the integrity of the PM . 

Reelin, a larg e glycoprotei n componen t o f th e 

marginal zon e ECM , i s produced an d secrete d b y 

Cajal-Retzius cell s (D'Arcangel o e t al, 1995 ; Hirot - 

sune e t al, 1995 ; Ogaw a e t al, 1995 ; Sod a e t al, 

2003). Reelin i s believed to act a s a stop signal causing 

migratin g neuron s t o en d migratio n an d detac h 

from thei r glia l guide s (Pearlma n an d Sheppard , 

1996; Marin-Padilla , 1998 ; Dulabo n e t al , 2000 ; 

Hack e t al. , 2002) . Immunohistocheniistr y studie s 

show that reeli n i s abnormally distributed i n ethano l 

induced-heterotopias (Moone y e t al. , 2004) . I t i s 

absent directly below heterotopias, but i t is distributed 

toward th e perimete r o f a heterotopia . Conceivably, 

the periphera l displacemen t o f reelin underlie s con - 

tinued migration of neurons into a heterotopia . 

Cytoskeleton 

Successful cel l adhesion an d release during neuronal 

migration depends o n the rapi d reorganization of the 

cytoskeleton. This reorganizatio n i s essential fo r th e 

initiation an d maintenanc e o f neurona l migratio n 

(Lambert de Rouvroit and Goffinet , 2001 ; Marin an d 

Rubenstein, 2003) . Th e tw o mai n cytoskeleta l ele - 

ments in neurons are actin and microtubules, proteins 

that ca n polymeriz e int o lon g chain s o f individua l 

subunits tha t compris e th e intracellula r scaffold . 

Actin filament s ar e enriche d a t th e leadin g edg e o f 

migrating neurons. Microtubules , in contrast, for m a 

cage-like structur e withi n th e cel l somat a an d ar e 

aligned longitudinall y in th e leadin g proces s (Riva s 

and Hatten, 1995) . The importanc e of both actin and 

microtubule assembly is emphasized b y findings that 

several geneti c NMD s ar e cause d b y defects in pro - 

teins tha t bin d an d organiz e cytoskeleta l protein s 

(Sapir et al, 1997; Gleeson et al., 1999) . 

Ethanol negativel y affects the cytoskeleton i n neu - 

ral and non-neural cells . Actin assembly is aberrant in 

cultured neura l cres t cell s treate d wit h ethano l out - 

growth (Hassle r and Moran , 1986a , 1986b) . Ethanol 

induces the formation of thick bundles o f actin fibers 

that prevent the normal process. The acti n cytoskele - 

ton i n astrocyte s is als o sensitiv e t o treatmen t wit h 

ethanol (se e Chapter 18) . The norma l organization of 

actin filament s i n culture d astrocyte s is severely disrupted 

b y ethanol i n a concentration-dependent man - 

ner (Allansso n e t al. , 2001 ; Guasc h e t al. , 2003 ; 

Tomas e t al, 2003) . Ethano l promote s th e transfor - 

mation o f actin stres s fibers into dysfunctiona l acti n 

rings. Interestingly , the effec t o f ethanol o n th e acti n 

cytoskeleton i s alleviated b y overexpression o f RhoA, 

a small GTPase that induce s actin stress fiber assembly 

(Guasc h e t al , 2003) . This effec t suggest s tha t 

ethanol exposure may disrupt signaling pathways regulating 

cytoskeleta l reorganization . Microtubul e 

polymerization is also affected b y ethanol exposur e i n 

astrocytes an d hepatocyte s (Yoo n et al., 1998 ; Toma s 

et al. , 2003) . Afte r treatmen t wit h th e microtubule - 

disrupting agen t nocodazole , th e spee d o f micro - 

tubule repolymerization is reduced in the presence of 

ethanol. 

Growth Factors 

Role in Neuronal Migration 

Growth factors are proteins tha t regulate cell ontogen y 

via extracellula r mechanisms . Growt h factor s tha t 

affect neurona l migratio n includ e member s o f th e 

transforming growt h facto r (TGF ) ß superfamil y 

(TGFßl an d bon e morphogeni c protei n [BMP ] 4 )

and brain-derive d neurotrophic facto r (BDNF) . Several 

studies imply that these factors are active endogenously. 

Eac h ligan d an d th e relevan t receptor s 

are expresse d i n proliferativ e zone s durin g cortica l 

development (Flander s et al, 1991 ; Fukumitsu et al, 

1998; L i et al, 1998; Miller, 2003). 

Functional studie s relyin g o n exogenou s growt h 

factor furthe r indicat e tha t TGFßl , BMP-4 , an d 

BDNF ar e involve d i n th e initiatio n and/o r mainte - 

nance o f neurona l migration . Bot h TGFß l an d 

BMP-4 decrease cel l proliferatio n and promot e neuronal 

migratio n (L i e t al. , 1998 ; Luo an d Miller , 

1999; Siegenthale r an d Miller , 2004 , 2005a) . Complimentary 

line s o f evidenc e indicat e tha t BDNF, 

though no t classicall y associate d wit h neurona l mi - 

gration, promote s th e initiatio n o f neurona l migra - 

tion. First , studies show that cerebella r granul e cell s 

from BDNF~ f ~ mic e ar e abl e to attac h t o glial fiber s 

but fai l t o begin migratin g (Borghesani et al., 2002). 

Also, addition of exogenous BDN F enables BDNF~ /granule 

cells to migrate properly. Finally, injection o f 

BDNF int o th e latera l ventricle s o f embryonic animals 

induces the premature initiatio n of migration of 

neurons destine d fo r dee p corte x (Ohmiy a e t al. , 

2002). 

Effect ofEthanol on Growth 

Factor-Mediated Neuronal Migration 

Ethanol effect s bot h th e expressio n o f the endoge - 

nous TGFß system and th e function of exogenously 

applied TGFßl i n vitro . Rat s prenatally exposed t o 

ethanol exhibi t altere d TGF ß ligan d an d recepto r 

expression (Miller , 2003 ) Specifically , ethano l in - 

creases TGFßl an d TGFß2 content in the prolifer - 

ative zone s prenatally , bu t i t diminishe s perinata l 

TGFß2 expressio n i n th e interna l segment s o f radial 

glia. In contrast, ethanol increase s TGFß receptor 

typ e 1 expressio n i n radia l glia l fibers . Th e 

presence of an endogenous TGFß system in the proliferative 

an d intermediat e zone s suggest s tha t 

TGFß i s importan t fo r a t leas t th e initiatio n an d 

propagation o f neuronal migratio n throug h th e in - 

ner half of the cerebral wall. Further, th e vulnerability 

o f thi s portio n o f th e TGF ß syste m t o ethano l 

may contribut e t o ethanol-induce d defect s i n neuronal 

migration. 

Ethanol disrupts TGFßl-mediated neuronal migration. 

I n slic e culture s o f cerebral cortex , ethano l im - 

pedes th e effect s o f exogenousl y applie d TGFß l o n 


both cel l migration and CAP expression (Siegenthaler 

and Miller , 2004 , 2005b) . In vitro studies o f dissociated 

cortica l cell s an d neura l tumorgeni c cell s de - 

scribe a simila r effec t o f TGFßl an d ethano l o n a 

specific CAP , nCAM. nCAM expression is increased 

with TGFßl treatmen t alon e bu t i s reduced t o control 

level s b y combine d treatmen t wit h ethano l 

(Luo an d Miller , 1999 ; Mille r an d Luo , 2002a , 

2002b). Collectively , thes e studie s sho w tha t (1 ) 

ethanol interfere s with TGFßl-mediated promotion of 

cell migratio n an d (2 ) i t ma y d o s o b y blockin g 

TGFßl-dependent increases i n expressio n of nCAM 

and other CAPs . 

BMP signaling is involved in the initiatio n of cell 

migration i n th e developin g cerebra l corte x (L i 

et al., 1998) . BMP- 4 specificall y is expressed by cells 

about to begin migration (e.g., at the ventricular surface). 

Althoug h th e effec t o f ethano l o n BMP - 

mediated initiatio n o f migratio n i s no t establishe d 

per se , ethanol doe s bloc k BMP-4-induce d cel l ad - 

hesion amon g culture d neuroglioblastom a cell s 

(Wilkemeyer e t al., 1999) . Presumably , such inhibi - 

tion may be involved in delay of the initiation of neuronal 

migration. 

BDNF promotes th e initiatio n o f glial-guided migration 

(Borghesan i e t al. , 2002) . Incidentally , th e 

endogenous BDN F syste m i s perturbed b y ethanol . 

Perinatal ethano l exposur e down-regulate s BDN F 

mRNA and it s receptor, trkB , in the developin g cere - 

bellum (Heato n e t al, 2000 ; Ligh t e t al, 2001) and 

olfactory bul b (Maie r e t al. , 1999) . A similar downregulation 

o f BDN F protei n accompanie d b y a 

ethanol-induced decreas e i n BDN F signaling is evident 

i n prenata l corte x (Climen t e t al. , 2002) . An 

ethanol-induced reductio n i n BDN F activit y i n 

the developin g brai n likel y affect s BDNF-regulate d 

processes includin g th e initiatio n o f neurona l 

migration. 


Neuronal migratio n i s a uniqu e an d critica l component 

o f neural development . I t provides a bridge be - 

tween th e birt h o f a cel l an d it s prope r integratio n 

into a n evolvin g structure . NMD s ar e cause d b y 

breakdowns i n th e commencement , completion , 

and/or cessatio n o f neuronal migration . Th e NMD s 

detected in case s of FAS are likely caused by ethanol 

interfering with each phas e of neuronal migration .


FIGURE 13- 5 Ethano l disrupts cortical migration a t 

many levels. A. Ethanol reduce s the rat e of neuronal 

migration i n the developin g cerebra l cortex . Migrat - 

ing neuron s depen d o n prope r cell adhesio n (e.g. , 

cell adhesion molecules an d integrins) and cytoskeletal 

reorganizatio n (e.g. , microtubule s an d actin ) fo r 

motility alon g glia l guides . Ethanol-dependen t in - 

creases in cell adhesion protein expression and abnormal 

cytoskeleta l organizatio n ar e a likel y caus e o f 

reduced cortica l cel l motility . B. Heterotopi a forma - 

tion has been linke d t o disruptions in both the extra - 

cellular matri x component s o f th e pia l membran e 

(PM) an d thei r organizatio n b y integri n receptors . 

The resultin g "holes" i n th e P M an d adjacen t M Z 

permit overmigratio n o f neuron s tha t woul d nor - 

mally sto p an d incorporat e int o th e cortica l plate . 

In particular , absenc e o f th e stop-signa l reeli n 

within th e breache s likel y contribute s t o hetero - 

topia formation . Evidenc e o f radia l glia l fiber s 

coursing into the heterotopia ma y mean tha t migrating 

neurons migrat e int o th e war t on overextende d 

glial fibers . C . Ethano l exposur e cause s prematur e 

transformation o f radial glia into astrocytes. Neurons 

still migratin g o n thes e retractin g fiber s woul d b e 

stranded inappropriatel y i n deepe r cortica l layers . 

This phenomeno n ma y explai n th e appearanc e o f 

layer II/III neurons are found in layers IV, V, and VI 

in ethanol-expose d brains . D. Variou s growth factor s 

(e.g., TGFßl , BMP-4 , an d BDNF ) ar e involve d i n 

Ethanol-induced defect s i n neurona l migratio n 

have seriou s consequence s fo r downstrea m develop - 

mental events . For example, exposure to ethanol dur - 

ing th e perio d o f neurona l migratio n ca n caus e 

neurons to land i n th e wron g cortical layers (Miller, 

1986, 1987) . Despite this, the neurons may retain their 

destined phenotype , fo r example , a s corticospina l 

(Miller, 1987 ) o r callosa l (Miller , 1997 ) projectio n 

neurons. As the cell bodies are distributed in inappropriate 

locations , the y integrat e int o th e synapti c web 

incorrectly (Al-Rabia i an d Miller , 1989) . Conse - 

quently, abnormalitie s i n cortica l metabolis m an d 

activity occur (Vinga n e t al, 1986 ; Mille r and Dow - 

Edwards, 1988 , 1993) . Flawe d synapti c connection s 

due t o heterotopia s ca n caus e a myriad of problems , 

the foremos t being epileps y (Chevassus-au-Louis an d 

Represa, 1999) , a neurological symptom prevalent in 

children with FAS (Burd et al, 2003). 

Over th e las t two decades, studies of neuronal mi - 

gration hav e identifie d a number of possible mecha - 

nisms fo r ethanol-induce d defect s i n migration . 

Ethanol alter s bot h th e microenvironmen t encoun - 

tered by the migrating neuron (e.g. , radial glial fibers, 

CAPs, EC M proteins , an d growt h factors ) an d th e 

motility o f neuron s withi n tha t environmen t (e.g. , 

CAPs an d cytoskeleta l proteins) (Fig. 13-5) . The di - 

versity of cellular pathways affected b y ethanol speak s 

to the complexit y of ethanol-induced disturbance s in 

neuronal migration. 

Abbreviations 


BMP bon e morphogenic protei n 

CAM cel l adhesion molecul e 

CAP cel l adhesion protei n 




G gestatio n day 


the initiatio n of neuronal migration . Ethano l disrupts 

the normal function of these growth factors. The dela y 

in initiatio n o f migration see n wit h ethano l exposur e 

likely result s fro m inhibitio n o f growt h factor-medi - 

ated activities.

nCAM neura l cell adhesion molecule 

NMD neurona l migration disorde r 


PM pia l membrane 

TGF transformin g growt h facto r 

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14 

Effects of Ethanol on Mechanisms 

Regulating Neuronal Proces s 

Outgrowth 

Establishment o f normal neura l circuitr y in the cen - 

tral nervou s syste m (CNS ) require s spatial and tem - 

poral contro l o f th e outgrowt h o f neuntes . Thi s 

outgrowth an d th e specificatio n of axon s an d den - 

drites ar e amon g th e earlies t event s i n th e develop - 

ment of postmigratory neurons. 

Three decade s o f researc h sho w tha t exposur e to 

ethanol disrupt s th e developmen t o f axon s an d den - 

drites an d alter s neura l circuitr y i n divers e brai n re - 

gions. Th e primar y actions o f ethanol o n developin g 

neurons are still largely unknown, however, in part, because 

th e endogenou s mechanism s and extracellular 

signaling pathway s regulating proces s outgrowt h an d 

guidance during normal development ar e not fully un - 

derstood (se e Chapter 4) . Fo r comprehensive descriptions 

of effects o f ethanol o n the microscopi c structure 

of the developing brain, the reader is referred to reviews 

by Pentney and Miller (1992) and Berman and Hannigan 

(2000), respectively. 

The presen t chapte r summarize s th e effect s o f 

ethanol on the outgrowth and maturation o f dendrites 

Tara A. Lindsley 

230 

and axons , focusin g o n CN S neuron s developin g 

in vivo or in vitro. The selecte d observation s highlight 

both well-establishe d an d emergin g insight s regarding 

th e effect s o f ethano l o n axona l an d dendriti c 

growth. Althoug h not exhaustive , one sectio n describes 

how recent advances in developmental neural 

cell biology are informing work aimed at understanding 

th e mechanism s underlyin g disruption o f neu - 

ronal development b y ethanol. 

EFFECTS OF ETHANOL ON 

DEVELOPMENT OF DENDRITES 

In Vivo Studies 

The comple x an d characteristi c shap e o f a dendriti c 

arbor, where neurons receive and integrate input fro m 

multiple synapti c partners , i s a critica l determinan t 

of th e electrophysiologica l propertie s o f a neuron . 

Ethanol-induced change s i n dendriti c morpholog y

could severel y affec t neurona l function . Result s of 

morphometric studie s in rodent s exposed to ethanol 

during development sugges t that regions of the brain 

are not equivalently affected b y ethanol. Eve n within 

a brain region vulnerable to ethanol-induced change s 

in dendriti c shape , th e spécifi e parameter s o f th e 

dendritic arbo r (e.g. , lengt h o f dendriti c segments , 

number o f primary dendrites, o r branching) ma y be 

differentially affected . 

The expans e o f dendritic arbor s is determined b y 

the numbe r an d lengt h o f it s primar y dendrite s 

(which exten d directl y fro m th e cel l body ) and den - 

dritic branches . I n youn g mic e expose d t o ethano l 

pre- and perinatally , pyramida l neurons i n th e CA 1 

region o f the hippocampu s hav e shorte r basa l den - 

drites, but th e lengt h o f apical dendrites is unaffected 

(Davies an d Smith , 1981 ; Smit h an d Davies , 1990) . 

Parallel results have been reported for the dendrites of 

pyramidal neurons i n the sensorimoto r cortex (Hammer 

and Scheibel , 1981 ; Fabregues et al., 1985) . The 

number o f primary dendrites an d th e exten t o f their 

branching are also decreased in other areas including 

chick spina l cor d serotonergi c neuron s (Mendelso n 

and Driskill , 1996) , rat dopaminergic neurons of the 

substantia nigra (Shett y et al., 1993) , and ra t cerebellar 

granul e neuron s (Smit h an d Davies , 1986) . Tha t 

ethanol may differentially affec t distinct parameters of 

dendritic morpholog y i s a potentiall y importan t ob - 

servation give n evidenc e tha t th e number , length , 

and branchin g o f dendrite s ma y b e independentl y 

regulated (Scott and Luo, 2001). 

Although man y report s emphasiz e th e inhibitor y 

effects o f prenata l ethano l exposur e o n th e siz e o f 

dendritic arbor s i n variou s regions o f immatur e ro - 

dent brain, studies indicate that ethano l ca n increase 

the siz e of dendritic arbors. For example, ethanol in - 

creases th e numbe r an d lengt h o f apical an d basilar 

dendritic branche s o f callosal projection neurons i n 

the ra t (Qiang et al., 2002). The stimulator y effec t of 

ethanol o n dendrite s appear s to involv e N-methyl-Daspartate 

(NMDA ) receptors , sinc e dendrite s are nor - 

mal i n ethanol-expose d transgenic mice with deletio n 

of the NR 1 subtype of NMDA receptor (Den g and El - 

berger, 2003). 

Notably, the dat a cite d abov e exemplif y effect s o f 

developmental exposur e t o ethano l observe d whe n 

neonatal or immature animals are examined. In some 

cases, however , whe n prenata l ethanol-expose d ani - 

mals ar e examine d a t other young ages, or as adults, 

areas that had stunted dendrite s are found to be either 

ETHANOL AND PROCESS OUTGROWTH 23 1 

normal (Pentne y e t al, 1984 ) o r hav e significantl y 

larger an d mor e comple x dendrite s tha n control s 

(Miller e t al. , 1990) . Thes e dat a ar e generall y be - 

lieved t o indicat e tha t ethanol-induce d reductio n i n 

dendritic arbo r siz e reflect s delayed dendriti c development 

o r that initial inhibition o f growth is followed 

by an abnormal compensatory growth response when 

the anima l is no longe r expose d to ethanol. Anothe r 

possibility i s tha t ethano l interfere s wit h dendriti c 

pruning, a proces s associate d wit h a glutamatergic - 

NMDA receptor mechanism (Kozlowsk i et al., 1997) . 

This concept i s supported b y evidenc e tha t ethano l 

can caus e axona l hypertrophy as well (se e Effect s o f 

Ethanol on Development of Axons). 

That dendriti c morpholog y coul d chang e s o significantly 

i s not surprising, given that dendrite development 

i s a highl y dynami c process . Dendriti c 

growth i n viv o is characterized by extensive remodeling, 

as exemplified by the postnata l retractio n o f the 

apical dendrite s o f some laye r V callosa l projection 

neurons, fro m cortica l layer I to layer IV (Koester and 

O'Leary, 1992) . Thus, assessment of prenatal ethanol 

effects o n dendrites ove r extended development , an d 

after withdrawa l fro m ethanol , i s important . Mor e 

studies are neede d to explor e the possibilit y tha t 

ethanol affects th e cellular event s whereby exuberan t 

early growth is followed b y partial pruning, or initially 

stunted developmen t i s later reverse d b y compensa - 

tory growth. 

Dendritic remodelin g ca n b e directl y visualize d 

by time-lapse imaging, as performed on neurons fro m 

frog opti c tectum, in which rapi d addition an d retrac - 

tion o f ne w branche s an d remodelin g o f existin g 

branches, a s well as the differentia l regulatio n of these 

events during maturation, has been documented (W u 

et al. , 1999) . I t i s now possibl e t o imag e neuron s i n 

complex environment s ove r extende d tim e period s 

(Danuser an d Waterman-Storer , 2003) . Suc h time - 

lapse studie s coul d hel p distinguis h whethe r ethano l 

delays o r alter s on e o r mor e feature s o f dendriti c 

growth in the intact developing brain (e.g., outgrowth, 

elongation, branching, or remodeling). 

In Vitro Studies 

Several neuron cultur e systems have been used to examine 

th e effect s o f ethanol o n developin g neurons. 

Some cell culture models are poorly suited to exploring 

effect s o f ethano l specificall y o n dendrite s o r 

axons. Fo r example , i n som e dissociate d cultures ,


(1) the neurons are too dense or there are various cell 

types present , limitin g bot h morphometri c an d mo - 

lecular analyses ; (2) th e neuron s d o no t surviv e o r 

develop long enough fo r researchers to investigate th e 

persistence o f ethano l effect s o r th e capacit y o f th e 

neuron to recover normal morphology, or (3) the neu - 

rons are not the type s sensitive to ethanol damage i n 

vivo. No t surprisingly , a n inconsisten t pictur e o f 

ethanol effect s o n neuromorphogenesis has emerged. 

Some report s sho w that ethano l inhibit s proces s out - 

growth (Do w an d Riopelle , 1985 ; Kentrot i an d Ver - 

nadakis, 1991 ; Heato n e t al. , 1994 ; Saunder s e t al. , 

1995; Beare r e t al. , 1999) , wherea s other s repor t a 

stimulatory effec t (Messin g e t al., 1991 ; Woote n an d 

Ewald, 1991 ; Roivaine n e t al , 1993 ; Zo u e t al , 

1993). The followin g discussion focuses on studies for 

which there is reasonable certaint y that the processe s 

being studied are either dendrites or axons. 

Unique characteristic s o f low-densit y culture s o f 

fetal ra t hippocampal pyramidal neurons make them 

a valuable model for examining the molecular mech - 

anism^) b y which ethano l interfere s wit h dendriti c 

and axonal growth. These cultures, detailed in Chapter 

4, are the mos t extensivel y characterized primar y 

culture of mammalian CNS neurons , and numerou s 

studies have been conducte d comparin g them t o pyramidal 

neuron s developin g i n sit u (Crai g an d 

Banker, 1994 ; Gosli n e t al, 1998) . Neurons in thes e 

cultures ar e nearl y homogeneous ; typically 94% ar e 

readily distinguished as pyramidal neurons. They live 

for u p t o 4 weeks , undergoin g stereotypical , nearly 

synchronous development, each neuron havin g a single 

axo n an d severa l dendrites of well-defined shap e 

and characteristi c molecula r constituents . Th e cul - 

tured neuron s for m synapti c relationship s tha t ar e 

representative o f those normall y present i n th e hip - 

pocampus (e.g. , Schaffe r collateral s interconnectin g 

pyramidal neurons). This pattern parallels hippocampal 

development i n situ, which i s largely complete by 

the en d o f th e thir d postnata l wee k (Minkwit z an d 

Holz, 1975). Moreover, the developmental progression 

that occurs during the elaboratio n o f axons, dendrites, 

and synapse s can b e readil y quantified by time-laps e 

microscopy o f individual neurons (Dott i e t al. , 1988 ; 

Goslin and Banker, 1989). 

Some of the inhibitory effects of ethanol on dendrite 

development ar e supporte d b y results from studie s using 

these cultures, in which definitive dendrites can be 

identified b y their characteristi c proximal-dista l tape r 

and b y immunolocalization o f microtubule-associated 

protein (MAP ) 2, a protein distribute d in the somato - 

dendritic compartment (Gosli n e t al., 1998) . I n hip - 

pocampal cultures , quantitativ e morphometri c 

analyses of MAP2-stained, pyramidal neuron s 6 days 

in vitro (DIV) , expose d continuousl y t o 43-8 7 mM 

ethanol, sho w concentration-dependent decrease s i n 

total dendritic length , in the number o f primary den - 

drites and dendritic branches, and in the length o f individual 

dendrite s (Yann i an d Lindsley , 2000 ; Yanni 

et al. , 2002) . Ethano l als o decrease s th e numbe r o f 

synapses formed by these neuron s (Yann i and Linds - 

ley, 2000) . Nevertheless , synapti c densit y i s no t af - 

fected. Hence , i t i s likel y secondar y t o effect s o n 

dendrites an d not a direct effect o n synapse formation 

per se. Delaying the additio n of ethanol unti l 1 DIV, 

when mos t neuron s hav e develope d a n axon , results 

in significantl y shorter, less-branche d dendrite s a t 6 

DIV, relative to the neurons exposed to ethanol a t the 

time of plating (Lindsley et al., 2002). Thus the maturity 

of hippocampal neuron s influences their vulnerability 

to morphoregulatory effects o f ethanol. 

The duratio n o f ethanol exposur e and th e timin g 

of withdrawal have variable effects o n dendrite development 

i n hippocampa l culture s (Lindsle y an d 

Clarke, 2004). When ethanol is added to the mediu m 

shortly after plating , continuous exposure for up t o 14 

DIV decrease s th e lengt h an d numbe r o f dendrite s 

formed, but it has no effect on neuron survival. When 

ethanol exposur e is limited to the first day post-plating 

and withdrawa l occurs before dendrites elongate , in - 

dividual dendrites achieve normal length b y 1 4 DIV. 

The arbors , however , ar e stil l smalle r tha n control s 

because o f a persisten t reduction i n th e numbe r o f 

primary dendrites. Thes e findings are consistent wit h 

in vivo observations that inhibitory effects o f prenatal 

ethanol o n dendriti c lengt h ma y no t endur e afte r 

withdrawal from ethanol (Pentne y et al., 1984; Lopez- 

Tejero e t al., 1986) , but th e effec t o n th e numbe r o f 

primary dendrites may persist even after limited expo - 

sure to ethanol (Duran d et al, 1989). 

The additio n of ethanol at any time point prior to 6 

DIV does not affec t neuro n survival . In contrast, addi - 

tion of ethanol on Day 6 (after rapid growth of dendrites 

and synapses has begun) triggers cell death, despite th e 

shorter duratio n o f ethanol exposur e (Lindsle y e t al. , 

2002). Developmenta l stage-dependen t sensitivit y to 

ethanol-induced cel l deat h ha s also been reporte d fo r 

cerebellar neuron s in vivo and i n vitro (Goodlet t an d 

Johnson, 1999 ) and may involve the nitric oxide-cyclic 

guanosine 5 / -nionophosphate-dependent protei n

kinase pathwa y (Bonthiu s e t al, 2004) . Elucidatin g 

the mechanisms by which the timing of ethanol exposure 

influences the nature and severity of its effects o n 

dendrites and o n neurona l surviva l is a goal o f ongoing 

research. 

EFFECTS O F ETHANOL ON 

DEVELOPMENT O F AXONS 


The effec t o f prenatal ethano l o n axona l growt h i n 

various brain regions is less well characterized than its 

effects o n dendrites . I t i s mos t ofte n reporte d a s a n 

overall hypertroph y o f axon s fro m projectio n neu - 

rons, such a s those comprisin g the corticospina l trac t 

and th e moss y fiber tract i n th e hippocampus . Thi s 

hypertrophie effec t o f ethano l i s no t a s consistentl y 

observed i n axo n tract s that cros s the midlin e i n th e 

brain, such a s the corpus callosum an d anterior com- 

Axons of Projection Neurons 

Prenatal exposur e t o ethano l markedl y alter s th e 

number an d distributio n of axons of projection neu - 

rons in various brain regions. I n mos t cases , change s 

in the number of axons cannot be attributed to corresponding 

change s i n th e numbe r o f neurons i n th e 

brain region fro m whic h th e tract extends. Fo r example, 

prenatal exposure to ethanol increase s the num - 

ber o f neuron s projectin g fro m ra t somatosensor y 

cortex to the spina l cord (Miller , 1987) , th e numbe r 

of axons in the cauda l pyramidal tract (Miller and Al - 

Rabiai, 1994) , an d th e tota l axoplasmi c volum e i n 

layer V o f the somatosensor y corte x (Al-Rabia i an d 

Miller, 1989) . Although the absolute number of corticospinal 

neuron s i s no t affected , becaus e th e tota l 

number of neurons in layer V is reduced by one-third 

(Miller an d Potempa , 1990) , th e relativ e numbe r i s 

substantially increased . I t ha s bee n suggeste d tha t 

these exuberan t projections are persistent, immature 

connections an d ma y indicat e tha t ethano l inhibit s 

the prunin g of axons that normally refines ove r time 

(Miller, 1987) . 

A hypertrophi e axona l projectio n ha s bee n ob - 

served i n axon s extendin g fro m granule cell s i n th e 

dentate gym s (moss y fibers ) t o apica l dendrite s o f 

the pyramida l neuron s i n th e CA 3 regio n o f th e 


hippocampus. Adul t rats prenatally exposed to ethano l 

have a conspicuous abnorma l ban d of infrapyramida l 

mossy fibers in hippocampal subfiel d CA3 a a t middle 

and temporal levels (West et al., 1981). In addition, th e 

band o f suprapyamidal mossy fibers in the stratu m lucidum 

is disorganized. This exposure period coincide s 

with th e birt h o f pyramidal neurons i n CA3 , but i t is 

before most granule cell s or their axons appear. Therefore, 

it has been postulated that alterations in the terminal 

field induce hypertrophie growt h of mossy fibers. 

As with the corticospina l tract, moss y fibers in th e 

hippocampus underg o stereotype d prunin g o f axonal 

branches tha t sculpts the connections int o the matur e 

pattern. Althoug h th e mechanism s controllin g mossy 

fiber axon pruning are not fully understood, recen t evidence 

points to a role for the repulsive guidance mole - 

cule Sema 3 F (Bagri et al., 2003). There are intriguing 

similarities between the abnormal mossy fiber trajectories 

of prenatal ethanol-exposed rats and mice deficient 

in the axon guidance molecule semaphorin 3F (Sahay 

et al. , 2003 ) o r i n member s o f th e holorecepto r fo r 

semaphorins, neuropilin- 2 (Che n e t al. , 2000 ) an d 

plexin A3 (Cheng et al., 2001). It is appealing to speculate 

that ethanol interferenc e with mossy fiber pruning 

is mediated by altered semaphorin signaling. 

Axons That Cross the Midline 

During brai n development , axon s fro m specifi c re - 

gions within each cerebral hemisphere ar e guided by 

extracellular signal s t o gro w acros s th e midlin e t o 

synapse on neurons in the opposite hemisphere. Th e 

largest axo n trac t crossin g the midlin e i s the corpu s 

callosum. I t consist s of axons projecting to th e con - 

tralateral cortex , thereby allowin g sensory and moto r 

integration betwee n th e lef t an d righ t side s o f th e 

brain. A higher than normal incidenc e o f corpus cal - 

losum abnormalities has been reported for individuals 

with feta l alcoho l syndrom e (FAS ) (Rile y e t al , 

1995). 

Magnetic resonanc e imagin g an d autops y stud - 

ies i n human s expose d prenatall y t o ethano l sho w 

that ethano l decrease s the cross-sectiona l area of the 

corpus callosum, especiall y the posterior region (sple - 

nium), an d displace s i t relativ e to othe r brai n structures 

(e.g. , Clarre n e t al, 1978 ; Peife r e t al, 1979 ; 

Pratt an d Doshi , 1984 ; Schaefe r e t al, 1991 ; Rile y 

et al, 1995; Roebuck et al, 1998 ; Swayz e et al., 1997 ; 

Sowell et al., 2001). In contrast, prenatal ethanol ca n 

increase th e siz e of the corpu s callosu m i n childre n


prenatally exposed t o ethanol (Bookstein et al, 2002) 

and i n a nonhuma n primat e (Macaca nemestrina) 

(Miller et al, 1999) . Indeed, th e tota l numbe r o f axons 

i s increased. The mos t labile segment i s the ros - 

tral portion (genii) , without affecting th e thicknes s of 

axons or their myelin sheaths. The difference s i n th e 

primates may reflect concentration-dependent effect s 

of ethanol: at lower exposures, ethanol leads to hypertrophic 

effects , wherea s at highe r exposure s i t i s full y 

depressive. Result s in roden t model s ar e similarl y in - 

consistent, with some studies showing ethanol-induced 

decreases in various measures of corpus callosum size 

(Chernoff, 1977 ; Zimmerber g an d Mickus , 1990 ; 

Moreland e t al., 2002) and other s showing no effec t 

(Livy and Elberger , 2001 ) or an increase i n the num - 

ber of callosal projection neurons (Miller, 1997). 

Noncallosal axo n tracts that cros s the midlin e are 

vulnerable to disruption by ethanol. Prolonged expo - 

sure to relatively high doses of alcohol can reduce th e 

size o f th e hippocampa l commissur e (Liv y an d El - 

berger, 2001) . Th e midsagitta l are a o f th e anterio r 

commissure i s reduced b y prenatal ethanol exposure 

in some inbred mic e (Cassell s et al., 1987) , however , 

this effec t ha s no t bee n observe d i n studie s o f rat s 

(Zimmerberg an d Mickus , 1990 ; Liv y and Elberger , 

2001). Granato et al. (1995) reported reduce d terminal 

axona l arbor s o f corticothalami c projections , i n 

which th e mos t severe damage i s in th e selectiv e absence 

o f corticothalami c projection s tha t cros s th e 

midline, a characteristi c o f ra t corticothalami c sys - 

tems that is not seen i n humans. 

Some o f thes e inconsistencie s ma y reflec t differ - 

ences in species, strain differences i n the developmen - 

tal sensitivit y of commissural axons, differences i n th e 

dose o r timin g o f ethano l administration , o r othe r 

méthodologie factors (Miller et al, 1999). In any case, 

the susceptibilit y o f crossin g fiber s t o disruptio n b y 

ethanol has some important mechanistic implications 

because the midlin e is a unique developmenta l field 

where the response of axons to attractive and repulsive 

cues i s finely regulated b y precise molecula r mecha - 

nisms no w bein g identifie d (Tessier-Lavign e an d 

Goodman, 1996 ; Hube r et al, 2003). 

In Vitro Studies 

Partial t o complet e agenesi s o f the corpu s callosum , 

as seen i n FAS, is also common i n people wit h mutations 

in LI, the so-called CRAS H syndrome (Franse n 

et al, 1995 ; Bearer , 2001) . Thi s findin g le d t o th e 

hypothesis tha t disruptio n o f L I functio n ma y con - 

tribute t o neuropathologic effect s o f prenatal ethano l 

exposure (Charnes s e t al. , 1994) . L I i s a neura l cell 

adhesion molecul e expresse d o n axons that facilitates 

axon growt h throug h bot h heterophili c an d ho - 

mophilic binding (Brummendorf and Rathjen, 1996) . 

Some i n vitr o evidence is consistent with ethanol 

disruption of Ll-mediated axon outgrowth from CN S 

neurons. Ra t cerebellar granule neuron culture s extend 

neunte s whe n culture d o n substrate s coate d 

with LI. Ethano l inhibit s Ll-mediated outgrowt h of 

the longes t neurit e forme d 1 2 hr afte r platin g (pre - 

sumably the axon) in these cultures, even at concen - 

trations a s lo w a s 3- 5 mM (Beare r e t al. , 1999) . 

Apparently, thi s i s no t du e t o inhibitio n o f Ll - 

mediated adhesio n pe r se, because th e sam e effec t of 

ethanol on axon growth is observed when the cells are 

plated o n poly-lysine substrates and treated wit h a soluble 

form o f the extracellula r domain of LI. This result 

wa s take n t o indicat e tha t ethano l disrupt s 

signaling downstream of LI tha t influences organization 

of the growth cone cytoskeleton. 

There i s growin g evidence fro m i n vitro experiments 

tha t second messenge r cascade s and cytoskele - 

tal reorganization are triggered by LI, either directly 

or vi a it s interaction s wit h co-receptor s (Burden - 

Gulley et al., 1997 ; Castellani et al, 2000; Watanabe 

et al, 2004). Other subsets of axons in the developin g 

CNS expres s LI i n high amounts, including pyramidal 

trac t axon s (Jooste n an d Gribnau , 1989) , whic h 

are sensitiv e t o ethano l disruptio n durin g develop - 

ment (se e Axon s o f Projectio n Neurons , above) . 

Thus, elucidating the mechanisms by which ethano l 

disrupts LI regulate d axo n growth is an importan t focus 

o f future studies . Various studies have shown that 

LI i s but on e o f many cell adhesion proteins affecte d 

by ethano l (Lu o an d Miller , 1999 ; Mille r an d Luo , 

2002; Siegenthaler an d Miller, 2004). These proteins 

are regulate d by factors suc h a s transforming growth 

factor ßl , whic h i s known t o promot e neurit e out - 

growth (Ishihara et al., 1994) and i s a target of ethanol 

(e.g., Lu o an d Miller , 1999 ; Mille r an d Luo , 2002 ; 

Siegenthaler and Miller, 2004). 

Although studie s in vivo have not focuse d o n th e 

effects o f ethanol on early stages of process outgrowth, 

some in vitro studies point to effects o f ethanol o n th e 

timing of initial axon outgrowth (axo n specification). 

In culture s o f hippocampal pyramida l neurons , th e

proportion o f neurons tha t reac h Stag e 2 (extende d 

neuntes, bu t befor e axo n specification ) an d th e 

proportion that reach Stag e 3 (axon specification) are 

both increased when ethanol is added to the medium, 

beginning shortly after platin g (Clam p an d Lindsley , 

1998). Subsequen t time-laps e studies, which includ e 

only cells that achieved Stag e 3 , show that ethanol delays 

axonal outgrowth (Lindsley et al., 2003). Both observations 

indicate that the increas e in the proportion 

of cell s wit h undifferentiate d neunte s i s responsibl e 

for th e fina l increas e in th e proportio n o f polarized 

cells at 1 DIV, and that ethanol ma y increase the likelihood 

of forming an axon even as it decreases the rate 

at which this occurs. 

Some caution is required when interpreting studies 

of ethanol effect s o n proces s outgrowt h tha t compar e 

neurite morpholog y a t a selecte d tim e afte r plating . 

Differences i n th e lengt h o f processes measure d a t a 

single time poin t d o not tak e int o accoun t effect s o n 

the time it takes for a neuron to initiate axon extension 

and th e rat e of elongation onc e the axo n is formed. A 

time cours e o f ethanol effect s durin g initial stage s of 

neurite outgrowth, or time-lapse analyse s on live cells, 

is needed to make this distinction. 

An even higher-resolution tim e cours e i s required 

to asses s the effect s o f ethano l o n th e rat e o f axon 

growth afte r a n axo n emerges . Thi s i s because onc e 

axons ar e formed , the y gro w i n a saltator y manner 

characterized b y cyclin g betwee n period s o f growth 

(when length increases ) and periods of relative quiescence 

("non-growth" ; whe n shor t retractio n occurs ) 

(Fig. 14-1) . Molecula r an d pharmacologi e treat - 

ments ca n selectivel y alter specific aspects of process 

outgrowth, suc h a s the relativ e duration o f periods of 

growth, the timing of axon establishment, o r the mag - 

nitude o f retraction durin g pauses (Esc h e t al., 1999 ; 

Ruthel and Banker , 1999 ; She a an d Beerman , 1999 ; 

Walker et al, 2001; Lindsley et al., 2003). These findings 

suggest that these feature s of process growt h dynamics 

ar e independentl y regulate d unde r norma l 

conditions an d coul d b e differentiall y sensitiv e t o 

ethanol. 

Time-lapse analysi s of living neurons ha s demon - 

strated saltator y axonal growt h i n dissociate d culture s 

(Katz et al., 1984; Aletta and Greene, 1988; Ruthe l and 

Banker, 1999 ; Lindsle y et al., 2003), tissue slices (Halloran 

and Kalil , 1994) , and the intac t brain (Kaethner 

and Stuermer , 1992) . I n som e axons , suc h a s thos e 

comprising th e corpu s callosum , thi s ca n b e quit e 


FIGURE 14- 1 Axona l growt h o f a hippocampa l py - 

ramidal neuron . Phase-contras t image s o f a ra t hip - 

pocampal pyramida l neuron , capture d a t variou s 

times postplating (shown in hr). The dista l ti p of the 

axon advance s and retract s during elongation. Scal e 

bar= IS |im. 

dramatic, with extensive lengths o f axon retracting or 

sprouting rapidl y (Dent e t al., 1999) . Dendrite s als o 

undergo significant retraction s during outgrowth an d 

remodeling, a s demonstrate d b y withdrawa l o f th e 

apical dendrite from subset s of pyramidal neurons i n 

the developin g corte x (Koeste r an d O'Leary , 1992) . 

Aside from affectin g ne t growt h rate, saltatory growth 

is associated with othe r importan t feature s o f neural 

development, includin g branc h formatio n (Szebeny i 

et al. , 2001) , branc h growt h alternatio n (Futerma n 

and Banker , 1996) , an d growt h cone responses to attractive 

and repulsiv e guidance cue s (Son g and Poo , 

1999; Ming et al, 2002). 

Time-lapse analysis of the effect s o f ethanol on th e 

saltatory growt h dynamic s i n hippocampa l culture s 

shows that ethanol increase s th e overal l rate o f axon 

elongation b y decreasin g th e exten t o f retraction s


during pauses i n growth . Ethano l doe s no t increas e 

the rat e of extension durin g growth spurts or the rela - 

tive time axons spend growing and pausin g (Lindsley 

et al., 2003). 

MECHANISMS UNDERLYING 

ETHANOL-INDUCED CHANGE S 

IN DENDRITIC AN D 

AXONAL MORPHOLOGY 

As described above, in vivo and i n vitro data indicate 

that specifi c morphologi c feature s that mak e u p th e 

overall shape o f dendrites an d specifi c features of axonal 

growth dynamics may not be equally affected b y 

ethanol. The y als o indicat e tha t th e morphoregula - 

tory effects o f ethanol ma y be correlated with the ear - 

liest stage s o f proces s outgrowth . Mor e studie s ar e 

needed t o identif y th e molecula r basi s for such spe - 

cific vulnerabilit y to ethanol-induce d damage . Ad - 

vances i n understandin g ke y cellula r regulator s o f 

growth con e motilit y provid e ne w opportunitie s t o 

speculate o n ho w ethanol exposur e migh t modulat e 

morphogenesis o f axons and dendrite s during devel - 

opment. Tw o of these emergin g idea s ar e discusse d 

below. 

Small Rho Guanosine Triphosphatases 

Although regulator y mechanism s underlyin g den - 

dritic remodeling ar e not fully understood , the mole - 

cules an d signal s involve d ar e though t t o influenc e 

the actin and microtubule cytoskeleton, sinc e signifi - 

cant cytoskeleta l rearrangemen t occurs . Th e Rh o 

family, smal l guanosin e triphosphatase s (GTPases) , 

Rac, Cdc42, and RhoA , are essentia l component s o f 

signaling pathways that transduce a variety of extracellular 

signal s (e.g., neurotrophin s an d integrins ) int o 

morphological changes . Rh o guanosin e S'-triphos - 

phatases (GTPases) d o this by regulating downstream 

kinases tha t alte r th e activit y o f acti n an d tubuli n 

binding protein s involve d i n actin-base d cytoskeleta l 

remodeling i n growt h cone s (Lu o e t al. , 1997 ; 

Mackay an d Hall , 1998 ; Luo , 2000 ; Redmon d an d 

Ghosh, 2001; Lundquist, 2003). 

Different member s o f Rho-famil y GTPase s regu - 

late distinct features of dendritic development, including 

initia l outgrowth, rat e o f growth , numbe r o f 

primary dendrites, and extent of branching (Redmond 

and Ghosh, 2001). For example, results of experimentally 

altere d Rac l o r Cdc4 2 expressio n i n cortica l 

neurons indicate s tha t thes e protein s ar e importan t 

for dendritic branching and remodeling, but that they 

have littl e effec t o n dendriti c growt h rat e pe r s e 

(Threadgill e t al, 1997 ; L i et al, 2000). I n contrast , 

overexpression o r activatio n o f Rho A inhibit s den - 

dritic growt h an d underexpressio n o r inactivatio n o f 

RhoA lead s t o a n increas e i n dendriti c lengt h (Le e 

et al, 2000; Li et al, 2000). 

Studies focused on the cellular mechanisms regu - 

lating extension and retractio n behavior of axons during 

growth an d i n respons e t o guidance factor s (e.g., 

semaphorins) als o point t o Rho famil y GTPase s an d 

their downstrea m effector s (Lu o e t al. , 1997 ; Küh n 

et al, 2000; Luo, 2000 ; Redmond an d Ghosh, 2001). 

In general , proces s extensio n i s correlate d wit h in - 

creased formation of lamellipodia and filopodia at the 

leading edge o f the growth cone, which require s acti - 

vation o f Cdc42 and Racl , wherea s retraction i s correlated 

wit h increase d Rho A signalin g (Hall , 1998 ; 

Luo, 2000). 

Two studies show that ethanol ca n dramatically rearrange 

F-actin structure s in some non-neuronal cells 

and that activation of small Rho GTPases may mediate 

these effects. Ethanol induce s actin reorganization and 

shape chang e i n astrocyte s in vitro, and i t has bee n 

suggested tha t these effect s are mediated b y decreased 

RhoA activity, since they could be reverted by expression 

o f constitutivel y activ e Rho A (Guasc h e t al. , 

2003). In the endothelial cell line, SVEC4-10, ethanol 

induces actin reorganization and motility. Since thes e 

effects ca n be blocked b y expression of dominant neg - 

ative Cdc42, it has been suggested tha t they are mediated 

b y activation of Cdc42 (Qia n e t al, 2003) . O n 

the othe r hand , the effec t o f ethanol o n F-actin distri - 

bution in the growth cones of developing neurons has 

not been reported . Som e preliminary data are consistent 

wit h altere d F-acti n organizatio n i n axona l 

growth cones of hippocampal neurons maintained for 

1 DIVwith ethanol (Sha h and Lindsley, unpublished 

observations). Figure 14- 2 contain s example s of confocal 

image s showing F-actin rich structures in axonal 

growth cone s o f contro l an d ethanol-treate d hip - 

pocampal neuron s 1 DIV that ar e fixed and staine d 

with phalloidin-Alex a 568 . Continuou s exposur e t o 

low doses of ethanol induce s extensive elaboration of 

lamellipodia an d numerou s thickene d filopodi a i n 

axonal growt h cones , an d hig h dose s onl y increas e

ETHANOL AND PROCESS OUTGROWTH 237 

FIGURE 14- 2 Effec t o f ethanol o n F-acti n distribution i n growth cones. Repre - 

sentative confoca l image s o f F-acti n distributio n i n axona l growt h cone s o f 

1-day-old hippocampa l neuron s culture d fo r 1 day i n vitr o withou t o r wit h 

ethanol (concentration s shown) . Images were captured a t 0.20 \im intervals using 

8-image jump averaging. Each imag e i s the lowest section fro m th e z-serie s 

stack. The lo w dose of ethanol increase d the exten t of lamellipodia an d filopodia 

compare d t o controls , an d th e hig h dos e increase d filopodi a only . Scal e 

bar= 5.0 Jim. 

filopodia formation . Moreover , th e involvemen t o f 

small GTPase s i n reorganizatio n o f growt h con e 

actin i n neuron s expose d t o ethano l i s indicate d 

by pull-down experiment s showin g change s i n Rac l 

activation i n whole-cel l lysate s o f hippocampa l 

cultures expose d t o ethano l fo r 1 da y durin g axo n 

elongation. 

Important advance s i n understandin g th e effect s 

of ethano l o n growth-relate d signalin g i n axon s an d 

dendrites ca n b e expected , give n th e availabilit y o f 

appropriate mode l system s amenabl e t o measurin g 

the effect s o f expressio n o f relevan t dominant - 

negative or constitutively active mutant Rh o GTPases 

on ethanol-induce d disruptio n o f morphogenesis . 

Interestingly, many o f the signalin g pathway s known 

to contro l growt h con e extension-retractio n an d at - 

traction-repulsion behavior s appea r t o b e differen - 

tially regulate d i n axon s an d dendrites . Som e 

evidence suggests that Rh o GTPase s ar e differentiall y 

distributed i n growt h cone s (Renaudi n e t al. , 1999) , 

and experimenta l manipulations t o over- or underexpress 

Rho-famil y protein s hav e differentia l effect s o n 

the number and length o f dendrites compared to their 

effects o n axona l growth (Threadgil l e t al., 1997 ; Lee 

et al., 2000; Luo, 2000). Thus, if Rho-family GTPase 

modulation i s indeed involve d in the morphoregula - 

tory effect s o f ethanol, the n differentia l effect s o f th e 

various Rho-famil y member s o n axona l vs . dendritic 

effects ma y help explain the various effects o f ethanol 

on these distinct process types. 

If ethanol-induce d change s i n growt h dynamic s 

and/or cytoskeleta l reorganizatio n d o indee d involv e 

altered GTPas e signaling , then on e coul d as k what 

upstream modulator s o f Rho GTPases are particularly 

important i n morphoregulator y effect s o f ethanol. I n 

NIH 3T 3 cells , growt h factor-induce d Rac l activa - 

tion i s sensitive to protein kinase C (PKC ) inhibition , 

placing PK C upstrea m o f Ra c i n tha t syste m 

(Buchanan e t al. , 2000) . Th e relationshi p betwee n 

Rho GTPase signaling and PKC s i s potentially quit e 

interesting. I n P C 12 cells, exposur e t o 25-20 0 mM 

ethanol fo r 2-8 day s stimulates neurite growth via upregulation 

o f PKCe (Messin g e t al, 1991 ; Hundle 

étal, 1997) . 

What rol e might downstrea m effector s o f Rho GT - 

Pases have in mediating th e effect s o f ethanol o n neuronal 

proces s growth ? Amon g a numbe r o f know n 

effectors, severa l are of particular interest. In hippocam - 

pal neuron cultures, axon specification is linked to activation 

of Cdc42 and Ra c 1 in a positive feedback loo p 

involving a highly localized comple x o f mPar3/mPar6, 

PKC, an d phosphoinositid e 3 kinas e (PI3K) , whic h 

modulates actin and microtubule cytoskeletal dynamics 

in th e growt h con e o f the nascen t axo n (Sh i et al. , 

2003). It is not yet clear, however, whether PI3K initiates 

signals that activate Racl/Cdc42 downstrea m or 

whether Racl/Cdc42 are upstream signals for PI 3K in 

this cascade . Othe r know n downstrea m effector s o f 

potential interes t include acti n cytoskeleton-regulating 

proteins, suc h a s ezrin , radixin , an d moesin , an d


regulators of mitogen-activated protein kinase pathways 

(Hall, 1998 ; Redmond and Ghosh, 2001). 

Some insigh t into th e regulatio n o f neurite out - 

growth may come from studies of neuronal migration , 

as commo n protein s ar e involve d i n bot h processe s 

(see Chapters 3 and 13) . Reduced F-acti n at the lead - 

ing edg e o f migratin g neuron s i s associate d wit h 

modulation of RhoA, Racl, and Cdc42 activity (Kholmanskikh 

et al., 2003). Since abnorma l neurona l mi - 

gration i s a ke y neuropathologi c featur e o f prenatal 

alcohol exposur e (Miller , 1986 , 1993 ; Hira i e t al , 

1999; Moone y et al, 2004 ; Siegenthale r an d Miller , 

2004), a n understandin g o f the effect s o f ethanol o n 

Rho GTPase signaling in neurons may shed ligh t on 

cellular mechanisms contributin g to prenatal ethanol - 

induced neuronal migration errors. 

Intracellular Calcium 

Results o f numerous studie s o f the molecula r path - 

ways linkin g activation o f specifi c Rho-famil y pro - 

teins and their effectors poin t to involvement of Ca 2+ 

influx (Küh n e t al, 1998) . Thus i t i s likely that effects 

o f ethano l o n dynami c growt h con e behavio r 

might ultimatel y be linke d mechanisticall y throug h 

Rho-family effector s actin g o n th e growt h con e cytoskeleton, 

eithe r directl y o r indirectly , t o Ca 2+ sig - 

naling. 

It ha s lon g bee n appreciate d tha t intracellula r 

Ca 2+ i s a critica l second messenge r tha t modulate s 

growth cone behavior during neuronal developmen t 

by communicating growth-related signal s to th e cy - 

toskeleton an d vesicula r apparatu s o f th e growt h 

cone (Kate r an d Mills , 1991 ; Letournea u an d 

Cypher, 1991 ; Neel y and Nicholls , 1995) . Ethano l 

can alter the function and expression of L-type Ca 2+ 

channels in neural cells (Walter and Messing , 1999) . 

The effect s o f ethanol o n Ca 2+ signaling i n neuron s 

have focused primarily on mature neurons, in which 

Ca 2+ channels an d receptors figur e importantl y int o 

cellular adaptation s t o ethano l an d t o withdrawalinduced 

neurona l damag e (Fadd a an d Rossetti , 

1998). Ethano l affect s Ca 2+ homeostasi s i n matur e 

neurons b y alterin g th e expressio n o r functio n o f 

voltage-dependent Ca 2+ channel s (VDCCs) , iono - 

tropic receptors, and Ca 2+ -induced Ca 2+ release from 

intracellular stores (Leslie et al., 1990 ; Bergamasch i et 

al., 1993) . Unfortunately, far fewer studie s have pursued 

th e ide a tha t ethano l disrupt s Ca 2+ signalin g 

regulating neuronal development. The potential importance 

o f this distinction i s suggested b y evidenc e 

that th e effec t o f ethano l o n VDCC s ma y no t b e 

constant durin g neurona l differentiatio n (Berga - 

mashi et al, 1995 ; Webb e t al., 1997) . 

The "set-poin t hypothesis " propose s tha t proces s 

growth occur s within a narrow range o f intracellular 

Ca 2+ concentratio n [Ca 2+ ]j, an d tha t sustaine d in - 

creases o r decrease s i n [Ca 2 " 1 "^ lea d t o growt h con e 

collapse and/or cell damage (Kate r and Mills , 1991) . 

In contrast , oscillation s o r transien t increase s i n 

[Ca 2+ ]j are associated with signaling induced by effec - 

tor pathways, similar to those though t t o be involve d 

in effect s o f ethanol o n process growth. Some o f these 

transient Ca 2+ signals are highly localized. For example, 

growt h cone s generat e transien t increase s i n 

[Ca 2+ ]j as they migrat e i n viv o (Gome z an d Spitzer , 

1999) an d i n vitro (Gome z e t al, 1995 ; Ciccolin i 

et al, 2003; Tang et al, 2003). The frequencie s and 

amplitudes of these [Ca 2+ ]j transients are inversely related 

t o th e rat e o f axo n elongatio n an d increas e 

several-fold whe n axons pause during periods o f nongrowth. 

Thes e transient s ar e likel y t o alte r growt h 

cone extension b y reorganizing the cytoskeleton , perhaps 

b y disassemblin g local acti n an d microtubul e 

networks. I n cortica l neurons , thes e transient s ar e 

spontaneous an d Ca 2+ -dependent an d ac t primarily 

through L-typ e VDCCs (Tang et al, 2003). Signaling 

to the cytoskeleton appears , at least in part, to involv e 

the phosphatase calcineri n (Lautermilch and Spitzer , 

2000). 

The result s o f time-series , confoca l imagin g o f 

Ca 2+ flux in axon s o f hippocampal neuron s expose d 

to ethanol unde r condition s tha t alte r axonal growth 

dynamics hav e bee n recentl y reporte d (Gilli s an d 

Lindsley, 2004) . I n thes e studies , th e frequenc y and 

kinetics o f spontaneous [Ca 2+ ]j transient s i n axona l 

growth cone s o f hippocampal neuron s a t 1 DIV i n 

control mediu m wer e compared t o those o f neurons 

maintained i n mediu m t o which ethano l (22 , 43, or 

87 mM) was added a t the time of plating. Figure 14- 3 

illustrates typical time-series image s o f axonal growt h 

cones from contro l and ethanol-exposed cultures, and 

corresponding plot s o f relative fluorescenc e intensit y 

in th e subscribe d regio n o f the growt h con e ove r a 

10 min recordin g period . A s describe d b y Gome z 

and colleague s (1995) , a transient i s defined b y a n 

increase in [Ca 2+ ]i of >200% of baseline, slow kinetics 

(~20 sec to peak), and return t o baseline.


FIGURE 14- 3 Time-serie s confoca l image s of axona l growt h cones . The 

growth cone s o f 1-day-ol d neuron s wer e loade d wit h Fluo-3 . A growth con e 

from a contro l cultur e (to p row ) and one fro m a culture expose d to 87 mM 

ethanol (botto m row ) are shown . Fo r each growth cone tw o images from th e 

time serie s are presented, one depictin g baselin e fluorescenc e (left) , an d th e 

other, peak fluorescence during a spontaneous calcium transient (right). Peak 

calcium elevation s are imaged i n white. The plot s on the righ t show the rela - 

tive fluorescenc e intensit y as a percent of baseline for the growt h cones in th e 

panels (left). Scal e bar =5.0 iim. 

Results of recording more than 10 0 neurons from 

10 cultures showed that only 18 % of axons in contro l 

cultures exhibited one o r more transients during the 

10 min recordin g period . Exposur e t o lo w dose s o f 

ethanol reduce d th e numbe r of neurons with one o r 

more transients to 8% (p < 0.005), but exposur e to the 

highest dos e o f ethanol increase d th e proportio n o f 

growth cone s wit h transient s t o 39 % (p < 0.005) . 

Ethanol, at any concentration tested , had no effect on 

the peak amplitude, time to peak, or total duration of 

the transients . Thes e finding s sugges t tha t ethano l 

modulates th e frequenc y of spontaneous Ca 2+ transients 

i n axona l growt h cones . I t i s yet t o b e deter - 

mined whether dendritic growth dynamics and Ca 2+ 

transients ar e similarl y altere d b y ethanol . I n hip - 

pocampal cultures , a globa l increas e i n [Ca 2+ ]j ca n 

inhibit dendritic growth but promotes or has no effec t 

on axona l growt h (Mill s an d Kater , 1990 ; Mattso n 

et al, 1988 ; Mattso n an d Kater , 1987) . Thi s finding 

indicates potentia l difference s i n Ca 2+ -dependent, 

growth-related signaling in axons and dendrites . Th e 

modulation o f transient Ca 2+ flux in the growth cone 

could be an important cellular target of ethanol that 

mediates it s differentia l effect s o n axon s an d den - 

drites, considerin g the sensitivit y o f growth cone s t o 

elevated fre e Ca 2+ and th e numbe r o f Ca 2+ -activated 

proteins expresse d differentiall y b y axon s an d den - 

drites. 


Descriptive studie s detailin g specifi c morphologica l 

features of neuronal development that are sensitive to 

disruption by ethanol in a variety of neuronal cell cultures 

are essential in laying the groundwork for identifying 

cellula r mechanism s underlyin g th e effect s o f 

ethanol o n neuronal morphogenesis . Man y ethanol - 

sensitive developmenta l phenomen a discovere d s o 

far—the altere d growt h dynamic s and stage-specific 

effects o f ethanol — could not have been so readily derived 

from fixed sections taken fro m animal s exposed 

to ethanol in utero. Neuronal development in vivo involves 

a diversity of interactions between neuron s and 

their environmen t tha t experimenta l condition s i n 

vitro cannot fully reproduce. Therefore, a n importan t 

goal for the future should be to test specific mechanistic 

hypothese s arisin g from cel l cultur e experiment s 

in mode l system s tha t preserv e th e endogenou s 

spatiotemporal expressio n o f extracellula r growth - 

regulating molecules. Such model systems might provide 

opportunitie s t o manipulat e th e expressio n o f


specific molecule s throug h th e us e o f geneticall y 

modified animal s an d optica l method s tha t enabl e 

the visualizatio n o f axon s an d dendrite s growin g 

in situ. 

Abbreviations 

[Ca 2+ ]j intracellula r concentration of Ca 2+ 

CNS centra l nervous syste m 

DIV day s in vitro 

FAS feta l alcoho l syndrom e 

GTPase guanosin e 5'-triphosphatase 

PBK phosphoinositid e 3 kinase 

PKC protei n kinas e C 

MAP microtubule-associate d protein 


VDCC voltage-dependen t Ca 2+ channel 

ACKNOWLEDGMENTS Th e author thanks Samit Shah 

and Jessica Gillis for their assistance in preparing the figures 

for this chapter an d agreein g to share their unpub - 

lished observations . Thi s wor k wa s supporte d b y th e 

National Institute of Alcohol Abuse and Alcoholism. 

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15 

Neuronal Survival Is Compromised 

by Ethanol: Extracellular Mediator s 

A salient characteristic of fetal alcoho l spectru m dis - 

order (FASD) is microcephaly an d th e associate d mi - 

croencephaly (Lemoin e e t al. , 1968 ; Jone s an d 

Smith, 1973 ; Stratton et al., 1996 ; se e Chapters 1 , 8, 

and 9). Major contributors to this microencephaly are 

ethanol-induced reductions i n the additive ontogenetic 

processe s (cell proliferatio n an d neurona l migration) 

and increase s in the amount of neuronal death. 

The effect s o f ethanol o n th e additiv e processe s ar e 

addressed i n Chapter s 11 , 12 , and 13 . The presen t 

and followin g chapte r (Chapte r 16 ) describe the ef - 

fects o f ethanol o n neurona l deat h and , conversely, 

neuronal survival . This chapter deal s with the effect s 

of ethano l o n th e incidenc e o f neuronal deat h an d 

the extracellular mediators that define this death, and 

the next chapter covers intracellular mechanisms that 

underlie this death. 

Neuronal deat h i s a natural process i n th e devel - 

oping nervous system (Jacobson, 1991; Martin , 2001; 

Lossi an d Merighi , 2003). It affects proliferatin g an d 

postmitotic, postmigrator y neuron s (se e Chapte r 5) . 


Maria B. Bruns 

Paula L. Hoffma n 

245 

The amoun t of neuronal deat h i n the nervous system 

can var y from 20 % to 80%. Factors that contribute t o 

the amount of death are the location and the maturational 

stat e of the vulnerabl e neurons. One exampl e 

is the cerebra l cortex, in which the incidenc e o f neuronal 

deat h i s lamina- an d neuro n type-dependen t 

(Miller, 1995a) . Two- to threefold more neurons die 

in superficia l laye r VI (o r layer Via) than i n layer IV 

and local circuit neurons (which, in part, differentiate 

on a later time line) are twice as likely to die as projection 

neurons in the same layer. 

EFFECTS O F ETHANOL ON 

BRAIN STRUCTURE S IN VIVO 

In viv o studie s sho w tha t ethano l affect s the spa - 

tiotemporal pattern of neuronal death throughout th e 

nervous system. Most studies have focused on the outcome, 

i.e. , th e effec t o f ethano l o n tota l neurona l 

number. Fe w have teased out the effect s o n neurona l


death fro m th e effect s o f ethanol o n othe r develop - 

mental events. The presen t review describes the limitations 

an d advance s i n ou r understandin g o f suc h 

effects o n fou r structures : th e principa l sensor y nu - 

cleus of the trigeminal nerve (PSN), cerebellum, hippocampal 

formation , and neocortex . Eac h structur e 

has a different developmenta l histor y that provide s a 

distinctive platfor m fo r understandin g th e mecha - 

nism of ethanol toxicity. 

Principal Sensor y Nucleus of the 

Trigeminal Nerv e 

Documentation o f neuronal death in vivo has consisted 

of two challenges: difficulties i n identifyin g dyin g neurons 

an d distinguishin g the effect s o f ethanol o n cel l 

proliferation from those on neuronal death. The PS N is 

an ideal structure for separating these issues because it 

is a small, pontine nucleus that lends itself to quantitative 

studies . Th e PS N i s largel y surrounded b y fibe r 

bundles, facilitatin g th e delineatio n o f border s an d 

comprises a relatively homogeneous population of neurons 

(Miller and Muller, 1989) . Furthermore , th e rol e 

of the PS N i n neurona l developmen t implicate s i t in 

FASD because it is a key site for the passag e of information 

fro m somatosensor y afférents arisin g in facia l ski n 

to highe r centers— a primar y characteristic o f FAS D 

is craniofacia l malformation s (Lemoine e t aL , 1968 ; 

Smith and Jones, 1973; Sulik et al., 1988 ; Johnston and 

Bronsky, 1995; Astley etal, 1999) . 

