SNS07 - Abstracts pdf file 35 Mb

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS 

TRABAJOS 

DEL 

INSTITUTO CAJAL 

TOMO LXXXI 

C O N T I N U A C I Ó N D E L A “ R E V I S T A T R I M E S T R A L M I C R O G R Á F I C A ” 

F U N D A D A P O R S. RAMÓN Y CAJAL 

4 th INTERNATIONAL MEETING 

STEROIDS AND NERVOUS SYSTEM 

TORINO, Italy, Villa Gualino 

February 17 - 21, 2007 

ABSTRACTS OF INVITED LECTURES 

AND FREE CONTRIBUTIONS 

G.C. Panzica and S. Gotti, editors 

MADRID – 2007

Organizers 

Roberto C. Melcangi 

GianCarlo Panzica 

(Milano, Italy) 

(Torino, Italy) 

International Scientific Committee 

Jacques Balthazart 

Luis M. García-Segura 

Allan E. Herbison 

Margaret McCarthy 

Roberto C. Melcangi 

GianCarlo Panzica 

Belgium 

Spain 

New Zealand 

USA 

Italy 

Italy 

Educational Committee 

Cheryl Frye 

USA 

Local Organizing Committee 

Aldo Fasolo 

Carla Viglietti Panzica 

Francesca Allieri 

Elisabetta Bo 

Daniela Grassi 

Stefano Gotti 

Mariangela Martini 

Désirée Miceli 

Elena Mura 

Honour Committee 

Ezio Pelizzetti 

Alberto Conte 

Aldo Fasolo 

Rector of the University of Torino 

Dean of the Faculty of Science 

President of the Research Committee of the University of Torino 

Visit us at our WWW site 

http://www.dafml.unito.it/anatomy/panzica/neurosteroids/ 

The Abstracts published here were reproced directly from author’s original text with little or no 

alteration. The Editors take no responsibility for their content.

Contents 

• Selective estrogen receptors modulators and the brain 5 

• New perspectives in the dosage of neuroactive steroids 17 

• Plenary lecture: Herbison A.E. 31 

• Effects mediated by classical steroid receptors 35 

• Round table I: Steroid hormones and sexually dimorphic brain circuits 49 

• Neuroactive steroids and neurogenesis 61 

• Plenary lecture: Mellon S.H. 71 

• Neuroprotective effects 75 

• Xenoestrogens and brain circuitries 87 

• Young investigators symposium 95 

• Effects mediated by membrane receptors 109 

• Plenary lecture: Swaab D.F. 123 

• Corticosteroid effects and stress 127 

• Posters’ exhibition 136

SATURDAY, 17 th February 2007 

10.00 – 13.00 

Satellite Symposium: 

Selective estrogen receptors 

modulators and the brain


Selective estrogen receptors modulators and the brain 

(Organizers: Marchetti B., Panzica G.C.) 

• R.D. Brinton, Zhao L. (USA) Mechanisms of neuroprotection by SERMs in the 

brain 

• Di Paolo T., Bourque M., Liu B., Dluzen D.E., Morissette M. (Canada) 

Tamoxifen and raloxifene neuroprotection in Parkinson's disease models: examples 

of MPTP and methamphetamine toxicities 

• Garcia-Segura L.M., Tapia González S, Diz-Chaves Y, Pernía O, Carrero P, 

Ciriza I (Spain) Selective estrogen receptor modulators, neuroprotection and glial 

cells 

• Frye C.A., Walf, A.A. (USA) Estrogen receptor beta is a target of estrogens’ and 

androgens’ for affective and cognitive behavior 

• Marchetti B, L’Episcopo F, Tirolo C, Testa N, Caniglia S, Giaquinta G, Gennuso 

F, Arcieri P, Serra P-A, Desole MS, Miele E, Delitala G, Morale MC. (Italy) 

Neuroimmune interactions and estrogen deficiency: innate immunity as a doubleedged 

sword in neurodegeneration and repair 

• Tena-Sempere M. (Spain) Effects of selective ER ligands and modulators on the 

gonadotropic axis

4 th International Meeting STEROIDS AND NERVOUS SYSTEM 

Villa Gualino, TORINO, Italy. February 17-21 2007 

MECHANISMS OF NEUROPROTECTION BY SERMS IN THE BRAIN 

Brinton R.D. and Zhao L. 

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, and 

Neuroscience Program, University of Southern California, Los Angeles, CA, USA 

Our scientific endeavors are a hybrid of basic science discovery and preclinical 

translational research. The goal of our basic science discovery is to elucidate fundamental 

cellular mechanisms of 1) neural defense and repair and 2) neural plasticity required for 

cognitive function. Our therapeutic development goal is to translate our cellular 

mechanistic insights into safe and efficacious therapeutics for the prevention of and 

rehabilitation from neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and 

stroke. To achieve these goals, we have investigated the mechanisms and neurobiological 

outcomes of estrogens, progestins, hormone therapies and neurosteroids. Results of these 

analyses have yielded insights into cellular strategies required for neural defense against 

degenerative insults that involve multifaceted cytoplasmic and nuclear signaling cascades 

that converge upon the mitochondria. Further, these signaling cascades are required for 

gonadal hormone regulation of morphogenesis and neurogenesis. Our data indicate a 

healthy cell bias of estrogen action for estrogen-inducible neuroprotective and neurotrophic 

outcomes. Our translational therapeutic development efforts target sites of estrogen action 

that promote neural defense and plasticity in brain while reducing untoward effects in 

reproductive tissue. Our novel NeuroSERM molecules are designed to target the 

membrane site of estrogen action whereas our natural source PhytoSERMs are designed to 

target estrogen receptor beta. Results of our in vitro analyses indicate that our novel 

NeuroSERM candidate molecule induces neuroprotective efficacy comparable to 17 β- 

estradiol. In addition, NeuroSERM candidate molecule induces neuronal plasticity markers 

consistent with 17 β-estradiol. In parallel, PhytoSERM formulations show a high degree of 

estrogenic efficacy in cultured hippocampal and cortical neurons and induces both 

neuroprotective and neural plasticity reponses. Results of in vitro analyses provide proof of 

principle that combination of ERβ selective NeuroSERM and PhytoSERMs provide 

neuroprotective efficacy which will be extended into in vivo analyses. The data thus far 

suggest that novel NeuroSERM molecules and select combinations of PhytoSERMs have 

the potential to be effective and safe estrogen alternative therapies for preventing estrogen 

deficiency-related cognitive decline and vulnerability to neurodegenerative insults such as 

those leading to Alzheimer’s disease in postmenopausal women. 

Acknowledgements: This work was supported by a grant from the Alzheimer’s Association 

to LZ and by the Kenneth T. and Eileen L. Norris Foundation to RDB 

7

Trabajos del Instituto Cajal. Tomo LXXXI, 2007 

TAMOXIFEN AND RALOXIFENE NEUROPROTECTION IN PARKINSON'S 

DISEASE MODELS: EXAMPLES OF MPTP AND METHAMPHETAMINE 

TOXICITIES 

Di Paolo T. 1 , Bourque M. 1 , Liu B. 2 , Dluzen D.E. 2 , and Morissette M. 1 

1 Molecular Endocrinology and Oncology Research Center, Laval University Medical 

Center, CHUL, Quebec City, G1V 4G2 and Faculty of Pharmacy, Laval University, 

Quebec City, G1K 7P4, Quebec, Canada. 

Fax: 418-654-2761, e-mail: therese.dipaolo@crchul.ulaval.ca 

2 Department of Anatomy, Northeastern Ohio Universities College of Medicine 

(NEOUCOM), Rootstown, Ohio 44272, USA. 

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by a 

selective depletion of dopamine (DA) neurons in the substantia nigra (SN). Accumulating 

evidence support a gender difference in PD with a relative risk 1.5 times greater in men 

[3,6]. Gender differences in the evolution of symptoms and responses to L-dopa are also 

reported [3]. A neuroprotective role of estrogens on DA activity is likely a contributing 

factor but the Women’s Health Initiative (WHI) study reported increased risk over benefits 

of equine estrogens in post-menopausal women [4]. Thus, it becomes of great interest to 

identify alternative estrogens with neuroprotectant potential. Selective estrogen receptor 

modulators (SERMs) could be an alternative for both men and women. The SERM, 

tamoxifen, is the most widely prescribed medication for prevention and treatment of breast 

cancer; it has estrogen antagonist activity in mammary tissue while it mimics the effects of 

estrogen in other tissues [1]. The second generation SERM, raloxifene, is an estrogen 

antagonist in mammary and uterine tissues while it is an estrogen agonist in bone and 

cholesterol metabolism [1]; it treats osteoporosis and is used in menopausal women. 

Tamoxifen and raloxifene have in vivo affinity for both estrogen receptors (ERs) [5]. 

It is well documented that estrogens modulate rat DA receptors and membrane DA 

transporters (DAT). We characterized the estrogenic specificity of this modulation by 

testing various estrogenic drugs. Ovariectomy decreased DA D2 receptor and DAT 

specific binding in the striatum. Estradiol and raloxifene, but not tamoxifen treatment 

prevented this decrease. Estradiol and raloxifene treatment decreased DA D3 receptor 

binding in the islands of Calleja, the nucleus accumbens and striatum, compared to 

ovariectomized rats. The ERbeta agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN), 

but not the ERalpha agonist 4,4’,4’’-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT), 

mimicked the estradiol increase of D2 receptor and DAT specific binding. Tamoxifen and 

raloxifene also corrected the decrease of DAT specific binding caused by ovariectomy. 

Neither ovariectomy nor estrogenic treatments modulated striatal specific binding to the 

monoamine vesicular transporters 2 (VMAT2). These results suggest that ERbeta mediates 

estradiol and SERMs increase of striatal D2 receptors and DAT. 

Similar gender differences are observed in animal models of PD with male mice 

displaying more nigrostriatal DA neuronal loss than females after administration of the 

toxins 1-methy-4-phenyl-1,2,3,6-tetrahypdropyridine (MPTP) [2] or methamphetamine 

(MA) [7]. Estradiol is neuroprotective against these toxicities. The prevention of MPTPinduced 

DA depletion in the striatum of male mice by estradiol is mimicked by raloxifene 

but not tamoxifen. Moderate doses of MPTP induce a decrease of striatal DAT specific 

binding (50% of control) and DAT mRNA in the SN (20% of control), suggesting that loss 

of neuronal nerve terminals is more extensive than cell bodies. The MPTP-induced 

decrease of striatal DAT specific binding is prevented by estradiol or raloxifene 

8



(5mg/kg/day) but not by a lower dose of raloxifene (1mg/kg/day). Striatal DAT specific 

binding is positively correlated with DA concentrations in intact, saline or hormone treated 

MPTP mice. This paradigm models early DA nerve cell damage and is responsive to 

hormones. 

Protective properties of tamoxifen against MA toxicity were compared to estradiol. 

Estradiol or tamoxifen were administered (estradiol: 1, 10 or 40 µg; tamoxifen: 12.5, 125 

or 500 µg) 24 hours prior to a MA injection (40mg/kg) in ovariectomized mice. Both 

treatments, at all concentrations, prevented the MA-induced decrease of striatal DA 

concentrations and VMAT2-binding. Only estradiol prevented the loss of DAT-binding in 

the lateral striatum and attenuated the MA-induced increase in striatal preproenkephalin 

(PPE) mRNA levels (at 1 or 40 µg). While both treatments prevented the DA decrease, 

estradiol protected more efficiently other dopaminergic parameters suggesting that overall 

it is more effective than tamoxifen as a neuroprotectant in female mice. By comparison, 

male mice are protected against MA toxicity by tamoxifen and not by estradiol. Intact male 

mice were administered 12.5 or 50 µg tamoxifen 24 hours before MA (40 mg/kg) 

treatment. MA reduced striatal DA concentration and its metabolites 3,4- 

dihydroxyphenylacetic acid and homovanillic acid, striatal and SN DAT and VMAT2 

specific binding as well SN DA transporter mRNA levels whereas tyrosine hydroxylase 

and VMAT2 mRNA levels were not significantly decreased. These MA effects were not 

altered by 12.5 µg tamoxifen except for increased striatal DA metabolites and turnover. 

Tamoxifen at 50 µg reduced the MA effect on striatal DA concentration and DA 

transporter specific binding; in the SN tamoxifen prevented the decrease of DA transporter 

mRNA levels. The present results show a dose–dependent protection of tamoxifen against 

MA-induced DA toxicity in male mice. MA is a potent and addictive psychostimulant of 

increasing use in humans and toxic to striatal nerve terminals. Tamoxifen is the only 

known hormonal protection of male mice against MA toxicity thus providing important 

new information on specific parameters of nigrostriatal dopaminergic function preserved 

by this SERM. 

Tamoxifen and raloxifene thus modulate and protect specific parameters of 

nigrostriatal dopaminergic function with similarities and differences compared to estradiol. 

Reference list 

[1] TA Grese, JA Dodge JA Selective estrogen receptor modulators (SERMs). Curr Pharm Des. 

4(1998):71-92. 

[2] D.B. Miller, S.F. Ali, J.P. O'Callaghan, S.C. Law, The impact of gender and estrogen on striatal 

dopaminergic neurotoxicity. Ann N Y Acad Sci, 844 (1998) 153-165. 

[3] L.M. Shulman, V. Bhat, Gender disparities in Parkinson’s disease, Expert Rev Neurother 6 (2006) 

407-416. 

[4] S.A. Shumaker, C. Legault, S.R. Rapp, L. Thal L, R.B. Wallace, J.K. Ockene, S.L. Hendrix, B.N. 

Jones 3rd, A.R. Assaf, R.D. Jackson, J.M. Kotchen, S. Wassertheil-Smoller, J. Wactawski-Wende; 

WHIMS Investigators. Estrogen plus progestin and the incidence of dementia and mild cognitive 

impairment in postmenopausal women : the Women’s Health initiative memory study: a randomized 

controlled trial JAMA 289 (2003) 2651-2662. 

[5] R.V. Weatherman., N.J. Clegg, T.S. Scanlan, Differential SERM activation of the estrogen receptors 

(ERalpha and ERbeta) at AP-1 sites. Chem Biol, 8 (2001) 427-36. 

[6] G.F.Wooten, L.J. Currie, V.E. Bovbjerg, J.K. Lee, J. Patrie, Are men at greater risk for Parkinson’s 

disease than women?, J Neurol Neurosurg Psychiatry 75 (2004) 637-639. 

[7] L. Yu, P.C. Liao, Sexual differences and estrous cycle in methamphetamine-induced dopamine and 

serotonin depletions in the striatum of mice. J Neural Transm, 107 (2000) 1139-1147. 

9


SELECTIVE ESTROGEN RECEPTOR MODULATORS, NEUROPROTECTION 

AND GLIAL CELLS 

Garcia-Segura L.M., Tapia González S., Diz-Chaves Y., Pernía O., Carrero P., and 

Ciriza I. 

Instituto Cajal, CSIC, Avenida Doctor Arce 37, E-28002 Madrid, Spain. 

E-mail: lmgs@cajal.csic.es; Fax: +34-915854754. 

Neuroprotective effects of estradiol are well characterized in animal experimental 

models. However, in humans, the outcome of estrogen treatment for cognitive function and 

neurological diseases is very controversial. Selective estrogen receptor modulators 

(SERMs) may represent an alternative to estrogen for the treatment or the prevention of 

neurodegenerative disorders. SERMs interact with the estrogen receptor and have tissuespecific 

effects distinct from those of estradiol, acting as estrogen agonists in some tissues 

and as antagonists in others. Previous studies have shown that some SERMs are 

neuroprotective. However, it is important to understand the mechanisms of action of these 

compounds before considering their possible therapeutic use for the treatment of 

neurodegenerative diseases. In our laboratory we are currently exploring the cellular 

targets that may be related with the neuroprotective actions of SERMs. Astroglia and 

microglia seem to play an important role in the neurodegenerative response since both cell 

types acquire a reactive phenotype after neural injury. To determine the effect of SERMs 

on glia activation we have assessed the effect of tamoxifen, raloxifene, lasofoxifene (CP- 

336,156), bazedoxifene (TSE-424) and estradiol in two experimental models: (i), the 

systemic administration of kainic acid (KA), an excitotoxin that induces neuronal loss and 

glial activation in the hippocampus and (ii), the systemic administration of 

lipopolysaccharide (LPS) from Escherichia coli, which induces microglia activation in the 

brain. Estradiol exerted dose-dependent neuroprotective effects, preventing neuronal loss 

in the hilus of the dentate gyrus of the hippocampal formation of adult ovariectomized rats 

injected with KA. In addition, estradiol reduced the activation of astrocytes and microglia 

in the hilus, assessed by the expression of vimentin and major histocompatibility complex 

class II (MHCII), respectively. Estradiol also reduced the number of MHCII microglia, 

assessed in the white matter of the cerebellum, after LPS administration. Tamoxifen, 

raloxifene and bazedoxifene prevented neuronal loss in the hilus after the administration of 

KA in a dose dependent manner and reduced microglia activation after KA or LPS 

administration. In contrast, none of these SERMs had a significant effect on the activation 

of astroglia. Lasofoxifene was not neuroprotective and did not affect glial activation. These 

findings suggest that SERMs act with cellular specificity in the brain and may 

differentially modulate the activation of microglia and astroglia. Reactive glial cells exert a 

mixture of positive and negative responses for neuronal survival and regeneration. 

Reactive astroglial cells release many different factors that promote neuronal survival and 

participate in the formation of the glial scar, which is important to maintain water 

homeostasis but prevents axonal regeneration. Reactive microglial cells exert important 

positive functions by remodeling the damaged tissue. However, they also release 

proinflammatory cytokines and their prolonged activation may exacerbate neuronal 

damage. Therefore, the selective action of tamoxifen, raloxifene and bazedoxifene on 

astroglia and microglia may be highly relevant for the control of neural damage after 

injury. 

Supported by Ministerio de Educación y Ciencia, Spain (SAF 2005-00272) and the 

European Union (EWA project: LSHM-CT-2005-518245). 

10



ESTROGEN RECEPTOR BETA IS A TARGET OF ESTROGENS’ AND ANDROGENS’ 

FOR AFFECTIVE AND COGNITIVE BEHAVIOR 

Frye C.A. 1-4 , and Walf A.A. 1 

Dept. of Psychology 1 , Biological Sciences 2 , and The Centers for Neuroscience 3 and Life Science 4 Research 

The University at Albany-SUNY, Life Sciences 01058, 1400 Washington Avenue, Albany, NY USA 12222; 

cafrye@albany.edu, 518-591-8823 

Estrogens, such as 17β-estradiol (E 2 ), and androgens, such as the 5α-reduced 

metabolite of testosterone, 3α-androstanediol (3α-diol), have anti-anxiety, antidepressant-like, 

and cognitive-enhancing effects in female and male rodents. Our 

laboratory has been investigating the β isoform of the estrogen receptor (ER) as a putative 

target for these effects. To accomplish this, we have utilized three approaches. First, the 

effects of systemic or intra-brain administration of selective ER modulators (SERMs) or 

selective androgen receptor modulators (SARMs) when administered to female and male 

rodents, respectively, on anxiety, depression, and cognitive behavior were assessed. 

Second, systemic or intra-brain administration of ER antagonists or ERα and/or ERβ 

antisense oligonucleotides to rodents administered SERMs or SARMs was utilized to 

determine effects of blocking or knocking down ERs on anxiety, depression, and cognitive 

behavior. Third, the effects of SERMs or SARMs administration to mice with targeted 

deletions of ERβ (BERKO) on anxiety, depression, and cognitive behavior was 

investigated. Utilizing these approaches has revealed the importance of steroids’ actions at 

ERβ in the hippocampus for anxiety, depression, and cognitive behavior. 

Whether E 2 ’s effects among female rodents require activity at ERβ was 

investigated. First, ovariectomized (ovx) rats were administered subcutaneous (SC) 17β- 

E 2 (equal affinity for ERα and ERβ), ERα-selective SERMs (PPT), ERβ-selective SERMs 

(coumestrol; DPN) or vehicle before testing in anxiety (open field, elevated plus maze) and 

depression (forced swim test) tasks, or following training in cognitive (object recognition, 

inhibitory avoidance, water maze) tasks. Subcutaneous administration of ERβ-specific 

SERMs, compared to vehicle, decreased anxiety (i.e. increased open field central entries, 

increased open arm time in the plus maze), decreased depression (decreased time immobile 

in the forced swim test) and improved performance in the object recognition, inhibitory 

avoidance, and watermaze tasks [6,8,12,13]. Similar anti-anxiety and anti-depressive 

effects were observed in ovx rats administered ERβ-selective SERMS directly to the 

hippocampus [10]. Second, the effects of blocking or knocking down ERs for anxiety, 

depression, and cognitive (conditioned place preference) behavior were determined. 

Administration of ER antagonists, SC or to the hippocampus, produce similar effects to 

attenuate the anti-anxiety and anti-depressant-like effects of SC 17β-E 2 or ERβ-SERMs 

administration [11,12]. SC or intra-nucleus accumbens administration of ER antagonists 

or antisense oligonucleotides attenuate E 2 -induced conditioned place preference [8]. These 

data suggest that ERs are important for the functional effects of E 2 , but the ER antagonists 

utilized in these studies were not ER isoform (ERα or ERβ)-specific. Therefore, the effects 

of infusions of ERα and/or ERβ antisense oligonucleotides to the lateral ventricle in ovx 

rats administered SC 17β-E 2 for anxiety and depression behavior was investigated. 

Infusions of ERβ, but not ERα, antisense oligonucleotides attenuates the anti-anxiety and 

anti-depressive effects of SC injections of E 2 to ovx rats and reduces ERβ expression in the 

hippocampus [7]. Third, the effects of natural fluctuations in E 2 , and the effects of SC 17β- 

E 2 or ERβ-SERMs administration to, BERKO mice for anxiety, depression, and cognitive 

performance were investigated. These studies revealed that BERKO mice do not respond 

11


like WT mice to endogenous variations in, and exogenous administration of, E 2 . Proestrous 

(high E 2 ) WT mice had decreased anxiety and depression behavior and improved cognitive 

performance across a variety of tasks compared to diestrous (low E 2 ) WT mice. This 

estrous cycle effect was not observed in ΒERKO mice. Furthermore, WT, but not BERKO, 

mice administered E 2 or DPN had decreased anxiety and depression behavior and 

enhanced learning across a number of tasks compared to ovx mice administered vehicle. 

Thus, these data suggest that ERβ is required for the anti-anxiety and anti-depressant-like 

and cognitive-enhancing effects of E 2 among female rodents. 

Whether androgens’s effects for anxiety and cognitive behavior among male 

rodents require activity at ERβ was investigated. First, gonadectomized (gdx) rats were 

administered SC SARMs that have different affinity for ERβ and anxiety and depression 

behavior were assessed. SC SARMs with high affinity for ERβ (3α-diol and 3β-diol) 

decreased anxiety in the open field and elevated plus maze, compared to vehicle or a 

SARM with low affinity for ERβ (androsterone). SC 3α-diol enhanced performance in the 

object recognition, conditioned contextual fear, conditioned place preference, and 

inhibitory avoidance tasks [3-5]. There are similar effects of 3α-diol when administered SC 

or to the hippocampus of gdx rats [2-3]. Second, the effects of knocking down ERβ for 

cognitive performance was assessed. Compared to vehicle, scrambled, or ERα antisense 

oligonucleotides, infusions of ERβ antisense oligonucleotides to the hippocampus of 3αdiol-administered 

rats attenuated performance in the inhibitory avoidance task [1]. Third, 

the effects of SARMs administration to gdx BERKO mice was investigated. SC 3β-diol to 

WT, but not BERKO, mice decreased anxiety behavior compared to that seen with vehicle. 

Thus, androgens may require ERβ for their anti-anxiety and cognitive-enhancing effects. 

In summary, ERβ is a putative target for the anti-anxiety, anti-depressant-like, and 

cognitive-enhancing effects of estrogens in females and androgens in males. 

Supported by: NSF (IBN03-16083) U.S.A. Army Dept. of Defense (BC051001). 

Reference List 

[1] K.L., Edinger, C.A. Frye, Androgens' effects to enhance learning may be mediated in part through actions at 

estrogen receptor-β in the hippocampus, Neurobiol Learn Mem. 87 (2007) pp. 78-85. 

[2] K.L. Edinger, C.A. Frye, Testosterone's anti-anxiety and analgesic effects may be due in part to actions of its 5α - 

reduced metabolites in the hippocampus, Psychoneuroendocrinology. 30 (2005) pp. 418-30. 

[3] K.L. Edinger, C.A. Frye, Testosterone's analgesic, anxiolytic, and cognitive-enhancing effects may be due in part to 

actions of its 5α -reduced metabolites in the hippocampus. Behav Neurosci. 118 (2004) 1352-64. 

[4] K.L. Edinger, B. Lee, C.A. Frye, Mnemonic effects of testosterone and its 5α-reduced metabolites in the 

conditioned fear and inhibitory avoidance tasks, Pharmacol Biochem Behav. 78 (2004) pp. 559-68. 

[5] C.A. Frye, Some rewarding effects of androgens may be mediated by actions of its 5alpha-reduced metabolite 3αandrostanediol, 

Pharmacol Biochem Behav. (in press). 

[6] M.E. Rhodes, C.A. Frye, ERβ -selective SERMs produce mnemonic-enhancing effects in the inhibitory avoidance 

and water maze tasks. Neurobiol Learn Mem. 85 (2006) pp. 183-91. 

[7] A.A. Walf , I. Ciriza, L.M. Garcia-Segura, C.A. Frye, , Antisense oligodeoxynucleotides for estrogen receptor β and 

α attenuate estrogen’s modulation of affective and sexual behavior, respectively, Neuropsychopharmacology (in 

revision). 

[8] A.A. Walf, M.E. Rhodes, C.A. Frye, Ovarian steroids enhance object recognition in naturally cycling and 

ovariectomized, hormone-primed rats, Neurobiol Learn Mem. 86 (2006) pp. 35-46. 

[9] A.A. Walf, M.E. Rhodes, J.R. Meade, J.P. Harney, C.A. Frye, Estradiol-induced conditioned place preference may 

require actions at estrogen receptors in the nucleus accumbens, Neuropsychopharmacology (in press). 

[10] A.A. Walf, C.A., Frye, Administration of estrogen receptor β-specific selective estrogen receptor modulators to the 

hippocampus decrease anxiety and depressive behavior of ovariectomized rats, Pharmacol Biochem Behav. (in 

press). 

[11] A.A. Walf, C.A., Frye, A review and update of mechanisms of estrogen in the hippocampus and amygdala for 

anxiety and depression behavior. Neuropsychopharmacology 31 (2006) pp. 1097-111. 

[12] A.A. Walf, C.A., Frye, ERβ-selective estrogen receptor modulators produce antianxiety behavior when 

administered systemically to ovariectomized rats. Neuropsychopharmacology.30 (2005) pp. 1598-609. 

[13] A.A. Walf, M.E. Rhodes, C.A. Frye, Anti-depressant effects of ERβ selective estrogen receptor modulators in the 

forced swim test. Pharmacol Biochem Behav. 78 (2004) pp. 523-9. 

12



NEUROIMMUNE INTERACTIONS AND ESTROGEN DEFICIENCY: INNATE 

IMMUNITY AS A DOUBLE-EDGED SWORD IN NEURODEGENERATION AND 

REPAIR 

Marchetti B. 1,2 , L’Episcopo F. 1,2 , Tirolo C. 1 , Testa N. 1 , Caniglia S. 1 , Giaquinta G. 1,2 , 

Gennuso F. 1 , Arcieri P. 1 , Serra P.-A. 1 , Desole M.S. 1 , Miele E. 1 , Delitala G. 3 , Morale 

M.C. 1 

1 Neuropharmacology, OASI Institute for Research and Care (IRCCS), Troina (EN), 

2 Department of Pharmacology and 3 Endocrinologic Clinic, University of Sassari, Italy; 

bianca.marchetti@oasi.en.it 

Over the past decade neuroinflammation has been increasingly recognized as a crucial 

contributory factor to neurodegeneration in normal aging, as well as in age-related 

neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases [1]. Hence, the 

therapeutic potential of steroidal and non-steroidal anti-inflammatory drugs (NSAIDs) for treating 

various neurodegenerative diseases including PD has recently been emphasized by various 

laboratories including our own [1]. Neurodegeneration caused by chronic inflammation involves 

activation of astrocytes and the brain’s resident immune cells, the microglia, which produce a large 

number of pro-inflammatory factors. Also acute brain insults, e.g. stroke and traumatic brain injury, 

are linked to inflammation, which contributes to the propagation of the neuropathological events 

[1,2]. Systemic inflammation may also impact on local inflammation in the diseased brain, leading 

to over-production of inflammatory mediators, which may, in turn, influence neuron survival, 

plasticity and repair [1,2]. Inflammation triggered by brain injury may either activate or impair 

neurogenesis, depending on the type of neuronal insult (i.e acute vs chronic) and degree of glia 

activation. Of particular mention, there are critical interactions between the hypothalamic-pituitary 

gonadal (HPG) and the hypothalamic-pituitary-adrenocortical (HPA) axes in modulating both 

central and systemic inflammation, with important implications for the brain’s ability to mount an 

efficient beneficial and protective response to injury [3-7]. Then, the net effect of glial-mediated 

inflammation may be beneficial, depending on quantitative and qualitative aspects of astrocytes 

and microglia activation, specificity of the brain region affected, coupled to sex, age, genetic 

predisposition and a number environmentally-mediated factors, which may, in turn dictate the 

severity of a neuronal insult, and/or influence the brain’s ability to recover. Indeed, crosstalk 

between blood-born cells, astrocytes and microglia uniquely contribute to an inflammatory 

signature critically modulated by the sex steroid hormone milieu [3-7]. Both experimental and 

clinical evidences clearly document that women are protected from many forms of neurological 

injury, in part attributable to endogenous estrogen. Here we emphasize estrogen modulation of 

central and peripheral innate mechanisms as potential contributing factors [3-7]. The onset of 

menopause is associated with spontaneous increases in cytokine production, whereas cytokine 

levels are lower in post-menopausal women on hormone replacement therapy and in estrogentreated 

ovariectomized rodents. However, estrogen background may influence different types of 

pro- and anti-inflammatory molecules in complex and distinct ways. Thus, whether estrogen and 

selective estrogen receptor modulators (SERMs) effects on inflammation have therapeutical 

potentials for neuroprotection, still remains to be elucidated. In our laboratory we have focused on 

the nigrostriatal dopaminergic (DA) neurons, the cells that degenerate in Parkinson’s disease (PD). 

The main pathological hallmark of PD is a selective and progressive death of DA neurons in the 

substantia nigra pars compacta (SNpc) leading to loss of DA afferents to basal ganglia and motor 

dysfunction, including tremor, rigidity, and bradykinesia. Idiopathic PD is one of the most common 

ageing-associated neurodegenerative disorders, with a prevalence in male gender. Using the MPTP 

mouse model of PD we documented activated astrocyte, macrophages and microglial cells as key 

elements of the neuroendocrine-immune dialogue involved in steroid hormone programming of 

brain response to neurotoxic challenge [4-7]. Hence, glia and its pro-/anti-inflammatory mediators 

were shown to represent a final common pathway through which genetic, hormonal and 

environmental influences modulate resistance or susceptibility to experimental PD [4-7]. 

13


Specifically, we have presented an hypothetical model whereby efficiency of the neuroimmune 

dialogue via steroid receptor-nitric oxide crosstalk represents a critical step in dictating 

susceptibility versus resistance to Parkinson’s and possibly other degenerative CNS diseases [4-7]. 