The PS N i n th e norma l rat has 28,00 0 neurons . 

Following prenatal exposure to ethanol, the nucleu s 

has only 19,60 0 neurons (Mille r and Muller , 1989) . 

This exposur e occur s durin g the period s whe n PS N 

neurons proliferate and migrate (Nornes and Morita, 

1979; Miller and Muller, 1989 ; Al-Ghoul and Miller, 

1993a). A longitudinal study of the tempora l effect s of 

prenatal exposure to ethanol showed that one-third of 

this differenc e i s a latent effec t o n neurona l survival 

(Miller, 1999 ) (Fig . 15-1) . Thi s findin g wa s docu - 

mented b y two changes in the neonate: (1) a decreas e 

in th e tota l number of neurons and (2 ) increases in 

the numbe r o f pyknotic and silver-staine d cells. PS N 

neurons als o di e whe n exposur e occur s durin g th e 

periods o f primary synapse formation and naturall y 

occurring neuronal death (NOND) (Miller , 1995b) — 

i.e., postnatally before postnatal da y (P ) 1 0 (Ashwell 

and Waite, 1991 ; Al-Ghoul and Miller, 1993b ; Miller 

and Al-Ghoul , 1993) . In contrast , ethano l exposur e 

during adolescence doe s not hav e a significant effect 

on neuronal number (Miller, 1995b) . Thus, the criti - 

cal window for ethanol-induced neuronal death in the 

PSN i s framed by the period s of neuronal generatio n 

and synaptogenesis. 

A prim e afferen t o f th e PS N i s th e infraorbita l 

nerve. A neonatal lesio n of this nerve causes massive 

neuronal death, confined to the ventral portion of the 

PSN wher e the infraorbita l nerv e terminates (Miller 

et al., 1991) . Exposure to ethanol doubles the amount 

of thi s frans-synapti c deat h (Miller , 1999) . Further - 

more, this death is rapid, as the expressio n of Fos (a 

marker o f cellular activity) an d ALZ-5 0 ( a marker of 

dying neurons) increases within 2 hr of placing the lesion 

(Miller and Kühn, 1997) . 

Cerebellum 

The cerebellu m i s susceptibl e t o ethanol-induce d 

damage in a dose-dependent manner; ethanol-exposed 

rats hav e smalle r cerebell a tha n control s (Dia z an d 

Samson, 1981 ; Wes t e t al, 1989) . A key factor con - 

tributing to th e degre e o f damage i s the pea k bloo d 

ethanol concentratio n (BEG ; Pierce an d West, 1986 ; 

Bonthitis et al., 1988) . Ra t pups expose d t o the sam e 

amount o f ethano l dail y d o no t hav e th e sam e 

amount of damage if the schedule of ethanol delivery 

differs. Exposure s t o relativel y large bol i o f ethano l 

are significantly more deleterious than a constant delivery 

of low doses of ethanol. 

The stud y of neuron number s i n the cerebellu m 

has centere d o n Purkinj e neuron s (Goodlet t an d 

Horn, 2001; Light et al, 2002). Adult rats exposed to 

ethanol during the perio d from P 4 to P10 have significantly 

fewer neuron s tha n controls . Th e amoun t o f 

Purkinje cel l loss can var y from 10 % to 70% depending 

on the developmental history and cerebellar compartment 

(Fig . 15-2) . Fo r example, lobule s I-V , IX, 

and X are more vulnerable to ethanol exposur e than 

lobules V I an d VI I (Pierc e e t al. , 1989 ; Goodlet t 

et al., 1990) . Th e developmen t o f neurons i n lobule s 

I, II, IX, and X precedes that of neurons i n the inter - 

vening lobules VI and VII (Miale and Sidnian , 1961 ; 

Altman and Das , 1966 ; Altaian and Bayer , 1978). Exposure 

to ethanol during the restricted period of P4 or 

P5 cause s a s much o f a differenc e a s does exposure 

between P4 and P10 (Light et al, 2002). This effect is 

concentration-dependent. Thus , exposur e t o a hig h 

BEG, eve n fo r a short time, ca n resul t in th e los s of 

vulnerable neuron s i n th e cerebellu m (Wes t et al. , 

1989; Miller, 1996b ; Thomas et al, 1996) .

EXTRACELLULAR MEDIATORS OF NEURONAL DEATH 24 7 

FIGURE 15- 1 Neurona l deat h i n the developin g principal sensor y nucleus of 

the trigeminal nerve (PSN). A. The number s of neurons in the PS N rises prenatally 

an d fall s durin g th e firs t 2 postnatal week s (left) . Thi s decreas e coin - 

cides wit h a n increas e i n th e frequenc y o f pyknoti c cell s (right) . Prenata l 

exposure to ethanol delays the period of neuronal death . B . Presumptive dyin g 

neurons (arrows ) can be identified in a silver-stained section fro m the pons of a 

3-day-old rat. They are distributed throughout th e PSN . C . Mor e argyrophili c 

neurons are evident in the PS N of an ethanol-treated 3-day-ol d pup. D . An argyrophilic 

cel l (arrow ) ha s a dense cor e an d ofte n i s surrounded by granules. 

These are presumed to be blebs and released nucleosomes. E. A pyknotic cell 

is characterized b y a condensed nucleu s that often is broken int o small bodies . 

Scale bar s = 10 0 ujn ( B and C ) an d 1 0 Jim (D and E) . (Source: Dat a i n A are 

taken with permission from Miller , 1999. ) 

Ethanol-induced difference s i n cel l numbe r ma y 

result from reduction s i n neuronal number and/o r increases 

i n neurona l death . Th e cerebell a o f rat s exposed 

to ethanol during gestation alone or during the 

pre- and early postnatal period weigh less and have no 

fewer Purkinj e neurons tha n rats exposed durin g th e 

postnatal perio d alon e (Wes t e t al. , 1994 ; Miller , 

1996b; Light e t al, 2002). These dat a hav e bee n interpreted 

a s evidenc e tha t ethano l doe s no t affec t 

neuronal generation . Thi s ma y i n par t b e true , a s 

ethanol may not affect the generation of Purkinje cell s 

which occur s prenatall y (Mial e an d Sidman , 1961 ; 

Altman an d Bayer , 1978) . Th e possibilit y still exists , 

however, that ethano l doe s reduc e th e generatio n of


FIGURE 15- 2 Effec t o f ethanol exposur e o n number s o f Purkinje neurons . 

Rats were exposed t o ethanol b y gavage on postnatal da y 4. The number s of 

Purkinje neurons in various cerebellar lobule s were determined. Th e dat a for 

two pairs of lobules are presented: lobule s I and IX , which ar e early developing 

lobules, and lobules VI and VII, which develop relativel y late. Each point 

describes th e change i n the number o f Purkinje neurons relativ e to the num - 

ber identified in controls on P4. No significant changes were detected for latedeveloping 

lobules . Bot h o f th e early-developin g lobules , however , wer e 

vulnerable to ethanol toxicity. Significant differences ( p < 0.05; noted b y asterisks) 

were detected onl y 24 hr or 1 0 days after exposure . (Source: Dat a taken 

from Ligh t and colleagues, 2002. ) 

granule neurons produced postnatally, which can secondarily 

lead to the degeneration of Purkinje neurons 

dependent on their afférent s (Reza i and Yoon , 1972 ; 

Herrup and Trenkner, 1987) . 

Recent evidence, from studie s of restricted ethanol 

exposure, supports the notion that ethanol causes the 

death of Purkinje neurons. It is unlikely that a 1-da y 

exposure to ethanol affect s granul e cel l generation s o 

profoundly an d rapidl y a s t o caus e th e short-ter m 

death o f Purkinje neurons . Moreover, various studies 

show that ethanol increase s th e expressio n o f two indices 

of apoptotic deat h b y Purkinje neurons (Ligh t et 

al., 2002)—th e expressio n of activated caspase-3 , a n 

enzyme involved in the clipping of DNA into packets 

during apoptoti c death , an d evidenc e o f termina l 

uridylated nick-en d labelin g (TUNEL) , a n inde x of 

DNA fragments . Moreover, exposur e t o ethano l o n 

P4 lead s to a n increas e in whol e cerebella r catalas e 

and reactiv e oxygen species (Horn, personal comm.) . 

Activated caspase- 3 expression is markedly increased 

in man y Purkinje cell s an d i n a smattering of small 

cells in the internal granule cell layer. The sensitivity 

of th e granul e neuron s pe r s e t o ethanol-induce d 

death remain s a question, as does th e mechanis m o f 

ethanol-induced loss of Purkinje neurons (West et al., 

1994; Goodlet t and Horn, 2001) . Suffice i t to say that 

ethanol ca n compromis e neurona l survival . Intracellular 

event s underlyin g thi s deat h ar e reviewe d i n 

Chapters 6 an d 16 . The molecula r mechanis m o f 

Purkinje cel l deat h deserve s mor e detaile d analysis, 

given recent evidence that Purkinje neurons ma y die 

by a proces s o f autophag y followin g remova l o f 

trophic support (Florez-McClure e t al., 2004). 

Hippocampal Formatio n 

The hippocampa l formatio n i s a comple x structur e 

containing a t leas t tw o majo r subdivisions , th e

hippocampus an d th e dentat e gyms . Th e axon s of 

granule cells in the dentate gyrus synapse with pyramidal 

neuron s i n th e hippocampus . Thes e neurona l 

populations develo p differently . Fo r example , hip - 

pocampal pyramida l neurons ar e generate d prena - 

tally i n th e ventricula r zone , wherea s mos t (85%) 

granule neuron s are generated postnatall y in th e in - 

trahilar zone (Schlessinge r e t al., 1978 ; Nowakowski 

andRakic, 1981). 

Prenatal an d postnata l exposur e t o ethano l ha s 

differential effect s o n th e number s o f cells in th e hip - 

pocampal formation. The amoun t o f DNA in the hippocampal 

formation is not affected followin g prenatal 

exposure; however, postnatal exposur e leads to an in - 

crease in DNA content (Miller, 1996a) . Although thi s 

result suggest s that ethano l doe s no t affec t neurona l 

survival, these biochemical measure s do not discriminate 

betwee n neuron s an d gli a o r differentiat e th e 

specific effect s o f ethano l o n th e hippocampu s o r 

dentate gyrus. Careful anatomica l studies have shown 

that prenatal exposure to ethanol reduces the number 

of neurons in th e hippocampu s (Miller , 1995c) . It is 

likely that som e o f this reductio n result s from a de - 

crease i n cel l proliferation . Earl y postnata l exposur e 

does no t affec t neurona l numbe r unles s th e ethano l 

exposure i s high (BE G >30 0 mg/dl; Miller , 1995c) . 

Thus, i t appear s tha t ethano l doe s no t induc e th e 

death o f neurons i n th e hippocampa l formatio n be - 

yond that occurring naturally. 

Neocortex 


Cortex contrast s wit h the cerebellu m an d hippocam - 

pus i n tha t th e generatio n o f its neuronal subpopulations, 

suc h a s projectio n an d loca l circui t neurons , 

occurs concurrently (Miller, 1985) . Likewise, the periods 

of NOND and synaptogenesi s are largely synchronized 

for all cortical neurons (Rakic et al., 1986; Miller, 

1988). In these aspects, neocortex is more like the PS N 

than the cerebellum and hippocampal formation . 

Pre- and postnata l exposure s to ethano l induc e a 

difference i n the number o f cortical cells . Overall, total 

DN A conten t i s 29 % lowe r i n th e cortice s o f 

ethanol-treated rat s tha n tha t o f control s (Miller , 

1996a). If the same mechanisms are at work in the developing 

corte x a s i n th e PS N (Miller , 1999) , one - 

third of the differenc e cause d b y prenatal exposure to 

ethanol i s due t o laten t death . Prenata l exposur e to 


ethanol cause s a -33 % differenc e i n th e numbe r o f 

neurons in somatosensory cortex (Miller and Potempa , 

1990) and n o difference i n the numbe r o f neurons in 

visual cortex (Mooney et al., 1996; Mooney and Nap - 

per, 2005). Apparently, not all parts of the pallium are 

equally affecte d b y ethanol . Postnata l exposur e t o 

ethanol als o cause s a significan t differenc e i n DN A 

content, althoug h thi s difference i s only 13% (Miller, 

1996b), a difference expecte d t o have equal effec t o n 

neurons and gli a (Mille r and Potempa , 1990) . Inter - 

estingly, the amoun t o f neuronal deat h (estimate d at 

29% x 1/ 3 = 10%) caused b y exposure during the pe - 

riod o f neuronogenesis (betwee n gestationa l da y [G ] 

11 and G21 ) is similar to that caused by exposure during 

early synaptogenesis (betwee n P 4 and P10). Does 

this mean tha t ethano l cause s an equivalen t amount 

of cell deat h regardles s of the tim e o f exposure, i.e., 

that ther e i s n o critica l perio d fo r ethano l toxicity ? 

No. Exposur e to ethanol afte r th e en d o f the period s 

of NON D an d synaptogenesi s doe s no t affec t th e 

number o f cortica l neuron s (Miller , unpublishe d 

results). 

Changes i n cel l numbe r ar e parallele d b y alterations 

i n th e expressio n o f protein s associate d wit h 

neuronal death . Thes e includ e ALZ-5 0 (Küh n an d 

Miller, 1998) , be l proteins , an d activate d caspase- 3 

(Mooney an d Miller , 2000). The expressio n o f eac h 

of these markers rises during the first and second postnatal 

weeks . Ethano l significantl y affect s eac h 

marker, mostl y by delaying the perio d o f expression 

(Kühn and Miller, 1996; Mooney and Miller, 200la). 

A prime sourc e o f cortical afférent s an d targe t of 

cortical efferents i s the thalamus. I t is noteworthy that 

although ethanol-induce d neurona l deat h occur s i n 

the neocortex , apparentl y no such deat h occur s in at 

least some thalamic nuclei (Liv y et al., 2001), including 

th e ventrobasa l thalamu s (VB ; Moone y an d 

Miller, 1999a) . No t onl y i s the numbe r o f VB neu - 

rons the sam e i n mature ethanol-treate d an d contro l 

rats, but there appears to be no difference i n the tem - 

poral chang e i n number s durin g th e period s o f 

NOND an d synaptogenesi s (Moone y an d Miller , 

200 Ib). Thi s phenomeno n i s particularly interesting 

in that the differential effec t of ethanol sets up a potential 

mismatc h i n thalamocortica l afférent s an d thei r 

target an d wit h corticothalami c afférent s an d thei r 

target. 

A series of studies b y Olney an d colleague s (e.g. , 

Ikonomidou et al., 1999 , 2000) describe the effect s of 

early postnata l exposur e t o ethano l o n th e deat h o f


cortical cells . The author s clai m tha t earl y develop - 

mental exposur e t o ethanol cause s massiv e neurona l 

death whe n th e exposur e selectivel y occur s durin g 

synaptogenesis. I n cortex , thi s proces s begin s o n P 3 

and peak s afte r P3 0 (Miller , 1988) . Allegedly , tran - 

sient exposur e t o ethano l cause s a wav e of neurona l 

death tha t i s initiall y greates t i n dee p lamina e an d 

progresses superficially . Thei r wor k suggests tha t th e 

vast majority of cortical neurons die as a consequenc e 

of earl y postnata l exposur e t o ethanol . Thes e dat a 

are i n conflic t with a wealth o f data (som e o f which 

are reference d above ) and mus t b e interprete d wit h 

caution. 

As th e dat a o f Olne y an d colleague s hav e re - 

ceived considerable attention, they deserve comment. 

The dat a ar e base d o n a silver-staining method (d e 

Olmos an d Ingram , 1971 ; Friedma n an d Price , 

1986) that purportedly identifies dyin g neurons. I t is 

difficult t o assess the applicabilit y of this method be - 

cause section s fro m th e brain s o f contro l animal s 

have virtuall y n o label . Thi s lac k o f labeling i s disturbing 

because th e consensu s i s that th e firs t post - 

natal wee k i s par t o f th e perio d o f NOND i n th e 

normal ra t brai n (e.g. , Finla y an d Slattery , 1983 ; 

Ferrer e t al, 1990 ; Miller , 1995a ; Spreafic o e t al, 

1995). Th e dat a fro m th e ethanol-treate d rat s ar e 

equally disturbing . Massiv e number s o f neurons i n 

the cortice s o f ethanol-treate d rat s ar e argyrophili c 

(Ikonomidou e t al. , 1999) . Recen t evidenc e show s 

that neuron s tha t exhibi t a "marker " o f cel l deat h 

(e.g., TUNEL or caspase immunoreactivity ) are no t 

obliged t o die (Gordon e t al., 2002; Cheng and Zo - 

chodne, 2003 ; Ishid a e t al, 2004 ; Oomma n e t al. , 

2004). Furthermore , i f al l o f th e cortica l neuron s 

that exhibi t ethanol-induce d silve r stainin g indee d 

do die , the n earl y postnata l exposur e t o ethano l 

would caus e a collapse of the cerebra l wall . Careful 

studies show, however, that less than one-sixt h o f all 

cortical neuron s di e a s a resul t o f ethanol exposur e 

(Miller, 1996a ; Moone y e t al, 1996 ; Moone y an d 

Napper, 2005). 

According to Olney an d colleagues , the ravage s of 

ethanol are not confined to cerebral cortex. For example, 

variou s thalamic nucle i als o exhibi t widespread 

neuronal degeneratio n followin g earl y postnata l ex - 

posure to ethanol. I t appears that most neurons in the 

anterior nuclei an d ventra l thalamus o f 8-day-old rat s 

are argyrophilic . Studies o f mature ra t thalami, how - 

ever, sho w tha t rat s expose d t o ethano l pre - o r post - 

natally hav e robus t anterio r nucle i an d stereologica l 

data sho w that ethanol doe s no t affec t th e numbe r of 

neurons i n thalami c nucle i (Moone y an d Miller , 

1999a;Livy et al, 2001,2002). 

Finally, Olne y an d colleague s conclude , incor - 

rectly, that the tempora l window of ethanol toxicit y is 

restricted t o the perio d o f synaptogenesis. Indeed, exposure 

t o ethano l durin g thes e ontogeneti c event s 

can cause neuronal death during the period of synaptogenesis 

(Miller , 1995b , 1999 ; Küh n an d Miller , 

1998; Moone y an d Miller, 1999b). Prenatal exposur e 

to ethanol can also cause neuronal death (se e above). 

Furthermore, ethano l ha s profoun d effect s o n cel l 

proliferation (se e Chapters 1 1 an d 12 ) and neurona l 

migration (se e Chapter 13). 

Cell Culture Studies 

Cultured cortica l neurons obtained fro m 17-day-ol d 

fetuses exhibi t a 25 % los s ove r th e firs t 2 day s post - 

plating (Fig . 15-3 ) (Seabol d et al., 1998 ; Jacob s an d 

Miller, 2001 ; Mille r e t al. , 2003 ; Ramachandra n 

et al., 2003). This los s is equivalent to the amoun t o f 

natural deat h occurrin g amon g post-migrator} ' neu - 

rons i n corte x in vivo (Finlay and Slattery , 1983 ; Fer - 

rer et al., 1990 ; Miller , 1995a; Spreafico et al, 1995). 

Ethanol exposur e cause s a n additiona l 25 % loss. Interpretation 

o f thes e dat a i s confounded becaus e a t 

least som e o f th e neuron s i n feta l cortica l culture s 

have th e abilit y t o proliferat e (Jacob s an d Miller , 

1999). Thi s confoundin g variabl e can b e addresse d 

by applyin g a mathematica l mode l tha t determine s 

the tota l amount s o f cel l proliferatio n an d deat h 

(Miller, 2003) . A particular strength o f this model i s 

that it does not rel y on a method fo r identifying dying 

cells, which can bias the results. Accordingly, ethanol 

causes a 30 % differenc e i n th e numbe r o f culture d 

cortical neurons or B104 neuroblastoma cells. Of this 

difference, two-third s resul t fro m decrease s i n cel l 

proliferation and one-thir d from ethanol-induce d cell 

loss. Thus, the i n vitro studies of cortical neurons con - 

cur with the i n vivo data. 

SURVIVAL-PROMOTING FACTORS 

Evidence tha t neurona l deat h occur s durin g the pe - 

riod o f synaptogenesi s support s th e hypothesi s tha t 

neuronal deat h i s driven by neurons failing t o com - 

pete fo r a n entit y ( a growt h facto r and/o r synapti c 

sites) i n limite d supply . Among th e factor s tha t hav e

FIGURE 15- 3 Effec t o f ethano l o n neurotrophin - 

supported neurona l survival . Cortical neurons derive d 

from 17-day-ol d fetuse s were cultured i n the presenc e 

of a neurotrophi n ( 0 o r 1 0 ng/ml) an d ethano l ( 0 o r 

400 mg/dl). Over 3 days postplating, one-quarter o f the 

untreated cortical neurons were lost. Nerve growth factor 

(NGF ) prevented thi s loss , wherea s brain-derived 

neurotrophic facto r (BDNF ) an d neurotrophi n 3 

most commonl y bee n implicate d a s promotin g 

neuronal surviva l ar e nerv e growt h facto r (NGF) , 

brain-derived neurotrophi c facto r (BDNF) , insulin - 

like growt h factor- 1 (ÏGF-1) , N-methyl-D-aspartat e 

(NMDA), an d pituitar y adenyly l cyclas e activatin g 

polypeptide (PACAP). 

Neurotrophins 

Neurotrophins ar e secreted , diffusibl e signalin g pro - 

teins know n t o exer t neuromodulator y effect s o n 

adaptive processes such as synaptic plasticity, required 

for learning and memory, and neuronal survival. This 

family o f protein s act s i n a n autocrin e an d antero - 

grade fashio n simila r t o tha t o f neurotransmitter s 

(Pitts and Miller , 1995 , 2000; Miller and Pitts , 2000; 

Neet an d Campenot , 2001) . A numbe r o f neu - 

rotrophic factor s hav e bee n identified , includin g 

NGF, BDNF, neurotrophin (NT)3, and NT-4/5, each 

of which supports distinct populations of neurons. To 

perform thei r functions, the neurotrophins bind to tyrosine 

kinase receptors (trks). NGF bind s to trkA with 

high affinity , wherea s BDN F an d NT-4/ 5 bin d t o 

trkB. NT-3 binds primarily to trkC, bu t ha s also bee n 


(NT-3) di d not . Ethano l exacerbate d th e "natural " 

death observe d i n untreate d cultures . Ethano l als o 

eliminated th e abilit y of NGF t o maintai n viability . 

Asterisks an d poun d sign s denot e significan t differ - 

ences relative t o the untreate d control s o n da y 0 and 

day 3, respectively. A single sign identifies data that are 

different a t a level oïp < 0.05 and a double sig n designates 

differences a t the p < 0.01 level of significance. 

associated with trkA or trkB in certain populations . A 

low-affinity receptor , p75, may also bind t o any of the 

neurotrophins. Thi s receptor ma y act in cooperatio n 

with or independently o f the trks. 

The tyrosin e kinase associated with the high-affinit y 

trks can activat e a t least three differen t signalin g path - 

ways to support differentiation, proces s outgrowth, cell 

survival, an d activity-dependen t plasticity (Bibel an d 

Barde, 2000). The p7 5 receptor has no kinase activity 

associated wit h i t per se , but i t is now known t o hav e 

signaling capability . The mechanism s o f p75 signal - 

ing are far less characterized. 

Depending o n the extracellula r environment an d 

the compositio n an d abundanc e o f receptors o n th e 

membrane surface , neurotrophin s ca n hav e benefi - 

cial o r deleteriou s effect s o n neurons . Th e trk s an d 

p75 for m homo - an d heterodimer s wit h eac h other , 

and thes e dynamic complexe s ca n cooperat e to exert 

varied effect s (Chao , 2003 ; Huan g an d Reichardt , 

2003). Receptor interactions can also be antagonistic, 

depending on which ligands are available. 

The expressio n o f neurotrophin s change s i n a 

time-dependent manne r durin g development . Tha t 

is, certain population s o f neurons ar e dependen t o n


one neurotrophi n fo r a period o f time. Then, afte r a 

critical period, these neuron s becom e dependen t o n 

another neurotrophin(s) . For example, Purkinje cell s 

switch from BDNF dependence t o NT-3 dependenc e 

between P 4 an d P 6 (Davies , 1997) . Coincidentally , 

this i s the perio d o f highest vulnerability of Purkinje 

cells t o ethano l (Ligh t e t al. , 2002 ; se e above) . 

Ethanol neurotoxicity may be mediated by changes in 

neurotrophin systems. 

The distributio n o f neurotrophin ligand s i n vari - 

ous brain tissues during normal development i s critical 

t o appropriat e signa l transductio n fo r survival . 

Alterations in the abundance of the neurotrophi n ligands 

and receptor s may result after ethano l exposure . 

In othe r words , withdrawa l fro m o r a n inadequat e 

supply of neurotrophins may underlie decrease d cel l 

survival. Conversely , ethano l ma y down-regulat e re - 

ceptor expressio n below a threshold require d to initiate 

signals, interfere with ligand-receptor binding , or 

render neurotrophi n receptor s nonfunctional . Mos t 

likely, several mechanisms ar e a t play, depending, o f 

course, on the population o f neurons and the time of 

ethanol exposure . The differentia l effect s o f ethano l 

on distinc t populations of neurons ca n offe r valuable 

insight t o th e norma l interaction s o f neurotrophin s 

during development. 

Nerve Growth Factor 

NGF prevent s NOND—i.e. , i t is an anti-thanatopi c 

factor (e.g. , Majda n an d Miller , 1999 ; Bibe l an d 

Barde, 2000 ; Morriso n e t al, 2002). This i s particularly 

evident among cultures of cortical cells in which 

NGF uniquel y support s cel l surviva l (Fig . 15-3 ) 

(Seabold e t al., 1998 ; Mille r et al, 2003). NGF ca n 

also affect othe r developmental phenomena , notabl y 

the cel l cycle and the surviva l of cells that exit the cycling 

population (Lopez-Sanchez and Frade , 2002). 

Ethanol ca n interfer e wit h NGF-mediate d func - 

tion bot h i n vitr o and i n vivo . It disrupt s th e NGF - 

mediated surviva l o f primar y culture d cortica l 

neurons (Seabol d e t al., 1998 ; Mille r e t al, 2003). A 

neuroprotective rol e o f NGF i s supported b y studies 

of NG F over-expressio n i n mic e (Heato n e t al , 

2000b). Earl y postnata l exposur e t o ethano l kill s 

fewer Purkinj e cell s i n thes e mic e tha n i n wild-type 

controls. Altered NGF abundanc e coul d hav e patho - 

logical consequences, fo r example, NGF ca n potentiate 

fre e radical-induce d apoptosi s i n vitro (Spea r 

etal, 1998). 

The effect s o f gestationa l ethano l exposur e o n 

NGF expressio n hav e als o bee n examine d i n vivo. 

The amoun t of NGF protei n was measured at PI and 

P10 afte r gestationa l ethano l exposure . NG F i s in - 

creased i n the corte x and striatum of ethanol expose d 

animals a t PI , bu t doe s no t diffe r fro m control s a t 

P10. No other neurotrophins are affected i n any brain 

region a t P I (Heato n e t al. , 2000e) . Expressio n o f 

NGF protei n i s also increase d i n th e hippocampa l 

formation transientl y at PI5 afte r gestationa l ethanol 

exposure (Angelucci etal., 1997) . 

In additio n t o affectin g neurotrophi n ligan d ex - 

pression, ethano l ca n alte r recepto r expression . 

Ethanol-induced cel l death i n vitro is associated with 

a selective decreas e i n p75 expression; the expressio n 

of al l tr k isoform s i s unaffected b y ethano l (Seabol d 

et al., 1998) . Thi s pattern reverberate s in other cells , 

including septohippocampa l neuron s (Angelucc i 

et al. , 1997 ) an d P C 12 pheochromocytom a cell s 

(Luo e t al., 1996 , 1999) . In cerebellar Purkinje cells, 

following earl y postnatal ethanol exposure , there i s a 

significant decreas e i n trk A and p7 5 recepto r expres - 

sion f? 7 vivo, despit e ther e bein g n o chang e i n th e 

amount o f NGF (Dohrma n e t al, 1997) . Selectiv e 

down-regulation o f th e neurotrophi n receptor s b y 

ethanol durin g th e earl y postnata l developmen t pe - 

riod is fatal t o Purkinje cell s (Light et al, 2002). Th e 

differential effec t o f ethanol o n receptor s ma y reflec t 

the distribution of these receptors in the plasma mem - 

brane. Conceivably , p7 5 i s distribute d i n lipi d raft s 

(Higuchi et al., 2003), the fluidity of which is affected 

by ethanol (People s et al., 1996), whereas trks are relatively 

protected fro m ethano l because they appear no t 

to be dispersed within rafts (Mutoh et al., 2004). 

In addition , gli a offe r a critica l suppl y o f neu - 

rotrophins. Unde r norma l homeostati c conditions , 

glia d o no t expres s NGF, however , they may elevat e 

NGF unde r pathological conditions (Pitts and Miller, 

1995; Miller and Pitts , 2000). This glial production of 

neurotrophins ma y be an attempt at neuroprotection . 

For example, astrocyte s increas e NG F expressio n after 

ethanol exposure in vitro (Vallès et al., 1994) . C6 

glioma cell s secret e increase d amount s o f NGF an d 

BDNF, quantitie s that ar e sufficien t t o protec t cere - 

bellar granul e neuron s fro m ethano l insul t (Pantazi s 

et al., 2000). Antibodies specific to neurotrophins and 

trk receptors block this ethanol-induced neuroprotec - 

tion o f the granul e cells . Interestingly, trkA receptors 

are not localized in cerebellar granule cells, and the y 

express only low amounts o f p75. The implicatio n i s

that NGF mus t be acting through a promiscuous trk 

isoform. 

A downstream effec t o f ethanol toxicity in cortica l 

cultures i s the quashin g o f the expressio n o f a gen e 

up-regulated b y NGF , ne g (nerv e growt h factor - 

stimulated, ethanol-depresse d gene ) (Mille r e t al, , 

2003). The functio n of the gene product is unknown, 

although i t appear s tha t Ne g i s involve d i n NGF - 

mediated cell survival . The gen e product ha s protein 

domains suggestin g tha t i t i s a niitochondria l trans - 

membrane protein (Nagas e et al., 1996). This finding 

is intriguing in ligh t o f evidence tha t ethano l affect s 

other niitochondria l protein s tha t regulat e cel l sur - 

vival, fo r example , bcl- 2 an d ba x (Moone y an d 

Miller, ZOOla) (see Chapter 16) . 

Brain-Derived Neurotrophic Factor 

BDNF promote s cel l surviva l i n viv o an d i n vitro. 

During critica l period s o f norma l development , 

BDNF i s necessary fo r bot h neurit e outgrowt h an d 

synaptic pruning . Developin g neurona l processe s 

compete fo r a limited suppl y o f BDNF. Thos e neu - 

rons that ar e unable t o obtain a sufficient amoun t o f 

BDNF o r tha t hav e insufficien t trk B receptor s un - 

dergo cell death . 

Like NGF, BDN F i s also thought to play a neuroprotective 

rol e i n respons e t o ethano l exposure . In - 

deed, BDN F ma y pla y a rol e i n th e tissue - an d 

age-dependent component s of ethanol-induced neu - 

ronal cel l death . Th e striatu m i s a good example . I n 

the normal striatum, NOND is more common o n P3 

than P1 4 (Heaton e t al., 2003). This sequence is the 

inverse o f the patter n o f endogenous BDN F expres - 

sion; striata l BDN F conten t i s highe r o n P1 4 

(Fig. 15-3) . Earl y postnatal exposur e to ethano l increases 

striatal BDNF content (Heato n e t al, 2000e). 

Cerebellar Purkinj e cell s revea l a contrastin g situa - 

tion: Purkinje cell s decrease BDN F expression afte r 

early postnata l ethano l exposur e (G e e t al. , 2004) . 

Within hour s o f ethanol exposure , there i s also de - 

creased expressio n o f cerebella r trkB . Afte r P9 , 

ethanol exposur e doe s no t affec t BDN F o r trk B expression. 

Compromised , time-dependen t BDN F 

support i n cerebellu m ma y underli e hig h rate s o f 

Purkinje cel l apoptosi s durin g it s period o f ethano l 

sensitivity (P4 to P5). 

Manipulation o f availabl e BDN F suppor t i n vivo 

can further demonstrat e neurotrophi n system-specifi c 

vulnerability t o ethanol . Transgeni c mic e lackin g 


BDNF expressio n in the cerebellum sho w significant 

losses o f Purkinj e cell s afte r earl y postnatal ethano l 

exposure (Heato n e t al. , 2002) . There ar e als o con - 

current decreases i n anti-apoptotic factor s (e.g., bcl-2) 

in these mice , suggesting that BDNF signals mediate 

neuronal survival . The contrastin g results observed in 

cerebellum an d striatu m illustrat e that ethano l ca n 

exert tissue-specific effects dependen t o n BDNF 1 regulation. 

In cortical tissue , the effect s o f ethanol o n BDNF 

and trk B expression are also specific to the time of exposure. 

Earl y postnata l exposur e cause s significan t 

increases i n neocortica l BDN F abundance , simila r 

to change s observe d i n hippocampu s an d striatu m 

(Heaton e t al., 2000e) . I n contrast , gestationa l expo - 

sure result s i n significan t decrease s i n BDN F an d 

trkB recepto r expressio n i n cortex , whic h correlat e 

with significant increase s in apoptosis of cortical neu - 

rons (Climent e t al., 2002). Both of these observations 

support th e hypothesi s tha t a sufficien t amoun t o f 

BDNF mus t be present to promote cel l survival in response 

to ethanol . 

Insulin-Like Growth Factor- 1 

IGF-1 is another survival-promoting factor that i s target 

of ethanol (Zhan g e t al, 1998) . Activatio n of the 

IGF-1 recepto r ca n inhibi t cel l deat h i n vitr o (W u 

et al, 1996 ) an d in vivo (Resnicoff et al., 1995) . An in 

vivo model i n which transplante d tumo r cell s overex - 

pressed IGF- 1 receptor s show s that IGF- 1 play s a major 

protectiv e role . I n contrast , a decreas e i n IGF- 1 

receptor expression results in heavy amounts of apoptosis 

in vivo. The cytotoxi c effects ar e more dramati c 

than when tumor cells are manipulated i n vitro. 

Like th e neurotrophins , IGF- 1 expressio n fluctu - 

ates durin g development . Norma l change s o f IGF- 1 

receptor distributio n indicat e tha t differen t popula - 

tions of cells have different period s of IGF-1 depend - 

ence durin g the postnata l period of brain maturation 

(Breese e t al. , 1991) . Norma l neonate s expres s th e 

greatest abundanc e o f IGF- 1 protei n a t birth ; IGF - 

lcontentfalls61%byP20. 

Gestational ethano l exposur e alter s IGF- 1 

abundance an d distributio n durin g postnata l brai n 

development (Brees e e t al. , 1994) . I n offsprin g o f 

ethanol-exposed mothers, the decrease in IGF-1 expression 

i s attenuated, falling onl y by 25% during the first 

20 day s afte r birth . Localizatio n o f IGF-1 an d IGF- 1 

receptors, however , i s not affected . Thus , ethanol ma y


disrupt th e regulatio n o f IGF-1 availabilit y (i.e., production 

and secretion) rather than th e ligand-receptor 

signals. 

Ethanol ca n revers e th e anti-apoptoti c effect s o f 

IGF-1 recepto r signalin g (Resnicoff e t al , 1996) . Fi - 

broblasts lacking IGF-1 receptor s are completely insensitive 

to ethanol-induced cell death in vitro. Cerebellar 

granule neurons can normally be rescued from apopto - 

sis i n th e presenc e o f IGF- 1 (D'Mell o e t al. , 1993) . 

Ethanol ca n inhibi t this neuroprotection eve n whe n 

excess IGF-1 i s available (Zhang et al., 1998) . 

The amoun t o f IGF- 1 i n materna l plasm a i s reduced 

afte r ethano l exposur e (Brees e an d Sonntag , 

1995). Insufficien t IGF- 1 ligan d o r receptor s ca n 

cause massiv e amounts o f apoptosis. Thus, the conse - 

quences o f insufficient plasm a IGF- 1 o n metabolis m 

and hormon e physiolog y ma y contribut e t o bod y 

growth deficiencie s tha t compoun d th e pathologica l 

microcephalic brain development associated with FAS. 

The vulnerabilit y of an individual IGF-1 recepto r 

to ethanol ca n var y in that the affinit y o f the recepto r 

for it s ligan d ma y b e altered . Th e enzym e activity 

of the IGF- 1 recepto r ca n als o be disrupted . Several 

subclones o f 3T 3 cell s generate d t o expres s equal 

amounts of IGF-1 recepto r demonstrated that not all 

IGF-1 receptors are created equal (Seiler et al., 2000). 

IGF-1 receptors in some subclones resisted the effect s 

of ethanol; ethanol di d not inhibit the autophospho - 

rylation of the IGF- 1 receptor. Like the tissue-specific 

distribution o f neurotrophi n receptor s an d variabl e 

expression of trk isoforms, relative abundance of IGF- 

1 receptor s an d thei r variabl e resistanc e t o ethano l 

may explain the differentia l effect s o f ethanol o n various 

cell populations . 

N-Methyl-D-Aspartate 

Glutamate i s a majo r excitator y neurotransmitte r i n 

the mammalia n nervous system. Its actions ar e medi - 

ated b y interactio n wit h bot h metabotropi c an d 

ionotropic receptors (Hollman and Heinemann, 1994 ; 

Ozawa et al., 1998) . The NMD A subtype of ionotropic 

glutamate receptor plays an important role in synaptic 

plasticity (e.g., learning and memory), but hyperactivation 

of NMDA receptors can lead to excitotoxic, primarily 

necrotic, death of mature neurons (Choi, 1992). 

During neurona l development , NMD A recepto r 

function i s necessary for the activity-dependent organization 

of synapses (Collingridge and Lester, 1989; Mc- 

Donald and Johnston, 1990). In addition, activation of 

NMDA receptors can hav e a trophic o r protective effect 

on developing neurons. This protective effect has 

been mos t extensively studied with cultured cerebel - 

lar granule neurons. Cerebellar granule neurons isolated 

fro m 7-day-ol d rat s requir e a growt h mediu m 

containing a depolarizing concentration of K + (25 mM 

KC1) fo r surviva l (Gall o e t al , 1987 ; Balâz s et al , 

1988). Th e dependenc e o f cell surviva l o n depolar - 

ization develop s ove r a narrow time frame . Thi s pe - 

riod includes the time when the glutamatergic mossy 

fibers innervate postmitoti c granul e neurons . Treat - 

ment of cerebellar granul e neurons with NMDA also 

can protect these postmitotic neuron s fro m cel l death 

when they are grown in medium containin g a physiological 

concentratio n o f K + ( 5 mM KC1 ) (Balâz s 

et al, 1988 ; Burgoyn e et al, 1993 ; Bhav e and Hoff - 

man, 1997) . Thi s neuroprotectiv e actio n o f NMD A 

apparently mimic s th e effec t o f innervatio n o f th e 

granule neurons by mossy fiber afférents i n vivo. 

Two model s o f culture d cerebella r granul e cell s 

have been use d t o explore the neuroprotectiv e property 

of NMDA. In the first model, neurons from post - 

natal rat s ar e culture d i n a mediu m containin g a 

physiological concentration of KC1 (see above). These 

cells underg o spontaneou s death . I n th e secon d 

model, th e cell s ar e grow n i n mediu m containin g 

25 mM KC 1 an d afte r severa l day s i n culture , th e 

medium i s replaced wit h mediu m containin g a low 

KC1 concentration and often i s serum-free (Yan et al , 

1994). Unde r bot h o f these conditions , neuron s di e 

over a period o f 1 or 2 days, and ca n b e protected b y 

the additio n o f NMDA (Yan et al, 1994 ; Bhav e an d 

Hoffman, 1997 ) o r polyaniine s (Harad a an d Sugi - 

moto, 1997) . Polyaniine s modulat e th e functio n o f 

NMDA receptors (Dod d e t al, 2000 ) and act in part 

through a n NMDA receptor-dependent mechanis m 

(Harada an d Sugimoto , 1997) . I n bot h instances , 

NMDA ha s a neuroprotectiv e effec t o n cerebella r 

granule neurons, however , the molecular mechanis m 

of cel l deat h differ s i n th e tw o model s (Kharlamov 

et al, 1995 ; Bhave et al, 1999 ; Bonn i et al, 1999) . 

It is well-established that ethano l acutel y inhibits 

NMDA recepto r function . Thi s actio n ha s bee n 

demonstrated i n viv o an d i n vitro , and i n recombi - 

nant systems where the NMD A recepto r i s expressed 

in heterologou s cell s (Hoffman , 2003) . These find - 

ings led to the proposal that ethanol ca n promote th e 

death o f cerebella r granul e neuron s b y interferin g 

with th e protectiv e effec t o f NMDA . Interestingly , 

ethanol no t onl y reduce s th e protectiv e effec t o f

NMDA o n cerebellar granule neurons grow n in th e 

presence o f a lo w concentratio n o f K + bu t als o re - 

duces th e toxi c effec t o f NMDA o n cerebella r gran - 

ule neuron s grow n i n a hig h concentratio n o f K + 

(Wegelius an d Korpi , 1995) . Neurons grow n in low 

K+-containing medium are thought t o have the characteristics 

o f immatur e neurons , thu s cerebella r 

granule neurons grown under these conditions represent 

a mode l i n whic h th e effect s o f ethano l an d 

NMDA o n neurona l surviva l durin g developmen t 

can be assessed in a mechanistic manner . 

Cerebellar granul e neuron s grow n i n lo w K + , o r 

subjected t o remova l o f high KC1 , exhibi t apoptoti c 

features, suc h a s DN A fragmentation , nuclea r con - 

densation, somati c blebbing , an d caspas e activation 

(Yan e t al. , 1994 ; Bhav e and Hoffman , 1997 ; Bhave 

et al. , 1999) . Th e deat h o f these neuron s i n cultur e 

appears to mimic the apoptosis observed in the developing 

ra t cerebellu m i n vivo betwee n P 5 an d Pl l 

(Wood e t al., 1993) . Many factors , such a s activation 

of variou s caspases , th e proteasom e an d accumula - 

tion of ubiquinated proteins, mitochondrial function, 

Bel-family proteins , and oxidativ e stress , hav e bee n 

implicated i n apoptoti c cel l los s (Atabay et al., 1996 ; 

Alavez e t al, 2000 ; Can u e t al, 2000 ; Vogel , 2002 ; 

Konishi an d Bonni , 2003) . Treatment o f cerebellar 

granule neuron s wit h NMDA prevent s this apoptoti c 

death (Ya n et al. , 1994 ; Bhav e an d Hoffman , 1997 ; 

Bhave et al, 1999) . 

Ethanol promote s apoptosi s b y interferin g wit h 

the protectiv e effec t o f NMDA (Bhav e and Hoffman , 

1997). Neurons grown in low KCl-containing medium 

for 4 days prior to addition o f ethanol and/or NMD A 

for 2 4 hr experienc e th e sam e amoun t o f apoptoti c 

death as neurons treated with ethanol alone . Ethano l 

also inhibit s th e neuroprotectiv e effec t o f NMD A 

when cerebella r granul e neuron s ar e switche d fro m 

high- t o low-KC l mediu m (Castold i e t al , 1998) . 

NMDA protect s cerebella r granul e neuron s fro m 

apoptosis b y increasin g th e expressio n o f BDNF , 

which i n tur n activate s phosphatidylinosito l 3 - 

phosphokinase (PI3K ) an d Ak t phosphorylatio n 

(Bhave et al., 1999) . Ethano l inhibit s the first step of 

this pathway , th e NMDA-stimulate d increas e i n 

BDNF expression , whic h i s mediate d b y NMDA - 

induced increases in intracellular calcium (Zafra e t al., 

1990, 1991 ; Ghosh et al, 1994). This action of ethanol 

is specific , i.e. , ethano l doe s no t affec t late r step s i n 

the neuroprotectiv e pathwa y (Bhav e e t al. , 1999) . 

Thus, ethanol apparentl y doe s no t interfere with the 

EXTRACELLULAR MEDIATORS OF NEURONA L DEATH 25 5 

protective effec t o f BDN F (Hoffma n e t al , 1994 ; 

Seabold e t al., 1998) . The sit e of action o f ethanol a t 

the NMD A recepto r i s further supporte d b y studies of 

cerebellar granul e neuron s showin g tha t th e in - 

hibitory effec t o f ethano l o n NMD A recepto r func - 

tion (measure d a s a n increas e i n intracellula r 

calcium) an d tha t o n th e protectiv e rol e o f NMD A 

are both attenuate d i n the presence of a high concen - 

tration of glycine, the co-agonis t at the NMDA recep - 

tor (Bhave and Hoffman , 1997) . 

Although ethano l acutel y inhibit s NMD A recep - 

tor function, chroni c exposure of mature neuron s t o 

ethanol up-regulate s NMDA recepto r function (Hoff - 

man, 2003) . This change, i n vivo, is thought t o con - 

tribute t o ethano l withdrawa l hyperexcitabilit y an d 

the brai n damage associate d with long-ter m ethano l 

ingestion. By contrast, chroni c treatmen t o f culture d 

"immature" cerebella r granul e neuron s (grow n i n 

5.0 mM KC1) with ethanol decreases the neuroprotective 

effec t o f NMDA , simila r t o th e effec t o f short - 

term ethano l treatmen t (Bhav e e t al. , 2000) . Th e 

effect o f chronic ( 3 day) exposure to ethano l persists 

even afte r ethano l i s removed (Castold i e t al. , 1998 ; 

Bhave et al., 2000), attributed to decreased expressio n 

of NMD A recepto r subunit s b y th e ethanol-treate d 

"immature" neurons , resultin g i n decrease d NMD A 

receptor-mediated stimulatio n o f BDNF expressio n 

(Bhave e t al. , 2000) . Similarly , chroni c prenata l 

ethanol treatmen t i n vivo reduces th e expressio n of 

NMDA recepto r subunit s i n th e offsprin g (Hughe s 

etal, 1998). 

The ag e of the culture d cerebella r granul e neu - 

rons defines the respons e to ethanol an d NMDA. Fo r 

example, ethanol exposur e induces the deat h o f cerebellar 

granul e neurons harveste d on P10 and treate d 

with ethano l (5 0 o r 20 0 mM) fo r 1 da y (Pantazi s 

et al., 1995) . This death ca n be prevente d b y adding 

NMDA to the cultures (Pantazis et al, 1995) . In contrast, 

i f cells ar e maintaine d i n cultur e (5. 0 mM o r 

25 mM KC1 ) for 7 days and then expose d t o ethano l 

for 2 4 hr, there i s no evidence o f increased cel l death . 

The underlyin g mechanism o f this phenomeno n i s 

unknown. I t mus t b e considere d tha t th e ethanol - 

induced death o f neurons o n the first day in cultur e 

could reflec t interferenc e with cellula r adhesio n t o 

the cultur e dishe s and thu s be a n artifac t o f the cul - 

ture system. Nevertheless, unde r condition s i n which 

ethanol alon e produces neuronal death, a number of 

anti-apoptotic agent s (e.g. , NMDA , neurotrophins , 

and PACAP ) ca n preven t ethanol-induce d deat h


(Pantazis e t al , 1995 ; Beato n e t al. , 1999 ; 2000a , 

ZOOOb; de la Monte et al, 2000; Vaudry et al, 2002). 

Whether thes e result s ar e indicativ e o f th e mecha - 

nism by which ethano l itsel f induces neuronal deat h 

or reflect algebraically additive effects o f ethanol an d 

protective agents has yet to be determined. Suc h studies 

of the tempora l vulnerabilit y of cultured cerebel - 

lar granul e neuron s t o ethanol-induce d deat h ar e 

reminiscent o f the i n vivo studies demonstrating windows 

o f vulnerability of th e cerebellu m t o ethanol - 

induced neurona l los s durin g developmen t i n viv o 

(e.g., Hamre and West, 1993) . 

An i n viv o interactio n o f ethanol an d th e NMD A 

receptor wit h respect t o neuronal deat h i s not well established. 

Studie s reporte d b y Olne y an d colleague s 

(2002b) suggest such an interaction. They found that a 

single treatmen t wit h ethano l produce s apoptoti c degeneration 

i n mous e o r rat brain (Ikonomido u e t al. , 

2000). On th e basi s of patterns o f neurodegeneration 

and comparison s with the effect s o f NMDA recepto r 

antagonists, the y propose d tha t th e pro-apoptoti c ac - 

tion o f ethano l i s mediate d throug h NMD A (an d yaminobutyric 

aci d A ) receptor s (Ikonomido u e t al , 

1999, 2000 ; Olne y et al, 2002a) . In the cerebellum , 

the fact that cerebellar granule neurons undergo apoptosis 

at a specifi c tim e durin g developmen t (Woo d 

et al., 1993 ) and th e possibilit y that this apoptosis i s a 

consequence o f the lac k o f innervation b y the gluta - 

matergic mossy fiber afférents (Balâz s et al., 1988) sug - 

gest tha t inhibitio n o f NMD A recepto r functio n b y 

ethanol a t this critical period o f development i n vivo 

would resul t in inappropriat e cel l loss . Although th e 

mechanism o f cell deat h propose d b y Olney an d col - 

leagues i s consistent with results from th e i n vitro studies, 

th e caveat s raised earlier in thi s chapter , tha t th e 

massive amount o f cell deat h reporte d b y Olney an d 

colleagues is not consistent with studies of the number 

of neurons i n adult animals treated pre - or postnatally 

with ethanol, cas t doubt on the interpretation o f the in 

vivo studies. 

Pituitary Adenylyl Cyclas e 

Activating Polypeptid e 

Activation of the transcription factor cyclic adenosine 

monophosphate (cAMP ) respons e elemen t bindin g 

protein (CREB ) ma y b e a downstrea m mediato r o f 

stimulus-dependent neurona l surviva l (Fig . 15-4 ) 

(Shaywitz an d Greenberg , 1999 ; Finkbeiner , 2000 ; 

Walton an d Dragunow , 2000) . Persisten t phosphory - 

lation of CREB is considered necessary to increase the 

expression o f gene s neede d fo r cel l survival . Therefore, 

activation of several signal transduction pathways 

that contribut e t o CRE B phosphorylatio n ma y pro - 

vide optima l condition s for survival. On e suc h path - 

way i s th e cAMP/protein kinas e A (PKA ) pathway . 

Activation o f the intracellula r messenge r cAM P ca n 

increase neurona l proliferation , differentiation , an d 

survival (Tojim a e t al, 2003 ; Fujiok a e t al, 2004) . 

Many studie s o f th e rol e o f th e cAMP/PKA/CRE B 

pathway in neuronal death hav e used cerebellar granule 

neurons as a model. Raisin g cAMP concentratio n 

in cerebellar granule neurons in many (D'Mello et al., 

1993; Chan g e t al, 1996 ; Kienle n Campar d e t al, 

1997; Mora n e t al., 1999 ; L i et al., 2000 ) but no t all 

(Yan et al., 1995 ) instances has prevented apoptosis . 

PACAP, a cAMP generator, i s a neuropeptide originally 

isolate d fro m a n ovin e hypothalami c extrac t 

(Miyata e t al. , 1989) . This peptide exist s in tw o isoforms 

(PACAP-2 7 and PACAP-38 ) tha t are member s 

of th e vasoactiv e intestina l polypeptid e (VIP) / 

secretin/glucagon family . Bot h isoform s o f PACA P 

have simila r biochemical effects . The y interac t wit h 

high-affinity PAC j receptor s (whic h hav e hig h spe - 

cific affinit y fo r PACAP) and wit h two forms of VPAC 

receptors (whic h have equa l affinit y fo r PACA P an d 

VIP; Harma r e t al., 1998) . Bot h PACA P and severa l 

splice variants of the PAC j recepto r (Spengle r e t al, 

1993) ar e found i n the cerebellu m an d expresse d b y 

cultured cerebella r granul e neuron s (Basill e e t al. , 

1995; Favit et al, 1995; Tabuchi et al, 2001) . In these 

neurons, activation of the PACj receptor leads both to 

cAMP accumulatio n an d polyphosphoinositid e hy - 

drolysis (Basille et al, 1995 ; Favit et al, 1995) . Treatment 

o f cerebella r granul e neuron s wit h PACA P 

attenuates apoptosi s produced eithe r b y growing th e 

neurons in low KCl-containing medium or by switching 

them from a high to low KCl-containing mediu m 

(Cavallaro et al, 1996 ; Campar d e t al, 1997 ; Villalba 

et al , 1997 ; Bhav e and Hoffman , 2004) . PACA P i s 

likely an endogenou s facto r tha t promotes cerebellar 

granule neuron surviva l because treatment of granule 

neurons grow n i n a hig h KCl-containin g mediu m 

with a PACA P receptor antagonist reduce s neurona l 

survival (Tabuchi etal, 2001). 

The mechanis m b y whic h PACA P promote s 

cerebellar granul e neuro n surviva l ha s bee n evalu - 

ated in several studies. The cAMP/PK A pathway and 

the mitogen-activate d protei n kinas e (MAPK ) path - 

way (Villalb a e t al , 1997 ) hav e bee n implicate d

Casuases 

FIGURE 15- 4 Neuroprotectiv e pathways in cerebellar granule neurons. Cerebel - 

lar granule neurons from postnata l rats , cultured i n mediu m containin g 5 mM 

KC1, undergo spontaneous apoptosis . These neurons can b e protecte d b y addition 

o f n-methyl-D-aspartat e (NMDA) , brain-derive d neurotrophi c facto r 

(BDNF), insulin-lik e growth factor 1 (IGF-1), or pituitary adenylyl cyclase activating 

polypeptide (PACAP) to the culture medium . Th e anti-apoptoti c effect of 

NMDA depend s o n NMDA-induced expressio n of BDNF, which i s inhibited by 

ethanol. The protectiv e effect s o f NMDA and BDNF are mediated throug h acti - 

vation o f phosphatidylinositol-3' hydroxy kinase (PBK) , which i n turn activate s 

Akt. NMDA has also been suggeste d to activate Akt directly via Ca 2+ /calrnodulindependent 

protein kinase kinase (CaM-KK) (dotted line) . BDNF can als o promote 

cerebella r granul e neuron surviva l by activation of microtubule-associated 

protein kinas e (MAPK ) extracellula r signal-related kinase (ERK) 1/2 . Activation 

o f this pathway results in phosphorylation of the pro-apoptoti c protei n Bad 

on th e Ser 112 , which i s mediated b y Rsk-2, a member o f the MAPK-activate d 

pp90 ribosomal S6 kinase family. Phosphorylation o f Bad results in dissociation of 

Bad from th e anti-apoptotic Bcl- 2 protein, allowin g the surviva l function of Bcl-2 

to be expressed. Activation of the PBK/Ak t pathway results in phosphorylation o f 

Bad o n Ser 136 , an d phosphorylatio n o n bot h site s ma y be necessar y t o reduc e 

apoptosis (Bonni et al., 1999) . Although treatmen t o f cells with NMDA activate s 

MAPK, a n inhibito r o f thi s pathwa y doe s no t bloc k th e protectiv e effec t o f 

NMDA. Ther e is also evidence tha t the anti-apoptoti c actions of the MAP K and 

PI 3-kinas e pathways may converge at the leve l of cyclic AMP response elemen t 

binding protein (CREB) . CREB activatio n can increas e th e expressio n of bcl-2 

mRNA. The overal l effect o f activation of these pathways is to release Bcl-2 from 

dimerization wit h Ba d and t o increas e Bcl- 2 levels. The anti-apoptoti c effec t o f 

PACAP in cerebellar granul e neuron s i s mediated throug h activatio n o f PKA by 

cAMP and by activation of the PBK pathway via a pertussis toxin-sensitive G protein. 

Ethanol enhance s bot h of these PACAP-stimulated pathways. Protein Kinase 

A (PKA) can phosphorylate Bad at Ser 112 while Akt phosphorylates Bad at Ser 136 , 

as discusse d above. Activation of PKA and PB K als o leads to CREB activation . 

Therefore, the protective effect o f PACAP may depend o n the convergence of two 

signaling pathways at the level of phosphorylation of Bad and/or CREB. 

257


(Campard e t al, 1997 ; Vaudr y et al, 1998) . Ethano l 

acutely enhances receptor-stimulate d cAM P produc - 

tion i n variou s neuronal an d non-neurona l system s 

(Tabakoff an d Hoffman , 1998) . I f ethano l als o en - 

hances PACAP-stimulate d cAM P production , on e 

could postulate tha t ethanol ha s a neuroprotective effect 

when granule neurons are treated simultaneousl y 

with ethanol and PACA P 

The signalin g pathway s tha t mediat e th e anti - 

apoptotic effec t o f PACAP on cerebellar granule neu - 

rons hav e bee n examined . I n cell s grow n i n th e 

absence o r presence o f ethanol i n a medium contain - 

ing 5 mM KC1 , th e anti-apoptoti c effec t o f PACA P 

was reduced b y inhibitors of the cAMP/PK A pathway 

and th e PB K pathwa y (Bhave and Hoffman , 2004) . 

The anti-apoptoti c effec t o f PACAP is evident within 

4 hr . Ethano l (lOOmM ) accelerate s thi s proces s a s 

the anti-apoptoti c effec t o f PACA P i s maximal afte r 

only 1 hr of exposure t o ethanol an d PACAP . The ef - 

fect of ethano l is blocke d by inhibitor s of bot h the 

cAMP/PKA an d th e PB K pathways , an d ethano l 

stimulates PACAP-induced production o f cAMP an d 

phosphorylation o f Akt (Bhave and Hoffman , 2004) . 

In addition , ethano l enhance s PACAP-induce d 

FIGURE 15- 5 Schemati c o f the relationshi p o f neurotrophi n expressio n an d 

neuronal ontogeny . Ther e ar e tw o period s o f cel l deat h durin g neura l on - 

togeny: during the period o f cell proliferation (in the germinal zone [GZ] ) an d 

during the perio d of neuronal differentiatio n (i n the gra y matter). At least th e 

latter i s associated with neurotrophin expression . Neurotrophin expressio n increases 

during the perio d o f neuronal differentiatio n t o support the growt h of 

neuntes an d promote synaptogenesis . Nevertheless, neurotrophin expressio n is 

limited, hence it defines the number o f neurons tha t can successfully compete 

for enoug h neurotrophi n t o maintain thei r survival . Ethanol delay s the num - 

bers of neurons tha t are generated an d successfull y migrate t o the CN S struc - 

ture an d reduce s th e amoun t o f available neurotrophin. Th e consequenc e of 

this reductio n i s that fewe r neuron s survive . Soli d an d broke n line s identif y 

changes for control and ethanol-treate d subjects, respectively. Thicker line s depict 

changes i n the number s of neurons i n a CNS structur e and thinner line s 

describe changes i n neurotrophin expression . The graph s at the botto m depic t 

changes in the numbers of cells involved in a particular ontogenetic event (for a 

control subject).

CREB phosphorylation . These result s indicate d tha t 

ethanol treatmen t o f cerehellar granul e neurons ca n 

increase neuroprotection induce d by PACAP. 

Although exposur e o f th e developin g brai n t o 

ethanol i s associated with a n overal l los s o f neurons , 

and the cerebellu m i s particularly sensitive to ethanolinduced 

neurona l los s (Goodlet t e t al. , 1990 ; Wes t 

et al, 1990 ; Miller , 1992 , 1996b ; Maier e t al, 1999) , 

the finding s fo r PACA P offe r a possibl e approac h fo r 

amelioration o f th e overal l deleteriou s effec t o f 

ethanol. Sinc e PACA P i s endogenousl y presen t i n 

cerebellar granule neurons , th e presenc e o f ethanol a t 

critical times o f development coul d enhanc e the pro - 

tective effec t o f this endogenous factor . Neurona l survival 

during development depend s o n the interpla y of 

various neurotrophi c factors . Ethano l ca n affec t th e 

balance amon g th e effect s o f thes e factors . On e ap - 

proach t o alleviatin g the damagin g effect s o f ethanol 

during centra l nervou s syste m developmen t i s to in - 

crease th e contributio n o f neuroprotectiv e pathway s 

that are targeted by ethanol. 


Ethanol-induced neurona l deat h occur s durin g 

defined period s o f th e developmenta l tim e lin e 

(Fig. 15-5) . These time period s coincide wit h thos e 

of neuronogenesi s an d primar y synaptogenesis , 

which ar e periods of NOND (see Chapter 5) . Exposure 

to ethanol at this time may exacerbate processes 

involved i n NOND. This pattern applie s to parts of 

the brai n i n whic h th e developmen t o f th e con - 

stituent neurons is relatively synchronized, as in th e 

PSN an d cerebra l cortex . A n interestin g counter - 

point i s th e cerebellum , i n whic h population s o f 

neurons follo w nonoverlappin g ontogeneti c tim e 

lines. Fo r example, th e proliferatio n of granule cel l 

precursors coincide s wit h th e synaptogenesi s o f 

Purkinje neurons . I n thi s case , th e windo w of vulnerability 

to ethano l i s narrow; it i s only 2 days (P 4 

and P5), early during the perio d o f Purkinje neuron 

synaptogenesis. 

Ethanol ha s multiple targets , including extracellu - 

lar component s o f growth factor systems , i.e., ligands 

and receptors . Ethano l i s no t a promiscuou s toxin ; 

rather, it affects th e actions of spécifie ligand s (e.g., for 

fetal cortica l neurons, NGF , bu t not BDNF or NT-3). 

Ligands ma y b e temporall y expresse d an d overlai d 

upon pre-existin g systems. This situation i s evident i n 


the developin g periphera l nervou s syste m (Davies , 

1997). Potentially, neurons ar e more susceptible whe n 

only a singl e neurotrophin syste m is active, but the y 

become more resistant to ethanol toxicity as additional 

intercellular communicatio n system s are established . 

How thi s affect s intracellula r processin g i s currentl y 

unknown. A n alternativ e but no t mutuall y exclusive 

mechanism i s that ethanol target s receptors (e.g. , p75, 

but no t trk isoform content) . Recepto r selectivit y may 

reflect th e differentia l distributio n o f p75 an d trk s in 

the plasma membrane, for example, in lipid rafts . 

In summary, ethanol vulnerability is determined by 

extracellularly initiate d events . Th e effect s o f ethano l 

on ligands and receptor s may be additiv e or synergistic. 

Regardless , th e differentia l effect s ar e instructiv e 

about th e rol e o f neurotrophin system s during early 

development. 

Abbreviations 


BEC bloo d ethanol concentratio n 

cAMP cycli c adenosine monophosphat e 

CREB cAM P response element binding protein 

ERK extracellula r signal-related kinase 



IGF-1 insulin-lik e growth factor- 1 

MAPK mitogen-activate d protein kinase 



NOND naturall y occurring neuronal death 



PACAP pituitar y adenyly l cyclas e activatin g 

polypeptide 

PI3K phosphatidylinosito l 3'-phosphokinase 

PKA protei n kinase A 

PSN principa l sensory nucleus of the trigeminal 

nerve 

trk tyrosin e kinase receptor 

TUNEL termina l uridylated nick-end labelin g 

VB ventrobasa l nucleus of the thalamu s 

VIP vasoactiv e intestinal polypeptid e


ACKNOWLEDGMENTS Thi s chapte r wa s written with 

the suppor t o f the Nationa l Institute s o f Alcohol Abus e 

and Alcoholism (MBB , PLH, an d MWM ) an d th e De - 

partment of Veterans Affairs (MWM) . 

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16 

Intracellular Events in 

Ethanol-Induced Neuronal Death 

Developmental exposur e to ethanol ca n significantly 

reduce the number o f neurons i n specific areas of the 

brain. This may result from a decrease i n the additive 

processes of cell generation an d migratio n (Chapters 

11, 12 , and 13) and/o r an increas e in th e subtractiv e 

process o f cell deat h (se e Chapter 15) . The presen t 

Chapter explore s the effect s o f ethanol o n intracellular 

pathways involved in neuronal death. 

Before embarkin g on a description o f the various 

pathways and th e effect s o f ethanol o n them, it is important 

t o acknowledge caveat s that ma y affect inter - 

pretation o f the relevan t data . Exposur e t o ethano l 

alters expressio n and/o r activatio n o f many protein s 

involved i n apoptosis . Evidenc e show s that ethano l 

can trigger intrinsic and extrinsic apoptotic pathways. 