Here we highlight neuroinflammation as a window to develop therapeutical strategies for 

neuroprotection and describe our experimental paradigm based on the dualistic role of 

inflammation in neurodegeneration and repair. In our approach, we investigate both central and 

systemic innate mechanisms underlying the selective vulnerability/resistance of nigral DA neurons 

to MPTP-induced PD in wild type and genetically modified mice, a. using different schedules and 

dose-regimens of the neurotoxic challenges to more closely mimick the slowly progressing late 

onset nature of PD; b. by studying the impact of aging, sex and estrogen deficiency in the crosstalk 

between systemic and central innate and adaptive response to brain injury; c. by investigating how 

these interactions may impact in the severity of the nigrostriatal lesion, in motor behaviour and 

neurogenesis. One aspect regards the identification of immununogenetic and inflammatory markers 

for early detection of neuronal damage, as indicators of disease onset and progression. In this 

ongoing research program integrating both in vivo findings with in vitro data and molecular 

screening in the search for ideal pharmacological targets, which include steroids, NSAIDs and 

SERMs, the overall data point to anti-inflammatory pathways amplifying drugs (AIPADs) as 

candidate disease modifying therapeutics. Cytokines variations in human peripheral blood 

monocytes (PBMC) or cytokine polymorphisms, which may influence susceptibility to neuronal 

damage, are also being actively investigated as diagnostic/prognostic indicators of disease 

progression [8,9]. In particular, early markers of neuronal deterioration may be exploited for 

identifying high-risk subpopulations and for assessing the usefulness of timely (combination) 

treatments [8,9]. Efforts aimed at identify integrated approaches will hopefully modify the natural 

course of the disease, by either preventing, slowing progression, or halting neuronal degeneration. 

Supported by Italian Ministry of Health (National Research Project RF-05/82) and Italian Ministry 

of Research. 

References List 

[1] Marchetti B and Abbracchio MP. To be or not to be (inflamed)--is that the question in anti-inflammatory drug 

therapy of neurodegenerative disorders? (2005) Trends Pharmacol Sci. Oct; 26(10):517-25. 

[2] Marchetti B, Kettenmann H, Streit WJ (Eds) (2005b) Glia-neuron crosstalk in neuroinflammation, 

neurodegeneration and neuroprotection. Brain Res Reviews Special Issue vol 48/2: 129-408. 

[3] Morale MC, Gallo F, Tirolo C, Testa N, Caniglia S, Marletta N, Spina-Purrello V, Avola R, Caucci F, Tomasi P, 

Delitala G, Barden N, Marchetti B. (2001) Neuroendocrine-immune (NEI) circuitry from neuron-glial interactions 

to function: Focus on gender and HPA-HPG interactions on early programming of the NEI system. Immunol Cell 

Biol. Aug 79(4):400-17. 

[4] Marchetti B, Serra PA, L’Episcopo F, Tirolo C, Caniglia S, Testa N, Cioni S, Gennuso F, Rocchitta G, Desole MS, 

Mazzarino MC, Miele E, Morale MC. (2005) Hormones are key Actors in gene x environment interactions 

programming the vulnerability to Parkinsons disease: Glia as a common final pathway. Ann. NY Acad. Sci.; 1057: 

296-318. 

[5] Morale MC, Serra PA, L'episcopo F, Tirolo C, Caniglia S, Testa N, Gennuso F, Giaquinta G, Rocchitta G, Desole 

MS, Miele E, Marchetti B. (2006) Estrogen, neuroinflammation and neuroprotection in Parkinson's disease: glia 

dictates resistance versus vulnerability to neurodegeneration. Neuroscience. 2006;138(3):869-78. Epub 2005 Dec 5. 

[6] Morale MC, Serra PA, Delogu MR, Migheli R, Rocchitta G, Tirolo C, Caniglia S, Testa N, L'Episcopo F, Gennuso 

F, Scoto GM, Barden N, Miele E, Desole MS, Marchetti B. (2004) Glucorticoid receptor deficiency increases 

vulnerability of the nigrostriatal dopaminergic system: critical role of glial nitric oxide. FASEB J. Jan;18(1):164-6. 

Epub 2003 Nov 20. 

[7] Marchetti B, Serra P-A, Tirolo C, L’Episcopo F, Caniglia S, Gennuso F, Testa N, Miele E, Desole MS, Barden N, 

Morale MC (2005). Glucocorticoid receptor-nitric oxide crosstalk and vulnerability to experimental Parkinsonism: 

pivotal role for glia-neuron interactions. Brain Res Reviews 48/2: 302-321. 

[8] Wirz SA, Morale MC, Marchetti B, Barr AM, Sotgiu S, Rosati G, Pugliatti M, Sanna MV, Giliberto O, Bartfai T, 

Conti B. (2004) High frequency of TNF alleles -238A and -376A in individuals from northern Sardinia. Cytokine 

26(4):149-54. 

[9] Sotgiu S, Zanda B, Marchetti B, Fois ML, Arru G, Pes GM, Salaris FS, Arru A, Pirisi A, Rosati G. Inflammatory 

biomarkers in blood of patients with acute brain ischemia. Eur J Neurol. 2006 May;13(5):505-13. 

14



EFFECTS OF SELECTIVE ESTROGEN RECEPTOR (ER) LIGANDS AND 

MODULATORS ON THE GONADOTROPIC AXIS 

Tena-Sempere M. 

Physiology Section, Department of Cell Biology, Physiology and Immunology. Faculty of 

Medicine, University of Córdoba. Avda. Menéndez Pidal s/n. 14004 Córdoba, Spain 

fi1tesem@uco.es 

The gonadotropic (or hypothalamic-pituitary-gonadal) axis is extremely sensitive to 

the regulatory actions of sex steroids during development and in adulthood [1]. Thus, 

acting during critical periods of maturation, estrogen is the molecular signal responsible for 

the sexual differentiation of the brain centers governing reproduction. In addition to these 

organizing effects, estrogen (either systemically-derived or locally-produced via 

aromatization of androgen) conducts a wide diversity of activational effects at the 

hypothalamus and pituitary; transient modulatory actions that contribute to the dynamic 

regulation of the gonadotropic axis at different physiologic and pathologic conditions. 

The complexity of the biological actions of estrogen upon the HPG axis is further 

determined by the diversity of mechanisms and receptors mediating these actions, which 

involve genomic and non-genomic (i.e., via surface receptors) effects, as well as 

interactions between classical (i.e., nuclear) and cell-surface signalling systems. Even 

concerning genomic actions, the effects of estrogen might be mediated, in a tissue-specific 

manner, by at least two major receptor isoforms (ERα and ERβ) with ability to interact 

with multiple co-modulators (either activators or repressors). This degree of complexity 

likely contributes to the plasticity of estrogen effects, at different levels of the HPG axis 

and during different stages of development. 

In order to provide a deeper insight into the mechanisms for the effects of estrogen in 

the control of key aspects of the development and function of the HPG axis, the 

consequences of in vivo administration of selective ER ligands (specific for ERα or ERβ) 

and modulators (SERMs) on different functional parameters of the gonadotropic system 

have been evaluated in our laboratory over the last years. Of note, these studies have 

targeted both organizing actions (i.e., administration during critical periods) and 

activational effects. 

Concerning developmental effects, we have evaluated the reproductive consequences 

of neonatal administration of the SERM raloxifene in the rat. The aims of these studies 

were two-fold: (i) to provide evidence for the mode of action of this SERM on the brain 

(i.e., estrogenic vs. anti-estrogenic), taking advantage of the well-known state of sensitivity 

to the effects of sex steroids during critical periods of development; and (ii) to illustrate on 

the complexity of estrogen actions in the control of related, but distinct, reproductive 

functions, such as the neuroendocrine regulation of gonadotropic secretion and sexual 

behaviour. Our results indicate that neonatal administration of raloxifene induces an array 

of reproductive defects both in male and female rats, which included delayed 

balanopreputial separation, reduced body weight and hyper-prolactinaemia in males, and 

advanced vaginal opening, decreased body weight, reduced gonadotropin secretion, 

blunted positive and negative feed-back between estradiol and LH, anovulation and 

infertility in females [2,3]. Such defects are grossly similar to those induced by neonatal 

administration of estradiol, thus suggesting an estrogen-like effect of raloxifene upon the 

functional organization of the gonadotropic axis. Interestingly, however, raloxifene did not 

act as an estrogen upon the organization of the system governing sexual receptivity in the 

female rat [4], thus reflecting a high degree of cell-specificity within the hypothalamus for 

15


the mechanisms of action of estrogen on different reproductive centers. Further studies on 

this phenomenon, evaluating the consequences of neonatal administration of estrogen, as 

well as selective ER ligands and modulators, on sensitive molecular markers, such as 

hypothalamic expression of Kiss1 gene, are currently in progress in our laboratory. 

Concerning activational effects, the impact of in vivo administration of selective ER 

ligands to adult animals on different molecular and functional parameters of the 

gonadotropic axis, at the pituitary and hypothalamic levels, has been evaluated at our 

laboratory. At the pituitary, the effects of short-term exposure to selective ERα (PPT) or 

ERβ (DPN) ligands on the expression of ER-related transcripts was studied in 

ovariectomized (OVX) rats. OVX resulted in increased pituitary expression of ERβ 

mRNA, but decreased TERP-1 and -2 levels without affecting those of ERα. Estradiol 

benzoate, as agonist of α and β forms of ER, fully reversed the responses to OVX; a 

phenomenon which was mimiked by PPT but not by DPN, which failed also to increase the 

pituitary expression of progesterone gene (sensitive marker for estrogen action) but evoked 

a moderate stimulation of TERP-1 and -2 mRNA levels. Of note, the SERM tamoxifen 

behaved as ERα agonist at the pituitary, albeit with a lower magnitude of responses [5]. In 

addition to expression tests, functional analyses suggested that ERα is the predominant 

subtype mediating the effects of estrogen on the negative feedback control of 

gonadotropins (likely via repression of Kiss1 gene expression at the arcuate nucleus), 

GnRH self-priming and stimulation of prolactin secretion. Intriguingly, the role of ERβ, 

which is the only ER expressed in GnRH neurons and is also present at the gonadotrope, in 

the control of the gonadotropic axis remains to be fully elucidated. 

In sum, we have summarized herein studies on the effects of in vivo administration of 

selective ER ligands and modulators on the maturation and function of the gonadotropic 

axis. These analyses have contributed to define the predominant mode of action (agonist 

vs. antagonist) of SERMs such as tamoxifen and raloxifene at the pituitary and 

hypothalamus, and have provided interesting clues for the receptor mechanisms mediating 

some of the pleiotropic effects of estrogen in the control of key aspects of reproductive 

function, from sexual differentiation to gonadotropin feedback control. 


[1] Tena-Sempere et al. Current Topics in Steroid Research (2000) 3:23-37. 

[2] Pinilla et al. Reproduction (2001) 121:915-924. 

[3] Pinilla et al. Journal of Endocrinology (2002) 172:441-448. 

[4] Pinilla et al. Neuroscience Letters (2002) 329:285-288. 

[5] Tena-Sempere et al. Biology of Reproduction (2004) 70:671-678. 

16

SATURDAY, 17 th February 2007 

15.00 - 18.00 


New perspectives in the dosage of neuroactive steroids


New perspectives in the dosage of neuroactive steroids 

(Organizers: Melcangi R.C., Mensah-Nyagan A.G.) 

• Schumacher M, Liere P, Labombarda F, De Nicola AF, Guennoun R, Baulieu EE 

(France) Analysis of steroids by gas chromatography/mass spectrometry: novel 

perspectives for understanding their significance in the nervous system. 

• Griffiths WJ, Wang, Y. (UK) Capillary Liquid Chromatography Combined with 

Tandem Mass Spectrometry for the Study of Neurosteroids and Oxysterols in Brain 

• Higashi T, Nagahama A., Ninomiya Y., Shimada K. (Japan) Analysis of stressinduced 

changes in rat brain neuroactive steroid levels using LC/MS coupled with 

derivatization. 

• Caruso D., Scurati S., Crotti S., Maschi O., Melcangi RC (Italy) Recent advances 

in liquid chromatography-mass spectrometry related to the evaluation of plasma 

and tissue levels of neuroactive steroids during neurodegenerative events 

• Reddy DS (USA) Mass Spectrometric Quantification and Physiological- 

Pharmacological Activity of Androgenic Neurosteroids 

• Romeo E, Rupprecht R., Pasini A., Manieri G., Bernardi G., Longone P. (Italy) 

Neurosteroids Determination in Biological Fluids and their Enzymes Expression in 

Lymphocytes of Patients with Neuropsychiatric Disorders



ANALYSIS OF STEROIDS BY GAS CHROMATOGRAPHY/MASS 

SPECTROMETRY: NOVEL PERSPECTIVES FOR UNDERSTANDING THEIR 

SIGNIFICANCE IN THE NERVOUS SYSTEM 

Schumacher M. 1 , Liere P. 1 , Labombarda F. 2 , De Nicola A.F. 2 , Guennoun R. 1 , and 

Baulieu E.E. 1 

(1) UMR 788 Inserm and Univ. Paris 11, 80 rue du Général Leclerc, 94276 Kremlin- 

Bicêtre, France. Fax : +33-1-45211940 e-mail :Michael.Schumacher@kb.inserm;fr. 

(2) Instituto de Biologia y Medicina Experimetal and University of Buenos Aires, 

Argentina. e-mail: denicola@dna.uba.ar 

The conventional view that the gonadal steroids just have reproductive functions, 

and that adrenal steroids only regulate fluid homeostasis, metabolic pathways and 

adaptative responses to stress, has completely changed over the past few years. A large 

number of experimental studies, most performed in rodents, have indeed demonstrated the 

multiple actions of progestagens, estrogens, androgens, glucocorticoids and 

mineralocorticoids throughout the nervous system, and their considerable influence on the 

functioning of neurons and glial cells. In particular their neurotrophic and neuroprotective 

effects have attracted much attention because of their therapeutic promises. In this respect, 

progesterone and its 5α-reduced metabolites are particularly interesting because they not 

only promote the viability and regeneration of neurons, but they also act on the 

myelinating glial cell and play an important role in the formation of myelin sheaths [6]. 

What makes steroids particularly attractive molecules for treating lesions and 

diseases of the nervous system is their property to easily cross the blood-brain and bloodnerve 

barriers and to very rapidly distribute throughout nervous tissues. In addition, some 

steroids can also be synthesized within the central nervous system (CNS) and the 

peripheral nervous system (PNS) by neurons and by glial cells. Steroids which are 

synthesized within the nervous system de novo from cholesterol have been named 

“neurosteroids” [1,2]. As both the endocrine glands and local production contribute to the 

pool of steroids present within nervous tissues, changes in their circulating levels do not 

necessarily reflect changes in their availability for neural cells. The physiological 

significance of neurosteroid synthesis in development, regeneration and aging is still 

poorly understood, and much remains to be done. 

Sensitive methods for the accurate analysis and quantification of steroids in plasma 

and nervous tissues have thus become a major issue. Indeed, the reliability of methods 

which continue to be used for the analysis of steroids remains a serious problem. This 

concerns both the methods for quantitative analysis, such as immunoassays, and the 

methods for pretreatment of the biological samples, including steroid extraction and 

separation. That is, specificity cannot be guaranteed even with carefully validated 

immunoassays, some chromatographic procedures for separating the different categories of 

steroids are questionable, and steroid sulfates are usually determined indirectly after 

removal of the sulfate group by solvolysis and subsequent analysis of the free steroids. The 

dangers of the latter method are well illustrated by our finding that the currently used solidphase 

extraction on C18 columns does not remove nonpolar steroid-containing lipids from 

the fraction which contains sulfated steroids. Moreover, it was shown that acidic solvolysis 

is not specific for 3β-hydroxy-Δ5 steroid sulfates and is able to release free steroids from 

other nonpolar components. As a consequence, sulfated steroids have been artefactually 

measured in rodent plasma and brain during decades [5]. 

19


Steroid sulfates can now be measured directly by liquid chromatography/mass 

spectrometry (LC/MS) and free steroids by gas chromatography/mass spectrometry 

(GC/MS) [4,5]. Advantages of these technologies are that : 1) they allow the chemical 

identification and accurate quantification of steroids ; 2) they are not dependent on the 

availability of antibodies with limited specificity ; 3) several steroids can be analyzed 

within a single tissue sample. 

We have also recently developed a new sample separation method coupled to 

GC/MS analysis for the quantification of sulfate esters of pregnenolone (PREGS) and of 

dehydroepiandrosterone (DHEAS) in the rat and human brain and plasma [5]. Using a 

solid-phase extraction recycling protocol, the results show that little or no PREGS and 

DHEAS is present in rat and mouse brain and plasma. These data are in agreement with 

studies in which steroid sulfates were analyzed without deconjugation. We suggest that 

discrepancies between analyses with and without deconjugation are caused by internal 

contamination of the brain extract fraction, supposed to contain steroid sulfates, by nonpolar 

components. However, in contrast to rodents, PREGS and DHEAS were shown to be 

present in human brain tissue. 

Analysis of free steroids by GC/MS has allowed us to provide reference values for 

steroid levels in the different compartments of the nervous system, and to show that an 

increase in the synthesis of neuroprogesterone and its 5α-reduced metabolites within the rat 

spinal cord is part of the mechanisms by which nerve cells cope with neurodegeneration 

[3]. In this study, it was shown that the levels of pregnenolone and progesterone are 

increased in the spinal cord of castrated and adrenalectomized male rats in response to 

transection. As the animals were deprived of their steroidogenic endocrine glands, these 

findings strongly suggest an increase in the local synthesis of the steroids. Moreover, 

increased levels of 5α-dihydroprogesterone and allopregnanolone were measured in the 

spinal cord of lesioned animals, whithout changes in their plasma levels, consistent with 

their local synthesis. 


[1] E.E. Baulieu, Neurosteroids: of the nervous system, by the nervous system, for the nervous system, 

Recent Prog. Horm. Res. 52 (1997) 1-32. 

[2] E.E. Baulieu, P.Robel, and M.Schumacher, Neurosteroids: beginning of the story, Int Rev Neurobiol 46 

(2001) 1-32. 

[3] F. Labombarda, A.Pianos, P.Liere, B.Eychenne, S.Gonzalez, A.Cambourg, A.F.De Nicola, 

M.Schumacher, and R.Guennoun, Injury elicited increase in spinal cord neurosteroid content analysed 

by gas chromatography mass spectrometry, Endocrinology 147 (2006) 1847-1859. 

[4] P. Liere, Y.Akwa, S.Weill-Engerer, B.Eychenne, A.Pianos, P.Robel, J.Sjovall, M.Schumacher, and 

E.E.Baulieu, Validation of an analytical procedure to measure trace amounts of neurosteroids in brain 

tissue by gas chromatography-mass spectrometry, J Chromatogr B 739 (2000) 301-312. 

[5] P. Liere, A.Pianos, B.Eychenne, A.Cambourg, S.Liu, W.Griffiths, M.Schumacher, J.Sjovall, and 

E.E.Baulieu, Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and absence of 

their sulfated counterparts in rodent brain, J Lipid Res 45 (2004) 2287-2302. 

[6] M. Schumacher, S.Weill-Engerer, P.Liere, F.Robert, R.J.Franklin, L.M.Garcia-Segura, J.J.Lambert, 

W.Mayo, R.C.Melcangi, A.Parducz, U.Suter, C.Carelli, E.E.Baulieu, and Y.Akwa, Steroid hormones 

and neurosteroids in normal and pathological aging of the nervous system, Prog Neurobiol 71 (2003) 3- 

29. 

20



CAPILLARY LIQUID CHROMATOGRAPHY COMBINED WITH TANDEM 

MASS SPECTROMETRY FOR THE STUDY OF NEUROSTEROIDS AND 

OXYSTEROLS IN BRAIN 

Griffiths W.J., and Wang Y. 

The School of Pharmacy, University of London, 29-39 Brunswick Square, WC1N 1AX, 

London, UK. Fax +44 20 7753 5964, e-mail: william.griffiths@pharmacy.ac.uk 

Cholesterol is present at a level of 7-8 microgram/milligram wet weight in human 

brain, where it is synthesised de novo, and corresponds to about 25% of the total body pool 

[1]. In brain, cholesterol is metabolised to oxysterols and neurosteroids. Both of these 

steroids are biologically active. Oxysterols can act as ligands to nuclear receptors e.g. liver 

X receptors (LXRs), while neurosteroids interact with neurotransmitter gated ion channels 

and modulate neural transmission. Neurosteroids exert their effects in seconds or 

milliseconds, in contrast to nuclear receptor whose physiological actions occur within 

hours or days. 

Oxysterols are present in brain at levels ranging from 20 nanogram/milligram to 30 

picogram/milligram, while neurosteroids are found at the picogram/milligram level. Both 

oxysterols and neurosteroids are present in brain in different isomeric forms, where 

different isomers have distinct biological activity. The low level and structural diversity of 

these cholesterol metabolites has made their analysis challenging. In this paper we will 

discuss the mass spectrometric (MS) methods we have employed for steroid analysis in 

brain and present new data recently obtained. 

In the early 1980’s Baulieu and colleagues reported the presence of steroid sulphates in rat 

brain [2,3]. It was subsequently found that these steroids act on neurotransmitter gated ion 

channels, e.g. pregnenolone sulphate inhibits GABA-mediated currents and enhances 

NMDA-activated currents in cultured rat hippocampal neurons [1]. Neutral steroids were 

also identified and found to be biologically active, e.g. allopregnanolone enhances 

GABAergic transmission and decreases NMDA transmission, while progesterone can bind 

to the progesterone receptor. In their early work on steroid sulphates, Baulieu and 

colleagues used GC-MS for steroid sulphate analysis after removal of the sulphate ester 

group. However, intact conjugate were not analysed, and recent data questions the earlier 

identification of steroid sulphates in brain [5]. 

Since the early 1980’s MS in many laboratories has shifted from GC-MS to LC-MS, which 

has the advantage of being able to analyse intact conjugates and give direct molecular 

weight information, and when in combination with MS/MS provide structural information. 

To maximise chromatographic performance and MS sensitivity we have applied a strategy 

based on miniaturised chromatography and low-flow rate electrospray (ES)-MS for steroid 

analysis in brain. Steroid sulphates are readily ionised by negative-ion ES and give 

informative MS/MS spectra with high sensitivity (0.1 pg on column detection limit) [6]. 

Neutral steroids can be more difficult to analyse by ES-MS/MS. Although steroids with a 

3-oxo-4-ene structure e.g. progesterone and testosterone, can be analysed with high 

sensitivity by positive-ion ES-MS, steroids with less basic functional groups e.g. 

dehydroepiandrosterone, pregnenolone, are ionised poorly. To counter this problem, we 

have incorporated a derivatisation step to our analytical method. Initially, we derivatised 

oxosteroids with hydroxylamine to give steroid oximes, which are more readily ionised 

than their underivatised analogues [6]. Now we use a different derivatisation reagent, the 

Girard P (GP) hydrazine reagent, which adds greater sensitivity to the assay and provides 

more informative fragmentation spectra. Using the GP derivative neurosteroids can be 

21


analysed on the sub-picogram level [4]. When derivatised with the GP reagent, 

neurosteroids are suitable for analysis at high sensitivity by both MALDI-MS and LC-ES- 

MS. 

24S-Hydroxycholesterol is the major cholesterol metabolite found in brain [7]. This 

oxysterol is difficult to analyse by LC-ES-MS or MALDI-MS, however by using 

microchemical oxidation and derivatisation with the GP reagent, this oxysterol and 

analougous metabolites can be analysed with high sensitivity [8]. Using a combination of 

oxidation and derivatisation we have been able to identify a series of dihydroxycholesterols 

with structures of potential ligands to LXRbeta, a nuclear receptor expressed in neurons. 


[1] M.R. Bowlby, Pregnenolone sulfate potentiation of N-methyl-D-aspartate receptor channels in 

hippocampal neurons, Mol. Pharmacol. 43 (1993) 813-9. 

[2] C. Corpéchot, P. Robel, M. Axelson, J. Sjövall, E.E. Baulieu, Characterization and 

measurement of dehydroepiandrosterone sulfate in rat brain, Proc. Natl. Acad. Sci. U S A. 78 

(1981) 4704-7. 

[3] C. Corpéchot, M. Synguelakis, S. Talha, M. Axelson, J. Sjövall, R. Vihko, E.E. Baulieu, P. 

Robel, Pregnenolone and its sulfate ester in the rat brain, Brain Res. 270 (1983) 119-25. 

[4] W.J. Griffiths, S. Liu, G. Alvelius, J. Sjövall, Derivatisation for the characterisation of neutral 

oxosteroids by electrospray and matrix-assisted laser desorption/ionisation tandem mass 

spectrometry: the Girard P derivative, Rapid Commun. Mass Spectrom. 17 (2003) 924-35. 

[5] P. Liere, A. Pianos, B. Eychenne, A. Cambourg, S. Liu, W. Griffiths, M. Schumacher, J. 

Sjövall, Baulieu E.E. Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone 

and absence of their sulfated counterparts in rodent brain, J. Lipid Res. 45 (2004) 2287-302. 

[6] S. Liu, J. Sjövall, W.J. Griffiths, Neurosteroids in rat brain: extraction, isolation, and analysis 

by nanoscale liquid chromatography-electrospray mass spectrometry, Anal. Chem. 75 (2003) 

5835-46. 

[7] D. Lutjohann, Cholesterol metabolism in the brain: importance of 24S-hydroxylation, Acta 

Neurol. Scand. Suppl. 185 (2006) 33-42. 

[8] Y. Wang, M. Hornshaw, G. Alvelius, K. Bodin, S. Liu , J. Sjövall , W.J. Griffiths. Matrixassisted 

laser desorption/ionization high-energy collision-induced dissociation of steroids: 

analysis of oxysterols in rat brain. Anal. Chem. 78 (2006) 164-73. 

22



ANALYSIS OF STRESS-INDUCED CHANGES IN RAT BRAIN NEUROACTIVE 

STEROID LEVELS USING LC/MS COUPLED WITH DERIVATIZATION 

Higashi T., Nagahama A., Ninomiya Y., and Shimada K. 

Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa 

University, Kakuma-machi, Kanazawa, Japan. Fax +81-76-234-4459 e-mail: higashi@p.kanazawa-u.ac.jp 

Neuroactive steroids possessing anxiolytic and anti-stress properties act through 

membrane receptors, mostly the γ-aminobutyric acid type A (GABA A ) receptor complex. 

For example, allopregnanolone (AP) positively modulates the action of GABA at these 

receptors to raise the threshold of brain excitability during the response to stressful stimuli. 

It is also known that the endogenous AP level in the animal brain is rapidly elevated from 

the trace level (practically none) to the ng/g tissue level by several acute stress paradigms. 

Thus, the stress-induced changes in the brain levels of AP and also its precursors have been 

fairly well studied and a great deal of knowledge has been accumulated regarding this 

matter. However, the stress-induced change in the brain and serum levels of 

epiallopregnanolone (EAP), the 3β-isomer of AP, has been poorly studied, although it can 

antagonize the GABAergic function of AP in rats [1]. Furthermore, several papers 

described that some androstane steroids, such as testosterone (T), elicit the anesthetic and 

anxiolytic effects in animal models [2], but their brain levels have not been elucidated. 

Based on this background information, we performed the following two studies; 1) 

analysis of the stress-induced level change of EAP in the rat brain and serum, and 2) 

elucidation of the influence of acute stress on the T and 5α-dihydrotestosterone (DHT) 

levels in the rat brain and serum. In order to clarify these matters, we chose LC/ESI- 

MS/MS as the analytical methodology. 

1. Analysis of the stress-induced level change of EAP in the rat brain and serum 

We began this study by developing the LC/ESI-MS/MS method for the simultaneous 

determination of AP, EAP and their precursor, 5α-dihydroprogesterone (DHP). The 

biggest problem encountered in the ESI-MS measurement of these 5α-reduced steroids is 

the lack of sensitivity; around 100 pg of steroids were required to obtain a signal to noise 

ratio of 5. To overcome this problem, we introduced the derivatization with a permanently 

charged reagent, 2-hydrazine-1-methylpyridine (HMP) [3]. This reagent quantitatively 

reacts with steroids having an oxo-group to give the ESI-active derivatives. The derivatives 

provided only their molecular cations, [M] + , with a high intensity in the positive-ESI-MS 

and also provided a characteristic product ion at m/z 108 in MS/MS. When the SRM mode 

with the transition from [M] + to this product ion was employed, the low femtomole level of 

the derivatives could be readily detected. The developed method was then applied to the 

animal experiment. Rats were divided into two groups, control (n=10) and immobilization 

stressed rats (n=10), and the latter was immobilized on their backs on a board for 20 min 

and then the brain and serum were collected. The brain homogenate corresponding to 20 

mg of tissue or a 20 µl of serum was purified using a Strata-X cartridge and the 

neuroactive steroid fraction was then treated with HMP. In the brain of the stressed rats, 

the concentrations of AP, EAP and DHP were 1.74±0.71, 0.58±0.30 and 2.74±1.12 ng/g 

tissue, respectively, but the levels in the control group were less than the quantitation limit 

(0.25 ng/g tissue). The serum AP (1.31±0.72 ng/ml) and DHP (0.94±0.36 ng/ml) levels 

were also drastically elevated by the stress (control: not detected), while EAP was not 

detected in the serum even in the stressed rats. In conclusion, this study demonstrated that 

23


the brain EAP level was rapidly elevated by immobilization stress like the AP and DHP 

levels. The study also found that EAP is the brain-specific product. 

2. Influence of acute stress on the T and DHT levels in rat brain and serum 

We first analyzed the stress-induced level change of T. Because T has the ESI-responsive 

3-oxo-4-ene structure, its quantification was performed without derivatization. As a result, 

we could not find a recognizable change in the brain T level between the control and 

stressed rats. There was also no statistical difference in the serum T level between the two 

groups. However, due to the large individual differences in the experimental animals, we 

could not develop a clear answer to the question of whether or not the T level is influenced 

by the stress. The fact that most of T found in the brain is derived from the peripheral 

organs [4] prompted us to use the ratio of the brain T concentration to the serum T 

concentration (BT/ST) as an alternative index for the more detailed analysis of the stressinduced 

level change in T. The BT/ST value in the stressed rat was significantly higher 

than that of the control rat. Thus, we found that the T level is also influenced by the acute 

stress. Although several mechanisms for the elevation of the BT/ST value due to the stress 

can be imagined, we thought that the most probable mechanism is the suppression of the T 

metabolism in the brains by the stress. T is metabolized into DHT by the catalysis of 5αreductase. 

The affinity of 5α-reductase is higher for progesterone than for T, thus 

progesterone may competitively inhibit the T metabolism, which may cause the decline of 

the DHT level and accumulation of T in the brain. To prove this point, we measured the 

brain and serum DHT using LC/ESI-MS/MS after conversion to its HMP derivative. The 

measured values obtained from a 100 mg of brain tissue revealed that there was no 

significant difference in the DHT level between the control (0.71±0.59 ng/g tissue) and 

stressed rats (0.58±0.39 ng/g tissue). In the serum, the DHT peak was observed neither in 

the control nor in the stressed rats. These results show that the brain always synthesizes a 

fair amount of DHT. Moreover, when the brain T concentration and DHT concentration 

were plotted on a scatter diagram, they lay almost on a straight line. These data 

demonstrate that the brain DHT level is not influenced by the stress and depends on the 

brain T level. Thus, our forecast that the suppression of the T metabolism in the brain 

causes elevation of the BT/ST value was found to be wrong. In conclusion, we found that 

the BT/ST value was significantly elevated by the immobilization stress, although the 

mechanism for this phenomenon is still unclear. 


[1] Bäckström T., Wahlström G., Wahlström K., Zhu D., Wang M.-D., 2005. 

Isoallopregnanolone; an antagonist to the anaesthetic effect of allopregnanolone in 

male rats. Eur. J. Pharmacol. 512, 15–21. 

[2] Edinger K.L., Frye C.A., 2005. Testosterone’s anti-anxiety and analgesic effects may 

be due in part to actions of its 5α-reduced metabolites in the hippocampus. 

Psychoneuroendocrinology 30, 418–430. 

[3] Higashi T., Yamauchi A., Shimada K., 2005. 2-Hydrozine-1-methylpyridine: a highly 

sensitive derivatization reagent for oxosteroids in liquid chromatography-electrospray 

ionization-mass spectrometry. J. Chromatogr. B 825, 214–222. 

[4] Alomary A.A., Vallée M., O’Dell L.E., Koob G.F., Purdy R.H., Fitzgerald R.L., 2003. 