For example, ethanol-induced cel l death i n vivo ca n 

be mediate d by Bel proteins , i.e. , the intrinsi c pathway 

(Mooney and Miller , 2001; Young et al, 2003). 

Studies with organotypic cultures , by contrast, show 

that Fa s receptor expressio n ca n als o b e affecte d b y 

ethanol (e.g. , Cheema et al., 2000; de la Monte and 



George I . Henderson 

267 

Wands, 2002) , thu s implicatin g th e extrinsi c path - 

way. Par t o f th e resolutio n o f thi s riddl e i s tha t 

ethanol ma y trigge r deat h throug h bot h pathways . 

The relevan t research is thus based on a variety of (in 

vivo an d i n vitro ) model s tha t encompas s multipl e 

variables, such a s the pea k concentration o f ethanol , 

the duration of the exposure, the type of cell or tissue 

being examined, and the time after exposur e that th e 

cells o r tissue s wer e examined . Althoug h thes e different 

model s ma y provid e complementar y ap - 

proaches, the y ma y also generat e nonphysiological , 

model-dependent responses . Thus , i t is challengin g 

to reconstruc t the mechanism s of ethanol-induce d 

apoptosis. 

When reviewin g the literatur e o n th e effect s o f 

ethanol o n neurona l death , i t must b e kep t i n min d 

that developmenta l exposur e t o ethano l interfere s 

with othe r developmenta l events , notably cell prolif - 

eration an d migratio n (se e Chapters 11 , 12 , and 13) . 

Without includin g th e effect s o f ethano l o n thes e 

early ontogenetic events the data ma y be confounde d


and misinterpretation s ma y result . Fo r example , 

longitudinal studies of the effect s o f ethanol on neural 

cell number in vitro (Jacobs and Miller, 2001; Miller, 

2003) o r i n viv o (Miller , 1999 ) documen t a signifi - 

cant reductio n i n cel l number . Two-third s o f thi s 

change is due to a depression in cell proliferation; the 

remaining thir d result s fro m ethanol-induce d cel l 

death. Th e consequen t lesso n mus t be heeded by alcohol 

investigator s examinin g cel l number : reduc - 

tions i n cel l numbe r ar e no t necessaril y due t o cel l 

death. 

CELL DEAT H PATHWAYS 

The effect s o f ethanol o n thre e pathway s of neuronal 

death ar e discusse d below : (1 ) th e intrinsi c caspase - 

dependent pathway, (2) the extrinsic caspase-dependen t 

pathway, an d (3 ) th e caspase-independen t pathwa y 

(Fig. 16-1) . These pathways are described i n greater 

detail i n Chapte r 6 , bu t a brie f outline i s included 

below. 

The intrinsi c pathway i s mitochondriall y medi - 

ated, typically in response to an apoptotic signal such 

as DN A damag e o r reactiv e oxyge n specie s (ROS) . 

An initia l even t i s releas e o f proapoptoti c protein s 

from th e niitochondria l intermembran e space . Per - 

meabilization o f the mitochondrial oute r membran e 

appears to be mediated b y Bcl-2 family protein s an d 

may includ e direc t binding o f p53 t o on e o r mor e 

anti-apoptotic proteins , suc h a s Bcl-X L o r Bax . Upregulation 

of Bax may allow insertion of Bax-Bax homodimers 

into the mitochondrial membrane, which 

alters membrane permeability and allows intermembrane 

substances to escape int o the cytoplasm . On e 

such substance , Cytochrom e C , associate s wit h 

apoptotic proteas e activatin g factor 1 and caspase- 9 

in th e presenc e o f adenosin e triphosphat e (ATP ) 

to for m a n apoptosome . Thi s activate s caspase-9 , 

which i n tur n activate s caspase-3. Activ e caspase-3 

inactivates poly-adenosin e diphosphat e ribos e poly - 

merase (PARP) 1 , thus repressing DNA repair mechanisms. 

I n combinatio n wit h activation of enzymes 

such a s DN A fragmentatio n factor 40 , th e resul t is 

DNase-mediated fragmentatio n o f DN A an d cel l 

death. 

The extrinsi c pathway is activated b y binding of a 

ligand t o a cel l surfac e receptor . On e exampl e i s 

the bindin g o f Fas ligand (FasL ) to it s receptor Fas . 

This bindin g cause s recepto r oligomerization , an d 

FIGURE 16- 1 Schemati c of three pathways leading to 

cell death . Thre e pathway s o f cel l death , outlinin g 

the flow of information fro m externa l effects t o nuclear 

response, are depicted: intrinsic and extrinsi c caspasedependent 

pathway s and a caspase-independent pathway. 

A subse t o f th e molecule s involve d i n thes e 

pathways is shown; a more comprehensiv e figur e de - 

tailing the pathways is shown in Figure 6- 1 i n Chapter 

6 . Factor s know n t o b e affecte d b y ethano l ar e 

identified. Smal l arrow s sho w th e effec t o f develop - 

mental exposur e t o ethano l o n eac h factor . Th e 

blunted line signifies an inhibitor y effect. AIF , apoptosis 

inducin g factor ; PARP , poly-adenosin e diphos - 

phate ribose polymerase; ROS, reactive oxygen species. 

See text for details. 

recruitment o f th e Fas-associate d deat h domai n 

(FADD) and procaspase s 8 and 1 0 (Benn and Woolf, 

2004). Th e FADD-caspase-8/1 0 comple x form s th e 

death-inducing signalin g comple x tha t cleave s an d 

activates caspase-3. As with the intrinsi c pathway, active 

caspase-3 inactivates PAR P and allow s DNA frag - 

mentation. I n addition , activ e caspase- 8 cleave s th e 

Bel family protein, Bid. Truncated Bi d translocates to 

the mitochondri a wher e i t can activat e the intrinsic 

pathway b y promoting insertio n of Bax-Ba x homod - 

imers into the mitochondrial membrane. 

Like th e intrinsi c pathway , the caspase-indepen - 

dent pathwa y is mitochondria-dependent. Followin g 

release o f apoptosi s inducin g facto r (AIF ) fro m th e 

mitochondrial intermembran e space , it can eithe r directly 

up-regulate DNase activity or cleave and inacti - 

vate PARP, thus repressing DNA repair and promotin g 

cell degeneration.

EFFECT O F ETHANOL ON APOPTOSIS 

Intrinsic Pathway s 

Bel Family Proteins 

Exposure t o ethano l alter s th e i n viv o expressio n o f 

members o f the Be l protein family. This is apparent i n 

changes of the mRNAs and the proteins. Changes ma y 

be rapid , as in the cas e of the mRN A (Moor e et al. , 

1999; Inoii e et al, 2002), or occur later , as detected i n 

protein expressio n (Mooney and Miller , 2001). Corti - 

cal expressio n of Bcl-2 , a prosurviva l membe r o f th e 

family, i s lowe r i n th e cortice s o f rat s expose d t o 

ethanol prenatally , most notably in the period aroun d 

postnatal day (P) 6. This finding is particularly interesting 

i n light of the lac k of change i n th e expressio n of 

bax in cerebral cortex , the net result being a reduction 

in th e rati o of Bcl-2 to Ba x on gestationa l day (G) 16 , 

P6, an d P12 . Thes e times , prenata l an d postnata l 

week, coincid e with th e period s o f both naturall y occurring 

and ethanol-induced cell death i n the prolifer - 

ative zone s an d i n th e immatur e corte x (Küh n an d 

Miller, 1998 ; se e Chapter 5) . It is noteworthy that an - 

other immunoblotting study of the cortice s of rats prenatally 

exposed to ethanol showed an increase in Bcl-2 

expression on PI an d no change betwee n P 7 and P35 

(Climent e t al. , 2002) . Th e difference s i n reporte d 

Bcl-2 expressio n i n th e tw o studie s ma y reflec t th e 

feeding paradigms . Although fetuse s were expose d t o 

ethanol i n both studies , i n the stud y by Climent and 

colleagues ethanol exposur e continued into the postnatal 

period. 

The chang e i n corte x contrast s wit h the lac k o f a 

change in the relative amounts of bcl-2 and bax in the 

thalamus. The thalamu s is a structure that exhibits no 

neuronal deat h followin g prenata l exposur e t o 

ethanol (Moone y and Miller, 1999 ; Liv y et al, 2001). 

Thus, these dat a indicat e tha t the chang e i n the bal - 

ance o f bcl- 2 an d ba x i s a ke y determinan t o f neu - 

ronal death an d survival. 

The expressio n o f Be l protein s i s altere d i n th e 

murine cerebellum followin g earl y postnatal exposure 

to ethanol (Heaton et al, 2002a, 2003a). Expression of 

the anti-apoptoti c protei n Bcl- 2 decreases an d o f proapoptotic 

Ba x an d Bcl-x s increase s i n concomitan t 

temporal manner s tha t correlat e wit h th e perio d o f 

Purkinje cell vulnerability (Heaton e t al., 2003a). That 

is, during the period of vulnerability, on P4, expression 

of Bax and Bcl-x s increases within 2 hr o f exposure to 

INTRACELLULAR EVENTS IN ETHANOL-INDUCED NEURONAL DEATH 26 9 

ethanol. I n contrast, ethanol exposur e on P7, a time of 

relative invulnerability , causes a n earl y up-regulatio n 

of th e survival-promotin g proteins, Bcl- 2 an d Bcl-xl , 

and a down-regulation of the pro-apoptotic Bcl-xs. 

The change s i n protein expressio n apparentl y follow 

earlie r changes i n transcrip t expression. For example, 

exposur e t o ethano l induce s a n immediat e 

change i n th e expressio n o f bd famil y transcript s i n 

the cerebra l corte x an d cerebellu m (Moor e e t al. , 

1999; Inoue et al., 2002). Ethanol up-regulate s the expression 

o f pro-apoptotic genes , ba x and bcl-xs, in th e 

cerebellum. Interestingly , however, the up-regulatio n 

of hax i n the cerebellu m occur s in the absenc e o f an 

effect o n cell survival . This finding concurs with data 

from a strain of bax knockout animals in which ther e 

was n o increas e i n cell deat h followin g exposur e 

to ethano l (Youn g et al. , 2003) . Overexpressio n o f 

bcl-2, by contrast, confer s invulnerability to ethanol - 

induced deat h o f cerebella r Purkinj e cells (Heato n 

etal, 1999) . 

Cytochrome C 

Release of cytochrome C from mitochondria i s a central 

even t i n th e intrinsi c apoptoti c pathwa y (Polste r 

and Fiskum, 2004). This release, combined with that 

of othe r proapoptoti c proteins , elicit s activatio n of 

initiator caspase s tha t ultimatel y produce s effecto r 

caspase activation . Thus , mitochondria l releas e o f 

Cytochrome C i s an early , feed-forward ste p i n th e 

apoptotic process , and combined wit h subsequent ac - 

tivation o f effecto r caspases , i s compelling evidenc e 

that apoptotic death is a work in progress. 

Several laboratorie s hav e use d Cytochrom e C release 

to address ethanol-mediated apoptotic brai n cell 

death followin g in viv o ethano l exposure . Appropriately, 

thes e observation s have been linke d t o activation 

o f caspase-3 . Mitochondri a isolate d fro m feta l 

brains, that have been expose d to ethanol in utero (on 

G17 an d G18) , exhibit enhanced releas e of both cytochrome 

C an d AI F (Ramachandra n e t al. , 2001) . 

This resul t parallel s th e activatio n o f caspase- 3 i n 

whole feta l brai n homogenates . Simila r response s 

have been reported followin g ethanol treatment o f 4to 

10-day-ol d ra t pup s (Ligh t e t al. , 2002 ; Carlon i 

et al, 2004) or infant mice (Youn g et al, 2003). 

The ethanol-induce d i n viv o releas e o f Cyto - 

chrome C ha s been replicate d i n vitro. Exposure o f 

cultured fetal rat cortical neurons to ethanol generates 

enhanced releas e o f Cytochrom e C withi n 2 hr o f


exposure (Ramachandra n e t al. , 2003) . Thi s i s followed 

b y activation o f caspase-3 within 1 2 hr o f treatment. 

Thus, neurons exposed to ethanol eithe r in vivo 

or i n cultur e ca n elici t mitochondrial releas e o f Cytochrome 

C in some, as-yet, undetermined manner . 

Caspases 

The researc h community has focused on the effects of 

developmental exposur e t o ethanol o n tw o caspases : 

caspase-3 and caspase-8. These are of interest because 

(a) the y ar e fro m tw o differen t molecula r subgroup s 

and (b ) they are involve d i n tw o pathways; caspase- 3 

is associated wit h the intrinsi c apoptotic pathwa y an d 

caspase-8 with the extrinsic pathway (Fig. 16-1) . 

There i s considerable i n vitro (Sait o e t al. , 1999 ; 

Jacobs an d Mille r 2001 ; Jan g e t al, 2001 ; Sohm a 

et al, 2002; Vaudry et al, 2002; Ramachandran e t al., 

2003) an d i n vivo (Moone y an d Miller , 2001 ; Ra - 

machandran et al, 2001; Climent e t al, 2002; Light 

et al., 2002; Olney et al, 2002a, 2002b; Mitchell an d 

Snyder-Kelly, 2003 ; Ragjopa l e t al. , 2003 ; Youn g 

et al., 2003; Carloni e t al, 2004) evidenc e that exposure 

t o ethano l increase s the activatio n of caspase-3. 

This activation ma y be model-dependent. Fo r exam - 

ple, Sohm a an d colleague s (2002 ) repor t tha t al - 

though bot h C 6 ra t gliom a cell s an d A54 9 huma n 

adenocarcinoma cell s show evidence of apoptosis following 

exposure to ethanol th e C6 cells increase thei r 

caspase activity, whereas the A549 cells do not. 

Ethanol cause s apoptosi s (a t least i n vitro ) whe n 

trophic factor s ar e i n limite d suppl y (Oberdoerste r 

and Rabin, 1999) . Cerebellar granul e neuron s grow n 

in serum-containin g mediu m supplemente d wit h 

high concentrations o f potassium chloride do not undergo 

apoptosi s i n respons e t o ethanol . Remova l o f 

the seru m result s i n increase d activatio n o f caspase- 3 

and apoptosis in response t o ethanol . 

To date, there ar e few data regardin g the effec t of 

ethanol o n othe r caspases . One i n vitr o study shows 

that ethanol-induced deat h of cerebellar granul e neu - 

rons i s accompanied b y an increas e i n activatio n o f 

initiator caspase s 2, 8, and 9, and effecto r caspase s 

3 and 6 (Vaudry et al, 2002). This deat h i s abrogated 

by the additio n o f pituitary adenylate cyclas e activating 

polypeptid e (PACAP ; se e Chapte r 15) . Additio n 

of exogenou s PACA P ameliorate s th e activatio n o f 

caspases 3 an d 6 , bu t no t caspase s 2 , 8 , o r 9 . Thi s 

implies that activation of an initiator caspase does not 

oblige a cell to die. 

Extrinsic Pathway s 

Although littl e is known about th e effec t o f developmental 

exposur e to ethanol on tumor necrosis factor a 

or Apo2/TRAIL systems in the brain , th e Fas L syste m 

has been studie d in vivo and i n vitro. Fas and Fas L are 

transiently expresse d i n developin g corte x during th e 

time o f naturally occurrin g neurona l deat h (Cheem a 

et al., 2000). Exposure t o ethanol induce s Fa s mRNA 

expression in vitro, but only at subapoptotic concentra - 

tions and no t at concentrations that induce apoptosis. 

Similarly, there i s an increas e i n Fa s receptor an d lig - 

and i n cultures o f cerebellar granul e cell s taken i n th e 

postnatal perio d fro m rat s expose d t o ethano l prena - 

tally (de la Monte an d Wands, 2002). Other evidence 

showing tha t ethano l activate s extrinsi c pathway s is 

that th e ethanol-induce d deat h o f cultured cerebella r 

granule cells is accompanied b y an increase in the ac - 

tivity of caspase-8 (Vaudry et al., 2002). 

Caspase-Independent Pathway 

In a numbe r o f neurona l cel l deat h paradigm s i n 

which caspase s are normall y activated, inhibitio n of 

caspase activity delays but does not prevent cell deat h 

from occurrin g (Mille r e t al. , 1997 ; Stefani s e t al. , 

1999; D'Mell o e t al, 2000 ; Keramari s e t al, 2000 ; 

Selznick e t al., 2000). Suc h dat a impl y that prenata l 

exposure to ethanol affect s caspase-independen t pathways, 

bu t fe w dat a directl y addres s thi s possibility . 

Ethanol exposur e doe s induc e th e releas e o f Cy - 

tochrome C and AIF from mitochondri a (Ramachan - 

dran e t al. , 2001) . Consequently, AI F ca n initiat e a 

feedback loop that further induce s Cytochrome C release 

fro m th e mitochondri a (Porter , 1999) . Thus , 

ethanol-induced initiatio n o f caspase-independen t 

death can be a self-perpetuating phenomenon. 

EFFECTORS OF CELL DEAT H 

p53-Related Factors 

p53 i s a critica l fulcru m definin g th e fat e o f cells : 

whether the y di e o r surviv e (Mille r e t al. , 2000 ; 

Morrison et al, 2002). The teeter-totter i s pushed on e 

way or the othe r b y the phosphorylatio n of serines on 

p53, and particularly by the positions of the phospho - 

rylated serines . Although a n effec t o f ethanol o n thi s 

process ha s been surmise d (Küh n and Miller , 1998) ,

direct data are lacking. Nonetheless, i t is known that 

ethanol ca n affec t kinas e activit y i n othe r system s 

(e.g., Resnicoff et al, 1994 ; Luo and Miller, 1999) . 

In additio n to potential effect s o f ethanol on posttranslational 

modification of p53, the effectivenes s o f 

p53 can be affected b y an ethanol-induced change in 

the amount of p53 available. Indeed, p5 3 expression 

in vivo is affected b y prenatal exposure to ethanol i n a 

spatiotemporal manne r (Küh n an d Miller , 1998) . 

Ethanol increases p53 expression in the fetu s and reduces 

it in the first and second postnatal weeks. Interestingly, 

followin g chroni c gestationa l exposur e t o 

ethanol, expression of p53 is increased in the rat cerebellum 

durin g th e earl y postnata l perio d (d e l a 

Monte an d Wands , 2002) . The increas e i n p5 3 occurs 

concurrent with increases in Fa s ligand an d re - 

ceptor, implying that the increas e in p53 is related to 

cell death. Although many ethanol-susceptible activities 

occu r coincidentall y i n th e developin g cere - 

bellum, tw o are wort h noting : th e reductio n i n th e 

population o f proliferatin g granul e cell s (Bauer - 

Moffett an d Altman, 1977; Kornguth et al., 1979) and 

the death of Purkinje neurons (e.g., Light et al, 2002; 

see Chapter 15) . Thus, the ethanol-induce d increase 

in cel l mortalit y among th e proliferatin g neurona l 

precursors in the corte x and cerebellu m ma y be signaled 

by an increase in p53 expression. 

A phosphorylated form o f p53 may be recognize d 

by th e antibod y ALZ-5 0 (Küh n an d Miller , 1998) . 

ALZ-50 immunoreactivit y i s associate d wit h dyin g 

central nervou s syste m (CNS ) neuron s (Al-Ghou l 

and Miller , 1989 ; Valverd e et al., 1990 ; Mille r e t al, 

1991). Interestingly, the expressio n profile o f ALZ-50 

in the developin g cortex parallels that of p53 expression 

(Kühn and Miller, 1998) . Moreover, prenatal exposure 

t o ethano l affect s bot h protein s similarly . I t 

reduces the amoun t o f peak ALZ-50 and p53 expression 

an d induce s this peak to occur earlier. The im - 

plication is that ethanol alters the timing of neuronal 

death in the developing cortex. 


Basic Findings 

Over the last four decades, studies have linked ethano l 

intake to oxidative stress (Di Luzio, 1963 ; Sha w et al., 

1983). The earlies t reports connecting maternal drinking 

t o feta l oxidativ e damage wer e b y Dreosti (1987 ; 

Dreosti and Patrick, 1987), who reported that maternal 


ethanol intak e increased malondialdehyde (MDA ) in 

fetal ra t live r mitochondria . Thi s increas e ha s als o 

been describe d i n culture d feta l hepatocyte s (Dev i 

et al. , 1994 , 1996) . Likewise , ethano l ca n increas e 

both MD A an d conjugate d diene s i n feta l brain . 

Chronic prenata l ethano l exposur e ca n reduc e glu - 

tathione (GSH ) conten t o f fetal brai n (Reye s e t al, 

1993) an d ethano l produce s fre e radica l damag e i n 

neural cres t cell s (Che n an d Sulik , 1996) . Thus , 

ethanol-mediated oxidativ e damag e t o feta l cell s 

occurs in vivo and this can be reproduced i n culture d 

fetal cells. These data sets laid the conceptual groundwork 

fo r numerou s subsequen t report s o n ethanol - 

related oxidativ e damag e t o feta l cells . The y ar e 

reviewed below in the context of mechanisms underlying 

ethanol-mediated apoptotic death of neurons. 

Ethanol induces oxidative stress and activates apoptotic 

pathways in the developing brain and i n cultured 

neurons (d e l a Monte e t al, 2001 ; de l a Monte an d 

Wands, 2001 ; Ramachandra n e t al. , 2001 , 2003 ; 

Heaton et al, 2002b, 2003a, 2003b, 2003e). The parallel 

observation s of these tw o ethanol-related phenom - 

ena are correlative and do not establish oxidative stress 

as a causative factor for neuronal death. Extensive documentation 

o f suc h concomitan t events , combine d 

with the effectivenes s o f anti-oxidant interventions and 

time lines illustrating that onset of oxidative stress ca n 

precede apoptosis , i s convincing evidence tha t oxidative 

damage is one mechanism underlying the ethanol - 

mediated loss of neurons in the developing brain. 

In utero exposure to ethanol o n G17 and G18 can 

elicit bot h oxidativ e damage t o brain mitochondria , 

such a s 4-hydroxynonenal (HNE) formation, an d in - 

duction of markers of mitochondria-mediated apopto - 

sis. Th e latte r include s change s o f mitochondria l 

permeability transition , activation o f caspase-3 , an d 

mitochondrial releas e of Cytochrome C and AIE (Ramachandran 

e t al. , 2001) . A mechanism underlying 

the ethanol-mediate d leakag e o f pro-apoptoti c pro - 

teins from mitochondri a i s activation of mitochondri - 

ally generated HNE . Mitochondri a isolate d from th e 

brains o f fetuse s treate d wit h HN E mimi c th e re - 

sponse observe d i n th e brain s o f rodents expose d t o 

ethanol. Th e abilit y o f HN E t o elici t apoptosi s i n 

neurons makes it an attractiv e mechanism, however, 

a clear causative link remains to be established . 

Ethanol-related increases in ROS have been examined 

in young rats acutely exposed to ethanol presented 

as a binge exposure . Increased amounts of ROS were 

detected in the cortices of these rats on P7 and in their


FIGURE 16- 2 Ethanol-induce d oxidativ e 

stress originating in mitochondria. Feta l cor - 

tical neurons, grow n on coverslips , were exposed 

t o ethano l (4. 0 mg/ml) fo r 2 hr a t 

37°C. Afte r ! 3 /4hr, hydroethidiu m (Het ) 

(400 nM) , whic h detect s generatio n o f 

Superoxide anio n radicals , an d 5 0 nm Mi - 

totracker Gree n (MG) , whic h label s mito - 

chondria, were added t o the medium . Liv e 

cerebella o n P4 , P7 , o r P1 4 (Heato n e t al , 2002b , 

2003c). This ethanol exposur e als o increased cortica l 

Bax, pAkt, and pJNK expression. Taken together, thes e 

data strongl y suppor t th e occurrenc e o f enhance d 

apoptotic deat h concomitan t wit h oxidativ e stress . 

Cerebella o f 2-day-old rat s exposed to ethanol i n utero 

are hypoplastic and exhibit increased amounts ofapop - 

tosis, beyond that occurring naturally (de la Monte and 

Wands, 2002) . I n culture d granul e neuron s isolate d 

from thes e cerebella , th e previou s ethano l exposur e 

generated increase d oxidativ e stress (dihydrorosarnine 

fluorescence) an d apoptosi s (evidenced by phosphorylation 

of GSK-3ß, Akt, and Bad), along with inhibitio n 

of insulin-stimulate d viabilit y an d pro-apoptoti c gen e 

expression (e.g. , increased ba d expression). Thus, ges - 

tational ethano l exposur e impair s neurona l surviva l 

systems and insuli n signaling mechanisms durin g th e 

early postnatal period . These ethanol effect s appea r to 

be mediated b y oxidative stress. 

The abov e reports document tha t ethanol ca n con - 

comitantly elicit oxidative stress and enhance apoptosi s 

in the developin g brain . Using in vitro models, several 

laboratories have shown that oxidative stress can play a 

direct role in ethanol-mediated neuro n death . One approach 

probe d th e causality of oxidative stress by determining 

i f thi s phenomeno n precede d o r followe d 

evidence o f apoptosis and/o r decrease d neuro n viabil - 

ity (Ramachandran e t al, 2003). In culture d feta l ra t 

cortical neurons, confocal microscopy of live cells preloaded 

wit h dichlorofluorescei n diacetat e illustrate d 

that ethano l ca n stimulat e RO S productio n withi n 

minutes o f exposure. This initia l ROS burs t persisted 

for 2 hr and wa s followed b y increased HN E i n neu - 

ron mitochondria (withi n 2 hr; Fig. 16-2) . By 2 hr, an 

cell image s were acquired through confoca l 

microscopy wit h a n argo n lase r t o excit e 

MG and an He-Ne543 laser to excite Het. A. 

This imag e identifie s enhance d generatio n 

of Superoxide anion radicals in discrete areas 

in th e cel l bod y an d i n th e processes . Th e 

Het fluorochrom e freel y crosse s th e plasm a 

membrane, i t is very specific for the superox - 

ide anio n radical , and , a t nanomola r con - 

centrations, i t preferentiall y localize s i n 

mitochondria. B . Mitochondri a ar e distributed 

i n th e perinuclea r regio n a s well as in 

the processes . C . Th e exten t of the neurite s 

can b e appreciate d i n a differential interfer - 

ence contrast image of the neuron.

increase in annexin V binding is seen, along with increased 

Cytochrom e C release . Thes e event s wer e 

followed b y caspase-3 activation. This time course of 

ethanol responses , wit h increase d RO S conten t oc - 

curring a s a n upstrea m event , suggest s causality . 

More convincin g evidenc e i s th e mitigatio n o f 

ethanol-mediated apoptosis by prevention of oxidative 

stress vi a normalizatio n o f neuron glutathion e con - 

tent (Ramachandra n e t al., 2003 ) and b y other antioxidant 

interventions (see Anti-oxident Interventions, 

below). 

Potential Role for Mitotoxic 

Effects of Ethanol 

The specifi c mechanism(s ) underlyin g ethanol - 

induced apoptosi s and oxidativ e stress remains undetermined 

an d ar e likel y rnultifactoria l an d cel l 

type-dependent. One consisten t finding from numer - 

ous laboratories , however , i s that ethano l i s a mito - 

toxin, and mitochondrial damage may be an important 

player i n both th e enhance d productio n of ROS an d 

apoptotic deat h associate d with ethanol exposur e (Hirano 

et al., 1992 ; Kukielka et al., 1994 ; Garcia-Rui z et 

al., 1995 ; Dev i e t al. , 1996 ; Che n e t al., 1998 , 2002 ; 

Goodlett and Horn, 2001; Mooney and Miller, 2001; 

Bailey and Cunningham, 2002). 

With respec t t o th e fetus , there i s abundant evi - 

dence tha t ethano l negativel y alter s mitochondria l 

function i n liver and brain . Among the earlies t fetal - 

related studie s are report s that , i n culture d feta l ra t 

hepatocytes, th e ethanol-relate d increase s i n RO S 

(H2O2, Superoxid e anio n radical ) an d membran e 

lipid peroxidatio n ar e parallele d b y morphologica l 

and biochemica l mitochondria l damag e suc h a s reduced 

comple x I an d I V activitie s an d decrease d 

synthesis of ATP (Devi et al, 1993, 1994) . Since inhibition 

o f respirator y chai n component s stimulate s 

production o f ROS , i t ha s bee n propose d tha t thi s 

could be the origin of the increased RO S levels. More 

recently, this phenomenon ha s been reporte d in feta l 

brain an d i n cultured cerebellar and cortica l neurons 

exposed to ethanol. Followin g an i n utero binge expo - 

sure to ethanol (G1 7 and G18), mitochondria isolated 

from feta l ra t brain hav e elevate d level s o f the highl y 

pro-apoptotic product of lipid peroxidation, HNE (Ramachandran 

et al., 2001). This i s accompanied by increased 

mitochondria l permeabilit y transitio n an d 

release o f pro-apoptotic proteins. Likewise, cerebellar 

neurons isolate d fro m ethanol-expose d 2-day-ol d ra t 


pups exhibit signs of oxidative stress and activate pathways 

o f apoptosi s alon g wit h altere d mitochondri a 

morphology and decreased cell number (d e la Monte 

and Wands , 2002). Furthermore, in vitro exposure of 

insulin-sensitive immatur e huma n PNET2 neurona l 

cells t o ethano l produce s a variet y o f mitotoxi c re - 

sponses, includin g a strikin g reduction o f comple x 

IV/Cytochrome C oxidas e (CytOx ) expression . Although 

n o measures of CytOx activitie s have been reported, 

inhibitio n of this complex ha s been shown to 

stimulate formation of ROS (Shigenaga et al., 1994). 

Parallel to impaired mitochondrial function is evidence 

of apoptosis. Similarly, following 24 hr of exposure 

t o ethanol , ra t postmitoti c cerebella r granular 

neurons sho w an inhibitio n o f multiple measure s of 

insulin-stimulated mitochondria l function , sign s o f 

apoptosis, an d generatio n o f H 2O2 (d e l a Mont e 

et al, 2001). Thus, disruption of mitochondrial func - 

tion i s a central event in the apoptoti c death o f cells, 

and oxidativ e stress, which i s often a product of mitochondrial 

damage , i s a proven initiator of apoptosis. 

There i s compelling evidenc e that th e mitotoxi c effects 

of ethanol play a role in ethanol-mediated apop - 

totic death of neurons in the developing brain. 

Anti-oxidant Interventions 

Many anti-oxidan t interventions aimed a t ameliorat - 

ing th e effect s o f ethano l o n th e developin g brai n 

have use d vitami n E (oc-tocopherol) , a lipid-solubl e 

anti-oxidant that preferentially concentrates i n lipoid 

compartments o f the cell , wher e it can bloc k mem - 

brane lipid peroxidation. I t is present i n mitochondr - 

ial membranes (approximatel y 1 molecule pe r 210 0 

molecules o f phospholipid) an d i s a highly effectiv e 

peroxyl radica l tra p wit h a one-electro n oxidatio n 

product tha t can underg o several reactions that con - 

sume (bu t ca n als o generate ) radical s (Lieble r an d 

Burr 1992) . As such, i t is a late-stage defense against 

the formatio n o f toxi c lipi d peroxidatio n product s 

such as HNE. This capability, combined with its relative 

low toxicity, makes its use an alluring anti-oxidant 

intervention strategy. 

Ethanol-induced deat h o f culture d neonata l ra t 

hippocampal cell s can be mitigated by treatment with 

vitamin E o r ß-caroten e (Mitchel l e t al. , 1999a , 

1999b). Ethano l activate s caspase-3 and increase s at 

least on e marke r o f apoptosi s (annexi n V binding) . 

Both vitami n E an d th e anti-oxidan t Pycnogeno l 

(a bioflavonoi d mixture ) bloc k thes e response s


(Siler-Marsiglio e t al , 2004) . Likewise , vitami n E 

enhances th e surviva l o f ethanol-expose d culture d 

cerebellar granule cells. This survival occurs concomitantly 

wit h increase d amount s o f th e anti-apoptoti c 

proteins Bcl-2 , Bcl-x l an d activate d Ak t kinas e 

(Heaton e t al. , 2004) . Th e mechanism s underlying 

the protective effects o f vitamin E remain to be established, 

bu t i t is tempting t o propose tha t blockad e of 

membrane (possibly mitochondrial) lipid oxidation is 

an important factor . The settin g i s complex an d vitamin 

E treatmen t ca n normaliz e cellula r GS H con - 

tent as well as prevent an ethanol-related reduction of 

neurotrophin secretio n (Heato n e t al., 2004). The former 

manipulatio n can , b y itself , bloc k th e ethanol - 

mediated apoptosi s o f culture d cortica l neuron s 

(Ramachandran etal., 2003). 

The ultimat e clinica l value o f anti-oxidant treatment 

fo r the neurotoxi c effects o f ethanol i n th e de - 

veloping brain remain s to be determined. A possible 

insight int o th e futur e o f suc h intervention s i s re - 

flected b y a study in which behavioral alterations induced 

b y ethanol wer e at best only partially alleviated 

by vitami n E treatmen t (Marin o e t al. , 2004 ; Tra n 

et al. , 2005) . Th e pathogenesi s o f FA S behavioral 

deficits i s complex an d oxidativ e damage t o neuron s 

is likely only one underlying mechanism. The abov e 

reports, however , d o suppor t th e concept s tha t 

ethanol ca n induc e oxidativ e stress i n neuron s fro m 

the developin g brain an d tha t thi s i s one mean s by 

which apoptotic deat h i s induced. 


Evidence i s abundant that exposure to ethanol i n vivo 

or in vitro can caus e neuronal death an d tha t i t does 

so via multipl e pathways : the caspase-dependen t in - 

trinsic an d extrinsi c pathways, and th e caspase-inde - 

pendent pathway . Th e integration , o r cross-talk , 

among thes e pathway s i s extensive . Nevertheless , 

there i s compelling evidenc e tha t eac h o f the thre e 

pathways i s targeted b y ethanol. Furthe r analyse s of 

the effect s o f ethanol migh t b e bes t focuse d o n ke y 

upstream players such as p53 and receptors that medi - 

ate death-relate d signals , a s wel l a s PARP , a player 

presumed t o be commo n t o all three pathways . Th e 

factors that define the effect s o f ethanol ar e the developmental 

stat e o f the cell s and th e concentratio n o f 

ethanol. Thes e issue s ar e addresse d i n th e previou s 

chapter. 

Although beyond the scop e of this chapter, i t also 

must be kept in mind that ethanol-induced cel l death 

may also occur by other mechanisms such as necrosis 

(Obernier e t al, 2002). Awareness of the diversit y of 

death mechanism s i s important whe n conceptualiz - 

ing possibl e interventions. It is nonetheless unrealis - 

tic t o expec t tha t blockin g th e activit y o f a singl e 

protein ca n rescu e a cel l fro m death . Indeed , i t has 

been show n tha t blockin g one mechanis m o f deat h 

may alter the time line and pathway of cell death, but 

not th e outcom e (Mille r et al. , 1997 ; Stefani s et al., 

1999; D'Mell o e t al, 2000 ; Keramari s et al. , 2000 ; 

Selznick et al, 2000) . 

Progress i n establishing a causal link between ox - 

idative stress and cell death has been made. Throug h 

documentation o f relevant tim e lines , the upstrea m 

effectors o f ethanol-mediate d oxidativ e stress in th e 

neuron ca n be pinpointed and opportunities create d 

for reversin g or preventing th e relate d apoptosi s with 

anti-oxidant treatments. The latte r venture is an especially 

excitin g research direction , a s i t ma y b e on e 

path t o an intervention tha t does not have an advers e 

impact o n norma l brai n development . A s laboratories 

narro w thei r focu s o n th e underlyin g mecha - 

nisms o f ethanol-mediate d deat h o f neurons , i t i s 

hoped tha t more potentia l therapeuti c interventions 

will evolve. 

Abbreviations 

AIF apoptosi s inducin g factor 

ATP adenosin e triphosphate 


CytOx cytochrome C oxidase 

FADD Fas-associate d death domain 

FasL Fa s ligand 


GSH glutathion e 


MDA malondialdehyd e 


PACAP pituitar y adenylat e cyclas e activatin g 

polypeptide 

PARP poly-adenosin e diphosphat e ribos e poly - 

merase 

ROS reactiv e oxygen specie s

ACKNOWLEDGMENTS Wor k on this chapter wa s supported 

by the National Institute o f Alcohol Abuse and Alcoholism 

and the Departmen t o f Veterans Affairs . 

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17 

Neural Crest an d Developmental 

Exposure t o Ethanol 

The realizatio n that th e developin g neura l cres t is a 

target o f ethanol date s t o the origina l description s o f 

the feta l alcoho l syndrom e (FAS ; Lemoin e e t al. , 

1968; Jone s et al. , 1973) . A key observation was th e 

finding o f a characteristi c facia l dysmorpholog y tha t 

often, bu t no t necessarily, accompanies the neurobe - 

havioral an d neurocognitiv e deficits . Earl y evidenc e 

that ethanol targets the neura l crest comes fro m a series 

o f mous e mode l studie s (Suli k e t al. , 1981).Ye t 

neural cres t encompasses man y more structure s tha n 

the face, includin g key components o f the peripheral 

nervous system. The presen t chapter summarize s the 

literature on neural cres t and ethanol, an d highlight s 

issues that can benefit from furthe r research. 

OVERVIEW OF NEURAL 

CREST DEVELOPMEN T 

The neura l crest , sometime s know n a s th e fourt h 

germ layer , is a transien t ye t critica l contributo r t o 

Susan M. Smit h 

Katherine A. Debelak-Kragtorp 

279 

embryogenesis. It is of neuroectodermal origin, yet i t 

differentiates int o populations a s diverse as peripheral 

neurons, glia , chondrocytes , melanocytes , an d con - 

nective tissue . Its accessibility and migrator y properties 

hav e mad e i t attractiv e fo r stud y fo r ove r 10 0 

years, especially in avian and amphibia n model s an d 

more recently i n mouse. Recen t wor k has greatly expanded 

ou r understandin g o f neura l cres t develop - 

ment a t th e cellula r an d molecula r levels . Befor e 

addressing ho w alcoho l affect s th e developmen t o f 

this comple x population , it is first helpful to under - 

stand ho w thes e originat e an d t o appreciat e wher e 

their progeny may be found in the adult. An in-dept h 

summary o f neural cres t ca n b e foun d i n th e excel - 

lent book by Le Douarin and Kachiem (1999). 

Neural cres t arise s at the border between th e pre - 

sumptive neura l plat e an d latera l ectoderm . I t ap - 

pears quit e earl y i n development , shortl y afte r th e 

onset o f neurulation. It s specification an d inductio n 

involve signal s between ectoder m an d neura l plat e 

and the underlyin g mesendoderm. These signal s are


incompletely understoo d an d appea r t o includ e 

members o f the Wnt, fibroblast growth factor (FGF) , 

and bone morphogenic protein (BMP ) families (Basch 

et al., 2004). Their induction occurs in a rostrocaudal 

manner commensurat e wit h overal l developmen t 

along that axis . Figur e 17- 1 illustrate s these popula - 

tions as defined by the expressio n of the transcriptio n 

factor slug. 

The upwar d rollin g o f th e neura l plat e durin g 

early somitogenesis place s neural crest populations at 

the dorsa l lip of the neura l folds. Shortl y before this, 

or as the neural folds fuse to form the neural tube (depending 

on the species), the neural crest undergoes a 

transformation from epithelia to mesenchyme. Subsequently, 

the cells delaminate an d migrate into the periphery 

o f th e embryo . Thei r migrator y route s ar e 

defined b y extracellula r matri x protein s suc h a s fibronectin 

an d specifi c integrin s (notabl y oclß4 ) o n 

the neura l crest Onc e neura l cres t cell s reach thei r 

targets they differentiate int o appropriate cell types. 

FIGURE 17- 1 Morphogeneti c sequenc e o f neura l 

crest induction an d migration . Step s describing chick 

neural cres t development ar e exemplified by changes 

in th e expressio n o f th e transcriptio n facto r slug. A. 

Prechordal plat e stage . At this time, neura l crest cell s 

are slug negative . Arrow s indicate thei r locatio n (als o 

see Fig . 17-2A) . Slug expressio n a t thi s stag e i s re - 

stricted t o migrating cells of the primitiv e streak. B. 3 

somite stage. The first pre-migratory neural cres t cell s 

have appeared at the dorsal margin of the folding neural 

plate (arrow). C. 6 somite stage. As the neural fold s 

fuse, neura l crest cells receive their cues to begin mi - 

gration. Younger populations are being induced mor e 

caudally, and these have a low slug expression (arrow). 

D. 1 0 somite stage. Neural crest migration occurs in a 

Three distinctiv e populations o f neural cres t ar e 

defined: crania l (o r cephalic), cardiac , an d truncal . 

Cranial neura l cres t originate s fro m th e forebrain , 

midbrain, an d selecte d hindbrai n segment s (rhom - 

bomeres 1/2 , 4, 6, 7/8). Transplantation studie s show 

that their rostrocaudal identity (for example, mandible 

vs. hyoid) is dictated b y the leve l of the neuraxi s from 

which they originate, as defined by their expression of 

segment polarit y genes (hox, emx, otx). Recen t evi - 

dence suggest s ther e i s some plasticit y in thi s aspect 

(Trainor e t al. , 2002) . Crania l neura l cres t cell s 

have the mos t divers e fates, and , depending o n thei r 

level o f origi n an d tim e o f emergence , the y differ - 

entiate t o for m th e facia l bone s an d cartilage , 

melanocytes, toot h papilla , sensor y component s o f 

certain crania l nerves , an d othe r feature s (Tabl e 

17-1). Cell s formin g the connectiv e tissue s emerg e 

first, followed b y neuronal an d melanocyti c lineages . 

Thus both th e positiona l identit y and differentiatio n 

fate o f crania l neura l cres t cell s ar e determine d 

rostral-to-caudal sequence. Cell s that are rostral to the 

otic vesicle have commenced migration. E. 1 5 somite 

stage. The segmenta i nature of neural crest migration 

is depicted. Arrow s indicate th e migrator y waves that 

emerge fro m rhombomere s 2 , 4 , an d 6 . These cell s 

are fated to form facial bones and cartilage. Within th e 

hindbrain, neural cres t cells that migrate slightly later 

will attai n neurona l fates . Rostra l neura l cres t cell s 

have entere d th e mesenchym e tha t form s th e uppe r 

facial region . F. 1 8 somite stage. Neural crest populations 

have reached thei r targets within the face . With 

their cessatio n o f migration, they down-regulat e slug 

(arrow) and begin to differentiate. Migration o f neural 

crest from trunk and cardiac regions is still on-going at 

this stage.

TABLE 17- 1 Neura l crest contributions to the adult 

NEURAL CREST AND EARLY DEVELOPMENTAL EXPOSURE TO ETHANO L 28 1 

Cranial 

Bones and cartilag e o f the face and skull* 

Cartilaginous bones: nasal capsule (ect-, mesethmoid, interorbita l septum), basipresphenoid, scleroti c ossicles, ethmoid, 

pterygoid, Meckel's cartilage, quadratoarticular, hyoid (basihyal, entoglossum, basi-, epi-, ceratobranchial), otic capsule 

(pars cochlearis [partly] , parotic process ) 

Membranous bones: nasal, vomer, maxilla, jugal, quadratojugal, palatine, mandibular 

Membranous bones of skull: frontal (partly) , parietal, squamosal, columell a 

Cranial nerves * 

Sensory ganglia: portions of nerves V (posterior portion of trigeminal ganglion), VII (facial, root ganglion), IX 

(glossopharyngeal superior ganglion), X (vagal, jugular ganglion) 

Neurons of the sympathetic an d parasympathetic ganglia : superior cervical, ciliary (otic, submandibular, lingual , ethmoid, 

spheropalatine) 

Glia and Schwann cells for all cranial ganglia and nerves 

Other crania l tissues 

Melanocytes of skin and internal organs (except the retinal pigmented epithelium ) 

Dermis of scalp 

Dermis, smooth muscle, an d adipose tissue of skin in face and ventral neck region 

Méninges of the forebrain (pachymeninx and leptomeninx); not its blood vessels 

Connective tissu e of cranial glands: lacrimal, pituitary, parathyroid, salivary, thymus, thyroid 

Connective tissues associated wit h facial muscle s 

Calcitonin-producing C cells 

Type I chemoreceptor cell s and type II supporting cells of the carotid body 

Portions of the teeth: odontoblasts, cementoblast s 

Eye: stromal and endothelial cells of cornea and orbit* 

Ciliary muscles 

Cardiac * 

Smooth muscl e o f the outflow tract 

Mesenchymal cells of the outflow tract 

Conotruncal cushion s of the outflo w tract 

Cardiac gangli a 

Truncal 

Sensory neurons and glia l cells of the dorsal root ganglia 1 

The entiret y of the autonomie nervous system: 

Catecholaminergic an d noncatecholaminergic neuron s and satellite cells of the sympathetic ganglia and plexuses 

Cholinergic an d noncholinergic neuron s and satellite cells of the parasympathetic ganglia and plexuses 

Enteric gangli a 

Schwann cells of the peripheral nerve s 

Neuroendocrine (adrena l chromaffin an d small intensely fluorescent) cells of the adrenal medulla t 

Parasympathetic gangli a of the pancrea s 

Merkel cells (mechanoreceptor cell s of the mammalian whisker follicle) 

Melanocytes of skin and internal organs 

* Affected b y ethanol, evidence based on in vivo work in animal models or humans. 

* Affected b y ethanol, evidence based on cell culture work. 

Source: Adapted from Le Douarin and Kacheim (1999); this list may be incomplete. 

predominantly prior to their emigration from the neu- smoot h muscl e an d connectiv e tissu e mesenchym e 

ral tube. o f the outflo w trac t and aorti c arches. They also par - 

Thé cardia c neural crest is a relatively small, spe- ticipat e i n th e remodelin g o f these earl y structure s 

cialized subpopulatio n tha t emerge s fro m neura l int o the aorta, pulmonary aorta, and affiliate d vascu - 

tube at the level between the otic vesicle and somite lature . Deficits in this neural crest population lead to 

4, includin g rhombomere s 6- 8 (Kirb y 1999) . Lik e sever e cardia c defects , suc h a s double-outle t righ t 

cranial crest, they migrate into the third, fourth, and ventricle , patent truncus arteriosus , and tetralog y of 

sixth pharyngeal arches, but instead contribute to the Fallot .

Z82 ETHANOL-AFFECTE D DEVELOPMENT 

Neural cres t populations cauda l t o somit e 5 contribute 

t o muc h o f th e periphera l nervou s syste m 

(both sympatheti c an d parasympatheti c neurons an d 

their supporting Schwann cells), the enteri c nervou s 

system, and rnelanocytes . Cells that follow ventromedial 

migratory routes contribute to the peripheral nervous 

system . Thes e cell s onl y migrat e throug h th e 

rostal hal f o f eac h sclerotome , an d thi s restrictio n 

contributes t o th e segmentatio n o f th e sympatheti c 

and dorsa l roo t ganglia . Cell s fate d t o becom e 

rnelanocytes follo w a dorsolatera l rout e an d migrat e 

somewhat later . Trun k neura l cres t cell s appea r t o 

have a much greater plasticity with respect to fate an d 

identity and henc e seem t o have a greate r regenera - 

tive capacity than that o f cranial and cardia c populations; 

when on e regio n i s ablated, neighborin g cell s 

migrate i n and replac e th e missin g cells . I n general , 

fairly larg e losse s must occu r befor e anatomical an d 

physiological changes are seen. 

A preponderance o f genetic and experimenta l dat a 

show that the basi c rules and signal s governing neural 

crest development ar e largel y conserved amon g vertebrates. 

This is best shown in the numerous human neu - 

rocristopathies that are recapitulated by specific mous e 

null mutants . Ther e ca n als o b e smal l difference s i n 

the timing or identity of regulatory molecules; fo r these 

finer details investigators are encouraged t o consult the 

primary literature on their species of interest. 

NEURAL CREST POPULATION S 

AFFECTED B Y ETHANOL 

For obvious reasons, the best evidence tha t ethanol exposure 

disrupts neural crest development come s fro m 

animal models. As discussed below, the critical window 

for human neura l cres t development i s between week s 

three an d eight postconception , an d embryo s tha t 

young ar e generall y unavailabl e for study . A furthe r 

limitation i s the apparen t lac k of systemic description s 

of neural crest derivatives and thei r fate i n humans exposed 

to heavy gestational alcohol exposure. Thus, this 

section emphasize s anima l models , incorporate s hu - 

man observation s whe n possible , an d highlight s in - 

stances wher e the huma n evidenc e i s suggestive an d 

would benefit from clarifying work. The reade r should 

further not e that findings in rodent and chick, the two 

most popular models for this work, are largely in agreement. 

Their concurrence enhance s thei r applicability 

and relevance for human development. 

Ethanol Targets Crania l 

Skeletal Derivative s 

The facia l change s observe d i n human s wit h heav y 

gestational alcohol exposur e are similar to known neurocristopathies, 

promptin g th e firs t hypothese s tha t 

neural crest was targeted by ethanol exposure (Lemoine 

et al, 1968 ; Jone s et al, 1973) . The facia l change s i n 

humans includ e midfacia l flattening , shortene d nos e 

with upturne d nares , micrognathia , an d hypoplasti c 

maxilla (Clarre n an d Smith , 1978 : Pria s et al, 1982 ; 

Johnson e t al., 1996 ; Mattso n et al, 1997) . Midfacial 

clefting an d degree s o f holoprosencephaly ar e some - 

times seen . Anthropométri e studie s (Moor e e t al. , 

2001, 2002) report that facial dimensions are reduced 

overall i n childre n wit h FA S (also known as dysmorphic 

fetal alcohol spectrum disorde r [FASD]) and to a 

similar, but lesser extent, in those with alcohol-related 

neurodevelopmental disorder (ARND; also known as 

nondysmorphic FASD) . Diagnostic s emphasiz e sof t 

tissue changes, particularly the tri o of thin upper lip, 

reduced o r absent philtrum , and smal l palpebra l fissures 

in affected individuals. The grea t value of these 

changes arises from thei r ease of assessment and thei r 

relative uniquenes s t o alcoho l (an d toluene ) expo - 

sure. No t surprisingly , they ar e importan t compo - 

nents of diagnostic screens for ARND and FA S (Astley 

and Clarre n 1996) . This emphasis on sof t tissu e may 

seem contradictor y because facia l skeleta l muscl e i s 

not neura l cres t derived. Sof t tissu e shape, however , 

is dictated b y the underlyin g skeletal elements (proof 

of this i s in th e forensi c scientist' s ability t o recreat e 

a person' s likenes s fro m th e bar e skull) . Thus , th e 

size and shape o f the philtrum, lip , and palpebral fissures 

ar e a direc t consequenc e o f dysmorpholo - 

gies i n th e underlyin g neural crest-derive d skeleta l 

elements. 

Several other conclusion s ca n b e draw n from th e 

human evidence . Th e facia l appearanc e transcend s 

racial characteristics, reinforcin g the robus t nature of 

ethanol-induced changes . Second , i n n o instanc e 

does ethano l exposur e alter the segmenta l identit y of 

the face. Effect s instea d are manifested in dimensional 

shifts i n otherwis e normal structures , indicating that 

ethanol target s aspects o f neural crest cellularity an d 

outgrowth, a finding that provide s important clue s to 

locating the molecular targets of ethanol action. 

Animal studie s confir m the hypothese s tha t alco - 

hol targets the developin g neural crest. Studies of rats 

prenatally expose d t o ethano l hav e the constellatio n

NEURAL CREST AND EARLY DEVELOPMENTAL EXPOSUR E T O ETHANO L 28 3 

of malformation s in th e craniofacia l skeleton tha t i s 

characteristic o f human s wit h FA S (Edward s an d 

Dow-Edwards, 1991) . Murin e studie s hav e refine d 

the tempora l windo w o f vulnerabilit y (Suli k e t al. , 

1981; Webste r e t al, 1983) . I n mic e o n gestationa l 

day (G) 7 , i.e., at the time of gastrulation, ethanol exposure 

(as two acute doses 4 hr apart) causes craniofacial 

defect s consisten t wit h huma n FAS . Thes e 

defects include midline reductions, reduced midface , 

and hypoplasti c primordi a o f th e maxill a an d 

mandible. Ethanol exposur e one day later (G8, head - 

fold stage ) also affects th e face , but cause s a facial ap - 

pearance more similar to that of DiGeorge syndrome 

(Sulik e t al. , 1986) . Fo r bot h treatmen t windows , 

these morphologica l change s ar e preceded b y significant 

cel l deat h withi n thei r respectiv e neura l cres t 

precursors a s well a s in adjacen t regions (Kotc h an d 

Sulik 1992a , 1992b ; Dunt y e t al, 2001) . These two 

critical windows for mouse correspon d t o weeks 3 and 

4, respectively, in human pregnancy. Identical ethanol 

doses at G9 or G10 d o not appreciably affect facial appearance 

(Webster et al., 1983) , reinforcing the obser - 

vation that ethanol target s early events in neural crest 

induction, expansion , and/o r migration , rathe r tha n 

later events of differentiation. 

A similar phenotype occur s i n chic k embryo s exposed 

to acute ethanol. A s with mouse, the precis e facial 

changes depend o n stage of treatment (Cartwright 

and Smith , 1995b) . Gastrulation-stag e (HH4 ) o r 

neurulation-stage (HH6 ) exposur e produces a wider, 

taller chic k fac e wit h a shorte r maxill a an d nasa l 

bone, wherea s exposur e a t th e onse t o f migratio n 

(HH10-12) produces an overal l hypoplastic face (De - 

belak-Kragtorp, Moyers , an d Smith , unpublishe d 

results). The facia l appearanc e i s preceded b y signifi - 

cant cel l deat h i n region s includin g th e crania l 

chondrogenic precursors of neural crest, and immunostaining 

corroborates the los s of these neural crest populations 

(Cartwrigh t an d Smit h 1995a ; Cartwrigh t 

et al. , 1998) . Pretreatmen t o f chick embryo s wit h a 

caspase-3 antagonis t prevente d bot h th e ethanol - 

induced cel l deat h an d th e change s i n bea k lengt h 

(Debelak-Kragtorp, Moyers , an d Smith , unpublishe d 

results). This finding reinforces the concep t that cel l 

death contribute s t o th e facia l dysmorphology . Sus - 

ceptibility to ethanol-induced apoptosis i s greatest for 

premigratory neura l crest ; migrator y an d postmigra - 

tory cell s ar e mor e resistan t (Cartwrigh t an d Smith , 

1995b). Thi s timin g suggest s tha t uniqu e develop - 

mental event s prio r t o neura l cres t migratio n ma y 

govern thei r sensitivity; some o f these target s ar e discussed 

below. 

These animal data predict that humans at weeks 3 

and 4 of gestation ar e the mos t sensitive to the facia l 

teratogenic effects . A stud y i n pregnan t macaque s 

reinforces thi s prediction . Onc e weekl y dose s o f 

alcohol (mea n bloo d ethano l concentratio n [BEG ] 

-220 mg/dl) are associated with facial dysmorphology 

in the offspring only when the exposure coincide wit h 

G19 or G20 (Astle y et al, 1999) . These findings suggest 

that a sufficiently high BEG during a critical window 

i s require d t o induc e th e characteristi c facia l 

changes o f FAS. The implicatio n i s that if a pregnan t 

woman ha s restricte d o r no acces s t o alcohol durin g 

the critica l window, her chil d ma y lack the facia l dysmorphology, 

despit e having profound nervous system 

deficits fro m late r exposure . The diagnosti c implica - 

tions of this are obvious for professionals and affecte d 

families. 

Ethanol Targets Neuronal Derivatives 

of Neural Crest 

The literatur e on this point is sadly scant with respec t 

to humans. I n mice, ethano l cause s significant apoptosis 

i n th e oti c regio n an d i n th e neura l cres t an d 

placodal component s o f th e crania l sensor y gangli a 

(Dunty e t al., 2001) . Ethanol-expose d chic k embryo s 

display reductions in cranial nerve cellularity and aberrant 

migration of their neuronal branche s (Cartwrigh t 

and Smith, unpublished results). The functional con - 

sequences o f suc h losse s hav e no t bee n studied . 

There are hints, however, that these population s also 

are targete d i n humans. Fo r example, a n infan t wit h 

FAS often ha s a reduced rootin g reflex when nursing , 

a behavio r partl y dependen t o n th e abilit y of facia l 

cranial nerves to sense and proces s a tactile stimulus. 

This tactil e los s ca n b e s o significan t tha t som e 

affected individual s requir e sof t o r liqui d diet s be - 

cause the y hav e troubl e sensin g foo d textur e an d 

when i t is appropriate t o swallow . There ca n als o b e 

significant hearin g losse s and persisten t ear cana l in - 

fections tha t ar e partl y attributed t o change s i n th e 

neural crest-derive d bon e an d cartilag e structure s 

and i n the acoustica l nerves , which ar e supported b y 

glia and Schwan n cell s originating from neura l crest 

(Church e t al , 1996 , 1997) . Furthe r investigatio n 

into crania l an d periphera l nerves , an d th e conse - 

quences o f any such dysfunctions , shoul d b e a high 

priority.


The situatio n regarding neural cres t precursors of 

the periphera l nervou s system i s even les s clear. Em - 

bryos treated at G7 and G8 have increased cel l deat h 

within regions populated b y trunk neural crest (Sulik 

et al , 1988 ; Kotc h an d Sulik , 1992b) . Th e conse - 

quences o f such losse s hav e ye t to b e detailed . Seg - 

mentation o f the dorsa l roo t gangli a appear s normal 

by gross inspection, althoug h cultur e studie s sugges t 

that ethano l impair s th e surviva l an d outgrowt h o f 

DRG neuron s (Do w an d Riopelle , 1990) ; reduce d 

synthesis of and response s to trophic factor s are note d 

(Dow and Riopelle, 1990 ; Beaton et al, 1992; Bradley 

et al., 1995) . Advers e affects o n th e sympatheti c an d 

parasympathetic nervou s syste m hav e no t bee n re - 

ported fo r humans an d i n anima l models . Impaire d 

balance i s observed , bu t follow-up testin g impli - 

cates targets in the central nervous system rather than 

the neura l crest-derive d periphera l nervou s syste m 

(Church e t al, 1997 ; Roebuc k e t al, 1998) . More - 

over, obstructive bowel problems characteristic of impaired 

enteri c nervou s syste m developmen t ar e no t 

widely reported. Whereas gli a and supportin g cells of 

the brain are appreciated a s a target of ethanol (Chap - 

ter 18) , littl e attentio n ha s bee n pai d t o supportin g 

cells o f the periphera l nervou s system . I n summary , 

our bes t answe r to this questio n i s that these populations 

ca n b e targete d b y ethanol, bu t th e functiona l 

consequences ar e essentially unexplored. 

Ethanol Targets Cardiac Neura l 

Crest Derivative s 

Outflow trac t anomalie s ar e mor e frequen t i n chil - 

dren with FAS than i n the genera l populatio n (Abel , 

1990), promptin g studie s int o potentia l effect s o f 

ethanol o n th e cardia c neura l crest . I n mous e an d 

chick models , ethano l exposur e cause s apoptosi s o f 

cardiac neura l cres t precursor s (Kotc h an d Sulik , 

1992b; Cartwright et al, 1995b). On the basis of extirpation 

studies (Kirby, 1999) , such losse s should result 

in aorticopulmonar y defect s suc h a s persistent trun - 

cus arteriosus , double-outle t righ t ventricle , an d 

tetralogy o f Fallot . Indeed , ethano l cause s suc h de - 

fects i n bot h mous e (Daf t e t al. , 1986 ) an d chic k 

(Fang e t al. , 1987 ) wit h a n incidenc e o f 10%-20% . 

These dysmorphologies are preceded by the expecte d 

malpositioning an d reduction s o f the cona l cushion s 

(Daft et al., 1986) and hypoplasia of the proximal bulbar 

ridge s (Fan g e t al. , 1987 ; Bruyèr e an d Stith , 

1993). The critica l window for these effect s i s during 

the tim e o f cardiac crest migration, invasion , and expansion 

int o the outflo w trac t and pharyngea l arches 

(G8.5 fo r mouse, Daf t e t al., 1986 ; 72-8 0 hr incuba - 

tion in chick, Fang et al., 1987) . But are these change s 

actually du e t o th e neura l cres t losses ? Our vie w is 

that the heart defects represent defect s in cardiac pop - 

ulations othe r tha n neura l crest . W e expose d chic k 

embryos to ethanol a t critical times of cardiac neural 

crest an d outflo w trac t development ; despit e signifi - 

cant losse s o f cardia c crest , inspectio n a t late r tim e 

points found normal neura l crest distributions within 

the outflo w tract , an d n o aorticopulmonary or septa l 

defects (Cavieres and Smith , 2000) . 

Bruyère and colleague s (1994a ) not e inconsisten - 

cies between th e heart defects commonly see n in human 

prenata l alcoho l exposur e an d thos e cause d i n 

the laborator y b y neura l cres t ablation . I n ethanol - 

exposed humans , th e mos t commo n cardia c defect s 

are no t aorticopulmonary . Rather , the y ar e isolate d 

ventricular septal defect s (VSDs) and atrial septal defects 

(ASDs ; -75% o f congenital cardia c anomalies) . 

These tw o septa ar e no t neura l cres t derived . VSDs 

and ASD s ar e generall y secondar y response s t o pri - 

mary changes i n flow dynamics (Kirby , 1999 ; Firull i 

and Conway, 2004). Significant effects i n cardiac output, 

heart rate, metabolism, an d cardiomyocyte differ - 

entiation ar e documente d i n model s o f prenata l 

ethanol exposur e (Nyquist-Batti e an d Fréter , 1988 ; 

Ruckman e t al, 1988 ; Adicke s et al, 1993 ; Bruyèr e 

and Stith , 1994a ; Bruyèr e et al, 1994b) . Fan g an d 

colleagues (1987 ) suggest that ethanol ha s a biphasi c 

effect o n the developing heart, with lower BECs caus - 

ing VSD s (perhap s targetin g cardiomyocytes ) an d 

higher concentration s targetin g aorticopulmonar y 

events (an d cardia c crest) . Lower ethano l exposures , 

though deletin g som e cardia c crest , ma y allo w th e 

survivors t o repopulat e an d replac e thei r missin g 

neighbors. Determinin g th e exten t t o whic h neura l 

crest losse s contribute t o cardia c defect s i n FA S will 

require additiona l research . Carefu l morphologica l 

studies using , fo r example , th e lacZ-wnf l reporte r 

mouse (i n whic h neura l cres t ca n b e followe d b y 

merit o f lac Z expression ) coul d nicel y resolv e thi s 

question. 

Ethanol Targets 

Melanocyte Derivative s 

Alcohol likel y ha s n o long-ter m consequence s o n 

melanocyte development , althoug h thi s conclusio n

NEURAL CREST AND EARL Y DEVELOPMENTAL EXPOSURE TO ETHANO L 28 5 

rests o n negativ e data . Melanocyte s ar e th e pig - 

mented cells within the dermis, and to our knowledge 

there ar e n o report s o f reduced pigmentatio n o r altered 

pigmenta l patterns in eithe r individual s prenatally 

exposed to alcohol o r exposed mice. Melanin i s 

still synthesize d i n salamande r neura l cres t culture d 

with ethanol , an d it s unusual perinuclea r depositio n 

may be secondar y to microfilament dysarrangement s 

(Hassler an d Moran , 1986) . Th e pigmente d chick s 

we have hatched sho w no sig n of altered feather col - 

oration (Smith , Tessmer, an d Debelak-Kragtorp , un - 

published results) . This lack of effect ma y reflect the 

lack of segmental identit y within melanocytes, which 

facilitates thei r ability to expand an d compensat e fo r 

losses, 

Other Neural Cres t Populations 

Targeted by Ethanol 

There ar e hints in the literature that additional neural 

cres t subpopulations may be affecte d b y ethanol. 

Carones an d colleague s (1992 ) an d Mille r an d 

Beauchamp (1988) document multiple case s of ocular 

defects involving the corne a endothelia , a neural 

crest-derived populatio n that contributes to the anterior 

segmen t o f th e eye . Toot h problem s ar e men - 

tioned, althoug h i t wa s no t indicate d whethe r thi s 

involves the neura l crest-base d papilla . Similarly , it 

is not know n whether problem s o f thymocyte maturation 

and thyroid hormone production are linked to 

aberrant strom a o f their respectiv e glands. A similar 

question ca n be raise d with the altere d signal s of the 

hypothalamic-pituitary-adrenal (HPA ) axis . I n tha t 

light, i t i s worth notin g that a cel l lin e popula r for 

studies o f ethanol-mediate d neurona l signaling , 

PC12 cells (Messing et al, 1986 ; Luo et al, 1999) , is 

an adrenochromaffi n tumo r originatin g from neura l 

crest lineage of the adrenal medulla. Again, a systematic 

stud y o f neura l cres t derivative s i n human s 

(Table 17-1) would provide important clues as to the 

effects o f ethanol o n nonclassi c neural crest populations. 

EVENTS OF NEURAL CREST 

DEVELOPMENT AFFECTED 

BY ETHANOL 

Data fro m anima l models , a s discussed above, poin t 

to earl y event s of neural cres t induction, determina- 

tion, an d migratio n a s the majo r target s for ethano l 

teratogenicity (Fig. 17-2) . Evidence summarize d be - 

low indicate s tha t n o singl e even t i s affected; rather , 

multiple target s likely lead t o a cumulative effect o n 

this cell population. 

Neural Induction 

A key, early event of embryogenesis is the anterio r extension 

o f the prechordal plat e and it s subsequent in - 

duction of the neural plate, including neural crest, at 

the midlin e o f th e overlyin g ectoderm. Prechorda l 

plate siz e affect s neuronal—an d neura l crest — 

population size . Ethano l reduce s th e migrator y ca - 

pacity o f thes e anterio r mesoderma l cell s i n bot h 

chick (Sander s et al. , 1987 ) and zebrafis h embryo s 

(Blader and Strahle , 1998) . Unfortunately, any consequences 

fo r neural cres t population siz e have yet t o 

be tested . Reduce d prechorda l plat e migratio n ma y 

also contribut e t o the holoprosencephali c aspect s o f 

acute prenata l alcoho l exposur e (Suli k e t al. , 1988 ; 

Blader and Strahle , 1998) , although a major midlin e 

mediator, sonic hedgehog (shh), does not appear to be 

a direct target of ethanol i n this model. 

Neural Cres t Migratio n 

Rovasio an d colleague s hav e detaile d ho w ethano l 

impairs neura l cres t migration . Co-culture o f chick 

embryos ( 3 somites ) with 0.25% ethanol cause s significant 

reduction s i n th e numbe r o f crania l an d 

truncal neura l cres t cells exiting the neura l tube , as 

visualized i n cross-section via immunostaining with 

the neura l crest-specifi c antibod y NC- 1 (Rovasi o 

and Battiato , 1995) . Primary cultures of neural crest 

cells treate d wit h 15 0 m M ethano l los e thei r mes - 

enchymal appearanc e an d becom e spindl e shaped . 

They hav e fewe r filopodia , increase d surfac e bleb - 

bing, an d pronounce d rearrangement s of the acti n 

cytoskeleton tha t ar e accompanie d b y dramati c re - 

ductions i n thei r migrator y capacit y (Rovasi o an d 

Battiato, 2002) . Cranial and truncal population s ar e 

similarly affected . Lowe r BECs (100-20 0 mg/dl) d o 

not affec t migration , but the y d o lea d t o thickene d 

actin and tubulin filaments in cultured primary neural 

cres t (Hassle r and Moran , 1986) . Cell-cel l con - 

tacts are disrupted. Migrator y pathways in the trun k 

are directed by the somites; the segmental anomalies 

in alcohol-expose d somite s (Sander s an d Cheung ,


A. Induction events 

B. Apoptotic events 

Reduced prêcherai plate 

extension would reduce the 

number of neural crest ceils 

induced at the neuroepithelial 

margin. 

Apoptosis due to: 

- oxidative stress; 

- altered Ça** signals; 

- loss of L1-mediated cell-cell 

interactions; 

- loss of retinoid signals; 

- loss of trophic signals 

C. Migration and differentiation 

Altered identity 

„ and/or delayed 

emergence from 

neural tube. 

Impaired migration 

and/or cellular 

expansion into 

target tissues 

Delayed or arrested 

- differentiation within 

target tissue regions. 

FIGURE 17- 2 Candidat e target s o f ethano l durin g 

neural cres t development. A . Targets durin g the window 

of neural cres t induction. A chick embry o at th e 

prechordal plat e stag e (HH5+ ) wa s prepare d t o vis - 

ualize slug transcript s b y i n sit u hybridization . Th e 

presumptive neural crest-forming regio n (shaded ) lies 

at the boundar y of the neuroectoder m and ectoderm . 

B. Targets that coul d caus e neura l cres t apoptosi s are 

summarized. Show n i s a chic k embry o o f th e 1 7 

somite stage. Apoptotic cells are visualized by the vita l 

dye acridine orange. This embryo was exposed to -60 

mM ethanol at gastrulation, as described by Cartwright 

and colleague s (1998) . C. Target s durin g the window 

of neural cres t migration and differentiation . Thi s im - 

age depicts a transvers e view throug h th e hindbrai n 

(level o f rhombomer e 6 ) o f a chic k embry o a t th e 

1990) ma y hav e deleteriou s effect s o n neura l cres t 

migration and fate . 

Neural Crest Expansion 

Reduced prechorda l plat e size and increase d apopto - 

sis could reduc e th e population size of neural crest. It 

should b e noted, however, that embryo s have robus t 

repair mechanisms; autoregulator y signals and group - 

effect response s ca n restor e populatio n siz e i n re - 

sponse t o additio n o r loss . Signal s tha t regulat e th e 

size of the neural cres t population includ e shh, bmp, 

fgf, an d wnt. The degre e t o which thes e ar e ethano l 

targets likely depends o n the timing and dos e of exposure. 

The homeoti c selecto r gene, msxl, is expressed 

in premigratory and migrator y neural crest and i s associated 

wit h both expansio n and apoptosis . One repor t 

notes nea r ablatio n o f msx2 expressio n i n ethanol - 

treated mous e embryos and ca l variai osteoblasts (Rifa s 

et al, 1997) , however , the BECs i n this study are very 

high. Preparation s expose d t o mor e physiologicall y 

relevant exposure s d o no t affec t msxl (Cartwrigh t 

et al, 1998) . Ethano l ma y affec t shh, a mediator o f 

directional outgrowth , an d suc h suppressio n migh t 

contribute t o neural cres t reduction s (Ahlgre n et al, 

2002). Sh h withi n th e fac e i s transiently reduced b y 

ethanol exposur e commensurat e wit h reduce d mes - 

enchymal proliferatio n an d elevate d fg f an d ms x 

expression (Smit h e t al , 2001) . Thes e change s 

are proportionat e t o late r shift s i n directiona l out - 

growth. Ye t ther e i s n o evidenc e tha t an y suc h 

changes ar e associate d with neura l cres t apoptosi s a t 

these time s (se e below). Much mor e wor k is neede d 

to understan d th e effect s o f ethano l o n thes e mor - 

phoregulatory cues. 

Apoptosis 

Cell deat h i s an importan t componen t o f ethanol ter - 

atogenicity. Sando r (1968 ) observe d a prominen t 

cellular "necrosis " withi n th e neuroepitheliu m o f 

ethanol-exposed chick embryos, a region that includes 

neural cres t precursors . Suli k an d colleague s (1981 ) 

documented cytoplasmi c an d nuclea r extrusion s from 

36 somite stage. The pharny x lies ventral to the hind - 

brain. Neural cres t cell s ar e visualize d b y in sit u hy - 

bridization for slug.

NEURAL CREST AND EARL Y DEVELOPMENTAL EXPOSURE TO ETHANO L 28 7 

neural folds of mice treated with ethanol at G8. Similar 

foci of necrotic and degenerating cells were observed in 

neuronal population s of the mous e o n G 9 (Banniga n 

and Burke , 1982; Bannigan and Cottell , 1984) . Sulik' s 

work using vital dye s and high-magnificatio n histology 

had establishe d tha t this "necrosis" actually represents 

pyknosis (Suli k e t al. , 1986 , 1988 ; Kotc h an d Sulik , 

1992a, 1992b) . Thi s pyknosi s i s par t o f a n ethanol - 

induced apoptoti c death ; caspas e inhibito r pretreatment 

prevents it (Cartwright et al, 1998 ) and the DNA 

is labele d b y termina l transferas e (TUNEL^positive ) 

(Cartwright et al, 1998; Dunty et al., 2001). 

Apoptosis i s a well-described , active , energy - 

dependent process that is a mechanism for the removal 

of unwanted cells (Reed, 2000). It is prominent i n th e 

developing embryo, for example, i n removing interdigital 

webbing. It also is used to eliminate cells that have 

aberrantly activate d thei r proliferativ e machinery , 

such a s self-recognizin g T cells ; tumor s represen t 

cells that fail t o apoptose whe n promitotic signals become 

activated . Teratogens frequentl y act by expanding 

existin g domain s o f endogenou s cel l death . 

Under thi s model, th e teratoge n recruit s susceptible 

populations by merit of their latent capacity for apoptosis. 

This certainly is the cas e for acute ethanol. Suli k 

and colleague s (Suli k et al. , 1988 ; Kotc h an d Sulik , 

1992b; Dunt y e t al. , 2001 ) hav e comprehensively 

mapped the cell death patterns for normal mouse embryogenesis 

an d followin g ethano l treatment . Thi s 

work elegantl y shows that ethanol-induce d apoptosis 

is selective . Populations ar e no t affecte d uniforml y 

and th e precis e target depends on th e stag e of treatment. 

Region s o f cel l deat h coincide d wit h tissue s 

and structure s affected b y ethanol treatment , including 

neural crest of cranial, cardiac, and trunk popula - 

tions. A critical implicatio n o f this work is that ther e 

are inheren t propertie s withi n certai n cel l popula - 

tions that govern their susceptibility to ethanol. These 

properties are reviewed in detail below . 

Our recen t work with chick embryo s emphasize s 

the mechanisms underlying ethanol-induced deat h of 

neural cres t cells . Crania l neura l cres t apoptosi s i n 

the chic k coincide s wit h th e endogenou s deat h o f 

neural cres t precursor s i n rhombomere s 3 an d 5 . 

Thus, we hypothesize that ethanol exposure might activate 

thos e endogenou s signals . Thes e signal s ar e 

well described and involv e the wnf-dependent activation 

o f bmp4 synthesi s within rhombomeres 3 and 5 . 

Bmp4 act s i n a n autocrine-paracrin e manne r t o 

stimulate msxl expressio n and the apoptosis sequenc e 

(Graham e t al , 1994) . Neithe r bmpi no r msxl, 

however, ar e elevate d i n th e hindbrain s o f ethanol - 

exposed chic k embryo s (Cartwrigh t e t al. , 1998) . 

Thus the initia l machinery of ethanol-induced apop - 

tosis must lie upstrea m o f the endogenou s deat h signals. 

We recently ascertained that these signals involve 

the activatio n by ethanol o f a pertussis toxin-sensitive 

G protei n an d intracellula r Ca 2+ transient s (Garic - 

Stankovic, et al., 2005; see below). 

HOW DOES ETHANOL KILL 

NEURAL CREST CELLS? 

Four majo r mechanism s hav e receive d considerabl e 

scrutiny: oxidative stress, LI-mediated cell adhesions , 

induction o f Ca 2+ transients, and retinoid-dependen t 

signals. It should be stressed that evidence for the first 

three i s quite good . Ethano l ha s pleiotropi c effects , 

and i t is quite likely that ultimately these mechanism s 

will prove to be interrelated . 


Ethanol exposur e leads to oxidative stress in the neu - 

ral crest . Migrator y an d postmigrator y neura l cres t 

cells lac k appreciabl e Superoxid e dismutas e (SOD ) 

activity (Davi s e t al. , 1990) , an d exposur e o f neural 

crest t o a free-radica l sourc e (suc h a s xanthine oxidase) 

is sufficient t o impede their survival (Chen an d 

Sulik, 1996) . Superoxide , hydroge n peroxide , an d 

hydroxy radical concentrations all increase after exposure 

to 50-100 mM ethanol, and upon supplementa - 

tion with SOD those concentrations return to normal 

(Davis et al., 1990 ; Che n and Suli k 1996) . I n whol e 

embryos treate d wit h ethanol , lipi d peroxidase s an d 

Superoxide radical s ar e selectivel y enriche d withi n 

the neura l cres t an d neuroepitheliu m (Kotc h e t al. , 

1995). Co-cultur e o f whol e embryo s (Kotc h e t al. , 

1995) or postmigratory neural crest (Davis et al., 1990 ; 

Chen an d Sulik , 1996 ) wit h free-radica l scavenger s 

(SOD o r catalase) improves cell survival and reduce s 

ethanol-induced cytotoxicity . I t shoul d b e noted , 

however, that thes e treatment s provide only a partial 

rescue. Iro n chelator s suc h a s deferoxamin e an d 

phenarthroline als o offe r protectio n agains t ethanolinduced 

cel l deat h (Che n an d Sulik , 2000). This is 

an interestin g finding because ethano l exposur e can 

elevate iro n concentration s a s i n th e brai n ste m


(Miller et al., 1995) . In two separate studies, however, 

vitamin E is less effective a t ameliorating these effects , 

a surprisin g outcome give n tha t thi s free-radical scav - 

enger preferentially acts on lipid and membrane com - 

partments (Davis et al, 1990; Chen and Sulik, 1996) . 

There is little question that free radicals contribute 

to neura l cres t death , jus t a s oxidativ e stres s con - 

tributes to man y instances of neuronal deat h i n th e 

fetus. Les s clear i s the precis e rol e o f oxidative stress 

in the sequence of events. Does ethanol itsel f directly 

cause this stress? Neural crest at these stages does not 

express CypZE l (Deltou r e t al, 1996) , bu t i t doe s 

have a n ADH 4 capable o f metabolizin g ethano l a t 

high concentration s (Deltou r et al., 1996 ; Haselbec k 

and Duester , 1998) . Th e resultin g imbalance s i n 

NADP/NADPH ratio s woul d invok e a metaboli c 

stress, an effec t wel l documented fo r adult heart an d 

liver. Alternatively, these increased free-radica l levels 

could simpl y reflect tha t the cell s are dying, perhaps 

through changes in the mitochondrial transition pore 

that ar e par t o f apoptosis itself . Studie s t o dat e hav e 

emphasized tim e point s o f 1 2 hr an d late r followin g 

ethanol treatment. Additional studies looking at much 

earlier times would clarify this question. Our curren t 

view i s that th e pro-oxidan t effects ar e likel y secondary 

t o the primar y action o f ethanol, becaus e o f this 

lack of earlier time point data and becaus e o f the effects 

on Ca 2+ signaling detailed below. 

Cell Adhesion and LI 

Ethanol also disrupts the development o f neural crest 

cells and promotes their apoptosis through LI, a cell 

adhesion molecul e (CAM ) o f the immunoglobuli n 

CAM superfamily. Ethano l inhibit s LI-mediated cell 

adhesion (Charnes s e t al., 1994 ; Ramanatha n e t al. , 

1996; Wilkemeyer and Charness, 1998) . LI is strongly 

expressed by neural crest (Chen et al., 2001), and i n 

this context it is noteworthy that disruptions of neural 

crest cell-cel l adhesion , a t leas t a t th e leve l o f gap 

junctions, result s i n thei r apoptosi s (Bannerma n 

et al, 2000). LI deficit s caus e craniofacial dysgenesis 

in humans (CRASH syndrome) that shares aspects of 

that wit h alcoho l exposur e (Franse n e t al. , 1995) . 

Ethanol likel y act s o n L I throug h it s abilit y t o si t 

within hydrophili c pocket s o f membran e proteins , 

stabilizing protei n structur e an d enhancin g o r re - 

pressing its activity (Mihic et al., 1997). In this model, 

ethanol would bind LI to disrupt crucial trophic cue s 

that the protein supplies to neural crest. 

Low concentrations o f n-octanol block the action s 

of LI; they block LI-mediated cel l adhesion (Wilke - 

meyer e t al., 2000) . Furthermore , n-octano l amelio - 

rates the toxi c consequence s o f ethanol o n culture d 

mouse embryo s (Che n e t al, 2001) . Their appear - 

ance i s mor e norma l an d the y exhibi t significantl y 

less cel l death , includin g neural crest-populate d re - 

gions. N-octanol may act by competitively inhibiting 

ethanol fro m bindin g t o th e hydrophili c pocket s 

of LI. 

Ethanol antagonism of LI i s prevented by the pep - 

tides NAPVSIPQ (NAP ) and SALLRSIP A (SAL), frag - 

ments o f th e large r glial-derive d activity-dependen t 

neuroprotective protei n (ADNP ) an d activity - 

dependent neurotrophi c factor (ADNF) , respectively. 

These peptide s confe r protectio n agains t numerou s 

neuronal insults , includin g ethano l (Spon g e t al. , 

2001). NAP reduces several of the advers e actions o f 

ethanol, suc h a s th e antagonis m o f L I adhesio n 

(Wilkemeyer et al., 2002, 2003), the depletion of glutathione 

(Spon g e t al., 2001) , and th e growt h inhibi - 

tion of embryos, including craniofacial regions (Spong 

et al, 2001; Wilkemeyer et al, 2003). How NAP does 

this is unclear . Charnes s and colleague s speculat e 

that disruption of LI adhesion s may trigger an oxidative 

death proces s calle d anoikis (Wilkemeye r et al. , 

2000, 2003). NAP may override these death signals as 

part of its broader neuroprotection. I t may also have 

direct effects on LI action. Further investigation s into 

LI will provide additional, important insights into the 

molecular mechanisms that underlie the sensitivity of 

neural crest to ethanol. 

Ca 2+ Signaling 

The earl y mous e blastocys t respond s t o ethano l 

(0.10%) with immediate generatio n o f an intracellu - 

lar Ca 2+ transient , th e sourc e o f whic h i s throug h 

PLC-mediated inosito l 1,4,5-triphosphat e (IP3 ) re - 

lease (Stacheck i et al, 1994 ; Rou t et al, 1997) . IP3dependent 

Ca 2+ transient s occu r spontaneousl y i n 

neural crest, and these are essential for the neural differentiation 

o f neura l cres t cell s (Care y an d Mat - 

sumoto 1999 , 2000) . Ethanol treatment of HH8 chick 

embryos stimulates an immediate (< 5 sec) intracellular 

Ca 2 " 1 " transient. Moreover, this transient is essential 

to induce neural crest apoptosis, because pretreatment 

with an intracellula r Ca 2+ chelator (BAPTA-AM ) prevents 

th e ethanol-induce d bu t no t endogenou s cel l 

death (Debelak-Kragtor p et al., 2003). Generation o f


this transien t require s th e activit y o f a pertussi s 

toxin-sensitive G protein , PLCß , an d ÏP 3 releas e 

(Garic-Stankovic et al, 2005). Gai2, but not Gail or 

Gaio, i s expressed in thes e cells . Extracellula r Ca 2+ 

contributes about 30 % of this Ca 2+ signa l (Debelak- 

Kragtorp e t al. , 2003) . We recentl y detecte d a Ca 2+ 

oscillation i n ethanol-treate d neura l crest , wit h a 

repeat ever y 1 5 t o 2 0 sec ove r 1 t o 2 min (Garic - 

Stankovic, Hernandez , an d Smith , unpublishe d 

results). A potentia l contributio n o f thi s oscillatio n 

is unde r investigation . Thes e event s ar e sensitiv e 

to inhibitio n b y decano l (Garic-Stankovi c e t al. , 

2006). 

The sourc e o f thi s Ca 2+ transien t involve s th e 

Gi/oßy stimulatio n o f a phosphoinositidyl-PLCß . 

Ethanol-mediated amplification of endogenous Giß y 

and phosphoinositidyl-PLC ß signal s i s implicated i n 

the dopaminergi c rewardin g properties o f ethanol i n 

neurons (Ya o et al. , 2002 , 2003) . Ethanol enhance s 

the activation of Gas, protei n kinase A, and cAM P response 

elemen t bindin g protei n signals , a s wel l a s 

Gißy, and synergis m through thes e pathways permits 

intracellular signalin g a t ligan d level s tha t normall y 

would no t transduc e signal . Th e identit y o f th e 

G-protein couple d recepto r and othe r participants in 

this transient is actively being investigated. It is of interest 

that two different cellula r target s and outcome s 

of ethanol, addiction/rewar d an d apoptosis , us e overlapping 

signaling pathways. 

Retinoic Acid Signaling 

One mechanis m propose d fo r ethanol-induced cran - 

iofacial dysmorpholog y involve s the perturbatio n o f 

retinoic aci d (RA ) signaling . Some isoform s o f alco - 

hol dehydrogenas e (ADH) , notabl y ADH4, will me - 

tabolize bot h ethano l an d retinol , a precursor o f RA 

(Chou e t al. , 2002) . Severa l group s hav e postulate d 

that ethano l compete s wit h retino l metabolism , an d 

thus R A production , i n th e developin g embry o 

(Duester, 1991 ; Grummer et al., 1993 ; Deltour et al, 

1996). R A signaling through th e nuclea r retinoi c aci d 

receptors (RARs ) directl y regulate s th e expressio n o f 

genes critical for the development of numerous embryonic 

structure s including neura l crest . These includ e 

the ho x genes, which define rostral-caudal patternin g 

of the embry o (Längsten and Gudas, 1994) ; the los s of 

retinoid signalin g leads t o ablation o f posterior hind - 

brain regions , including their neural cres t derivatives 

(Maden e t al, 1996) . R A also sustain s th e identit y 

(Plant e t al. , 2000 ) and outgrowt h (Schneide r e t al. , 

2001) o f the frontonasa l primordial , i n par t throug h 

its support o f fgß an d sh h pro-proliferativ e cues . RA 

deficiency a t thes e stage s lead s t o failur e o f fron - 

tonasal an d forebrai n outgrowth a s well a s apoptosis 

in craniofacial precursors (Zile, 2001). 

Until recently , thi s hypothesi s wa s challenging t o 

investigate, because o f the lo w circulating concentra - 

tions o f R A (K d o f R A fo r RAR s i s i n th e 0. 5 n M 

range) and th e hig h retino l store s of standard roden t 

models. To circumvent these difficulties, Dueste r and 

colleagues generate d null-mutan t mic e t o identif y 

ADH1 as a major ethanol dehydrogenase an d ADH4 

as a majo r retino l dehydrogenas e (Deltou r e t al. , 

1999). Clinically relevant ethanol concentration s can 

compete fo r retino l oxidatio n fo r bot h enzymes ; 

Molotkov an d Dueste r (2002 ) cit e K { value s o f 

0.040-3.8 mM fo r ADH1 an d 6-1 2 m M fo r ADH4. 

They furthe r sho w tha t bot h ethano l an d adhl nul l 

mutation prolong s retino l half-lif e throug h suppres - 

sion o f RA synthesis rather than R A catabolism. On e 

hundred m M bu t no t 1 0 mM ethano l inhibit s R A 

synthesis i n G7. 5 bu t no t G8. 5 mous e embryos , a s 

measured b y a lacZ-RAR E reporte r construc t (Del - 

tour e t al. , 1996) . I n ra t whole-embry o cultures , 

retinol and ethano l ha d synergisti c effects o n dysmorphology, 

bu t n o effec t o n tissu e R A and retina l con - 

tent (Che n e t al, 1996a) . Usin g liqui d diet s (36 % 

calories as ethanol; BEG ~260 mg/dl) and i n utero exposure, 

however, Zachman and colleagues report that 

tissues fro m 12-day-ol d mous e fetuse s ha d two - t o 

three-fold highe r retino l an d lowe r RA expression, as 

well as altered RA R and cellula r retino l bindin g pro - 

tein transcrip t level s (Zachma n an d Grummer , 

1998). In the developing quail, 10 nM R A rescued the 

cardiac dysmorpholog y cause d b y exposur e t o 1.0 % 

ethanol (Twal and Zile, 1997) . 

Evidence o f a specific interaction betwee n ethanol 

and endogenou s retinoi d signalin g i n neura l cres t is 

lacking. ADH may be expressed in premigratory neural 

cres t cells , althoug h thi s particular expressio n is 

not always observed an d doe s not appear t o correlat e 

with RA production (An g et al., 1996 ; Haselbeck an d 

Duester, 1998) . Mor e certai n i s th e expressio n o f 

ADH4 (an d the subsequen t production o f RA) in migratory 

neura l cres t an d th e developin g craniofacial 

mesenchyme (Ang et al, 1996) . I t is likely, therefore, 

that any effects o f ethanol on retinoid signaling would 

act on th e migrator y and postmigrator y neural crest , 

rather tha n affectin g premigrator y populations wit h


respect t o thei r survival . Indeed , ethanol-exposed , 

RARE-lacZ reporter mice have reduced expressio n of 

this RA-dependent construct, an outcome mos t likely 

explained b y a reductio n o f RA-mediate d signalin g 

(Deltouretal., 1996) . 

A major cavea t is that endogenous R A specifically 

acts o n frontonasa l development . Onc e patterned , 

there is no apparent retinoid requirement for maxilla, 

mandible, and hyoi d outgrowth (e.g. , Wedden , 1987 ; 

Dickman et al., 1997 ; Schneide r e t al, 2001). At this 

time, the retinoid hypothesi s does no t adequately explain 

the sensitivity of neural crest to ethanol-induced 

apoptosis, nor does it wholly account for the change s 

in maxillar y an d lowe r ja w development. Nonethe - 

less, this remains an attractive hypothesis and it merits 

additional, careful investigation . 

GENETIC FACTORS 

Genetic factor s modulat e neura l cres t sensitivit y to 

ethanol, jus t as they modulate th e ris k o f other con - 

genital anomalie s an d neurobehaviora l outcomes . 

We screene d 1 1 leghor n chicke n strain s and foun d 

that they differed widel y in their sensitivity to ethanolinduced 

apoptosi s withi n neura l cres t and th e subse - 

quent craniofacia l outcom e (Debela k an d Smith , 

2000; S u e t al, 2001) . Culture d neura l cres t cell s 

from inbre d C57B1/6 J mic e ar e mor e sensitiv e t o 

ethanol-induced toxicit y tha n thos e fro m outbre d 

ICR mice (Chen e t al., 2000). Their sensitivit y correlates 

positivel y with indicator s of membrane fluidit y 

and negatively with GM1 gangliosid e content. Moreover, 

GM 1 applicatio n attenuate s ethanol-induce d 

damage t o C57B1/6 J neura l cres t (Che n e t al. , 

1996b). Chick strains also differ wit h respect to aorticopulmonary 

defects associated with the cardiac neural 

cres t (Bruyèr e an d Stith , 1993) . I n al l thre e 

models, ethanol metabolism is not appreciably differ - 

ent, whic h suggest s tha t factor s intrinsic t o th e em - 

bryo and neural crest are responsible . 

Maternal an d feta l capacit y for ethano l metabo - 

lism affect s ris k fo r ethanol-induce d malformation s 

of the fetus . Th e mor e efficien t alcoho l dehydroge - 

nase allele IB* 3 confers increased protection against 

teratogenic effect s o f ethano l i n a huma n cohor t 

(McCarver e t al. , 1997) , an d contribution s o f Cy - 

tochrome P45 0 2E 1 ma y b e protectiv e (Rashee d 

et al., 1997) . These findings extend to facial dysmorphology 

and reinforce the notion that alcohol rathe r 

than acetaldehyd e i s th e proximat e teratoge n (Da s 

et al., 2004). 

FUTURE DIRECTIONS 

It is now clear that the effect s o f ethanol on facia l ap - 

pearance represen t a respons e a t a relatively narrow 

developmental window . Facial appearance remain s a 

highly useful diagnosti c tool, and the study of its neural 

crest precursors continues to provide important insights 

int o th e molecula r mechanism s tha t underlie 

neurotoxic effect s o f ethanol. One suc h avenu e i s to 

investigate whethe r ethano l exposur e ultimatel y affects 

common signaling pathways within otherwise diverse 

cell types. 

It i s disconcertin g tha t th e consequence s o f 

ethanol exposur e to neura l crest-derive d structure s 

other tha n th e fac e remai n largel y unstudied , 

because thes e population s contribut e t o critica l activities 

suc h a s hearing , vision , textur e sensation , 

chewing, th e autonomi e nervou s system , an d hor - 

monal action s tha t include th e adrena l medulla , pituitary, 

parathyroid , an d thyroi d glands . Studie s o f 

these less-appreciate d neurochristopathie s an d thei r 

consequences fo r health i s an importan t gap i n FAS 

research. The addressin g of this question shoul d be - 

come a high priority in the assessment of affected in - 

dividuals and i n detaile d investigatio n usin g anima l 

models. 

ABBREVIATIONS 

ADH alcoho l dehydrogenase 

ADNF activity-dependen t neurotrophi c facto r 

ADNP activity-dependen t neuroprotective protein 

ARND alcohol-relate d neurodevelopmenta l disorder 

ASDs atria l septal defects 

BEG bloo d ethano l concentratio n 

BMP bon e morphogenic protein 

CAM cel l adhesion molecule 


FASD feta l alcohol spectrum disorder 


G gestationa l day


IP3 inosito l 1,4,5-triphosphat e 

RA retinoi c acid 

RAR retinoi c acid receptor 

SOD Superoxid e dismutase 

VSDs ventricula r septa l defect s 


ACKNOWLEDGMENTS Thi s work was supported by the 

National Institut e of Alcohol Abuse and Alcoholis m and 

National Institute for Environmental Health Sciences, and 

the March of Dimes Birth Defects Foundation. 

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18 

Glial Targets of Developmental 

Exposure t o Ethanol 

The wor d glia is derived from th e Gree k wor d gliok, 

meaning glue , whic h Vircho w (Letterer , 1958 ) ap - 

plied i n the sense of nerve glue. The traditiona l point 

of view of glia a s a passive component of the centra l 

nervous system (CNS) has changed drasticall y during 

the pas t decade . Gli a ar e no w recognize d a s active 

partners wit h neuron s a s participants i n neurotrans - 

mission and they play essential roles in axonal conduction, 

synapti c plasticity , an d informatio n processin g 

(Nagler et al, 2001; Ullian et al., 2001, 2004 ; Fields - 

Graham, 2002) . Further, gli a ca n respon d t o insults 

and ar e capabl e o f proliferatin g throughou t lif e 

(Chen and Swanson, 2003). 

In th e adul t huma n brain , gli a outnumbe r neu - 

rons by one order of magnitude. There are two classes 

of glia : microgli a (whic h mediat e inflammator y re - 

sponses in the CNS ) an d macroglia . The latte r cell s 

are oligodendrocyte s (whic h for m th e insulatin g 

niyelin sheaths) and astrocytes, the most paradigmatic 

glia (whic h are abundant an d ubiquitou s within th e 

CNS). The present chapter focuses on astrocytes. 

Consuelo Guerri 

Gemma Ruber t 

Maria Pascual 

295 

ROLE OF GLIA IN THE DEVELOPIN G 

CENTRAL NERVOUS SYSTE M 

Glia ar e presen t an d integra l throughou t CN S de - 

velopment. Glial-neurona l interaction s play critical 

roles in multiple developmental events. For example, 

radial glia (RG) provide physical and chemical guid - 

ance fo r th e migratio n o f young neuron s fro m th e 

embryonic proliferativ e zone s int o th e developin g 

cortex (Rakic , 1972 ; Hatte n an d Mason , 1990 ) (se e 

Chapter 3) . When the migratio n is completed, mos t 

RG transform into astrocytes. RG also serve as a rnultipotential 

precurso r cell , a s the y ar e abl e t o self - 

renew and generate neuron s (Malatest a e t al., 2000 ; 

Noctor e t al, 2001 , 2002 ; Göt z e t al. , 2002 ; Sana i 

étal., 2004). 

Astroglia and glial-derive d factors ar e key to synaptogenesis, 

since they promote th e formation of mature 

functional synapse s (Nagler et al., 2001; Ullian et al., 

2001, 2004). Disturbances o f glia or of neuronal-glial 

communication durin g th e well-establishe d critica l


periods o f brain developmen t ca n caus e irreversible 

deficits i n CN S functio n (Hatten , 1999 ; Lammens , 

2000; Ros s an d Walsh , 2001 ; Crespe l e t al, 2002). 

Such findings strongly support the crucia l role of glia 

in development and the notion tha t their dysfunction 

underlies anomalies in brain development . 

An increasin g number o f studies shows (a) that a 

prime target of ethanol within the developing brain is 

astrocytes (Guerri and Renau-Piqueras , 1997 ; Guerr i 

et al, 2001) and (b) that ethanol impairs astrogliogenesis. 

The presen t chapter reviews clinical, experimental, 

and in vitro evidence about the actions of ethanol 

on astrogli a and thei r functions. Specia l attentio n i s 

paid to the effect s tha t prenatal ethanol exposur e has 

on neura l ste m cell s (e.g. , RG) and t o the action s of 

ethanol o n astroglia l proliferatio n an d cel l survival . 

Since astrogli a regulate synaptogenesi s and synapti c 

transmission, the potentia l effect s o f ethanol o n thes e 

processes are also discussed. 

GLIAL ABNORMALITIES CAUSE D 

BY ETHANOL 

Since th e first neuropathological studies on childre n 

with feta l alcoho l syndrom e (FAS ) (Clarre n et al , 

1978), abnormalitie s in glial development hav e been 

suspected a s contributin g t o th e advers e effect s o f 

ethanol o n th e developin g brain . Ethanol-expose d 

brains exhibit abnormal glial placement primaril y associated 

wit h th e méninge s (meningea l neuroglia l 

heterotopias) (Clarren e t al., 1978 ; Peiffe r e t al, 1979 ; 

Clarren, 1986) , gyra l malformations, cerebral dysgenesis, 

and, in some cases, reactive gliosis (Peiffer e t al., 

1979; Wisniewski et al, 1983). White matter, a repository 

o f glia , appear s t o b e affecte d b y prenata l 

ethanol exposur e (PEE). Severa l studies usin g mag - 

netic resonanc e imagin g and singl e photon emissio n 

computed tomograph y have shown that children with 

FAS have hypoplasia of the corpu s callosum an d an - 

terior commissure (the anläge of which are formed by 

glia) (Rile y et al., 1995 ; Johnson e t al., 1996 ; Swayz e 

et al. , 1997) . PE E ca n lea d t o a reductio n i n whit e 

matter an d delaye d myelinatio n (Riikone n e t al. , 

1999;SowelletaL, 2001b). 

Animals expose d t o ethano l durin g brai n devel - 

opment exhibi t alteration s i n glia-relate d develop - 

ment, including aberrant neuronal migration, delayed 

astrogliogenesis, an d a reductio n o f cortica l astro - 

cytes (Miller, 1986, 1988 ; Mille r and Potempa , 1990 ; 

Gressens e t al, 1992 ; Mille r an d Robertson , 1993 ; 

Vallès e t al. , 1996) . Moreover , studie s o f a primat e 

model o f FAS implicate glial involvement. Ethanolexposed 

monkey s hav e glia l heterotopia s (Clarre n 

and Bowden , 1984 ) an d dysgeni c corpor a callos a 

(Miller e t al., 1999) . Thes e alterations ar e consisten t 

with th e concep t tha t on e o f th e developmenta l 

stages most vulnerable to ethanol i s the brain-growt h 

spurt (Bonthius and West, 1991) . This period i s characterized 

b y majo r developmen t o f glia an d myeli n 

structures. 

ETHANOL AFFECT S 

ASTROGLIAL ONTOGENY 

Radial Glia 

Normal Ontogeny of Radial Glia 

Among th e firs t cell s t o differentiat e fro m th e neu - 

roepithelium are RG (Misson et al, 1991) , which appear 

befor e th e onse t o f neuronogenesi s (Cavines s 

et al., 1995). RG have a bipolar morphology: each RG 

has a soma i n the ventricula r or subventricular zon e 

and bear s a lon g apica l proces s tha t extend s toward 

the pia l surfac e an d a shorter basa l process tha t con - 

tacts the ventricular wall. The ter m radial glia was introduced 

by Rakic (1972) in his classic description of 

neural migration in the fetal primate neocortex. 

Most RG transform into mature, stellate-appearin g 

astrocytes afte r th e neurona l migratio n i s complete d 

(Schmechel an d Rakic , 1979 ; Pixle y an d DeVellis , 

1984; Voigt , 1989; Camero n an d Rakic , 1991; Misson 

et al, 1991 ; Mille r and Robertson, 1993) . Changes i n 

the morphology of RG are linked to changes in the expression 

of specific proteins. Vimentin an d nesti n (in - 

cluding its immunorecognized antigenic determinant s 

RC1 and RC2) are prenatally present in RG of rodent 

CNS, wherea s glia l fibrillary acidi c protei n (GFAP ) 

appears in neonates (e.g. , Hockfield and McKay, 1985 ; 

Millerand Robertson, 1993; Sancho-Tello etal, 1995; 

Chanas-Sacreetal.,2000). 

Effects of Ethanol on 

Radial Glia Development 

Cerebral Cortex . Ethano l exposure during embryogenesis 

induce s irreversibl e alteration s i n th e CN S 

(Guerri, 1998 , 2002). The first suggestion that ethanol

impairs migratio n originate s fro m neuropathologica l 

examination a t autops y o f brains fro m childre n wit h 

FAS (Clarre n e t al, 1978 ; Wisniewsk i e t al, 1983) . 

Most notable among the findings were neuroglial heterotopias 

located nea r the pia l surface o f the cerebral 

cortex. Alteration s observe d i n human s hav e bee n 

reproduced i n studie s o f animals expose d t o ethano l 

in utero . Prenata l exposur e t o moderat e amount s o f 

ethanol (100-20 0 mg/dl) cause s leptomeningea l het - 

erotopias wit h breache s i n th e gli a limitan s an d re - 

duces th e numbe r an d alter s the morpholog y o f RG 

(Kotkoskie an d Norton , 1988 ; Gressen s e t al. , 1992 ; 

Komatsu et al, 2001; Mooney et al, 2004). At least in 

the rat , heterotopias persis t into adulthoo d (Komats u 

et al., 2001). 

Cultured R G harveste d fro m 12-day-ol d fetuses are 

affected b y pre - an d postconceptio n exposur e t o 

ethanol (Vallè s e t al, 1996) . Thes e R G exhibi t shor t 

glial processes, reduce d number s o f cells, and delays in 

their transformation into GFAP-positive astrocyte s (Vallés 

et al., 1996 ) (Figs . 18- 1 an d 18-2) . A developmen - 

tal delay in GFA P expressio n has also been describe d 

in rat whole brains in vivo. During normal rat brain development, 

GFA P mRN A appear s o n gestationa l da y 

(G) 14 , then bot h GFA P transcript and protein expression 

increas e durin g lat e feta l an d earl y postnata l 

development. Prenata l ethanol exposur e delays the appearance 

o f GFA P mRN A unti l G1 9 an d decrease s 

GFAP expression (Vallès et al., 1996 , 1997) . A comparable 

delay is evident among astrocyte s proximal to th e 

dorsal and medial raphe (Tajuddi n et al, 2003). 

An in vivo study focusing on the RG and astrocytes 

in th e cortica l plate-derive d lamina e o f cortex, i.e. , 

layers II—Via , show s that GFAP expressio n i s ubiquitously 

delayed (Mille r an d Robertson , 1993) . I n con - 

trol rats , nestin-positiv e R G ar e presen t throug h th e 

period o f neuronal generatio n (pas t postnatal da y [P] 

8). GFAP-positiv e astrocytes , thoug h common i n th e 

intermediate zon e (th e anläg e o f the whit e matter) , 

only appear i n deep layer Via on P3. With time, they 

progressively appear i n more superficial positions . I n 

contrast, i n ethanol-treate d rat s R G begi n t o regres s 

by P 5 and GFA P immunoreactivit y appears i n corti - 

cal gray matter on PI. From th e effect s o f ethanol o n 

the paire d change s i n nesti n an d GFA P expression , 

the compellin g conclusio n i s that ethano l cause s th e 

premature transformatio n o f R G int o astrocyte s i n 

cortex—this underlies the migratio n of late-generated 

neurons t o ectopi c site s (Miller , 1986 , 1988 , 1997) . 

The differen t timin g o f GFA P expressio n i n whol e 

GLIAL TARGETS OF DEVELOPMENTAL EXPOSURE TO ETHANO L 29 7 

control PEE 

FIGURE 18- 1 Effec t of ethanol on nestin and glial fibrillary 

acidi c protei n (GFAP ) expression . Corona l 

sections o f 21-day-ol d contro l (A ) an d prenata l 

ethanol-exposed (PEE ) (D ) fetuses , staine d wit h 

hematoxilin-eosin. B, C, E, F. Higher magnificatio n 

of boxed regions to show nestin-positive glia (RG) and 

GFAP-positive astrocytes. Brain sections from PE E fe - 

tuses show a reduction i n the corpu s callosum (CC) , 

alternations i n the morpholog y o f RG fibers, and re - 

duction i n the generation of astrocytes. 

brain vs. cortex and th e effec t o f ethanol on that tim - 

ing (cf . Mille r an d Robertson , 1993 ; Vallè s e t al , 

1996) likel y reflect the effec t o f ethanol on astrocyte s 

in th e intermediat e zone , th e primar y (sole ) sit e o f 

GFAP expression i n the fetus. 

Cerebellar Cortex . Th e cerebellu m i s anothe r 

brain regio n tha t i s sensitive to ethano l toxicit y dur - 

ing developmen t (Guerri , 1998) . Th e cel l bodie s o f 

Bergmann glia , the cerebella r subpopulatio n o f R G 

(Sievers et al, 1994 ; Yuasa , 1996) , initiall y assume a 

position periventricula r t o th e Purkinj e cel l layer . 

Postnatally, thei r cel l bodie s translocat e peripherall y 

so that they ca n guid e th e inwar d migration o f granule 

neurons.


control PEE 

FIGURE 18- 2 Radia l glia a t 2 and 7 days of culture. 

Radial gli a were isolate d fro m th e brain s of 12-day - 

old contro l o r prenata l ethanol-expose d (PEE ) fe - 

tuses. A, B. During the initial days of culture, control 

cells expresse d mainl y nesti n (R G marker ) an d 

formed aggregate s resemblin g neurosphere s (A ) 

composed o f newly born neuron s (Tujl + ) an d radia l 

glia (nestin + ). C-F. Th e neurosphere-lik e aggregate s 

increased in number and siz e with culture time (C), 

and radia l gli a wer e transformed into mor e matur e 

neurons (MAP2+ ; D , arrowheads ) and int o GFAP - 

positive astrocyte s (E , F) . Culture d radia l glial cell s 

isolated fro m PE E fetuse s displaye d morphological 

alterations an d impaire d neurogeni c potential , a s 

demonstrated by the decreas e in number of neurons 

and astrocyte s generated . Nucle i wer e staine d wit h 

Hoechst ( g Rubert, r Miïlana, and c Guerri, unpub - 

lished results). 

Ethanol, given eithe r acutel y or chronically dur - 

ing fetal and neonatal development , delay s the maturation 

o f Bergman n glia , decrease s th e numbe r o f 

GFAP-positive fibers, and induce s the growt h of abnormal 

glia l processe s (e.g. , shorter, thinner, an d ir - 

regular) (Shett y e t al. , 1994 ; Perez-Torrer o e t al. , 

1997). Moreover , gestationa l exposur e t o ethano l 

causes defect s i n th e cerebella r glia l limitan s 

(formed b y Bergmann glia l en d fee t o n th e cerebel - 

lar surface ) o f the ra t and th e appearanc e o f solitary 

or clustered ectopic granule cells (Sakata-Haga et al., 

2001). Damag e t o th e glia l limitan s ma y underli e 

the fusio n o f th e foli a V an d V I o f th e cerebella r 

vermis an d th e disruptio n of the cerebella r cortical 

structure. 

Factors Regulatin g Radia l Glia l Transformation . 

Our curren t understanding o f mechanism(s) and factors 

that regulate the transformation of RG into astro - 

cytes o r neuron s developmen t i s limited . Extrinsic 

cues suc h a s ciliar y neurotrophi c factors , epiderma l 

growth factors , neureguli n l-erbB 2 signalin g an d 

members o f the bon e morphogeneti c protei n famil y 

may induc e cortica l progenitor s t o differentiat e int o 

astrocytes (Hughe s e t al, 1988 ; Gros s e t al, 1996 ; 

Johe e t al. , 1996 ; Küh n an d Miller , 1996 ; Burrows 

étal., 1997 ; Schmid et al, 2003). 

Critical determinants of GFAP expression and as - 

trocyte differentiatio n i n feta l brai n ar e change s i n 

DNA methylation and chromatin structure (Takizawa 

et al, 2001 ; Song and Ghosh, 2004). These change s 

are regulate d b y hormones an d growt h factors . Th e 

methylation o f genomic DN A at CpG dinucleotide s 

regulates cell - o r tissue-specifi c gen e expressio n 

(Razin,1998; Bird and Wolffe, 1999) . During embryogenesis 

an d differentiation , mos t tissue - an d cell - 

specific genes are almost fully methylated and undergo 

programmed demethylatio n at the moment of activation 

and transcription (Barresi et al., 1999) . 

Chronic i n uter o ethano l exposur e decreases th e 

demethylation o f GFAP gene, affectin g bot h it s transcription 

and expressio n (Vallès et al., 1997) . Ethano l 

exposure interfere s wit h th e releas e an d actio n o f 

growth factor s (Luo an d Miller , 1998) , whic h migh t 

affect th e developmentally regulated epigenetic mod - 

ifications, leadin g t o th e change s i n bot h DN A 

methylation and GFAP expression. 

Cell Adhesion Proteins. Ethano l affect s glia l elab - 

oration o f cell adhesio n protein s an d growt h factors 

(e.g., transforming growth factor [TGF ] ßl ) mediatin g 

neuronal-glial interactions. Among the cel l adhesion 

proteins, neural cell adhesion molecules (nCA M an d 

LI), integrins, and astrotactin mediate neuronal-glia l 

attachments an d communicatio n (Edelman,1994 ; 

Hatten, 2002 , Nadaraja h an d Parnavelas , 2002 ; 

Schmid and Anton, 2003). 

Ethanol affects th e expressio n and function of several 

neural cell adhesion proteins both in vitro (Miller 

and Luo, 2002; Minana et al, 1998 , 2000) and in situ 

(Siegenthaler an d Miller , 2004) . Ethano l als o dis - 

rupts (a) LI-dependent cell adhesion and homophilic 

interactions among LI molecule s (Wilkemeyer et al.,

1999, 2000 ; Bearer , 2001 ) an d (b ) integri n expres - 

sion in fetal brain organotypic cultures (Siegenthaler 

and Miller, 2004). Likewise , TGFßl, which promotes 

neuronal migratio n i n corte x an d increase s expres - 

sion o f adhesio n proteins , i s profoundly affected b y 

ethanol (Mille r an d Luo , 2002 ; Siegenthale r an d 

Miller, 2004). 

Radial Gli a as Neuronal Precursors . Th e recentl y 

described rol e o f RG a s neuronal precursors (Malatesta 

etal, 2000; Noctor etal., 2001, 2002; Götz etal., 

2002) dramaticall y change s ou r understandin g o f 

CNS developmen t unde r norma l an d pathologica l 

conditions. Takin g int o accoun t th e contributio n of 

RG a s a neurona l progenito r cell , i t i s plausible to 

speculate tha t alteratio n o f R G b y ethano l migh t 

cause dysgeneratio n o f both astrocyte s and neurons . 

The numbe r o f neuron s an d astrocyte s generate d 

from culture d R G isolated fro m ethanol-expose d fe - 

tuses is reduced (Rupert, Minana and Guerri, unpublished 

results) (Fig. 18-2) . In vivo studies have shown 

that prenata l exposur e to ethanol cause s a reductio n 

in neuronogenesis and gliogenesis (e.g., Miller, 1986 , 

1988; Gressen s e t al , 1992 ) (se e Chapte r 13) . Although 

i t has been interprete d that this reduction results 

fro m ethanol-induce d change s i n th e outpu t 

from th e cortica l proliferativ e zones, a contributio n 

from R G must be considered . 

It has been suggeste d tha t RG (a ) comprise a het - 

erogeneous population , (b ) var y i n growt h facto r ex - 

pression accordin g to their location withi n the CNS , 

and (c ) diffe r i n th e type s o f cell s the y generat e 

(Kriegstein and Götz , 2003). In contrast , othe r inves - 

tigators argue that RG i n all parts of the CN S serv e as 

neuronal progenitors (Anthony et al., 2004). In either 

case, controlle d studie s o f th e potentia l effec t o f 

ethanol o n th e ste m cel l rol e o f RG ar e require d t o 

clarify whether ethanol decrease s the pool of stem cells 

or onl y induce s abnormalitie s i n th e RG-astrocyte 

lineage. 

Astroglial Proliferation 

Effects of Ethanol on Cell Cycle 

The mos t activ e perio d o f glia l proliferatio n i s th e 

brain growth spurt, a time when the brain undergoes 

its mos t rapi d growt h (Dobbin g an d Sands , 1993) . 

In humans , thi s perio d occur s durin g th e fina l 

trimester o f gestation an d earl y infancy, wherea s in 


the ra t i t occur s durin g the firs t 2 postnatal weeks. 

During th e brain growt h spurt , neural developmen t 

is particularl y sensitiv e t o th e toxi c effect s o f envi - 

ronmental agent s suc h a s methylmercury , lead , 

and ethano l (Cost a e t al. , 2004) . An implicatio n is 

that gli a ar e a target fo r this neurotoxicity (Aschner 

etal., 1999) . 

Ethanol exposure during gestation ca n impair th e 

proliferation an d differentiatio n o f astroglia. The cor - 

tices o f rats prenatally expose d t o ethano l hav e one - 

third fewe r gli a (an d als o o f neurons) tha n control s 

(Miller an d Potempa , 1990) . Prenata l exposur e t o 

ethanol reduces the expression of the astrocyte marker 

GFAP durin g postnata l brai n developmen t (Vallè s 

et al, 1996 ; Tajuddi n e t al, 2003) . Cultured astro - 

cytes fro m th e pup s o f ethanol-fe d rat s exhibi t re - 

duced [ 3 H]thymidine and [ 3 H]leucine incorporation 

and GFAP-positiv e cell s (Guerr i e t al., 1990 ; Guerr i 

and Renau-Piqueras, 1997) . These data are consistent 

with a n ethanol-induce d decreas e i n glia l prolifera - 

tion and differentiation . 

Direct demonstratio n tha t ethano l affect s th e pro - 

liferation o f astroglial cell s come s fro m experiment s 

using primary cultures of astrocytes prepared from th e 

brains of neonatal rats . Ethanol (110-88 0 mg/dl) im - 

pairs cell proliferation , inhibits th e increas e i n th e 

astrocyte number , an d reduce s [ 5 H]thymidine incorporation 

(Kenned y an d Mukerji , 1986 ; Davie s an d 

Cox, 1991 ; Snyde r e t al. , 1992 ; Aroo r an d Baker , 

1997; Guizzett i e t al, 1997 ; Lu o an d Miller , 1998 , 

1999; Mille r and Luo , 2002) . Ethanol affect s th e cycling 

population i n multiple ways : it inhibits the cel l 

cycle i n the GO/G 1 phas e o f the cel l cycle , impede s 

the progressio n of cells int o S phase, an d decrease s 

the numbe r o f mitoti c cell s (Guerr i e t al. , 1990 ; 

Mikami e t al, 1997 ; Lu o and Miller , 1999) . Impair - 

ment o f cel l proliferatio n through ethano l exposur e 

has als o bee n show n i n glia l tumo r cells , principall y 

C6 astrocytom a cell s (Wazir i e t al. , 1981 ; Isenber g 

et al, 1992 ; Lu o an d Miller , 1996 , 1998 ; Guizzett i 

et al , 1997 ; Mille r an d Luo , 2002) . This effec t i s 

concentration-dependent. 

Mechanisms of Ethanol Toxicity among 

Proliferating Astroglial Populations 

Astrocytic proliferatio n i s regulate d b y mitogeni c 

growth factors and growt h inhibitor y agents (Lu o an d 

Miller, 1998 ) (se e Chapte r 11) . I n turn , thes e pro - 

teins trigge r signalin g pathway s involvin g tyrosin e


kinases, protei n kinas e C (PKC) , an d mitogen - 

activate protein kinase (MAPK). Ethanol inhibit s the 

proliferative effect s o f several growth factors , includ - 

ing basi c fibroblas t growt h factor , platelet-derive d 

growth factor (PDGF) , an d insulin-lik e growth factor 

1 (Resnicof f e t al , 1994 ; Lu o an d Miller , 1996 , 

1999). These factors affec t bot h MAP K and PKCs . I n 

addition, ethano l potentiate s th e antiproliferativ e activity 

of TGFßl (Mille r and Luo, 2002) . Neural cell s 

actively stimulated b y growth factors are more suscep - 

tible t o th e antiproliferativ e effect s o f ethanol , al - 

though ethano l doe s not affect the action of all growth 

factors equivalently (Luo and Miller, 1998 , 1999) . 

Ethanol potentl y inhibit s mitogeni c signal s initiated 

by binding (by carbachol or acetylcholine) to astroglial 

muscarinic receptors, particularly M3 subtype 

(Catlin e t al , 2000 ; Cost a an d Guizzetti , 2002) . 

Binding of M3 receptors increase s DN A synthesis by 

glia (Costa e t al, 2001). Ethanol (46-46 0 mg/dl) prevents 

thes e mitogeni c effect s i n th e huma n 1321N 1 

astrocytoma cells, fetal human astrocytes , and ra t cortical 

astrocyte s (Guizzett i e t al, 1998 , 2003 ; Cost a 

et al., 2001). Ethanol affect s at least two pathways triggered 

by activation of M3 receptors. First, ethanol targets 

the activatio n of phospholipase D (PLD) , which 

hydrolyzes phosphatidylcholin e t o cholin e an d PA . 

Thus ethano l decrease s phosphatidi c aci d (PA ) concentration. 

P A stimulates downstrea m target s such a s 

protein kinases , includin g PKC , an d i s a mitogeni c 

signaling cascade i n astrocytes (Schatter et al., 2003). 

When ethano l i s present, PL D ca n catalyz e a transphosphatidylation 

reactio n generatin g phosphatidy l 

ethanol (PEt ) rathe r tha n P A (Kotte r an d Klein , 

1999). Thi s diversio n essentiall y block s th e signa l 

transduction cascad e becaus e PE t doe s no t activat e 

the downstrea m targets of PA (Schatter e t al, 2003). 

The secon d pathway affected b y ethanol i s the inhibi - 

tion o f atypica l PKCÇ , Subsequently , thi s down - 

regulates th e activatio n o f both the p70s 6 kinas e and 

the nuclear factor KB (Costa an d Guizzetti, 2002) . 

A prime metabolite o f ethanol, acetaldehyde , ca n 

inhibit cel l growt h (Holowni a e t al. , 1996) . Astro - 

cytes expose d t o ethano l ca n produc e acetaldehyd e 

(Eysseric e t al. , 1997) . Ethano l ca n b e metabolize d 

into acetaldehyde via alcohol dehydrogenase (ADH), 

catalase, and cytochrom e P45 0 2E 1 (CYP2E1) . Th e 

effects o f ethano l ma y no t b e transduce d throug h 

ADH, a s ethanol-induce d cel l growt h inhibitio n 

is observe d i n gli a treate d wit h th e AD H inhibito r 

4-methylpyrazole (Isenber g e t al. , 1992) . O n th e 

other hand , catalas e an d CYP2E 1 ar e presen t i n astrocytes 

(Montoliu et al, 1995 ; Eysseric et al, 2000). 

These enzymes may be key players in the toxic effect s 

of ethanol on the developing CNS. 

Astrocytic Death 

Cell death i s a normal featur e o f CNS developmen t 

(e.g., Oppenheim, 1991 ; Raffe t al, 1993) . I t is regulated 

b y growth factors , cytokines, neurotransmitters , 

and cell-cel l signaling , including glial-neurona l in - 

teractions (Johnson and Deckwerth, 1993 ; Raffetal. , 

1993). Althoug h mainl y ascribe d t o neurons , deat h 

among developin g astrocyte s i s also commo n (Sori - 

ano et al, 1993). 

Developmental ethano l exposur e affects mos t nor - 

mal regulatory factors, exacerbating the processe s associated 

wit h naturally occurring neural deat h i n the rat 

developing cortex (Mooney and Miller, 2001; Climent 

et al., 2002; see Chapters 1 5 and 16) . This ethanol ex - 

posure increases the numbe r of neurons and astroglial 

cells tha t di e b y necrosi s an d b y apoptosi s (Climen t 

et al., 2002). Ethanol can also induce necrotic (Holownia 

et al., 1997) or apoptotic (Pascual et al., 2003) death 

in cultured astrocytes. These effects ar e concentration - 

dependent. 

Astrocytes die as a result of ethanol-induced activation 

o f sphingomyelinas e (SMase ) (Pascua l e t al. , 

2003), leading t o the hydrolysi s of sphingomyelin t o 

ceramide (Ohania n an d Ohanian , 2001) . Treat - 

ment wit h lo w o r moderat e ethano l concentration s 

(46-230 mg/dl) either in vitro or in vivo increases astrocyte 

susceptibility to cell deat h vi a tumor necrosis 

factor (TNF)-o t (D e Vito e t al, 2000) . Conceivably , 

ethanol shifts the balance of the sphingolipid metabolism 

by decreasing the mitogeni c signal s in favo r o f a 

pathway (SMase/ceramide ) tha t increase s astrocyt e 

susceptibility to the cytotoxi c effect o f TNFot. 

Sphingomyelins ar e cel l membran e phospho - 

lipids that contains ceramid e and phosphorylcholine . 

Ceramide i s an importan t regulator o f cell prolifera - 

tion, differentiation , an d apoptosis . It has trophi c ef - 

fects a t lo w concentrations an d trigger s apoptosis a t 

high concentration s (Hannu m an d Luberto , 2000) . 

Increased ceramid e productio n participate s i n th e 

cell deat h occurrin g durin g earl y neural differentia - 

tion (Herge t e t al. , 2000 ) and i n neurona l apoptosi s 

induced b y nutrient deprivation (Toma n et al., 2002). 

A variet y o f stimul i an d Stressor s (e.g. , oxidant s 

and cytokines ) stimulat e SMas e (Andriu-Abadi e and

Levade, 2002) , leadin g t o ceramid e generation . 

Ethanol induce s a stres s respons e (e.g. , oxidativ e 

stress) by astrocytes (Montoliu e t al., 1995) . Stimula - 

tion o f SMas e activit y an d ceramid e generatio n 

appears to be a key event i n the subsequen t ethanol - 

induced cel l death (Pascua l et al., 2003). Further in - 

vestigation i s require d t o evaluat e th e quantitativ e 

participation o f this mechanis m i n ethanol-induce d 

astroglial cell death during in vivo brain development 

(Climent e t al., 2002) an d t o clarif y i f other mecha - 

nisms are involved. 

GLIA AND NEURONA L SYNAPSIS 

Pivotal Rol e of Glia in Synaptogenesis 

It has been suggeste d tha t gli a pla y a role in synapse 

formation becaus e astrocyte s an d synapti c terminal s 

are intimatel y relate d (Ventur a an d Harris , 1999 ; 

Schikorski an d Stevens , 1999 ; Grosch e e t al, 2002) 

and glia l developmen t an d synaptogenesi s ar e spa - 

tiotemporally relate d (Aghajania n an d Bloom , 1967 ; 

Miller and Peters , 1981 ; Parnavela s et al, 1983) . For 

example, i n ra t cortex , neuron s for m mos t o f thei r 

synapses during the third postnatal wee k after the differentiation 

o f astrocytes is largely complete. Furthermore, 

culture d retina l ganglio n cells , hippocampa l 

neurons, an d spina l moto r neuron s for m several-fold 

more functional synapses when astrocyte s are presen t 

(Ullian etal, 2001, 2004; Song et al, 2002). Thus, astrocytes 

appea r t o promot e synaps e formation . In - 

deed, astrocyte s ar e considere d activ e partner s tha t 

participate with the pre- and postsynaptic terminals in 

tripartite synapti c structure s (Araqu e e t al. , 1999 , 

2001;Haydon,2001). 

Various glial-derived factors mediat e synaptogene - 

sis. One suc h factor is cholesterol, which is produced 

by astrocyte s an d secrete d i n apolipoprotei n E-con - 

taining lipoprotein s (Mauc h e t al. , 2001) . Anothe r 

glial factor , activity-dependen t neurotrophi c facto r 

(ADNF), i s released b y astrocytes i n respons e t o vasoactive 

intestina l polypeptide . ADN F ma y pla y a 

role in synaptic development an d neuronal differenti - 

ation (Blondel et al., 2000) and i s also a neuroprotector 

(Gozes and Brenneman, 2000). 

Not only are glia important for synapse formation, 

they ar e als o key players in synapti c function . The y 

enhance postsynaptic responsiveness and help to maintain 

efficient synapti c connectivity. In the presence of 


glia, retina l ganglio n cell s hav e large r glutamate - 

induced miniatur e postsynapti c current s (Ullia n e t 

al., 2001, 2004). Experiments conducted o n both cultured 

an d intact-tissu e preparation s hav e demon - 

strated tha t transmitter s release d fro m neuron s ca n 

stimulate an d caus e th e gli a t o releas e glutamate , 

ATP, an d othe r neuroactiv e substance s (Bezz i an d 

Volterra, 2001 ; Field s an d Stevens-Graham , 2002) . 

These neuroactive substances can feed bac k onto the 

presynaptic terminals , enhancin g o r depressin g fur - 

ther releas e o f neurotransmitters . Moreover , astro - 

cytes ca n respon d t o electrica l neurona l activity , 

elevating thei r intracellula r Ca 2+ concentration an d 

hence triggerin g glutamat e releas e (Araqu e e t al. , 

1998; Innocent i e t al, 2000 ; Pascua l e t al, 2001 ; 

Pasti e t al. , 2001 ) throug h a Ca 2+ -dependent exocy - 

totic proces s (Araque et al, 2001; Bezzi et al, 2004) . 

Confirmation that this release occurs through such a 

process i s provided b y the abolitio n o f glutamate re - 

lease b y tetanu s toxi n (e.g. , Pascua l e t al. , 2001) . 

Glutamate releas e fro m nerv e terminal s an d astro - 

cytes ca n b e triggere d b y potassium chlorid e (KC1 ) 

and brain-derive d neurotrophi c facto r (BDNF ) 

(Fig. 18-3). 

Glia ca n indirectl y modulat e synapti c transmis - 

sion b y regulatin g th e ioni c extracellula r environ - 

ment, clearin g neurotransmitter s fro m th e synapti c 

cleft (Bergle s and Jahr, 1998; Anderson and Swanson, 

2000), an d respondin g t o th e metaboli c demand s o f 

synaptic transmission (Shulman et al., 2001). Glia ca n 

also affect glutamatergi c neurotransmission b y releasing 

co-factors. Glia (astrocyte s in the brain and Müller 

cells i n th e retina ) ar e th e onl y source o f D-serine in 

the CNS . D-serine , rather than glycine, appears to be 

the endogenou s agonis t tha t activate s th e glycine - 

binding site at the N-methyi-D-aspartat e (NMDA ) re - 

ceptors (Wolosker et al, 1999; Steve n et al, 2003). 

Effects of Ethanol 

Ethanol exposur e durin g brain ontogen y alter s brain 

synaptology, decrease s synapti c formation , an d im - 

pairs development and maturation o f synapses in both 

the hippocampa l formatio n an d neocorte x (Guerri , 

1987; Al-Rabiai and Miller, 1989 ; Tanaka etal., 1991 ; 

Kuge et al, 1993 ; Sutherlan d e t al, 1997) . Althoug h 

specific studie s have not yet broached the rol e of glia 

in these ethanol-induced synaptogenesis , various data 

compel us to consider glial-mediate d synaptic formation 

as a target of ethanol.


/y.. 

FIGURE 18- 3 Induce d glutamate release from synaptosome s and cortical astrocytes. 

A spectrofluorimetric assa y was used t o determine th e Ca 2+ -dependent 

release o f glutamate evoke d b y potassiu m chlorid e (KC1 ; 30 mM) o r brain - 

derived neurotrophi c facto r (BDNF ; 10 0 ng/ml) fro m nerv e terminal s an d 

from contro l or prenatal ethanol-exposed (PEE) astrocytes (4-day-old cultures). 

Data ar e means ± standard deviations (n = 3). An asterisk signifies a significant 

(p < 0.01) difference betwee n PE E an d contro l astrocyte s treated with KC 1 or 

BDNF. 