Acutely administered ethanol participates in testosterone synthesis and increases 

testosterone in rat brain. Alcohol. Clin. Exp. Res. 27, 38–43. 

24



RECENT ADVANCES IN LIQUID CHROMATOGRAPHY-MASS 

SPECTROMETRY RELATED TO THE EVALUATION OF PLASMA AND 

TISSUE LEVELS OF NEUROACTIVE STEROIDS DURING 

NEURODEGENERATIVE EVENTS 

Caruso D.# 1 , Scurati S. 1 , Crotti S. 1 , Maschi O. 1 and Melcangi R.C. 2 

1 Dept. of Pharmacological Sciences, 2 Dept. of Endocrinology, University of Milano, 

Milano, Italy. 

#Presenting author: Dept. of Pharmacological Sciences, University of Milano, via 

Balzaretti 9, 20133, Milano Italy, donatella.caruso@unimi.it 

Nervous system represents an important target for the action of neuroactive 

steroids. Indeed, these molecules exert a broad spectrum of actions regulating 

neuroendocrine events, reproduction, feeding, behavior etc. Moreover, as recently 

demonstrated, neuroactive steroids also exert important protective effects at the level of 

central (CNS) and peripheral nervous system (PNS), suggesting that they might represent a 

new therapeutic strategy to counteract neurodegenerative events. In this context, to know 

the levels of the neuroactive steroids present in nervous structures and how these are 

modified during neurodegenerative events is absolutely necessary. This means the set up of 

a sensitive and specific methodology that gives a complete information of steroid levels in 

target nervous structures and in plasma. The advent of robust and analytically reliable 

techniques based on the combination of liquid chromatography (LC) and mass 

spectrometry (MS) by means of atmospheric pressure ionization (API) techniques (e.g., 

electrospray, ESI and atmospheric pressure chemical ionization, APCI) and in particular 

the improvements brought about by tandem MS (MS/MS), has opened new perspectives in 

terms of mass spectrometric identification and quantification of steroids that are difficult to 

analyse by gas chromatography-MS. With respect to the ionization mode, APCI is mainly 

applied to rather less polar compounds than ESI but is less susceptible to ion suppression 

due to the presence of several interferences as in biological tissues. For quantitative assays 

employing MS detection, triple quadrupole systems are most commonly used, while the 

new generation of ion trap, namely the linear trap, exerts similar performance, as 

demonstrated also by our results. In addition, when an APCI-linear trap is operated in the 

MS/MS mode, the identification and quantification of the analyte are based on both 

precursor and product ions, giving higher selectivity and best sensitivity than for any other 

MS system. On these remarks, we set up an analytical method mass spectrometry-based for 

the identification and quantification of pregnenolone (PREG), progesterone (PROG), 

dihydroprogesterone (DHP), tetrahydroprogesterone (THP), testosterone (T), 

dihydrotestosterone (DHT), 5α-androstan-3α17β-diol (3α-diol), and 17β estradiol (17β- 

E2). After validation of the HPLC-APCI-MS/MS procedure, the method was applied to the 

detection and determination of these neuroactive steroids in plasma and nervous structures 

of control rat and we have compared these levels to what occurring in an experimental 

model of neuropathy, such as streptozotocin (STZ)-induced diabetes. Indeed it is well 

known, that diabetes is associated to a spectrum of functional and structural changes in 

PNS and CNS. 

Neuroactive steroids in plasma, in cerebellum (representative of CNS) and in brachial 

nerves (representative of PNS) were extracted by solid phase technique and quantified by 

means of calibration curves using deuterated internal standards, since they are expected to 

possess similar ionization efficiencies as the correspondent analyte. Our results indicate 

that all neuroactive steroids here considered are present in the two nervous structures and 

25


in plasma, with the exception of THP and DHT, which in plasma are under the detection 

limit. Interestingly, tissue and plasma levels of these neuroactive steroids are significantly 

modified in the experimental model of STZ-induced diabetes. In particular, PREG, PROG 

and T levels are significantly decreased in cerebellum. The same trend is shown by their 

metabolites without reaching statistical significance. Moreover, in brachial nerves, not only 

PREG and T, but also metabolites of PROG (i.e., DHP and THP) and of T (i.e., DHT and 

3α-diol) show a significant decrease. Furthermore, our data indicate that diabetes causes 

alterations in the plasma levels of steroids that, in some cases, do not completely reflect the 

changes observed in nervous structures. For instance, in contrast to what observed in 

nervous structures, plasma PREG, DHP and DHT levels are unaffected by diabetes while 

3α-diol levels show a significant increase. 

Taken together these data demonstrate that LC-MS/MS method allows the assessment of 

neuroactive steroids in plasma and in structures of central and peripheral nervous system 

with high sensitivity and specificity and that neurodegenerative processes affect their 

levels. 

26



MASS SPECTROMETRIC QUANTIFICATION AND PHYSIOLOGICAL- 

PHARMACOLOGICAL ACTIVITY OF ANDROGENIC NEUROSTEROIDS 

Doodipala S.R. 

Department of Molecular Biomedical Sciences, North Carolina State University College of 

Veterinary Medicine, Raleigh, NC 27606,USA. 

Testosterone modulates seizure susceptibility in animals and humans, but the underlying 

mechanisms are obscure. Here, I present evidence that testosterone-derived “androgenic 

neurosteroids”, 3α-androstanediol and 17β-estradiol, mediate the testosterone effects on 

neural excitability and seizure susceptibility. The presentations will primarily be focused 

on: (1) development and validation of mass spectrometric determination of the androgenic 

neurosteroid 3α-androstanediol; and (2) investigations on the molecular mechanisms 

underlying the testosterone modulation of seizure susceptibility in animals models of 

epilepsy. 

Although 3α-androstanediol (5α-androstane-3α,17-diol) could act as a key 

neuromodulator in the brain, there is no specific and sensitive assay for quantitative 

determination of the 3α-androstanediol in biological samples. We have established a liquid 

chromatography-tandem mass spectrometry assay to measure 3α-androstanediol in rat 

plasma. Standard 3α-androstanediol added to rat plasma has been successfully analysed 

with excellent linearity, specificity, sensitivity, and reproducibility. 

Testosterone modulation of seizure susceptibility is hypothesized to occur through its 

conversion to neurosteroids with “anticonvulsant” and “proconvulsant” actions, and hence 

the net effect of testosterone on neural excitability and seizure activity depends on the 

levels of distinct testosterone metabolites. Testosterone undergoes metabolism to 

neurosteroids via two distinct pathways. Aromatization of the A-ring converts testosterone 

into 17β-estradiol. Reduction of testosterone by 5α-reductase generates 5αdihydrotestosterone, 

which is then converted to 3α-androstanediol, a powerful GABA A 

receptor-modulating neurosteroid with anticonvulsant properties. Systemic doses of 

testosterone decreased seizure threshold in rats and increased the incidence and severity of 

pentylenetetrazol-induced seizures in mice. These proconvulsant effects of testosterone 

were associated with increases in plasma 17β-estradiol and 3α-androstanediol 

concentrations. The 5α-reduced metabolites of testosterone, 5α-dihydrotestosterone and 

3α-androstanediol, had powerful anticonvulsant activity. 

The 3α-androstanediol assay is an important tool in this area because of the growing 

interest in the potential to use adjuvant hormonal therapy to improve treatment of epilepsy. 

The testosterone-derived neurosteroids 3α-androstanediol and 17β-estradiol could 

contribute to the net cellular actions of testosterone on neural excitability and seizure 

susceptibility. Because of its powerful GABA-A receptor-modulating properties, the 

androgenic neurosteroid 3α-androstanediol is proposed as an endogenous modulator of 

seizure susceptibility in men with epilepsy. 

Acknowledgements: Supported partly by NC State CVM grant. 

27


NEUROSTEROIDS DETERMINATION IN BIOLOGICAL FLUIDS AND THEIR 

ENZYMES EXPRESSION IN LYMPHOCYTES OF PATIENTS WITH 

NEUROPSYCHIATRIC DISORDERS 

Romeo E. a,b , Rupprecht R. d , Pasini A. a , Manieri G. c , Bernardi G. a,b , and Longone P. b 

a Department of Neuroscience Tor Vergata University; b Experimental Neurology Santa 

Lucia Foundation; c Clinica Neurologica, La Sapienza University, Rome Italy; d Department 

of Psychiatry Ludwig Maximilian University, Munich Germany. 

Steroids action is on the basis of concentration, as well as the specificity of tissue 

receptors. Steroids affect several different types of body processes, that taken together 

lead to a highly specific function. This might explain how steroids came to symbolize 

precise body’s events and necessities. 

In the nervous system (NS) steroids elicit important glial and neuronal cell responses by 

stimulating gene expression and by modulating different neurotransmitters receptors [2]. 

Nevertheless a precise physiological role in the NS has not yet been established. We and 

others have found that steroids levels can be altered in many neuropsychiatric and 

neurodegenerative disorders as: depression, anxiety, schizophrenia, panic disorder and 

Alzheimer [11,12,13,15]. This indicate that their dysregulation it is maybe related to 

psychopathological dimensions present in different pathologies more than being linked to a 

particular syndrome. Central and peripheral fluids show the same concentrations trend of 

steroids and one of the reasons is that the most of them can easily cross the blood brain 

barrier allowing steroids in the two compartments to reach an equilibrium [5]. 

Yet, if we consider the biosynthesis of steroids, there are some differences between the 

periphery and the NS: i) in the NS, steroids biosynthesis is not restricted to specialized 

brain areas or cells endowed of their synthesis, as it happens in the peripheral endocrine 

organs; ii) steroids, in the NS, can have alternative pathways, like the biosynthesis of 

cholesterol [3]; iii) the enzymes isoforms, expressed in the NS, are often present 

periferally, as in the case of 3alpha hydroxysteroidehydrogenase (3HSD) [14], that rapidly 

clears steroids from the circulation, differently from the NS where 3HSD synthetizes 

3alpha,5alpha tetrahydroprogesterone (THP) [9], one of the most potent modulator of 

numerous neurotransmitter receptors such as GABA A receptors [8]. Therefore, the 

expression of steroidogenic enzymes and the synthesis of steroids in the NS seems to relay 

either on the crosstalk with the periphery, and on a direct communication between the NS 

cells. Thus, to relate steroids quantification and their enzymes expression, we have 

quantified steroids involved in the GABAergic system, in cerebro spinal fluid (CSF) and 

plasma of parkinsonian patients along with the mRNA of their enzymes and isoforms in 

peripheral blood mononuclear cells (PBMC) [4,7]. We have measured the steroids 

[progesterone (PROG), 3alpha, 5alpha THP and 3beta, 5alpha THP, 5alpha 

dihydroprogesterone (DHP) and dehydroepiandrosterone(DHEA)] in CSF and plasma of 

parkinsonian patients and age matched controls with the GC/MS technique [4]. Moreover 

we have quantified by means of comparative RT-PCR the mRNA expression of the 

enzymes 5alpha reductase type I (mainly central), 3HSD type I (mainly peripheral) and II 

(mainly central) [10] and the peripheral benzodiazepine receptor (PBR) [7]. As a source of 

mRNA, for the quantification of these enzymes, we have used PBMC because they express 

both central and peripheral isoforms of many neurosteroidogenic enzymes [6,16] and of 

many receptors such as GABA A [1]. Steroids levels had a similar trend in plasma and CSF 

of parkinsonian patients treated with L-DOPA, showing a decrease of 3alpha, 5alpha THP 

and 5alpha DHP and unchanged levels of PROG, 3beta, 5alpha THP and DHEA. The 

28



5alpha reductase expression was decreased, while PBR expression was unchanged. 

Interestingly the two 3HSD isoforms behaved differently, and while the mRNA expression 

of HSD I was unchanged compared to control, the HSD II was increased. Our data indicate 

that the enzymes isoforms present also in the NS are differently regulated from those only 

peripheral and further studies are required. 

In conclusion we consider important to evaluate steroids levels in biological fluids along 

with the expression of their biosynthetic enzymes, in order to have a more inclusive 

comprehension of the role that steroids may have in the NS and in the cross talk with the 

periphery. 


[1] Alam S, Laughton DL, Walding A, Wolstenholme AJ., 2006. Human peripheral blood mononuclear 

cells express GABAA receptor subunits. Mol Immunol. 43, 1432-42. 

[2] Baulieu EE, Robel P, Schumacher M., 2001. Neurosteroids: beginning of the story. Int Rev 

Neurobiol.46,1-32. 

[3] Bjorkhem, I., Meaney, S., 2004. Brain cholesterol: long secret life behind a barrier. Arterioscler Thromb 

Vasc Biol. 24, 806-815. 

[4] di Michele F, Longone P, Romeo E, Lucchetti S, Brusa L, Pierantozzi M, Bassi A, Bernardi G, 

Stanzione P., 2003. Decreased plasma and cerebrospinal fluid content of neuroactive steroids in 

Parkinson's disease. Neurol Sci. 24, 172-3. 

[5] Dubrovsky, B., 2006. Neurosteroids, neuroactive steroids, and symptoms of affective disorders. 

Pharmacol Biochem Behav. 84, 644-655. 

[6] Leb C.R., Hu F..Y, Murphy B.E., 1997. Metabolism of progesterone by human lymphocytes: 

production of neuroactive steroids. J Clin Endocrinol Metab 82, 4064-8 

[7] Luchetti S., di Michele F., Romeo E., Brusa L., Bernardi G., Cummings B.J., Longone P., 2006. 

Comparative non-radioactive RT-PCR assay: an approach to study the neurosteroids biosynthetic 

pathway in humans. J Neurosci Methods. 153, 290-8. 

[8] Magnaghi V, Ballabio M, Consoli A, Lambert JJ, Roglio I, Melcangi RC., 2006. GABA receptormediated 

effects in the peripheral nervous system: A cross-interaction with neuroactive steroids. J Mol 

Neurosci. 28, 89-102. 

[9] Martini L., Celotti F., Melcangi R.C., 1996. Testosterone and progesterone metabolism in the central 

nervous system: cellular localization and mechanism of control of the enzymes involved. Cell Mol 

Neurobiol. 16, 271-82. 

[10] Penning TM, Burczynski ME, Jez JM, Hung CF, Lin HK, Ma H, Moore M, Palackal N,Ratnam 

K.,2000. Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto 

reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and 

formation of male and female sex hormones. Biochem J. 351, 67-77. 

[11] Romeo E., Strohle A., Spalletta G., di Michele F., Hermann B., Holsboer F., Pasini A.,Rupprecht R., 

1998. Effects of antidepressant treatment on neuroactive steroids in major depression. Am J Psychiatry 

55, 910-3. 

[12] Schaeffer V, Patte-Mensah C, Eckert A, Mensah-Nyagan AG., 2006. Modulation of neurosteroid 

production in human neuroblastoma cells by Alzheimer's disease key proteins. J Neurobiol. 66, 868-81. 

[13] Schumacher M, Weill-Engerer S, Liere P, Robert F, Franklin RJ, Garcia-Segura LM, Lambert JJ, Mayo 

W, Melcangi RC, Parducz A, Suter U, Carelli C, Baulieu EE, Akwa Y., 2003. Steroid hormones and 

neurosteroids in normal and pathological aging of the nervous system. Prog Neurobiol. 71 ,3-29. 

[14] Stoffel-Wagner, B. 2001. Neurosteroid metabolism in the human brain. Eur J Endocrinol. 145, 669-679. 

[15] Strohle A., Romeo E., di Michele .F, Pasini A., Hermann B., Gajewsky G., Holsboer F., Rupprecht R., 

2003. Induced panic attacks shift gamma-aminobutyric acid type A receptor modulatory neuroactive 

steroid composition in patients with panic disorder: preliminary results. Arch Gen Psychiatry. 60, 161-8. 

[16] Zhou Z., Shackleton C.H., Pahwa S., White P.C., Speiser P.W., 1998. Prominent sex steroid metabolism 

in human lymphocytes. Mol Cell Endocrinol. 138, 61-9. 

29

SUNDAY, 18 th February 

12.00 - 13.00 

Plenary Lecture: 

Herbison A.E. (Dunedin, New Zealand)



ESTROGEN REGULATION OF GONADOTROPIN-RELEASING HORMONE 

NEURONS 

Herbison A.E., Porteous R., Clarkson J., Romano N., and Campbell R.E. 

Centre for Neuroendocrinology and Department of Physiology, University of Otago, 

Dunedin, New Zealand. Fax +64-34797323 e-mail: allan.herbison@otago.ac.nz 

Estrogen exerts critical feedback actions upon multiple neuronal networks within 

the brain. One of the most important targets for estrogen is the gonadotropin-releasing 

hormone (GnRH) neuronal network that controls pituitary gonadotropin secretion and, 

thus, fertility, in all mammalian species. However, the GnRH cell bodies exhibit a 

scattered topography within the basal forebrain and this has greatly hindered progress in 

understanding their biology. Recent transgenic approaches, targeting GnRH neurons with 

fluorescent and calcium-sensitive reporter molecules, have been of benefit in 

characterizing the cellular and molecular characteristics of adult GnRH neurons in situ. 

Furthermore, mice with cell type-specific knockouts of selective receptors have been of 

great use in refining information obtained from earlier global knockout mice. In addition, 

exciting new transgenic approaches are now being used to define primary and higher-order 

inputs to GnRH neurons within the GnRH neuronal network. The use of these transgenic 

approaches in elucidating the mechanisms of estrogen feedback regulation of GnRH 

neurons will be presented. Data from global and neuron-specific estrogen receptor (ER) 

mutant mice demonstrate that ERalpha-expressing neurons are critical for estrogen positive 

feedback. As GnRH neurons do not express ERalpha, estrogen feedback must occur 

through ERalpha-expressing neuronal afferents to GnRH neurons. Experiments using Credependent 

Pseudorabies virus in GnRH-Cre transgenic mice to trace these afferents, 

indicate that ERalpha-expressing neurons innervating GnRH neurons are located 

predominantly in the periventricular preoptic area [1]. On-going electrophysiological and 

imaging studies suggest that neurons expressing the newly discovered neuropeptide, 

kisspeptin, provide a critical estrogen-sensitive neuronal population regulating the activity 

of GnRH neurons 


[1] T.M. Wintermantel, R.E. Campbell, R. Porteous, D. Bock, H.J. Grone, M.G. 

Todman, K.S. Korach, E. Greiner, C.A. Perez, G. Schutz and A.E. Herbison, 

Definition of estrogen receptor pathway critical for estrogen positive feedback to 

gonadotropin-releasing hormone neurons and fertility, Neuron 52 (2006) 271-280. 

33


15.00 - 18.00 

Symposium: 

Effects mediated by classical steroid receptors

Symposium: 

Effects mediated by classical steroid receptors 

(Chairs: Mani S., Tena-Sempere M.) 

• Smith J (Australia) Steroid regulation of kisspeptin signalling in the brain 

• Bass AH (USA) Steroid-dependent modulation of vocal motor systems 

• Handa RJ (USA) Estrogen receptor beta in the brain: from form to function 

• Bodo C, Rissman EF (USA) A role for the androgen receptor in the sexual 

differentiation of the olfactory system in mice. 

• Ishunina T.A., Swaab D.F. (Russia) Canonical and alternatively spliced 

estrogen receptor α in the human mamillary bodies and hippocampus in aging 

and alzheimer’s disease 

• Klein S., Grossmann R. (Germany) Female specific activation of galanin in the 

supraoptic nucleus of hens after oviposition related upregulation of arginine 

vasotocin (AVT) 

• Sica M., Martini M., Verzè L., Viglietti-Panzica C., Panzica G.C. (Italy) 

Expression of nitric oxide synthase in the male mouse limbic system is mediated 

by estrogen receptors.



STEROID REGULATION OF KISSPEPTIN SIGNALLING IN THE BRAIN 

Smith J.T. 

Department of Physiology, Monash University, Building 13F, Clayton, Victoria 3800, 

Australia. E-mail: jeremy.smith@med.monash.edu.au Fax: +61 3 99052547 

The KiSS-1 gene encodes a family of peptides called kisspeptins. Post-translational 

processing of an initial 145 amino acid peptide results in the formation of smaller C- 

terminal peptides (kisspeptin-54, -14, -13 and -10), which activate with equal efficacy the 

G protein-coupled receptor, GPR54. In both humans and mice, inactivating mutations in 

GPR54 result in the failure to initiate puberty and hypogonadotropic hypogonadism [1,7], 

indicating that these peptides play a vital role in the regulation of GnRH secretion. In many 

species, centrally administered kisspeptin stimulates gonadotrophin secretion in a GnRH 

dependant manner [4]. Moreover, virtually all GnRH neurons co-express GPR54 [5,6]. In 

the hypothalamus, the vast majority of kisspeptin producing cells (those expressing KiSS-1 

mRNA) also express sex steroid receptors, particularly oestrogen receptor alpha [9]. Thus, 

sex steroids are able to directly regulate the expression of KiSS-1 mRNA, implicating 

kisspeptin as the missing link between sex steroids and GnRH feedback. Kisspeptin 

producing neurons (or KiSS-1 neurons) have been localised to various regions of the 

forebrain in rodents, primates and most recently sheep [2-4,8]. In the arcuate nucleus 

(ARC) of the rodent, sex steroids inhibit the expression of KiSS-1 mRNA, suggesting that 

the kisspeptin secreting neurons here are the conduit for the negative feedback regulation 

of GnRH secretion [9]. However, in the anteroventral periventricular nucleus (AVPV), sex 

steroids induce the expression of KiSS-1 mRNA, implying that these kisspeptin neurons 

play a role in the positive feedback regulation of rodent GnRH secretion [9]. In sheep, 

KiSS-1 neurons appear robustly within the ARC, and a smaller population is present in the 

preoptic area (POA)[2,3]. Recent studies in the ewe demonstrate that KiSS-1 mRNA in the 

ARC is inhibited by oestrogen (see Figure 1.) and progesterone, but is stimulated 

immediately prior to the preovulatory luteinising hormone surge. Interestingly, the AVPV 

of the ewe is void of KiSS-1 mRNA expression and the population of KiSS-1 neurons in 

the POA is not regulated by sex steroids. Thus, kisspeptin neurons in the ovine ARC 

appear well placed to play a role in the negative and positive feedback regulation of GnRH 

exerted by sex steroids. 

Intact 

OVX 

OVX+E 

3V 

3V 

3V 

Figure 1 

Dark-field photomicrographs of the ovine ARC showing KiSS-1 mRNA-expressing cells 

(as shown by the presence of silver grain clusters) from gonad-intact, ovariectomised 

(OVX), and OVX & oestrogen replacement (E) ewes. 3V = third ventricle. 

37



[1] de Roux, N., Genin, E., Carel, J.C., Matsuda, F., Chaussain, J.L. and Milgrom, E., 

Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor 

GPR54, Proc Natl Acad Sci U S A, 100 (2003) 10972-6. 

[2] Estrada, K.M., Clay, C.M., Pompolo, S., Smith, J.T. and Clarke, I.J., Elevated KiSS-1 Expression in 

the Arcuate Nucleus Prior to the Cyclic Preovulatory Gonadotrophin-Releasing 

Hormone/Lutenising Hormone Surge in the Ewe Suggests a Stimulatory Role for Kisspeptin in 

Oestrogen-Positive Feedback, J Neuroendocrinol, 18 (2006) 806-9. 

[3] Franceschini, I., Lomet, D., Cateau, M., Delsol, G., Tillet, Y. and Caraty, A., Kisspeptin 

immunoreactive cells of the ovine preoptic area and arcuate nucleus co-express estrogen receptor 

alpha, Neurosci Lett, 401 (2006) 225-30. 

[4] Gottsch, M.L., Cunningham, M.J., Smith, J.T., Popa, S.M., Acohido, B.V., Crowley, W.F., 

Seminara, S., Clifton, D.K. and Steiner, R.A., A role for kisspeptins in the regulation of 

gonadotropin secretion in the mouse, Endocrinology, 145 (2004) 4073-7. 

[5] Han, S.K., Gottsch, M.L., Lee, K.J., Popa, S.M., Smith, J.T., Jakawich, S.K., Clifton, D.K., Steiner, 

R.A. and Herbison, A.E., Activation of gonadotropin-releasing hormone (GnRH) neurons by 

kisspeptin as a neuroendocrine switch for the onset of puberty, J Neurosci (2005). 

[6] Irwig, M.S., Fraley, G.S., Smith, J.T., Acohido, B.V., Popa, S.M., Cunningham, M.J., Gottsch, 

M.L., Clifton, D.K. and Steiner, R.A., Kisspeptin Activation of Gonadotropin Releasing Hormone 

Neurons and Regulation of KiSS-1 mRNA in the Male Rat, Neuroendocrinology, 80 (2005) 264- 

272. 

[7] Seminara, S.B., Messager, S., Chatzidaki, E.E., Thresher, R.R., Acierno, J.S., Jr., Shagoury, J.K., 

Bo-Abbas, Y., Kuohung, W., Schwinof, K.M., Hendrick, A.G., Zahn, D., Dixon, J., Kaiser, U.B., 

Slaugenhaupt, S.A., Gusella, J.F., O'Rahilly, S., Carlton, M.B., Crowley, W.F., Jr., Aparicio, S.A. 

and Colledge, W.H., The GPR54 gene as a regulator of puberty, N Engl J Med, 349 (2003) 1614-27. 

[8] Shahab, M., Mastronardi, C., Seminara, S.B., Crowley, W.F., Ojeda, S.R. and Plant, T.M., Increased 

hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates, Proc 

Natl Acad Sci U S A, 102 (2005) 2129-34. 

[9] Smith, J.T., Cunningham, M.J., Rissman, E.F., Clifton, D.K. and Steiner, R.A., Regulation of Kiss1 

gene expression in the brain of the female mouse, Endocrinology, 146 (2005) 3686-92. 

38



STEROID-DEPENDENT MODULATION OF VOCAL MOTOR SYSTEMS 

Bass A.H. 

Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, 14853, 

U.S.A. ahb3@cornell.edu, Fax 607-254-1303 

Vocal communication is not unique to humans, but rather is a trait shared with most 

non-mammalian vertebrates. A practical way to address questions of vocal signal 

production and encoding has been to identify mechanisms in non-mammalian model 

systems that use acoustic communication signals in their social behavior. A major goal of 

this presentation is take a broad phylogenetic view of How and Why steroid hormones have 

been adopted as neuromodulators shaping both long-term and short-term plasticity in the 

neural mechanisms of vocal behaviors. It is in this context that I will first review my 

laboratory’s studies of neuroendocrine mechanisms leading to vocal-auditory plasticity 

among teleost fishes. 

Teleosts make up nearly half of all living vertebrate species [7]. Vocal teleosts have 

a simple repertoire of acoustic communication signals, and auditory and vocal pathways 

organized like those of birds and mammals [4]. They also present the simplest and perhaps 

primordial example of how a vertebrate nervous system is organized to both produce and 

detect social, context-dependent sounds [2, 4]. Studies of vocal species have recently 

begun to elucidate a suite of neuroendocrine adaptations for both the production and the 

perception of acoustic signals that are essential to reproductive success [3, 4]. I review our 

studies of both auditory and vocal mechanisms because both the production and perception 

of vocal signals is “in the time domain … the time varying acoustic waveform is the 

physical signal that is actually produced by the temporally patterned action of the motor 

system under ongoing control of the central nervous system. …. The two systems [vocal 

and auditory] co-evolved and we should expect them to share the same underlying code for 

signal generation and recognition” [5]. 

The closely related midshipman fish and toadfish (same family and order) produce 

simple, highly stereotyped vocal signals that are essential to social interactions, including 

reproduction and aggression [4]. Behavioral studies demonstrate that the temporal features 

within a call, including pulse duration, rate and number, can all be important to a call’s 

communicative value; hence, our focus on temporal processing [4]. How does the auditory 

system encode vocal signals? Neurophysiological studies show that the saccule division of 

the inner ear is the main auditory end organ in most teleosts; single neuron recordings 

show that afferents within the saccular branch of the eighth cranial nerve encode acoustic 

waveforms in the temporal pattern of their action potentials [4]. We recently discovered 

that the temporal encoding of frequency via phase-locking by midshipman saccular 

afferents exhibits reproductive state and steroid-dependent plasticity [10]. This leads to 

enhanced sensitivity to the higher harmonics of male advertisement calls during the 

breeding season that likely also contributes to improved mechanisms of sound localization. 

We are now studying the cellular and molecular events leading to steroid modulation of 

auditory encoding mechanisms in the context of natural shifts in plasma and brain hormone 

concentrations [see 9, 11]. 

Midshipman and toadfish produce sound by the simultaneous contraction of a pair 

of sonic muscles attached to the walls of the gas-filled swim bladder. The physical 

attributes of the acoustic waveform are a direct translation of the temporal attributes of a 

hindbrain-spinal vocal pattern generator (VPG) that shares developmental origins with the 

vocal pacemaker circuits of birds and mammals [1, 2]. The VPG is part of a more 

39


expansive vocal-acoustic network that most closely resembles the organizational pattern in 

mammals [6]. We refer to the rhythmic, oscillatory-like output of the VPG (i.e., the sonic 

motor volley) as a “fictive vocalization” because its temporal properties set the 

fundamental frequency and duration of natural vocalizations [1, 8]. Fictive calls are easily 

monitored using intracranial recordings from occipital nerve roots (homologous to 

hypoglossal nerve of tetrapods) that give rise to the sonic nerve that innervates each of the 

swim bladder muscles [1]. Several studies now show that steroids can induce changes in 

the duration and rate of production of fictive calls within minutes [e.g., 8]. Importantly, 

field studies establish a causal relationship between rapid shifts in plasma levels of steroids 

and vocal behaviors [8]. 

In sum, several studies in teleost fish now show how naturally occurring steroids 

modulate the response properties of auditory neurons and vocal motor neurons that are 

essential to, respectively, the encoding and production of vocalizations. As will be 

discussed, the principles identified here can be generalized across vertebrates due to the 

conserved pattern of auditory-vocal and neuroendocrine mechanisms between vocal 

teleosts and tetrapods [e.g., 4, 6]. 

Acknowledgements. Research support from the U.S. National Institutes of Health 

(DC00092) and National Science Foundation (IBN9987341, IOB-0516748). 


[1] Bass, A.H. and Baker, R., Sexual dimorphisms in the vocal control system of a teleost fish: 

morphology of physiologically identified neurons, J Neurobiol, 21 (1990) 1155-1168. 

[2] Bass, A.H. and Baker, R., Phenotypic specification of hindbrain rhombomeres and the origins of 

rhythmic circuits in vertebrates. Brain, Behav Evol, 50 (1997) 3-16. 

[3] Bass, A.H. and Forlano, P.M., Neuroendocrine mechanisms of alternative reproductive tactics: the 

chemical language of social plasticity. In R. Oliveira, M. Taborsky and J. Brockmann (Eds.), 

Alternative Reproductive Tactics: An Integrative Approach, Cambridge University Press, 

Cambridge, UK, in press. 

[4] Bass, A.H. and McKibben, J.R., Neural mechanisms and behaviors for acoustic communication in 

teleost fish, Prog Neurobiol, 69 (2003) 1-26. 

[5] Capranica, R.R., The untuning of the tuning curve: is it time? Sem Neurosci, 4 (1992) 401-408. 

[6] Goodson, J. L., and Bass, A.H., Vocal-acoustic circuitry and descending vocal pathways in teleost 

fish: Convergence with terrestrial vertebrates reveals conserved traits. J Comp Neurol, 448 (2002) 

298-322. 

[7] Nelson J.S., Fishes of the world. (1994) New York: Wiley & Sons. 

[8] Remage-Healey, L.H. and Bass, A. H., From social behaviour to neurons: Rapid modulation of 

advertisement calling and vocal pattern generators by steroid hormones. Horm Behav 50 (2006) 

432-441. 

[9] Schlinger, B.A., Greco, C. and Bass, A.H., Aromatase activity in the hindbrain vocal control region 

of a teleost fish: divergence among males with alternative reproductive tactics, Proc Roy Soc Lond 

B-Biol Sci, 266 (1999) 131-136. 

[10] Sisneros, J.A., Forlano, P.M., Deitcher, D.L. and Bass, A.H., Steroid-dependent auditory plasticity 

leads to adaptive coupling of sender and receiver, Science, 305 (2004) 404-7. 