Astrocytes from prenatall y ethanol-exposed fetuse s 

release less glutamate after being stimulated with KC1 

(Fig. 18-3) . This finding indicates that PEE compro - 

mises th e KCl-stimulate d elevatio n o f intracellula r 

Ca 2+ . Thu s prenata l ethano l exposur e likel y affect s 

the glia l response to neuronal activity and cause s abnormalities 

i n bot h synapti c functio n an d synapti c 

stabilization. Severa l studie s hav e show n tha t feta l 

and/or neonata l ethano l exposur e not onl y alters ligand 

binding to the NMD A receptor s but als o affect s 

NMDA receptor function (Savage et al., 1991 ; Vallès 

et al., 1995; Spuhler-Phillips e t al, 1997; Gruol et al., 

1998). Th e compellin g inferenc e i s tha t ethanol - 

induced alteration s i n gli a affec t D-serin e release , 

thereby altering the function of the NMDA receptors 

during brain development. 

GLIAL DEVELOPMENT IN TH E 

CORPUS CALLOSUM 

The corpus callosum (CC ) i s a larg e fibe r trac t 

formed fetall y tha t interconnect s neuron s i n th e 

right and lef t cerebra l hemispheres. In humans, th e 

CC appear s durin g th e sixt h prenata l wee k an d 

grows i n a rostral-to-cauda l direction. Midlin e glia l 

populations a s wel l a s axona l guidanc e molecule s 

play critica l role s i n C C developmen t (Richards , 

2002). Agenesis , dysgenesis , an d change s i n th e 

shape o f the C C ar e observed in 7% of children prenatally 

expose d t o alcohol (Rile y e t al., 1995 ; Book - 

stein étal., 2001, 2002; Sowell étal, 2001a) which is 

more tha n 20-fol d tha t fo r the genera l population . 

Since th e midlin e gli a ar e involve d in th e develop - 

ment of the CC, ethano l targeting of these cells may 

initiate CC dysrnorphology . 

Glia positione d a t the midlin e produce a number 

of different molecules that regulate axonal pathfinding 

at th e midlin e an d determin e whic h axon s projec t 

ipsilaterally an d whic h projec t contralaterall y (Fig. 

18-4). These gli a ar e hypothesize d t o participat e i n 

the adhesio n an d closur e o f the interhemispheri c fissure 

(Silve r et al, 1993) . Concurrently , the y secret e 

repellent and growth-suppressive molecules that cause 

callosal axons to turn awa y from th e midline. The im - 

portance o f the midline glial populations i n the development 

of the CC i s further supported by experiments 

showing tha t whe n thes e glia l structure s are excise d 

and replace d b y cortica l graft s tha t d o no t contai n 

midline glia, the callosal axons actually fail to turn and 

cross the midline (Shu and Richards, 2001; Richards, 

2002). 

Hypothetically, ethano l alter s glia l productio n 

and secretion of chemoattractive and chemorepellent

FIGURE 18- 4 Midlin e glia l populations . Thi s 

schematic vie w o f a corona l sectio n represent s th e 

various population s o f gli a a t th e leve l o f the septa l 

nuclei. GS , glia l sling ; GW , glia l wedge ; IGG , gli a 

within indusiu m griseum , MG, midlin e zippe r glia. 

Source: Adapted from Richards , 2002. 

molecules that affect the guidance of midline callosa l 

axons tha t lead s t o disrupte d C C formation . Thes e 

molecules ar e secrete d i n th e glia l wedge an d indu - 

sium griseu m an d mediat e pre - an d postcrossin g axonal 

guidance ; thei r depletio n ha s bee n foun d t o 

cause axon s t o defasciculat e o r t o ente r th e septu m 

aberrantly (Shu e t al., 2003). We have recently found 

abnormalities in the glial wedge and in indusium griseum 

glia l cel l populations , alon g wit h hypoplasi a 

of th e CC , i n th e brai n o f 21-day-ol d fetuse s fro m 

ethanol-fed mother s (Ruber t et al., 2003). Alterations 

in the CC wer e accompanied b y cortical atrophy and 

microcephaly (Fig. 18-1) . 

In contras t wit h thes e findings, studies of rats an d 

nonhuman primate s sho w that th e numbe r o f axons 

in the CC i s increased following ethanol exposur e between 

Gl 1 and G21 (Qiang et al., 2002) or 1 day per 

week throughou t gestatio n (Mille r e t al. , 1999) , re - 

spectively. Result s fro m thes e studie s contras t wit h 

the agenesi s an d dysgenesi s note d i n childre n wit h 

FAS. This discrepancy likel y reflect s the pea k bloo d 

ethanol concentratio n i n th e childre n (estimate d 

at >3Ö Ö mg/dl) vs . tha t obtaine d i n th e animal s 

(100-300 mg/dl). In fact, a dose-response relationship 

in th e amoun t o f hypertrophy ha s been describe d for 

monkeys (Mille r e t al. , 1999) . Difference s i n th e 

amount o f ethanol exposur e and/o r nutritiona l defi - 

ciencies occurrin g durin g critica l stage s of CC devel - 

opment (e.g. , embryogenesis) ma y also contribut e t o 

the variabilit y of effect s (e.g. , agenesis , dysgenesis . 

GLIAL TARGETS OF DEVELOPMENTA L EXPOSURE TO ETHANO L 30 3 

and changes in shape) reported i n children prenatally 

exposed to alcohol . 

GLIAL-DERIVED FACTORS AND 

THE NEURON: THE POTENTIA L 

EFFECTS O F ETHANOL 

Astrocytes synthesiz e an d releas e a larg e variet y of 

compounds, includin g growt h factor s (Lafon-Caza l 

et al., 2003). These substances have myriad effects o n 

neurons an d CN S function . Proliferatin g astrocyte s 

release mor e growt h factor s tha n d o differentiatin g 

glia (e.g. , Vallès et al., 1994) , perhaps becaus e o f the 

considerably highe r (two-fol d higher ) numbe r o f 

genes expresse d b y neonatal tha n b y adult astrocyte s 

(Nakagawa and Schwartz , 2004) . A particularly interesting 

factor released by glia i s the activity-dependen t 

neuroprotective protein (ADNP ) (Bassa n et al., 1999). 

This protein, its active fragment NAP (asn-ala-pro-valser-ile-pro-gln), 

an d th e activ e fragment s o f ADNF 

are strongly protective against neural insults (Brenneman 

et al, 1998 ; Goze s et al, 2000 ; Beni-Adani et al , 

2001), includin g ethanol-induce d insult s (Spon g 

et al, 2001 ; Wilkemeyer e t al, 2003) . 

Given the actions of ethanol o n glia and the num - 

ber an d importanc e o f th e trophi c an d signalin g 

functions o f glia, i t i s reasonable t o propos e tha t a t 

least som e o f the detrimenta l developmenta l effect s 

of ethanol ar e mediated b y defects in the productio n 

of glial factors , suc h a s S100B (Eriksen e t al, 2002 ) 

and ADN P (Pascua l e t al , 2004) . SIOO B i s a glia l 

trophic facto r tha t i s essentia l fo r th e developmen t 

of serotonergic neurons (Liu and Lauder, 1992) , an d 

ADNP ha s potent growth-promotin g an d neuropro - 

tective action s (Bassa n e t al , 1999 ; Goze s e t al , 

2003). 

Astrocytes raise d i n a mediu m conditione d b y 

ethanol-treated astrocyte s stunt proces s outgrowt h b y 

neurons and reduce the survival of serotoninergic neu - 

rons (Ki m an d Druse , 1996 ; Erikse n an d Druse , 

2001). Moreover, astrocyte s treated wit h ethano l ca n 

increase or reduce the secretion o f soluble factors tha t 

influence dendritic growth (Yanni et al, 2002) . 

Secretion o f growt h factor s i s reduce d i n astro - 

cytes isolate d fro m fetuse s o f ethanol-fe d femal e 

rats (PEE ) an d culture d i n th e absenc e o f ethanol . 

Ethanol alter s th e intracellula r vesicula r transpor t 

in astrocyte s (Guerri e t al, 2001) , leading to impairment 

of both th e expressio n o f neurotrophic factor s


FIGURE 18- 5 Co-cultur e of cortical neurons with control 

or prenatal ethanol exposed (PEE) astrocytes. Neurons 

grown on control (A , C) or PEE (B , D) astrocytes 

were staine d th e neurona l marke r MAP 2 an d wit h 

the synapti c vesicle protein marke r SNAP25 (arrows , 

A, B) . Neurons co-culture d wit h PE E astrocyte s (B, 

D) showed a marked reduction in neuronal differentia - 

tion and synaptic connections compared to co-cultures 

with contro l astrocyte s (A, C). Scal e bar s = 50jlm (A , 

B) and 1 0 |im (C, D) . 

receptors an d th e productio n an d releas e o f growt h 

factors suc h a s nerv e growt h facto r (NGF ) (Vallè s 

et al, 1994 ; Climen t e t al, 2000) . PE E astrocytes 

also exhibit a remarkable reduction i n cellular NG F 

(Vallès et al, 1994 ) an d ADNP (Pascua l et al, 2004) 

mRNA. Co-cultur e o f PE E astrocyte s wit h cortica l 

neurons fro m contro l rat s lead s t o a reductio n i n 

neuronal differentiatio n an d synapti c connection s 

(Fig. 18-5). Interestingly, these effect s ar e blocked by 

NAP (Pascual et al, 2004). ADNP and its active peptide 

NA P antagoniz e ethanol-induce d inhibitio n 

of LI adhesio n an d protec t agains t ethano l embry - 

otoxicity (Spon g e t al , 2001 ; Wilkemeye r e t al , 

2003). These findings may form the basi s for future 

therapeutic approache s t o t o protec t neuron s fro m 

the toxi c effects o f ethanol b y means o f glial-derived 

factors. 


Clinical an d experimenta l studie s provid e com - 

pelling evidenc e that feta l and/o r neonata l exposur e 

to ethano l profoundl y affect s astroglia . Amon g th e 

critical period s o f brain developmen t i n whic h glia l 

cells are particularly susceptible to ethanol i s embryogenesis. 

Ethano l exposur e durin g embryogenesis , a 

stage durin g whic h R G ar e generated , no t onl y im - 

pairs neuronal migratio n bu t als o can affec t th e gen - 

eration and maturation of astrocytes. Importantly, the 

novel rol e o f RG a s neural ste m cell s (Nocto r e t al. , 

2001; Götz e t al, 2002 ; Anthony et al, 2004 ) raises 

questions as to whether th e effect s o f ethanol o n R G 

underlie the reduction i n numbers of neurons an d astrocytes 

generated . 

The secon d critica l perio d durin g which ethano l 

can affec t gli a i s the brai n growt h spurt. Ethanol an - 

tagonizes th e proliferativ e effect s o f astroglia l mito - 

gens, interfering with signaling transduction pathways 

associated wit h th e activatio n o f growt h factor s an d 

neurotransmitters importan t fo r glia l proliferation . 

Exposure t o ethano l durin g thi s perio d ca n als o en - 

hance neurona l an d astrocyti c apoptotic death . Th e 

ability of ethanol t o interfere with astroglial mitogens 

and th e increas e i n cel l deat h ma y contribut e t o 

ethanol-induced microencephaly . Exposur e t o hig h 

concentrations o f ethano l durin g th e brai n growt h 

spurt can induce reactiv e astrogliosis (Goodlett et al., 

1993), thereb y activatin g gli a t o releas e toxi c com - 

pounds tha t damag e neuron s (Lu o e t al. , 2001 ; 

Blanco et al., 2004). Ethanol-induced astroglia l damage 

ca n affec t man y developmental process , such a s 

guidance an d availabilit y o f trophi c suppor t mole - 

cules; modulatio n o f the formation , maturation, an d 

maintenance o f synapses; an d regulatio n o f synapti c 

transmission (Field s an d Stevens-Graham , 2002 ; 

Nedergaardetal.,2003). 

We are just starting to understand the role of glia in 

many processe s o f th e adul t an d developin g brai n 

(Ransom et al., 2003 ). The importanc e of glia is illustrated 

b y the severit y of the mor e commo n for m o f 

Alexander disease, a rare disorder that results from mu - 

tations i n th e gen e fo r GFAP (Brenne r et al. , 2001) . 

The geneti c modificatio n of Drosophila melanogaster 

resulting i n glia l ablatio n cause s dramati c defect s 

in neurona l proliferatio n and differentiation , axona l 

growth, and neuronal deat h (Jones et al., 1995 ; Boot h 

et al., 2000) . This finding stresse s the importanc e o f 

glia for neuronal ontogeny . Mor e studie s ar e needed 

to confir m th e roles o f astrocyte s an d t o identif y 

molecular target s o f ethano l o n astroglia . Recen t 

advances i n thes e topic s hav e relie d o n studie s 

performed i n cel l culture , where glial network organization 

ma y no t exactl y mirro r tha t i n th e intac t 

brain.

Abbreviations 

ADNF activity-dependen t neurotrophic factor 

ADNP activity-dependen t neuroprotectiv e protei n 

ADH alcoho l dehydrogenas e 

BDNF brain-derive d neurotrophic factor 

CC corpu s callosu m 


CYP2E1 cytochrom e P450 2E1 




KC1 potassiu m chlorid e 

MAPK mitogen-activate d protein kinas e 

NAP asn-ala-pro-val-ser-ile-pro-gl n 

nCAM neura l cel l adhesion molecul e 

NGF nerv e growt h factor 



PA phosphatidi c acid 

PDGF platelet-derive d growt h facto r 

PEE prenata l ethanol exposure 

PEt phosphatidy l ethano l 

PKC protei n kinas e C 

PLD phospholipas e 

RG radia l glia 

SMase sphingomyelinas e 

TGF transformin g growt h facto r 

TNF tumo r necrosis facto r 

ACKNOWLEDGMENTS Th e author s than k Dr . Vi - 

cente Rubi o for his valuable help i n the critica l readin g 

of this manuscript. Researc h wa s supported b y Spanish 

MCYT (project s BF I 2001-0123-01 ) an d SA F (2003 - 

06217) an d b y Conselleria Sanida d (Direc . Gen . Aten - 

cion a la Dependencia). 

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Ill 

NICOTINE-AFFECTED DEVELOPMENT


19 

Tobacco Use During Pregnancy : 

Epidemiology and Effect s 

on Offsprin g 

Tobacco us e durin g pregnanc y affect s feta l develop - 

ment on multipl e levels. Nicotine readil y crosses the 

placenta, concentratin g in feta l tissue , and result s in 

fetal concentration s o f nicotin e tha t ca n b e 15 % 

higher than those in the pregnant woman (Luck et al., 

1985). The direct action s o f nicotine o n th e fetu s affect 

growt h an d neura l developmen t durin g th e in - 

trauterine period an d hav e long-term effect s o n brain 

function, cognition, and behavior (Abreu-Villaca et al , 

2004a, 2004b). 

Tobacco us e durin g pregnanc y ha s indirec t 

effects o n feta l development . Prenata l tobacc o us e 

affects th e nutritional status of the mother , leading to 

an increase d risk of having an infan t wit h lo w birth 

weight (LBW ) (Hast e e t al, 1990) , an d cause s in - 

creased vascular resistance in the placenta, resultin g 

in reduce d oxyge n flow to th e fetu s (Lamber s an d 

Clark, 1996) . Intermitten t feta l hypoxia , secondary 

to exposure to carbon monoxid e and the metabolit e 

carboxyhemoglobin, ma y als o affec t oxyge n flo w t o 

the fetus . 

Jennifer A. Willford 

Nancy L. Day 

Marie D. Corneliu s 

315 

The presen t chapte r review s the epidemiolog y o f 

tobacco us e during pregnanc y an d the n focuse s o n 

the effect s o f prenatal tobacco exposure (PTE). 

SCOPE O F THE PROBLEM 

Epidemiology of Tobacco Use 

During Pregnancy 

In genera l population s surveys , 10 % t o 20 % o f 

women repor t tha t the y smoke d durin g pregnanc y 

(National Institut e o n Dru g Abus e [NIDA] , 1996 ; 

LeClere and Wilson, 1997 ; Center s for Disease Con - 

trol and Preventio n [CDC] , 2002 , 2003) . Dat a col - 

lected o n th e U.S . Standar d Certificate s o f Liv e 

Births sho w tha t th e proportio n o f wome n wh o 

smoked durin g pregnanc y decline d fro m 19.5 % t o 

11.4% between 198 9 an d 200 2 (Martin et al, 2003). 

This decrease mirror s a general decreas e i n smokin g 

among wome n o f reproductiv e age , althoug h on e

316 NICOTINE-AFFECTE D DEVELOPMENT 

group, young women aged 18-20 , increased their use 

slightly fro m 198 7 t o 1996 (Ebrahim e t al, 2000) . 

This overall decline in smoking during pregnancy results 

from lo w rates of smoking initiation in women , 

rather tha n change s i n smokin g befor e o r durin g 

pregnancy (Wisbor g e t al. , 1996 ; Cnattingius an d 

Haglund, 1997 ; Department o f Health an d Huma n 

Services [DHHS], 2001). 

Smoking durin g pregnanc y i s mor e prevalen t 

among Caucasia n wome n tha n amon g African - 

American o r Hispanic wome n (NIDA , 1996 ; CDC, 

2002). The rate s vary by age (Cornelius et al., 1999b ; 

Kahn e t al. , 2002) , with younge r wome n smokin g 

more cigarettes . Us e als o varies by trimester o f pregnancy. 

A Maternal Health Practices and Child Development 

(MHPCD ) study of teenage mother s report s 

that th e rat e o f smoking during pregnancy i s 47% in 

the first trimester and 58 % in the third trimester (Cornelius 

e t al. , 2004) . I n general , women wh o smok e 

during pregnanc y ar e younge r an d les s likel y to b e 

married, have less education an d lower incomes, an d 

attend fewe r prenata l visit s compare d wit h wome n 

who do not smok e durin g pregnanc y (Da y et al. , 

1992; Corneliu s e t al. , 1994 ; DHHS , 2001 ; CDC , 

2003). Wome n wh o smok e durin g pregnanc y als o 

have highe r rate s o f antisocia l behavior , mate s tha t 

have mor e antisocia l behaviors , highe r rate s o f de - 

pression, an d lowe r socioeconomi c statu s (SES) 

(Maughanetal., 2004). 

Women are less likely to decrease their tobacco us e 

during pregnancy than they are to decrease othe r substance 

use, including alcohol, marijuana, and other illicit 

drugs (Cornelius e t al, 1995 ; Day et al, 2000), 

and are more likely to continue t o smoke i n the post - 

partum period (Corneliu s et al., 1999a ; Leech e t al., 

1999). I n a Nationa l Pregnanc y an d Healt h Stud y 

(NIDA, 1996), approximately two-thirds of the wome n 

who smoke prior to their pregnancy continue smoking 

through th e las t trimester . B y contrast , onl y onequarter 

o f the wome n wh o us e alcoho l prio r to conception 

continu e t o drin k i n th e thir d trimester . 

Women who continue to smoke during pregnancy are 

more likely to have had previous pregnancies, to have 

a younger age at smoking onset, to be heavy smokers, 

and to have less education (Cnattingius, 2004). 

Women wh o smok e durin g pregnanc y als o continue 

to smoke in the postpartum period. This means 

that the offsprin g o f a woman who uses tobacco during 

pregnanc y ar e expose d continuousl y t o tobacc o 

smoke i n th e household . Thi s environmental , o r 

passive, exposur e als o affect s chil d development . I n 

the MHPC D cohor t o f adult mothers , for example, 

the correlation between prenata l tobacco us e and tobacco 

us e 1 0 years later wa s 0.63 (Cornelius e t al. , 

2000). I n addition , a s many as 60 % o f women wh o 

quit smokin g durin g pregnanc y bega n agai n withi n 

6 months o f delivery (Mullen et al., 1997; Hajek et al., 

2001). Significan t predictors o f relaps e afte r preg - 

nancy include living with another smoker, less education, 

and lower income (Kah n et al., 2002). 

Women who smoke during pregnancy have higher 

rates of other substance use as well. Among women in 

the MHPCD project, 76% of the women over the age 

of 1 8 who smoke d durin g the firs t trimeste r o f pregnancy 

dran k alcohol durin g this period (Day et al. , 

1992). Among pregnant teenagers , 61 % of those wh o 

smoked in the first trimester drank alcohol (Corneliu s 

et al , 1995) . Tobacc o us e i s als o highl y associated 

with th e us e o f illici t drug s durin g pregnancy . I n 

the Nationa l Pregnanc y an d Healt h Surve y (NIDA, 

1996), wome n wh o smoke d cigarette s durin g preg - 

nancy reporte d usin g illicit drug s (26%) , drinkin g 

(16%), o r bot h (32%) . Therefore , i t i s important t o 

control fo r the covariate s of PTE an d othe r prenata l 

substance exposures , as well as current maternal tobacco 

an d othe r substanc e use . In th e absenc e o f 

considering these othe r ris k factors, it is not possibl e 

to identif y accuratel y the uniqu e effect s o f prenatal 

tobacco exposure. 

Mortality and Morbidity 

A meta-analysi s estimate s tha t PT E wa s responsibl e 

for u p t o 480 0 infan t deaths , 61,00 0 infant s wit h 

LBW, and 26,00 0 neonatal intensive care admissions 

per year (DiFranza and Lew, 1995) . An earlier report 

estimates tha t materna l smokin g was responsible fo r 

approximately 10 % of fetal an d infan t death s (Kleinman 

e t al. , 1988) . Thi s report , alon g wit h anothe r 

(Singleton et al., 1986), find that the impact of PTE is 

greater amon g Africa n American s tha n fo r Caucasians 

an d i s greate r amon g th e offsprin g o f olde r 

mothers (Cnattingius et al., 1988) . 

PTE i s a significan t risk facto r fo r sudde n infan t 

death syndrom e (SIDS ) (Nationa l Cance r Institut e 

[NCI], 1999) . SID S result s fro m th e PTE-relate d 

chronic hypoxi a (Bulterys et al., 1990) . Postnatal passive 

exposure of the infan t als o significantly increases 

the ris k of SIDS (Klonoff-Cohe n e t al., 1995 ; Dwye r 

étal, 1999) .

GROWTH AND MATURATION 

Human Studie s 

PTE ha s lon g bee n identifie d a s a significan t ris k 

factor fo r intrauterin e growt h restrictio n (DHHS , 

1980, 2001 ; Stillman e t al., 1986 ; Floy d e t al., 1993 ; 

NCI, 1999 ; CDC , 2003) . Birt h weigh t decrease s i n 

direct proportion t o the numbe r o f cigarettes smoke d 

(Yerushalmy, 1971 ; Persso n e t al. , 1978) , an d off - 

spring of smokers are 15 0 to 250 g lighter than the off - 

spring of non-smokers (DHHS , 1980) . Th e effect s o f 

PTE ca n b e seen eve n a t lower levels of exposure: in 

one report , 11.5 % o f infant s bor n t o ligh t smoker s 

(


over the lon g term (Hard y and Mellitus , 1972 ; Frie d 

and O'Connell , 1987 ; Vi k e t al , 1996) . Mor e re - 

cently, i t ha s bee n argue d tha t th e associatio n be - 

tween PTE and reduced height at 6.5 and 1 3 month s 

postpartum i s attributable to prenatal exposure to alcohol 

(Jacobson et al., 1994), highlighting the impor - 

tance of controlling for the effect s o f other drugs. 

Recent reports demonstrate that PTE leads to an increase 

in weight at older ages. Power and Jefferis (2002) 

note that althoug h PT E i s associated with lower birth 

weight, thi s relatio n reverse d by adolescence, an d b y 

age 33 , exposed offsprin g hav e 1. 5 greater odd s of being 

obese . I n a MHPC D cohor t o f teenage mother s 

and thei r offspring, a positive association is evident between 

PT E an d increase d skinfol d thickness , an d 

higher body mass index an indication that the children 

are overweight for their height (Cornelius e t al, 2002). 

Similar finding s hav e been reporte d b y other authors 

(Vik e t al, 1996 ; Frie d e t al. , 1999 ; Toschk e e t al, 

2003). Thus , th e preponderanc e o f evidenc e fro m 

longitudinal studie s that have controlled fo r passive tobacco 

exposur e and othe r covariate s of pre- and post - 

natal tobacc o exposur e i s that th e growt h deficits that 

are eviden t a t birth ar e not maintained , an d ove r th e 

long term there appears to be an increase in weight. 

The mea n ag e a t menarch e i s approximatel y 6 

months earlier among the female offspring exposed to 

a pac k or more o f cigarettes per da y during gestatio n 

than that of girls unexposed during pregnancy (Windham 

e t al, 2004). Girls with bot h hig h prenata l an d 

childhood passiv e exposur e to tobacco hav e onse t of 

menarche 4 months earlie r than the girls who are not 

exposed a t eithe r time . Earl y onse t o f menarch e i s 

present afte r adjustin g for alcohol, coffee , an d te a exposure, 

mothers' age at menarche, parity , race, education, 

and income . 

The complexit y of relations between prenatal nico - 

tine exposure and growth are demonstrated in animal 

models. Navarr o and colleague s (1989 ) repor t effect s 

of PTE o n viability, growth, and nervous system development 

o f th e offsprin g a t hig h dose s o f nicotin e 

(>6mg/kg/day). A t lower dose s ( 2 mg/kg/day), whic h 

are approximatel y equivalent t o

IQ i s also evident at ages 9 to 1 2 years (Fried, 2002 ) 

and 1 3 to 1 6 years (Fried e t al, 2003) for the OPP S 

cohort. Anothe r prospectiv e stud y (Streissgut h e t al., 

1989), however , show s n o effec t o f PT E o n I Q i n 

4-year-olds. 

PTE produces deficits in visual memory and verbal 

learning o n th e Wid e Rang e Assessmen t o f Memory 

and Learnin g (Sheslow and Adams, 1990) among th e 

10-year-old offsprin g o f adult mothers i n a study fro m 

the MHPCD (Cornelius e t al, 2001). These findings 

are significant afte r controllin g for SES, maternal psychological 

status , hom e environment , othe r prenata l 

substance exposures , an d curren t materna l substanc e 

use. In the OPPS cohort, when the offsprin g ar e 9 to 

12 years old, PTE produce s visua l perceptual deficit s 

(Fried and Watkinson, 2000). A later report on this cohort 

a t age s 1 3 t o 1 6 years shows that PT E produce s 

poorer auditory functioning (Fried et al., 2003). 

Poor languag e developmen t i s reporte d i n 2 - 

(Fried and Watkinson, 1988) , 3-, and 4-year-olds with 

PTE (Frie d and Watkinson , 1990) . Among 9 - to 12 - 

year-old children i n this study, PTE i s negatively associated 

wit h languag e an d readin g abilitie s (Frie d 

et al., 1997) . Smokin g during pregnancy is also significantly 

relate d t o poore r performanc e o n spelling , 

reading, an d arithmeti c assessment s amon g 5 - to 11 - 

year-olds in another study (Batstra et al., 2003). 

In animals, learning and memory deficits are seen 

on a numbe r o f complex cognitiv e behaviora l tasks, 

including decreased performance on a two-way avoidance 

tas k (Vaglenov a et al. , 2004) , deficit s i n spatial 

learning and memory (Sorenso n e t al., 1991 ; Yanai et 

al, 1992 ; Levi n e t al, 1993 , 1996) , opérant learnin g 

(Martin and Becker, 1971), and discrimination learning 

(Johns et al., 1982) . These impairments in cogni - 

tive functio n ar e consisten t wit h th e learnin g an d 

memory deficits observed in children with PTE . 

Behavior 

Associations between PT E an d errors of omission and 

commission, measures o f inattention an d impulsivity , 

respectively, are detectable in 4-year-olds (Streissgut h 

et al, 1984) . Ther e i s a significant relation betwee n 

PTE an d impulsivity among 6-year-old s in the OPPS 

cohort (Frie d et al, 1992) . In a MHPCD study of off - 

spring o f adult mothers , PT E significantl y predicte d 

increased error s of commission, o r inattention , o n a 

continuous performanc e tes t i n 6-year-old s (Leec h 

EPIDEMIOLOGY OF MATERNAL TOBACCO USE 31 9 

et al, 1999) . I n the MHPCD, however, the mother' s 

current tobacc o us e i s so highly correlate d wit h th e 

prenatal exposur e tha t the effect s o f the prenata l an d 

current exposure s could no t b e separated . Kristjans - 

son an d colleague s (1989 ) sho w that PT E produce s 

impulsivity and increase s overal l activity among 4 - to 

7-year-olds. A prospective stud y o f 9000 childre n i n 

Finland (Kotima a e t al , 2003 ) found a 30 % higher 

rate in hyperactivity among 8-year-old s who had PTE 

than i n thos e wh o di d not , afte r controllin g fo r gender, 

family structure, SES, maternal age, and prenatal 

alcohol exposure . I n th e OPP S cohor t o f 9 - t o 12 - 

year-olds, there was a dose-dependent associatio n be - 

tween PT E an d impuls e contro l (Fried , 2002) . 

Eskenazi an d Trupi n (1995) , however , repor t tha t 

there is no association betwee n PT E an d activit y levels 

at 5 years of age in their study. 

In a case-control study of children with and without 

attentio n defici t hyperactivity disorder (ADHD) , 

Mick an d colleague s (2002 ) report that 6 - to 7-year - 

old children with ADHD are 2.1 times more likel y to 

have been expose d t o tobacco during gestation, eve n 

after controllin g fo r prenatal alcoho l an d dru g expo - 

sures. There i s a positive association between mater - 

nal smoking during pregnancy and the ris k of ADHD 

in stud y childre n betwee n th e age s o f 6 an d 1 7 

(Milberger e t al , 1996) . Materna l smokin g durin g 

pregnancy i s significantl y associate d wit h ADH D 

symptoms i n twins , controlling fo r the heritabilit y of 

ADHD (Thapar et al, 2003). 

The effect s o f prenatal nicotin e exposur e on mo - 

tor activity in laboratory animals are equivocal. Som e 

studies sho w increase d activit y (John s e t al , 1982 ; 

Fung an d Lau , 1988 ; Richardso n an d Tizabi , 1994 ; 

Tizabi e t al , 1997 ; Ajare m an d Ahmad , 1998 ; 

Thomas et al, 2000) , wherea s other s sho w no effec t 

(Martin an d Becker , 1970) . O n th e othe r hand , pre - 

natal nicotin e exposur e is linked to abnormal development 

o f spontaneous alternation , a behavior that is 

associated with cholinergic neurotransmitter function 

and hyperactivit y (Fung, 1989 ; Shack a e t al , 1997 ; 

Tizabi e t al, 2000) . Studie s als o sho w tha t prenata l 

nicotine exposur e change s cellula r communicatio n 

(Maggi e t al, 2003) , electrophysiology (Ehlers et al , 

1997; Slaweck i e t al , 2000) , an d neurotransmitte r 

function (Tizab i e t al , 2000 ) i n th e hippocampus , 

changes that could affec t activity levels. 

There is consistent evidence that children expose d 

to tobacco during pregnanc y ar e at an increase d ris k


for behaviora l problems, specificall y externalizin g behaviors. 

I n a n MHPC D cohor t o f adul t mothers , 

3-year-old offspring expose d prenatally to tobacco hav e 

significantly mor e oppositiona l an d aggressiv e behaviors 

and are more immature (Day et al., 2000). These 

effects persis t after controllin g for SES, factor s in the 

current hom e environment , materna l psychologica l 

status, and other prenatal and current drug exposures. 

In anothe r stud y o f 3-year-old s (Orlebek e e t al. , 

1997), there is a significant effec t o f PTE o n external - 

izing behaviors, primaril y higher aggression , i n bot h 

genders. In a prospective study of 5-year-olds, Williams 

and colleague s (1998 ) describ e a relativ e risk o f 2. 6 

for externalizin g behavior in children wit h PTE com - 

pared t o unexpose d children . Anothe r stud y report s 

a significan t positiv e associatio n betwee n PT E an d 

externalizing behaviors i n 5 - to 11-year-old s (Batstra 

et al. , 2003) . B y contrast, Weitzma n an d colleague s 

(1992) do not fin d significan t effect s o f PTE o n children 

wh o ar e expose d onl y durin g gestation . The y 

show that children whos e mothers smoked both during 

an d afte r pregnanc y hav e mor e behavio r prob - 

lems. I n th e latte r group , th e rat e o f behavio r 

problems i s increased by 1.1 7 times among thos e exposed 

t o less than on e pac k of cigarettes per da y and 

2.04 times among thos e expose d t o one pack or more 

per day. 

These behaviora l effect s persis t int o th e adoles - 

cent and adult years. Among children whos e mother s 

smoked a t leas t 1 0 cigarette s pe r da y durin g preg - 

nancy, there i s a fourfold increas e i n the rat e o f prepubertal 

onse t o f conduct disorde r amon g boy s (

Abnormalities i n cel l developmen t an d respon - 

sivity associated with prenatal nicotin e exposur e may 

lead t o permanent functiona l deficits whe n brai n regions 

and the relate d circuitry mature in adolescenc e 

and adulthoo d (Erns t et al., 2001) . An up-regulation 

of nicotinic acetylcholine receptors i s evident amon g 

adult rat s expose d t o nicotin e i n th e immediat e 

neonatal period , a tim e comparabl e t o th e thir d 

trimester i n human s (Slotki n e t al. , 1991) . A recen t 

study (Klei n e t al. , 2003 ) show s that peri-adolescen t 

male mic e expose d t o gestationa l nicotin e hav e in - 

creased nicotin e preference . Prenata l nicotin e expo - 

sure leads to cholinergic hypoactivity , decreased cel l 

numbers i n th e midbrain , and increase d cel l siz e i n 

the hippocampu s i n adolescen t animal s (Abreu - 

Villaca e t al, 2004a , 2004b) . Kan e an d colleague s 

(2004) sho w decrease d responsivit y t o dopamin e i n 

the nucleu s accumbens , a brai n regio n associate d 

with rewar d an d th e reinforcin g effect s o f drugs, i n 

adolescent rats . Thus, there are direct biological associations 

that parallel the smokin g behavior outcome s 

in human studies . 

Psychological Status 

There are few reports of effects of PTE on psychological 

symptoms . Mother s wh o smoke d durin g preg - 

nancy ar e significantl y mor e likel y to hav e toddler s 

who display negativity than mothers who only smoked 

after deliver y (Brook et al, 2000) . Among 16 - to 18year-olds 

in another cohort , PTE predict s higher rates 

of depression (Fergusson etal., 1998) . 

Recent anima l studie s identify link s between pre - 

natal nicotin e exposur e an d anxiety . The open-fiel d 

and elevate d plus-maze test s have been use d to evaluate 

activity, anxiety, and emotionalit y in animals. Pre - 

natal nicotin e i s associated with poor adaptation an d 

anxiety i n a nove l environmen t (Vaglenov a e t al. , 

2004). Femal e offsprin g wit h prenatal nicotin e expo - 

sure d o no t sho w a normal increas e i n activit y in a n 

open-field tes t across multiple days of testing (Romero 

and Chen , 2004) . This suggests an inabilit y to adapt 

to a novel environment, lear n contextual cues , o r retrieve 

learne d informatio n o n th e familiarit y o f th e 

testing environment. I n an open-field test, male mic e 

exposed i n utero to nicotine sho w increased locomo - 

tor hyperactivity, whereas females have an attenuate d 

behavioral respons e (Paul y e t al, 2004) . Thes e ani - 

mal studies demonstrate a link between prenata l nicotine 

exposure and anxiety , or the inabilit y to adapt to 

EPIDEMIOLOGY OF MATERNA L TOBACCO USE 32 1 

new environments, and they may provide an explanation 

fo r some o f the mechanism s underlying the be - 

havioral outcomes i n humans. 

Exposure t o Environmental 

Tobacco Smoke 

Pregnant wome n wh o liv e wit h o r spen d tim e wit h 

smokers expos e thei r childre n t o environmenta l to - 

bacco smoke (ETS). The level s of newborn exposur e 

correlate significantly wit h the level s of nicotine an d 

cotinine i n the mother s (Eliopoulo s et al., 1994 ; Os - 

treaetal, 1994) . 

As with PTE, ther e ar e growth, cognitive , and be - 

havioral effect s fro m exposur e t o ET S durin g preg - 

nancy. ET S exposur e i n earl y pregnanc y (20-2 4 

weeks) lead s t o a decreas e i n biparieta l diameter , a 

measure usefu l i n predictin g feta l weigh t (Hank e 

et al., 2004). Kharrazi and colleague s (2004 ) demon - 

strate a dose-respons e relatio n betwee n ET S expo - 

sure an d birt h weight ; increasin g levels o f materna l 

cotinine ar e associate d wit h declinin g birt h weight . 

Other studies have also found that birth weight is negatively 

associated wit h ET S exposur e (Marti n an d 

Bracken, 1986 ; Rebagliat o et al, 1995) . The relativ e 

risk for LBW i s 2.17-fold highe r amon g th e offsprin g 

of nonsmoking mothers who are exposed to ETS, an d 

the expose d infants , o n average , are 24 g lighter than 

nonexposed infant s (Marti n and Bracken , 1986) . Another 

study , however , describe s effect s o f ET S o n 

birth weigh t an d preter m deliver y onl y i n infant s o f 

older mothers (Ahluwali a et al., 1997) . 

Makin an d colleague s (1991 ) examined th e long - 

term effect s o f ETS durin g gestation o n 6 - to 9-yearolds. 

They show that on test s of speech an d languag e 

skills, intelligence , visual/spatia l abilities and o n th e 

mother's rating s o f offsprin g behavior , th e perfor - 

mance of offspring o f passive smokers is intermediate 

to tha t o f th e childre n o f activ e smoker s an d non - 

smokers. 

Exposure t o ETS i s prevalent i n the postnata l pe - 

riod an d ca n negativel y affec t chil d outcomes . I n 

one study , 71 % o f inner-cit y childre n o f lo w SE S 

lived i n household s wit h smoker s (Corneliu s e t al. , 

2003). Postnata l exposur e t o ET S increase s th e ris k 

of SIDS, asthma, respirator y problems, and otiti s media 

(DHHS, 1999) . Postnata l ETS i s associated with a 

reduction i n I Q scores i n 3-year-old s (Johnson et al., 

1999) an d developmenta l languag e impairment s i n 

kindergartners (Tomblin et al., 1997) . Note, however,


that neither of these analyses controlled for the effect s 

of PTE. ET S i s also associated with decreased perfor - 

mance o n measure s of receptive vocabulary and rea - 

soning abilit y in a stud y o f 10-year-old s (Bauma n e t 

al., 1991) . Among 6 - to 11-year-ol d childre n o f non - 

smoking mothers , postnata l exposur e t o ET S result s 

in deficit s o n centra l auditor y processing task s (Mc - 

Cartney et al., 1994) . 

Two studies evaluate the relation s betwee n expo - 

sure to ETS and child outcomes controllin g for PTE . 

In th e MHPC D cohor t o f adolescen t mother s an d 

their 6-year-old children, the children's current urin e 

cotinine level s inversel y correlat e wit h score s o n a 

measure o f receptiv e languag e controllin g fo r PT E 

(Cornelius et al., 2000). Five-year-olds wit h postnata l 

ETS have significantly lower scores on the Raven Test 

and Peabod y Pictur e Vocabular y Test , measure s o f 

cognitive development , an d ar e rate d a s more activ e 

by their mothers , compare d t o children wh o ar e no t 

exposed to ETS (Eskenaz i and Trupin, 1995) . 


There ar e significan t effect s o f PT E o n th e growth , 

cognitive development, an d behavior of exposed children. 

Children with PTE ar e smaller at birth, and recent 

research reports suggest that these children hav e 

a greate r ris k o f obesit y a s the y reac h adolescence . 

Cognitive deficit s i n reasonin g an d memor y hav e 

been describe d an d the expose d children do less well 

on test s o f language , reading , an d vocabular y tha n 

nonexposed children . PTE predict s higher rates of activity, 

inattention, an d impulsivity . Increases i n oppo - 

sitional an d aggressiv e behavio r an d highe r rate s of 

delinquency an d criminalit y i n adolescenc e an d 

adulthood have been found i n a number of studies. In 

general, thes e outcome s ar e consisten t whethe r th e 

mother i s a smoker or th e exposur e t o the mothe r i s 

through ETS. I n addition, there appear to be differen - 

tial effect s tha t depend on duration an d patter n o f exposure 

and on whether the offspring are exposed only 

during gestation , durin g gestatio n an d afte r birth , o r 

only after birth . Fo r most o f the identifie d outcome s 

of PTE, ther e are parallel data from laboratory studies 

on animals that lend credenc e to the human data . 

Women wh o us e tobacc o durin g pregnanc y ar e 

often differen t fro m wome n wh o d o no t i n man y 

ways, includin g i n thei r SES , race/ethnicity , us e o f 

other substances , an d psychiatri c disorders . I n addi - 

tion, even women who do not smoke tobacco ca n be 

exposed t o ETS , resultin g i n exposur e t o th e fetus . 

Many o f the studie s currently in the literatur e fail t o 

control fo r these significan t covariate s of tobacco us e 

or for ETS, makin g definitiv e conclusions abou t th e 

effects o f exposure mor e difficult . Ther e ar e severa l 

studies, however , tha t ca n asses s th e competin g effects 

of other substance us e during pregnancy as well 

as the rol e of the other covariate s and ETS. Th e gen - 

eral conclusions ar e validated by the result s of these 

studies. 

Characteristics o f th e rearin g environmen t ar e 

also significantly associate d with both PT E an d ETS , 

as well as with the outcomes i n the offspring . Wome n 

who smok e durin g pregnanc y ar e highl y likel y t o 

smoke afte r delivery . Thi s mean s tha t childre n wh o 

are prenatally exposed to tobacco ar e at higher risk for 

continued exposur e du e t o ET S exposur e fro m th e 

mother and/o r other househol d smokers . Heredit y is 

another important factor to consider in the interpreta - 

tion of these findings. As the mothe r an d chil d shar e 

genetic components , the y shar e factor s tha t migh t 

predict thei r respons e t o exposur e o r coul d increas e 

vulnerability for specific outcomes . Studie s that hav e 

controlled for role of heredity, nonetheless, find independent 

effect s o f PTE . 

For al l o f the abov e reasons , i t i s difficult t o enu - 

merate definitivel y th e effect s o f PTE whethe r i t b e 

from a smokin g mothe r o r fro m exposur e t o ETS . 

Nevertheless, it is imperative that the short - and long - 

term effect s o f both prenatal and postnatal exposure to 

tobacco o n th e growt h an d neurobehaviora l develop - 

ment o f children b e identified. Although man y of the 

effects o f PTE ar e subtle , the y have important effect s 

on the long-term functionin g of the expose d offspring . 

Future studie s mus t evaluat e th e separat e an d 

combined effect s o f exposur e t o tobacc o an d othe r 

substance exposure s during gestation. I t is also critica l 

to measure the othe r factor s that coexist with tobacc o 

use, a s well a s to evaluat e the co-occurrenc e o f PT E 

and ET S bot h durin g pregnancy and i n the postpar - 

tuni period. Studie s that measur e al l of these factor s 

across time allows a more specifi c definition of the effects 

o f prenatal an d postnata l tobacc o exposur e o n 

the offspring . 

Abbreviations 

ADHD Attentio n Defici t Hyperactivity Disorde r 


ETS environmenta l tobacco smok e

G gestationa l da y 

LBW lo w birth weigh t 

MHPCD Materna l Healt h Practice s and Chil d 

Development 

ODC ornithin e decarboxylase 

OPPS Ottawa Prospective Prenata l Stud y 

P postnata l da y 

PTE prenata l tobacco exposur e 

SES socioeconomi c statu s 

SIDS sudde n infant death syndrom e 


the Nationa l Institute of Alcohol Abuse and Alcoholism 

(JAW, NLD) , th e Nationa l Institut e o f Dru g Abus e 

(NLD, MDC) , an d th e Nationa l Institut e o f Chil d 

Health and Human Developmen t (NLD) . 

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20 

Prenatal Nicotin e Exposure 

and Animal Behavio r 

Cigarette smokin g affect s prenata l brai n develop - 

ment and subsequent behavior . Mothers wh o smoke 

cigarettes durin g pregnanc y ar e mor e likel y tha n 

nonsmoking mother s t o bea r childre n wh o ar e hy - 

peractive, impulsive, and have impairments in learning, 

memory, and attentio n (Naeye , 1992; DiFranz a 

and Lew , 1995 ; Eskenaz i an d Trupin , 1995 ; Erns t 

et al, 2001; Thapar et al, 2003). Children expose d 

to tobacc o smok e durin g pregnanc y ar e als o mor e 

likely to smoke cigarette s late r i n lif e (Kande l e t al., 

1994; Abreu-Villac a e t al , 2004a ; 2004b) . Thes e 

effects ar e i n additio n t o th e retarde d feta l growth , 

premature births , stillbirths , and increase d neona - 

tal mortalit y associated with cigarett e smokin g during 

pregnancy (Butler and Goldstein , 1973 ; Denso n 

et al. , 1975 ; Kristjansso n e t al. , 1989 ; Buk a e t al. , 

2003). 

Although cigarette s contai n roughl y 400 chemi - 

cals an d cigarett e smok e contain s >400 0 chemicals , 

Brenda M. Elliott 

Neil E. Grunberg 

329 

nicotine is the mos t widely studied chemical . Various 

properties o f nicotine mak e i t a neurotoxin. Nicotine 

crosses th e blood-brai n barrier , bind s t o specifi c 

receptors i n th e brain , an d affect s man y behaviors , 

including addiction, appetite, stress responses, and attention 

(Grunberg , 1982 , 1985 ; Luck and Nau, 1985 ; 

Levin, 1992 ; Scheufel e e t al, 2000 ; Traut h e t al , 

2000; Slaweck i an d Ehlers , 2002 ; Slotkin , 2002 ; 

Slawecki e t al , 2003 ; Farada y e t al , 2003) . Anima l 

models o f nicotin e an d tobacc o exposur e provid e 

valuable information on the behavioral effects of prenatal 

smoking. 

The presen t chapte r discusse s knowledge gaine d 

from studie s of the effect s o f prenatal nicotine and to - 

bacco exposur e on th e behavio r and developmen t of 

the offspring . I t focuse s o n rodent s becaus e mos t of 

the researc h on th e effect s o f prenatal nicotine expo - 

sure has been don e i n these animal s and the findings 

parallel research on humans.


VALUE OF ANIMAL MODELS TO ASSESS 

THE EFFECT S O F PRENATA L SMOKING 

Animals hav e bee n studie d fo r centurie s t o lear n 

about anatom y an d physiology . I n ancien t Greece , 

Galen dissecte d goats , pigs , and monkey s t o explor e 

the structur e o f the bod y and to test specifi c hypothe - 

ses about ho w the bod y worked (Nutton , 2002). Dur - 

ing the eighteent h an d nineteent h centuries , anima l 

experimentation contributed to our knowledge of physiology, 

pharmacology , bacteriology , and immunolog y 

(Nutton, 2002). 

Darwin's landmark Origin of the Species (1859 ) in - 

cluded several concepts that further expanded the study 

of animals: (1) the continuit y of species, (2) natural selection, 

an d (3 ) spontaneous mutation . Darwi n argued 

that the senses and intuitions, various emotions, an d aspects 

of cognition suc h a s memory, attention, curiosity , 

and reason (attributes once thought to be unique to humans) 

migh t b e foun d i n a well-developed for m i n 

lower animals . Darwin' s compellin g argument s le d t o 

the study of animal biology and behavior to learn mor e 

about humans . Animal s were use d t o stud y attention, 

learning, emotions, and social interaction. 

Darwin's argumen t tha t ther e i s a continuit y 

of specie s i n th e anima l kingdo m provide s the ra - 

tionale fo r studyin g infrahuma n specie s t o lear n 

about huma n behavior . Ethologists , includin g 

Lorenz an d Tinbergen, observed th e behavior o f animals 

i n thei r natura l habitat s an d provide d com - 

pelling evidenc e tha t supporte d Darwin' s idea s 

(Dewsbury, 2003) . Pavlov, Thorndike, an d Skinne r 

separately examine d anima l behavio r i n controlle d 

laboratory setting s i n a n attemp t t o understand hu - 

man learnin g and provide d empirica l evidenc e tha t 

the study of animal behavior is relevant to the huma n 

condition. 

Pavlov (1927 ) develope d th e concep t o f classica l 

conditioning o n th e basi s o f hi s stud y o f gastri c re - 

flexes in dog s (Kimbl e an d Schlesinger , 1985) . H e 

observed tha t dog s respon d t o cue s othe r tha n food , 

cues tha t ha d bee n previousl y associated wit h food . 

He labele d thes e ne w response s conditioned (o r 

conditional) responses , to distinguish them fro m th e 

naturally occurrin g responses , tha t occu r withou t 

prior training . Classica l conditionin g i s a pilla r o f 

learning principle s an d applie s t o humans a s well as 

to animals. 

Around th e sam e tim e tha t Pavlo v wa s experi - 

menting wit h dogs , Thorndik e (1911 ) wa s studying 

cats to explore how animals learn tricks. As part of his 

investigation, Thorndike constructe d th e puzzle box , 

a bo x containin g severa l lever s an d handle s fro m 

which a cat must learn how to escape . Fro m carefu l 

observations o f th e cats ' behaviors , Thorndik e pro - 

posed th e La w o f Effect . Thi s behaviora l la w state s 

that an animal wil l repeat a n action tha t bring s desirable 

consequences , bu t i t wil l no t repea t a n actio n 

that brings undesirabl e consequences . Thi s law also 

holds for humans. 

Skinner studie d th e behavio r of rat s an d pigeon s 

and expande d Thorndike' s findings (Skinner, 1935). 

Building on the concept that animals operate on their 

environment t o obtai n desirabl e consequences o r to 

avoid negativ e consequences , Skinne r propose d th e 

concept o f opérant conditioning . Specifically , Skin - 

ner foun d tha t th e frequenc y o f a give n behavio r 

could b e increased by following the occurrence o f the 

behavior wit h positiv e consequences (reinforcement ) 

or decreased b y following th e behavio r with negative 

consequences (punishment) . Together, th e studie s by 

Pavlov, Thorndike, an d Skinne r laid the groundwork 

for modern-da y theories of learning and provid e convincing 

evidenc e tha t anima l model s ar e a usefu l 

representation o f human behaviora l an d psychologi - 

cal processes. 

Animal studie s ar e valuabl e fo r assessin g dru g 

effects, includin g th e effect s o f nicotin e o n devel - 

opment an d behavior . Anima l model s allo w inves - 

tigators t o evaluat e an d manipulat e separatel y th e 

components o f tobacco smokin g (e.g. , nicotine) tha t 

might affec t prenata l development . Investigator s can 

control aspect s o f drug deliver y (e.g., dosage , timin g 

of exposure , duration o f exposure, rout e o f administration, 

and environmental context). In addition, they 

can isolat e variable s to focu s o n nicotin e an d avoid 

variables, suc h a s prenatal care , socioeconomi c sta - 

tus, nutrition , o r us e o f othe r substances , tha t con - 

found huma n studies . Anima l model s offe r uniqu e 

advantages for studying drug effects , includin g thos e 

of nicotine, on behavior. 

ANIMAL MODELS OF NICOTINE 

AND TOBACCO ADMINISTRATION 

It is well establishe d tha t tobacc o smoking ha s deleterious 

effect s o n health. A single cigarette, a n objec t 

smaller tha n a standar d pencil , deliver s mor e tha n 

4000 chemical s tha t separatel y o r togethe r ma y

contribute t o thes e effect s (Dub e an d Green , 1982 ; 

Department o f Healt h an d Huma n Service s 

'DHHS], 1989) . Nicotin e ha s been identifie d as the 

addictive componen t i n tobacc o smokin g and i s the 

centra] dru g contributin g t o th e maintenanc e o f tobacco 

us e (DHHS , 1988 ; Fieldin g e t al. , 1998 ; 

Center fo r Diseas e Contro l an d Prevention , 2002) , 

Nicotine act s i n th e brai n an d result s i n man y behavioral 

an d psychologica l action s includin g en - 

hancement o f reward , decrease d appetite , focuse d 

attention, an d change s i n activity . Further , nicotine 

and it s metabolites readily cros s the placenta l mem - 

brane, penetrat e th e preimplantatio n blastocyst , an d 

cross th e blood-brai n barrie r (Fabr o an d Sieber , 

1969; Shiverickand Salafia, 1999) . 

In rodents , tobacc o ca n b e administere d in man} ' 

different ways : through tobacco-extract administration, 

tobacco-smoke exposure , an d topica l applicatio n o f 

tobacco tars . Tobacco-extract administration refer s t o 

daily subcutaneou s injection s (SC ) o f nicotine-free 

tobacco. Som e studie s usin g thi s metho d hav e re - 

ported significan t degenerativ e change s i n variou s 

organs, includin g th e thyroid , pituitar y gland , an d 

pancreas (Suematsu, 1931) , or gangrene i n the toe s of 

male rat s (Friedlande r e t al, 1936 ; Harkavy , 1937) . 

The tobacco-smoke exposure model involve s exposing 

rats to cigarette smoke for days or years and ha s bee n 

used t o simulat e th e huma n smokin g experience . 

Kulche and colleague s ( 1952) report that rats exposed 

to tobacc o smok e exhibi t increase d tea r secretion , 

salivation, an d copiou s bowe l movements . Othe r 

studies sho w that rodent s expose d t o tobacc o smok e 

either los e weigh t o r gro w les s tha n contro l animal s 

(Pechstein and Reynolds , 1937; Passey, 1957). Tobacc o 

tar, whe n applie d topicall y t o rats , retard s growth , 

slows weigh t gain , an d cause s gastri c lesion s (Mc - 

Nallv, 1932 ; Roffo, 1942) . 

Although tobacco-smok e exposur e i s mean t t o 

simulate the huma n smokin g experience, nicotine is 

the actual substanc e i n tobacco smok e that exert s the 

addictive and psychobiologica l effect s an d maintain s 

self-administration (DHHS , 1988 ; Stolerma n an d 

Jarvis, 1995) . Therefore, nicotin e administration paradigms 

have been th e focus of animal studies in recen t 

decades. Nicotin e ca n b e administere d b y oral nico - 

tine administration (e.g., nicotine i n drinking water), 

single o r repeate d nicotin e injection s (S C o r intra - 

peritoneal [IP]) , intravenou s self-administration , or 

sustained nicotin e administratio n (e.g. , a n osmoti c 

pump). Th e effect s o f nicotin e var y wit h dosage , 

PRENATAL NICOTINE EXPOSUR E AND ANIMAL BEHAVIOR 33 1 

timing, duration of exposure, route of administration, 

and chemica l for m o f nicotin e (Levi n e t al. , 1996 ; 

Slotkin, 1998 ; Phillips et al, 2004). 

Oral Nicotine Administration 

Nicotine is ingested by animals via drinking fluids or 

forced ora l administration . Som e studie s repor t tha t 

consumption o f nicotine retard s the growt h o f mic e 

and rat s (Nice, 1913 ; Wilson an d De Eds, 1936 ) or reduces 

litte r size o f pregnan t mic e (Wilson , 1942) , 

but othe r studies report no such effect s (Nice , 1912). 

The utilit y o f oral-administratio n metho d i s limite d 

by th e difficult} 7 i n gettin g rat s an d mic e t o drin k 

nicotine-containing solution s (Murrin et al., 1987 ; Le 

Houezec et al., 1989) . Another disadvantage of using 

this metho d i s tha t nicotin e i s poorl y absorbe d 

through th e gastrointestinal tract , leading to lower con - 

centrations of nicotine in the blood. Therefore, mor e 

nicotine may be neede d t o obtain desirabl e amount s 

of nicotine t o serv e a s a mode l o f human consump - 

tion. SC injection s and deliver y via a subcutaneously 

implanted pump are more commonly use d than oraladministration 

paradigm s becaus e th e amoun t o f 

nicotine administere d ca n b e bette r controlle d an d 

the absorptio n o f the dru g i s not limite d b y passag e 

through the gastrointestinal tract. 

Repeated Nicotine Injections 

Most studie s examinin g the effect s o f chronic expo - 

sure t o nicotin e o n behavio r have use d on e o f two 

methods: (1 ) repeate d dail y injection s o r (2 ) sus - 

tained nicotin e release . Repeated dail y injections involve 

parentera l injection s —SC o r I P a fe w times a 

day o r once a day for several days (Pietila and A h tee, 

2000). Thi s approac h ha s bee n widel y use d t o ex - 

amine th e effec t o f nicotine o n behavio r (e.g. , loco - 

motion, acousti c startl e respons e (ASR) , prepuls e 

inhibition [PPI ] o f the ASR , socia l interaction , ele - 

vated plus maze, and nociception) and generates reliable 

an d reproducibl e result s (Levi n e t al. , 1997 ; 

Hahn an d Stolerman , 2002 ; Farada y e t al , 2003 ; 

Elliott e t al. , 2004 ; Olausso n e t al. , 2004) . Whe n 

nicotine i s injecte d repeatedl y int o pregnan t dams , 

similar behavioral disruptions in offspring ar e observed 

(Slotkin, 2004) . I t i s possibl e tha t thes e behaviora l 

changes reflec t th e consequence s o f uteroplacenta l 

vasoconstriction an d feta l hypoxi a associate d wit h th e 

high pea k plasm a concentration s o f nicotin e tha t


occur wit h repeate d injection s (Jonsso n an d Hall - 

man, 1980 ; Seidle r an d Slotkin , 1990 ; Carlo s e t al. , 

1991; Slotkin, 2004) . That is, nicotine injection s may 

result in high peak plasma concentrations o f nicotine 

accompanied b y brie f hypoxi c episode s associate d 

with eac h dos e (McFarlan d e t al , 1991 ; Slotkin , 

1992). These episodic hypoxic events can alte r brain 

development (Jonsso n an d Hallman , 1980 ; Slotkin , 

1986; Seidle r and Slotkin , 1990) . Consequently , an y 

observed effect s canno t b e full y attribute d to nicotine 

per se. The osmoti c pump provide s an alternate an d 

preferable way of administering nicotin e prenatally . 

Osmotic Pump 

Osmotic pump s ar e miniature , subcutaneousl y im - 

plantable pump s use d t o continuousl y delive r drugs 

or other agents at controlled rates up to 4 weeks. Th e 

pump i s a small object , resemblin g a pill capsul e i n 

shape but larger in size. The pum p ha s two compartments: 

a n inne r capsul e an d an outer chamber . Th e 

inner capsul e i s filled with the dru g or control liqui d 

to be delivered. The oute r chamber, whic h surrounds 

the capsule , contain s a sal t an d i s referred to a s th e 

salt sleeve. When placed underneath th e skin or in an 

aqueous medium , a n osmoti c pressur e differenc e de - 

velops between thi s outer salt filled chamber an d th e 

tissue environment, pulling water from th e tissue into 

the pump throug h a semipermeable membrane tha t 

forms th e surfac e o f the pump . Th e pressur e o f th e 

water i n thi s outsid e chambe r put s pressur e o n 

the impermeabl e capsule , displacin g th e dru g fro m 

the inne r cor e int o th e bod y a t a controlle d an d 

steady rat e muc h lik e the slo w squeezing o f a tooth - 

paste tube . This method produces a constant admin - 

istration of nicotine an d a steady-state level of plasma 

nicotine (Carr et al, 1989 ; Paul y et al, 1992 ; Slotkin , 

1998), therefore , avoiding th e stres s associate d wit h 

and th e hypoxi a induced b y repeated nicotin e injec - 

tions (Levin etal, 1996 ; Slotkin , 1998) . 

Grunberg (1982 ) wa s the first to us e a n osmoti c 

minipump to subcutaneously administer nicotine con - 

tinuously to rats for a week and t o examine their bod y 

weight and food consumption . Th e findings from thi s 

experiment paralle l result s fro m a stud y o f cigarett e 

smoking i n humans . Suc h parallel s betwee n dat a 

generated fro m osmoti c pump-delivere d nicotin e 

models an d finding s fro m researc h o n huma n ciga - 

rette smokin g hav e bee n describe d i n variou s othe r 

studies a s well (Bowe n e t al. , 1986 ; Grunber g e t al. , 

1986; Murri n e t al. , 1987 ; Richardso n an d Tizabi , 

1994; Levi n e t al., 1996 ; Farada y et al, 2001 ; Irvine 

et al., 2001; Malin, 2001) . The osmoti c pump i s now 

widely use d t o examin e long-term effect s o f nicotine 

in rodents. 

Prenatal Nicotine Models 

Studies examinin g prenata l effect s o f nicotin e hav e 

used mos t o f the method s describe d above. The tw o 

most commo n approache s t o evaluatin g prenata l 

smoking effects ar e repeated injection s of nicotine int o 

the pregnan t da m an d S C administratio n of nicotin e 

in the pregnant dam via an osmotic pump. Administration 

o f nicotine t o pregnan t dam s b y repeate d injec - 

tions ha s provide d consisten t evidenc e tha t prenata l 

nicotine exposur e cause s cellular damage i n th e feta l 

brain that may explain subsequen t behaviora l and cog - 

nitive deficits (Slotki n et al., 1986) . Repeated nicotin e 

injections, however , als o caus e pea k plasm a concen - 

trations o f drug tha t are associate d wit h ischemia , a n 

effect that has the potentia l to disrupt fetal brain development 

(McFarlan d et al, 1991 ; Slotkin, 1992) . 

The osmoti c pum p ha s becom e th e standar d 

choice for examining prenatal effects o f nicotine (Murrin 

e t al, 1987 ; Ulric h et al, 1997) . I t delivers drugs or 

other agents at controlled rate s for up to 4 weeks, avoids 

peak plasm a concentration s associate d wit h repeate d 

injections, allows for better contro l over nicotine delivery, 

an d eliminate s th e ris k o f hypoxic episodes. Th e 

continuous infusio n o f nicotine via osmotic minipump 

is analogou s t o nicotin e patc h deliver} ' bu t differ s 

markedly fro m nicotin e self-administratio n throug h 

smoking tobacco . 

Overview o f Behavioral Assessmen t 

in Animals 

The stud y of animal behavio r ha s become commo n 

practice an d behaviora l model s ar e frequentl y in - 

cluded i n studie s o f psychology, pharmacology , an d 

behavioral and cognitiv e neuroscience. Animal mod - 

els provide informatio n abou t norma l behavio r an d 

responses to drugs, disease, or adverse environmental 

conditions. Th e behaviora l pattern s observe d rang e 

from studie s o f basic sensory (e.g., olfactory, taste, auditory, 

and tactile) , motor (e.g. , balance), and neuro - 

logical (e.g., eye blink, righting reflex, and ea r twitch) 

functions to unconditioned behaviors (e.g. , food con - 

sumption, locomotor activity, and sexual behavior), to

more complex assessments of attention, learning, and 

memory (e.g., Morris water maze, T-maze, radial arm 

maze, an d PPI) . The followin g section discusse s key 

findings from prenata l nicotine studies in which some 

of thes e behaviora l paradigms were use d an d offer s 

suggestions for future studies. 

EFFECTS OF PRENATAL EXPOSURE 

TO NICOTINE IN ANIMAL MODELS 

Outcome measure s o f prenatal effect s includ e birt h 

weight and feta l liability , locomotor activity, and cog - 

nition. Th e effec t o f nicotine o n thes e variable s is a 

function o f th e amoun t o f nicotin e administered , 

route of administration, timing of administration, and 

animal specie s (Erns t et al., 2001). Prenatal nicotin e 

exposure compromises th e health of the newborn an d 

can hav e long-lasting deleterious effect s o n offsprin g 

behavior. 

Studies o f prenata l nicotin e effect s dat e bac k t o 

the earl y 1900 s (Nakasawa , 1931 ; Sodano , 1934 ; 

Grumbecht an d Loeser , 1941) . Mos t o f these earl y 

studies wer e conducte d i n rabbit s and dogs . Gener - 

ally, thes e studie s focuse d o n effect s o f nicotin e o n 

pregnancy duratio n and th e siz e an d viabilit y of the 

young. Earl y studie s i n th e rabbi t repeatedl y sho w 

that dail y injection s o f tobacco-smok e solutions , 

chronic nicotine infusions, or nicotine administration 

caused spontaneous abortions or premature deliveries 

(Guillain and Gy, 1907 ; Benigni , 1911 ; Perazzi , 

1912). Tissue s fro m fetuse s o f rabbit s tha t receive d 

daily SC injection s of nicotine fo r up t o 1 2 weeks are 

damaged (Var a an d Kinnunen , 1951) . Hofstatte r 

(1923) note s that pregnancies occurring during nicotine 

administration frequently abort. 

Body Weight 

Animals expose d t o nicotin e prenatall y consistentl y 

weigh les s than animal s not expose d t o nicotine pre - 

natally (Bass i e t al. , 1984 ; Paulso n e t al , 1993 ; 

Ajarem and Ahman , 1998 ; Vaglenov a et al, 2004) . 