[11] Sisneros, J.A., Forlano, P.M., Knapp, R. and Bass, A.H., Seasonal variation of steroid hormone 

levels in an intertidal-nesting fish, the vocal plainfin midshipman, Gen Comp Endocrinol, 136 

(2004) 101-116. 

40



ESTROGEN RECEPTOR BETA IN THE BRAIN: FROM FORM TO FUNCTION 

Handa R.J. 

Department of Biomedical Sciences, College of Veterinary Medicine, Colorado State 

University, Fort Collins, Colorado, USA 

Estrogens have numerous effects on the brain both in adulthood and during 

development. These actions of estrogen are mediated by two distinct estrogen receptor 

(ER) systems, ERα and ERβ. In brain, ERα plays a critical role in estrogen regulating 

reproductive neuroendocrine function and behavior, however, a definitive role for ERβ in 

any neurobiological function has been slow in forthcoming. Clues to the function of ERβ 

in the CNS can be gleaned from the neuroanatomical distribution of ERβ and the 

phenotypes of neurons that express ERβ. ERβ immunoreactivity has been found in 

populations of GnRH, CRH, vasopressin, oxytocin and prolactin containing neurons. We 

have also utilized subtype-selective estrogen receptor agonists to determine the roles for 

ERβ in non-reproductive behaviors in a rat model. ERβ selective agonists were found to 

exert potent anxiolytic activity when animals were tested in a number of behavioral 

paradigms. Consistent with this, ERβ selective agonists also inhibited the ACTH and 

corticosterone response to stress. In contrast, ERalpha selective agonists were found to be 

anxiogenic and correspondingly increased the hormonal stress response. Taken together, 

our studies implicate ERβ as an important modulator of some non-reproductive 

neurobiological systems. The molecular and neuroanatomical targets of estrogen that are 

mediated by ERβ remain to be determined. 

In the course of these studies we have identified a number of splice variants of 

ERβ mRNA in brain tissue. Imaging of eGFP labeled receptor proteins in transfected cell 

lines has demonstrated that ERβ splice variation can alter trafficking patterns. The 

originally described ERβ (herein termed ERβ1) is characterized by possessing a high 

affinity for estradiol. Similar to ERα, it is localized in the nucleus and is trafficked to 

nuclear sites termed “hyperspeckles” following ligand binding. In contrast, ERβ2 contains 

an 18aa insert within the ligand binding domain and as a result can be best described as a 

low affinity form of ERβ. A delta3 variant of ERβ has a deletion of the 3 rd exon (coding 

for the second half of the DNA binding domain) and as a result does not bind an estrogen 

response element in DNA. Delta3 variants are trafficked to a unique low abundance and 

larger nuclear site following ligand binding. A delta 4 variant lacks exon 4 and as a result 

is localized to the cytoplasm. The amount of individual splice variant mRNAs varies 

depending upon brain region. Examination of neuropeptide promoter regulation by ERβ 

and its splice variants demonstrate that ERβ functions as a constitutively active 

transcription factor. Moreover, it appears that splice variation of ERβ alters its ability to 

regulate transcription in a promoter-dependent and ligand-dependent fashion. 

41


A ROLE FOR THE ANDROGEN RECEPTOR IN THE SEXUAL 

DIFFERENTIATION OF THE OLFACTORY SYSTEM IN MICE 

Bodo C. 1 and Rissman E.F. 2 

1 Graduate Program in Neuroscience and 2 Department of Biochemistry and Molecular 

Genetics, University of Virginia, Charlottesville, VA 22908 

Phone: 01 434 982 4742; Fax: 01 434 243 8433 

Olfactory signals play a central role in the identification of a mating partner in 

rodents, and the behavioral response to these cues varies markedly between the sexes. As 

several other sexually dimorphic traits, this response is thought to differentiate as a result 

of exposure of the developing individual to gonadal steroids, but both the identity of the 

specific steroid signal and the neural structures targeted for differentiation on this 

particular case are largely unknown. Using genetic males affected by the testicular 

feminization syndrome (Tfm) as our experimental model, we have identified a potential 

role for non-aromatized gonadal steroids acting through the androgen receptor (AR) in the 

differentiation of olfactory cues processing in mice. In contrast with their WT male 

littermates, Tfm males spend more time investigating bedding soiled by other males rather 

than by receptive females, and they do not a show a clear preference for females as 

potential partners. Moreover, when cFos expression on the central projections of the 

accessory olfactory system in response to male odors was measured on these individuals, 

they exhibited a feminized pattern of activation. Conversely, when WT females were 

treated neonatally with the non-aromatizable androgen dihydrotestosterone, their 

preference for soiled bedding was masculinized, confirming the hypothesis that AR 

activation is solely responsible for the masculinization of the neural circuit that regulates 

the detection and processing of non-volatile olfactory cues in mice. We are currently 

working to obtain data on partner preference and c-fos response to olfactory signals on 

neonatally-androgenized individuals. Considered together, the pieces of data to be 

presented represent a good example of the specificity of function of steroids receptors in 

the overall process of sexual differentiation of neural circuits in mammals. 

This work was supported by NIH grants R01 MH57759 and K02 MH01349. 

42



CANONICAL AND ALTERNATIVELY SPLICED ESTROGEN RECEPTOR α IN 

THE HUMAN MAMILLARY BODIES AND HIPPOCAMPUS IN AGING AND 

ALZHEIMER’S DISEASE 

Ishunina T.A. 1,2 , and Swaab D.F. 2 

1 Department of Histology, Kursk State Medical University, Kursk, Russia 

2 

Neuropsychiatric diseases, Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 

BA, Amsterdam, The Netherlands. Fax +31 20 6961006, e-mail: T.Ishunina@nin.knaw.nl 

Beneficial role of estrogens in brain aging and neurodegenerative diseases remains 

a controversial topic of research. In addition to multiple mechanisms of estrogen action, 

the subject is complicated by a diversity of estrogen receptor isoforms that may mediate 

estrogen effects or influence the wild type receptor function. 

In the present study we aimed at determining whether estrogen receptor α (ERα) 

mRNA and protein are affected in aging and Alzheimer’s disease (AD) in the human 

mamillary bodies (MB) and the hippocampus. These brain areas were chosen for this 

investigation since they are involved in the regulation of memory, learning, emotions [1]. 

They are joined within the mamillothalamic circuit and are severely affected in AD [2]. 

We also addressed the question whether specific ERα mRNA splice variants are present in 

these brain areas and change with advanced age and in AD. 

ERα protein levels were determined by quantitative immunocytochemistry with 

MC-20 antibody (Santa Cruz) [3]. Wild type (wt) ERα mRNA and ERα splice variants 

were amplified in RT-PCR with subsequent sequencing and relative quantification in Q- 

PCR [4, 5]. 

Canonical ERα mRNA and protein levels were increased in the MB and were 

decreased in the hippocampus of AD patients compared to elderly control cases [3-5]. 

Exon-skipping ERα variants were common in both brain areas [4, 5]. In the MB the major 

ERα splice form was del. 7 [4], lacking exon 7 that encodes a significant portion of the 

ligand-binding domain (LBD) [6]. Del. 4 (missing exon 4 encoding a part of the LBD [7]) 

and del. 2 (lacking exon 2 leading to alterations in the DNA-binding domain [7]) were 

present in the MB to a lesser extent than del. 7. In the hippocampus the most abundant 

splice variant was del. 4 [5] that does not bind to estradiol and to the estrogen responsive 

elements [8]. The del.7 and del. 2 were expressed in the hippocampus to a significantly 

lesser extent (del. 4 > del. 7 > del. 2). Interestingly, we have identified two novel ERα 

mRNA splice variants: MB1 (mamillary body, exon 1) [4] and TADDI (from the 

hippocampus) [5]. MB1 is characterized by a 168 nucleotide deletion in exon 1 encoding 

the major portion of the transactivation function 1 domain of the ERα [4]. TADDI is 

missing 31 bp at the junction between exons 3 and 4 with an insertion of 13 nucleotides 

from the middle of the exon 2 into this splice site [5]. In general, ERα mRNA splice 

variants showed the same direction of changes in AD as the wt ERα mRNA amplicons. 

While no aging-related changes were observed for the wild type and alternatively 

spliced ERα mRNA in the hippocampus of elderly subjects (≥ 46 years of age), canonical, 

del. 7 and del. 2 ERα mRNA amplicons declined during aging in elderly control patients ≥ 

61 years of age in the MB1. 

Interestingly, ERα protein levels as judged from quantitative immunocytochemistry 

were increased in the CA4, hilus and dentate granular layer of postmenopausal women (58- 

83 years of age) compared to young women (34-50 years old). Moreover, young women 

(34-50 years of age) showed significantly more ERα in all hippocampal subregions than 

43


men of the same age. Since no sex differences were found in aromatase immunoreactivity, 

this finding is related to higher circulating estrogen levels in young women rather than to 

locally synthesized estrogens. 

Our data show that ERα splice variants in the brain are region-specific, that they 

may change in aging and AD and that they should be considered as potential mediators of 

estrogen effects in the human brain and/or as confounders of the normal receptor function. 


[1] D.F. Swaab, The human hypothalamus: basic and clinical aspects. Part I. Nuclei of the human 

hypothalamus. In M.J. Aminoff, F. Boller and D.F. Swaab (series eds.): Handbook of Clinical 

Neurology. Elsevier, 2003, vol.79, 3 rd series, vol. 1, pp.502 

[2] D.F. Swaab, The human hypothalamus: basic and clinical aspects. Part II. Neuropathology of the human 

hypothalamus and adjacent structures. In M.J. Aminoff, F. Boller and D.F. Swaab (series eds.): 

Handbook of Clinical Neurology. Elsevier, 2004, vol.80, 3 rd series, vol. 2, pp.632 

[3] T.A. Ishunina, W. Kamphorst, D.F. Swaab, Changes in metabolic activity and estrogen receptors in the 

human medial mamillary nucleus: relation to sex, aging and Alzheimer's disease, Neurobiol Aging 24 

(2003) 817-828. 

[4] T.A. Ishunina, D.F. Swaab, D.F. Fischer, Estrogen receptor-α splice variants in the medial mamillary 

nucleus of Alzheimer’s disease patients: identification of a novel MB1 isoform, J Clin Endocrinol 

Metab 90 (2005) 3757-3765. 

[5] T.A. Ishunina, D.F. Fischer, D.F. Swaab, Estrogen receptor α and its splice variants in the hippocampus 

in aging and Alzheimer’s disease, Neurobiol Aging (2006): in press. 

[6] J.M. García Pedrero, P. Zuazua, C. Martínez-Campa, P.S. Lazo, S. Ramos, The naturally occurring 

variant of estrogen receptor (ER) ERΔe7 suppresses estrogen-dependent transcriptional activation by 

both wild type ERα and ERβ, Endocrinology 144 (2003) 2967-2976 

[7] S. Hirata, T. Shoda, J. Kato, K. Hoshi, Isoform/variant mRNAs for sex steroid hormone receptors in 

humans, Trends Endocrinol Metab 14 (2003) 124-129 

[8] S.G. Koehorst, J.J. Cox, G.H. Donker, S. Lopes da Silva, J.P. Burbach, J.H. Thijssen, M.A. 

Blankenstein, Functional analysis of an alternatively spliced estrogen receptor lacking exon 4 isolated 

from MCF-7 breast cancer cells and meningioma tissue, Mol Cell Endocrinol 101 (1994) 237-245 

44



FEMALE SPECIFIC ACTIVATION OF GALANIN IN THE SUPRAOPTIC 

NUCLEUS OF HENS AFTER OVIPOSITION RELATED UPREGULATION OF 

ARGININE VASOTOCIN (AVT) 

Klein S., and Grossmann R. 

Institute for Animal Science Mariensee (FAL), Dept. Functional Genomics and 

Bioregulation, Höltystrasse 10, D-31535 Neustadt, Germany, klein@tzv.fal.de, 

fax: +49-5034-871 247 

The neuroendocrine AVT system in birds comprises homeostatic function as it does 

argi-nine vasopressin in mammals and was additionally shown to be activated for 

reproductive functions. Oviposition in birds is accomanied by about sevenfold increase of 

plasma AVT concentrations to the unstimulated values with a short half life time of 13 min 

and no influence on plasma osmolality [11] [12]. From the nonapeptides of birds only 

AVT, but not mesotocin, was found with oviposition related changes in plasma [5]. Thus, 

AVT is proposed to perform functions as they are known from oxytocin in mammals. 

However, little is known about AVT functions of magnocellular neurons. Oxytocin 

secreted from magnocellular supraoptic neurons (SON) in mammals was shown to be 

involved in regulation of female reproductive behaviour [9]. The positive feedback mechanism 

of oxytocin during birth and lactation needs additional neuromodulatory regulation to 

stabilize the system. Galanin was found colocalized with oxytocin in female rats [8] and is 

colocalized with vasopressin and oxytocin in lactating rats [7]. Because of its 

hyperpolarizing action, galanin was suggested as neuromodulator to curb estrogenic 

stimulated dendritic oxytocin release via GABA-ergic inhibition [10] and by presynaptic 

action on synaptic inputs [6]. Variable galanin immunoreactivity was found in hens, those 

ovulatory cycle was not determined [3]. 

Previously, we have shown sex differences for galanin immunoreactivity (ir) in 

magnocellular SON neurons in chickens [4]. Fluorescent immunohistochemistry revealed 

no galanin in male supraoptic neurons as it was previously reported from male quails [1]. 

Galanin-ir was found in up to 50% of AVT neurons in the rostral half of the SON in hens 

perfused within three hours after lights on [4]. In the current study, we investigated the 

time course of AVT and galanin-ir in the SON of female chickens from four hours before 

until four hours after oviposition. Multitrack confocal imaging (LSM510, Zeiss, Germany) 

was performed to localize FITC labeled guinea pig anti-galanin (Chemicon) and Alexa 555 

labeled rabbit anti-AVT [2] in perfused brains of adult hens of 25 to 45 weeks of age. The 

rostral half of SON, known from mammals to contain most oxytocin synthesizing neurons, 

was investigated for the number of neurons and intensity of ir for galanin and AVT. The 

intensity of AVT-ir within neurons increased from four hours to 30 minutes before 

oviposition by one third. The significant decrease of AVT intensity at 30 minutes after 

oviposition implies an oviposition related AVT release from these neurons. Galanin-ir was 

found in about 10% of neurons with AVT labeling at four hours and 30 minutes before 

oviposition with low intensities. At 30 minutes after oviposition, a significant increase of 

the intensity of galanin labeling in an increased number of AVT neurons was seen. Thus, 

the proportion of colocalization for galanin and AVT in SON neurons increased to 25 - 

30%. At four hours after oviposition, no galanin was detectable in SON of female chickens 

with intensities at least three times above background. 

The data suggest that AVT-ir is strongly upregulated in the SON neurons from four hours 

before oviposition to 30 minutes before oviposition. The decreased intensities of AVT ir at 

30 minutes after oviposition indicate a release of AVT at the time of oviposition from SON 

45


neurons. Galanin synthesis in magnocellular supraoptic neurons is stimulated after the 

release of AVT at oviposition. Taken together, these observations indicate that galanin-ir is 

upregulated in SON neurons delayed to previous strong stimulation of the AVT system. 

Therefore, the data are in agreement to the findings of galanin function in negative 

feedback control of the upregulated oxytocinergic system in mammals and indicate similar 

mechanisms of oxytocin regulation for reproductive behaviour during birth in mammals 

and AVT during oviposition in birds. 


[1] Y. Azumaya and K.Tsutsui, Localization of galanin and its binding sites in the quail brain, Brain Res. 

727 (1996) 187-195. 

[2] D.A. Gray and E.Simon, Mammalian and avian antidiuretic hormone: studies related to possible species 

variation in osmoregulatory systems., J Comp Physiol [A] 151 (1983) 241-246. 

[3] R. Jozsa and B.Mess, Galanin-like immunoreactivity in the chicken brain, Cell Tiss Res. 273 (1993) 

391-399. 

[4] S. Klein, A.Jurkevich, and R.Grossmann, Sexually dimorphic immunoreactivity of galanin and 

colocalization with arginine vasotocin in the chicken brain (Gallus gallus domesticus), J Comp Neurol 

499 (2006) 828-839. 

[5] T.I. Koike, K.Shimada, and L.E.Cornett, Plasma levels of immunoreactive mesotocin and vasotocin 

during oviposition in chickens: relationship to oxytocic action of the peptides in vitro and peptide 

interaction with myometrial membrane binding sites, Gen Comp Endocrinol 70 (1988) 119-126. 

[6] M.G. Kozoriz, J.B.Kuzmiski, M.Hirasawa, and Q.J.Pittman, Galanin modulates neuronal and synaptic 

properties in the rat supraoptic nucleus in a use and state dependent manner, J Neurophysiol 96 (2006) 

154-164. 

[7] M. Landry, D.Roche, E.Angelova, and A.Calas, Expression of galanin in hypothalamic magnocellular 

neurons of lactating rats: coexistence with vasopressin and oxytocin, J Endocrinol 155 (1997) 467-481. 

[8] M. Landry, A.Trembleau, R.Arai, and A.Calas, Evidence for a colocalization of oxytocin mRNA and 

galanin in magnocellular hypothalamic neurons: a study combining in situ hybridization and 

immunohistochemistry, Mol Brain Res 10 (1991) 91-95. 

[9] I. Neumann, A.J.Douglas, Q.J.Pittman, J.A.Russell, and R.Landgraf, Oxytocin released within the 

supraoptic nucleus of the rat brain by positive feedback action is involved in partuition-related events, J 

Neuroendocrinol 8 (1996) 227-233. 

[10] S. Papas and C.W.Bourgue, Galanin inhibits continuous and phasic firing in rat hyphothalamic 

magnocellular neurocsecretory cells, J Neurosci 17 (1997) 6048-6056. 

[11] G.E. Rice, S.S.Arnason, Z.Arad, and E.Skadhauge, Plasma concentrations of arginine vasotocin, 

prolactin, aldosterone and corticosterone in relation to oviposition and dietary NaCl in the domestic 

fowl, Comp Bioch Physiol A 81 (1985) 769-777. 

[12] B. Robinzon, N.Sayag, I.T.Koike, S.Kinzler, and H.L.Neldon, Effects of sex and gonadal steroids on 

arginine vasotocin and mesotocin int he pineal gland and neurohypophysis of white leghorn fowls, Brit 

Poult Sci 31 (1990) 843-849. 

46



EXPRESSION OF NITRIC OXIDE SYNTHASE IN THE MALE MOUSE LIMBIC 

SYSTEM IS MEDIATED BY ESTROGEN RECEPTORS 

Sica M., Martini M., Verzè L., Viglietti-Panzica C., and Panzica G.C. 

Dept. Anatomy, Pharmacology and Forensic Medicine. Neuroscience Institute of Torino. 

University of Torino. Corso M. D’Azeglio 52 10126 Torino Italy. 

Nitric oxide (NO) is involved in the control of reproductive functions, and sexual 

behavior [3]. NADPH-diaphorase-positive and neuronal nitric oxide synthase (nNOS) 

immunoreactive neurons are located in medial preoptic area (MPOM), bed nucleus of stria 

terminalis (BST), paraventricular nucleus (PVN) and ventromedial nucleus of the 

hypothalamus (VMH) [1]. nNOS expression in the hypothalamus changes by treatments 

with testosterone as well as with estrogens, suggesting that effects of T on NOS expression 

are controlled by aromatase, the enzyme that catalyzes the biosynthesis of estrogens from 

precursor androgens [2,4,5]. The endocrine specificity of nNOS control could thus be 

similar to the specificity of the activation of male sexual behavior: both could depend more 

on the action of estrogens produced by aromatization than on the action of testosterone 

itself. 

So far the majority of the studies concerning the effect of E 2 and/ or T on NO synthase 

(NOS) expression have been performed mainly in castrated or ovariectomized subjects. To 

better describe how the E 2 modulate the expression of NOS immunoreactive system in 

mouse hypothalamus we studied the modifications in NOS expression in two mutant 

strains: the estrogen receptor α knock out (ERαKO) and the aromatase knock out (ArKO) 

mice. 

In both models we have detected variations in the expression of neural NOS (nNOS), as 

detected by means of immunocytochemistry and quantitative analysis. 

In particular in MPOM and PVN we have observed a significant decrease of 

immunoreactivity in both ERαKO and ArKO mice. ERαKO mice show also a significant 

decrease in the arcuate nucleus, whereas in ArKO mice a significant decrease was 

observed at the level of VMH. No changes have been observed at the level of BST and of 

the caudate-putamen (a region lacking of estrogen receptors, that was analyzed as a control 

nucleus). 

These data are a further confirmation that at least part of the hypothalamic nitrinergic 

system of male mice is under the control of brain estrogens and that the decrease of NO in 

nuclei involved in the control of sexual behavior could be one of the main factors 

responsible of the impairment of sexual behavior displayed by these mutant mice. 

Moreover, these data indicate that ER-alpha is involved in the control of nNOS expression 

is some limbic regions, but not in the whole system. Differences observed at the level of 

VMH may indicate a major involvement of ER-beta in this region. 

Acknowledgements – The authors want to acknowledge Julie Bakker (Liege) and Emilie 

Rissmann (Charlottesville) for having provided the animals used in this study. Supported 

by PRIN and University of Torino grants. 

47


References list 

[1] S. Gotti, M. Sica, C. Viglietti Panzica and G.C. Panzica, The distribution of nitric 

oxide sythase immunoreactivity in the mouse brain, Micr. Res. Techn. 68 (2005) 13- 

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effects of estrous cycle on the nitrinergic system in mouse hypothalamus, Horm. 

Behav. 46 (2004) 95-96. 

[3] G.C. Panzica, C. Viglietti-Panzica, M. Sica, S. Gotti, M. Martini, H. Pinos, B. Carrillo 

and P. Collado, Effects of gonadal hormones on central nitric oxide producing 

systems, Neuroscience 138 (2006) 987-995. 

[4] S. Sato, C.S. Braham, S.K. Putnam and E.M. Hull, Neuronal nitric oxide synthase and 

gonadal steroid interaction in the MPOA of male rats: co-localization and testosteroneinduced 

restoration of copulation and nNOS-immunoreactivity, Brain Res. 1043 

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453 (2002) 336-344. 

48


21.00 - 23.00 

Round table I: 

Steroid hormones and sexually dimorphic brain circuits

Round table I: 

Steroid hormones and sexually dimorphic brain circuits 

(Chair: Guillamon A., Panzica G.C.) 

• Guillamon A, Segovia S (Spain) Sex differences in the olfactory system of 

mammals 

• Swaab DF (Netherlands) Human brain sex differences in relation to gender, 

sexual orientation and brain disorders 

• Patisaul H.B. (USA) Assessing the functional disruption of brain sexual 

differentiation by endocrine disrupting compounds 

• Micevych P, Phoebe Dewing P. (USA) Sexual differentiation: it’s not just for 

development anymore 

• Bass A.H. (USA) Sexually polymorphic neuroendocrine phenotypes: examples 

from singing fish 

• Bakker J (Belgium) Are estrogens required for the development of the female 

brain? 

• Balthazart J (Belgium) Sexual dimorphism in the avian limbic system



SEX DIFFERENCES IN THE OLFACTORY SYSTEM OF MAMMALS 

Guillamon A., and Segovia S. 

UNED, Madrid, Spain. aguillamon@psi.uned.es 

At the beginning of the eighties, and working with neurohistological techniques and 

rats, we found that the rat vomeronasal organ is a sexually dimorphic structure 

differentiated by gonadal hormones early after birth ( ) and we suggested that the whole 

vomeronasal system (VNS) could be sexually dimorphic. Sex differences in the VNS have 

functional importance since VNS structures are implicated in the control of sexual and 

maternal behavior. The structures that that receive vomeronasal input, such as the 

accessory olfactory bulb, medial amygdala, (Me) posteromedial cortical amygdala (C 3) , 

medial posterior region of the bed nucleus of the stria terminalis, , medial preoptic area 

(MPA), the ventromedial hypothalamus (VMH) and the premammillary nucleus (PMV) 

have androgen and estrogen receptors and present sexual dimorphism (1, 2). Interestingly, 

in all these structures the male shows greater morphological measurements (volume and or 

number of neurons) than the female rats. From this view we suggested that the VNS of 

Rodent and, probably of Mammals could be sexually dimorphic. Recently, we have 

investigated the VNS structures of the rabbit, as representative species of Lagomorpha, and 

found sexual dimorphism. Females have greater number of mitral and dark and light 

granule cells in the accessory olfactory bulb while the males. In the medial amygdala and 

in it dorsal and ventral subdivisions , males show greater values than females in volume 

and number of neurons while in the posteromedial cortical amygdala females show greater 

density of neurons than males. However, in the posteromedial division of the bed nucleus 

of the bed nucleus of the stria terminalis males have more neurons than females (3). 

More recently, and using voxel-based morphometry, we have investigated the human 

olfactory system (Price, ) that still retains functions related to sexual physiology and 

sexual behavior. Women have a higher concentration of gray matter in the orbitofrontal 

cortex involving Brodmann’s areas 10, 11 and 25 and temporomedial cortex (bilateral 

hippocampus and right amygdala), as well as their left basal insular cortex. In contrast, 

men show a higher gray matter concentration in the left entorhinal cortex (Brodmann´s 28), 

right ventral pallidum, dorsal left insular cortex and a region of the orbitofrontal cortex (4). 

Taking into account the existence of sex differences in the olfactory system of rodents, 

lagomorphs and humans, and the fact that some structures that receive olfactory input such 

as the sexually dimorphic nucleus of the medial preoptic areas and the posteromedial 

division of the bed nucleus of the stria terminalis are sexually dimorphic in a wide range of 

species, we suggest that the mammalian olfactory system is a sexually dimorphic network, 

with species specifics characteristics. This could provide a theoretical framework for the 

morphofunctional approach to sex differences in mammals. Such an approach will help to 

exploit the abundant literature of animal data on sex differences and sexual behavior and 

facilitate the construction of a neurobiological motivational model that could explain the 

function of the sex differences found in the human brain. 

51



(1) Segovia, S and Guillamon, A., Sexual differences in the vomeronasal pathway and 

sex differences in reproductive behavior, Brain Res. Review. 18: 51-74, 1993. 

(2) Simerly, R.B., Wired for reproduction: organization and development of sexually 

dimorphic circuits in the mammalian brain. Ann. Rev. Neurosci., 25: 507-536, 

2002. 

(3) Segovia, S., Garcia-Falgueras, A., Carrillo, B., Collado, P., Pinos, H, Perez-Laso, 

C., Vinader-Caerols, C., Beber, C., and Guillamon, A., Sexual dimorphism in the 

vomeronasal system of the rabbit, Brain Res. 1102: 52-62, 2006. 

(4) Garcia-Falgueras, A., Junque, C., Jiménez, M., Caldú, X., Segiovia, S., and 

Guillamon, A., Sex differences in the human olfactory system. Brain Res. 1116: 

103-111, 2006. 

52



HUMAN BRAIN SEX DIFFERENCES IN RELATION TO GENDER, SEXUAL 

ORIENTATION AND BRAIN DISORDERS 

Swaab D.F. 

Netherlands Institute for Neuroscience, Amsterdam, The Netherlands. 

(d.f.swaab@nin.knaw.nl) 

Sex differences in behaviour are present from early childhood onwards, e.g. in our 

playing behaviour and drawings, followed by cognition, reproduction, gender (the feeling 

to be male or female) and sexual orientation, and the incidence of neurological and 

psychiatric disorders. All these differences are presumed to be based upon structural and 

functional sex differences that are present all over the human brain. They arise during 

development by an interaction of sex hormones and the developing neurons, although 

direct genetic effects are probably also involved. Factors influencing structural en 

functional sex differences in the brain are genetic factors like mutations or polymorphisms 

in the sex hormone receptors, abnormal prenatal hormone levels and compounds that 

interact with the action of sex hormones on the brain during early development such as 

anticonvulsants, DES and environmental endocrine disrupters. An influence of postnatal 

social factors on gender or sexual orientation has not been established. In rodents, 

masculinization of the brain in development is due to estrogens that are formed by 

aromatization of testosterone. In sexual differentiation of the human brain direct effects of 

testosterone seem to be of primary importance as is clear e.g. from subjects with mutations 

in the gene for the androgen receptor, estrogen receptor or aromatase. 

In transsexuals we observed a reversal of the sex difference in the Bed Nucleus of 

the Stria terminalis (BSTc). The size, type of innervation and neuron number agreed with 

their gender and not with their genetic sex. 

Various brain differences related to sexual orientation have now also been reported. 

In addition, there are sex differences present in the way the brain ages and in Alzheimer 

neuropathology. The field is becoming extra complex by the presence of splice variants 

and isoforms of estrogen receptor-α and the local production of steroid hormones in the 

brain. This means that sex differences may be expected in many functions in all stages of 

life, in heath as well as in disease. 

53


ASSESSING THE FUNCTIONAL DISRUPTION OF BRAIN SEXUAL 

DIFFERENTIATION BY ENDOCRINE DISRUPTING COMPOUNDS 

Patisaul H.B. 

North Carolina State University, Department of Zoology, 127 David Clark Labs, Raleigh, 

NC 27695 USA, fax: (919) 515-5327, heather_patisaul@ncsu.edu 

Developmental disruption of the reproductive neuroendocrine system is likely to be 

expressed as subtle changes in adult behaviors and functions, rather than overt changes in 

brain anatomy and reproductive physiology. Therefore, in developing predictive strategies 

for evaluating the ultimate effects of early endocrine active compound (EAC) exposure, it 

is critical to employ a comprehensive approach that assesses neuronal function and 

reproductive physiology across the lifespan. We have found that neonatal exposure to 

EACs such as Bisphenol-A and genistein can affect sexually dimorphic brain morphology 

and neuronal phenotypes in adulthood with regional and cellular specificity. We have also 

found that EACs can simultaneously enhance and interfere with the effects of endogenous 

estrogen on the developing brain, making it difficult to extrapolate effects seen in one 

region to more global inferences about potential EAC effects on sexually dimorphic 

development and behavior. However, subtle impairments to sex-specific behaviors, 

fertility, and cyclicity could collectively have significant ramifications for the fitness of an 

affected population, including human populations. Therefore it is imperative that the 

effects of EAC exposure during development, when sexually dimorphic systems are 

organizing, are appreciated. 

54



SEXUAL DIFFERENTIATION: IT’S NOT JUST FOR DEVELOPMENT 

ANYMORE 

Micevych P., and Dewing P. 

Dept of Neurobiology, MRRC and Lab of Neuroendocrinology of the Brain Research 

Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 

Brain sex differences are the result of events beginning with sex-determining genes 

and continuing with the actions of sex steroids throughout the lifetime of the mammalian 

species. In particular, gonadal steroid hormones from the developing embryo are the 

primary signals which initiate brain sexual differentiation. As a result, anatomical and/or 

morphological dimorphisms within the brain arise, which then translate into differences in 

brain function and behavior. Neurochemical sex differences, unlike the permanent 

morphological sex differences, co-vary with the steroid hormone environment that 

influences reproduction. One example is the neuropeptide cholecystokinin (CCK), which 

has been a useful marker to study sex differences and the action of steroids on the brain. 

CCK is found in the limbic system and hypothalamic circuitry that regulate reproductive 

behavior. Animals are born with a sexual dimorphism in the number of CCK cells that 

favor males. This slight sex difference, in the absence of hormones, becomes dramatic with 

the rise of sex steroid hormone levels during puberty. Estradiol in male and female rats 

increases CCK expression, resulting in a neurochemical sexual dimorphism favoring males 

throughout the limbic-hypothalamic lordosis regulating circuit. Despite this sex difference, 

CCK does not affect male copulatory behavior but rather mediates female sexual 

receptivity. In fact, CCK can augment lordosis behavior in castrated adult male rats treated 

with estradiol. In these male rats displaying lordosis, CCK expression in the lordosis 

regulating circuitry resembles that of female rats. Treatment with testosterone induces a 

greater expression of CCK but no female behavior, suggesting that testosterone, in addition 

to its actions on CCK expression, induces a circuit in male brains that is inhibitory to the 

display of lordosis behavior. 