Animals receivin g 4. 0 mg/kg/day o f nicotin e prena - 

tally (ora l administration ) weig h les s tha n controls , 

and thes e effect s persis t unti l postnata l da y (P ) 2 9 

(Paulson e t al, 1993) . Ajarem an d Ahmed (1998 ) administered 

nicotin e (0.5 0 mg/kg) or saline to pregnan t 

mice via SC injection s for 9-10 day s and assesse d offspring 

development . Th e nicotine-expose d animal s 

PRENATAL NICOTINE EXPOSURE AND ANIMAL BEHAVIOR 33 3 

gained weigh t a t a slowe r rate tha n tha t o f controls. 

Such findings are interesting because they parallel reports 

from human s and suggest that prenatal smokin g 

affects prenata l an d postnata l development . Specifi - 

cally, wome n wh o smok e durin g pregnanc y have a 

greater number o f spontaneous abortions , premature 

births, an d infant s with low-birt h weight s (Eskenaz i 

etal, 1995; Hofhuis etal, 2003). In addition, nicotineinduced 

reductio n i n postnata l weigh t gai n als o has 

been reporte d i n huma n offsprin g (Marti n e t al. , 

1977). 

Hyperactivity 

Several studies have reported hyperactivit) ' in the offspring 

o f nicotine-treate d animal s (Marti n e t al. , 

1976; Lichtensteige r e t al, 1988; Ajarem and Ahmad, 

1998; Fun g and Lau, 1998 ; Newma n e t al, 1999 ; Tizabi 

and Perry , 2000). In rats , hyperactivity is indexed 

by measurin g open-field activit y o r activit y i n othe r 

novel environments such as the elevate d plus maze. 

Open-field activity refers to the behavio r of an animal 

whe n i t i s placed i n a nove l environment . Ini - 

tially, activity levels are high when an animal explores 

its ne w environment . A s the environmen t become s 

familiar, ambient activity decreases. Persistent heightened 

activity is interpreted a s evidence o f hyperactivity, 

a behavior frequently observed i n th e offsprin g o f 

mothers who smoked during pregnancy. 

Rats exposed to nicotine (1. 5 mg/kg/day via SC in - 

jections) throughout gestation hav e increased spontaneous 

locomoto r activit y compare d t o tha t o f 

saline-exposed control s (Fung and Lau , 1988) . Simi - 

larly, Tizabi and colleagues (1997), who administered 

nicotine (9. 0 mg/kg/day) vi a osmoti c minipump s t o 

pregnant dams throughout gestation, show that 20-24day-old 

offsprin g prenatall y expose d t o nicotin e ex - 

hibit mor e locomoto r activit y tha n tha t o f salin e 

controls. 

The elevate d plus maze i s used to test anxiety and 

to measur e activit y and impulsivity . Accordingly , an 

animal is placed i n the center of an elevated four-arm 

maze tha t include s tw o ope n an d tw o close d arms . 

The tota l number of times an animal enters any of the 

four arm s provides an inde x of activity and impulsiv - 

ity. Ajarem an d Ahmad (1998 ) administered nicotine 

(0.50 mg/kg) or saline to pregnant mic e for 9-10 days 

and measure d activit y on th e elevate d plu s maz e of 

weaned offspring . Nicotine-expose d offsprin g mad e 

more entrie s in the arm s of the maz e than di d saline


controls. This finding suggests that prenatal exposure 

to nicotin e promote s hyperactivit y an d impulsivity . 

Sobrian an d colleague s (2003 ) report simila r effect s 

of prenata l nicotin e exposur e o n activit y i n a n ele - 

vated plus maze. They administere d nicotin e (2. 5 or 

5.0 mg/kg) or saline via an osmotic pump t o pregnant 

dams throughout gestation . Adult rats exposed to high 

doses of nicotine prenatall y ar e les s timi d an d mor e 

impulsive than controls. These findings are consistent 

with reports from huma n studies , that prenatal smoking 

t o nicotin e result s i n hyperactivit y i n offsprin g 

(Milberger e t al, 1996 ; Mic k e t al, 2002 ; Thapa r 

et al, 2003). 

Attention 

Prenatal smokin g disrupt s attentio n i n expose d offspring 

(Kristjansson et al., 1998). In rats, attention can 

be measure d usin g the PP I of the ASR . PPI refers t o 

the reductio n o f a startle respons e whe n th e startlin g 

stimulus is preceded b y a nonstartling stimulus. Con - 

trol animal s exhibi t a reduce d startl e respons e t o a 

sudden, loud nois e if the nois e i s preceded b y a softe r 

sound. Disorders that disrupt attention, such as schizophrenia 

an d attentio n defici t hyperactivit y disorder 

(ADHD), disrupt PPI (Swerdlo w et al, 2000 ; Geye r 

et al., 2001). 

Reports o f prenatal nicotin e o n attentio n i n rat s 

are limited, but dat a that ar e available show that rat s 

respond to prenatal nicotine exposure. Prenatal nicotine 

exposur e (6mg/kg/da y vi a osmoti c minipump ) 

throughout gestatio n reduce s the PP I of the ASR to a 

98 dB stimulu s i n femal e offsprin g (Popk e e t al. , 

1997). Thes e finding s ar e consisten t wit h th e ob - 

served attentiona l deficit s reporte d i n human s ex - 

posed t o nicotin e prenatally . Futur e studie s usin g 

other attentional paradigms (e.g., five-choice serial reaction 

time test) will further hel p t o characterize the 

attentional domain s affecte d b y prenata l nicotin e 

exposure. 

Learning an d Memor y 

Prenatal nicotin e exposur e alter s learning and mem - 

ory (Cornelius et al., 2000). In rats , the effect s o f prenatal 

exposur e to nicotine o n learnin g and memor y 

have been evaluate d primaril y by use of a radial arm 

maze, whic h present s a challeng e tha t assesse s th e 

ability of an animal to learn and remembe r multipl e 

spatial locations. The anima l i s placed i n the cente r 

of a maz e containin g eigh t o r mor e radiatin g arms. 

Food i s placed a t th e en d o f four arms . The anima l 

must ente r a n arm to retrieve the food . The number s 

of repeat entries by a rat into the arms already entered 

are recorded as working memory errors (Olton, 1987 ; 

Hodges, 1996) . Th e number s o f reentries int o arm s 

never havin g containe d foo d ar e recorde d a s refer - 

ence memory errors. 

Several studie s repor t impairment s o n th e radia l 

arm maz e followin g prenatal nicotin e exposure . Fol - 

lowing administration o f nicotine (6. 0 mg/kg) to pregnant 

dam s i n thei r drinkin g wate r throughou t 

gestation, offsprin g exhibi t impaired performance in 

the radia l arm maz e (Sorenso n e t al., 1991) . Severa l 

other studie s describ e simila r finding s (Yana i e t al. , 

1992; Levin et al., 1996). Levin and colleagues used a 

radial arm maz e to assess the effect s o f prenatal nico - 

tine exposur e (2.0mg/kg/da y via osmotic minipump ) 

on learnin g an d memory . The y report that nicotine - 

exposed animals do not diffe r fro m control s in the acquisition 

of the task , but thes e rat s make more errors 

when spatia l cue s ar e changed, henc e addin g t o th e 

demands o f the task. 

Prenatal effect s o f nicotin e hav e bee n evaluate d 

using the two-wa y active avoidance (shuttle box) task. 

In thi s task , classica l conditionin g procedure s ar e 

used t o asses s learning an d long-ter m memory . Th e 

apparatus consist s of two equall y divide d compart - 

ments connecte d b y a cente r opening . Durin g th e 

training th e anima l i s presente d wit h a combine d 

light an d tone (conditioned stimuli) , followed by the 

presentation o f shock o n a grid floo r (unconditione d 

stimulus [UCS]) . Following presentation of the con - 

ditioned stimulus , th e anima l ca n avoi d the UC S by 

passing fro m on e compartmen t t o another . Thi s 

avoidance behavior i s evidence o f learning, whereas 

failure t o avoi d th e UC S i s evidence o f no learning . 

Memory i s assessed b y re-exposing the anima l t o th e 

apparatus several days later and measuring avoidanc e 

activity. Greate r avoidanc e behavio r is evidenc e of 

long-term memory . 

Two-month-old mal e offsprin g o f dam s injecte d 

with nicotin e (0.50mg/kg/day ) durin g pregnanc y exhibit 

deficit s i n learnin g th e avoidanc e respons e 

(Genedani e t al. , 1983) . Mor e recently , Vaglenova 

and colleague s (2004 ) administere d nicotin e 

(6.0 mg/kg/day) vi a osmoti c minipum p t o pregnan t 

dams an d measure d activ e avoidanc e behavio r o f

offspring o n P45. Animals of both sexes prenatally exposed 

t o nicotin e hav e a significantl y lowe r numbe r 

of avoidance behavior s during all days of training an d 

during the retention test . These data are indicative of 

impaired learnin g and memory . Th e result s of these 

animal studie s parallel reports of memory an d learn - 

ing problems in humans and provid e further suppor t 

for th e valu e o f animal s fo r investigatin g prenata l 

smoking effects o n memor y and learnin g in offsprin g 

(Naeye an d Peters , 1983 ; Rantakalli o and Koiranen , 

1987; DiFranza an d Lew , 1995 ; Slotkin, 1998) . 

Addiction Liability 

Prenatal tobacco smokin g increases the likelihoo d of 

smoking amon g expose d offsprin g (Kande l e t al. , 

1994; Niaura et al, 2001; Chassin et al, 2002; Abreu- 

Villaca et al, 2004a, 2004b). In humans, prenatal exposure 

to nicotine is an important predictor of smoking 

in adolescents . (Corneliu s e t al., 2000 ; Niaura et al., 

2001). Interestingly , thi s vulnerabilit y exists , regard - 

less of whether parents continue t o smoke. 

Recent anima l studie s examinin g th e effect s o f 

prenatal nicotin e o n late r biologica l response s t o 

nicotine paralle l th e finding s o f human studie s an d 

provide underlying mechanisms fo r these observed effects 

(Abreu-Villac a et al, 2004a, 2004b). It has been 

suggested tha t th e increase d likelihoo d o f smoking 

among exposed offspring reflect s long-lasting changes 

in neural cell number, neural cell size, and cell surface 

area. These changes are evident in the neonate and become 

exaggerate d whe n nicotin e i s re-administere d 

during adolescence . Anothe r propose d mechanis m 

for th e relationshi p between prenata l nicotin e expo - 

sure and adolescen t smokin g is that prenatal nicotine 

exposure disrupt s th e programmin g o f cholinergi c 

function (Abreu-Villac a et al., 2004b). 

After prenata l nicotin e exposure , there i s impairment 

o f nicotin e acetylcholin e recepto r (nAChR ) 

up-regulation an d recepto r desensitizatio n whe n 

nicotine i s administered durin g adolescence . Thes e 

factors ma y contribute to increased cigarette smoking 

by adolescent s (Abreu-Villac a e t al, 2004b) . A relationship 

betwee n gestationa l nicotin e exposur e an d 

nicotine preferenc e i n peri-adolescen t offsprin g ha s 

also been examine d in mice (Klein et al., 2003). Pregnant 

female s wer e administere d saccharin-flavored 

water containin g 5 0 [Ig/ml nicotin e o r saccharin - 

flavored water without nicotine fro m th e nint h da y of 


gestation throug h P21 . Th e nicotin e preferenc e o f 

adolescent mal e offsprin g expose d t o nicotine prena - 

tally i s increase d compare d t o tha t i n age-matche d 

controls. The mechanis m fo r this effect ha s not bee n 

established. 

CONCLUSIONS AND 

FUTURE IMPLICATIONS 

Animal models of nicotine administration and animal 

studies of behavior provide investigators with valuabl e 

ways t o examin e th e relationshi p betwee n prenata l 

smoking (especiall y prenatal nicotine ) an d cognitiv e 

and behaviora l outcomes i n affecte d offspring . Th e 

fact that findings from prenatal nicotine studies in animals 

parallel and predic t findings from human s support 

th e validit y o f thes e studies . These model s ar e 

well establishe d an d hav e provide d reliabl e result s 

that hel p expan d ou r understandin g of the effect s o f 

tobacco. Despite this knowledge, man y question s re - 

main unanswered . Fo r example , althoug h w e no w 

have a better understanding o f specific brain region s 

affected b y prenata l nicotin e exposure , th e mecha - 

nisms by which thes e effect s occu r ar e poorly understood. 

Littl e researc h ha s bee n conducte d o n 

higher-animal species ; mos t studie s examinin g th e 

effects o f prenata l exposur e t o nicotin e hav e bee n 

conducted i n rodents . Studie s conducte d i n anima l 

species that more closel y parallel humans (i.e. , mon - 

keys) ma y provide additional information about rele - 

vant mechanisms . Ou r understandin g ma y offe r 

opportunities fo r th e developmen t o f intervention s 

that reverse damaging effects o f nicotine on the fetus . 

Future studie s shoul d includ e othe r behaviora l 

measures. In humans, prenata l nicotin e exposur e results 

in attentio n deficit s i n offspring , however , mod - 

eling anima l studie s tha t examin e attentio n ar e 

limited. Comple x measures of attention d o exis t and 

should b e include d i n futur e studie s of the effect s o f 

prenatal exposur e to nicotine. 

Examination o f sensor y an d moto r function s 

would be instructive , bu t fe w animal studie s hav e in - 

cluded suc h behaviora l measure s (Ajare m an d Ah - 

mand, 1998) . Human offsprin g prenatall y exposed t o 

nicotine exhibi t impairment s i n sensor y processin g 

and basi c moto r function s tha t persis t eve n a yea r 

postdelivery. Saxto n (1978) , fo r example , examine d 

babies born to mothers who smoked with the Brazelton


Neonatal Behavioral Assessmen t Scale . These infants 

have reduce d auditor y acuit y compare d to that of infants 

of nonsmoking mothers . Further , Gusell a and 

Fried (1984) report a decrease i n motor scores, as mea - 

sured b y the Bayley Mental and Motor Scales, among 

13-month-old offspring whose mother s smoked in during 

pregnancy. Thes e findings are important because 

they sugges t tha t prenata l smokin g affect s no t onl y 

complex behaviors suc h a s learning an d memory , bu t 

also fundamenta l sensor y an d moto r processes . Fur - 

ther studie s examinin g thes e relationship s ar e impor - 

tant, a s disruptions i n sensor y an d moto r processin g 

can explai n learnin g problems , behaviora l problem s 

such a s hyperactivity, an d perhap s even sensitivit y t o 

drug exposure i n adolescence or adulthood. 

Abbreviations 

ADHD attentio n deficit hyperactivity disorde r 

ASR acousti c startl e respons e 

IP intraperitonea l 

nAChR nicotin e acetylcholine receptor 

P postnata l da y 

PPI prepuls e inhibition 

SC subcutaneou s 

UCS unconditione d stimulu s 


the Rober t Wood Johnso n Foundatio n an d the U.S . De - 

partment of Defense. 

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21 

Neuronal Receptors for Nicotine: 

Functional Diversity and 

Developmental Changes 

The nicotini c acetyleholine receptor (nAChR ) is the 

principal target-mediato r o f nicotin e fro m smokin g 

and chewing of tobacco (Hog g et al., 2003; Picciotto, 

2003; Gott i an d dementi , 2004 ; Wonnacot t e t al, 

2005). Th e effect s o f nicotin e o n offsprin g o f preg - 

nant smoker s ar e widespread , rangin g fro m altere d 

neural development t o increased susceptibility to addiction 

i n adolescenc e (Kande l e t al. , 1992 ; Broide 

and Leslie , 1999 ; Everitt e t al, 2001 ; Frank e t al, 

2001; Walsh e t al, 2001; Slotkin, 2002, 2004; Abreu- 

Villaca e t al , 2004a , 2004b ; Cohe n e t al , 2005 ; 

Slotkin e t al. , 2005) . Thes e facts , couple d wit h epidemiological 

findings that >20 % of pregnant women 

smoke, underscor e the importanc e o f understanding 

nAChR function s i n th e prenata l mammalia n brai n 

(Chassin e t al, 1996 ; Ebrahim e t al, 2000 ; Wals h 

et al, 2001; Cohen et al, 2005). 

Intrinsic cholinergi c signalin g participates in be - 

haviors a s fundamental as memory, motivation , an d 

movement (Picciott o an d Corrigall , 2002 ; Picciotto, 

2003; Gotti an d dementi, 2004 ; Laviolett e an d van 

Huibert D. Mansvelder 

Lorna W. Role 

341 

der Kooy , 2004; Wonnacot t e t al, 2005). Alterations 

in nACh R number s an d profile s ar e associate d with 

numerous disorders , includin g Alzheime r demen - 

tia, schizophrenia , and , not surprisingly , addictio n 

(Cordero-Erausquin e t al. , 2000 ; Marubi o an d 

Changeux, 2000 ; Changeu x an d Edelstein , 2001; 

Sabbagh e t al, 2002 ; Freedma n e t al, 2003 ; Hog g 

et al, 2003 ; Gott i an d Clementi , 2004 ; Newhous e 

et al, 2004b; Sacc o e t al, 2004). In view of the central 

role of nAChRs in such divers e conditions, it may 

seem surprisin g that mor e i s not know n o f the precise 

composition , localization , an d overal l rol e o f 

nAChRs i n synaptic signaling in th e centra l nervou s 

system (CNS) . 

A majo r obstacl e t o advancin g th e fiel d i s tha t 

CNS cholinergi c synapses , i n contras t t o thei r 

cousins i n th e periphera l nervou s syste m (PNS) , 

have largel y elude d direc t electrophysiologica l 

scrutiny. In defense of efforts t o date, several difficul - 

ties should b e noted . First , centra l cholinergi c neurons 

ar e fe w i n numbe r an d thei r projection s ar e


diffuse (Woolf , 1991) . Second , ther e ar e multipl e 

subunit-combination variants of nAChRs. This structural 

medle y i s reflected i n a comparable functiona l 

diversity that includes differences i n nACh R sensitivity 

to both endogenou s agonist s and exogenousl y administered 

nicotin e (L e Novere e t al., 2002a, 2002b ; 

Hogg e t al, 2003 ; Gott i an d Clementi , 2004) . Th e 

cellular targetin g o f nAChR s i s als o diverse . Som e 

nAChRs ar e shippe d t o presynapti c o r pretermina l 

sites, other s t o postsynapti c an d perisynapti c locales , 

and al l nAChR subtypes are expressed at strikingly low 

amounts compare d wit h ionotropic receptor s fo r glutamate 

o r y-aminobutyri c aci d (GABA ) (Colquhou n 

and Patrick, 1997; Tembumi et al, 2000; Le Novere et 

al., 2002a, 2002b; Hogg et al, 2003). Perhaps most discouraging 

i s how quickly nAChRs transi t into close d 

(desensitized) state s i n respons e t o eve n minut e con - 

centrations o f agonist . Thi s trait , too , varie s amon g 

neurons and among nAChR subtype s (Lu et al., 1999 ; 

Quick an d Lester , 2002 ; Wooltorto n e t al , 2003 ; 

Alkondon and Albuquerque, 2004, 2005). 

Although suc h feature s present seriou s obstacle s 

to investigators , these sam e characteristic s underli e 

the important contribution of nAChRs to the compu - 

tational flexibilit y o f circuits where signalin g is subject 

to modulation, rathe r than jus t "on/off" control . 

Recent molecula r biological , biochemical, an d phar - 

macological studie s have generated a powerful armamentarium 

o f ne w probe s an d approache s fo r 

analyzing nAChR subuni t compositions and subtypes 

(Gotti an d Clementi , 2004 ; Laviolett e an d va n de r 

Kooy, 2004 ; Nick e e t al , 2004 ; Wonnacot t e t al , 

2005). The challeng e no w is to advanc e th e curren t 

understanding o f ho w cholinergi c synapses , i n gen - 

eral and pre - and postsynapti c nAChR s i n particular, 

contribute t o th e function s and dysfunction s of th e 

developing nervous system. 

The las t decad e ha s witnesse d considerabl e ad - 

vances i n basi c knowledg e o f the function , develop - 

ment, an d plasticit y o f nAChRs . Severa l recen t 

reviews have presented nove l perspective s on nACh R 

signaling mechanism s an d o n emergen t view s o n 

nAChR involvemen t i n neuropsychiatri e disorder s 

(Jones et al, 1999 ; Picciott o et al, 2001; Hogg e t al, 

2003; Picciotto, 2003 ; Dajas-Bailador an d Wonnacott , 

2004; Fucile, 2004 ; Gotti and Clementi, 2004 ; Laviolette 

and van der Kooy, 2004; Lester et al, 2004; Newhouse 

e t al , 2004a , 2004b ; Sacc o e t al , 2004 ; 

Wonnacott e t al., 2005) . The presen t chapte r focuses 

on fundamenta l aspect s o f th e acetylcholin e (ACh ) 

receptors with which nicotine interacts in the context of 

potential effects on neural development and plasticity. 

STRUCTURAL DIVERSITY OF NEURONAL 

NÏCOTINIC ACETYLCHOLINE 

RECEPTORS 

Twelve separat e gene s encodin g distinc t neurona l 

nicotinic receptor subuni t proteins have been identi - 

fied an d iterativel y labeled a s oc2 , ot3, cc4, oc5 , ot6 , Ot7 , 

cx8, cc9 , an d oclO , o r ß2 , ß3 , an d ß 4 (L e Nover e an d 

Changeux, 1995 ; L e Nover e e t al , 2002b ; Hog g 

et al., 2003; Gotti an d Clementi , 2004) . Each o f the 

five subunits that comprise the assembled oligomeri c 

complex i s a four-transmembran e domai n protei n 

with considerabl e homolog y amon g th e membran e 

spanning regions (Fig. 21-1 A, B). All nicotinic recep - 

tor subunits are characterized b y a tw o C-loop con - 

taining N-termina l domain , th e signatur e o f thei r 

membership i n th e broade r famil y o f ligand-gate d 

ion channels, along with GABA A, glycine, and mus - 

cle nicotini c recepto r subunit s (L e Nover e an d 

Changeux, 1995 ; Le Novere et al, 2002a; Hogg et al., 

2003; Gotti an d Clementi, 2004 ; Leste r et al, 2004). 

Similar t o the muscl e nicotini c Ot l recepto r subunit , 

neuronal a subunit s are identified by the presence of 

vicinal cystein e residue s withi n th e N-termina l do - 

main, a uniqu e ta g o f th e ligand-bindin g regio n 

(Brejc e t al, 2001 ; Karlin , 2002; L e Nover e e t al, 

2002a, 2002b) . The assemble d recepto r protei n i s a 

pentamer with a hydrophili c cor e line d primaril y by 

the secon d transmembran e domai n o f each subuni t 

(Fig. 21-1B , C) . Th e contributio n o f specifi c sub - 

units t o assemble d receptor s an d ke y features of th e 

ligand-binding domains and ion channel por e are being 

scrutinize d wit h "knock-down, " single - an d 

double-iiAChR subuni t gen e knockou t mic e (L e 

Novere e t al., 2002b; Champtiaux et al, 2003; Hog g 

et al, 2003 ; Leste r e t al., 2003) . The ligand-bindin g 

domain i n neurona l nAChRs , lik e muscle-typ e 

nAChRs, i s formed b y th e N-termina l domain s o f o e 

subunits an d a t th e interfac e of adjacent subunits in 

the assemble d pentamer . Th e identificatio n o f se - 

quences involve d in th e ioni c "gate" of nAChRs ha s 

emerged fro m studie s using ingenious combination s 

of mutagensis t o incorporate cystein e residues at key 

sites, an d subsequen t biochemica l an d biophysica l 

probing wit h specia l cysteine-reactiv e reagent s (Karlin, 

2002; Jensen et al, 2005).

DEVELOPMENT OF NICOTINE RECEPTORS 34 3 

FIGURE 21- 1 Curren t model s of neuronal nicotinic receptor (nAChR ) subunit 

complexes. Panel s A and B illustrate singl e a - o r ß-type nicotinic recepto r sub - 

unit protein in the membrane bilayer . A. Each o f the neuronal nicotinic recep - 

tor subuni t gene s encode s a protei n tha t traverse s the membran e fou r time s 

(labeled 1-4) . Th e N-termina l domain is on the external face of the membrane, 

the first and secon d transmembran e domains (TM ) ar e linked by short amino 

acid sequence s o n th e interna l face , an d th e secon d an d thir d T M ar e con - 

nected b y a short external linker. The cytoplasmi c loop between T M 3 and 4 is 

the mos t variabl e portion o f the subuni t sequences . I t has been implicate d i n 

both th e modulatio n an d membran e targetin g o f assemble d nACh R com - 

plexes. The C termina e are short extracellula r domains . B . Disposition o f the 

TM withi n a single o c subunit relative to each othe r and to the Hydrophobie surrounds. 

Th e linke r betwee n TM 1 an d TM 2 an d th e intracellula r sid e TM 2 

amino acids comprise the io n channel gat e (se e text and Karlin , 2002). Panels 

C an d D sho w model s o f the arrangemen t o f five nAChR subunits (e.g. , oc 7 

alone or a an d ß subunits) assembled as neuronal nACh R complexes . C . This 

simplified structura l model o f a neuronal-type nAChR pentame r i s largely inferred 

fro m studie s of the muscl e an d torped o electropla x nAChR. Ke y features 

include th e "stave s o f a barrel " arrangement o f each subuni t aroun d th e hy - 

drophilic pore, an d the contributio n o f the TM2 t o the linin g of the channel . 

D. Example s o f propose d subuni t composition s o f neurona l nACHR s an d 

experimentally derive d difference s i n Ca 2+ :Na permeabilit y of the illustrate d 

subtypes. 

The multiplicit y of nAChR-subunit types begs the 

question, "why so many ?" . This issu e received a con - 

siderable researc h effor t ove r the las t decade resultin g 

in th e conclusio n tha t differen t subuni t combination s 

are functionall y an d pharmacologicall y distinc t fro m 

one anothe r (Fig . 21—ID ; se e Diversit y of Nicotini c 

Acetylcholine Receptors, below). The physiologica l justification 

fo r the subuni t diversity, however , raise s th e 

difficult questio n of how t o identif y th e combination s 

and stoichiometrie s o f subunits tha t compris e native 

nAChRs (Colquhoun an d Patrick , 1997 ; Champtiau x 

et al, 2003 ; Na i e t al, 2003 ; Alkondo n an d Albu - 

querque, 2005) . Despit e a concerte d effort , thi s 

issue remains largely unresolved. Nevertheless , sev - 

eral generalization s abou t nACh R compositio n 

have held u p t o scrutiny: there i s reasonable agree - 

ment that the oc4 , ß2, and oc 7 subunits are the majo r 

players i n nAChR s expresse d i n th e CNS , wherea s


oc3, ß4 , an d a? subunit s ar e mor e typica l compo - 

nents o f PN S nAChR s (Cordero-Erausqui n e t al. , 

2000; Picciotto et al, 2001; Le Novere et al, 2002b; 

Hogg et al, 2003; Gotti and démenti, 2004). Likewise, 

i t i s generally agreed tha t (1 ) a2, oc3 , an d oc 4 

require co-assembly with a ß subunit to make func - 

tional channels ; (2 ) ß2 and ß 4 requir e co-assembl y 

with an a subuni t to make functional channels; (3) 

oc5, ot6 , an d ß 3 requir e co-assembly with bot h an - 

other a an d ß subunit to make functional channels; 

and (4 ) onl y oc7 , oc8 , oc9 , an d ocl O ca n for m func - 

tional homopentamers (Wang et al., 1996 ; Cordero - 

Erausquin e t al. , 2000 ; Changeu x an d Edelstein , 

2001; Ber g an d Conroy , 2002 ; L e Nover e e t al , 

2002b; Hogg et al, 2003; Gotti and Clementi, 2004; 

Nicke, 2004). 

This i s what nAChR subunit s can d o i n term s of 

co-assembly. Wha t happen s i n viv o i s les s clear , al - 

though a cleare r pictur e i s emergin g a s nativ e 

nAChRs becom e bette r define d throug h combine d 

molecular, pharmacological , an d biophysica l ap - 

proaches (Sivilott i e t al, 1997 ; Y u and Role , 1998a , 

1998b; Cueva s et al, 2000; Champtiaux e t al, 2003; 

Nai et al, 2003; Fucile, 2004 ; Fischer et al, 2005). In 

recognition o f compositiona l uncertainty , a consor - 

tium o f "nicotinologists " hav e adapte d a nomencla - 

ture in which the inclusion of a subunit in an nACh R 

complex i s indicate d b y a n asteris k (Luka s e t al , 

1999) Fo r example, the designatio n oc4ß2* is used for 

nAChR complexe s that include oc 4 and ß 2 subunits, 

regardless o f stoichiometry, i.e. , (oc4) 2 (ß2) 3 o r (oc4) 3 

(ß2)2, and allows for the possibility that other subunits 

may participate in the nativ e complex. S o far, reports 

on nativ e nACh R compositio n includ e everythin g 

from th e simples t configuratio n (i.e. , homopen - 

tameric, such a s (oc7) 5 t o th e mos t complicate d ver - 

sions (i.e., different a an d ß subunits) of nAChRs (Le 

Novere et al., 2002b). Mercifully, studies of the over - 

laps in nAChR-subunit gene expressio n patterns sug - 

gest that the subunit composition of native nAChRs is 

not as horrific as the simple mathematical calculatio n 

with 1 2 subunits in groups of five would predict (>10 5 

possible outcomes!) . I n addition , the expressio n patterns 

for several subunits are relatively restricted (e.g., 

oc5, a6, and ß3 ) (Le Novere et al., 1996 ; Klin k et al, 

2001; Le Novere et al, 2002b; Zoli e t al, 2002); cx8 

has no t bee n identifie d in mammalia n system s an d 

oc9 an d ocl O appea r t o b e largel y restricte d t o inner - 

vated sensory epithelia (Elgoyhen et al., 2001; Keiger 

et al. , 2003) . In sum , th e subuni t compositio n an d 

stoichiometry o f nativ e neurona l nAChR s i s mor e 

complicated than can be resolved with any one of the 

current analyti c techniques , bu t i s probabl y les s 

baroque tha n straigh t combinatoria l analyse s migh t 

suggest. 

DIVERSITY OF NEURONAL NICOTINIC 

ACETYLCHOLINE RECEPTORS 

Functional Group s 

The functiona l distinctions among nAChRs of differ - 

ent compositio n are not trivial. Each i s an importan t 

determinant o f how th e gatin g o f a particula r nicotinic 

receptor could contribute to synaptic excitability 

and/or be affected b y exogenously delivered nicotine. 

In th e beginnin g o f the 1990s , biochemical an d bio - 

physical analyses combined t o rescue oc5 , oc6 , and ß 3 

from thei r orpha n statu s (L e Nover e e t al. , 1996 ; 

Ramirez-Latorre e t al , 1996 ; Wan g e t al , 1996 ; 

Groot-Kornielink et al, 1998; Lena et al, 1999 ; Kury - 

atov et al., 2000). More recen t wor k has honed thes e 

techniques t o better defin e the functiona l properties 

that distinguis h on e nACh R subtyp e fro m anothe r 

(Nai e t al., 2003). It is now clear that the inclusio n of 

each o f thes e subunit s i n nACh R complexe s ca n 

change th e kinetics , unitar y conductance , an d cal - 

cium permeabilit y o f th e resultan t channel s 

(Ramirez-Latorre e t al., 1996 ; Wan g e t al, 1996 ; Yu 

and Rôle , 1998a , 1998b ; Nai et al, 2003). For example, 

biophysica l studie s sho w tha t Ot3ß4 * an d 

a3oc5ß4* nAChRs , whic h ar e presen t a t man y pe - 

ripheral site s and involve d in th e cardiovascula r effects 

of nicotine pass more picoamps per millisecond 

than Ot4ß2 * nAChR s (Gerzanic h e t al, 1998) . Th e 

activation profile s an d desensitizatio n rates of thes e 

nAChRs are also dissimilar from on e another and are 

very differen t fro m oc 7 homomeri c nAChR s (Ber g 

and Conroy , 2002 ; Quick an d Lester , 2002 ; Khiroug 

et al, 2004). 

Rectification propertie s o f th e variou s channe l 

subtypes a s wel l a s agonis t an d antagonis t bindin g 

properties are distinct, but perhaps the most functionally 

importan t difference amon g nACh R subtype s is 

in their relative Ca 2+ permeability (e.g., Fig. 21-1D). 

The subjec t o f Ca 2+ conductanc e an d nAChR s re - 

quires an extra comment on

(otBgTx) sites . The dogge d effort s o f several investigators 

brought th e beguilin g finding that th e arche - 

typal muscl e aBgTx-bindin g sit e wa s peppere d al l 

over the CNS, to the ultimate resolution of a unique 

set of nicotine-gated io n channels (Wonnacot t e t al., 

1982; Clark e e t al, 1985 ; Dajas-Bailador an d Wonnacott, 

2004 ; Fucile , 2004 ; Nicke et al, 2004). The 

calcium permeabilit y o f oc7 * nAChR s i s greate r 

than tha t o f othe r neurona l nAChR s an d muc h 

larger tha n tha t o f the muscl e nAChR . I n fact , th e 

fractional curren t throug h a? * nAChR s tha t i s attributable 

t o Ca 2+ i s comparabl e t o N-methyl-D - 

aspartate (NMDA)-typ e glutamat e receptors , bu t 

without the extra control afforded b y the voltage and 

Mg-dependent gatin g that constrain s NMDA recep - 

tors (Dajas-Bailador and Wonnacott, 2004). I n other 

words, a7* nAChR s ar e essentially Ca 2+ flood gate s 

just waiting for ligand to happen! Judicious targeting 

of suc h receptor s t o presynapti c domain s ca n (and 

apparently does ) provid e a loca l trigge r fo r Ca 2+ - 

dependent transmitte r release . Mor e subtl e t o a n 

electrophysiologist, bu t no t t o th e nAChR-bearin g 

neurons, ar e th e man y downstrea m molecula r targets 

o f calcium influ x (Dajas-Bailado r and Wonna - 

cott, 2004) . Thes e sort s o f Ca 2+ -dependent tuning , 

by both oc 7 an d non-oc 7 nAChRs , appea r t o b e im - 

portant loc i o f nicotine' s effect s o n th e developin g 

nervous system (see Nicotinic Acetylcholin e Recep - 

tors, Synapti c Plasticity , an d Brai n Development , 

below). 

The essentia l contributio n o f oc2ß4 * nAChR s a t 

synapses an d circuit s relate d t o nicotine-elicite d re - 

sponses ha s bee n repeatedl y an d compellingl y 

demonstrated (Picciott o an d Corrigall , 2002 ; Leste r 

et al , 2003 ; Picciotto , 2003 ; Gott i an d dementi , 

2004; Laviolett e an d va n der Kooy , 2004; Wonnacot t 

et al. , 2005) . Sinc e Changeu x an d colleague s (Picciotto 

et al., 1995 ) first reported th e altered avoidance 

learning i n knockou t mic e o f th e high-affinit y ß2 * 

nAChRs, th e choru s has mounted o n the ke y role of 

these nAChR s i n CN S function , culminatin g wit h 

the recen t identificatio n of a singl e sit e deeme d es - 

sential t o Ot2ß4*-mediate d addictio n (Tappe r e t al, 

2004). The rol e of changes i n the number of nAChRs 

(not confined to, but exemplified by, oc2ß4* nAChRs) 

in th e effect s o f long-ter m nicotin e exposur e i s still 

under debat e (Gentr y an d Lukas , 2002 ; Wonnacot t 

et al., 2005). 

In addition to compositional diversit y in nAChRs , 

other factor s an d condition s that may differ fro m cel l 


to cell and fro m synaps e to synapse regulate the func - 

tional efficac y o f nAChRs. Thus, marked difference s 

in the physiological profile of native nAChRs vs. their 

recombinant version s expressed i n heterologou s systems 

underscor e th e potentia l rol e o f loca l o r cell - 

specific regulator y molecules in determining nACh R 

function i n vivo. Extracellular modulators of nAChRs 

abound, includin g endotoxins , endocannabionoids , 

and neuropeptide s (Miw a et al., 1999 ; Di Angelantonio 

e t al , 2003 ; L e Foi l an d Goldberg , 2004 ; O z 

et al, 2004). 

A striking example of native receptors being more 

functionally comple x tha n thei r heterologous equivalents 

ha s emerge d fro m studie s o f oe7-containin g 

nAChRs (Y u an d Role , 1998b ; Ber g an d Conroy , 

2002; Khirou g e t al. , 2002 ; Drisde l e t al , 2004) . 

Comparison o f many in vitro expression studies with 

neuronal studie s has led to the conclusio n tha t mos t 

native oc7 * nAChR s ar e homopentameric complexe s 

with ver y rapi d desensitization , lo w agonis t affinity , 

and long-lastin g otBgT x binding . Othe r wor k indi - 

cates that a subset of native a7* nAChRs may include 

other subunits , o r oc 7 subunit s tha t hav e bee n subjected 

t o post-translationa l modifications , yieldin g 

nAChRs wit h ver y differen t agonist-induce d activa - 

tion an d desensitizatio n fro m (oc7) 5 (Y u an d Role , 

1998a; Cueva s et al, 2000; Khiroug et al, 2002; Drisdel 

étal, 2004). 

Diversity in Targeting o f Nicotinic 

Acetylcholine Receptors i n the 

Central Nervous System 

The developmen t of better pharmacological tools and 

new molecula r probe s t o nAChR s hav e unveile d 

impressive intricacie s in the cellula r traffickin g of 

nAChRs (William s e t al. , 1998 ; Temburni e t al , 

2000, 2004; Berg and Conroy, 2002; Brumwell et al, 

2002; Blank et al., 2004; Liu et al., 2005). Dependin g 

on subuni t compositio n (an d in th e cas e o f oc3 , o n 

identified subuni t sequences ) a n nACh R comple x 

can b e selectively targeted t o somatic-dendritic vs. axonal 

domains , a s well a s t o synaptic , pre-, or perisy - 

naptic subdomains within a neuron. 

As ACh an d nicotine can influence synaptic transmission 

via pre- and postsynapti c nAChRs , and sinc e 

"extrasynaptic" nAChRs ma y exert important regula - 

tory effect s o n neurona l activity , the traffickin g t o all 

loci is likely important (Fig. 21-2) (Hogg et al., 2003; 

Dajas-Bailador an d Wonnacott , 2004 ; Gott i an d


A. POSTSYNAPTIC NiCOTINIC ÄChRs 

B. PRE-SYNÄPTIC NICOTINiC AChRs 

FIGURE 21- 2 Propose d disposition and physiological 

effects o f pre-, post - and perisynapti c nicotini c recep - 

tors. A . This imag e depict s neuron-neuro n connec - 

tions where postsynaptic nAChRs participate i n direc t 

transmission. Acetylcholin e (ACh ) act s a s a primary 

synaptic transmitte r i n periphera l autonomi e gangli a 

and a t some CN S sites . ACh i s released fro m closel y 

apposed presynaptic elements, leadin g to the direct activation 

o f postsynapti c nAChRs . Not e tha t despit e 

their perisynaptic location o n ciliary ganglion neurons , 

oc7* nAChR s participat e i n direc t synapti c responses . 

Administration of nicotine could activate and/or desensitize 

receptor s a t both peri - an d directl y postsynapti c 

locales. B . Presynapti c (and/o r preterminal) nAChR s 

contribute t o the modulation o f synaptic transmission. 

ACh may be released from nearby cholinergic projec - 

tions, leading to the activatio n of nAChRs localize d a t 

or near presynaptic terminals. Depending o n the position 

an d subtyp e of nAChR, as well as the ambien t vs. 

stimulated concentratio n o f ACh , th e spontaneou s 

and/or evoked release of transmitter is facilitated o r depressed 

b y adde d nicotine . Th e ambiguit y o f thes e 

statements, and those in the text, as well as the fancifu l 

nature of the diagram reflect the very real difficulties i n 

assessing the actions of nicotine in vivo. 

Clementi, 2004) . Man y group s hav e advance d th e 

proposal that , despit e th e relativel y lo w concentra - 

tions of nAChRs in the CNS, nAChR s do play an important 

role in fine-tuning CNS synaptic transmission 

because o f their strategi c localization t o presynapti c 

sites (Dani, 2001; Fucile, 2004 ; Sher et al, 2004). 

Early demonstration s o f presynapti c effect s o f 

nicotine recepto r activatio n cam e fro m studie s o f 

synaptosome preparation s b y Wonnacott , Collins , 

and colleague s (Rapie r e t al , 1988 ; Wonnacot t 

et al, 1989 ; Grad y e t al, 1992 ) an d fro m electro - 

physiological studie s o f Changeu x an d colleague s 

(Vidal an d Changeux , 1993 ; Gioann i e t al, 1999) . 

The presynapti c modulatio n o f central glutamater - 

gic transmissio n by nAChRs a t several sites was initially 

distinguishe d b y it s ocBgT x sensitivit y and it s 

ablation followin g knock-dow n o f oc 7 subunit s 

(McGehee e t al, 1995 ; Gray et al, 1996) . The hig h 

Ca 2+ permeabilit y o f th e oc7 * nAChR s serve s t o 

escort Ca 2+ into axon terminals, thus facilitating spon - 

taneous an d stimulus-evoke d transmissio n at numer - 

ous site s o f glutamate release . Non-ot7-containin g 

presynaptic nAChR s ha s bee n implicate d i n th e 

regulation o f glutamat e an d GAB A releas e (Vida l 

and Changeux , 1993) . Th e cumulativ e literatur e 

suggests tha t th e recepto r pharmacolog y an d pre - 

sumed compositio n o f presynaptic nAChR s appea r 

to b e jus t as elaborat e a s those o f peri- an d postsy - 

naptic nAChR s (Mull e e t al. , 1991 ; Jone s e t al , 

2001; Jone s an d Wonnacott , 2004 ; Fische r e t al, 

2005). 

Despite evidenc e tha t nAChR s can participate i n 

the modulatio n o f transmitter releas e i n th e CNS , 

one should not get too carried away. There is increasing 

evidenc e fo r direc t synapti c transmissio n (i.e. , 

mediated b y ACh releas e an d postsynapti c nACh R 

activation) i n severa l CN S regions , includin g th e 

hippocampus, nigrostriata l system , an d i n sensor y 

cortex (Jone s and Yakel , 1997 ; Alkondo n an d Albu - 

querque, 2004 ; Matsubayash i et al, 2004). The vari - 

ety o f nACh R subtype s tha t hav e bee n accuse d o f 

mediating postsynapti c transmissio n i n th e CN S i s 

also ver y broad , includin g bot h Ot7 * an d non-a7 * 

nAChRs. Last , bu t no t least , regardles s o f whethe r 

the nACh R populatio n densit y i s sufficien t t o sup - 

port a synaptic current, activation of pre- and/or post - 

synaptic nAChRs may be sufficient to elicit important 

downstream signalin g and metabolic response s (Ber g 

and Conroy , 2002 ; Dajas-Bailado r an d Wonnacott , 

2004). 

The mechanism s underlyin g th e release , an d the 

ambient vs. stimulated concentrations , o f ACh i n th e 

CNS remai n a bit o f a mystery . Both publishe d an d 

unpublished report s hint at less than classica l mecha - 

nisms o f ACh releas e (d e Rove r et al. , 2002 ; Lester , 

Jo, an d Role , persona l communication) . Electro n

microscopy studie s examinin g th e localizatio n o f 

cholinergic synaptic terminal s hav e reveale d consid - 

erable distanc e fro m cholinoceptiv e terminals , 

consistent with a volume mod e o f cholinergic trans - 

mission (Descarrie s e t al. , 1997 ; Zol i e t al. , 1999 ; 

Jones and Wonnacott, 2004) . Suc h vagarie s in the local 

concentration s o f AC h a t pre - o r postsynapti c 

cholinocepetive site s in th e CN S mus t be addressed 

before th e contribution of nAChR activation (and inactivation) 

t o th e developin g nervou s syste m ca n b e 

understood. 

DEVELOPMENTAL EXPRESSION 

OF NICOTINI C 

ACETYLCHOLINE RECEPTORS 

Prenatal Expressio n o f Neuronal 

Nicotinic Acetylcholine Receptor s 

Constituents of the Cholinergic System 

Acetylcholine an d nAChR s pla y critica l role s i n 

virtually al l phase s o f brai n maturation , durin g 

embryogenesis a s wel l a s postnata l development . 

Choline acetyltransferase (ChAT), the syntheti c enzyme 

for ACh, appear s very early during embryoge - 

nesis (Smit h e t al , 1979) , an d AC h influence s 

development a s early as gastrulation (Fig. 21-3). Initially, 

AC h promote s cel l divisio n a t a stag e when 

ACh synthesi s occur s i n preneura l undifferentiate d 

cells. Durin g gastrulatio n an d postgastrulation , 

ACh regulate s cel l movement s that are blocke d b y 

ACh antagonist s i n se a urchi n embryo s (Falugi , 

1993; Laude r an d Schambra , 1999) , and se a urchin 

oocytes an d earl y embryo s contai n nAChR s (Ivon - 

net an d Chambers , 1997 ; Buzniko v an d Rakich , 

2000). Thi s findin g suggest s tha t i n additio n t o 

ChAT, other proteins involved in cholinergic signaling 

ar e expresse d b y embryoni c cell s durin g thes e 

stages. Indeed, acetylcholinesterase (AChE), the enzyme 

tha t break s dow n ACh , i s present durin g se a 

urchin gastrulatio n (Falugi, 1993) . Messenge r RN A 

coding for nAChR subunit s is detected in cultures of 

premigratory neura l cres t cell s (Howar d e t al. , 

1995), which i n the fetu s will give rise to most neu - 

rons in the PNS . Thus, even before neuronal differ - 

entiation, cholinergi c signalin g throug h nAChR s 

occurs an d ma y eve n contribut e t o development o f 

non-neuronal cells. 


In prenata l neura l tissue , bot h muscarini c acetyl - 

choline receptor s (mAChRs ) an d nAChR s ar e de - 

tected, bu t nAChR s ar e expresse d earlie r tha n 

mAChRs (Laude r an d Schambra , 1999) . nACh R ex - 

pression as well as ChAT immunoreactivity and ACh E 

activity show a caudorostra l gradient o f appearance i n 

the fetal brain (Zoli et al., 1995; Lauder and Schambra , 

1999). Functiona l nACh R expressio n ha s bee n de - 

tected i n ste m an d progenito r cell s of the ra t cerebral 

cortex at gestational day (G) 1 0 (Atluri et al., 2001) and 

in mesencephalon of developing brai n at Gil (Zol i et 

al., 1995) . Earl y expression o f subunit mRNA s seems 

characteristic o f nAChR, sinc e othe r ligand-gate d io n 

channels becom e expresse d somewha t later . GAB A 

and NMD A receptor mRNA s are no t detecte d i n ra t 

brain unti l G14-G15 (Lauri e e t al, 1992 ; Monye r e t 

al., 1994) . Durin g this stage, ACh promote s the switch 

from cel l divisio n to neuronal differentiatio n (Slotkin , 

1998), an d guidance of nerve growth cones appear s to 

be on e o f th e role s o f cholinergi c signalin g throug h 

nAChRs (Hum e e t al., 1983 ; Zheng e t al, 1994) . At 

the onse t o f neurona l nAChR-subuni t expression , 

innervation-independent factor s ar e responsibl e fo r 

neuronal differentiatio n before cholinergic projection s 

arrive (Zoli et al., 1995) . Indeed, although cholinergi c 

neurons are among the first neurons to differentiate i n 

the CNS , i n ra t fetuses , cholinergi c neuron s ar e no t 

generated befor e Gil an d ar e generate d firs t i n th e 

spinal cord , brai n stem , an d basa l ganglia . I n mos t 

other brai n regions , cholinergi c neuron s ar e no t de - 

tected until G12 to G16 (Lauder and Schambra, 1999) . 

ChAT-immunoreactive nerv e terminal s ar e de - 

tectable during the first postnatal weeks , especially i n 

the forebrain . Th e majo r cholinergi c innervatio n 

originating fro m th e nucleu s basali s o f Meynert en - 

ters th e cerebra l corte x at abou t postnata l da y (P ) 0 

(Hohmann an d Berger-Sweeney, 1998) , bu t i s ini - 

tially confine d t o th e subplate , a transient , synapse - 

rich laye r o f neuron s locate d directl y unde r th e 

cortical plate . Thus , th e initia l profil e o f nAChR ex - 

pression may be induced by local endogenous differen - 

tiation signals rather than by cholinergic input s per se. 

This contrast s wit h finding s o n cholinergi c synapse s 

in the developin g autonomi e nervou s syste m (Deva y 

et al. , 1999) . Here , nAChR-subuni t expressio n i n 

neuronal cholinergi c synapse s is regulated b y signals 

from th e autonomi e targe t tissu e tha t i s innervated. 

Depending o n th e innervate d tissu e type , differen t 

types of nAChRs ar e expresse d tha t hav e different ki - 

netic properties (Devay et al., 1999) .


FIGURE 21- 3 Developmenta l expressio n of nicotinic acetylcholine receptors 

(nAChRs) i n ra t forebrai n and cortex . nACh R subunit s ar e expresse d i n ra t 

forebrain an d corte x at differen t site s and a t differen t time s durin g development. 

Interestingly , the expressio n of nAChRs precedes th e birt h of cholinergic 

cell s wit h a neurona l phenotype , suggestin g tha t cholinergi c signalin g 

through nAChR s ma y be involve d i n th e differentiatio n o f neurons. Durin g 

early postnatal development, when cholinergic projections arrive in the cortex, 

different nACh R subtype s show different expressio n dynamics , indicatin g that 

cholinergic signalin g through nAChR s i s involved in synapti c refinement of 

the neuronal circuitry. 

Spatiotemporal Patterns 

Expression of nAChRs is not constant during fetal development; 

i t varie s amon g region s an d subunit . At 

G10, when the mouse cortex consists of dividing stem 

and progenito r cells, these cortica l cell s expres s oc3, 

oc4, an d oc 7 recepto r subunit s that giv e rise to ACh - 

evoked inward currents (Atluri et al., 2001). Although 

these nAChR subunits can be detected fro m G1 0 until 

birth, the incidence o f these subunits declines with 

increasing gestational age. I n feta l ra t brain, oc3 , oc4 , 

ß2, an d ß 4 nACh R subunit mRNA expression is detectable 

in the hindbrain and midbrain, but not fore - 

brain, at Gl 1 (Zoli et al, 1995) . In forebrain, mRNA 

for oc3 , oc4 , an d ß 2 subunit s become s detectabl e a t 

G12. ß 2 mRN A seem s t o b e th e mos t widel y expressed 

an d a t relatively stable level s throughout th e 

remaining prenatal development. oc4-subuni t mRNA 

is at lower levels than ß2 mRNA throughout gestation, 

but show s a similarl y wid e distribution. In contrast , 

Ct3 and ß 4 mRNA are less abundantly distributed and 

expression can be transient in some brain regions. For 

instance, oc 3 mRNA is present in the ra t forebrain o n 

G12, G13 , an d G15 , bu t i s not detecte d ther e anymore 

a t G17 , G19 , an d P O (Zoli e t al, 1995) . ß 4 

mRNA i s absent from forebrai n durin g embryogenesis, 

but show s transient expressio n in othe r brain areas. 

It is detected i n midbrain on G12 and G15, but is 

not detected i n midbrain nucle i a t G19 and P O (Zoli 

et al. , 1995) . I n developin g chic k brain , simila r regional 

specificit y an d transien t expressio n occu r 

throughout gestationa l stage s (Torra o e t al. , 2000) . 

oc2, oc3 , oc5 , an d ß 2 mRNA s are expresse d very early 

on i n gangli a a t embryonic da y 4, and ar e expresse d 

throughout th e chic k brai n a t G18 . oc 7 mRN A was 

detected i n Purkinje neuron s of chick cerebellum a t 

G12, bu t expressio n decrease d afte r G1 8 (Kanek o 

et al., 1998). Taken together, the early onset, regional

specificity, an d transien t nature o f nAChR expression 

suggest that they may play important roles during embryonic 

development o f rodent an d chick brain. This 

theory i s underscored b y findings in transgeni c mic e 

with inducible expression o f oc2 subunits. Restoratio n 

of ß2 subunit expression during development i n thalamocortical 

projections of ß2 knockout mice normal - 

izes passive-avoidanc e learnin g i n thes e animals , 

suggesting that ß2 expression in these projections during 

developmen t i s critical fo r norma l developmen t 

of this behavior (King et al, 2003). 

In fetal human brain, nAChR gen e transcripts and 

proteins ar e als o detecte d a t ver y earl y gestationa l 

stages. nAChR protei n an d gen e transcript s are readily 

detecte d b y 4 t o 5 weeks o f gestation (Hellstrom - 

Lindahl et al, 1998) . Messenger RNA s of a3, a4? a5, 

oc7, ß2, ß3 , and ß 4 ar e foun d widel y distributed over 

the feta l brai n betwee n 4 and 1 2 weeks of gestation . 

In mos t brain areas, radio-ligand binding of nAChR s 

by [ 5 H] epibatidine and [ 3 H] cysteine increase d fro m 

weeks 4 to 12 . The highes t specifi c bindin g of these 

ligands wa s detecte d i n spina l cord , pons , an d 

medulla oblongata , wherea s bindin g b y th e oc 7 

subunit-specific prob e [ 125 I]aBgTx was found t o b e 

strongest i n pons , medull a oblongata , an d midbrai n 

(Hellstrom-Lindahl and Court, 2000) . Periods of transient 

high nACh R densit y were found i n frontal cortex, 

hippocampus , cerebellum , an d brainste m o f 

humans durin g mid-gestatio n an d neonata l period s 

(Kinney et al, 1993; Court et al, 1997). A significant 

positive correlatio n betwee n gestationa l ag e and th e 

expression o f oe7 mRNA was observed i n al l brain re - 

gions excep t corte x (Fal k e t al., 2002). From th e lat e 

fetal stage , human brai n nAChR expressio n has been 

shown t o fal l wit h increasin g ag e (Hellstrom-Lindah l 

etal, 1998; Hellstrom-Lindahl an d Court, 2000; Falk 

et al, 2003) . Thus , nAChR s als o appea r t o play an 

important role in human development , neurogenesis , 

and synaptogenesis. 

Response to Prenatal Nicotine Exposure 

Depending o n th e developmenta l phase , AC h 

promotes o r prevents neuronal apoptosis : when cell s 

are poorl y differentiated , th e effec t i s primarily proapoptotic, 

wherea s i n differentiate d cells , i t i s anti - 

apoptotic (Hohman n an d Berger-Sweeney , 1998) . 

The earl y expression of nAChRs during fetal develop - 

ment give s rise to a vulnerability of the huma n fetu s 


to exogenou s nicotin e exposure . A substantia l percentage 

o f pregnant woman continue smoking during 

pregnancy. I n th e Unite d States , nearl y hal f o f al l 

women smoker s continue t o smoke during their pregnancies 

(Ebrahi m e t al, 2000) , o r abou t 12 % of all 

women wh o giv e birth (Mathews , 2001) . Mor e tha n 

500,000 infant s eac h yea r ar e expose d t o cigarett e 

smoke i n utero . 

Fetal exposur e t o nicotin e damage s th e develop - 

ing brain, interferin g with cel l replicatio n an d differ - 

entiation. I t als o evoke s apoptosis , culminatin g i n 

reduced cel l numbe r an d aberran t cel l proportion s 

(Slotkin, 2002; Abreu-Villaca e t al, 2004a). In rat fetus, 

durin g neurulatio n i n th e neura l tub e stag e a t 

G9.5, nicotin e exposur e cause s cytotoxicit y i n al l 

brain area s an d incidence s o f apoptosi s sharpl y in - 

crease (Ro y et al. , 1998) . Prenata l exposur e t o nico - 

tine increase s cortica l mRN A expressio n o f oc4 , ß2 , 

and


rearrangement persis t int o adolescence . Cholinergi c 

signaling continues to play an important role in postnatal 

brai n development , whic h i s emphasize d b y 

brain region-specific an d transient expression profiles 

of nAChR subunits (Fuchs, 1989 ; Broide et al, 1995 , 

1996; Zhan g et al., 1998) . Disruptio n o f cholinergic 

innervation durin g earl y postnatal developmen t re - 

sults i n delaye d cortica l neurona l developmen t an d 

permanent change s i n cortica l cytoarchitectur e an d 

cognitve behavior s (Hohmann an d Berger-Sweeney , 

1998). oc3 , a.7, an d ß 4 subuni t mRN A expressio n is 

low durin g mid-gestatio n an d increase s durin g lat e 

gestation and early postnatal life (Winzer-Serha n and 

Leslie, 1997) . Durin g th e firs t fe w postnatal weeks , 

various subunit s ar e transientl y expresse d i n selec t 

sites i n th e brain : a 2 i n th e hippocampu s an d i n 

brain stem; cx 3 i n the striatum , cerebellum, an d cor - 

tex; cc 4 i n hippocampus , striatum , an d cerebellum ; 

oc7 in the cerebellu m an d cortex ; and ß 2 in the striatum 

(Broid e et al, 1995, 1996 ; Zhang et al, 1998). In 

rat cortex , oc 4 mRNA is maximal b y P14 after whic h 

its expressio n remains high. ß 2 mRN A is low on P 7 

but peak s a t P14 . ot 7 mRN A expressio n reache s a 

maximum o n P 7 (Shack a an d Robinson , 1998b) . 

These expressio n pattern s sugges t tha t nACh R sub - 

units pla y specifi c role s durin g postnata l develop - 

ment. Geneti c silencin g o f oc4 , ß2 , an d ß 3 nACh R 

subunits shows that each subunit is involved in particular 

neuronal an d behaviora l phenotypes, hinting a t 

their uniqu e role s i n brai n developmen t (Cordero - 

Erausquin e t al., 2000) . Only th e oc3-subuni t knockout 

mice have a high fractio n of mortality during th e 

first postnatal week (Xu et al, 1999) , mos t likel y because 

thi s subuni t i s a n essentia l componen t o f 

nAChRs that mediate fas t synaptic transmission i n the 

autonomie nervou s system, and, a s such, i s essential 

for perinata l survival. T o illustrat e the contribution s 

of cholinergi c signalin g t o postnata l brai n develop - 

ment, developmenta l change s i n nACh R expression 

and function in primary sensory cortex are describe d 

below. 

Cholinergic transmissio n is presen t i n th e earl y 

developing ra t parieta l corte x a t birt h (Descarrie s 

et al., 1997 ; Mechawa r and Descarries , 2001). In th e 

first 2 week s after birth , th e cholinergi c innervation 

increases greatly in number o f varicosities and num - 

ber o f branches pe r axon . Th e associatio n o f thes e 

new cholinergic terminals with postsynaptic densities 

is lo w (

cortical oc 7 mRN A follow s th e arriva l o f AChE - 

labeled thalamocortical afférents. Preventin g these afférents 

fro m reachin g the corte x strongly reduces th e 

a? subunit mRNA and [ 125 I]ocBgTx binding i n layers 

IV and VI (Broide et al, 1996) . Thes e data sug - 

gest tha t th e expressio n o f oc 7 subunit-containin g 

nAChRs i s regulate d b y thalami c inputs . Thes e oc 7 

subunit-containing nAChR s could b e located postsynaptically 

as well as presynaptically on thalamic affér - 

ents. I t ha s bee n hypothesize d tha t presynapti c oc 7 

nAChRs i n primar y auditory corte x ar e involve d i n 

the maturatio n o f glutamate synapse s by facilitatin g 

the conversio n o f "silent synapses " (defined a s those 

containing onl y NMD A receptors ) int o matur e ot - 

amino-3-hydroxy-5-methyl-4-isoxazole-propionic aci d 

(AMPA) an d NMD A receptor-containin g synapse s 

(Metherate an d Hsieh , 2003 ; Metherate, 2004) . Th e 

authors o f this hypothesi s sugges t tha t throug h thi s 

mechanism o f nAChR-induced maturatio n o f glutamate 

synapses , the expressio n o f oc 7 subunit s i n th e 

auditory corte x coul d defin e a critica l perio d o f sensory 

corte x developmen t i n whic h synapti c refine - 

ment of cortical circuitry and tuning to sensory inputs 

takes place. nACh R activation has been shown to alter 

synapti c strength o f glutamatergic synapti c transmission 

i n othe r brai n area s a s well , a s describe d 

below. 

In developin g hippocampus, nAChR s containin g 


Changeux, 1993 ; Gil etal, 1997; Gioanni etal., 1999; 

Lambe e t al., 2003). Nieotinic modulation o f thalamocortical 

projections by oc4ß2-containing nAChRs appears 

t o b e a genera l phenomeno n throughou t th e 

cortex (Metherate, 2004) . 

An elegant study in the ra t motor cortex identified 

distinct LC N subtype s tha t bot h expres s nACh R 

mRNA for oc4, a5, and ß 2 subunits and sho w somatic 

nicotinic currents (Porter et al., 1999) . PNs, as well as 

LCNs expressing either parvalbumin or somatostatin , 

showed n o effec t o f agonist application i n thi s study. 

LCNs expressin g vasoactive intestinal peptide (VIP) 

and cholecystokini n (CCK) , b y contrast , di d sho w 

nicotinic currents , and pharmacologica l analysis implicated 

non-oc7 nAChRs. I n human cerebra l cortica l 

slices, bipolar and multipola r LCNs exhibited eithe r 

a? o r a4ß 2 nAChR-rnediate d current s (Alkondo n 

etal., 2000). 

An interestin g bu t seldo m addresse d aspec t o f 

cholinergic signalin g in the cortex is the role of cholinergic 

LCNs in cortical microcircuit function. Cholinergic 

LCN s i n th e nucleu s accumben s (NAc ) can 

affect GABAergi c transmission within th e NA c itsel f 

via nAChR activatio n (de Rover et al, 2002). In addition, 

these cholinergic LCN s could be important i n 

the productio n o f lasting changes i n microcircuitries 

that affect anima l behavior, since their intrinsic firing 

properties are altered during behavioral sensitization 

to amphetamin e (d e Rove r e t al. , 2004) . Further - 

more, immunohistochemica l dat a sho w that a smal l 

fraction o f bipolar LCNs i n laye r II/II I of the roden t 

sensory and motor cortices are cholinergic (Houser et 

al., 1985) . A mor e recen t stud y usin g single-cel l 

reverse-transcriptase polymeras e chai n reactio n 

found a subgrou p o f cortica l LCN s tha t ar e im - 

munopositive for VIP an d calretini n and als o express 

ChAT transcript s (Caul i e t al. , 1997) . Thes e sam e 

VIP-positive cells are the main LCN subtyp e expressing 

nicotini c current s an d nACh R mRN A (Porte r 

et al., 1999) . Th e Nationa l Institut e o f Neurological 

Disorders an d Strok e (NINDS ) GENSA T BA G 

Transgenics Projec t show s tha t ChAT-positlv e stain - 

ing i s eviden t i n almos t ever y par t o f th e cortex , 

including th e PF C (http://www.gensat.org/makecon - 

nection.jsp). Th e putativ e presenc e o f cholinergi c 

LCNs in the corte x introduces the possibility of cortical 

nAChR activatio n independent o f cholinergic terminals 

tha t originat e fro m lowe r brai n areas . Sinc e 

the PFC i s implicated in behavioral sensitization (see 

below), intrinsic properties of cholinergic LCN s an d 

ACh releas e in the PF C coul d als o be modified during 

behaviora l sensitizatio n followin g th e intak e o f 

drugs o f abuse , a s describe d i n th e NA c (d e Rove r 

et al, 2004). 

NICOTINIC ACETYLCHOLINE 

RECEPTORS, SYNAPTI C PLASTICITY, 

AND BRAI N DEVELOPMEN T 

Activation o f oc 7 nAChR s i n primar y sensory cortex 

during postnata l developmen t ma y alte r synapti c 

strength of glutamatergic synapses (Metherate, 2004) . 

In recent years, several studies in different brai n area s 

have reveale d lastin g effect s o f nicotin e o r AC h o n 

synaptic connection s (Mansvelde r an d McGehee , 

2000; Dan i e t al. , 2001; Girod an d Role , 2001 ; J i et 

al., 2001; Mansvelder et al., 2002). Long-term modu - 

lation o f glutamatergi c an d GABAergi c synapti c 

connections by nAChRs can alter functioning of neuronal 

circuits for many days and possibl y induce lasting 

change s tha t coul d b e importan t fo r postnata l 

development o f these brain areas. These properties of 

cholinergic signaling through nAChR s ar e discusse d 

in the contex t of findings on nicotini c modulation o f 

the mesolimbic DAergic system. 