Although the effects of sex steroid hormones are undeniable, there are instances 

where genetic determinants influence the brain causing sex differences that are 

independent of hormones. For example, Sry, the critical gene for sex determination in 

mammals has been identified in male mouse and human brain, but not in female brain. In 

the adult mouse brain, transcripts of Sry have been found specifically in the dopaminergic 

neurons of the substantia nigra. Down regulation of Sry expression in the brain 

significantly reduces the levels of dopamine in these nigro-striatal neurons ultimately 

altering mobility and limb-usage. These studies underscore the action of a gene, Sry, 

encoded only in the male genome to mediate a direct male-specific effect on the brain, 

independent of gonadal hormones. 

Together these studies demonstrate the complex interaction between genes and sex 

steroid hormones on the one hand, and the structure and neurochemistry on the other that 

determine the sexual dimorphism of the brain. These interactions influence brain functions 

as obviously dimorphic as reproduction and as opaque as motor function. 

55


SEXUALLY POLYMORPHIC NEUROENDOCRINE PHENOTYPES: EXAMPLES 

FROM SINGING FISH 

Bass A.H. 

Department of Neurobiology and Behavior, Cornell University, Ithaca, New York, 14853, U.S.A. 

ahb3@cornell.edu, Fax 607-254-1303 

Teleost fishes comprise nearly half of all living vertebrate species. They also show 

the greatest range of reproductive plasticity, including species with individuals that show 

sex and role change during their lifetime to species with two male morphs that permanently 

adopt alternative reproductive tactics (ARTs). We have extensively studied behavioral, 

neural and endocrine polymorphisms in the midshipman fish, Porichthys notatus, which 

has male ARTs [see 1, 2]. While the term sexual dimorphism applies, strictly speaking, to 

morphological dimorphisms, we as others have used the term to encompass behavioral and 

physiological mechanisms that diverge both within and between the sexes. 

Type I male midshipman build nests under rocky shelters in the intertidal zone and 

produce very long duration (mins - > 1h) advertisement calls (“hums”) to attract females to 

their nest. Smaller, non-humming, non-territorial type II males sneak- or satellite-spawn to 

steal fertilizations from a resident type I. Type I males also generate long trains of brief 

grunts (msec) during nest defense and egg guarding. So far, type II males and females 

(neither of which participate in nest/egg defense) have only been found to produce isolated 

grunts. We identified a parallel suite of intra- and intersexual dimorphisms in vocal traits 

along multiple dimensions, from neuromuscular junctions to the dimensions of individual 

neurons in a hindbrain-spinal vocal pattern generator (VPG) that establishes the temporal 

properties of natural vocalizations. Developmental studies of vocal motor traits support the 

hypothesis that type I and II males follow have distinct life history trajectories. Type II 

males become sexually mature at an earlier age than type I’s that essentially tradeoff early 

reproduction and gonadal maturation for a larger body size and an expansive vocal motor 

system. Type II males and females are convergent, both of which are divergent from type 

I’s, in vocal traits. These and other studies show an uncoupling of gonadal and behavioral 

(vocal) sex from neural mechanisms and an evolutionarily adaptable patterning of these 

traits (i.e., gonad, behavior and nervous system). Thus, type II males, like females and 

juveniles, essentially lack the behavioral, neurophysiological and morphological traits that 

allow type I males to produce a more dynamic vocal repertoire that functions in female 

courtship and territorial defense. This includes studies of the rapid neuromodulatory-like 

actions of neuropeptides [2] and androgenic steroids [see 3] on the vocal system. 

Might comparable dimorphisms shape the widespread evolution of ARTs among 

teleost fish [4] as well as the expression of intrasexual behavioral and phenotypes among 

other vertebrate taxa, including birds and mammals? 

Acknowledgements. Research support from U.S. NIH (DC00092), NSF (IOB-0516748). 


[1] Bass A.H., Dimorphic male brains and alternative reproductive tactics in a vocalizing fish. Trends 

Neurosci, 15 (1992)139-145. 

[2] Bass, A.H., Shaping brain sexuality. Amer Sci, 84 (1996) 352-363. 

[2] Goodson, J.L. and Bass, A.H., Forebrain peptide modulation of sexually polymorphic vocal motor 

circuitry. Nature, 403 (2000) 769-772. 

[3] Remage-Healey, L.H. and Bass, A.H., Rapid, hierarchical modulation of vocal patterning by steroid 

hormones. J Neurosci, 24 (2004) 5892-5900. 

[4] Mank, J.E. and Avise J.C., Comparative phylogenetic analysis of male alternative reproductive tactics in 

ray-finned fishes. Evol, 60 (2006) 1311-1316. 

56



ARE ESTROGENS REQUIRED FOR THE DEVELOPMENT OF THE FEMALE 

BRAIN? 

Bakker J. 

Center for Cellular & Molecular Neurobiology, University of Liège, Liège, Belgium 

The classic view of sexual differentiation in mammalian species holds that sex 

differences in the brain and behavior develop under the influence of estrogens derived 

from the neural aromatization of testosterone: the brain develops as male in the presence of 

estrogens and as female in their absence. In agreement with this view, it has been 

proposed 1 that the female brain needs to be protected from estrogens produced by the 

placenta and that alpha-fetoprotein (AFP) - a major fetal plasma protein present in many 

developing vertebrate species and produced transiently in great quantities by the 

hepatocytes of the fetal liver among other sources – is the most likely candidate to achieve 

this protection because of its estrogen-binding capacity. However, the idea that the female 

brain develops in the absence of estrogens and the role of AFP in protecting the brain 

against the differentiating action of estrogens have been challenged. First, there is 

accumulating evidence that the normal development of the female brain might actually 

require the presence of estrogens. The most recent evidence comes from our study 

revealing that female aromatase knockout (ArKO) mice that are deficient in estrogens due 

to a targeted mutation in the aromatase gene showed reduced levels of female sexual 

behavior 2 . Second, the presence of AFP within neurons in the absence of any evidence for 

local AFP synthesis suggests that AFP is transported from the periphery into the brain. It 

was thus proposed 3 that AFP acts as a carrier, which actively transports estrogens into 

target brain cells and, by doing so, has an active role in the development of the female 

brain. 

The recent introduction of an AFP mutant mouse model (AFP-KO 4 ) now finally 

allowed us to resolve this longstanding controversy concerning the role of AFP in brain 

sexual differentiation, and thus to determine whether prenatal estrogens contribute to the 

development of the female brain. We 5 found that AFP-KO females showed no female 

sexual behavior at all, and that normal levels of this behavior could be induced by blocking 

estrogen action during embryonic development, demonstrating that the principal action of 

prenatal estrogen exposure is to defeminize individuals (i.e. to decrease their capacity to 

display female sexual behavior later in life) and that AFP normally binds estradiol 

circulating in the female fetus and thereby protects the developing brain from 

defeminization. Thus our findings 5 corroborate the hypothesis originally proposed 1 and 

argue against the model that considers AFP a carrier delivering estrogen to the brain 3 . Why 

AFP is present in brain cells in some brain regions but not in others remains unclear, 

however. Likewise, the question whether estrogens are actually needed for the 

development of the female brain has not been resolved yet. Behavioral data from the AFP- 

KO 5 and ArKO 2 mouse models suggest that estrogens can have both defeminizing and 

feminizing effects on the developing brain mechanisms that control sexual behavior. 

Therefore, we suggest here that the defeminizing action of estradiol normally occurs 

prenatally in males and is avoided in fetal females because of the protective actions of 

AFP. Furthermore, the feminizing action of estradiol normally occurs in genetic females 

between birth and the age of puberty, when the ovaries start to produce estrogens and AFP 

no longer plays a role of significance. 

57



1. McEwen BS, Plapinger L, Chaptal C, Gerlach J, Wallach G (1975). Brain Res. 96: 

400-406. 

2. Bakker J, Honda S, Harada N, Balthazart J (2002). J. Neurosci. 22: 9104-9112. 

3. Toran-Allerand CD (1984). Prog. Brain Res. 61: 63-98. 

4. Gabant P, Forrester L, Nichols J, Van Reeth T, De Mees C, Pajack B, Watt A, Smitz 

J, Alexandre H, Szpirer C, Szpirer J (2002). PNAS 95: 6965-6970. 

5. Bakker J, De Mees C, Douhard Q, Balthazart J, Gabant P, Szpirer J, Szpirer C (2006). 

Nat. Neurosci. 9:220-226. 

58



SEXUAL DIMORPHISM IN THE AVIAN LIMBIC SYSTEM 

Balthazart J. 

University of Liege, Center for Cellular and Molecular Neurobiology, Research Group in 

behavioral Neuroendocrinology, 1 avenue de l’Hopital (B36), B-4000 Liege, Belgium 

Sex differences in reproductive behavior can be activational in nature, i.e. reflect 

sex differences in the endocrine milieu of the adult subjects, or organizational, i.e. 

represent irreversible effects of sex steroids during a critical period of the early 

development. The Japanese quail (Coturnix japonica) provides an excellent model to study 

these sex differences because both types co-exist in the same species. Sexually mature 

males exhibit a pre- and post-copulatory display called strutting and attract females with 

the use of a loud distinctive vocalization, the crow. Females never show these behaviors 

but this sex difference reflects exclusively the higher plasma concentration of testosterone 

in males as compared to females. Castration eliminates these behaviors in males and 

females treated with testosterone will display the behaviors exactly like males. Sexually 

mature males exposed to a female will also display a characteristic sequence of behaviors 

ultimately leading to the actual copulation, the male-typical copulatory sequence. These 

behaviors are never observed in females and cannot be activated in ovariectomized females 

by treatments with testosterone even in doses much larger than the minimally active dose 

in males. This sex difference in responsiveness to testosterone is organized before day 12 

of embryonic life by ovarian steroids. Indeed male embryos treated before day 12 of 

incubation with estradiol will be unable as adults to show the copulatory behavior in 

response to testosterone and conversely, females treated before day 12 with an aromatase 

inhibitor (that prevents secretion of estrogens by the ovary) will display as adult the full 

masculine behavioral phenotype in response to testosterone. 

If the endocrine control of the ontogeny of these sex difference is well understood, we have 

in contrast a very poor understanding of the brain mechanisms that mediate these sex 

differences, in other words we do not know what is different between the brain of a male 

and of a female that is organized by early estrogen action and justifies the sex difference in 

responsiveness to testosterone. 

Three lines of investigations have approached this problem. First neuroanatomical 

differences were investigated and volumetric differences between corresponding brain 

regions in males and females were identified. The medial preoptic nucleus (POM) is for 

example larger in males than in females but this difference is essentially activational and 

disappears in gonadectomized subjects of both sexes treated with similar doses of 

testosterone. The size of neurons in the dorso-lateral part of this nucleus is however also 

larger in males than in females and this difference seems to result from organizational 

effects of embryonic estrogens. 

A second line of research has investigated sex differences in neurochemical features of 

specific brain regions. A number of sex differences in peptide distribution were identified 

but again most of these differences appear activational in nature and thus cannot explain 

sex differences in responsiveness to testosterone. The density of the vasotocinergic 

innervation of the POM, bed nucleus of the stria terminalis and lateral septum is, however, 

higher in males than in females, demasculinized by embryonic treatment of males with 

estrogen and masculinized in females treated in ovo with an aromatase inhibitor. This 

neurochemical difference, which is controlled by the same endocrine mechanisms as 

sexual behavior is thus a likely candidate to explain the sex difference in behavior. The 

59


specific role of vasotocin in the control of male sexual behavior is however poorly 

understood. 

Finally a third line or research has tried to determine whether sex differences in behavior 

could result from differences in connectivity between brain regions. The quail POM 

projects to a premotor nucleus, the periaqueductal central gray (PAG) and this connection 

probably plays an important role in the control of sexual behavior. Retrograde tract tracing 

recently showed that males have more aromatase-immunoreactive neurons in the POM 

projecting to the PAG than females and this difference was most prominent in the caudolateral 

part of the nucleus that has been specifically implicated in the control of male 

copulatory behavior. These data therefore support the hypothesis that sex differences in 

POM-PAG connectivity are causally linked to the sex difference in the behavioral response 

to testosterone. 

60

MONDAY, 19 th February 2007 

08.30 - 12.00 

Symposium: 

Neuroactive steroids and neurogenesis

Symposium: 

Neuroactive steroids and neurogenesis 

(Chairs: Herbison A.E., Micevych P.) 

• Galea L.A.M., Barha C., Barker J.M., Pawluski J.L. Spritzer M.D. (Canada) 

Gonadal hormone regulation of adult hippocampal neurogenesis 

• Wang Z, Fowler CD (USA) Estrogen, amygdala, and adult neurogenesis 

• Brinton RD, Wang J, Irwin R, Liu L, Chen S, Chung E (USA) Allopregnanolone 

regulation of proliferation of human neural stem cells and neurogenesis in triple 

transgenic Alzheimer's disease mice 

• Abrous N (France) Neurogenesis and age-related-cognitive functions: implication 

of steroids 

• Herbert J (UK) control of neurogenesis in the adult hippocampus by corticoids and 

serotonin 

• Lecanu L., Ibrahim A., Yao W., McCourty A., Greeson J., Papadopoulos V. 

(USA) In vitro and in vivo induced neurogenesis by the naturally occurring steroid 

solasodine is associated with GAP-43/HuD pathway activation and increase of the 

translocator protein (18 kDa) (TSPO) expression



GONADAL HORMONE REGULATION OF ADULT HIPPOCAMPAL 

NEUROGENESIS 

Galea L.A.M., Barha C., Barker J.M., Pawluski J.L. and Spritzer M.D. 

Program in Neuroscience, Department of Psychology and Brain Research Centre, 

University of British Columbia, 2136 West Mall, Vancouver B.C., V6T 1Z4, CANADA, 

Fax: 001-604-822-6923, email: lgalea@psych.ubc.ca 

Gonadal hormones modulate neurogenesis in the dentate gyrus differentially in 

male and female adult rodents. Neurogenesis is comprised of both cell proliferation (the 

production of new cells) and cell survival (the number of new cells that survive to 

maturity). Acute estradiol initially enhances and subsequently suppresses cell proliferation 

in the dentate gyrus of adult female rodents, but has limited effects in male rodents. Lowlevel 

progesterone appears to attenuate the estradiol-induced enhancement of cell 

proliferation in female rodents. Intriguingly the estradiol-induced upregulation of 

hippocampal neurogenesis seems to be limited to 17-β estradiol and not extended to other 

forms of estrogens such as estrone or 17α-estradiol. Chronic levels of estradiol (15-30 d) 

also modulate hippocampal neurogenesis and cell death in adult female, but not male, 

rodents. However short-term estradiol treatment (5 days) in males enhances new cell 

survival in the dentate gyrus but only when administered during the ‘axon-extension’ 

phase. Testosterone and dihydrotestosterone upregulate hippocampal neurogenesis (via 

cell survival), but not cell proliferation, in adult male rodents. These effects of gonadal 

hormones on adult neurogenesis are not limited to exogenous manipulations but are also 

observed during endogenous fluctuations in hormones, such as during the breeding versus 

non-breeding seasons in males and females and during pregnancy and lactation in the 

mother. Pregnancy and motherhood differentially regulate adult hippocampal neurogenesis 

in the adult female rodent, with primiparous rats displaying lower levels of hippocampal 

cell proliferation and survival after parturition and multiparous rats displaying enhanced 

levels of hippocampal cell survival at this time. Thus, gonadal hormones and hormones 

associated with pregnancy and lactation appear to alter hippocampal neurogenesis. There 

are very few studies comparing males and females but those that have indicate that there is 

a sex difference in the response to hormone-regulated hippocampal neurogenesis in the 

adult. Clearly more work needs to be done to elucidate the effects of gonadal hormones on 

neurogenesis in the dentate gyrus of both male and female rodents, especially if we are to 

use our knowledge of how adult neurogenesis is regulated to develop strategies to repair 

neuron loss in neurodegenerative diseases. 

63


ESTROGEN, AMYGDALA, AND ADULT NEUROGENESIS 

Wang Z.X., and Fowler C.D. 

Department of Psychology and Neuroscience Program, Florida State University, 

Tallahassee, FL 32306, USA 

In addition to the dentate gyrus of the hippocampus (DG) and subventricular zone, 

neurogenesis has been documented in other brain regions, including the amygdala. Given 

the role of the amygdala in sensory processing and social behavior, our goal was to 

examine adult neurogenesis in this brain region in species differing in social behaviors. 

Male exposure and mating increased cell proliferation in the amygdala, but not the DG, of 

female prairie voles (Microtus ochrogaster). Since such male experience leads to increased 

levels of estrogen, we next investigated the effects of estrogen on neurogenesis. Estradiol 

treatment was ineffective in female prairie voles, but increased the density of new cells in 

the amygdala of female meadow voles (M. pennsylvanicus). The majority of these new 

cells also co-expressed estrogen receptor α, suggesting a potential direct effect of estrogen 

on proliferation. In male meadow voles, treatment of testosterone or estradiol, but not 

dihydrotestosterone, enhanced cell proliferation in the amygdala, further supporting the 

notion of estrogen-mediated adult neurogenesis. Finally, preliminary data has shown that 

anti-mitotic drug treatment decreases the number of new cells in the amygdala and inhibits 

the formation of a pair bond (a behavior mediated by the amygdala) in female prairie voles. 

Together, these data indicate that steroid hormones affect adult neurogenesis in the 

amygdala in a species-specific manner, and new cells may have functional significance in 

the vole’s social behavior. 

64



ALLOPREGNANOLONE REGULATION OF PROLIFERATION OF HUMAN 

NEURAL STEM CELLS AND NEUROGENESIS IN TRIPLE TRANSGENIC 

ALZHEIMER'S DISEASE MICE 

Brinton R.D., Wang J.M., Irwin R., Liu L., Chen S. and Chung E. 

Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, and 

Neuroscience Program, University of Southern California, Los Angeles, CA, USA 

The neuroendocrine status of the brain has been linked to the quality of the aging 

process, to risk of Alzheimer’s disease and to progression of neurodegenerative pathology. 

Data from multiple levels of analysis ranging from in vitro cellular investigations to in vivo 

studies in animal models of aging and disease to observational investigations of health 

outcomes in humans, indicate that gonadal steroid hormones and their metabolites can 

promote neural health whereas their decline or absence are associated with decline in 

neural health and increased risk of neurodegenerative disease including Alzheimer’s. 

Among the steroids in decline, is allopregnanolone (APα), a neurosteroid metabolite of 

progesterone, which was found to be reduced in the serum and plasma and brain of aged 

vs. young subjects. Further, Alzheimer disease (AD) victims showed an even further 

reduction in plasma and brain levels of APα relative to age-matched neurologically normal 

controls. Our earlier work demonstrated that APα is a neurogenic agent for rodent 

hippocampal neural progenitors and for human neural progenitor cells derived from the 

cerebral cortex. Our ongoing research seeks to determine the neurogenic potential of APα 

in the triple transgenic mouse model of Alzheimer’s disease (3xTgAD) as AD related 

pathology progresses from imperceptible to mild to severe. Initial analyses suggest that 

neurogenic potential changes with age in nontransgenic mice and that the neurogenic 

profile differs between non-transgenic and 3xTgAD mice. Comparative analyses indicate 

that APα modifies neurogenesis in both nontransgenic and 3xTgAD mice. Preliminary 

data suggest that APα may modify Alzheimer’s pathology progression. Together the data 

suggest that APα could maintain the regenerative ability of the brain and modify 

progression of AD related pathology. 

Acknowledgements: This work was supported by a grant from the Institute for Study of 

Aging and the Kenneth T. and Eileen L. Norris Foundation to RDB. 

65


NEUROGENESIS AND AGE-RELATED COGNITIVE FUNCTIONS: 

IMPLICATION OF STEROIDS 

Abrous D.N. 

INSERM U588, Institut F Magendie, University of Bordeaux 2, 146 rue Léo-Saignat, 

Bordeaux Cedex 33077, France. 

Aging is associated with cognitive dysfunction, which has been correlated to an 

alteration of hippocampal functioning Indeed, the hippocampal formation (HF) plays a 

crucial role in controlling cognitive functions, and is the brain region most vulnerable to 

ageing processes. The mammalian HF, in particular the dentate gyrus (DG), is an important 

site for the production of new neurons during adulthood. The aim of our work is to 

determine the role of adult neurogenesis in the appearance of age-related cognitive deficits. 

We have found that cognitively-impaired senescent rats display lower levels of 

neurogenesis than cognitively-unimpaired old rats. We have further shown that these interindividual 

differences result from early deleterious life events. Indeed, prenatal stress 

orients neurogenesis in pathological ways for the entire life, and precipitates age-related 

cognitive impairments. More importantly, we have recently fond that the consequences of 

prenatal stress on hippocampal plasticity can be reversed by a form of postnatal 

environmental stimulation, neonatal handling. Finally, we have highlighted that inhibition 

or stimulation of neurogenesis is one of the mechanisms by which glucocorticoids may 

fragilize and neurosteroids may protect, respectively, the cognitive functions during aging. 

Altogether these data strengthen that hippocampal neurogenesis plays a pivotal role in the 

development of pathological aging and reinforce the hypothesis of an early 

neurodevelopmental origin for psychopathological vulnerabilities in aging. 

66



CONTROL OF NEUROGENESIS IN THE ADULT HIPPOCAMPUS BY 

CORTICOIDS AND SEROTONIN 

Herbert J. 

University of Cambridge UK 

Neurogenesis in the dentate gyrus of the hippocampus in the adult is not only 

highly active but also highly labile. Glucocorticoids are a potent regulators of progenitor 

cell proliferation, maturation of new neurons, and their survival to form new connections 

with the established circuitry that links the dentate gyrus with the entorhinal cortex and the 

CA3 region of the pyramidal layer of the hippocampus. Since corticoids are themselves 

labile and are driven by events external to the animal, this means that neurogenesis is 

responsive to both predictable changes in the environment (eg time of day) as well as to 

more adventitious events (eg stressors). Corticoids interact with serotonin, a second 

powerful modulator of neurogenesis. The presence of the diurnal rhythm in corticosterone 

(in the rat) seems particularly essential for some of the other control systems to regulate 

neurogenesis. For example, abolishing this rhythm prevents the ability of fluoxetine (an 

SSRI that increases serotonin) stimulating progenitor cell division rates. The mechanism 

for this interaction is under investigation. Corticoids also interact with nitric oxide (NO), 

an intracellular regulator of neurogenesis, at least partly by altering the activity of nitric 

oxide synthases (NOSs). Current studies point to the possibility that BDNF may be a 

common endpoint for these control systems. The current interest in linking major 

depression in man with rhythmic changes in cortisol, neurogenesis in the dentate gyrus, 

activity of serotonin and polymorphisms in BDNF suggest that unraveling the control 

systems that regulate neurogenesis may have clinical as well as experimental interest. 

67


IN VITRO AND IN VIVO INDUCED NEUROGENESIS BY THE NATURALLY 

OCCURRING STEROID SOLASODINE IS ASSOCIATED WITH GAP-43/HUD 

PATHWAY ACTIVATION AND INCREASE OF THE TRANSLOCATOR PROTEIN 

(18 kDa) (TSPO) EXPRESSION 

Lecanu L. *§ , Ibrahim A. *§ , Yao W. *§ , McCourty A. *§ , Greeson J. ** and Papadopoulos 

V. *§ 

* Georgetown University Medical Center, Department of Biochemistry, Molecular and 

Cellular Biology, 3900 Reservoir Rd NW, Washington, DC 20057, USA. § Samaritan 

Research Laboratories, Georgetown University Medical Center, Washington, DC 20057, 

USA. ** Samaritan Pharmaceuticals Inc., 101 Convention Drive, Las Vegas, NV 89109, 

USA. 

Laurent Lecanu, ll55@georgetown.edu, Fax (202) 687-2354 

Repairing brain damage by replacing neuronal losses and restoring the associated 

functions is an ambitious challenge. In that aspect, the concept of stem cell therapy is 

extremely promising. The graft of stem cells differentiated into dopaminergic neurons has 

already been successfully applied to treat patients suffering from Parkinson’s disease. 

However, in disease or conditions during which the neuronal loss could be much more 

important, like Alzheimer’s disease (AD), brain stroke or traumatic brain injury, the 

transplantation of differentiated stem cells, although critical, might not be enough to 

compensate for the existing brain damage and to restore the hampered functions. Under 

these conditions one could hope that for maximal recovery, the transplantation might have 

to be associated to a stimulation of the in situ neurogenesis. We present herein the naturally 

occurring steroid (20α)-25ξ-methyl-(22R,26)-azacyclofurost-5-en-3ξ-ol (solasodine) was 

able to trigger in vitro the differentiation of neural stem cells into cholinergic neurons and 

to activate in vivo the neurogenesis from the stem cells present in the subventricular zone 

(SVZ) of rat brains. 

Solasodine was identified as potential candidate based on is silico screening for 

structural homologues of 22R-hydroxycholesterol, an intermediate of steroid biosynthesis 

that we found to induce neurogenesis in vitro but as an intermediate of steroid biosynthesis 

is rapidly metabolized. Mouse embryonic teratocarcinoma P19 cells were grown on glass 

cover-slip. When cells reached 70% confluence, the medium was replaced fresh medium 

containing 90 µM solasodine. P19 cells were then incubated for 2 days before solasodine 

was washed out and replaced by standard medium. The culture medium was changed every 

2 days for 5 days or every 2 days for 30 days before cells were fixed for 

immunocytochemistry. For in vivo studies, solasodine at 375 µM was infused for 2 weeks 

in the left ventricle of male Long-Evans rats (300-325 g) using an Alzet osmotic. Rats were 

sacrificed 3 weeks after the end of the infusion by intracardiac perfusion and brains fixed 

before being paraffin embedded for immunohistochemistry. To study neural stem cell 

proliferation, rats were injected daily with a BrdU solution at 100 mg/kg. The first 

injection took place the day following the surgery and the last injection was performed the 

day prior to euthanasia. To determine whether solasodine could be metabolized by 

steroidogenic enzymes thus affecting steroid formation mouse MA-10 tumor Leydig cells 

were treated with the compound and progesterone formation was monitored by 

radioimmunoassay. The affinity of solasodine for the various human steroid receptors was 

determined using High Throughput Screening technology in various steroid receptor 

specific cell and tissue models. 

Solasodine treatment induced P19 cell differentiation into neurons. Differentiated 

P19 cells displayed strong choline-acetyltransferase immunoreactivity showing a 

68



cholinergic phenotype. Significant sprouting processes were formed that expressed the 

specific neuronal markers βIII-tubulin and MAP2 suggesting axon formation. Solasodine 

also induced the expression of synaptophysin indicating that synaptogenesis was also 

triggered. The synapse formation and axonal growth kept evolving even 1 month after the 

treatment suggesting that the effect of solasodine was not transient. Neuronal 

differentiation was further confirmed by following the expression of doublecortin, a 

marker shown to be expressed specifically in newly formed neuroblasts. The continuous 

perfusion of solasodine inside rat left ventricle for 2 weeks induced a dramatic increase of 

the BrdU uptake by cells in SVZ. An increase of BrdU labeling was observed in the 

ependymal and sub-ependymal cells demonstrating that solasodine induced the 

proliferation of both cell populations. Although controversial, a theory already proposed is 

that the ependymal cells would serve as a progenitor cell reserve that would be activated 

only when massive neuronal cell replacement is required. Interestingly, solasodine greatly 

increased the expression of doublecortin that co-localized with BrdU immunostaining. 

Doublecortin is not expressed in proliferating cells but only in differentiating neural stem 

cells suggesting that solasodine increased the proliferating rate of the ependymal and subependymal 

cells before pushing them towards neuronal differentiation. These results 

support previous data showing that ependymal cells are capable of neurogenesis. 

Interestingly, solasodine did not bind to any of the classic steroid receptors and does not 

serve as a precursor for steroid synthesis, thus ruling out any contributing direct steroidlike 

pharmacological effect to the induced neurogenesis. 

In an attempt to identify pathways activated by solasodine, we observed that P19 

cells treated with solasodine over-expressed the neurite outgrowth-associated protein 

GAP43 and its associated post-transcriptional regulatory element HuD. Another interesting 

finding is the presence of a robust translocator protein (18 kDa; TSPO) immunostaining in 

the ependymal cells in which BrdU immunostaining was also dramatically increased, 

suggesting that solasodine increased the expression of TSPO in the proliferating 

ependymal cells. These data are consistent with previously reported findings that TSPO 

expression is increased during axonal regeneration and stem cell differentiation and the 

role of TSPO in mitochondrial membrane biogenesis, a requirement for rapidly 

proliferatings cells. 

Drug-induced activation of resident neural progenitor cell differentiation is the less 

controversial aspect of the stem cell therapy concept. The results presented herein suggest 

that the steroidal compound solasodine, naturally found in plants from the solanaceae 

family, induced neurogenesis both in vitro and in vivo. Although further studies are 

required to determine its mechanism of action, these data indicate that small molecules like 

solasodine could constitute an interesting approach to pharmacologically induce in situ 

neurogenesis as part of neuron replacement therapy. 

69

MONDAY, 19 th February 

12.00 - 13.00 

Plenary Lecture: 

Mellon S.H. (USA)

NEUROSTEROIDS IN HEALTH AND DISEASE 

Mellon S.H., Gong W., Schonemann M. 



Department of Obstetrics, Gynecology & Reproductive Sciences, The Center for 

Reproductive Sciences, 513 Parnassus Avenue, University of California, San Francisco, 

San Francisco, CA 94143-0556 USA. FAX 1-415-502-7866, 

email: mellon@cgl.ucsf.edu 

The functions for neurosteroids during development and in response to nervous 

system injury are beginning to be identified. We took several approaches to studying the 

function of some neurosteroids in vivo. Transgenic mice were created by us and by several 

other laboratories, in which the genes encoding steroidogenic enzymes were universally 

ablated. Unfortunately, none of these transgenic mice provided concrete evidence for 

functions of neurosteroids during embryonic development or during development of the 

nervous system. Hence, we chose to focus on another mouse model in which we believed 

neurosteroid production would be altered, and which had a neurodegenerative phenotype. 

Niemann Pick Type-C (NP-C) is an autosomal recessive neurodegenerative disease caused 

by mutations in NPC1 (95%) or NPC2 (5%), resulting in lysosomal accumulation of 

unesterified cholesterol and glycolipids. The NIH mouse model of NP-C has a mutation in 

the NPC1 gene, and exhibits several pathological features of the most severe NP-C 

patients. How lysosomal storage and trafficking defects lead to neurodegeneration is 

unknown. We found that these mice had normal neurosteroidogenic enzyme activity 

during development, but lost this activity in the early neonatal period, prior to onset of 

neurological symptoms. The most severely affected enzyme was 3α hydroxysteroid 

dehydrogenase, and decreased 5α reductase activity was also seen throughout the neonatal 

brain. Neurons that expressed P450scc, 3ß HSD, as well as those that expressed 3α HSD 

and 5α reductase were lost in adult NP-C brains, resulting in diminished concentrations of 

allopregnanolone. We treated NP-C mice with allopregnanolone using different regimens, 

and found that a single dose in the neonatal period resulted in a doubling of lifespan, 

substantial delay in onset of neurological symptoms, survival of cerebellar Purkinje and 

granule cell neurons, and reduction in cholesterol and ganglioside accumulation. The 

mechanism by which allopregnanolone elicited these effects is unknown. Our in vitro 

studies showed that Purkinje cell survival promoted by allopregnanolone was lost by 

treatment with bicuculline, suggesting GABA A receptors may play a role. Studies in 

collaboration with Dan Ory at Washington University using a GABA A -inactive 

entantiomer of allopregnanolone demonstrated survival and neurological benefits in NP-C 

mice, similar to allopregnanolone, suggesting a lack of GABA A -receptor involvement. 