In rat , the D A reward system show s considerabl e 

plasticity during postnatal life an d doe s not complet e 

maturation unti l late adolescence or early adulthood 

(Benes et al., 2000). Since this system exhibits a similar 

make-up i n the huma n brain , adolescence migh t 

well represent a time window prone to aberrations by 

exposure to drugs of abuse. The mammalia n brai n is 

particularly sensitive t o developing addiction s durin g 

adolescence. Th e uniqu e behaviora l features of adolescence 

ar e drive n largely by maturational change s 

in the CNS, a process prone to environmental influ - 

ences such as drug exposure. Epidemiological studies 

show that exposur e t o addictive drug s durin g adoles - 

cence increase s th e ris k o f becoming a dru g addict 

later i n lif e (Kande l et al, 1992 ; Grant an d Dawson , 

1997). Adolescent smokin g is clearly correlated with 

smoking i n late r life . Moreover , adolescen t smokin g 

is a strong predictor o f the occurrence of adult depression 

(Chassin et al., 1996) . 

In th e VTA , nAChRs ar e expresse d o n DAergi c 

neurons, GABAergi c neurons , an d glutamatergi c 

terminals (Fig . 21-4 ) (Pidoplichk o e t al. , 1997 ;

Charpentier et al., 1998 ; Mansvelde r an d McGehee, 

2000; Klin k e t al, 2001 ; Champtiau x e t al, 2002 ; 

Mansvelder e t al. , 2002 ; Pidoplichk o e t al, 2004) . 

VTA DAergi c neuron s expres s thre e pharmacologi - 

cally identifiable nAChRs—one that is likely a homomeric 

oc 7 nAChR an d tw o that do not contain th e oc 7 

subunit. Mos t DAergi c neuron s expres s nAChR tha t 

can b e blocked by mecamylamine, whereas less tha n 

half of the DAergi c neurons expres s a7 nAChRs (Pi - 

doplichko et al., 1997 ; Klin k et al, 2001; Wooltorto n 

et al , 2003 ; Pidoplichk o e t al. , 2004) . GABAergi c 

neurons i n th e VT A expres s a simila r variet y o f 

FIGURE 21- 4 Nicotini c modulatio n o f synaptic com - 

munication i n ra t ventra l tegmenta l are a (VTA) . I n 

the VTA , nAChR s ar e present o n dopamine (DA)ergic 

neurons, GABAergic neurons, and glutamatergi c 

terminals. Arrival of low concentrations o f nicotine, as 

experienced b y smokers, rapidly activates and desen - 

sitizes non-cc7 nAChRs, wherea s al nAChR s o n glutamatergic 

terminal s ar e activate d bu t suffe r muc h 

less fro m desensitization . (Source: Modifie d fro m 

Mansvelder and McGehee, 2002) 


nAChR subunits . A s wit h D A neurons , onl y a mi - 

nority o f GABAergi c neuron s expres s oc 7 nAChR s 

(Wooltorton e t al, 2003) . Th e majorit y o f the VTA 

GABAergic neurons expres s nAChRs tha t mos t likely 

contain a 4 an d ß 2 subunits , whic h ar e blocke d b y 

dihydro-ß-erythroidine (DHßE ) (Mansvelde r e t al. , 

2002). 

Glutamatergic transmissio n ont o DAergi c neuron s 

is enhance d b y activatio n o f presynapti c nAChR s 

(Mansvelder an d McGehee , 2000 ; Jone s and Wonna - 

cott, 2004). Interestingly, cholinergic synaptic terminals 

are no t foun d i n clos e vicinit y to glutamatergi c termi - 

nals expressing Ot7-containing nAChR, consisten t with 

a "volume " mode of cholinergic signalin g (Descarrie s 

et al. , 1997 ; Zol i e t al. , 1999 ; Jone s an d Wonnacott , 

2004). When nicotine arrives in the VTA, i t stimulates 

glutamatergic terminals as well a s dopamine neurons , 

thereby favorin g condition s o f pre - an d postsynapti c 

paired activation and a Hebbian typ e of synaptic plasticity 

7 . Nicotine-induced pairin g resulted i n long-ter m po - 

tentiation (LTP ) o f glutamatergic input s (Mansvelde r 

and McGehee , 2000) . Nicotine als o induced LT P i n 

vivo, measured a s an increase i n AMPA/NMDA recep - 

tor ratio (Saal et al., 2003). Taken together , thes e find - 

ings suggest tha t synapti c plasticit y in the VTA may be 

induced after smoking a single cigarette and most likely 

underlies th e persistent effect s o f the dru g o n D A re - 

lease in the NAc and PFC . 

The oc7-containin g nAChR s involve d i n thi s 

mechanism ar e not desensitized b y low nicotine con - 

centrations associate d wit h tobacc o smokin g (Mans - 

velder et al, 2002; Wooltorton e t al, 2003). However, 

the non-ot7 nAChRs on GABAergic neurons underg o 

rapid desensitizatio n withi n minute s afte r th e star t of 

nicotine exposur e and, a s a consequence, reduc e th e 

inhibitory inpu t t o th e D A neuron s (Mansvelde r 

et al, 2002; Wooltorton e t al, 2003). Desensitizatio n 

of the nAChR s o n GABAergi c neurons no t only prevents 

further activation by nicotine but also precludes 

the contributio n o f thes e receptor s t o endogenou s 

cholinergic transmissio n (Mansvelde r e t al. , 2002) . 

VTA dopamine neuron s ar e thus disinhibite d by desensitization 

o f non-oc7-typ e nAChR s (Mansvelde r 

et al., 2002). Despite rapi d desensitization properties , 

genetically engineered mice lacking ß2 subunits (Pic - 

ciotto e t al. , 1998 ) o r expressin g oc 4 subunit s hyper - 

sensitive t o nicotine (Tappe r e t al. , 2004 ) hav e bee n 

used t o sho w that thes e subunit s are key in nicotin e 

addiction.


NICOTINIC ACETYLCHOLINE 

RECEPTORS AND SYNAPTI C PLASTICIT Y 

IN OTHER BRAIN AREAS 

Currently, it is unknown whether long-term modula - 

tion o f synapti c connection s b y nicotin e occur s 

within th e PFC . Dat a o n long-ter m modulatio n o f 

synaptic contact s b y nicotine anywher e i n th e neo - 

cortex are lacking. On the other hand, nicotin e doe s 

modulate synaptic plasticity in brain areas other than 

the VTA . In ra t spinal cord, the tx 7 subunit contain - 

ing nAChR affect s th e inductio n o f synaptic plasticity 

(Genzen an d McGehee, 2003). In the absence of 

nicotine, pairin g of pre- and postsynapti c activity in 

dorsal hor n neuron s induce s LT P i n som e o f th e 

neurons. Wit h nicotine , th e prevalenc e o f LTP in - 

duction i s enhanced . I n th e hippocampus , i n a 

minority o f the glutamatergi c synapse s nicotine in - 

duces LT P b y itself , bu t i n mos t o f glutamatergi c 

synapses nicotine modulate s th e inductio n o f synaptic 

plasticity (Fujii e t al, 1999 ; J i et al., 2001; Man n 

and Greenfield , 2003) . Activatio n o f postsynapti c 

nAChRs on CA 1 PNs can boos t short-term plasticity 

into LT P i n Schaffe r collatera l synapse s (J i e t al. , 

2001). Activation of nAChRs o n LCN s tha t synapse 

on PN s ca n preven t LT P i n glutamatergi c synapses 

(Ji e t al. , 2001) . Thus , timin g an d localizatio n o f 

nAChR activit y in th e hippocampu s ca n determin e 

whether LTP will occur o r not. These types of nicotinic 

mechanisms o n LTP induction ma y contribute 

to the well-known effects o f nicotine on learning and 

memory. 


Over the past 30 years, research o n cholinergic signal - 

ing in the developin g nervous system has shown that 

nAChR pla y an importan t rol e i n shapin g neurona l 

circuits. The subuni t compositio n of nAChR s expressed 

i n different brain regions changes during preand 

postnata l development ; th e subuni t mRNA profiles 

are highly dynami c durin g thi s time. Given th e 

variety of biophysical and pharmacological properties 

conferred b y differences in nAChR composition, th e 

types of signals mediated b y nAChRs ar e mos t likely 

subject to important change s throughou t normal de - 

velopment. In thi s regard, cholinergic signaling during 

gestatio n an d postnata l developmen t change s 

from facilitating cell division, cell differentiation, an d 

axon guidanc e t o fine-tunin g neurona l circuits , ac - 

tivating silen t synapses , an d modulatin g synapti c 

strength. Th e sam e hold s tru e fo r nicotine exposur e 

during development: dependin g o n the developmen - 

tal stage , the magnitud e o f damage induced an d th e 

specific targets affected b y smoking during pregnancy 

will vary. 

The finding s reviewe d i n thi s chapte r describ e 

part of what is surely a much bigger picture of the rol e 

of nAChR-signalin g i n development ; man y gap s i n 

this picture remain. Molecular and electrophysiological 

techniques, despit e their great sensitivity, still lack 

the flexibility to precisely define which combination s 

of nACh R subunit s combin e t o determin e th e ful l 

spectrum o f responses to ACh. Cholinergi c signalin g 

during developmen t i s the culminatio n o f the inte - 

grated actio n o f different nAChRs , acting i n somato - 

dendritic vs . axona l domains , transducin g eithe r 

electrical informatio n o r calciu m signaling . Futur e 

studies wil l nee d t o addres s thes e question s befor e 

sketches of cholinergic signaling in development ca n 

emerge i n more vibrant detail. 


Abbreviations 

AChE acetylcholinesteras e 

OcBgTx oc-bungarotoxi n 

AMPA a-amino-3-hydroxy-5-methyl-4-isoxazole - 

propionic aci d 

CCK cholecystokini n 

ChAT cholin e acetyltransferas e 

CNS centra l nervou s system 

DA dopamin e 

DHßE dihidro-ß-erythroidin e 



LCN loca l circuit neuron 

LTP long-ter m potentiation 

mAChR muscarini c acetylcholine recepto r 

NAc nucleu s accumbens 

nAChR nicotini c acetylcholine recepto r 



PFC prefronta l cortex

PN projectio n neuron 

PNS periphera l nervous syste m 

VIP vasoactiv e intestina l peptide 

VTA ventra l tegmental are a 


grants from th e Netherland s Roya l Academy of Arts and 

Sciences (KNAW ) an d th e Netherland s Counci l fo r 

Medical Researc h (Zon-MW ) t o H.D.M . an d fro m th e 

National Institut e of Neurological Disorder s and Strok e 

to L.W.R . 

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22 

Neural Precursors a s 

Preferential Target s for Drug Abuse: 

Long-Term Consequences 

and Latent Susceptibility to Central 

Nervous System Disorders 

The productio n o f ne w neuron s an d gli a occur s 

throughout lif e an d i s exceedingly sensitive to disruption 

by drug abuse. Ne w neural cells originate within 

specialized region s o f th e centra l nervou s syste m 

(CNS) that maintain an environmental milieu neces - 

sary for cell proliferation, and they appear to be essential 

fo r th e maintenanc e o f ste m cell s (Fig . 22-1) . 

The germina l zone s als o provide spatial cues that establish 

cel l polarit y an d organization . Th e appear - 

ance o f germinal zones i s highly ordered an d follow s 

precise spatiotempora l guideline s tha t aris e i n th e 

ventricular zon e (VZ ) an d ar e sustaine d i n th e sub - 

ventricular zone (SZ), cerebellar external granular (or 

germinal) laye r (EGL), and intrahila r zone (IHZ ) of 

the dentat e gyru s (Ramo n y Cajal , 1960 ; Boulde r 

Committee, 1970 ; Goldman , 1998 ; Alvarez-Buyll a 

and Garcia-Verdugo, 2002; Taupin an d Gage, 2002). 

Considerable evidenc e indicate s tha t drug s wit h 

abuse liabilit y disrupt developmen t b y affectin g th e 

production o f new neuron s an d glia . Moreover , th e 

effects o f drug abus e ar e no t limite d t o a particular 

Kurt F. Hauser 

Nazira El-Hage 

Shreya Buch 

Gregory N. Barnes 

Henrietta S . Bada 

James R. Pauly 

363 

cell type, germinal zone, or stage of development, bu t 

rather the y uniquel y affec t individua l neuronal an d 

glial precurso r type s withi n variou s germinal zones . 

Germinal zone s ar e targete d t o varyin g degrees b y 

most substance s wit h abus e liabilit y (Lauder e t al. , 

1998; Luo and Miller, 1998 ; Hildebrand t et al., 1999 ; 

Teuchert-Noodt e t al, 2000 ; Buzniko v et al, 2001 ; 

Duman e t al, 2001; Radley and Jacobs, 2002; Crew s 

and Nixon , 2003 ; Lin and Rosenthal , 2003 ; Gordon 

and Hen , 2004 ; se e Chapters 1 1 an d 12) . Like thei r 

progeny, neura l precursor s expres s a n inexplicabl y 

rich variet y o f recepto r an d neurotransmitte r types , 

which seemingl y contribute s t o thei r pronounce d 

sensitivity to multiple neurotransmitter s and drug s of 

abuse that mimic the endogenous neurochemica l systems. 

Drug s o f abuse tha t effec t th e genesi s o f ne w 

neural cell s likel y includ e cocaine , methampheta - 

mine, opiates, nicotine , alcohol , an d perhap s ecstasy 

(via serotonergi c actions ) (Hause r an d Mangoura , 

1998; Laude r e t al, 1998 ; Hildebrand t e t al, 1999 ; 

Teuchert-Noodt e t al, 2000 ; Buzniko v et al, 2001 ;


FIGURE 22- 1 Schemati c diagra m showin g site s o f 

neural precurso r productio n throughou t roden t on - 

togeny. Neural cells are initially produced i n the ventricular 

zon e (VZ ) an d the n i n th e subventricula r 

zone (SZ) . The cerebella r externa l granula r (o r germinal) 

laye r (EGL) i s a secondary proliferative zon e 

that arises from th e brain stem an d exclusively generates 

neurons (Hauser et al., 2003). The S Z becomes a 

major source of supragranular neurons an d macrogli a 

relatively earl y durin g maturatio n (approximatel y at 

birth in rodents and during the third trimester i n humans), 

wherea s th e intrahilala r zone (IHZ ) o f th e 

dentate gyru s is a major sit e of adult neuronogenesis. 

There i s compelling evidenc e tha t opiate s an d nico - 

tine affect germinal cell s in all four of the zones . 

Duman et al., 2001; Radley and Jacobs, 2002; Hauser 

et al. , 2003 ; Li n an d Rosenthal , 2003 ; Gordo n an d 

Hen, 2004) . Although w e will focu s o n th e effect s o f 

opiates an d nicotin e o n th e genesi s o f neural cells , 

most other drugs with abuse potential also seem t o affect 

neura l developmen t t o varying degrees throug h 

direct actions on the CNS . 

As noted earlier , it is assumed tha t drugs of abuse 

disrupt the genesi s of neurons an d gli a b y disrupting 

the endogenou s neurochemica l system s tha t nor - 

mally regulate maturatio n (Hause r et al., 2003). It is 

also important to consider that, beside s mimicry , the 

unique pharmacologica l action s o f drug s o f abus e 

may activate novel signaling events and genes , resulting 

i n qualitativel y differen t response s fro m thos e 

seen with normal neurotransmitte r systems. 

OPIATES 

Opiates, which w e will define as being derive d fro m 

the opium poppy (e.g., heroin and morphine), act by 

mimicking endogenous "opioid " peptides and prefer - 

entially activating (i-opioid receptor s (MOR) . Opiat e 

exposure can modulate the production o f neural cells 

in th e V Z an d S Z (Dodg e Mille r e t al , 1982 ; 

Reznikov et al, 1999 ; Stiene-Marti n et al, 2001) and 

the EG L (Zago n an d McLaughlin , 1983 , 1987 ) dur - 

ing maturation in experimental animal models. Mor e 

recently, opiates have been additionally implicated i n 

affecting neurogenesi s i n the adult IHZ (Eisch et al, 

2000; Mandyam et al, 2004), which suggest s that the 

ability of opiates t o disrupt the genesi s of neural pre - 

cursors i s lifelong . Th e exten t t o whic h cellula r 

changes in the germinative zone s translate s int o neu - 

rocognitive defect s i n opiate-expose d childre n i s un - 

certain. Exposur e t o opiate s i n utero , however , i s 

highly correlate d wit h lo w infan t birt h weigh t an d 

length, reduce d hea d circumference , as well as alterations 

i n cognitive , motor , and/o r behaviora l mile - 

stones during the first 3 years (Messinger et al., 2004 ; 

Shankaranetal., 2004). 

Although providin g essentia l evidenc e tha t opi - 

ates effec t CN S developmen t irrespectiv e of a variety 

o f psychosocia l factors , whole-anima l studie s 

present som e limitation s i n approachin g certai n 

mechanistic questions . For example, the endogenou s 

opioid syste m —i.e., endogenou s peptides an d MOR , 

0- (DOR ) an d K-opioi d receptors (KOR)—regulate s a 

variety of physiological functions that may secondarily 

effect developmen t (Hause r an d Mangoura , 1998) . 

Besides direc t effect s o n opioid-recepto r expressin g 

neural progenitors , opioi d exposur e ca n induc e 

changes in , fo r example , respiration , nutrition , an d 

endocrine functio n (Martin , 1983 ; Aki l e t al, 1984 ; 

Simon, 1991 ; Simo n an d Hiller , 1994 ; Roger s an d 

Peterson, 2003) , whic h ma y i n tur n affec t neura l 

development. Dissociatin g th e direc t fro m indirec t 

effects o f opiate s i n vivo ca n b e quit e challengin g 

(Hammer, 1993) . Fo r this reason , tissu e cultur e ha s 

been prove n a n invaluabl e too l i n isolatin g differen t 

neural cel l type s and studyin g the rol e o f opioids i n 

the regulatio n o f the proliferation , differentiation, o r 

death o f neura l cell s (Hause r an d Stiene-Martin , 

1993; Hauser and Mangoura, 1998) . 

Endogenous opioid s and opiate drugs have marke d 

effects o n cel l neurona l differentiatio n (Zago n an d 

McLaughlin, 1986a , 1986b ; Vernadakis e t al, 1990 ; 

Mangoura an d Dawson , 1993) . I n particular , Ham - 

mer ha s show n tha t morphine , th e majo r bioactiv e 

form o f heroin i n th e brain , inhibit s pyramidal cel l 

differentiation i n th e cerebra l cortica l pyramida l 

neurons (Hamme r e t al. , 1989 ; Ricald e an d Ham - 

mer, 1991) . Althoug h th e effect s o n differentiatio n

during development ar e significant , man y aspects of 

the effect s o f opioid on CNS maturatio n has been extensively 

reviewe d i n th e pas t (Zagon , 1987 ; Ham - 

mer an d Hauser , 1992 ; Hammer , 1993 ; Hause r an d 

Stiene-Martin, 1993 ; Hause r e t al, 2003 ; Eisch an d 

Mandyam, 2004; Slotkin , 2004). For this reason, the 

present chapter focuses on the effect s o f opiates as related 

to the genesis of neurons and glia in the CNS. 

Neuronogenesis 

Alterations i n th e endogenou s opioi d system , eithe r 

by manipulating the endogenou s opioid s themselves 

or throug h opiat e dru g treatment , caus e profoun d 

changes i n th e developin g nervou s system . Opioid s 

affect multipl e regions , includin g th e cerebellu m 

(Zagon and McLaughlin, 1986a , 1987; Hause r étal, 

1989; Lorbe r et al, 1990 ; Hauser and Stiene-Martin , 

1991) an d forebrai n region s such a s the cerebra l cortex 

an d hippocampu s (Zago n an d McLaughlin , 

1986b; Hauser et al., 1989; Hauser and Stiene-Martin , 

1991). In excess, opiates reportedly inhibit the prolifer - 

ation o f neuroblasts and/or glia (Steel e an d Johannesson, 

1975 ; Vertes et al., 1982 ; Zagon and McLaughlin , 

1986a, 1986b , 1987 ; Kornblum etal., 1987b ; Hamme r 

et al, 1989 ; Schmah l e t al., 1989 ; Lorber e t al, 1990; 

Seatriz an d Hammer , 1993) , althoug h thes e initia l 

studies relie d o n labelin g indice s wit h thymidin e 

analogs t o asses s cel l proliferation , which ma y onl y 

yield a partia l understandin g o f cel l cycl e change s 

(Mandyam e t al. , 2004) . B y contrast , continuou s 

treatment with opioid receptor antagonists during development 

generall y has th e opposit e effect s o f con - 

tinuous agonis t treatment . Continuou s opioi d 

receptor blockad e increases (1) the rat e of thymidine 

incorporation int o DNA , (2 ) the are a and volum e of 

cortical cel l layers , (3) neuronal an d glia l numbers / 

unit area, (4) dendritic size and the number of spines/ 

unit length , an d (5 ) increase d differentiatio n an d 

synaptogenesis (Zago n an d McLaughlin , 1986a , 

1986b, 1987 ; Hauser etal., 1987, 1989) . 

Many o f th e abov e developmenta l studie s suggested 

that opiate drugs could modulate the genesis of 

neurons an d gli a i n th e V Z o r SZ . Initia l studie s 

showed that opiate exposure (morphine or methadone) 

markedly reduced cel l numbers in the forebrain whe n 

the genesi s o f th e cell s originate s fro m th e VZ/S Z 

(Steele and Johannesson, 1975; Zagon and McLaugh - 

lin, 1977b , 1982b) . Initially, it was uncertain whethe r 

neural progenitor s expresse d opioi d receptor s o r 

NICOTINE, OPIATES, AND THE GENESI S OF NEURAL CELLS 36 5 

whether th e effect s o f opioids were direct. Later studies 

used radioligand binding (Kornblum et al., 1987a ; 

Leslie an d Loughlin , 1993) , i n sit u hybridizatio n 

(Leslie et al, 1998 ; Zhu et al, 1998) , and immunocy - 

tochemistry (Rezniko v e t al. , 1999 ; Stiene-Marti n 

et al, 2001 ) t o identif y MOR , DOR, and KO R on 

germinal cell s in th e VZ/SZ. Conversely, i t was later 

shown tha t chroni c opioi d recepto r blockad e in - 

creased thymidin e incorporatio n an d cel l number s 

(Zagon and McLaughlin, 1983 , 1986b, 1987) , which 

was generally the opposit e effec t o f opiate drug treatment 

Importantly, the findings from opioi d antagonist 

studies additionally provided circumstantial evidenc e 

that the endogenou s opioi d syste m i s tonically active 

and inhibits development. 

Opioid peptide s an d receptor s ar e highl y ex - 

pressed b y immatur e EG L cells , whic h consis t o f 

neuroblasts an d immatur e post-mitoti c neurons , bu t 

are no t expresse d b y mor e matur e granul e neuron s 

derived fro m thi s germinativ e laye r (Zago n e t al. , 

1985;KinneyandWhite, 1991; Osborne étal, 1993). 

This finding suggests that the expression of the opioi d 

system b y EG L cell s i s unrelate d t o th e onse t o f 

expression of adult neurochemical systems and therefore 

likel y t o b e involve d i n maturationa l processe s 

(Hauser e t al., 2003) . In th e cerebellum , manipula - 

tion o f th e opioi d syste m alter s th e proliferatio n of 

cerebellar EGL neuroblast s and/or their granule neuron 

progen y (Zago n an d McLaughlin , 1983 , 1986a, 

1987; Hause r et al, 1987, 1989; Lorber et al, 1990) , 

as well as their differentiation (Zago n an d McLaugh - 

lin, 1986a ; Hause r e t al. , 1989 , 2000) an d surviva l 

(Hauser et al., 2000). Similarly, exposure to preferential 

MOR-actin g opiate s suc h a s morphin e o r 

methadone cause s decreased thymidine incorporation 

and/or reduction s i n cel l number s (Zago n an d 

McLaughlin, 1977a , 1977c , 1982a) . I n cell s isolate d 

from th e murin e EGL , morphine exposur e signifi - 

cantly reduces neuroblast numbers an d DN A synthesis 

an d th e effect s o f morphine ca n b e prevente d b y 

the selectiv e MOR antagonis t ß-funaltrexamine, findings 

suggesting that MOR regulate s cell proliferation. 

By contrast , DO R agonist s selectively reduce neurit e 

outgrowth fro m granul e neuron s a s they differentiat e 

from EG L precursors , while morphin e ha s n o effec t 

on this measure (Häuser et al., 2000). This action suggests 

that individua l opioi d recepto r type s may regu - 

late different aspect s of development i n the same cell. 

The deat h o f neuronal precursor s is an occasion - 

ally reporte d consequenc e o f exposur e t o opiate s


alone (Merine y et al. , 1985) . Opioid s ca n modulat e 

the effect s o f existing apoptotic signal s (especially in 

immune cell s [Singha l e t al., 2001 , 2002]) , and opiates 

may be co-factor s i n regulating neural cell deat h 

(Gurwell e t al, 2001 ; Khurdaya n et al, 2004) . Cel l 

death i s evident in cultured Purkinj e cell s with more 

chronic exposur e (Hauser et al., 1994) . Purkinje cell 

losses hav e bee n reporte d i n chroni c heroi n abuser s 

who are HIV seronegative (Oehmichen et al., 1996) . 

Although the mechanisms underlying opiate-induced 

cell death ar e incompletely understood an d likely differ 

amon g cell types , opiate-induce d modulatio n o f 

cell death has been proposed to involve phosphoinos - 

itol 3-phosphokinas e an d extracellula r signa l regu - 

lated kinase (ERK) (Polakiewicz etal, 1998). Singhal 

and coworker s report tha t p3 8 mitogen-activate d pro - 

tein kinase (MAPK), p53, and caspase s 3 and 8 medi - 

ate morphine-induced deat h i n macrophages (Singha l 

et al, 2002), whereas morphine-induced T cell losses 

involve c-jun-N-terminal kinase activation and reduc - 

tions i n ER K phosphorylation (Singha l e t al., 2001) . 

By contrast, cell death i s not seen with concentrations 

of morphine exceedin g 1 jim in cultured mous e EG L 

cells (Hause r e t al. , 2000 ) o r i n astrocyte s (Gurwell 

and Hauser , 1993) . Thus , i n some instances , opiate s 

may affec t cel l surviva l i n addition to their effect s o n 

proliferation, although this is not a consistent observation. 

Gliogenesis 

There are considerable indication s that glial-restricted 

precursors (GRPs) , a s wel l a s thei r astroglia l an d 

oligodendroglial progeny, are a direct target of opiates 

during maturation . Mos t glia l type s in th e forebrai n 

arise from the S Z and gliogenesis typically follows th e 

production o f neuron s i n a particula r brai n regio n 

(Fig. 22-2). 

Many hav e propose d tha t astrogli a ar e target s for 

drugs wit h abus e liabilit y (Eriksso n e t al. , 1991 ; 

Stiene-Martin an d Hauser , 1991 ; Beitner-Johnson e t 

al, 1993 ; Hauser and Mangoura, 1998 ; Fattor e et al, 

2002; Belchev a e t al, 2003) . Astroglial growt h i s inhibited 

b y opioid s i n cell cultur e an d i n vivo 

(Schmahl e t al , 1989 ; Stiene-Marti n an d Hauser , 

1990, 1991 ; Hauser and Stiene-Martin , 1991 ; Stiene - 

Martin e t al, 1991 , 2001 ; Zago n an d McLaughlin , 

1991; Hause r e t al. , 1996 ; Hause r an d Mangoura , 

1998). Astrogli a ca n expres s MOR , DOR , and/o r 

FIGURE 22-2 Presenc e o f opioid and nicotinic acetylcholine 

receptor s (nAChRs ) o n neurona l an d glia l 

precursors, fi- , Ô- , an d K-opioi d receptor s (MOR , 

DOR, an d KOR , respectively ) ar e variably expresse d 

(±) o n neurona l an d astroglia l precursors , an d 

nAChRs ar e variabl y expressed b y neuronal precur - 

sors (±). IHZ, intrahilar zone; O2A, oligodendrocytetype 

2 astrocyte progenitor; SZ, subventricular zone. 

KOR (Eriksson et al, 1990 , 1991 ; Stiene-Marti n an d 

Hauser, 1990 , 1991 ; Ruzick a e t al., 1995 ; Gurwell e t 

al, 1996 ; Hause r e t al. , 1996 ; Stiene-Marti n e t al, 

1998, 2001 ; Thorlin et al., 1998) , and opioid recepto r 

expression is developmentally regulated and/or differ s 

among brai n region s (Hause r an d Stiene-Martin , 

1991; Eriksso n e t al , 1992 ; Ruzick a e t al , 1994 , 

1995; Gurwell e t al, 1996 ; Stiene-Marti n e t al, 1998 ; 

Thorlin e t al, 1999) . Activatio n o f MOR, KOR , o r 

DOR cause s reduction s i n cel l proliferatio n an d in - 

creased cellula r hypertroph y (Stiene-Marti n an d 

Hauser, 1991 ; Gurwel l e t al, 1996 ; Hause r e t al, 

1996). Th e inhibitio n o f cel l proliferatio n an d as - 

troglial hypertroph y are mediate d b y opioid-induce d 

increases i n intracellula r concentratio n o f Ca 2+ 

([Ca 2+ ]i) (Gurwel l e t al, 1996 ; Hause r e t al, 1996) . 

The "reactive " cellula r hypertroph y i s accompanie d 

by increased glial fibrillary acidic protein (GFAP ) immunoreactivity 

and aspect s ma y mimic reactiv e gliosis 

in vivo (Hauser et al, 1996 , 1998) . 

Unlike in neurons and astroglia, where MOR acti - 

vation inhibits cell replication, in immature oligoden - 

droglia, MO R activatio n i s mitogenic (Knap p et al. , 

1998). By contrast to MOR, KO R is expressed in more 

mature, nondividin g oligodendroglia. KOR blockad e 

increases th e deat h o f culture d oligodendrocytes ,

inferring tha t KO R couple s t o enhance d surviva l i n 

this cell typ e (Knapp et al., 2001). Similar to granule 

neurons, differen t aspects o f oligodendroglia l devel - 

opment appea r t o b e independentl y regulate d b y 

MOR an d KOR . As mentioned, unlik e granule neu - 

rons and their EGL precursors , which do not express 

KOR in vitro (Hauser et al, 2000), MOR activation is 

mitogenic i n oligodendrogli a (Knap p e t al. , 1998) . 

Collectively, the findings in neurons and gli a suggest 

that opioi d effect s ar e highly complex—affecting th e 

maturation of each cel l type differently . 

Adult hippocampa l progenitor s (AHPs ) fro m th e 

rat IH Z expres s ß-endorphi n ( a post-translationa l 

product o f the proopiomelanocorti n gene ) (Persso n 

et al., 2003a). ß-endorphin stimulate s the maturatio n 

and perhap s cel l fat e decision s o f IHZ cell s through 

autocrine/paracrine mechanisms o f action (Persso n et 

al, 20 0 3a). Interestingly , AHP s expres s MO R an d 

DOR, an d blockad e o f these receptor s b y naloxone 

increases the AHPs, resultin g in increases in neurob - 

last replicatio n wit h apparen t compensator y reduc - 

tions i n th e genesi s o f astrogli a an d oligodendrogli a 

(Persson et al., 2003a, 2003b). These findings suggest 

that, i n additio n t o modulating th e proliferatio n and 

death o f neuronal an d glia l precursors , opioids ma y 

influence cel l fat e decision s o f bi- o r multipotentia l 

neural cell precursors. 

Importantly, opioids can modulate basic fibroblast 

growth factor (bFGF ) and/or epidermal growt h facto r 

(EGF) activatio n of ERK in C6 glioma cells or in primary 

astrocyte s (Boh n e t al. , 2000 ; Belchev a e t al. , 

2002, 2003). The proliferativ e effect s o f ß-endorphin 

in adul t hippocampa l progenitor s wa s mediate d 

through a signalin g pathwa y involvin g PI3-kinase , 

[Ca 2+ ]j, and MAPK activation (Persson et al, 2003a), 

which overlap s wit h event s associate d wit h MOR - 

mediated cel l deat h i n Chines e hamste r ovar y cells 

(Polakiewiczetal, 1998) . 

NICOTINE 

In adults, acetylcholine is a significant neurotransmitter 

i n severa l neura l pathway s (Rand , 1989 ; Luetj e 

et al, 1990 ; Heineman n e t al., 1991a ; Albuquerqu e 

et al., 1995) . During development, acetylcholin e (and 

perhaps relate d substances ) additionall y provide s 

trophic suppor t to guide th e maturatio n an d surviva l 

of incipien t cholinergi c neuron s (Zahalk a e t al. , 


1992; Role and Berg , 1996) . Nicotin e i s known to alter 

adul t neura l function . I n th e maturin g nervou s 

system, ther e i s considerable evidenc e tha t nicotin e 

exposure (as a component of cigarette smoke) disrupts 

the norma l interaction s betwee n acetylcholin e an d 

the cellula r targets that express subtypes of nicotini c 

acetylcholine receptors (nAChRs) . 

Cigarette smokin g durin g pregnanc y ca n hav e 

multiple direc t an d indirec t effect s o n feta l develop - 

ment an d i s strongly associate d wit h developmenta l 

and behaviora l abnormalitie s i n newborn s (Econo - 

mides an d Braithwaite , 1994 ; Mclntos h e t al, 1995 ; 

Shu et al., 1995; Lambers and Clark, 1996) . Epidemi - 

ological studie s sugges t that lo w infant birt h weight , 

delayed feta l development , an d prematur e birth s are 

closely correlate d wit h smokin g (Opanashu k e t al. , 

2001). Neurobehaviora l developmen t i s also altered . 

In addition , children bor n t o smoking mothers hav e 

an increase d incidence o f attention defici t hyperactivity 

disorder (Mclntosh e t al. , 1995) . Smok e exposur e 

may als o increas e th e incidenc e o f sudde n infan t 

death syndrom e (Slotkin et al., 1995) . Although ciga - 

rette smoke contains over 4000 chemical compounds , 

many studies have show n tha t nicotin e i s the princi - 

pal insulting agent in development . 

In animal models , prenata l exposur e to tobacco o r 

nicotine disrupt s somati c (Paulso n e t al. , 1991 ) an d 

CNS developmen t an d cause s defects in morphologi - 

cal, neurochemical, an d behavioral indices (Slotkin et 

al., 1987, 1993 ; Fuxe étal, 1989; Paulson étal., 1994a, 

1994b; Penningto n e t al, 1994 ; Witsch i e t al, 1994 ; 

Court e t al, 1995 , 1997 ; Ro y et al., 1998 ; Paul y et al, 

2004). Interestingly, nicotine ca n affec t brai n develop - 

ment in the absence o f overt changes i n body weight, 

which suggest s that effec t o f nicotine o n th e brai n is 

direct (Zahalka et al, 1992) . Additional evidence suggests 

tha t th e effect s o f nicotin e ar e gender-specifi c 

(Pauly e t al, 2004) . Although assessin g the effec t o f 

in uter o exposur e t o cigarett e smok e i s potentiall y 

confounded by reduced maternal-placental-feta l circulation 

(Birnbau m e t al. , 1994 ) o r amin o aci d up - 

take (Sastry , 1991), studies in chic k embryo s suggest 

that nicotin e retard s neurobehaviora l developmen t 

(Pennington e t al. , 1994) ; studie s i n vitr o similarl y 

suggest tha t nicotin e ca n directl y affec t developin g 

neural cells (Opanashuk et al., 2001). Perinatal expo - 

sure t o nicotin e cause s lastin g change s eviden t i n 

adult brains (Nordberg et al, 1991 ; Miao et al, 1998 ; 

Eriksson et al, 2000).


Epidemiological and/o r experimenta l studie s o f 

Parkinson an d Alzheime r disease s hav e show n tha t 

nicotine alon e (o r fro m cigarett e smoking , chewin g 

gum, and/or dermal patches) ma y have neuroprotec - 

tive properties (Baron, 1986; Sershen et al., 1987 ; van 

Duijn and Hofman, 1991 ; Jones et al., 1992 ; Ishikawa 

and Miyatake , 1993) . Som e interes t originate s fro m 

studies showing that ther e i s a selective reductio n i n 

neuronal nAChRs , i n compariso n t o age-matche d 

controls, i n severa l neurodegenerative disease s (Perry 

et al. , 2002) . Acute exposur e to nACh R agonist s en - 

hances som e measure s o f cognitiv e function , an d 

chronic exposur e t o nicotin e agonist s paradoxically 

increases th e numbe r of neuronal nAChR s (Jone s e t 

al, 1992 ; Wilson e t al., 1995) . Nicotine ca n protec t 

against ß-amyloi d toxicit y i n vitr o (Kihar a e t al. , 

1997), prompting a n interes t in attempting to understand 

interaction s betwee n nAChR s an d th e patho - 

physiology of these neurodegenerative diseases. In th e 

aging nervous system, nicotine appear s to be trophi c 

in neurons that express nAChRs and perhaps second - 

arily in other non-nAChR-expressing cells . Although 

the trophic effects o f nicotine in aging may be neuro - 

protective, th e developmenta l trophi c action s o f 

nicotine may be detrimental to the fetus , and i t is important 

to note that an inappropriate blockade of normal 

programme d cel l deat h o r a n abnormall y hig h 

rate of cell proliferation is also likely to be detrimen - 

tal (Opanashuk et al, 2001). 

Nicotine ha s lon g bee n propose d t o modulat e 

neural developmen t b y influencing the proliferation 

and/or surviva l o f neura l precursor s (Slotki n e t al. , 

1987, 1993 ; Slotkin, 2004). Recent findings that perinatal 

exposur e t o nicotin e ha s long-lastin g conse - 

quences o n behavio r in rats , combined wit h clinical 

observations of low infant birth weight , the potentia l 

for psychiatric problems, and predilection toward substance 

abus e (Lamber s and Clark , 1996 ; Erns t et al., 

2001), underscor e th e importanc e o f examinin g 

effects o f nicotine i n development. Targete d deletio n 

of specifi c nACh R subuni t genes , includin g oc3 , ß2 , 

and ß 4 subunits, can b e lethal earl y during development, 

wherea s deletio n o f man y othe r nACh R 

subunits significantl y modifie s adul t behavior s 

(Cordero-Erausquin e t al. , 2000) . Finally , nicotin e 

decreases neurogenesis in the IH Z of adult male rats 

(Abrous e t al., 2002), a findin g suggesting tha t nico - 

tine's ability to influence the production o f new neu - 

ral cells is lifelong. 


The appearanc e o f cholinergi c syntheti c enzyme s 

and multipl e nACh R subtype s in the germinal zones 

of th e developin g CN S suggest s tha t acetylcholin e 

might potentiall y influenc e CNS maturatio n i n th e 

VZ/SZ an d EG L (Goul d an d Butcher , 1987 ; Clo s 

et al , 1989 ; Perr y e t al, 1993 ; Cour t e t al, 1995 ; 

Atluri e t al, 2001 ; Opanashu k e t al, 2001 ; Hause r 

et al, 2003; Gai et al, 2004). For example, thé matu - 

ration o f cholinergi c system s i n th e cerebellu m 

(Brooksbank et al, 1989 ; Perr y et al, 1993 ; Court et 

al., 1995 ; Jaarsma et al., 1997 ) coincides with critical 

periods o f neurogenesi s i n thi s brai n regio n i n ro - 

dents (Mial e an d Sidman , 1961 ; Altman , 1972a , 

1972b). The mechanism s b y which nicotin e modu - 

lates maturatio n ar e likel y to be comple x an d diffe r 

among cel l types . nAChR s ar e member s o f th e 

ligand-gated ion channel recepto r superfamily. Binding 

to nAChRs induce s a conformational chang e i n 

an intrinsic ion channel, whic h increase s Na + influ x 

(Heinemann e t al. , 1991b ; Dan i an d Heinemann , 

1996). Althoug h nicotin e ma y acutely activate cells , 

desensitization and/o r possibl y permanen t inactiva - 

tion follow s th e activatio n o f th e recepto r (Collin s 

and Marks , 1996) . I t has been show n that individual 

neurons ca n expres s multiple nACh R subtype s (i.e., 

differing subuni t composition) , wit h eac h servin g 

unique function s (Conro y an d Berg , 1995) . I t ha s 

long been known tha t nicotin e induce s inwar d Ca 2+ 

currents, especiall y throug h oc 7 nACh R subunit - 

containing complexes, as well as through som e othe r 

nAChR subtype s (Dajas-Bailado r an d Wonnacott , 

2004). [Ca^J i ha s well-documente d effect s o n cel l 

proliferation, differentiation , and survival . Interest - 

ingly, th e expressio n o f som e nicotini c recepto r 

types ma y b e linke d t o ke y regulator s with neura l 

cell development. So x 10, an important transcription 

regulator o f neura l maturatio n an d cel l fat e deci - 

sions, activate s ß 4 an d oc 3 subunit s i n a cel l 

type-specific manner , and So x 10 directly affects reg - 

ulatory regio n o f the ß 4 nACh R subuni t promote r 

(Liu et al, 1999) . 

Nicotine increase s the rat e of proliferation o f neuroblasts 

in the EGL (Opanashu k et al, 2001), which 

are predecessor s o f granule neurons . When isolate d 

EGL precursor s are continuousl y expose d fo r 7 days 

to selectiv e oc3/oc 4 (epibatidine ) o r ct 4 (cytisine ) 

nAChR agonists or to partial agonists, epibatidine, but

not eytisine , concentration-dependen t increase s i n 

DNA synthesis and content are produced (Opanashu k 

et al, 2001) . Thi s resul t indicate s tha t Ot 3 iiACh R 

stimulation is mitogenic to cerebellar granule neuroblasts. 

Moreover , significan t effect s wer e significantl y 

attenuated b y concurren t administratio n o f th e 

iiAChR antagonis t dihydro-ß-erythroidin e (DHßE) , 

suggesting th e involvemen t o f specifi c nAChRs . I n 

summary, thes e dat a provid e nove l evidenc e tha t 

nAChRs directl y affec t th e developmen t o f granul e 

cell precursor s an d furthe r sugges t tha t th e effect s 

are mediate d throug h specifi c oc 3 nACh R subtype s 

(Opanashuk e t al , 2001) . Importantly , oc 7 nACh R 

subunits ar e expresse d b y differentiatin g granul e 

neurons, thu s othe r nACh R subtype s ma y regulat e 

alternative aspects o f development suc h a s differen - 

tiation, includin g synaptogenesi s and/o r surviva l 

(Didier e t al. , 1995 ; Fucil e e t al, 2004) . Th e rol e 

of nicotin e i n th e EG L o f the developin g cerebel - 

lum ha s been reviewe d in detai l elsewher e (Hauser 

et al., 2003). 

Importantly, beside s regulatin g cel l proliferation , 

nicotine can modulate programmed cel l death (Ren - 

shaw et al, 1993 ; Roy et al, 1998 ; Opanashu k e t al, 

2001; Prendergas t e t al, 2001 ; Abrous et al, 2002). 

Chronic nicotin e exposur e i s neuroprotectiv e i n 

organotypic culture s o f the ra t hippocampu s b y up - 

regulating calbindi n expression , whic h buffer s toxi c 

increases i n intracellula r Ca 2+ (Prendergas t e t al. , 

2001) an d modulate s nAChR-specifi c membran e 

currents in neural stem cell s (Cai et al., 2004). Similarly, 

i n culture d cerebella r EG L neuroblast s 

(Opanashuk e t al. , 2001) or their progen y (Fucile e t 

al., 2004), chronic activatio n o f oc3 nAChRs prevent s 

cell deat h an d th e activatio n o f oc 7 nACh R subunit s 

protects spina l cor d neuron s agains t arachidoni c 

acid-induced deat h (Garrid o e t al. , 2003) . By contrast, 

th e activatio n o f


progenitors in the S Z and triggers nAChR expression 

in skeleta l muscle an d neura l cells (Vartania n et al., 

1994). ARIA exposure causes O2A progenitors to develop 

along an oligodendroglial fat e in vitro, althoug h 

the maturationa l change s resul t fro m neureguli n sig - 

naling and i t is unclear whether ARIA drives nAChR 

expression i n oligodendroglia. Taken together , thes e 

findings suggest tha t nicotin e ma y hav e a profound 

influence o n th e maturatio n o f GRP s an d direc t 

them t o a n oligodendroglia l fate . Importantly , un - 

timely exposur e t o nicotin e ma y disrupt gliogenesi s 

and, in particular, the production of oligodendroglia. 

DRUG INTERACTIONS WITH 

DISEASE OR TRAUMA 

Considerable evidenc e her e an d elsewher e suggest s 

that drug abuse can influence neurogenesis and gliogenesis 

an d affec t long-ter m organizationa l change s 

in the brain. On th e basi s of this insight, we question 

whether th e maladaptive , organizationa l change s 

might predispose the CNS t o failure when confronte d 

with non-drug-related insults . A remarkable aspec t of 

the CN S i s that despite significan t insults during critical 

developmenta l periods , opposin g neuroadaptiv e 

processes ar e capabl e o f considerabl e recovery . Th e 

neuroplastic respons e to perinatal drug exposure may 

itself be maladaptive when the CNS i s challenged wit h 

other insult s unrelate d t o dru g abuse . Fo r example , 

does exposure to nicotine or opiates before disease onset 

or concurrently increase the risk and/or severity of 

diseases not typically attributed to deficits i n the gene - 

sis o f ne w cells , suc h a s Alzheime r o r Parkinso n 

disease, neurological acquired immunodeficiency syndrome 

(AIDS) , o r neurotrauma? As with dru g abuse, 

disruptions i n adul t neurogenesi s hav e bee n impli - 

cated i n Alzheime r diseas e (Haughe y e t al. , 2002) , 

traumatic brai n injur y (Brau n e t al. , 2002 ; Chiru - 

mamilla e t al. , 2002) , neuropsychiatrie disorders (Jacobs, 

2002 ; Kempermann , 2002 ; McEwen , 2002) , 

and us e of psychotropic drugs (Duman e t al., 2001) , 

as well as in schizophreni a (Corfa s et al. , 2004 ; Ste - 

fansson e t al., 2004), depression (Jacob s et al., 2000) , 

and seizur e disorders (Parent and Lowenstein , 2002 ; 

Ribak an d Dashtipour , 2002) . Moreover , althoug h 

neural progenitor s may be vulnerabl e t o a single insult, 

whe n multipl e insult s ar e combined , suc h a s 

drug abuse and disease, this combination ma y exacerbate 

losses in neural progenitors . 

Assuming dru g abus e modulate s CN S organiza - 

tion and plasticity, might the developmental effect s of 

opiates and/o r nicotin e exposur e o n CN S organiza - 

tion b e reveale d o r exaggerate d b y aging, disease , o r 

environmental Stressors ? Although thi s i s an impor - 

tant issue , i t i s difficul t t o addres s because o f chal - 

lenges i n modeling drug abuse an d CN S disease s in 

the laboratory. 

HIV-Drug Interaction s 

To address whether dru g exposur e might exacerbat e 

CNS disorders , we have been explorin g opiate-HIV 

interactions i n several murine and huma n model s of 

HIV. There are several key reasons for choosing HI V 

as a diseas e t o explor e dru g abuse-diseas e interac - 

tions. First , dru g abuse an d HI V are interlinke d epi - 

demics. I n th e Unite d States , AID S i s no w largel y 

spread through injectio n dru g use or through th e exchange 

o f sex for drugs (Nath et al, 2000, 2002). Second, 

opiat e drug s exacerbat e th e pathogenesi s an d 

neurological complication s o f HIV (Bell et al., 1998 ; 

Nath e t al., 2002). Opiates likel y exacerbate th e neu - 

ropathogenesis o f HIV indirectl y b y modulating im - 

mune functio n (Peterso n e t al. , 1998 ; Roger s an d 

Peterson, 2003 ; Donahoe, 2004) and directly by modulating 

th e intrinsi c respons e o f neuron s an d gli a 

to HI V and/o r vira l product s (Gurwel l e t al. , 2001 ; 

Khurdayan et al, 2004; Ei-Hage e t al, 2005). 

The HIV gene regulatory protein Tat has been implicated 

a s a mediator o f HIV-induced neurotoxicity . 

Tat i s a transactivating , nonstructura l vira l nuclea r 

regulatory protein compose d o f 10 1 amino acid s en - 

coded b y two exon s (Atwoo d e t al. , 1993) . Ta t i s released 

by infected lymphoid cells (Ensoli et al, 1993) 

and glia (Tardieu et al, 1992). Tat is intrinsically cytotoxic 

t o neurons (Sabatie r e t al, 1991 ; Magnuson et 

al, 1995 ; Week s e t al. , 1995 ; Marago s e t al, 2002 ; 

Singh e t al., 2004). Extracellular Tat is neurotoxic, i n 

part through interaction s wit h excitator y amino aci d 

receptors (Magnuso n e t al, 1995 ; Nath e t al., 1996 ; 

Haughey an d Mattson , 2002) , whic h ar e accompa - 

nied b y increase s i n [Ca 2+ ]j an d reactiv e oxyge n 

species (Nat h e t al , 1996 ; Kruma n e t al , 1998 ; 

Bonavia et al, 2001). 


Although HIV- 1 infectio n result s i n neurodegenera - 

tion, the neurons themselve s ar e not directly infected.

Instead, HIV-1 affects microgli a and astroglia , subsequently 

contributing t o neurodegeneration throug h 

inflammatory signalin g and th e releas e o f toxic viral 

products b y glia (Brack-Werner , 1999 ; Nath , 1999 ; 

Kaul e t al, 2001; Garden , 2002) . A novel finding is 

that multipotentia l neura l progenitor s ca n becom e 

infected an d serv e as a reservoir for HIV-1 (Lawrence 

et al., 2004). Interestingly, if the infecte d neura l pro - 

genitors ar e allowe d t o differentiat e towar d a n as - 

troglial phenotype , ther e ar e marke d increase s i n 

viral production . Thi s resul t suggest s that neurona l 

progenitors ca n becom e infected , bu t commitmen t 

to a neuronal fat e i s incompatible wit h viral expres - 

sion. Beside s bein g a sit e fo r viral infection , neura l 

precursors expres s severa l ke y chemokine receptor s 

that are co-factor s involved in HI V binding, fusion , 

and infectio n (Lazarin i e t al., 2000 ; Ka o and Price , 

2004; Pen g e t al. , 2004) , includin g CXCR 4 an d 

CCR5 (Pen g e t al, 2004) . I n addition , immatur e 

neurons expres s majo r histocompatibilit y comple x 

(MHC) clas s I and other key immune signalin g molecules 

that blur distinctions between neuroplasticit y 

and neuropatholog y (Boulange r an d Shatz , 2004) . 

Interestingly, bot h monotropi c an d T-tropi c strain s 

of HIV, respectively , inhibit the proliferation of neural 

progenitors through CCR5- or CXCR4-mediate d 

activation o f ER K (Krathwoh l an d Kaiser , 2004) . 

Although ther e i s evidence that neuroblasts are a target 

for HIV, no studies have examined the combine d 

effects o f opiates and HI V on neurogenesis. 

Gliogenesis 

Unlike neurona l precursors , preliminar y i n vitro evidence 

suggest s tha t opiate s ca n exacerbat e th e cyto - 

toxic effect s o f HI V i n glia l precursor s (Khurdaya n 

et al., 2004). O n th e basis of findings that neural pro - 

genitors ar e target s o f HIV , w e hav e bee n assessin g 

whether drug abuse might further disrup t the action s 

of HIV i n neural progenitors . Interestingly , glia l pro - 

genitors appea r t o b e especiall y vulnerabl e t o th e 

combined effec t o f opiates an d HIV , resultin g in in - 

creases i n activate d caspase- 3 an d increase d cel l 

death as assessed by a failure of cells to exclude ethidium 

monoazide (Khurdayan et al., 2004). Most of the 

dying cells are glia l precursors having characteristics 

of O2 A glia l progenitor s (Raf f e t al. , 1983 ; Fulto n 

et al., 1992) , whereas there i s a tendency fo r some im - 

mature oligodendrocyte s an d immatur e astrocyte s t o 

be preferentially lost. A vast majority o f cultured O2 A 


progenitors woul d hav e develope d int o oligoden - 

droglia wit h furthe r maturation , thus , i t appears tha t 

cells committe d t o a n oligodendroglia l fat e ma y b e 

preferentially vulnerabl e t o combine d opiate-HIV- 1 

toxicity (Khurdaya n et al. , 2004) . Interestingly , ther e 

tended t o b e greate r number s o f dying oligodendro - 

cytes wit h combine d morphine-Ta t exposure , al - 

though thi s tren d wa s no t statisticall y significan t a t 

96 hr. Since morphine an d Tat in combination preferentially 

kil l O2 A cells , significan t oligodendrocyt e 

losses migh t becom e apparen t wit h exposur e tha t i s 

more prolonged. It can be predicted that gliogenesis is 

affected an d tha t oligodendroglia l progenitor s ar e especially 

vulnerabl e i n HIV-infecte d individual s wh o 

abuse opiates. 

The consequence s o f losin g glia l precursor s ar e 

uncertain. Fate d glia l precursor s ma y have lifespan s 

lasting years, so that deficits in gliogenesis would only 

become eviden t with time. A tentative notion i s that a 

sustained destruction o f glial precursors wit h chroni c 

opiate abus e i n HIV-infecte d individual s and subse - 

quent los s in total glia l numbers migh t contribut e t o 

accelerated neurocognitiv e defects . Thus , chroni c 

opiate abuse may have debilitating effects o n the longterm 

stabilit y of glial populations an d CN S functio n 

in HIV-infected individuals. 


Collectively, the abov e findings suggest that glia l pre - 

cursors are novel targets for HIV and opiate abuse, an d 

disruptions in the genesi s and fat e o f new neural cells 

may contribut e t o th e pathogenesi s o f neuro-AIDS . 

The exten t t o which finding s i n opiat e an d HI V Tat - 

treated glia can be generalized to other drugs of abuse 

or disease s is uncertain, however , considerin g th e ap - 

parent susceptibilit y o f neura l progenitor s t o sub - 

stances with abuse liability and pathologi c insults , it is 

likely that precursors will be affected. Furthermore , assuming 

the los s of precursors inevitably contributes t o 

the pathology of the disease, the prognosis for slow, progressive 

cognitive decline may be assured. 

Abbreviations 

AHP adul t hippocampal progenito r 

AIDS acquire d immunodeficiency syndrome 

ARIA acetylcholin e receptor-inducing protei n


bFGF basi c fibroblast growth facto r 

[Ca 2+ ]A intracellula r concentration o f Ca 2+ 

CNS centra l nervou s syste m 

DHßE dihydro-ß-erythroidin e 

EGF epiderma l growth facto r 

DOR 0-opioi d receptor s 

EGL externa l granula r layer of the cerebellum 

ERK extracellula r signa l regulated kinas e 


GRP glial-restricte d precursor 


KOR K-opioi d receptor s 

MAPK mitogen-activate d protei n kinas e 

MHC majo r histocompatibity complex 

MOR ji-opioi d receptors 

nAChR nicotini c acetylcholine receptor 

SZ subventricula r zon e 



the Nationa l Institut e o f Dru g Abus e (KFH) , th e Na - 

tional Institut e o f Child Healt h an d Huma n Develop - 

ment (HSB), and the National Institut e o f Neurologica l 

Disorders and Strok e (JRP). 

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exposure: differential effect s o n choline acetyltrans - 

ferase activit y an d [ 3 H]hemicholinium-3 binding. 

Neurotoxicol Teratol 14:375-382 . 

Zhu Y , Hsu MS , Pinta r J E (1998 ) Developmenta l ex - 

pression o f the \Ji, K, and 0 opioid receptor mRNA s 

in mouse. J Neurosci 18:2538-2549 .

23 

Nicotinic Receptor Regulation of 

Developing Catecholamine System s 

Central catecholamine systems , consisting of dopaminergic, 

noradrenergic, and adrenergi c cell groups , serve 

numerous integrativ e neura l function s an d criticall y 

regulate action , emotion , motivation , an d cognitio n 

(Berridge an d Robinson , 1998 ; Berridg e and Water - 

house, 2003 ; Nieoullon and Coquerel , 2003) . These 

neural pathway s ar e functiona l a t a n earl y stage o f 

brain developmen t an d hav e importan t role s in neu - 

ronal maturatio n an d behavior s tha t ar e critica l for 

neonatal survival (Levitt et al., 1997 ; Sullivan , 2003). 

Dysregulation of these systems has been implicated in 

numerous diseas e states , particularly those i n whic h 

there ar e cognitive , emotional , and/or moto r deficit s 

(Aston-Jones e t al. , 2000 ; Nieoullo n an d Coquerel , 

2003). 

Numerous clinical studies have shown that smoking 

during pregnancy can lead to long-standing neurobehavioral 

deficit s in th e offsprin g tha t may result 

from catecholaminergi c dysfunction , includin g at - 

tention defici t hyperactivit y disorde r (ADHD) , con - 

duct disorder, cognitive deficits, an d substance abuse 

Frances M. Leslie 

Layla Azam 

Kathy Gallardo 

Kathryn O'Leary 

Ryan Franke 

Shahrdad Lotfipou r 

381 

(Ernst et al., 2001; Fried and Watkinson, 2001; Buka 

et al, 2003 ; Kahn e t al., 2003 ; Thapar e t al, 2003). 

Whereas tobacc o smok e contain s ove r 400 0 con - 

stituents, many of which may be harmful to the developing 

fetus , anima l studie s hav e focuse d o n an d 

confirmed a neuroteratogeni c rol e fo r nicotin e 

(Slotkin, 1998) . Gestationa l nicotin e exposur e in ro - 

dents result s i n cognitiv e impairment s (Levi n e t al. , 

1996), enhance d spontaneou s locomoto r activit y 

(Fung, 1988 ; Newman et al., 1999 ; Pauly et al, 2004), 

and increase d nicotine-induced locomotio n (Shack a 

et al. , 1997) . Neurochemica l studie s provide furthe r 

evidence o f a n advers e effec t o f prenata l nicotin e 

exposure o n centra l catecholamin e developmen t 

(Ribary and Lichtensteiger, 1989 ; Slotkin , 1998 ; Mu - 

neoka e t al, 1999) , wit h norepinephrine (NE ) more 

highly affected tha n dopamine (DA). Route of administration 

is a critical variable in such studies , however , 

with continuou s administratio n producing result s opposite 

to those of intermittent injection (Navarro et al., 

1988). Such findings have led Slotkin and colleague s


to propos e tha t nicotine-induce d hypoxi a ma y pro - 

duce man y of the observe d deficit s i n catecholamine 

development (Slotkin, 1998) . 

Nicotine bind s to specfic cholinergi c receptors , A 

nicotinic acetylcholine receptor (nAChR ) is a ligandgated 

catio n channel . Eac h recepto r consist s o f five 

subunit proteins surrounding a channel por e tha t mediate 

man y o f the biologica l effect s o f acetylcholin e 

(ACh) (Leonar d an d Bertrand , 2001 ; Picciotto et al., 

2001). Radioligan d bindin g an d mRN A expressio n 

studies have shown substantial expression o f nAChRs 

in feta l huma n an d roden t brai n (Naef f e t al, 1992 ; 

Zoli e t al , 1995 ; Ospin a e t al. , 1998 ; Hellstrom - 

Lindahl an d Court, 2000) . Therefore, thes e receptor s 

may pla y a critica l physiologica l rol e i n regulatin g 

brain development (Rol e and Berg , 1996 ; Broid e and 

Leslie, 1999 ; Metherat e an d Hsieh , 2004 ) an d ma y 

also serv e a s a targe t fo r th e neurotoxi c action s o f 

nicotine on developing brain. As outlined i n the pres - 

ent chapter , a combination o f biochemical, anatomi - 

cal, an d behaviora l approache s hav e bee n use d t o 

evaluate th e hypothesi s tha t functiona l nAChR s ar e 

expressed o n catecholamin e neuron s durin g critica l 

phases of brain development . 

DOPAMINE NEURONS 

Adult 

The forebrai n dopaminergi c syste m arise s fro m tw o 

main cel l group s locate d i n th e midbrain , th e sub - 

stantial nigra (SN) and ventral tegmental area (VTA). 

The nigrostriata l system , arisin g fro m th e SN , con - 

trols sensorimoto r integratio n an d moto r output . 

Mesolimbic an d mesocortica l projections , arisin g 

from th e VTA , regulat e reward and motivate d behav - 

ior. A wide variet y o f nACh R subunits , oc3-ot 7 an d 

ß2-ß4, are expressed on DAergic neurons both in the 

SN an d VT A o f adul t rodent s (Klin k e t al, 2001 ; 

Azam et al., 2002), giving rise to a rich pharmacologi - 

cal complexity . At least two classes of nAChR are expressed 

o n DAergi c terminal s withi n th e striatum , 

which ar e discriminate d o n th e basi s of their affinit y 

for th e antagonis t a-CtxMI I (Kula k e t al , 1997 ; 

Kaiser e t al, 1998 ; Gu i e t al, 2003 ; Marubio e t al, 

2003). oc-CtxMII-sensitive nAChRs consis t of Ot6ß2ß3 

and oc4oc6ß2ß 3 subunits . Thos e no t blocke d b y oc - 

CtxMII contai n oc4ß 2 an d oc4oc5ß 2 subunit s (Salmi - 

nen e t al. , 2004) . A separat e populatio n o f a ? 

FIGURE 23- 1 Expressio n o f nicotini c recepto r sub - 

units i n dopaminergi c (DAergic ) neurons . Th e pho - 

tomicrographs sho w expressio n o f oc 3 (A ) and oc 4 (B ) 

nAChR subuni t mRNA within substantial nigra (SN) 

and ventral tegmental are a (VTA) at Gl 5. mRNAs for 

nAChR subunit s (silve r grains) are expresse d i n bot h 

DAergic cell s expressin g tyrosin e hydroxylas e (TH ) 

mRNA (dar k cells ) an d non-DAergi c neurons . Not e 

the differentia l distributio n o f cc 4 mRN A withi n S N 

and VTA at this age.

nAChRs ha s als o bee n identifie d o n DAergi c cel l 

bodies (Pidoplichko et al, 1997 ; Klink et al, 2001). 

Perinatal Period 

DA-containing neurons of the S N and VTA are born 

between gestationa l da y (G ) 1 2 an d G1 4 (Voorn 

et al. , 1988) , wit h nACh R bindin g appearing i n th e 

anläge shortly thereafter (Naeffet al. , 1992 ; Zoli et al, 

1995). B y G15, severa l nAChR subuni t mRNA s ar e 

expressed by DAergic neurons including a3, a4, oc5 , 

oc7, ß2, and ß4 (Fig . 23-1, Table 23-1) . At this age, 

3 

both high-affinit y [ H]nicotine bindin g an d oc 4 

mRNA expressio n are highe r i n th e S N tha n i n th e 

VTA. A t later stage s o f feta l an d postnata l develop - 

ment, however, no differences are detectable betwee n 

nAChR expressio n in DAergi c neuron s o f these tw o 

nuclei (Aza m e t al., 2002) . The postnata l declin e i n 

[ 3 H] nicotine binding to lower adult concentrations in 

the S N an d VT A parallels a decreas e i n th e expres - 

sion o f mRNAs for oc3 , oc4 , and ß 2 subunits . I n con - 

trast, ot6 and ß3 mRNAs are expressed at low amounts 

in feta l DAergi c neurons , wit h transcrip t level s in - 

creasing durin g the postnata l period . Suc h findings 

suggest tha t th e type s o f nAChR s expresse d o n D A 

ergic neuron s chang e wit h age . Functiona l studie s 

support this view (Azam et al., 2005). 

By examinin g i n vitr o neurotransmitte r releas e 

from striata l slices , th e presenc e o f functiona l 

nAChRs o n ra t striatal DA terminals a s early as G17 

has bee n show n (Aza m e t al. , 2005 ) (Tabl e 23-2) . 

NICOTINIC RECEPTO R REGULATIO N OF CATECHOLAMINE 38 3 

3 

Maximal nicotine-stimulate d [ H]DA releas e de - 

clines by 50 % at birth, then graduall y increases during 

th e postnata l perio d t o reac h adul t level s b y 

postnatal day (P) 21. There is a concomitant increase 

in nicotine potency, as indicated by the concentratio n 

inducing a half maximum response (EC50) at P21 being 

almost an order of magnitude lowe r than that observed 

i n fetal brain . Thus, during the first postnatal 

month ther e i s a switch i n th e typ e of nAChR(s) expressed 

on striatal DAergic terminals from thos e with 

low affinity fo r nicotine to those with high affinity . 