Pregnane-X-receptors may mediate the effects of allopregnanolone and its enantiomer, as 

NP-C mice treated with these compounds increased expression of PXR-regulated genes in 

their cerebella. We showed that an additional feature of neuropathology of NP-C mice, 

region- and time-specific onset of neuroinflammation and its pathologic sequelae, is also 

ameliorated by allopregnanolone treatment, suggesting further functions for this 

neurosteroid. Thus, mouse models of neurodegeneration may be beneficial in establishing 

both physiologic and pharmacologic actions of neurosteroids. These animal models further 

establish the wide range of functions of these compounds, which may ultimately be useful 

for treatment of human diseases. 

73

MONDAY, 19 th February 2007 

15.00 - 18.30 

Symposium: 

Neuroprotective effects

Symposium: 

Neuroprotective effects 

(Chairs: Garcia-Segura L.M., Mellon S.H.) 

• Simpkins JW, Dykens JA (USA) Mitochondrial mechanisms of estrogen 

neuroprotection 

• Rosario ER, Carroll JC, Oddo S, LaFerla FM, Pike CJ (USA) Androgen 

regulation of neuropathology in a triple transgenic mouse model of AD 

• Stein DG (USA) Neurosteroids as protective factors in TBI: from laboratory 

bench to the bedside 

• Mensah-Nyagan AG, Meyer L., Kibaly C, Schaeffer V and Patte-Mensah C. 

(France) Neurosteroids and nociceptive sensitivity in neuropathic rats 

• Melcangi RC (Italy) Neuroactive steroids and peripheral neuropathy 

• Ritz M.-F., Hausmann O. (Switzerland) Neuroprotective effects of estradiol in a 

rat model of spinal cord injury. 

• Kondo S., Imaizumi K (Japan) BBF2H7, a novel transmembrane bZIP 

transcription factor, is a new type of ER stress transducer



MITOCHONDRIAL MECHANISMS OF ESTROGEN NEUROPROTECTION 

Simpkins J.W. and Dykens J.A. 

Department of Pharmacology & Neuroscience 

Institute for Aging and Alzheimer’s Disease Research 

University of North Texas Health Science Center 

Fort Worth, TX 76102 

Oxidative stress, bioenergetic failure and mitochondrial dysfunctions are all 

implicated in the etiology of neurodegenerative diseases such as Alzheimer’s disease (AD). 

The mitochondrial involvement in neurodegenerative diseases reflects the regulatory 

position mitochondrial failure plays in both necrotic cell death and apoptosis. The potent 

feminizing hormone, 17 β-estradiol (E2), is neuroprotective in a host of cell and animal 

models of stroke and neurodegenerative diseases. The discovery that 17α-estradiol, an 

isomer of E2, is equally as neuroprotective as E2 yet is >200-fold less active as a hormone, 

has permitted development of novel, more potent analogs where neuroprotection is 

independent of hormonal potency. Studies of structure-activity-relationships and 

mitochondrial function have led to a mechanistic model in which these steroidal phenols 

intercalate into cell membranes where they block lipid peroxidation reactions, and are in 

turn recycled. Indeed, the parental estrogens and novel analogs stabilize mitochondria 

under Ca 2+ loading otherwise sufficient to collapse membrane potential. The 

neuroprotective and mitoprotective potencies for a series of estrogen analogs are 

significantly correlated, suggesting that these compounds prevent cell death in large 

measure by maintaining functionally intact mitochondria. This therapeutic strategy is 

germane not only to sudden mitochondrial failure in acute circumstances, such as during a 

stroke or myocardial infarction, but also to gradual mitochondrial dysfunction associated 

with chronic degenerative disorders such as AD. (Supported by NIH grants AG10485 and 

AG22550) 

77


ANDROGEN REGULATION OF NEUROPATHOLOGY IN A TRIPLE 

TRANSGENIC MOUSE MODEL OF AD 

a Rosario E.R., a Carroll J.C., c Oddo S., c LaFerla F.M., b Pike C.J. 

a Neuroscience Graduate Program, b Davis School of Gerontology, University of Southern 

California, c Department of Neurobiology and Behavior, University of California, Irvine 

Normal, age-related, testosterone depletion in men is a recently identified risk 

factor for the development of AD. To investigate the relationship between testosterone 

depletion and the development of AD, we compared indices of pathology under different 

androgen conditions using the 3xTg-AD triple-transgenic mouse model. Male 3xTg-AD 

mice were gonadectomized (GDX) to deplete endogenous levels of androgens at 3 months 

of age, and then exposed to subcutaneous, slow-release drug delivery pellets containing 

either 10 mg dihydrotestosterone (DHT) or placebo. After 4 months of androgen depletion 

(7 months of age), mice were evaluated for behavioral deficits and severity of AD-like 

neuropathology. In comparison to gonadally intact 3xTg-AD mice, GDX mice exhibited 

robust increases in accumulation of β-amyloid (Aβ), the protein implicated as the primary 

causal factor in AD pathogenesis, in both hippocampus and amygdala. In parallel to 

elevated levels of Aβ, GDX mice exhibited significantly impaired performance in a 

spontaneous alternation Y-maze task, indicating deficits in hippocampal function. 

Importantly, DHT treatment of GDX 3xTg-AD mice prevented both neural accumulation 

of Aβ and hippocampal performance deficits. These data provide the first definitive 

evidence in an animal model linking androgen depletion to the development of AD 

pathology. These findings suggest that androgen-based hormone therapy may be a useful 

strategy for the prevention and treatment of AD in aging men. 

78



NEUROSTEROIDS AS PROTECTIVE FACTORS IN TBI: FROM LABORATORY 

BENCH TO THE BEDSIDE 

Stein D.G. 

Emory University School of Medicine, Department of Emergency Medicine, Atlanta, 

Georgia 30322 

e-mail: donald.stein@emory.edu 

phone: 404-712-9704 

Background: At present, there are no clinically effective treatments for traumatic brain 

injury (TBI). Many of the clinical trials seeking agents for the treatment of stroke and TBI 

have ended in failure (e.g., the CRASH trial with methylprednisolone and more recently 

the magnesium sulfate trial, which resulted in a 25% increase in mortality compared to 

patients given placebo). There is speculation that treatment with serum albumin may be 

efficacious, but the studies are still in progress and nothing definitive can be said yet. 

Hypothermia has been given considerable attention in the treatment of TBI, but the results 

of several clinical trials have yet to prove any substantial efficacy over controls. So, what 

is left? A growing contingent of investigators are now proposing that, after TBI and stroke, 

progesterone plays a much more important role in CNS repair, organization and function 

than has previously been realized. 

Why progesterone? Mainly because after more than 15 years of pre-clinical investigation, 

this hormone and its metabolite, allopregnanolone, appear to meet almost all the criteria for 

a safe and effective clinical treatment for TBI. The trajectory of research from the 

laboratory bench to the patient’s bedside will be the subject of my presentation. For well 

over a decade, our laboratory and others have been examining the role of progesterone and 

its precursors and metabolites to determine their specific molecular, physiological and 

functional mechanisms of action in repair and protection of the damaged central nervous 

system. There is now substantial experimental evidence that progesterone plays a vital role 

in reducing inflammatory disorders of the brain and that it enhances morphological and 

functional repair in the victims of TBI and stroke. 

A principal effect of progesterone treatment, given after injury, is to reduce cerebral edema 

and the inflammation that accompanies TBI. Cerebral edema also occurs after stroke and 

can be very problematic for patients undergoing open-heart surgery. Thus, an agent 

capable of resolving inflammation, lipid peroxidation, edema, necrosis and programmed 

cell death that also has a good safety profile with few side effects, could be of major 

benefit in a clinical setting. After presenting the background experimental data, the results 

of a recently completed, National Institutes of Health (NINDS) sponsored, Phase II (a), 

single-center trial for safety and efficacy of progesterone will be discussed. 

Summary points: 

• In laboratory animals, females with traumatic brain injury have better functional 

and morphological outcomes than males with the same extent of injury. 

79


• Treatment with exogenous progesterone and its’ metabolite, allopregnanolone in 

both adult males and females, enhances the rate and extent of recovery from 

traumatic brain injury and stroke. 

• The reparative molecular and receptor mechanisms of action of these neurosteroids 

on nerve cells, glia and vascular tissue are becoming better understood and may be 

directly applicable to clinical trial and further study. 

• Progesterone and its metabolites act by reducing immune-inflammatory reactions, 

membrane lipid peroxidation, cerebral edema, apoptosis and necrosis; thus 

preventing the slow but steady death of nerve cells long after the initial injury 

itself. 

• Progesterone and allopregnanolone stimulate soft-tissue wound repair, improve 

vascular responses to brain injury and enhance the remyelination of damaged 

axons. The neurosteroids regulate gene expression and protein synthesis involved 

in glial activity, apoptosis and regenerative repair. 

• New research is showing that progesterone and its related metabolites may also 

prove effective in the treatment of neurodegenerative disorders such as transient 

and ischemic stroke, multiple sclerosis and spinal cord injuries. 

Objectives of the Presentation: 

1. Provide a history and a better understanding of the role of neurosteroids in the early 

treatment of traumatic brain injury and stroke. 

2. Consider the data showing that sex differences in CNS functions led the way to 

research showing that so-called “sex hormones” can play a critical role in 

enhancing brain repair. 

3. Recognize that early intervention with neurosteroid treatments during the most 

acute phase of the TBI/stroke injury cascade, may enhance the efficacy of 

subsequent 

80



NEUROSTEROIDS AND NOCICEPTIVE SENSITIVITY IN NEUROPATHIC 

RATS 

Mensah-Nyagan A.G.*, Meyer L., Kibaly C., Schaeffer V. and Patte-Mensah C. 

Institut des Neurosciences Cellulaires et Intégratives, UMR7168/LC2 CNRS-ULP, Equipe 

« Stéroïdes et Système Nociceptif », 67084 Strasbourg Cedex, France. 

* E-mail : gmensah@neurochem.u-strasbg.fr 

Chronic neuropathic pain constitutes a major public health concern in many 

countries all around the world. This category of pain, which is refractory to the currently 

available analgesics including opioids, provokes persistent suffering in several thousands 

of patients and socio-economical problems such as substantial costs due to disability, 

decreased productivity and medical expenses. Therefore, development of novel therapeutic 

strategies against neuropathic pain has become a real challenge for biomedical research. 

Neuropathic pain is generated by injuries of the nervous system or by disturbances in the 

activity of spinal and supraspinal neural networks controlling nociception. Thus, it appears 

that the compounds to be characterized for the treatment of stubborn neuropathic pain must 

necessarily be capable of modulating the activity of spinal and supraspinal neuronal 

pathways. Various family of molecules are currently explored among which are 

neurosteroids that strongly modulates GABA A , NMDA and P2X receptors intervening in 

nociception and pain control. 

To obtain exploitable results on the role of neurosteroids in neuropathic pain modulation, 

we developed a multidisciplinary project allowing integration of data from molecular and 

cellular levels to behavioral components using a model of chronic pain generated in rat by 

sciatic nerve ligatures. Prior to the study with neuropathic animals, we investigated the 

occurrence of neurosteroid biosynthesis in the adult rat spinal cord (SC), a crucial structure 

involved in painful message transmission. We observed that the SC dorsal horn (DH), a 

pivotal nociceptive center, contains key steroidogenic enzymes such as cytochrome 

P450side-chain-cleavage, cytochrome P450c17 (P450c17), 5a-reductase and 3ahydroxysteroid 

oxido-reductase (3a-HSOR). Afterwards, molecular analyses using the 

real-time polymerase chain reaction after reverse transcription were used to determine 

changes occurring in the expression of genes encoding steroid-synthesizing enzymes in the 

DH during the chronic neuropathic pain situation. Reversed-phase HPLC analysis was 

coupled with flow scintillation detection to compare enzymatic activities leading to 

neurosteroid production in SC slices of control and neuropathic-pain rats. 

Radioimmunoassays allowed assessments of changes of endogenous neurosteroid levels in 

the SC during neuropathic pain state. 

The results revealed an up-regulation of enzymatic pathways (P450scc, 5a-reducatse and 

3aHSOR) leading to the biosynthesis of allopregnanolone or 3a,5a-THP (a potent allosteric 

activator of GABA A receptors) in the DH of neuropathic rats. In contrast, the biosynthetic 

pathway responsible for DHEA synthesis (P450c17) was down-regulated in the DH during 

the chronic painful state. 

Behavioral analyses were performed using the Hargreaves’ method and Von Frey filament 

test to assess respectively the thermal and mechanical nociceptive thresholds in naïve, 

sham-operated and neuropathic-pain rats. Two main symptoms characterizing chronic 

neuropathic pain in humans, namely a thermal hyperalgesia and a mechanical allodynia, 

were detected in rats submitted to sciatic nerve-evoked peripheral neuropathy. Intrathecal 

injection of 3a,5a-THP in the lumbar SC, which increased the thermal and mechanical 

sensitivity thresholds in naïve and sham-operated rats, provoked analgesia in neuropathic- 

81


pain animals by suppressing the thermal hyperalgesia and mechanical allodynia. Unlike 

3a,5a-THP, Provera, a pharmacological inhibitor of 3a-HSOR, decreased both thermal and 

mechanical nociceptive thresholds in naïve and sham-operated animals. In neuropathic 

rats, intrathecal administration of Provera potentiated both thermal hyperalgesia and 

mechanical allodynia. 

Subcutaneous administration of DHEA induced a biphasic action on pain sensitivity: a 

short term pro-nociceptive effect followed by a late analgesic action probably due to 

DHEA conversion into androgenic metabolites. The proper action of DHEA seems 

effectively to be pro-nociceptive because in vivo blockade of DHEA biosynthesis in the 

SC by intrathecal injection of ketoconazole, a pharmacological inhibitor of P450c17, 

induced analgesia in neuropathic rats. Unlike ketoconazole, intrathecal administration of 

DHEA rapidly potentiated both thermal hyperalgesia and mechanical allodynia 

characterizing the neuropathic pain. 

Since neurosteroids are endogenous compounds capable of interacting with various 

neurotransmitters involved in pain control, we hope that our results may open new 

perspectives for the development of efficient therapy against neuropathic pain based on the 

selective modulation of neurosteroidogenic pathways. 

82



NEUROACTIVE STEROIDS AND PERIPHERAL NEUROPATHY 

Melcangi R.C. 

Dept. of Endocrinology and Center of Excellence on Neurodegenerative Diseases, 

University of Milan, via Balzaretti 9, 20133, Milano Italy. 

roberto.melcangi@unimi.it 

It is now clearly demonstrated that peripheral nerves are target for the action of 

neuroactive steroids. Indeed, both classical steroid receptors, (e.g., progesterone receptor, 

PR, androgen receptor, AR, etc.), and non-classical steroid receptors (e.g., GABA-A and 

GABA-B receptors, sigma 1 receptor, NMDA receptor 1 subunit, etc.) have been 

demonstrated [see for review, 1]. On this basis, we have assessed whether treatment with 

neuroactive steroids had protective effects in experimental models of peripheral 

neuropathy, such as nerve injury (transection or crush), aging and diabetic neuropathy. 

Data we have so far obtained are very promising because they have indicated that 

neuroactive steroids, such as progesterone, testosterone and their metabolites (i.e., 

dihydroprogesterone, tetrahydroprogesterone, dihydrotestosterone and 5alpha -androstan- 

3alpha, 17beta-diol) exert important protective effects on several components of peripheral 

nerve. As will be reported, depending by experimental models considered, morphological 

parameters of the nerve, intraepidermal nerve fiber density, expression of myelin proteins, 

Na + ,K + -ATPase activity, thermal nociceptive threshold, nerve conduction velocity, which 

are affected by neurodegenerative events are a target of protective effects of neuroactive 

steroids [2-5]. These observations suggest that neuroactive steroids themselves or synthetic 

ligands of their receptors might represent an interesting therapeutic perspective for 

acquired peripheral neuropathies. Interestingly, an alternative to this therapeutic strategy 

might be represented by ligands of peripheral-type benzodiazepine receptor (PBR). This is 

a protein predominantly located in the mitochondrial outer membrane that plays an 

important role in the steroidogenesis and neurosteroidogenesis, and in the regulation of cell 

survival and proliferation. Indeed, recent observations have indicated that also PBR ligands 

may be considered as protective agents for peripheral neuropathy [6] 

(PRIN-2005060584_004 and FIRST from University of Milan). 


[1] Melcangi R.C., Cavarretta I.T., Ballabio M., Leonelli E., Schenone A., Azcoitia I., Garcia-Segura L.M., 

Magnaghi V. Peripheral nerves: a target for the action of neuroactive steroids. Brain Res. Rev. 48:328- 

338, 2005. 

[2] Melcangi R.C., Magnaghi V., Galbiati M., Ghelarducci B., Sebastiani L., Martini L. The action of steroid 

hormones on peripheral myelin proteins: a possible new tool for the rebuilding of myelin? J. 

Neurocytol., 29:327-339, 2000. 

[3] Azcoitia I., Leonelli E., Magnaghi V., Veiga S., Garcia-Segura L.M., Melcangi R.C. Progesterone and its 

derivatives dihydroprogesterone and tetrahydroprogesterone reduce myelin fiber morphological 

abnormalities and myelin fiber loss in the sciatic nerve of aged rats. Neurobiol Aging 24:853-860, 2003. 

[4] Veiga S., Leonelli E., Beelke M., Garcia-Segura L.M., Melcangi R.C. Neuroactive steroids prevent 

peripheral myelin alterations induced by diabetes. Neurosci. Lett. 402:150-153, 2006. 

[5] Leonelli E., Bianchi R., Cavaletti G., Caruso D., Crippa D., Garcia-Segura L.M., Lauria G., Magnaghi 

V., Roglio I., Melcangi R.C. Progesterone and its derivatives are neuroprotective agents in experimental 

diabetic neuropathy: a multimodal analysis. Neuroscience, in press, 2006, 

doi:10.1016/j.neuroscience.2006.11.014. 

[6] Leonelli E., Yague J.G., Ballabio M., Azcoitia I., Schumacher M., Garcia-Segura L.M., Melcangi R.C. 

Ro5-4864, a synthetic ligand of peripheral benzodiazepine receptor, reduces aging-associated myelin 

degeneration in the sciatic nerve of male rats. Mech. Ageing Dev. 126:1159-1163, 2005. 

83


NEUROPROTECTIVE EFFECTS OF ESTRADIOL IN A RAT MODEL OF 

SPINAL CORD INJURY 

Ritz M.-F. 1 , Hausmann O. 1,2 

1 

University Hospital, Department of Research, Neurosurgery Laboratory, 

Klingelbergstrasse 50, 4056 Basel, Switzerland. e-mail: marie-francoise.ritz@unibas.ch 

Fax:+41 61 267 1628, 

2 

Hirslanden Clinic St. Anna, Neurosurgery, Lucerne, Switzerland. 

Spinal cord injury (SCI) occurs mostly in young people as a result of traffic or sportsrelated 

accidents and leads to severe neurological deficits such as paraplegia and 

quadriplegia. After the initial mechanical deformation of the spinal cord, a cascade of 

biochemical and cellular processes initiate further cellular damage and cell death, known 

as the secondary injury. The secondary injury mechanisms include vascular changes 

(including ischemia, vasospasms, haemorrhages and thrombosis), ionic disturbances, 

neurotransmitter (glutamate) accumulation [1], generation of free radicals (NO) [2], 

edema, depletion of energy substrates, and activation of a variety of proteases including 

caspases, phospholipases, endonucleases and metalloproteinases [3]. This active and 

progressive spread of damage results from a process that begins within minutes and 

continues for weeks after the initial injury. Unfortunately, this secondary segmental 

neuronal loss is responsible for an often deleterious secondary functional worsening. 

Inflammation is one key-player that may exacerbate the spreading of the initial lesion. 

After experimental SCI, transcripts of pro-inflammatory cytokines such as interleukin 1 

(IL-1 etaand IL-1 lpha and tumor necrosis factor lphaare upregulated within the first 

few hours [3-5] in the injured environment. Inhibiting these processes is thought to 

potentially lead to a better functional outcome after SCI, however the beneficial effects of 

these cytokines are now also considered for improving some protective actions during 

secondary injury in SCI. 

17beta-Estradiol (E2) has been shown to possess neuroprotective activities and to modulate 

brain neurotransmitter transmission. Studies using in vivo and in vitro models of 

neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases suggest that 

estrogens provide neuroprotection of the central nervous system (CNS). Protective effects 

of E2 in stroke has also been extensively studied and include anti-apoptotic, anti-excitatory 

and anti-inflammatory actions. 

Therefore, the use of E2 to reduce secondary injury after SCI was performed in this study. 

We evaluated the effects of an immediate treatment with a physiological and a supraphysiological 

dose of E2 on the rat locomotor function recovery and on the lesion size over 

time after a mild spinal cord compression injury. In addition, the influence of this treatment 

on the inflammatory response, by measuring the release of various pro- and antiinflammatory 

cytokines was assessed. The activation of astrocytes and the size of the 

lesions were also followed at the compression site over time. 

A low physiological dose (0.1 mg/kg) and a supra-physiological dose (4 mg/kg) of E2 

were injected i.p. into male rats immediately after spinal cord compression at the T8-T9 

level. Functional outcome was assessed using the BBB score and the narrow beam walk 

test from day 3 to 4 weeks post-injury. The expression of both types of IL-1 and IL-6 as 

well as the anti-inflammatory cytokines IL-4 and IL-10 in the injured spinal cord and 

adjacent rostral and caudal segments were evaluated at 6 hr, 3 days and 1 week post-injury. 

The expressions of the glial fibrillary acidic protein (GFAP) and vimentin in astrocytes 

84



were visualized by immunohistology on spinal cord horizontal sections containing the 

compressed spine segment. 

After SCI, high-dose E2-treated rats improved more rapidly and had a better final score 

than the low-dose E2-treated group and the controls in the BBB test. These rats were also 

able to walk on the narrow beam at earlier time points compared to controls or low-dose 

treated rats and on a longer distance. However, both E2-treated groups showed the best 

performances in the narrow beam test at 4 weeks. In the lesion sites, both doses of E2 

enhanced the trauma-induced expression of IL-1alpha, IL-1beta, IL-6 and IL-1ra at 6 hr 

post-injury. The releases of anti-inflammatory cytokines were also slightly up-regulated by 

both doses of E2 but only after 3 d in the caudal segments. 

Astrogliosis, characterized by elevated expressions of GFAP and vimentin in the astrocytes 

was followed during the 4 weeks post-SCI period. The increased expression of both 

proteins was seen as early as 3 days after SCI in rats treated with 4 mg/kg E2, but only 

after 2 weeks in controls and in the low-dose E2-treated rats. Moreover, the expression of 

both proteins was higher in the high-dose E2-treated rats until 4 weeks. The sizes of the 

spinal lesions were quantified after 2 and 4 weeks post-injury. Rats treated with 4 mg/kg 

E2 showed significantly smaller lesions after 2 weeks as the low-dose treated or control 

rats. However, the lesion sizes were similar in all groups after 4 weeks, a time point where 

acute phase is already finished. 

Altogether, these results suggest that the acute treatment with a supra-physiological dose of 

E2 at the onset of spinal cord injury activates the early expression of pro-inflammatory 

cytokines involved in astroglial activation. In parallel, the lesion sizes were reduced at 2 

weeks and an improvement of the locomotor activity was observed with this treatment. A 

low physiological showed some effects on the induction of inflammatory cytokines, but the 

effects on the lesion size and functional improvement were small. The use of E2 as a 

therapy during the acute phase in spinal cord injured patients may be an option to 

significantly reduce secondary damage. 


[1] Liu, D, Thangnipon, W, McAdoo, DJ: Excitatory amino acids rise to toxic levels upon impact 

injury to the rat spinal cord. Brain Res 1991;547:344-8. 

[2] Diaz-Ruiz, A, Ibarra, A, Perez-Severiano, F, Guizar-Sahagun, G, Grijalva, I, Rios, C: 

Constitutive and inducible nitric oxide synthase activities after spinal cord contusion in rats. 

Neurosci Lett 2002;319:129-32. 

[3] Wang, CX, Olschowka, JA, Wrathall, JR: Increase of interleukin-1beta mRNA and protein in 

the spinal cord following experimental traumatic injury in the rat. Brain Res 1997;759:190-6. 

[4] Bartholdi, D, Schwab, ME: Expression of pro-inflammatory cytokine and chemokine mRNA 

upon experimental spinal cord injury in mouse: an in situ hybridization study. Eur J Neurosci 

1997;9:1422-38. 

[5] Hayashi, M, Ueyama, T, Nemoto, K, Tamaki, T, Senba, E: Sequential mRNA expression for 

immediate early genes, cytokines, and neurotrophins in spinal cord injury. J Neurotrauma 

2000;17:203-18. 

85


BBF2H7, A NOVEL TRANSMEMBRANE BZIP TRANSCRIPTION FACTOR, IS A 

NEW TYPE OF ER STRESS TRANSDUCER 

Kondo S., and Imaizumi K. 

Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of 

Medicine, University of Miyazaki, Japan. e-mail: sh-kondo@med.miyazaki-u.ac.jp 

Endoplasmic reticulum (ER) stress transducers IRE1, PERK and ATF6 are well 

known to transduce signals from the ER to the cytoplasm and nucleus when unfolded 

proteins accumulate in the ER. Recently, we identified OASIS as a novel ER stress 

transducer expressed in astrocytes. 

We report here that BBF2H7, originally identified as a novel human protein whose C- 

terminal part was fused to the FUS genes in low grade fibromyxoid sarcoma, is structurally 

homologous to OASIS. BBF2H7, an ER-resident transmembrane protein with the bZIP 

domain in the cytoplasmic portion, is cleaved at the membrane in response to ER stress. 

The cleaved fragments of the BBF2H7 translocate into the nucleus and can bind directly to 

CRE site to activate transcription of target genes. Interestingly, although BBF2H7 protein 

is not expressed in normal conditions, it is markedly induced at the translational level 

during ER stress, suggesting thatBBF2H7 might contribute to only the late phase of UPR 

signaling. In a mouse model of focal brain ischemia, BBF2H7 protein is prominently 

induced in neurons in the peri-infarction region. Taken together, our results suggest that 

BBF2H7 is a novel ER stress transducer and could play important roles in preventing 

accumulation of unfolded proteins in damaged neurons. 

86

TUESDAY, 20 th February 2007 

08.30 - 11.30 

Symposium: 

Xenoestrogens and brain circuitries

Symposium: 

Xenoestrogens and brain circuitries 

(Chair: Celotti F., Panzica G.C.) 

• Ottinger MA, Lavoie E, Thompson N, Whitehouse K, Barton M, Abdelnabi M, 

Quinn M, Jr. (USA) Neuroendocrine and behavioral consequences of embryonic 

exposure to endocrine disrupting chemicals 

• Patisaul HB, Polston EK (USA) Influence of endocrine active compounds in the 

developing brain 

• Maggi A (Italy) The ERE-Luc reporter mouse: twenty years of basic research to 

be applied to the study of endocine disrupters 

• Kawato S, Ogiue-Ikeda M., Tanabe N., Tsurugizawa T., Hojo Y., Mukai H. 

(Japan) Rapid modulation of long-term depression and spinogenesis by endocrine 

disrupters in adult rat hippocampus 

• Corrieri L., Della Seta D., Paola Materazzi, Dessì-Fulgheri F., Farabollini F., 

Fusani L. (Italy) Environmental-like exposure to xenoestrogen affects sexual 

differentiation of brain and behavior in female rats 

• Ponzi D, Palanza P , Maruniak J, Parmigiani S , Vom Saal F (USA) Sexual 

dimorphism in the number of TH-immunostained neurons in the Locus Coeruleus of 

young mice is eliminated by prenatal exposure to Bisphenol A



NEUROENDOCRINE AND BEHAVIORAL CONSEQUENCES OF EMBRYONIC 

EXPOSURE TO ENDOCRINE DISRUPTING CHEMICALS 

Ottinger M.A., Lavoie E., Thompson N., Whitehouse K., Barton M., Abdelnabi M., 

and Quinn M. Jr 1 . 

Department of Animal and Avian Sciences, University of Maryland College Park, 

Maryland 20742 USA and 1 Department of the Army, Aberdeen, MD. 

A number of endocrine disrupting chemicals (EDCs) have been characterized and 

generally appear to act via steroid-like mechanisms. However, it is not clear if avian 

species respond in a parallel manner to effects described in mammals. Moreover, there are 

also effects due to ancillary toxic actions which often vary with species. We have 

conducted comparative study of a variety of classes of EDCs that have potentially different 

mechanisms of action in an embryo bioassay, using the precocial Japanese quail model. 

The EDCs examined included estradiol, androgen active compounds, and atrazine and we 

studied effects on hypothalamic neuroendocrine systems and behavior. Fertile quail eggs 

(n=85-95/group) were injected with 17β estradiol, trenbolone, or DDE into the yolk at 

embryonic day 4 and atrazine at embryonic day 0. Quail were sampled at hatch or raised 

to maturity to determine consequences of early exposure on later reproductive maturation 

and function. Birds were behaviorally tested at 1 week of age on an adapted runway and 

adult males were tested at maturity to assess male sexual behavior. Hypothalamic 

aromatase (AROM), catecholamines, and GnRH-I were measured. Behavior proved to be 

a sensitive index of exposure for all EDCs. In young chicks, trenbolone exposure impaired 

vocalization in week old chicks. Male sexual behavior was impaired by estradiol, 

androgenic EDCs, and atrazine treatment, especially mount latency. GnRH-I was sexually 

dimorphic in adult controls; atrazine affected hypothalamic GnRH-I in hatchlings and 

adults; other EDCs had variable effects. Atrazine decreased dopamine in hatchlings and 

adults. Hypothalamic neurotransmitters that modulate reproductive function may provide 

valuable indices of endocrine disruption associated with later consequences of embryonic 

exposure to EDCs. In addition, plasma steroid hormones were affected by some of the 

EDCs; atrazine impacted thyroid gland hormone content. Finally, we have evidence for 

immune system impacts from embryonic exposure to the EDCs. In summary, the Japanese 

quail embryo provides an excellent avian model for determining effects of a variety of 

EDCs in development and for ascertaining long term impacts on adults. Attention should 

be paid to consequences of embryonic exposure to EDCs on adult health, reproductive 

function, and immune function as these types of effects are likely to impair fitness of field 

birds. 

Research supported by EPA R826134010 (Star Grant), NSF 9817024, and EPA R- 

2877801(MAO). 

89


INFLUENCE OF ENDOCRINE ACTIVE COMPOUNDS IN THE DEVELOPING 

BRAIN 

Patisaul H.B. 1 , and Polston E.K. 2 

1 North Carolina State University, Department of Zoology, 127 David Clark Labs, Raleigh, NC 

27695 USA, fax: (919) 515-5327, heather_patisaul@ncsu.edu 

2 Howard University College of Medicine, Department of Physiology, 520 W St. NW, Washington, 

DC, 20059 USA 

Changes in the volumes of sexually dimorphic brain nuclei are often used as a biomarker 

for developmental disruption by endocrine-active compounds (EACs). However, these gross, 

morphological analyses do not reliably predict disruption of cell phenotype or neuronal function. In 

the present experiments, we used a more comprehensive approach to assess whether postnatal 

exposure to the EACs genistein (GEN) or bisphenol-A (BPA) affected the development of two 

sexually dimorphic brain regions in male and female rats: the anteroventral periventricular nucleus 

of the hypothalamus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN). The 

AVPV and SDN are both structurally and functionally differentiated in rodents. In females, the 

AVPV relays hormonal and environmental signals to the gonadotropin-releasing hormone (GnRH) 

neurons that regulate ovulation. The female AVPV is larger than that of the male and contains 

higher numbers of tyrosine hydroxylase (TH) expressing neurons. The SDN is 2 – 4 times larger in 

males than females and is thought to play a role in the display of male sex behaviors. Recently, a 

sexually dimorphic subpopulation of neurons within the SDN that expresses calbindin-d28k 

(CALB) was described. The volume of this region, the CALB-SDN, is also larger in males. We 

hypothesized that neonatal exposure to BPA or GEN in the first few days of life, when both the 

AVPV and the SDN are undergoing steroid hormone directed sexual differentiation, would disrupt 

the anatomical structure, cellular phenotype, and function of these nuclei. Male and female rat pups 

were given 4 subcutaneous injections of sesame oil (control), 50 µg 17β-estradiol (E2), 250 µg 

GEN, or 250 µg BPA at twelve hour intervals over postnatal days (PND) 1 and 2. Half of the 

animals were sacrificed on PND 19 and the other half were allowed to grow to adulthood. In the 

PDN 19 animals, TH expression was unaltered by either BPA or GEN in the female AVPV, but 

both compounds demasculinized TH expression in the male AVPV. This observation suggests that 

BPA and GEN blocked the masculinizing effect of endogenous estrogen on this endpoint. 

Interestingly, in the adult animals GEN and E2, but not BPA, disrupted AVPV volume in both 

sexes, and neither EAC feminized GnRH secretion patterns in the adult males. GEN, also 

disrupted estrus cyclicity in the females. Over 80% of the GEN and E2 treated females were 

acyclic 6 weeks post-puberty, but the BPA females retained normal cycles through this period. 

These data demonstrate that the disruption of nuclear volume does not necessarily coincide with 

disruption of cellular phenotype or neuroendocrine function. SDN volume and phenotype were 

only examined in the adult males. SDN and CALB-SDN volume were unchanged by treatment, but 

the number of neurons immunoreactive for CALB in the SDN was significantly increased by both 

BPA and GEN. In this case the results suggest that BPA and GEN augmented the masculinizing 

effects of endogenous estrogen. Collectively, our results demonstrate that neonatal exposure to 

EACs such as BPA and GEN can affect sexually dimorphic brain morphology and neuronal 

phenotypes in adulthood with regional and cellular specificity. They also illustrate that EACs can 

simultaneously augment and interfere with the effects of endogenous estrogen on the developing 

brain. These findings emphasize the need to employ a comprehensive approach that addresses both 

anatomical and functional endpoints when evaluating the potential effects of EAC exposure. 

90



THE ERE-LUC REPORTER MOUSE: TWENTY YEARS OF BASIC RESEARCH 

TO BE APPLIED TO THE STUDY OF ENDOCRINE DISRUPTERS 

Maggi A. 

Center Of Excellence CEND University Of Milan, Via Balzaretti 9, I-20133 Milan, Italy. 

E-Mail: adriana.maggi@unimi.It. Fax +39-0250318290 

In view of the newly acquired awareness on the complexity and tissue specificity of IR 

mechanism of action and functions there is a growing concern on the validity of the 

methodologies so far applied to identify EDs and to predict their harmful effects. Current 

in vivo and in vitro methodologies restrict the analysis to very specific organs or cell types 

and, therefore, are not sufficient to predict the potential consequences of EDs after short or 

long-term exposure. The recent emphasis on generating in vitro model systems for 

toxicological analysis has certainly discouraged the development of models that are 

suitable to envision the whole spectrum of effects on the body, which is required when 

dealing with endocrine disruption that is, by definition, a living organism. We took 

advantage of novel molecular imaging techniques applied to animal engineering create a 

model system enabling to measure in living animals the state of trancriptional activity, the 

ERE-Luc reporter mouse. This model represents a first paradigmatic reporter mouse 

provided major insights on ER physiology and has been utilized to asses its suitability to 

the study of the systemic effects of EDs. 

With comparative studies involving the quantitative analysis of photon emission in vivo 

and luciferase enzymatic activity ex vivo we demonstrate that imaging is a method 

applicable to obtain a map of the effects of different concentrations of a given 

xenoestrogen in space and time. The faithfulness of luciferase as reporter of ER 

transcriptional activity is shown by comparing the effect of administration of oestrogens of 

different origin (the natural hormone 17β-oestradiol; the phytoestrogen genistein and the 

synthetic oestrogens p,p’-DDT and BHC) on the accumulation of luciferase or transcripts 

from endogenous ER target genes (such as progesterone receptor, CYP17 and the 

oestrogen receptors themselves). We also appied imaging-based methodology to the study 

of the effects of long-term exposure to food oestrogens like the phytoestrogen genistein or 

to complex mixture of oestrogens such as soy milk. Our experiments showed that 

phytoestrogens accumulate in the body and in long-term exposures interfere with ER 

signalling in a tissue selective manner. Most interestingly, treatment with soy milk 

generates a state of activation of ERs which differs significantly from that which was 

shown with genistein: in long-term treatment we observed a slight activation of ERs in 

liver, but a very significant activation in testis, indicating that the pure substance, genistein, 

may have effects significantly different than mixtures of phytoestrogens even if enriched in 

genistein itself. The model system proved particularly sensitive to exposure to different 

concentrations of xenoestrogens because it could show different responses to 

administration of pure or dilute soy milk and the state of ER activity was directly 

proportional to the amount of soy milk ingested. These data point to the necessity to better 

investigate the potential ED activity of soy milk, particularly when administered to infants 

of both sexes. 

In our view, the reporter mouse technology is an excellent candidate to REPLACE the 

existing tests that, for their nature, are unable to provide an overall view of oestrogenic 

activity in the whole organism. By means of non-invasive in vivo imaging technology, the 

methods provide the opportunity to REDUCE the number of animals to be used in the in 

vivo tests first of all because: animal sacrifice is not needed, and secondly the possibility to 

91


follow the endocrine effects in time in the same animal reduces the need to use large 

numbers of animals in each experimental group to reach significant data and also reduces 

the need for control groups (the effects of a treatment is evaluated versus the baseline state 

of activity of the receptor on the same animal). Last, but not least, the technology will 

REFINE current methods by providing for the first time the possibility to study the effect 

of EDs systemically and, after long-term exposure even to low doses, our methodology 

will eliminate the pain created by animal testing and abolish the necessity of animal 

sacrifice. 


[1] Ciana P, Brena A, Sparaciari P, Bonetti E, Di Lorenzo D, Maggi A. Estrogenic 

activities in rodent estrogen-free diets. Endocrinology. 2005, 146:5144-50. Epub 2005 

Sep 8. 

[2] Di Lorenzo D, Villa R, Biasiotto G, Belloli S, Ruggeri G, Albertini A, Apostoli P, 

Raviscioni M, Ciana P, Maggi Isomer-specific activity of 

dichlorodyphenyltrichloroethane with estrogen receptor in adult and suckling estrogen 

reporter mice. Endocrinology. 2002, 143:4544-51. 

[3] Maggi A, Ciana P. Reporter mice and drug discovery and development. Nat Rev Drug 

Discov. 2005, 4:249-55. 

[4] Villa R, Bonetti E, Penza ML, Iacobello C, Bugari G, Bailo M, Parolini O, Apostoli P, 

Caimi L, Ciana P, Maggi A, Di Lorenzo D. Target-specific action of organochlorine 

compounds in reproductive and nonreproductive tissues of estrogen-reporter male 

mice. Toxicol Appl Pharmacol. 2004, 201:137-48 

92



ENVIRONMENTAL-LIKE EXPOSURE TO XENOESTROGEN AFFECTS 

SEXUAL DIFFERENTIATION OF BRAIN AND BEHAVIOR IN FEMALE RATS 

°Corrieri L., *Della Seta D., *Materazzi P., °Dessì-Fulgheri F., *Farabollini F., and 

*Fusani L. 

*Dipartimento di Fisiologia, Sezione di Neuroscienze e Fisiologia Applicata, Università di 

Siena, via Aldo Moro, 53100, Siena, Italy, e-mail: fusani@unisi.it, fax: +39-0577-234037; 

°Dipartimento di Biologia Animale e Genetica, Università di Firenze. 

Estrogens act on the brain during development to regulate sexual differentiation of brain 

and behavior. In the adult female, estrogens are involved in the regulation of sexual behavior by 

acting on estrogen-sensitive brain structures. In particular, the sexually dimorphic nuclei of the 

preoptic area (SDN-POA) and the anteroventral periventricular nucleus (AVPV) are sexually 

dimorphic and are involved in the regulation of sexual behavior and the estral cycle in female rats. 

Environmental estrogens or xenoestrogens, entering the body through contaminated food and 

water, have the potential of interfering with the differentiation of brain and behavior and thus 

induce permanent physiological, behavioral and neural alterations. There is little experimental 

evidence for such effects in mammals, and the biological relevance of several studies has been 

question because the modalities of treatment did not mimic environmental exposure. In addition, 

most ‘xenoestrogens’ have additional toxic effects, therefore it is difficult to identify true 

xenoestrogenic modulation. 

We studied the effects of an environmental-like exposure to the pure, synthetic estrogen, 

ethynylestradiol (EE), on the sexual differentiation of brain and behavior in Sprague-Dawley 

female rats. EE is found in surface waters for its widespread use – it is the main estrogen of 

anticonceptional pills. The rats were treated from conception to puberty with either of two doses of 

EE: a lower dose (EEL, 4 ng/kg/day) equivalent to concentrations found in contaminated waters, 

and a higher dose (EEH, 400 ng/kg/day) equivalent to that of anticonceptional pills. Rats were 

treated indirectly from GD 5 to PND 21 by oral administration in peanut oil (EEL, EEH, or vehicle 

only = OIL) to the mothers and directly from PND 22 to PND 30. At 12 weeks of age, females 

were tested for sexual behavior with a standard sexually active male. After testing, the rats were 

deeply anesthetized and perfused with 4% formaldehyde. The brain was dissected, postfixed for 2 

hr in 4% formaldehyde and then cryoprotected by impregnation with 10% and then 20% sucrose in 

phosphate-buffer saline. Forty-micron sections were cryocut, mounted, and stained with thionin. 

We took digital microphotographs of the preoptic-hypothalamic region at 40X and measured the 

areas of interest. 

Exposure to the higher EE dose (EEH) caused loss of estral cyclicity with permanent 

vaginal estrus. Proceptive behavior was altered in females treated with the lower dose of EE (EEL) 

compared to controls (OIL). The EEL females also showed a larger volume of the medial preoptic 

nucleus compared to both EEH and OIL females. Thus, although exposure to pharmacological 

doses of EE throughout development results in major physiological alterations in adulthood, effects 

on sexually dimorphic brain structures appears to be limited. On the contrary, an exposure to lower, 

environmental-like doses of EE does not disrupt the estral cycle, but both sexual behavior and 

sexually dimorphic nuclei are altered. This work shows that xenoestrogen exposure during 

development can affect the differentiation of sexual behavior and sexually dimorphic nuclei in 

female rats, and that concentrations of xenoestrogen with no evident effects on physiological 

markers can in fact induce significant alterations in brain morphology and behavior. 

93


SEXUAL DIMORPHISM IN EXPLORATION AND IN THE NUMBER OF TH- 

IMMUNOSTAINED NEURONS IN THE LOCUS COERULEUS OF YOUNG MICE 

IS ELIMINATED BY PRENATAL EXPOSURE TO ENVIRONMENTAL 

ESTROGENS 

Ponzi D.(1,2), Maruniak J.(2), Parmigiani S.(1), Vom Saal F.S.(2), and Palanza P.(1) 

(1) Dipartimento di Biologia Evolutiva e Funzionale, Università di Parma, Parco Area 

delle Scienze 11A, 43100 Parma, Italy; (2) Div. Biol. Sci. Univ. of Missouri, Columbia- 

Mo, USA 

Bisphenol A is a man made chemical used in the production of polycarbonate, 

epoxy resins lining food storage cans and dental sealants. Because of its estrogenic 

activity, very high production volume, and presence in virtually all humans there is 

concern about its ability to disrupt the endocrine system, especially the neuroendocrine 

system. During fetal life the intrauterine environment is critical for the normal 

development, and even small changes in the levels of hormones, such as estradiol, or 

estrogen-mimicking chemicals, such as BPA, lead to changes in the brain structure, 

function and consequently in the behavior. It has thus been proposed that exposure during 

fetal life to chemicals such as BPA could contribute to mental diseases expressed later in 

life. In this experiment we administered to CD-1 pregnant mice 50 micrograms/kg body 

weight or to 5 mg/kg body weight doses per day of BPA from day 11-18 of gestation. 0.5 

microgram/kg of diethylstilbestrol (DES) and corn oil were used as positive and negative 

controls respectively. The aim of this experiment was to examine the effect of the prenatal 

exposure of BPA or DES on the patterns of exploration and on the number of neurons 

producing tyrosine hydroxylase (TH) in the locus coeruleus of 30 days old mice by 

performing an immunohistochemical assay. We choose to examine LC because of its 

sexual dimorphism in rodents and because the noradrenergic system (and more widely the 

monoaminergic system) is altered in several neurological diseases such as ADHD, anxiety 

related disorders and Parkinson's disease. Mice perinatally exposed to BPA and DES 

showed altered patterns of exploratory behavior; specifically, sex differences in 

exploratory activity were eliminated. We found that control animals showed sex difference 

in the number of TH-stained neurons in the LC, with females having significantly more 

stained neurons, but the exposure to the 5mg dose of BPA eliminate this difference as did 

DES. This study provides further evidence that fetal exposure to doses of BPA that are far 

below the “safe” , no effect dose can alter behavioral and brain development. 

94

TUESDAY, 20 th February 2007 

11.30 - 12.30 

Young Investigators Symposium

Young Investigators Symposium 

(Chairs: Frye C., Mensah-Nyagan A.G.) 

• Belloni V., Alleva E., Dessì-Fulgheri F., Zaccaroni M., Santucci D. (Italy) Effects 

of low doses of atrazine on the neurobehavioral development of mice 

• Forlano P.M., Bass, A.H. (USA) Substrates for plasticity: brain aromatase, 

estrogen and androgen receptors in sexually dimorphic, vocal-acoustic and 

auditory pathways in a teleost fish 

• Meyer L., Patte-Mensah C., Mensah-Nyagan AG. (France) The enzyme 3alphahydroxysteroid 

oxidoreductase is a key regulator of nociceptive mechanisms in the 

rat spinal cord 

• Romeo, R.D., McEwen, B.S. (USA) Stress during adolescence leads to depressivelike 

behaviors and changes in hypothalamic-pituitary-adrenal axis function in 

adulthood 

• Taziaux M., Keller M., Bakker J., Balthazart J. (Belgium) Brain estradiol rapidly 

regulates in a neurotransmitter-like fashion male sexual behavior in mice 

• Paris J.J., Rhodes M.E., Frye C.A. (USA) Inhibition of 3α,5α-THP formation 

decreases exploratory/anti-anxiety and socio-sexual behavior in sexually receptive 

female rats



EFFECTS OF LOW DOSES OF ATRAZINE ON THE NEUROBEHAVIORAL 

DEVELOPMENT OF MICE 

Belloni V. * , Alleva E. § , Dessì-Fulgheri F. * , Zaccaroni M. * , and Santucci D. § 

* Department of Animal Biology and Genetics, University of Firenze, Via Romana 17, 

I-50125 Firenze, Italy 

§ 

Section of Behavioral Neurosciences, Dep. Cell. Biol. and Neuroscience, Istituto Superiore di 

Sanità, viale Regina Elena 299, I-00161 Rome, Italy 

Atrazine (ATZ) is a triazine herbicide widely used and frequently detected in ground and 

surface water. The extensive use of atrazine has made this compound a focus for environmental 

impact studies [6]. 

Recent studies suggest that ATZ is an environmental risk factor able to affect estrogen production 

by inducing aromatase [7], the enzyme that converts androgen into estrogen [2], an essential 

transformation occurring at the CNS level for maturation and expression of behavior. Then, since it 

is well known that availability of sexual steroids, testosterone and estradiol, at CNS level, is 

essential for maturation and expression of sexually dimorphic behaviors [3], we consider that 

behavior may be an efficient marker of subtle effects of low ATZ concentrations. 

In the present study we evaluated the effects of environmentally-relevant doses of ATZ on somatic 

growth and early behavioral ontogeny, a crucial stage in shaping future behavior. For this purpose 

we observed mice born to mothers exposed to 1 or 100 µg/kg ATZ during pregnancy and lactation. 

We studied, between postnatal day 2 to 15, righting reflex, cliff aversion, forepaw grasping, 

auditory startle, eyelid and ear opening [5], and ultrasound vocalizations vocalizations [1, 4]. In 

both sexes effects of ATZ were evident on body weight at birth, on the maturation of righting and 

grasping reflexes, and on the rate of emission and the spectrographic characteristics of ultrasound. 

At birth (PND2) the offspring of ATZ treated mothers was lighter than controls (Fig.1), but this 

difference is compensated from PND4 on. 

Moreover, ontogenesis of cliff aversion and of auditory startle were not affected by ATZ, while 

righting reflex maturation was delaied in both sexes and grasping reflex maturation was accelerated 

in male pups (Table 1). 

Finally, the rate of emission of vocalizations showed a tendency to decrease at PND9 both in males 

and in females (Fig.2). A spectrographic analysis was conducted at PND9, revealing an increase of 

the interval between vocalizations in females (Fig.3) and an increase of the peak frequency 

(maximum amplitude frequency) in ATZ pups. 

These findings are also consistent with an additional study conducted to evaluate long-term effect 

of perinatal administration of ATZ on behavior from weaning to the adult. Infact, our results 

showed that ATZ is able to affect explorative and social behavior in males and cognitive behavior 

in both sexes. 

Dosage level appeared also particularly relevant since, in some cases the lowest ATZ exposure was 

more effective than the highest one in modifying behavior. This suggest that this compound, 

similarly to many others endocrine disruptors [8], does not follow a linear dose-response curve [6], 

and that, as a consequence, its effects should be studied by employing low, environmentallyrelevant 

exposure levels. Our results, compatible with ATZ properties, suggest caution in the use of 

a chemical agent that may, even at low doses, interfere with brain development and differentiation, 

inducing alterations of developmental trajectories of behavior. 


[1] Alleva, E., Laviola, G., Tirelli, E., Bignami, G. Short-, medium-,and long-term effects of prenatal oxazepam on 

neurobehavioural development of mice. Psychopharmacology, 1985;87:434-441. 

[2] Amateau, S.K., Alt, J.J., Stamps, C.L., McCarthy, M.M. Brain estradiol content in newborn rats: sex differences, 

regional heterogeneity, and possible de novo synthesis by the female telencephalon. Endocrinology, 

2006;145:2906-2917. 

[3] Arnold, A.P., Gorski, R.A. Gonadal steroid induction of structural sex differences in the central nervous system. 

Ann Rev Neurosci, 1984;7:413-442. 

97


[4] Branchi, I., Santucci, D., Alleva, E. Ultrasonic vocalization emitted by infant rodents: a tool for assessment of 

neurobehavioral development. Behav Brain Res, 2001;125:49-56. 

[5] Fox, M. Reflex-ontogeny and behavioural development of the mouse. Anim Behav, 1965;13:234-241. 

[6] Hayes, T.B., Haston, K., Tsui, M., Hoang, A., Haeffele, C., Vonk, A. Feminization of male frog in the wild. 

Nature, 2002;419:895-896. 

[7] Sanderson, J.T., Letcher, R.J., Heneweer, M., Giesy, J.P., van den Berg, M. Effects of Chloro-s Triazine Herbicides 

and Metabolites on Aromatase Activity in Various Human Cell Lines and on Vitellogenin Production in Male Carp 

Hepatocytes. Environ Health Perspect , 2001;109:1027-1031. 

[8] Welshons, W.V., Nagel, S.C., vom Saal, F.S., Large effects from small exposures. III. Endocrine mechanisms 

mediating effects of bisphenol A at levels of human exposure. Endocrinology, 2006;147: 56-69. 

Table 1. Assessment of somatic growth and neurobehavioral development (day of maturation) of 

atrazine-treated pups and their controls from PND2 to PND15 (n of subjects). 

Day of adult-like response 

Males 

Righting 

Cliff 

aversion 

Forepaw 

grasp 

Auditory 

startle 

Eyes 

opening 

Ears 

opening 

Control (20) 4,20±0,40 4,20±0,40 7,50±0,53 11,45±0,37 14,90±0,10 14,35±0,13 

ATL (17) 4,65±0,73 4,12±0,49 5,18±0,53** 11,18±0,42 14,65±0,17 14,05±0,13 

ATH (20) 6,35±0,69 3,50±0,20 5,25±0,50** 11,25±0,40 14,95±0,08 14,30±0,10 

Females 

Control (20) 4,70±0,67 4,40±0,37 6,05±0,65 11,72±0,27 14,95±0,11 14,30±0,01 

ATL (18) 5,00±0,71 4,22±0,45 6,77±0,60 10,38±0,43 14,89±0,08 14,17±0,09 

ATH (20) 6,85±0,68 * 4,35±0,51 6,65±0,62 10,71±0,41 15,00±0,00 14,35±0,11 

ATL, low dose atrazine; ATH, high dose atrazine. **p



SUBSTRATES FOR PLASTICITY: BRAIN AROMATASE, ESTROGEN AND 

ANDROGEN RECEPTORS IN SEXUALLY DIMORPHIC, VOCAL-ACOUSTIC 

AND AUDITORY PATHWAYS IN A TELEOST FISH 

Forlano P.M., and Bass A.H. 

Department of Neurobiology and Behavior, Cornell University, Ithaca, NY, U.S.A. 

pmf4@cornell.edu, Fax 607-254-1303 

The plainfin midshipman fish, Porichthys notatus, is a well-established 

neuroethological model system that has provided insights into neural and endocrine 

mechanisms of vocal-acoustic communication that are conserved throughout vertebrates. 

During the breeding season, males emit a mate call generated by a hindbrain-spinal cord 

pattern generator which innervates sonic musculature, and this signal is necessary and 

sufficient for females to localize potential mates [4]. Furthermore, this vocal-motor system 

is inter-and intrasexually dimorphic, i.e. individual neurons and nuclear volume are larger 

in courting (type I) males compared to non-courting (type II) males and females [1,3]. 

Androgens are known to masculinize dimorphic vocal motor circuitry and muscle, and 

both androgens and estrogen rapidly modulate vocal patterning, and are elevated during 

gonadal recrudescence in males and females and vocal courtship in males [2,5,11,12,15]. 

Since the enzyme aromatase could provide estrogen and/or regulate how much androgen 

(testosterone) reaches specific populations of neurons, we wanted to determine the sites of 

action of aromatase as well as estrogen and androgen receptors in the function of this 

behaviorally significant circuitry. 

We investigated the neuroanatomical localization of aromatase mRNA and protein 

using in situ hybridization (ISH) and teleost-specific antibodies [9]. The cellular basis of 

aromatase production in the teleost brain was discovered by showing abundant expression 

throughout the central nervous system in radial glia rather than neurons. Highest numbers 

of cells expressing aromatase were found in the forebrain, along ventricular areas 

throughout the brain and in the sexually dimorphic vocal-motor center of the hindbrainspinal 

cord. Quantitative ISH showed inter and intrasexual dimorphism in aromatase 

mRNA expression within the vocal hindbrain-spinal cord, consistent with differences in 

aromatase activity and other dimorphisms in this region between alternative male 

reproductive/vocal phenotypes [6,13]. 

In order to identify potential sites of action of local produced estrogens as well as 

sites of interaction between androgens and aromatase, ISH was employed to identify 

estrogen receptor alpha (ER) and androgen receptor (AR) mRNA distribution. AR 

expression was found to be more widespread than ER alpha in the brain, and robustly 

expressed in descending vocal motor nuclei and vocal-acoustic integration centers. 

Furthermore, while ER expression in the dimorphic sonic motor nucleus appear to be over 

the motor neurons themselves, AR mRNA follows a similar pattern to aromatase 

expression in glial cells, surrounding the motor nucleus [8,10]. Thus, while ARs may act 

directly on glial cells to regulate aromatase expression, estrogen produced by glia likely 

acts in a paracrine fashion to reach nearby motor neurons which express nuclear and/or 

membrane ERs. Together, this neuroanatomical evidence demonstrates that aromatase in 

glial cells within vocal circuitry can provide a local estrogen source for rapid modulation 

of vocal behavior. Furthermore, aromatase may function to regulate male morph-dependent 

differences in the availability of estrogen to act as a neurosteroid throughout the forebrain 

and in brain regions that support divergent reproductive and, in this case vocal, 

phenotypes. 

99


Males and females have distinct seasonal patterns of circulating steroids which 

correspond to changes in gonadal development, and reproductive and vocal behavior [15]. 

Frequency encoding by the inner ear of females changes seasonally, such that they are 

better adapted to detect the mate call of the male during the breeding season. This plasticity 

in audition was demonstrated experimentally to depend on elevated plasma levels of either 

testosterone or estradiol that naturally accompany the seasonal occurrence of reproduction 

[14]. Furthermore, these same manipulations upregulate and mimic seasonal changes in 

expression of aromatase, throughout the brain [7]. In the peripheral auditory system, 

aromatase-ir was identified in ganglion nerve cells in the branch of the eighth nerve 

adjacent to the sensory epithelium of the main auditory end organ (saccule) of the inner ear 

of females. Furthermore, ERα mRNA expression was found in unidentified cells just 

outside the saccular hair cell layer [8]. Localization of aromatase and ER in the inner ear 

support the hypothesis that estradiol alone could account for effects of circulating steroids 

on hearing. Thus, the ear itself may provide a local source of estrogen independent of the 

gonad to modulate plasticity of hearing. 

Research support from the U.S. National institutes of Health (NIDCD DC00092) and National Science 

Foundation (IBN9987341, IOB-0516748) to A.H.B., NIMH T32MH015793 to P.M.F. 


[1] Bass, A.H. and Baker, R., Sexual dimorphisms in the vocal control system of a teleost fish: morphology of 

physiologically identified neurons, J Neurobiol, 21 (1990) 1155-1168. 

[2] Bass, A.H. and Forlano, P.M., Neuroendocrine mechanisms of alternative reproductive tactics: the chemical 

language of social plasticity. In R. Oliveira, M. Taborsky and J. Brockmann (Eds.), Alternative Reproductive 

Tactics: An Integrative Approach, Cambridge University Press, Cambridge, UK, in press. 

[3] Bass, A.H. and Marchaterre, M.A., Sound-generating (sonic) motor system in a teleost fish (Porichthys 

notatus): Sexual polymorphisms and general synaptology of sonic motor nucleus, J Comp Neurol, 286 (1989) 

154-169. 

[4] Bass, A.H. and McKibben, J.R., Neural mechanisms and behaviors for acoustic communication in teleost fish, 

Prog Neurobiol, 69 (2003) 1-26. 

[5] Brantley, R.K., Marchaterre, M.A. and Bass, A.H., Androgen effects on vocal muscle structure in a teleost fish 

with inter-sexual and intra-sexual dimorphism, J Morphol, 216 (1993) 305-318. 

[6] Forlano, P.M. and Bass, A.H., Seasonal plasticity of brain aromatase mRNA expression in glia: Divergence 

across sex and vocal phenotypes, J Neurobiol, 65 (2005) 37-49. 

[7] Forlano, P.M. and Bass, A.H., Steroid regulation of brain aromatase expression in glia: Female preoptic and 

vocal motor nuclei, J Neurobiol, 65 (2005) 50-8. 

[8] Forlano, P.M., Deitcher, D.L. and Bass, A.H., Distribution of estrogen receptor alpha mRNA in the brain and 

inner ear of a vocal fish with comparisons to sites of aromatase expression, J Comp Neurol, 483 (2005) 91-113. 

[9] Forlano, P.M., Deitcher, D.L., Myers, D.A. and Bass, A.H., Anatomical distribution and cellular basis for high 

levels of aromatase activity in the brain of teleost fish: aromatase enzyme and mRNA expression identify glia 

as source, J Neurosci, 21 (2001) 8943-55. 

[10] Forlano, P.M., Marchaterre, M.A., Deitcher, D.L. and Bass, A.H., Distribution of androgen receptor mRNA in 

vocal and non-vocal circuitry of a teleost fish. Society for Neuroscience, Society for Neuroscience Online, 

Washington, D.C., 2005, pp. 1001.6. 

[11] Knapp, R., Marchaterre, M.A. and Bass, A.H., Relationship between courtship behavior and steroid hormone 

levels in parental male plainfin midshipman fish, Horm Behav, 39 (2001) 335. 

[12] Remage-Healey, L. and Bass, A.H., Rapid, hierarchical modulation of vocal patterning by steroid hormones, J 

Neurosci, 24 (2004) 5892-900. 

[13] Schlinger, B.A., Greco, C. and Bass, A.H., Aromatase activity in the hindbrain vocal control region of a teleost 

fish: divergence among males with alternative reproductive tactics, P Roy Soc Lond B Bio, 266 (1999) 131- 

136. 

[14] Sisneros, J.A., Forlano, P.M., Deitcher, D.L. and Bass, A.H., Steroid-dependent auditory plasticity leads to 

adaptive coupling of sender and receiver, Science, 305 (2004) 404-7. 

[15] Sisneros, J.A., Forlano, P.M., Knapp, R. and Bass, A.H., Seasonal variation of steroid hormone levels in an 

intertidal-nesting fish, the vocal plainfin midshipman, Gen Comp Endocrinol, 136 (2004) 101-116. 

100



THE ENZYME 3ALPHA-HYDROXYSTEROID OXIDOREDUCTASE IS A KEY 

REGULATOR OF NOCICEPTIVE MECHANISMS IN THE RAT SPINAL CORD 

Meyer L., Patte-Mensah C. and Mensah-Nyagan A.G.* 

Institut des Neurosciences Cellulaires et Intégratives, UMR7168/LC2 CNRS-ULP, Equipe 

« Stéroïdes et Système Nociceptif », 67084 Strasbourg Cedex, France. 

* E-mail : gmensah@neurochem.u-strasbg.fr 

Neuroactive 5α,3α-reduced steroids are potent activators of GABA-A receptors 

which play a crucial role in the modulation of nociceptive sensitivity. The biosynthesis of 

these steroids requires the activity of 3α-hydroxysteroid oxidoreductase (3α-HSOR) which 

converts 5α-reduced steroids such as dihydroprogesterone (5α-DHP) into 5α,3αmetabolites 

as tetrahydroprogesterone (5α,3α-THP) also called allopregnanolone. 

Immunohistochemical investigations revealed a strong expression of 3α-HSOR in 

the rat spinal cord (SC), particularly in the dorsal horn which controls nociceptive 

transmission. Incubation of SC slices with [3H]progesterone yielded the formation of 

[3H]5α-DHP and [3H]5α,3α -THP indicating that 3α-HSOR located in the rat SC is an 

active form of the enzyme. Moreover, in the SC of rats submitted to neuropathic pain 

generated by sciatic nerve ligature, 3α-HSOR activity was up-regulated and enhanced 

5α,3α-THP endogenous concentration. Direct intrathecal injection of 5α,3α-THP in the 

lumbar SC of naive rats increased the thermal and mechanical sensitivity thresholds which 

were assessed by the Hargreaves’ method and the Von Frey filament test, respectively. 

These sensitivity thresholds were both decreased by Provera, a pharmacological inhibitor 

of 3α-HSOR activity. We observed that the neuropathic-pain rats were characterized by 

thermal hyperalgesia and mechanical allodynia. Treatment of these neuropathic animals 

with 5α,3α-THP significantly increased both thermal and mechanical thresholds 

suggesting a potential analgesic action of neuroactive 5α,3α-reduced steroids in the control 

of neuropathic pain. 

Taken together, our results provide the first neurochemical and behavioral evidence 

for an effective role of 3α-HSOR in the modulation of nociceptive mechanisms in the 

spinal cord. 

101


STRESS DURING ADOLESCENCE LEADS TO DEPRESSIVE-LIKE 

BEHAVIORS AND CHANGES IN HYPOTHALAMIC-PITUITARY-ADRENAL 

AXIS FUNCTION IN ADULTHOOD 

Romeo R.D., and McEwen B.S. 

Rockefeller University, Laboratory of Neuroendocrinology, 1230 York Ave., New York, 

NY USA, romeor@rockefeller.edu, fax:212-327-8634 

Adolescence is a period of significant developmental vulnerabilities [1,2,7], marked by 

increases in the morbidity of, and susceptibility to, numerous psychological disorders (e.g., 

anxiety and depression) [4]. The mechanisms mediating the adolescent (pubertal) increase 

in these disorders are presently unknown. In adulthood, it is well established that exposure 

to stressors can lead to the onset and exacerbation of psychological disorders [5]. 