Data on the functional influence of a4-containing 

nAChRs on developing DAergi c neuron s are consis - 

tent wit h thos e fro m studie s o n a mutan t mous e 

model i n whic h th e a 4 subuni t wa s modified to be 

hypersensitive t o nicotin e an d endogenou s ligand s 

(Labarca et al., 2001). Whereas fetuses of these transgenie 

mice have the same number o f midbrain DAergic 

neurons as wild-type controls on G14, they exhibit 

a dramati c decline i n D A cell numbe r b y G16 an d 

G18. Thus, fetal DAergi c neurons in these transgenic 

lines degenerate following hyperactivation of the mu - 

tant


TABLE 23- 2 Nicotine-induce d [ 5 H] dopamin e re - 

lease fro m striata l slice s durin g th e firs t 3 postnata l 

weeks 

Age 

G17-G18 

PI 

P4 

P7 

P14 

P21 

EC50 fmM ) 

92*** 

(5.7-15) 

4.0 

(0.4-42) 

3.09 

(1.5-6.6) 

5.78** 

(2.0-17) 

2.48 

(1.3-4.8) 

1.65 

(0.8-3.3) 

Maximum Release (%) 

4.8410.55 

2.50 ±0.58** 

2.59 ±0.30** 

3.15±0.56** 

3.76 ±0.48* 

6.19 + 0.86 

EC50 values (95% confidence intervals) and maximum release (mean± 

SEM) ar e fro m thre e t o fou r independen t experiments . G , gesta - 

tional day; P, postnatal day. 

*/> < 0.05, **p < 0.01, ***p < 0.001 values significantly different on P21. 

initiation of addictive behaviors. There is increasing 

evidence fro m bot h roden t and primat e studie s that 

DAergic systems continue t o mature throughout this 

developmental period . DAergi c innervatio n o f prefrontal 

corte x increase s unti l youn g adulthoo d 

(Benes et al, 2000; Lamb e e t al, 2000), with som e 

evidence o f a n "overshoot " a t pubert y an d subse - 

quent prunin g o f nonspecifi c innervatio n (Lewis , 

1997). Both Dl an d D2 receptor level s in prefrontal 

cortex reac h pea k concentration s i n youn g mal e 

adult rats , the n declin e substantiall y (Andersen e t 

al., 2000 ; Tarazi an d Baldessarini , 2000). Overpro - 

duction o f Dl an d D 2 receptor s als o occur s i n th e 

caudate nucleu s an d putamen , wit h pea k conten t 

reached at puberty followed by substantial declines. 

A gende r differenc e ha s bee n note d i n thi s effect , 

with striata l recepto r prunin g occurrin g onl y i n 

males an d no t i n female s (Anderse n an d Teicher , 

2000). 

Whereas th e majorit y o f nAChR mRNA s i n th e 

SN and VT A reach matur e expressio n prior to ado - 

lescence, transcrip t amount s fo r oc5 , Ot6 , an d ß 4 

subunits continu e t o decreas e towar d lowe r adul t 

concentrations (Tabl e 23-1) . Nicotine-stimulate d 

DA releas e i n th e ventra l striatum , whic h include s 

the nucleu s accumbens , i s also higher durin g early 

adolescence (P30 ) tha n durin g lat e adolescenc e 

(P40) o r adulthoo d (Fig . 23-2) . Thi s transien t 

< £ 

Q o 

F* 

FIGURE 23-2 Releas e of dopamine (DA) from striatal 

slices. Thes e dose-respons e curve s depic t nicotine - 

stimulated [ 3 H]DA release fro m dorsa l (A ) and ventral 

(B ) striata l slice s fro m adolescen t mal e rat s o n 

P30 and P4 0 and fro m adults . Maximal release is significantly 

higher from ventra l striatum at P30 than a t 

P40 or in adults. *p < 0.05, Dunnett's post-hoc analysis 

with adult as control. 

increase i n nicotin e efficac y i s no t observe d i n th e 

caudoputamen. Suc h findings suggest an enhance d 

sensitivity o f motivational circuitr y to th e activatin g 

effects o f nicotin e durin g earl y adolescence . Al - 

though a recen t microdialysi s study suggest s tha t a 

similar enhanced effec t o f nicotine o n the releas e of 

DA fro m th e nucleu s accumben s i s not detectabl e 

in viv o (Badanic h and Kirstein , 2004), othe r neuro - 

chemical finding s ar e consisten t wit h evidenc e tha t

early adolescence i s a period o f unique sensitivit y to 

the rewardin g effects o f nicotine (Vastol a et al., 2002; 

Laviola et al, 2003; Belluzzi et al, 2004, 2005 ; Rezvani 

and Levin , 2004). 

NOREPINEPHRINE NEURON S 

Adult 

The noradrenergi c system projects extensively throughout 

the central nervous system (CNS) and modulates a 

variety of cognitive and viscera l functions. NE projections 

stem from nucle i in the pons and medulla, eac h 

with a rol e i n regulatin g response s t o sensor y input 

and t o environmental an d viscera l Stressors. Nicotinic 

receptors are widely expressed throughou t these cate - 

cholamine nuclei . 

Locus Coeruleus 

A dorsa l pontin e tegmenta l nucleus , th e locu s 

coeruleus (LC) , i s the CN S structur e wit h th e mos t 

widespread projection s (Loughli n e t al. , 1986) . I t 

gives ris e to a networ k o f fibers that exten d through - 

out the neuraxis . The L C provide s th e exclusiv e N E 

input t o th e majorit y o f brain structures , includin g 

the neocortex , hippocampus , an d cerebellum , an d 

plays a critical role in attention an d arousal. 

A wealth o f nAChR subunit s ar e expressed i n th e 

adult L C (Len a e t al, 1999) . I n sit u hybridization 

studies sho w high expressio n o f several nACh R sub - 

unit mRNAs , includin g a6, ß2, and ß3 , with lower 

levels o f expressio n o f oc4 , oc5 , an d oc 7 (Gallard o 

et al. , 2005) . Consistent wit h th e lo w expressio n of 

oc4 and ot 7 subunits, ver y low amounts o f [ 3 H]nicotine 

an d [ 125 I]oc-bungarotoxin binding , respectively , 

are present in the adult LC. Bot h oc 3 and ß4 mRNA s 

are topographicall y distributed , wit h muc h highe r 

expression i n dorsa l tha n i n ventra l cells . Thes e 

findings are consistent with a single-cell reverse transcriptase-polymerase 

chai n reactio n stud y i n 

which ot 3 and ß 4 mRNAs were found to be expressed 

in les s than hal f o f all L C cell s teste d (Len a e t al. , 

1999). 

LC efferen t projection s are organized topographi - 

cally, with dorsal cells projecting to more rostral brain 

regions (Loughlin e t al, 1986) . Th e topographi c dis - 

tribution of nAChR mRNA s in LC cell s suggests that 

different termina l field s expres s nAChRs wit h differ - 

NICOTINIC RECEPTO R REGULATIO N OF CATECHOLAMINE 38 5 

TABLE 23. 3 Nicotin e EC 50 values for stimulation of 

[ 3 H] norephinephrine release from brai n slices in the 

neonate and adult 

Brain region 

Cortex 

Cerebellum 

Hippoeampal formation 

Hypothalamus 

neonate 

1.4 

9.1 

26 

20 

Nicotine EC 50 (|lM) 

adult 

14.1 

10.3 

39 

75 

ing pharmacologica l properties . Thi s issu e ha s no t 

been examine d systematically . A preliminary comparison 

of nicotine potenc y t o stimulate [ 3 H]NE release 

in slice preparations from fou r brain regions suggests 

that ther e ma y b e pharmacologica l difference s i n 

nAChR regulatio n (Tabl e 23-3) . Immunoreactivit y 

for oc 6 an d ß 3 subunit s ha s bee n show n i n th e hip - 

pocampal formation , leadin g t o th e suggestio n tha t 

the nACh R o n LC terminal s in this structure consis t 

of a n oc6ß2ß 3 subuni t combinatio n (Len a e t al. , 

1999). O n th e othe r hand , nAChR s regulat e hip - 

pocampal [ 3 H]NE release b y both direct and indirec t 

mechanisms, wit h nicotine-stimulate d GAB A release 

leading t o an anomalou s excitatio n o f LC terminal s 

(Leslie e t al. , 2002) . I n organotypi c slic e culture s 

of parieta l cortex , i n contrast , nicotine-stimulate d 

[ 3 H]NE releas e i s mediated entirel y by nAChR s lo - 

cated o n LC terminals (dat a not shown). 

Medullary Nuclei 

Medullary catecholamin e neuron s cell s ar e foun d 

within th e nucleu s tractu s solitariu s (NTS) an d th e 

ventrolateral medulla (VLM ) an d consist of both NE - 

and epinephrine-containin g cells . Th e NT S i s th e 

initial site of integration for central and peripheral au - 

tonomie systems , with afférents projectin g from mul - 

tiple visceral modalities (Andrese n and Kunze, 1994). 

The NT S send s reciproca l projection s to numerou s 

brain stem nucle i tha t mediat e periphera l autonomi e 

outflow an d t o th e hypothalamus , wher e i t serve s a 

critical rol e i n regulatin g plasm a glucos e vi a activation 

of the hypothalamic-pituitary-adrena l (HPA) axis 

and food intake (Ritter et al, 2001, 2003). The cauda l 

and rostra l VLM generall y have opposin g effect s o n 

autonomie output , suc h a s central flui d an d cardio - 

vascular homeostasis (Heslop et al, 2004). The NEer - 

gic neurons of the rostral VLM also provide the major


inhibitory input to the LC, thus providing coordinate 

regulation o f cognitiv e an d autonomi e response s t o 

external stimuli (Aston-Jones et al., 1992) . 

Data from i n situ hybridization studies provide evidence 

fo r expression of nAChRs o n adult medullary 

catecholamine neurons (Gallard o e t al., 2005). Unlike 

the LC , wher e mos t neuron s exhibi t a rich complex - 

ity of subunit mRNAs, fewer nACh R subunits are expressed 

withi n thes e neuron s (dat a no t shown) . I n 

contrast to the LC , ther e is no expression of oc6 and ß 3 

subunits i n th e NTS . Th e predominan t subunit s expressed 

i n catecholaminergic neuron s o f this nucleu s 

FIGURE 23- 3 Quantitativ e analysis of nicotine binding site and nAChR sub - 

unit mRNA expressio n i n the locu s coemleus as a function o f age. A . Spe - 

cific [ ? H]nicotine bindin g (fmol/m g tissue) . B . oc 4 (triangles ) an d ß 2 

(squares) mRNA expression. C. a5 mRNA expression. D. oc 6 (triangles) and 

ß3 (squares) mRNA expression. E. Ot 3 mRNA expression, dorsal (d; triangles) 

and ventral (v; squares) subdivisions. F. ß4 in dorsal (d; triangles) and ventral 

(v; squares) subdivisions. The da y of birth wa s G22.

are Ot3 , oc5, and ß2/4 . I n the VLM , ther e i s expression 

of ß 3 mRN A i n scattere d cells , wit h a mor e wide - 

spread expressio n o f oc 6 mRN A i n -50 % o f cate - 

cholamine neurons. I n contrast to both LC and NTS , 

almost al l cell s o f the VL M expres s both a? and ß 2 

subunit mRNAs , wit h 30%-50 % als o expressin g 

Ot3-a5. Suc h finding s ar e consisten t wit h functional 

studies i n whic h nicotin e microinjection s into brain 

stem nuclei have been reported t o increase NE release 

in the hypothalamu s (Fu e t al., 1997 ) and t o regulate 

both HP A axi s (F u e t al. , 1997 ) an d cardiovascula r 

functions (Ferreir a et al., 2000). 

Ontogeny 

Locus Coeruleus 

Neurons o f the L C ar e born betwee n G1 0 an d G1 3 

in rat , and first exhibit the biosyntheti c enzyme tyrosine 

hydroxylas e (TH ) o n G1 2 (e.g. , Lande r an d 

Bloom, 1974 ; Spech t et al, 1981 ; Konig et al, 1988) . 

These neuron s rapidl y extend axon s int o immatur e 

brain structures , where the y are believe d t o serve an 

NICOTINIC RECEPTOR REGULATION OF CATECHOLAMINE 38 7 

important morphogeneti c rol e (Rui z e t al. , 1997 ; 

Naqui e t al, 1999) . L C cell s are also highly sensitive 

to earl y sensory input (Nakamur a e t al. , 1987 ; Mar - 

shall e t al. , 1991 ) an d regulat e function s critica l to 

the surviva l o f the neonat e suc h a s olfactory imprinting 

(Sullivan , 2003). A number o f studies sho w tha t 

the hyper-reactivit y of the L C t o innocuou s sensor y 

stimuli decrease s afte r birth , an d tha t thes e change s 

are accompanie d b y alteration s i n th e propertie s of 

adrenoceptors expressed by LC neuron s (Kimura and 

Nakamura, 1987 ; William s an d Marshall , 1987 ; 

Nakamura e t al. , 1988) . Interestingly , little attentio n 

has been give n to date t o possible changes i n the expression 

of other recepto r types. 

Anatomical an d functiona l studie s demonstrat e 

major change s i n th e expressio n o f nAChR subunit s 

in the L C ove r the cours e o f development (Gallard o 

et al. , 2005) . Feta l L C neuron s exhibi t high-affinit y 

[ 3 H]nicotine bindin g tha t decrease s t o lo w adul t 

amounts durin g th e earl y postnata l perio d (Figs . 

23-3, 23-4 , an d 23-5) . Consisten t wit h earlie r findings 

tha t high-affinit y [ 3 H]nicotine binding site s ar e 

comprised o f a4 and ß 2 subunits (Zoli e t al., 1998) , 

Age 

FIGURE 23- 4 Stimulatio n o f norepinephrine (NE ) i n developin g forebrain. 

Developmental change s i n nicotine-stimulate d [ 3 H]NE fro m slice s o f hip - 

pocampus (A) , cerebellum (B) , parietal corte x (C) , an d hypothalamu s (D) . 

Significant difference s ( p < 0.01, Dunnett's test) relative to the adul t are signified 

by asterisks.

NICOTINE-AFFECTED DEVELOPMENT 

FIGURE 23- 5 Propertie s o f nAChR s tha t stimulat e 

[ 3 H]norepinephrine (NE ) releas e fro m locu s 

coeruleus terminals in developin g parieta l cortex. A. 

Nicotine concentration-respons e curves on PI (triangles), 

P4 (diamonds) , an d P6 0 (adult ; circles). Nico - 

tine potenc y an d maxima l releas e value s diffe r wit h 

age. B . DHß E inhibitio n o f nicotine-induce d 

[ 5 H]NE release from cortica l slices on P4 (diamonds) 

and P6 0 (adult; squares). Antagonist inhibition on P4 

is biphasic, indicating that nicotine act s at two differ - 

ent type s of nAChR. In contrast , th e antagonis t inhi - 

bition curve is monophasic i n adults. 

high amounts o f expression of ot4 and ß 2 mRNAs occurs 

i n th e L C durin g th e perinata l period , alon g 

with ot3 , a5, an d ß 4 mRNA s (Fig . 23-3 ) (Gallard o 

et al, 2005) . Expressio n o f a4 an d a 5 mRNA s declines 

during the first postnatal week , in parallel with 

the los s of high-affinity [ 3 H]nicotine binding. Wherea s 

perinatal expressio n o f oc 3 and ß 4 subunit mRNA s is 

homogeneously distribute d throughout th e entire nu - 

cleus, there i s an emergenc e o f a topographic expres - 

sion durin g th e earl y postnata l period , wit h highe r 

levels of expression in the dorsal LC. At the same time, 

the phenotyp e o f th e matur e L C nACh R emerges , 

with increasin g postnata l expressio n o f ot 6 an d ß 3 

mRNAs. 

Fetal L C cell s dissecte d fro m dorsa l rhomben - 

cephalon on G14 and cultured fo r 4 days have been 

shown t o tak e u p an d releas e [ 3 H]NE i n respons e 

to appropriat e physiologica l stimul i (Raymo n an d 

Leslie, 1994) . Nicotin e stimulate s [ 3 H]NE releas e 

from feta l culture d L C cell s vi a activatio n o f a re - 

ceptor wit h hig h affinit y fo r nicotine an d propertie s 

consistent wit h that o f an a4ß2 * nACh R (Gallard o 

and Leslie, 1998) . The EC 50 value for nicotine stim - 

ulation o f thi s nACh R wa s foun d t o b e < 1 jilM, 

which i s within th e rang e o f bloo d nicotin e level s 

found i n moderat e t o heav y smoker s (Benowitz , 

1990). 

In orde r to evaluate possibl e postnata l change s in 

the propertie s o f nAChR s tha t regulat e N E release , 

similar studie s wit h slice s take n fro m L C termina l 

fields were conducted (Fig . 23-4) (Lesli e et al, 2002). 

Using thi s approach , a remarkabl e regiona l hetero - 

geneity i n nACh R regulatio n o f transmitte r releas e 

from L C terminal s durin g th e postnata l perio d wa s 

shown. I n th e hippocampus , a late-maturin g struc - 

ture, ther e i s n o majo r developmenta l switc h i n 

nAChR regulatio n of NE releas e (Leslie et al, 2002). 

There is a monotonie increas e in nicotine-stimulate d 

NE releas e fro m hippocampa l slice s durin g th e first 

postnatal wee k to reach adul t concentrations b y P10. 

There i s als o n o chang e i n th e pharmacologica l 

profile o f hippocampal nAChRs , wit h majo r contri - 

butions o f both direc t and indirec t pathways at eac h 

postnatal stag e (Lesli e e t al, 2002) . In contrast , an - 

other structur e tha t mature s largel y during the post - 

natal period in rat, the cerebellum, exhibit s substantial 

postnatal change s i n nAChR regulation of NE releas e 

(O'Leary and Leslie, 2003). During the first postnatal 

week, nicotin e i s highly efficacious , releasin g almos t 

40% o f cerebella r N E store s a t a maxima l effectiv e 

concentration. Ther e is a gradual decline i n nACh R 

efficacy during the next month to reach low adult levels. 

Pharmacological analysi s has shown tha t neona - 

tal an d adul t nAChR s o n cerebella r L C terminal s 

have different subuni t compositions , wit h decreasin g 

involvement o f ß 2 subunit s i n th e adul t recepto r 

(O'Leary and Leslie, 2003).

In parieta l cortex , ther e i s increase d maxima l 

nicotine-induced releas e in the neonatal perio d with a 

subsequent declin e t o lowe r adul t concentration s b y 

P7 (Fig. 23-4C) (Gallardo etal., 2005). More detailed 

pharmacological analysi s ha s show n tha t ther e i s a 

switch in the properties of nAChRs on cortical LC ter - 

minals durin g th e firs t postnata l wee k (Fig . 23-5 ) 

(Gallardo e t al , 2005) . Wherea s a t birt h nicotin e 

stimulates cortical NE releas e with high affinity, ther e 

is an orde r o f magnitude decreas e i n potenc y b y P4 . 

At this age, nAChRs on cortical LC terminals are in a 

period o f transitio n betwee n th e neonata l recepto r 

that ha s hig h affinit y fo r th e Ot4ß2-selectiv e antago - 

nist, DHßE, and the adult low-affinity form . 

Hypothalamus wa s examined because , unlik e th e 

other brai n region s studied , i t ha s substantia l inpu t 

from th e medullar y catecholamine nucle i a s well as 

the LC . Thi s region als o shows enhanced efficac y o f 

nicotine t o release [ 3 H]NE during the first postnatal 

week (Fig . 3-4D) . Subsequently , ther e ar e comple x 

changes in nicotine-induced transmitte r release , with 

adult profile s no t reache d unti l adolescenc e (P30) . 

Pharmacological comparison s o f nACh R propertie s 

at P7 and adul t have shown that, similar to the cere - 

bellum and neocortex, the neonatal receptor in hypothalamus 

ha s distinc t propertie s fro m tha t o f adult . 

The neonata l for m o f the recepto r i n al l thre e ter - 

minal field s appear s t o hav e a predominanc e o f ß 2 

subunits compare d t o adul t amounts , however , th e 

neonatal recepto r i s not identica l acros s regions . I n 

the hypothalamus and cerebellum , unlik e neocortex , 

the neonata l recepto r doe s no t hav e hig h affinit y fo r 

nicotine (Tabl e 23-3) . Althoug h hypothalamu s re - 

ceives mixed inputs from NEergi c neurons o f both the 

pons and medulla, lesion studies in adult show that hypothalamic 

nAChR s selectivel y regulat e N E releas e 

from L C terminal s (Sperlag h e t al. , 1998) . Similarly , 

nAChRs are exclusively localized on hypothalamic LC 

terminals i n th e neonate , sinc e nicotine-stimulate d 

pH]NE release is abolished by the LC-selective neurotoxin 

DSP-4 (O'Leary and Leslie, 2005). 

These finding s ar e consisten t wit h a n importan t 

and changing rol e of nAChRs i n regulating release of 

NE fro m L C terminal s throughou t development . 

Unique pattern s o f nicotinic regulation o f individual 

LC termina l field s indicat e tha t th e specifi c rol e o f 

nAChRs may vary by brain region. Despite thes e individual 

differences , ther e i s a consistent tren d i n mos t 

brain region s fo r enhance d nACh R efficac y durin g 

the perinata l perio d whe n L C i s hyperreactiv e t o 


external stimuli. Thus cholinergic input s may provide 

an importan t mechanis m fo r regulating L C respon - 

siveness during this critical neonatal period . 

Medullary Nuclei 

The VLM, whic h provides the major inhibitory input 

to the L C (Aston-Jone s et al, 1992) , exhibit s coordinated 

developmenta l regulatio n o f nACh R subuni t 

expression. There i s high perinata l expressio n o f oc 4 

and oc 5 mRNAs, with a slow decline t o adult concentrations 

after birth. There is also a parallel increase in 

oc6 and ß 3 mRNA expression during the postnatal period, 

indicatin g tha t som e cell s o f th e VL M ma y 

switch their nAChR phenotype . In contrast to the L C 

and VLM, th e NT S exhibit s less dynamic alteration s 

in nACh R subuni t expressio n throughou t develop - 

ment. Other than a brief up-regulation o f oc4 mRNA 

in feta l NTS , n o transien t nACh R expressio n ha s 

been observed. Rather, there is a slow maturation during 

the postnatal period to adult concentrations . 

The absenc e o f nAChRs o n medullary hypothalamic 

terminals precludes th e typ e of functional analysis 

o f developmenta l change s i n th e propertie s o f 

nAChRs describe d for the LC (Gallardo e t al, 2005). 

More complex in vivo studies are required to evaluate 

this issue. Whereas nicotin e administration in vivo activates 

the HP A axis via stimulation of nAChRs within 

the medull a (F u et al., 1997) , n o suc h stimulatio n is 

evident durin g earl y adolescenc e (dat a no t shown) . 

Such finding s sugges t tha t ther e i s a lat e postnata l 

maturation o f nACh R regulatio n o f HP A function , 

consistent wit h the lat e developmenta l maturatio n of 

nAChR expression withi n the NTS . 

DEVELOPMENTAL EXPOSURE 

Gestation 

Although ther e ar e functionally active nAChRs o n fetal 

D A terminals, they have relatively low affinit y fo r 

nicotine (~10|iM ) (Aza m e t al. , 2005) . Thus, given 

the lo w concentratio n o f nicotin e i n bloo d 

(-500 nM), one would expect there to be minimal occupancy 

o f these sites . In contrast , nAChR s o n feta l 

LC neuron s an d neonata l cortica l terminal s expres s 

nAChRs wit h high affinit y fo r nicotine tha t would be 

activated by blood concentration s o f nicotine i n pregnant 

femal e smokers . Suc h finding s predic t tha t


prenatal exposure to nicotine has a more harmful effect 

o n developin g NEergic system s than o n DAer - 

gic systems . Although a numbe r o f studie s sugges t 

that gestationa l nicotin e treatmen t doe s affec t th e 

development o f the striata l DAergic pathwa y (Olif f 

and Gallardo , 1999) , chroni c prenata l infusio n o f 

nicotine i n moderat e dose s more profoundl y affect s 

postnatal ontogen y o f NEergi c system s (Slotkin , 

1998). 

Gestational treatmen t wit h nicotin e infusio n 

(3 mg/kg/day) induces a long-term change i n the effi - 

cacy of nAChRs on cortical LC terminals (Fig. 23-6 ) 

(Gallardo e t al., 2005). Maximal nicotine-stimulate d 

[ 3 H]NE releas e i s greatly increased durin g th e firs t 

postnatal month and, although reduced, is still signif - 

icant in the adult. This does not result from a n overall 

increase i n th e excitabilit y of L C terminals , sinc e 

potassium-stimulated transmitte r releas e i s un - 

changed b y gestationa l nicotine . No r doe s i t resul t 

from a delayed transition in the properties of terminal 

nAChRs; bot h anatomica l an d functiona l studie s 

have demonstrate d tha t ther e i s a norma l postnata l 

switch i n nAChR phenotype on LC cell s and corti - 

cal terminal s i n gestationall y treated animals . On e 

change i n nACh R properties , however , i s a muc h 

greater acut e nicotine-induce d recepto r desensitiza - 

tion, that i s most eviden t at low drug concentration s 

(data not shown). This finding may explain an apparent 

discrepancy between i n vitro data (Gallardo et al., 

2005) an d thos e o f an earlie r i n viv o stud y (Seidle r 

et al, 1992 ) o f NE turnover , whic h foun d prenata l 

nicotine treatmen t t o induc e hypoactivit y i n th e re - 

sponse o f N E system s t o acut e postnata l nicotin e 

treatment. 

FIGURE 23- 6 Effec t o f gestationa l treatmen t wit h o f nicotine - (triangles ) o r saline - (squares ) treate d 

nicotine (3. 0 mg/kg/day) o n subsequen t nicotine - dam s o n P 4 (A) , P14 (B) , P3 0 (C) , o r P6 0 (adult ) 

induced [ 

3 

H]norepinephrine (NE ) releas e fro m (D) . Significan t difference s relativ e t o th e saline - 

locus coeruleu s terminal s i n parieta l cortex . Nico - treate d grou p ar e identifie d b y **(£ < 0.001).

Adolescence 

It is well established that catecholamine systems, particularly 

DA , ar e no t full y mature d b y adolescenc e 

(Spear, 2000 ; Chamber s e t al , 2003) . Functiona l 

studies sho w tha t nACh R regulatio n o f mesolimbi c 

DAergic an d medullar y NEergic system s has not yet 

fully mature d i n adolescen t rat s (Aza m e t al. , 2005). 

Such finding s sugges t tha t brai n catecholamin e systems 

are vulnerable to nicotine exposur e during adolescence. 

A growin g body o f evidenc e support s thi s 

view. Exposur e t o dru g fo r relativel y shor t period s 

during adolescenc e i n roden t induce s permanen t 

changes i n behavior s mediate d b y forebrain DA systems, 

includin g locomotio n an d rewar d (Slotkin , 

2002; Adriani e t al., 2003 ; Faraday et al, 2003 ; Belluzzi 

e t al., 2004) . Simila r change s ar e no t observe d 

following equivalent exposure in adult. Neurochemical 

studie s confirm tha t chroni c nicotin e treatmen t 

during adolescenc e produce s permanen t alteration s 

in D A an d N E system s i n ra t brai n (Traut h e t al. , 

2001). Thus , catecholamin e system s withi n adoles - 

cent brain may be particularly vulnerable to the neuroteratogenic 

effects o f nicotine. 

PHYSIOLOGICAL AND 

CLINICAL IMPLICATIONS 

Methodological Issue s 

Whereas earl y i n viv o studie s hav e show n lastin g 

changes induce d b y gestationa l nicotin e treatmen t 

(Slotkin, 1998 ; Erns t e t al , 2001) , th e underlyin g 

mechanisms ar e unclear . A systematic in vitro analysis 

wa s performe d t o clarif y mechanism s underlying 

nAChR regulatio n o f developin g catecholaminergi c 

neurons. Accordingly, it was demonstrated that nAChR 

are ke y regulator s o f developin g catecholaminergi c 

neurons. Interpretation o f these data must be qualifie d 

because nACh R pharmacolog y i s extremely complex, 

and subunit composition o f native receptors is difficult 

to assess (Cordero-Erausquin e t al., 2000 ; Whiteaker 

et al. , 2000) . Furthermore , severa l type s o f nACh R 

may b e expresse d within a singl e cell , wit h differen - 

tial localizatio n of receptor subtype s in terminal an d 

somatodendritic region s (Champtiau x e t al, 2003). 

Given these caveats, it is difficult t o draw conclusions 

about the nature of nAChR regulatio n of somatodendritic 

area s tha t hav e bee n show n t o hav e critica l 

functional roles . 


Physiological Implications 

nAChRs are expressed on immature DA and NE neu - 

rons and function to regulate transmitter release in fetal 

brain. Suc h finding s sugges t a critical interaction 

of cholinergic an d catecholamin e brai n systems during 

the earlies t phase o f brain development. Ther e is 

a lat e postnata l maturatio n o f mos t cholinergi c systems 

(Hohmann and Ebner, 1985 ; Dinopoulos e t al., 

1989; Kis s and Patel , 1992), however, a brief perinatal 

phase o f cholinergi c neurona l expressio n ha s bee n 

described i n several brain regions, includin g neocor - 

tex and cerebellu m (Goul d an d Butcher , 1987 ; Dor i 

and Parnavelas, 1989). Thus, transient cholinergic innervation 

ma y provide critical regulation o f developing 

catecholamine system s via activation of nAChRs. 

Although ther e ar e regiona l difference s i n th e 

properties o f nAChR s tha t regulat e termina l cate - 

cholamine releas e during the perinatal period , ther e 

are som e commonalities . Mos t feta l catecholaminer - 

gic cells , bot h DAergi c an d NEergic , expres s hig h 

amounts of [ 3 H]nicotine binding and a4/ß2 mRNAs. 

Many cell s als o expres s th e oc 5 structura l subunit , 

which combine s wit h othe r subunit s t o alte r agonis t 

affinity, calciu m permeability , an d agonis t desensiti - 

zation (Wang et al, 1996 ; Gerzanich et al, 1998) . Although 

quit e rare in adult brain, nAChRs comprise d 

of oe4oc5ß 2 subunit s ar e highl y expresse d during th e 

perinatal perio d an d ma y serv e uniqu e functiona l 

roles (Conro y an d Berg , 1998) . Afte r birth , ther e i s 

a declin e i n oc 4 an d oe 5 subuni t expressio n an d o f 

[ 3 H]nicotine binding. During thi s period, there i s increased 

expressio n of oe 6 and ß 3 subunits. These subunits 

do not appear to contribute to fetal nAChR s but 

are importan t constituent s o f nAChRs i n adul t cate - 

cholamine termina l field s (Len a e t al. , 1999) . Concomitant 

change s i n the pharmacologica l propertie s 

of nAChRs tha t regulat e termina l catecholamine re - 

lease ar e observed . Thus, i n bot h N E an d D A neu - 

rons ther e i s a postnata l switc h i n th e propertie s o f 

terminal nAChR s t o th e matur e phenotype . Thes e 

findings suggest that nAChR s serv e critical regulatory 

roles fo r catecholamine pathway s at al l stage s of on - 

togeny bu t tha t th e natur e o f the functiona l interac - 

tion changes with age. 

Clinical Implication s 

Fetal L C neuron s an d thei r cortica l terminal s hav e 

nAChRs with sufficiently hig h affinit y fo r nicotine t o


be activate d b y materna l smoking . Chroni c gesta - 

tional exposure to nicotine, at concentrations approximating 

bloo d concentration s i n smokers , induce s 

long-term change s i n th e propertie s o f nAChR s o n 

cortical L C terminal s suc h tha t thei r efficac y i s in - 

creased, but the y are more readily desensitized by agonist 

exposure. Given th e critica l role o f central N E 

in modulatin g cognitiv e an d viscera l functions an d 

the apparen t functiona l importanc e o f nAChR s i n 

regulating NEergic systems throughout development , 

such findings have substantial implications for understanding 

the etiolog y of many of the advers e effects of 

maternal smoking . In particular, altered sensitivit y of 

central NEergic systems to nAChR stimulation and to 

perinatal hypoxia has been implicated i n the etiology 

of sudde n infan t deat h syndrom e (Slotki n e t al. , 

1995). Given th e critica l role of the L C i n cognitio n 

and attentio n a t al l stage s o f developmen t (Aston - 

Jones et al, 1999 ; Moricea u an d Sullivan , 2004), it is 

likely that disrupted development o f forebrain NEer - 

gic system s may also contribute t o the cognitiv e dys - 

function an d ADHD that are associated with maternal 

smoking. 

DA system s are les s severel y disrupted b y gesta - 

tional nicotin e exposure , however , there i s evidence 

for long-ter m alteration s i n forebrai n DAergi c func - 

tion (Slotkin , 1998 ; Muneok a e t al, 1999) . Striata l 

DAergic terminal s expres s a lower-affinit y nACh R 

during the fetal period, which would not be activated 

by blood-born e nicotin e i n th e pregnan t femal e 

(Azam et al., 2005). Some type s of nAChR, however , 

are desensitize d b y nicotin e a t fa r lowe r concentra - 

tions than thos e require d to activate the recepto r (L u 

et al, 1999 ; Wooltorto n e t al, 2003) . Furthermore , 

somatodendritic nAChRs, whic h ar e critically implicated 

in the effect s o f systemic nicotine on DA release 

(Champtiaux et al., 2003) , have no t been evaluated. 

Thus, gestationa l nicotin e exposur e ma y directly influence 

th e ontogen y of DAergic system s via modulation 

of nAChR function. 

Adolescence i s also a critical time for maturatio n 

of forebrai n limbi c system s an d thei r afferen t cate - 

cholamine input s (Spear , 2000 ; Chamber s e t al. , 

2003). DAergi c systems , i n particular , are stil l quit e 

immature an d termina l nAChR s i n ventra l striatu m 

are more highly activated by nicotine a t this age than 

in the adult. Biochemical and behavioral studies indicate 

tha t bot h acut e an d chroni c nicotin e hav e 

unique effect s o n catecholamine systems during adolescence 

tha t ar e no t observe d i n adul t (Slotkin , 

2002). Thus, early smoking i n adolescence may reinforce 

subsequen t addiction , a s has been suggested b y 

epidemiological studie s (Kande l an d Logan , 1984 ; 

Kandel e t al. , 1992) , b y modifyin g forebrai n cate - 

cholamine development . 

Tobacco smoke contain s thousands o f chemicals, 

many o f which disrupt development. Thi s has led t o 

the recommendation tha t nicotine replacemen t ther - 

apy (NRT ) b e use d extensivel y a s a n alternativ e t o 

smoking by pregnant women (Dempsey and Benowitz, 

2001). Some studies , however, have questioned th e efficacy 

of NRT as a smoking cessation ai d for pregnant 

women an d adolescent s (Hur t et al, 2000; Wisborg 

et al , 2000) . Furthermore , a growin g bod y o f evi - 

dence show s (a ) that nAChR s pla y a critica l rol e i n 

modulating neura l developmen t an d (b ) tha t expo - 

sure o f immature brai n t o nicotin e ca n disrup t normal 

cholinergi c regulatio n o f ontogenetic processes . 

Thus, the safety of NRT as a therapeutic regimen dur - 

ing pregnancy and adolescence need s to be evaluated 

more thoroughly . 


Abbreviations 

ADHD attentio n defici t hyperactivity disorder 


DA dopamin e 

EC 5o concentratio n effectin g a half maximal response 


HPA hypothalamic-adrenal-pituitar y 

LC locu s coeruleus 

nAChR nicotini c acetylcholine recepto r 


NRT nicotin e replacement therap y 

NTS nucleu s tractus solitarius 


SN substantia l nigra 

TH tyrosin e hydroxylase 

VLM ventrolatera l medulla 

VTA ventra l tegmental area 

ACKNOWLEDGMENTS Thi s wor k was supported by a 

grant from th e Nationa l Institute of Drug Abuse and by

graduate fellowship s fro m th e Californi a Tobacco - 

Related Diseas e Researc h Program . W e woul d lik e t o 

thank Drs . Tilin g Chen , Ursul a Winzer-Serhan , an d 

Sandra Loughlin for their helpful contributions. 

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Academic performance, 133 

mathematics, 133 , 134 

Acetylcholine (ACh), 155 , 300, 346, 347 , 352, 367, 368 

acetylcholinesterase, 347, 351 , 369 

cholinergic neurotransmission, 341-354 

receptors. See Receptors 

Acquired immunodeficienc y syndrom e (AIDS ) 370-371. 

See also Human immunodeficienc y virus (HIV) 

Actin. See Cytoskeleton 

Activity-dependent neurotrophic protei n (ADNP) , 303 

Activity-dependent neurotrophic facto r (ADNF), 

301,303 

Adolescence, 126 , 131 , 134, 135, 316, 341, 350, 352 

Adrenalectomy (ADX), 159-162, 169 

Adrenal gland, 153-155, 159 , 160,281,285 

Adrenocorticotrophic hormone (ACTH) , 156 , 158 , 

160-165, 168 , 169 , 171 

Alcohol dehydrogenase (ADH), 106, 288-290, 300 

Alfred E . Newman, 4- 6 

Anxiety. See Mood disorders 

Apoptosis (programmed cell death), 73, 73, 76-79, 81, 85, 

92-99, 163 , 171, 206, 207, 248, 253-255 , 

267-274, 349, 366 , 368 , 369 . See also DNA, 

fragmentation 

apoptosis inducing factor (AIF) , 95-98, 268-271 

Index 

399 

apoptosis protease-activating factor (APAF) , 74, 78, 

93-98, 268 

neural crest, 286-290 

tumor necrosi s factor (TNF), 95, 97, 98, 167 , 169 , 171 , 

268, 269 , 272 , 300 

Arginine vasopressin (AVP), 156 , 161, 162 , 171 

Astrocyte. See Glia 

Astrotactin, 31 

Attention, 129 , 130 , 134 , 319 , 320 

Attention deficit hyperactivity disorder (ADHD), 129 , 130, 

134,319,367,381,392 

Autophagy, 74, 92, 246 

Axon 

bundling, fasciculation , 51 , 55 

effect o f ethanol, 233, 234 

fate, specification, 46, 48, 234 

growth, 47, 49, 50 , 58, 111, 233, 235, 238 

growth con e 

chemotaxis, directed growth, 50, 54 

filopodia, 28, 45,48-50,58, 236, 237 

lamellipodia, 28, 45, 48, 49, 236, 237 

motility, 48, 49 

structure, 48, 347 

guidance, 50-56,233,235 , 354 

pruning, 60, 233, 384

400 INDE X 

Basal forebrain, 17 

Basal ganglia (including caudate nucleus, putamen, and 

striatum), 9, 17-21, 145 , 148-150, 210, 346, 347 , 

350, 382-384 

Basement membrane, 32 

bel, 74, 79, 84, 93,96-98 

bax isoform, 74, 94-97, 255 

bcl-2 isoform, 74, 85, 93, 97, 98, 249, 253, 255, 257 

bcl-2 homology (BH), 74, 93, 94 

Behavior 

cognition. See Cognitio n 

conduct disorder, 134-136, 320 

aggression, 320 

externalizing, 320 

oppositional, 320 

delinquency, 13 5 

executive function, 130-132, 134 

impulsivity, 319 

motor, 128 , 319, 321 

sensory, 128 , 129 

social, 134 

emotional functioning, 134 

Bergmann glia. See Cerebellu m 

Brain-derived neurotrophic factor. See Neurotrophins 

Bromodeoxyuridine (BrdU), 15 , 19, 78, 206-211 

Cadherin. See Cell adhesion molecul e 

Caenorhabditis elegans, 74, 92-94 

Cajal-Retzius neuron, 50 , 57, 60, 91, 222 

Calcium, 208 , 209, 222, 238, 239, 288, 289, 301, 302, 

344-346, 353, 366-368 

Cancer cell line, 300 

B104 neuroblastoma cell, 188-190, 192 

C6 astrocytoma cell, 188 , 189 , 192 , 270, 299, 367 

PC 12 pheochromocytoma cell, 252 

Caspase, 74, 84, 92-99, 208, 248-250, 255, 257, 268-271, 

273, 283, 289, 366, 371 

Catecholamine, 59 , 15 6 

dopamine (DA), 231, 352, 353, 381-384, 389-392 

epinephrine, 155 , 171 

norepinephrine (NE), 155, 162, 163 , 171 , 381, 385-392 

Catenin, 62 , 95 

Caudate nucleus . See Basal ganglia 

CD133. SeeProminin 

ced, 74, 93, 94, 

Cell adhesion molecule (CAM) , 49, 55, 114, 219, 224 

cadherin, 55, 58-60,62 

integrin, 31-33, 35, 55, 59, 109, 220-222, 224, 

236,299 

LI, 55,219,234,288,299 

neural cell adhesion molecule (nCAM) , 31, 219, 

220,299 

Cell cycle, 77 

GO phase, exit, 10, 13, 15-17, 187 , 188 , 202, 208, 

218,299 

Gl phase, 10 , 11, 14, 15, 19, 20, 187 , 188 , 202-207, 

211,299 

kinetics, 10 , 14-18, 187-192, 201, 205, 299 

M phase, mitosis, 14, 15, 83, 84, 187 , 202-206, 210, 

211,299 

S phase, 10 , 11, 14, 15, 19,20,84,187, 188 , 

202-207, 299 

Cell fate, 15 , 74, 75, 79, 113 , 114 , 200, 201, 347, 366, 371 

Cerebellum, 114 , 127 , 144-147, 150, 208, 217, 269, 

271-273, 297, 298, 348-350 , 365-369, 385, 

387, 390 

Bergmann glia, 298 

external granular layer (EGL), 187 , 207, 363-366, 

368, 36 9 

granule neuron, 114 , 187 , 248, 207, 231, 234, 254-257, 

271,272,297,367-369 

neuronogenesis, 186—18 7 

Purkinje neuron, 78, 114 , 144 , 147 , 246, 247, 252, 253, 

259,269,271,272,297,348,366 

Cerebral cortex , 9, 11 , 13 , 17, 18, 20, 76, 78, 81, 83-85, 

91, 146 , 205, 206, 210, 231, 233, 269, 271-273, 

296, 297, 301 , 346-352, 385, 387, 389, 391 . For 

developmental references, se e also Cerebral wall 

death, 246 , 249-250, 252 

neuronogenesis, 187 , 249 

Cerebral wall 

cortical plate (CP), 10 , 11, 28, 29, 32, 24, 75, 76, 80, 84, 

297, 348 

intermediate zone (IZ), 10 , 11, 28, 30, 75, 76, 80, 84, 

218,220,297 

marginal zone (MZ), 10 , 11, 13 , 20, 28-30, 32, 34, 35, 

51,75,76,91,220-222,224 

preplate, 29, 32 

subplate, 29, 32, 51,76,91 

subventricular zone (SZ) 

normal, 10 , 11, 13, 17, 19-21, 28-30, 33, 35, 36, 

75, 76, 84 

ethanol-affected, 184-193 , 200, 205, 206, 208, 210, 

211,216,297 

nicotine-affected, 363-366 , 368, 369 

ventricular zone (VZ) 

normal, 9, 10 , 13, 19-21, 28, 29, 33, 75, 76, 80, 84 

ethanol-affected, 184-193 , 200, 205, 216, 297 

nicotine-affected, 350 , 363-365, 368 

Chemotaxis 

Cholinergic receptor. See Receptor, acetylcholine 

Cobblestone complex, 3 4 

Cocaine, 363 

Cognition, 107 , 108 , 143 , 148 , 150 , 315, 381, 383, 364 , 

385, 386, 392 

intelligence quotient (IQ), 128 , 129 , 132 , 143 , 148 , 

318,349 

learning, 129 , 143,319,322 

memory, 130-132, 319, 322, 341 

intentional, 13 2 

metamemory, 131 , 13 2 

working, 132 , 13 4 

test 

California Verbal Learning Test, 131 , 132 

Continuous Performance Test, 31 9 

McCarthy Scales , 131 

Peabody Picture Vocabulary Test, 322 

Ravens Test, 32 2 

Stanford Bine t Intelligence Scale , 31 8 

Tower of London, 13 3

Wisconsin Car d Sor t Task, 13 3 

Wide Range Assessment of Memory and Learning , 

131,319 

visuospatial, 129 , 13 4 

Collapsin respons e mediato r protein (CMRP) , 47 

Conduct disorder . See Behavior 

Congenital muscula r dystrophy, 34 

Corpus callosu m 

fiber bundle, 127 , 144, 145 , 147 , 148 , 150 , 233, 234, 

297, 302, 303 

neuronogenesis, 18 4 

neurons of origin, 18 4 

Cortical plate. See Cerebral wall 

Cortisol, 156-160 , 162 , 171 

Corticosterone (CORT), 156 , 158-162, 169 , 170 , 171 

Corticotropin releasing hormone (CRH) , 155 , 156, 158, 

160-162, 164 , 165 , 168,170, 171 

Cotinine 321 , 322 

Craniofacial malformation. See Facial dysmorphia 

Critical period, 60, 115 , 252, 282-284, 286, 351 , 368, 370 

Cyclic nucleotide (cAMP and cGMP), 20, 50, 54, 208, 256 

Cyclin. 13 , 14, 188, 191 , 201-205, 207, 208, 21 1 

Cyclin-dependent kinase (cdk), 13 , 14, 17, 32, 33, 188 , 

201,202,204-208,211 

Cyclin-dependent kinase inhibitor 

"p!7,pl8,p57, 202, 204, 206 

P21, 14,17, 96, 191 202, 204-208, 211 

P27, 13 , 14, 17, 20, 113 , 188 , 191 , 202 , 204, 205, 

207,211 

Cytochrome C, 94-98, 268-271, 273 

Cytochrome P450, 290, 300 

Cytokine, 156 , 168-171 

Cytoskeleton, 285 

"actin, 33, 47-50, 54, 58, 60, 202, 222, 224, 236-238 

microtubule, 34, 47-50, 202, 224, 236, 238 

regulation, 236-238 

Death 

ethanol-induced 

in vitro, 232, 250, 252-259, 267 , 268, 270, 271-274, 

286-290 

in vivo, 188, 245-250, 252-259, 267-274 , 

extracellular regulation, 288 

intracellular regulation, 255-258, 287-289 

naturally occurring, 92, 246, 249, 250, 253, 259, 269, 

270,287 

nicotine-induced, 368, 369 

opiate-induced, 364-367 

pathway 

intrinsic, 96-99, 267-269, 274, 287 

extrinsic, 96-99, 267, 268 , 270, 274 

caspase-independent, 97-99 

Dendrites 

effect o f ethanol, 230-233 

fate, specification, 46 

growth, 58,235,239 

remodelling, 231, 236 

Depression. See Mood disorders 

Disabled, 32, 33 

Distalless, dix, 4, 18 , 30 

INDEX 40 1 

DNA 

aneuploidy, 81, 82 

endjoining, 79-8 1 

fragmentation, 73 , 77, 79, 80, 81-92, 94-96, 98, 206, 

207,248,250,255,268 

Dopamine. See Catecholamine s 

Dose-response, 124 , 126 

Doublecortex syndrome, 33, 34 

Doublecortin (dcx), 32-34 , 36 

Endocrine, 153 , 364 

Ephrin, 52, 53, 58,61 

Epidermal growth factor. See Growth factor s 

Epigenetic/epigenomic, 172 , 173 

Epilepsy, 2 7 

Epinephrine. See Catecholamines 

Ethanol 

metabolism, 10 6 

threshold of consumption, 123 , 124 

genetic influences, 290 

Evolution, 104-106 , 109-112, 115 , 116 

duplicate and vary , 110—111 

redundancy. 11 1 

Excitotoxicity, 115 , 254 

Extracellular matrix (ECM), 55 , 58-60, 171 , 202, 219, 

221,222,224 

chondroitinsulfate proteoglycan , 36 , 58, 60 

dystroglycan, 35 

filamin A , 3 3 

fukutin, 3 5 

laminin, 32, 35 

mannosyl glycans, 35 

polysialic acid, 3 1 

reelin (r/n) , 4, 32, 33, 36, 114,224 

Extracellular signal-regulated kinase (ERK). See Mitogen - 

activated-protein kinase 

External granular layer. See Cerebellu m 

Facial dysmorphia, 4, 94, 107 , 109 , 123-126 , 143 , 148 , 

246, 282-290 

Fasligand,97,98,268,270,271 

Fetal alcoho l spectrum disorder s (FASD) 126 , 129 , 131, 

134, 135 , 143-150,153, 156 , 158, 159, 166 , 167 , 

199,211,282 

alcohol-related neurobehavioral disorder (ARND), 124 , 

134,135 

fetal alcohol syndrome (FAS), 4-6, 107 , 123 , 124 , 

126,130,132-135, 143-146, 148-150, 217, 223, 

233,234,274,296 

Fetal programming, 153 , 154 , 157 , 162 , 172 , 173 

Filopodia. Se e Axon, growth con e 

4-hydroxynonenal, 96, 271-273 

Fukuyama congenital muscular dystrophy, 34 

Gl phase. See Cell cycle 

y-aminobutyric acid (GABA), 9, 13 , 17, 18, 20, 21, 31, 

59-61, 155, 162, 163 , 342, 346, 350-354 

Ganglionic eminenc e (GE), 12 , 13, 17-19, 28, 30, 36 

Ganglioside, 290, 300 

Gap junction , 1 1

402 INDE X 

Glia, 113,252,281,283,28 4 

astrocyte, 17 , 29, 31, 78, 184 , 188-190 , 200, 210 , 223, 

236, 295-304, 366 , 367, 369, 371 

ependyma, 200 

glial-derived factor, 60 

oligodendrocyte, 369—37 1 

radial glia, 191,200,209,210 

Bergmann cell . See Cerebellum 

guides for migration, 29, 31, 51, 55, 219, 220, 224, 

295, 296, 29 8 

Müller cell. See Retina 

neural stem cell. See Stem cel l 

transformation to astrocytes, 29, 220-224, 295, 

297, 298 

Gliogenesis 

effects o f ethanol, 29 6 

effects o f nicotine, 369, 37 0 

effects o f opiates, 366 , 367 , 37 1 

Glucocorticoid, 154,155, 158 , 167-173 

Glutamate, 60 , 164 , 301 , 302, 342, 346, 350 , 351, 353, 354 

a-amino-3-hydroxy-5-methyl-4-isoxazole propionate 

(AMPA) 20 

N-methyl-D-aspartate (NMDA) 20, 31, 221, 231, 

254-256 

receptors. See Receptor 

Gonadotropin releasin g hormone (GnRH) , 165 , 171 

Growth cone . See Axon 

Growth factors 

bone morphogenic protei n (BMP), 222, 223, 280, 

286,287 

epidermal growth factor (EGF), 30 , 200, 298, 367 

fibroblast growth factor (FGF), 83, 109 , 188-190 , 192 , 

200,203,280, 286, 300, 367 

hepatocyte growt h factor (HGF), 30 , 31 

insulin-like growth factor (IGF) , 109 , 188 , 190 , 192 , 

203,251,253-254,300 

platelet-derived growth factor (PDGF) , 188 , 189, 192, 

193,203,300 

transforming growth factor (TGF ) a 30 

TGF ß 18, 31,188-193, 203, 222-224, 234, 298, 300 

Growth 

obesity, 318, 322 

retardation, low birth weight, 123, 126 , 134 , 315, 317 

Hippocampal formation, 246, 248-249, 301, 364 

death, 249 

dentate gyrus, 60, 186 , 249, 364 

normal, 47, 60, 12 , 114 

ethanol-affected, 148 , 156 , 162-164, 172, 185, 186 , 

200,231,233,236,237,239,249 

nicotine-affected, 319 , 321, 346, 349-351, 365, 

385,387 

neuronogenesis, 182 , 185, 186 

hippocampus, 24 9 

intrahilar zone (IHZ), 12, 182, 185, 192, 206, 249 , 

363, 364 , 36 7 

Homeostasis, 155 , 156, 158, 168, 238, 385 

Human immunodeficienc y virus (HIV), 370, 371 

neuronogenesis, 370 , 371 

gliogenesis, 37 1 

Hypothalamus, 153, 154, 156, 158, 160, 162-164 , 171 , 

172,385,387, 389 

hypothalamic-pituitary adrenal (HPA) axis, 153-165 , 

168, 169 , 171-173 , 285, 385, 387 , 389 

hypothalamic-pituitary gonadal (HPG) axis, 161, 164 , 165 

paraventricular nucleus (PVN) , 156 , 161 , 163 , 164 , 17 2 

Imaging, 12 7 

confocal, 238, 239, 272 

diffusion tenso r imagin g (DTI), 12 7 

computed tomography , 149 , 150 

magnetic resonanc e imaging (MRI), 34, 127, 145 , 

147-149,233,296 

positron emissio n tomograph y (PET) , 149 , 29 6 

ultrasonography, 149 

voxel-based morphometry , 145 , 146 

Immune cells 

B cells, 165-16 9 

T cells, 76, 165-167, 169-171 

Immune response , 154 , 163 , 165 , 168 , 17 1 

Immunity, adaptive, 166 

cell-mediate (CMI), 166-168, 170 

humoral, 166 , 168 , 17 0 

In situ end labeling (ISEL) . See DNA fragmentation 

Insulin-like growth factor. See Growth facto r 

Integrin. Se e Cell adhesion molecul e 

Interleukin (IL), 94, 164 , 169-17 1 

Intelligence quotient . Se e Cognition 

Intermediate zone. See Cerebral wall 

Ki-67,187,210,211 

LI. See Cell adhesion molecule s 

Lamellipodia. See Axon, growth cone 

Leading process, 28, 34 

Lipopolysaccaharide (LPS) , 161 , 164 , 168 , 16 9 

Lisl, 32, 34 

Lissencephaly, Se e Migration, defect s 

Local circuit neuron (LCN) , 17 , 18, 30, 31, 35, 76, 

245,351 

Locus coeruleus, 155, 156, 165, 168, 385-392 

Luteinizing hormone (LH), 165 

Luteinizing hormone releasin g hormone (LHRH) , 27, 30 

Lysophosphatidic aci d (LPA) , 83-85 

Major histocompatibility complex (MHC) , 165 , 169 , 

171,371 

Marginal zone . Se e Cerebral wal l 

Mental retardation , 27, 128 , 13 4 

Microtubules. Se e Cytoskeleton 

Microtubule-associated protein (MAP), 34, 47, 232 

Migration, 124,238,24 6 

defect 

ectopia/heterotopia, 33 , 34, 114, 144 , 216 , 217 , 

220-224, 296, 297 

lissencephaly, 216 , 217 

neural crest, 27, 166 

radial, 29, 114 , 115 

rate, 29, 218, 219 

tangential, 3 0

Mitogen-activated protein kinase (MAPK), 300, 366 

extracellular signal regulated kinase , 189 , 190 , 192 , 

256-257, 366 

jun kinase, 366 

Monoamines. See Catecholamines 

Mood disorder s 

anxiety, 32 1 

depression, 32 1 

Motor activity , 128 

M phase. See Cell cycle 

Müller cell. See Retina 

Muscle-eye-brain disease, 34 

Necrosis 73, 92, 99, 254, 274, 286 

Neostriatum. See Basal ganglia 

Nerve growth factor. See Neurotrophi n 

Neural cell adhesion molecule . Se e Cell adhesion 

molecules 

Neural crest 

cardiac development, 281, 284 

derivatives, 166 , 170,280-28 5 

effects o f ethanol, 222 , 271, 279-290, also see facial 

dysmorphia 

homeobox genes, 280 , 282 , 286, 287 , 289 

neural induction, 285 

origin, 280-282 

peripheral nervou s system (PNS), 280-284 

Neuroblastic layer. See Retina 

Neuroendocrine, 153, 154 

Neurogenesis, 17 , 18, 112, 113, 205, 206, 209 , 21 0 

Neuromuscular junction. 5 9 

Neuronogenesis, 365 , 366, 368 , 36 9 

effects o f ethanol, 218 , 249, 29 9 

effects o f nicotine, 368 , 36 9 

effects o f opiates, 364-366 

Neuropilin, 36, 52 

Neurotrophin, 236 , 251-253, 258 , 274, 288 

nerve growth factor (NGF), 74, 76, 251-253 

brain-derived neurotrophic factor (BDNF), 30, 

50,54, 60, 74, 76, 223, 224, 251-253, 255, 

301,302 

neurotrophin 3 (NT-3), 74, 83, 251, 252 

neurotrophin 4 (NT-4), 30 , 74, 251 

Neurotrophin receptor . See Receptor 

Nicotinamide-adenine dinucleotide (NAD), 95 

Nicotine, 4, 108 , 341-354 , 367-370 

Nicotinic receptor. Se e Receptor, acetylcholin e 

Nitric oxide (NO), 162-164 , 232 

N-methyl-D-aspartate (NMDA). See Glutamat e 

Norepinephrine. Se e Catecholamine 

Nucleus tractus solitarius (NTS), 385, 386-389 

Olfactory bulb, 35 , 36, 200 

Opioids, 15 6 

ß-endorphin, 156 , 160 , 162 , 169 , 36 7 

effects o n neuronal developmen t 

death (apoptosis) , 365-367, 371 

differentiation, 36 5 

proliferation, 211 , 365-367 

Optic nerve, 111 , 148 , 14 9 

INDEX 40 3 

Ornithine decarboxylase , 318, 321 

Oxidative stress, 95, 96, 108 , 115 , 271-274, 287, 288 

P53, 80, 95-98, 202-204, 207, 208, 268 , 270 , 271, 

274, 36 6 

Phosphotidylinositol 3 / -phosphokinase (PI3K), 237, 255, 

257,258,367 

Pial basement membrane , 32 , 34, 35, 221, 222, 224 

Pituitary adenylyl cylcase activating polypeptide (PACAP) , 

251,255-259,270 

Placenta, 153 , 156, 158, 159 

enzymes, 15 8 

feto-placental unit , 159 , 170 

Platelet-activating factor acetylhydrolase isoform Ib . 

See Lis l 

Polarity, 45-48, 234, 235 

Poly-adenosine diphosphate ribos e polymerase (PARP), 95, 

97, 98, 268, 274 

Poly-drug use, 124,316,32 2 

Preplate. See Cerebral wall 

Principal sensory nucleus of the trigeminal nerve, 186 , 

188,246,247,249 

Process outgrowth , 230-24 0 

axon, 45, 48 

dendrite, 45, 48 

Proliferation. See Cell cycle ; Stem cell s 

neuronal site s of origin. See Cerebral wall; Cerebellum ; 

Hippocampus 

Programmed cell death. See Apoptosis 

Projection neuron, 17 , 18, 60, 76, 231-234, 245, 351, 354 

Proliferation, 182-193 , 246, 250 , 363-371, See also Cell 

cycle; Cerebellum; Cerebral wall ; Hippocampal 

formation, Neuronal site s of origin; Stem cell s 

Pro-opiomelanocortin (POMC), 155, 156, 160-162, 

169, 17 0 

Protein kinase A (PKA), 256-258 

Protein kinas e C (PKC) , 189 , 237 

Protein-O-mannosyl transferase (POMT), 34 

Pseudostratified ventricular epithelium (PVE) , 9-1 8 

Pyknosis, 246 

Pyramidal (corticospinal) tract 184 , 233 

Pyramidal neurons. See Projection neuron s 

Radial glia, 200, 209 , 21 0 

guides for migration. See Gli a 

neural stem cell . See Stem cel l 

Reactive oxygen species (ROS) , 96-98, 248, 268, 271, 273, 

287, 28 8 

Receptor 

desensitization, 344 , 345 , 350, 353, 368, 390 , 39 1 

growth factor 

epidermal growt h factor (EGFr), 3 0 

insulin-like growt h factor , 191 , 253 , 254 

platelet-derived growt h factor, 189 , 192 , 19 3 

transforming growt h factor , 189 , 191 , 19 3 

neurotransmitter 

acetylcholine, 59 , 367 

muscarinic (mAChR), 207, 300, 321, 347, 382-39 2 

nicotinic (nAChR), 341-354, 368-370 

acetylcholine receptor-inducin g protein , 369 , 370

404 INDE X 

Receptor (continued) 

dopamine (DI and D2), 19-21 , 384 

y-aminobutyric acid, 20,21, 347 

glutamate 

a-amino-3-hydroxy-5-methyl-4-isoxazole propionate 

(AMPA), 20, 21,58,59, 351, 353 

N-methyl-D-aspartate (NMDA), 20, 21, 58-61, 164 , 

221, 231, 301, 302 , 345, 347 , 351 , 353 

opioid 

5-opioid receptors (DOR), 364-367 

K-opioid receptors (KOR), 364-367 

H-opioid receptors (MOR), 364-367 

neurotrophin 

p75, 74, 75, 251-253, 25 9 

trk30, 74,75, 223,251-253,259 

others 

corticotropin releasin g hormone typ e l (CRH-R1) , 

161,162,171 

glucocorticoid (GR), 161, 162 , 170, 172 

mineralocorticoid (MR), 161 , 162 

phosphatidylserine, 73, 78, 84 

very low density lipoprotein (VLDL) , 3 2 

Reeler mouse, 32,91, 11 4 

Retina, 79 , 111,113, 149 

ganglion cells, 301 

Müller cells , 113,301 

neuroblastic layer 

topographic maps , 50-53 , 79 

Retinoblastoma protein (Rb), 13, 14 , 202, 204, 20 6 

Retinoic acid, 289, 290 

Robo, 36,51, 52,55 

Schwann cells , 280-284 

Secondary proliferative population (SPP) , 10-15, 17, 

18, 36 4 

Serotonin, 155 , 162, 163, 36 3 

Slit, 36,51,52, 55 

Socioeconomic status (SES), 107 , 108 , 124 , 129 , 133,134, 

316,318-320,322 

Sonic hedgehog, 51,286 , 289 

S phase. See Cell cycle 

Stem cell , 76, 79-83, 85 

embryonic stem cell, 199 , 203-205, 208 , 211 

marker 

nestin, 29, 185, 200, 201, 209, 210, 296, 297 

prominin(CD133), 201,209 

vimentin, 210,296 

neural stem cell (NSC) , 11 , 17 , 18, 29, 185 , 192 , 

199-211,295-299,347,363,369 

Stress, 154 , 155, 162, 164 , 168-17 3 

concept, 15 4 

response, 154 , 160 

Subplate. See Cerebral wall 

Substantiel ragra , 231 , 346, 382 

Subventricular zone. See Cerebral wall 

Sudden infan t death syndrom e (SIDS), 316, 321 , 367, 392 

Superior colliculus , 52 

Sympathetic adrenal medullary system, 15 5 

Synapse 

chemical, 56-61 , 342 

electrotonic, 56 , 61, 62 

extra-synaptic communication, 35 0 

formation, synaptogenesis, 56-62, 76, 111 , 232, 246, 

249, 250 , 259, 295, 296 , 301 , 351 , 354 

molecular constituents, 57 , 58 

post-synaptic density , 56-5 8 

"silent," 59, 351,354 

structure, 56 , 61 

synaptosome associated protein, 58 

vesicular traffic, 57 , 58 

Telomerase, 200, 207, 208, 211 

Teratogen, 109 , 127,315-32 2 

Terminal deoxynucleotidy l transferase mediate d 

deoxyuridine nick end labeling. See 

DNA fragmentation 

Thalidomide, 109 

Thalamus, 149 , 349 , 35 0 

death, 92, 109,249,250 

cortical connections, 51,55 , 249, 350, 351 

Thymidine, 14 , 192, 217, 218, 221 , 299, 36 5 

Thymus, 154 , 165 , 167 , 170 

Tobacco, 108 , 124 , 315-322, 341 , 349 , 35 3 

composition, 329-331, 367 

effect o n growth, 317 , 318, 367 

effect o n behavior, 318-322 

environmental exposure, 316, 317, 321, 322, 367 

Topographic maps , 52 , 53 

Transcription factor s 

activating protein 1 , 207, 20 8 

nuclear factor KB , 207, 300 

sox, 201,368 

Transforming growth factor. See Growth factor s 

Transplantation, 114 , 201, 209, 210 

Trapezoid body , 60 

Tumor necrosi s factor. See Apoptosis 

UDP-N-acetylglucosamine protein-O-mannos e 

ßl ,2-N-acetylglucosaminyltransferase, 34 

Vasoactive intestinal polypeptide , 3 0 

Ventral tegmental area , 351-354, 382-384 

Ventricular zone. See Cerebral wall 

Vitamin E, 288 

Walker-Warburg syndrome, 34

Brain Development: Normal Processes and the Effects of Alcohol ...

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