Interestingly, recent studies in adolescent boys and girls have suggested that pubertal 

exposure to stress may be a particularly relevant environmental factor contributing to an 

individual’s vulnerability to various psychopathologies later in life [3,8]. In an effort to 

model how adolescent stress exposure affects neurobehavioral function in adult animals, 

the present study investigated whether physical and/or psychological stressors (e.g., 

restraint stress and/or social isolation) experienced during puberty leads to changes in 

depressive-like behaviors and hypothalamic-pituitary-adrenal (HPA) axis function in 

adulthood. 

To these ends, 40 male Sprague-Dawley rats were randomly assigned to one of four 

experimental groups (n=10): (i) GROUP HOUSED (3 per cage), (ii) GROUP HOUSED + 

RESTRAINT (3 per cage + 1 h of restraint stress every other day), (iii) ISOLATION 

(housed alone) and (iv) ISOLATION + RESTRAINT (housed alone + 1 h of restraint 

stress every other day). Animals were exposed to these conditions from 28 to 50 days of 

age, encompassing the pubertal period of development in this species. Animals were 

weighed weekly to examine growth rates. At 52 and 53 days of age all animals were 

administered the Porsolt forced swim test (FST), a pharmacologically validated behavioral 

test to assess learned helplessness and depressive-like behaviors [6]. To investigate 

possible changes in basal and stress-induced HPA reactivity, 48 h after the behavioral tests, 

each group was further divided into two (n =5) and blood was collected from the animals 

either immediately before or after a 30 min session of restraint stress. We found that both 

groups of animals exposed to restraint stress during the pubertal period demonstrated less 

weight gain during adolescence compared to animals either group housed or socially 

isolated without restraint. Our analyses on the FST data revealed that compared to group 

housed controls, animals undergoing the pubertal physical and/or social stressors showed a 

shorter latency to become immobile and spent significantly less time struggling/swimming 

and more time immobile, all indicating greater learned-helplessness behavior. Finally, 

basal plasma corticosterone (CORT) levels were significantly elevated in the animals 

exposed to the physical and/or social stressors during puberty compared to group housed 

controls. These data indicate that pubertal exposure to physical and/or social stressors lead 

to reduced weight gain during adolescence and increased depressive-like behaviors and 

basal CORT levels in adulthood. Importantly, these results lend construct and face validity 

to this model of pubertal stress and depressive-like behaviors in adulthood and recapitulate 

three key features of typical, melancholic depression: weight loss, feelings of learnedhelplessness 

and elevated basal HPA function. These data highlight how stress and 

pubertal development can interact to affect adult behavioral, physiological and endocrine 

102



function and provide a model to study stress-induced neurobehavioral changes during 

adolescent development. 

Acknowledgements: 

This work was supported by NIH grants MH 065749 and MH 41256 


[1] S.L. Andersen, Trajectories of brain development: point of vulnerability or window of 

opportunity., Neuroscience and Biobehavioral Reviews 27 (2003) 3-18. 

[2] R.E. Dahl, Adolescent brain development: a period of vulnerabilities and opportunities., 

Annals of the New York Academy of Sciences 1021 (2004) 1-22. 

[3] K.E. Grant, B.E. Compas, A.F. Stuchlmacher, A.E. Thurn, S.D. McMahon, J.A. Halpert, 

Stressors and child and adolescent psychopathology: moving from markers to mechanisms 

of risk., Psychological Bulletin 129 (2003) 447-466. 

[4] A. Masten, Toward a developmental psychopathology of early adolescence. In M. Levin 

and E. McArnarny (Eds.), Early adolescent transitions, Heath, Lexington, 1987, pp. 261- 

278. 

[5] B.S. McEwen, Mood disorders and allostatic load., Biological Psychiatry 54 (2003) 200- 

207. 

[6] R.D. Porsolt, M. Le Pichon, M. Jalfre, Depression: a new animal model sensitive to 

antidepressant treatments., Nature 266 (1977) 730-732. 

[7] L.P. Spear, The adolescent brain and age-related behavioral manifestations., Neuroscience 

and Biobehavioral Reviews 24 (2000) 417-463. 

[8] R.J. Turner, D.A. Lloyd, Stress burden and the lifetime incidence of psychiatric disorder in 

young adults., Archives of General Psychiatry 61 (2004) 481-488. 

103


BRAIN ESTRADIOL RAPIDLY REGULATES IN A NEUROTRANSMITTER- 

LIKE FASHION MALE SEXUAL BEHAVIOR IN MICE 

Taziaux* M., Keller* 1 M., Bakker* J. and Balthazart* J. 

*University of Liège, Center for Molecular and Cellular Neurobiology, Research Group in 

Behavioral Neuroendocrinology, 1 Avenue de l’Hôpital (Bat. B36), B-4000 Liège, 

Belgium. Fax : 32 (0)4- 366.59.71. E-mail : mtaziaux@student.ulg.ac.be 

1 Now at: Laboratoire de Physiologie de la Reproduction et des Comportements, 

CNRS/INRA/University of Tours, Nouzilly, France. 

Steroid hormones exert their physiological and behavioral effects predominantly by 

regulating the transcription of a variety of hormone-sensitive genes. In addition to these 

relatively “slow” genomic effects (hours to days), steroid hormones also exert rapid 

(within seconds or minutes after administration), non-genomic effects by acting at the 

membrane level in many cellular, in particular neural, systems. For example, estradiol-17β 

(E 2 ) modulates in this manner brain electrical activity, ion channel function or G-protein 

coupled receptor activity (see [7] for review). At present, the impact of these rapid cellular 

effects of E 2 on the functioning of the whole-organism is poorly documented. 

The presence of estrogen synthase (aromatase) in the brain was demonstrated more than 

30 years ago [8] and more recently the enzyme was shown to be present and active at the 

pre-synaptic level [9]. Changes in brain aromatase activity (AA) are mediated largely by 

slow steroid-dependent changes in enzyme transcription [10]. In addition, brain AA can be 

rapidly (within minutes) modulated by non-genomic mechanisms including Ca 2+ - 

dependent phosphorylations [1], suggesting that local brain estrogen concentrations could 

also be modified more rapidly than previously thought. 

Male sexual behavior offers excellent opportunities to investigate the relative 

contribution of genomic and non-genomic effects of estrogen. This behavior is activated in 

many vertebrate species by estrogens derived from testosterone aromatization in the brain 

that act largely by activating the transcriptional activity of nuclear estrogen receptors, but 

is also enhanced within 15-25 min in rats and quail by a single injection of a high dose of 

E 2 [5, 3]. Conversely, studies in quail indicated that the acute inhibition of AA (by 

injection of a specific inhibitor) rapidly decreases aspects of male sexual behavior (15 min 

post-injection; [4]) as well as significantly modifies the reaction latency to nociceptive 

stimuli (1-5 min post injection; [6]). Although these acute effects of aromatase inhibition 

could be partly (sexual behavior) or completely (nociception) reversed by a concomitant 

injection of estradiol, their specificity, and more specifically their link to aromatase 

inhibition could not be fully established. 

In the present experiments, we used male wild-type (WT) and aromatase knock-out 

(ArKO) mice to establish this specificity and further investigate rapid behavioral effects of 

fast up-and down-regulations of brain estrogen concentrations that should occur following 

activation (by dephosphorylation) or inactivation (by phosphorylation) of aromatase 

activity. 

With the use of ArKO mice that are presumably more sensitive to estrogen action due to 

their constitutive lack of exposure to this steroid, we first showed that most aspects of male 

sexual behavior are stimulated within 15 min after a single peripheral injection of E 2 . This 

rapid behavioral effect of E 2 was best observed if ArKO mice were sexually experienced 

and pre-treated for about one week with a small dose of EB, suggesting an interaction 

between slow genomic and fast non genomic actions of estrogen in the activation of this 

behavior. 

104



Conversely, a single injection of three different aromatase inhibitors (Vorozole TM , ATD 

or its metabolite 17OH-ATD) almost completely suppressed male sexual behavior 

(decrease in mount and intromission frequencies and increase in the latency of these 

behaviors) expressed 10 to 20 min later by WT mice. ATD and 17OH-ATD had in contrast 

no significant effect on the mounts and intromissions activated in male ArKO mice by a 

chronic treatment with estradiol benzoate (EB) confirming that the rapid behavioral 

inhibitions are, from the neuroendocrine point of view, specifically due to the blockade of 

aromatase activity. At the behavioral level, this idea is also supported by the fact that the 

behavioral inhibition observed in male WT mice following injection of ATD and 17OH- 

ATD could be partly reversed by the simultaneous injection of a single dose of E 2 . 

Moreover, the rapid inhibitory effects on male WT sexual behavior following 

administration of aromatase inhibitors were not systematically accompanied by a 

significant decrease in ano-genital olfactory investigations, suggesting that treated male 

mice were still socially and possibly sexually interested in the stimulus females. Finally, no 

changes in locomotor activity measured in the open field as well as no changes in odor 

preference for estrous females were detected following treatment with the aromatase 

inhibitors in male WT mice. Overall, these data strongly indicate that this behavioral 

inhibition is highly specific and does not simply result from a non-specific detrimental 

effect of the inhibitor. 

Taken together, the present experiments demonstrate that both up- and downregulations 

of brain estrogen concentrations rapidly affect in parallel the expression of 

male sexual behavior in ArKO and WT mice. These results, associated with previous 

reports demonstrating that brain E 2 is produced at the pre-synaptic level in a manner that 

can be rapidly modulated via calcium-dependent phosphorylations by inputs to aromataseexpressing 

cells [1] supports the notions that E 2 produced by aromatase in the brain 

displays most if not all the functional characteristics of neurotransmitters, or at least 

neuromodulators (see [2] for further elaboration of this notion). 


[1] Balthazart, J., Baillien, M., Charlier, T. D. & Ball, G. F., 2003. Calcium-dependent phosphorylation 

processes control brain aromatase in quail. Eur. J. Neurosci. 17, 1591-1606. 

[2] Balthazart, J. & Ball, G. F., 2006. Is brain estradiol a hormone or a neurotransmitter? Trends Neurosci. 

29, 241-9. 

[3] Cornil, C. A., Dalla, C., Papadopoulou-Daifoti, Z., Baillien, M. & Balthazart, J., 2006. Estradiol rapidly 

activates male sexual behavior and affects brain monoamine levels in the quail brain. Behav. Brain Res. 

166, 110-23. 

[4] Cornil, C. A., Taziaux, M., Baillien, M., Ball, G. F. & Balthazart, J., 2006. Rapid effects of aromatase 

inhibition on male reproductive behaviors in Japanese quail. Horm. Behav. 49, 45-67. 

[5] Cross, E. & Roselli, C. E., 1999. 17beta-estradiol rapidly facilitates chemoinvestigation and mounting in 

castrated male rats. Am. J. Physiol. Regul. Integr. Comp .Physiol. 276, R1346-R1350. 

[6] Evrard, H. C. & Balthazart, J., 2004. Rapid regulation of pain by estrogens synthesized in spinal dorsal 

horn neurons. J. Neurosci. 24, 7225-7229. 

[7] McEwen, B.S., 2001. Invited review: Estrogens effects on the brain: multiple sites and molecular 

mechanisms. J. Appl. Physiol. 91, 2785-2801. 

[8] Naftolin, F., Ryan, K. J., Davies, I. J., Reddy, V. V., Flores, F., Petro, Z., Kuhn, M., White, R. J., 

Takaoka, Y. & Wolin, L., 1975. The formation of estrogens by central neuroendocrine tissues.Rec. Prog. 

Horm .Res. 31, 295-319. 

[9] Peterson, R. S., Yarram, L., Schlinger, B. A. & Saldanha, C. J., 2005. Aromatase is pre-synaptic and 

sexually dimorphic in the adult zebra finch brain. Proc. Biol. Sci. 272, 2089-96. 

[10]Roselli, C.E & Resko, J.A., 1984. Androgens regulate brain aromatase activity in adult male rats through 

a receptor mechanism. Endocrinology 114(6):2183-9. 

105


INHIBITION OF 3α,5α-THP FORMATION DECREASES 

EXPLORATORY/ANTI-ANXIETY AND SOCIO-SEXUAL BEHAVIOR IN 

SEXUALLY-RECEPTIVE FEMALE RATS 

Paris, J.J. 1 , Rhodes, M.E. 1 , Frye, C.A. 1-4 

Dept. of Psychology 1 , Biological Sciences 2 , and The Centers for Neuroscience 3 and Life 

Sciences 4 Resesarch; The University at Albany-SUNY, Life Sciences 01058, 1400 

Washington Avenue, Albany, NY USA 12222; paris.j@gmail.com, 518-591-8823 

The progesterone (P 4 ) metabolite and neurosteroid, 5α-pregnan-3α-ol-20-one 

(3α,5α-ΤΗP) has actions in the midbrain ventral tegmental area (VTA) to modulate the 

intensity and duration of lordosis [3]. Other studies have demonstrated that 3α,5α-THP can 

also produce anti-anxiety effects through actions in the hippocampus [9,10]. 3α,5α-THP 

administration to VTA or hippocampus increases time spent on the open arms of an 

elevated plus maze, latency to bury in response to a shock, and time spent interacting with 

a novel conspecific [2,5,6]. 

3α,5α-THP is increased rapidly in brain in response to stressful stimuli to levels 

that produce agonist-like effects at GABA A receptors and dampen hypothalamic-pituitaryadrenal 

activity [1]. Administration of 3α,5α-THP in conjunction with an acute stressor 

decreases the adrenocorticotropic hormone response and can attenuate overexpression of 

corticotrophin releasing hormone mRNA following adrenalectomy [7,8]. 3α,5α-THP 

increases in midbrain, hippocampus, diencephalon, and cortex in response to reproductive 

behaviors (lordosis, proceptivity, aggression, pacing of sexual contacts) [4]. How blockade 

of 3α,5α-THP formation in midbrain VTA would effect exploratory/anti-anxiety and sociosexual 

behaviors of sexually-receptive female rats was investigated. 

Female, Long-Evans rats (n=33) were implanted with bilateral guide cannulae 

aimed at the VTA and were behaviorally screened for sexual receptivity daily. Rats that 

demonstrated lordosis in response to male mounting were considered to be in behavioral 

estrus. Sexually-receptive rats received infusions (1µg) of either a mitochondrial 

benzodiazepine receptor (MBR) blocker (PK11195), which acts by inhibiting translocation 

of neurosteroid precursor across the MBR membrane, a 3β-hydroxysteroid oxidoreductase 

inhibitor (indomethacin) that blocks P 4 metabolism to 3α,5α-ΤΗP, a combination of both 

inhibitors, or an infusion of β-cyclodextrin vehicle to VTA. 

As shown in Figure 1, rats infused with PK11195, indomethacin, or both to the 

VTA had significantly reduced midbrain 3α,5α-ΤΗP levels compared to vehicle-infused 

rats. Infusions of inhibitor, compared to infusions of vehicle, decreased exploratory/antianxiety 

behavior, indicated by a significant reduction in central entries in an open field and 

time spent on the open arms of an elevated plus maze, reduced social responding in a 

partner preference task and significantly decreased social interaction with a conspecific. 

Likewise, inhibitor infusions attenuated reproductive behaviors in a paced mating 

paradigm compared to rats infused with vehicle. Inhibition of metabolism of P 4 to 3α,5α- 

THP (which occurs both centrally and peripherally with indomethacin administration) was 

observed to attenuate reproductively-relevant behaviors to a greater degree than did 

inhibition of central production alone (PK11195). The greatest decrements in behavior 

were observed when rats received a combination of both inhibitors. These findings suggest 

that formation of 3α,5α-ΤΗP in midbrain is essential for increased exploration/anti-anxiety 

and socio-sexual behaviors associated with sexual receptivity and that expression of these 

behaviors may be dependent on both de novo production of 3α,5α-ΤΗP and metabolism 

from ovarian sources. 

106



Supported by: National Institute of Mental Health (MH06769801). 

Figure 1: Rats receiving VTA infusions of PK11195 (n=9), indomethacin (n=8), or both 

(n=8) demonstrate decreased exploratory/anti-anxiety and socio-sexual behavior compared 

to rats receiving vehicle infusions (n=8), * indicates p

WEDNESDAY, 21 th February 2007 

8.30 - 12.00 

Symposium: 

Effects mediated by membrane receptors

Symposium: 

Effects mediated by membrane receptors 

(Chairs: Melcangi R.C., Schumacher M.) 

• Micevych P., Hariri O., Soma K., Sinchak K. (USA) Neuroprogesterone: essential 

trigger for estrogen positive feedback? 

• Belcher S.M., Le H.H., Spurling L., Zsarnovszky A. (USA) Integrated signaling 

mechanisms of estrogen in developing neurons and neuroectodermal derived tumors 

• Guennoun R., Meffre D, Labombarda F, Gonzalez S.L, Gonzalez Deniselle M.C, 

Stein DG, De Nicola AF, Schumacher M (France) The membrane-associated 

progesterone-binding protein 25-Dx: expression, cellular localisation and upregulation 

after brain and spinal cord injuries 

• Valenzuela C.F., Mameli M., Carta M., Zamudio, P.A. (USA) Modulation of 

glutamatergic synaptic transmission by neurosteroids 

• Biggio G., Mostallino MC, Pisu MG, Talani G, Carta M, Sanna E, Serra M 

(Italy) Neurosteroid responses and GABA A receptor plasticity during chronic stress 

• Cambiasso M.J., Gorosito S.V. (Argentina) Axogenic effect of oestrogen in male 

rat hypothalamic neurons involves Ca 2+ , PKC and ERK signaling 

• Nyberg S., Bäckström T., Zingmark E., Sundström Poromaa I. (Sweden) 

Allopregnanolone decrease with symptom improvement during placebo and GnRH 

agonist treatment in women with premenstrual dysphoric disorder



NEUROPROGESTERONE: ESSENTIAL TRIGGER FOR ESTROGEN POSITIVE 

FEEDBACK? 

Micevych P. 1* , Hariri O. 1 , Soma K. 2 , and Sinchak K. 3 

1* Department of Neurobiology, David Geffen School of Medicine; Laboratory of 

Neuroendocrinology, Brain Research Institute at UCLA, 10833 LeConte Ave, Los 

Angeles, CA 90095-1763; pmicevych@mednet.ucla.edu, Tel: 310.825.2224, FAX 

310.825-2224. 2 Depts. of Psychology & Zoology, University of British Columbia, 

Vancouver, BC CANADA. 3 Dept of Biological Sciences, California State University, , 

Long Beach CA. 

In the gonadally intact rat, the surge of luteinizing hormone (LH) needed for 

ovulation, reproductive behaviors and implantation of fertilized ova, is dependent on 

estradiol and progesterone. Estradiol of ovarian origin induces progesterone receptors in 

the preoptic area and hypothalamus. Sequential activation of estrogen and progesterone 

receptors coordinates reproductive physiology and behavior. In ovariectomized and 

adrenalectomized (ovx/adx) rats, exogenous administration of estradiol alone is sufficient 

to initiate an LH surge suggesting that an endogenous source of progesterone remains in 

these animals. This is idea is supported by the observation that in ovx/adx rats, 

progesterone levels in the hypothalamus increase prior to the LH surge and inhibition of 

progesterone synthesis prevents the LH surge. These results suggest that the increase in 

pre-surge progesterone levels in the hypothalamus is a necessary aspect of the estrogen 

positive feedback mechanism that initiates the LH surge. Estradiol increases the expression 

of the progesterone synthesizing enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD) in 

the hypothalamus. Previous studies had indicated that astrocytes may be the most active 

progesterone synthesizing cells in the CNS. Experiments with neonatal cortical and postpubertal 

hypothalamic astrocytes in vitro demonstrated that estradiol stimulates 

progesterone synthesis. Estradiol stimulates progesterone synthesis through rapidly 

increasing in free cytoplasmic calcium ([Ca 2+ ] i ). Astrocytes were demonstrated to express 

both membrane and nuclear estrogen receptors (ER), but the effects on [Ca 2+ ] i were 

consistent with the activation of a membrane ER in terms of the time course of the 

response (seconds) and its activation with a membrane impermeable estradiol construct (E- 

6-BSA). Blockade of phospholipase C (PLC) or the inositol trisphosphate (IP 3 ) receptor 

prevented the estradiol-induced [Ca 2+ ] i transients, indicating that intracellular stores of 

Ca 2+ were mobilized. In neurons, estradiol stimulation of the PLC - [Ca 2+ ] i pathway is 

dependent on type 1a metabotropic glutamate receptors (mGluR1a). Similarly, in 

astrocytes, blockade of the mGluR1a, prevented estradiol-induced [Ca 2+ ] i transients. 

Thapsigargin, a sesquiterpene lactone, which releases IP 3 receptor-sensitive Ca 2+ stores 

rapidly increased [Ca 2+ ] i levels and mimicked the effect of estradiol treatment – increasing 

[Ca 2+ ] i and progesterone synthesis in astrocytes. One hour treatment with thapsigargin was 

as effective as estradiol at increasing progesterone levels in astrocyte cultures suggesting 

that estradiol induces progesterone synthesis in the hypothalamus by acting on membrane 

ER that act through mGluR1a to activate the PLC-IP 3 pathway stimulating the synthesis of 

progesterone. The steroid signals regulating reproductive behavior and ovulation are the 

same, estradiol and progesterone. To determine whether blocking neuroprogesterone 

synthesis prevented the display of proceptive and receptive sexual behaviors, ovx/adx rats 

were treated with aminoglutethimide (AGT), a blocker of P450side chain cleavage enzyme 

the first enzymatic step of steroidogenesis. Proceptive behaviors induced by estradiol were 

blocked in the AGT treated animals, but sexual receptivity was unaffected. Further, AGT 

111


infused into the III ventricle of gonadally and adrenally intact, cycling rats disrupted the 

cycle apparently by preventing the LH surge and ovulation. However, circulating levels of 

estradiol were unaffected by central administration of AGT, indicating that AGT did not 

affect peripheral steroidogenesis. 

In summary, the estrogen positive feedback mechanism involves estradiol acting on 

a membrane ER that requires mGluR1a signaling to increase [Ca 2+ ] i levels and induce 

progesterone synthesis in astrocytes. This neuroprogesterone acts on estradiol-induced 

progesterone receptors to initiate the cascade of events culminating in the surge release of 

LH and facilitation of proceptive behaviors. However, neither progesterone nor 

progesterone receptors are needed when lordosis is activated by estradiol alone, suggesting 

that estradiol + progesterone activate different circuits regulating sexual receptivity than 

estradiol alone. 

112



INTEGRATED SIGNALING MECHANISMS OF ESTROGEN IN DEVELOPING 

NEURONS AND NEUROECTODERMAL DERIVED TUMORS 

Belcher S.M., Le H.H., Spurling L., and Zsarnovszky A. 

University of Cincinnati College of Medicine, Department of Pharmacology and Cell 

Biophysics. 231 Albert Sabin Way, Cincinnati, OH USA 45267-0575. e-mail: 

scott.belcher@uc.edu; Fax: (513) 558-4329 

The steroid hormone 17β-Estradiol (E2) regulates the normal function and 

development of the mammalian nervous system. Many of estradiol’s effects are mediated 

via the nuclear hormone estrogen receptor (ER) α and ERβ. In addition to regulating 

estrogen-responsive gene expression, E2 also acts in an immediate and cell-specific 

fashion to regulate various intracellular signal transduction pathways. Whereas 

mechanisms underlying E2-responsive gene expression are fairly well understood, the 

signaling mechanisms of rapid estradiol-mediated signal transduction are less clear. In 

vitro analysis employing neonatal rat cerebellar granule cell culture, a model of nonreproductive 

actions of E2 on neuronal development, was used to investigate the signaling 

mechanisms active in developing and mature populations of cerebellar neurons. We 

demonstrated that rapid E2 activation of extracellular signal regulated kinase (ERK) 

signaling regulates oncotic, but not apoptotic programmed cell death in a subpopulation of 

sensitive granule cell precursors. Furthermore, it was demonstrated that exposure to low 

concentrations of E2 and/or the xenoestrogen bisphenol A, transiently modulates granule 

cell ERK signaling in vitro and in vivo. Pharmacological inhibition of Gαi- or protein 

kinase A (PKA)-mediated signaling blocked E2- and ICI182,780-mediated ERK activation 

in vitro, revealed a requirement for Gαi and PKA signaling. Moreover, E2 exposure 

following pretreatment with pertussis toxin or H-89 depressed basal ERK levels, 

suggesting that E2 also rapidly activates a Gαi- and PKA-independent phosphatase 

activity. Additional studies revealed that E2 specifically stimulates protein phosphatase 2A 

(PP2A) activity by an independent rapid intracellular mechanism. This first demonstration 

of a rapid effect on protein phosphatase activity could account for the transient nature of 

E2-induced ERK activation in these neurons. Additional molecular and pharmacological 

inhibitor studies revealed that activation of c-Src, but not the activation of the epidermal 

growth factor receptor (EGFR) is required for E2-mediated ERK signaling. The results 

presented reveal a distinctive cell specific mechanism for rapid E2-induced modulation of 

ERK signaling and a concerted activation of PP2A. 

Because medulloblastomas (MD), the most common malignant brain tumor in 

children, arise from cerebellar granule cell-like precursors we investigated the impact of 

rapid and nuclear hormone receptor mediated actions of E2 on these invasive 

neuroectodermal tumors. As a result of their shared developmental lineage, we 

hypothesized that like normal granule cell precursors, malignant MD cells would express 

ERβ and their growth or migration would be estrogen-responsive. To test this hypothesis 

immunohistochemical studies were done to determine whether ERs were expressed in 

human MD tumors from both male and female patients. We found evidence of ERβ 

expression in all medulloblastoma tumors analyzed. Highly focal expression of ERα was 

detected in 40% of the tumors. Western blot analysis was also used to determine whether 

ERs are expressed in a cerebrocortical primitive neuroectodermal tumor (PNET) cell line 

(PFSK1), and two MD-derived cell lines (Daoy; D283Med) representing the “glial” and 

“neuronal” phenotypic profiles of MD. Results of those studies indicated that specific 

113


isoforms of ERβ- and low levels of ERα-like proteins are expressed in each cell line. The 

physiological significance of the expressed ERs was also examined; physiological 

concentrations of E2 stimulated Rho-dependant migration in each MD/PNET line. The 

growth of D283Med cells, but not the growth of Daoy or PFSK1 cells, was also stimulated 

by E2. In all cases, the ER-antagonist ICI182,780 blocked E2-induced stimulation of 

growth and migration. Xenograft studies in nude mice have confirmed that the growth of 

medulloblastoma cells is markedly stimulated by the estradiol. These in vitro and in vivo 

studies reveal that ERs contribute to the growth and invasive phenotypes of some PNET 

and MD tumors, and demonstrate that MD are estrogen-responsive tumors. 

114



THE MEMBRANE–ASSOCIATED PROGESTERONE BINDING PROTEIN 25- 

DX: EXPRESSION, CELLULAR LOCALISATION AND UP-REGULATION 

AFTER BRAIN AND SPINAL CORD INJURIES 

Guennoun R. 1* , Meffre D. 1 , Labombarda F. 2 , Gonzalez S.L. 2 , Gonzalez Deniselle 

M.C. 2 , Stein DG 3 , De Nicola A.F 2 , and Schumacher M. 1 

(1) UMR788 Inserm and University Paris 11, Kremlin-Bicêtre, France; 

(2) Instituto de Biologia y Medicina Experimental and Department of Human 

Biochemistry, University of Buenos Aires, Argentina 

(3) Department of Emergency Medicine, Emory University, Atlanta, USA 

Phone : 33 1 49 18 95 Fax : 33 1 45 21 19 40 e-mail : Rachida.Guennoun@kb.inserm.fr 

Progesterone has neuroprotective effects in the injured and diseased spinal cord and after 

traumatic brain injury (TBI). In addition to intracellular progesterone receptors, membrane 

binding sites of progesterone may be involved in neuroprotection. A first putative 

membrane receptor of progesterone, distinct from the classical intracellular PR isoforms, 

with a single membrane spanning domain, has been first cloned from porcine liver. 

Homologous proteins were cloned in rat (named 25-Dx), cattle and humans. We shall refer 

to this progesterone binding protein as 25-Dx, to distinguish it from the progesterone 

membrane receptors (mPRs) which have later been cloned. The distribution and regulation 

of 25-Dx in the nervous system may provide some clues concerning its functions. 

In brain, 25-Dx is present in the microsomal and mitochondrial fractions. 25-Dx is 

particularly abundant in the hypothalamic area, circumventricular organs, ependymal cells 

of the ventricular walls, and in the meninges. 25-Dx is co-expressed with vasopressin in 

neurones of the paraventricular, supraoptic and retrochiasmatic nuclei. In spinal cord, 25- 

Dx is localised in cell membranes of dorsal horn and central canal neurones. In response to 

TBI, 25-Dx expression was up-regulated in neurones and induced in astrocytes. The 

expression of 25-Dx in structures involved in cerebro spinal fluid production and in 

osmoregulation, and its up-regulation after brain damage, point to a potentially important 

role of this progesterone-binding protein in the maintenance of water homeostasis after 

TBI. A role of 25-Dx in mediating protective effects of progesterone in the spinal cord is 

supported by the observation that its mRNA and protein are up-regulated by progesterone 

in dorsal horn of the injured spinal cord. On the contrary, the classical PR was downregulated 

under these conditions. Our observations point to the possibility that 

progesterone actions may involve different signalling mechanisms depending on the 

pathophysiological context and that 25-Dx may be involved in the neuroprotective effect of 

progesterone in the injured brain and spinal cord. 

115


MOUDULATION OF GLUTAMATERGIC TRANSMISSION BY 

NEUROSTEROIDS 

Valenzuela C.F., Mameli M., Carta M., Zamudio, P.A. 

Department of Neurosciences, University of New Mexico School of Medicine, 

MSC08 4740, Albuquerque, NM, U.S.A. 

Fax 1(505) 272-8082 e-mail: FValenzuela@salud.unm.edu 

A major question in neuroscience is how neuronal circuits develop. Studies have 

demonstrated that neuronal connections undergo profound remodeling after their initial 

formation and that this is mediated by activity-dependent synaptic plasticity. Despite the 

developmental importance of this process, its cellular mechanisms in immature neurons are 

not fully understood. We found that the excitatory neurosteroid pregnenolone sulfate 

(PREGS) induces long-term potentiation (LTP) of AMPA receptor (AMPAR)-mediated 

postsynaptic currents in hippocampal neurons during a restricted developmental period [3]. 

We recorded AMPAR miniature excitatory postsynaptic currents (mEPSCs) in the wholecell 

patch-clamp configuration from CA1 pyramidal neurons in hippocampal slices from 

postnatal day (P) 3-4 rats. Brief (5 min) exposure to 25 µM PREGS, induced LTP of in the 

frequency but not the amplitude of these events. This effect was robust in slices from P3-4 

rats and gradually decreased at P5 to become undetectable by P6. We evaluated the timecourse 

of the PREGS effect on the paired-pulse ratio (PPR) of AMPAR EPSCs evoked by 

stimulation of Schaffer collaterals. Application of PREGS significantly decreased the 

PPR. However, this effect was transient and EPSC amplitude continued to increase even 

after the PPR had returned to baseline. We next evoked single AMPA EPSCs using a 

stimulation intensity that yielded a mixture of synaptic transmission successes and failures. 

A few minutes after application of PREGS, but not vehicle, we could only record 

successes, consistent with an increase in glutamate release probability. At later time points 

(~20 min after PREGS application), EPSC amplitude gradually increased and remained 

elevated. Currents evoked by exogenous AMPA were dramatically increased ~20 min 

after brief exposure to PREGS. Based on these results, we conclude that PREGS induces a 

transient increase in glutamate release at the presynaptic level (early phase) that triggers 

LTP of postsynaptic AMPAR function (late phase). 

Studies have demonstrated that LTP of AMPA receptor-mediated responses 

involves changes in phosphorylation and/or trafficking of this receptor that are triggered by 

Ca 2+ influx via postsynaptic NMDARs. Therefore, we tested if postsynaptic NMDARmediated 

Ca 2+ influx was required for the late phase of PREGS-induced plasticity. We 

found that this phase could not be observed in neurons intracellularly dialyzed with the 

Ca 2+ chelator BAPTA or the NMDAR blocker MK-801. Bath application of ifenprodil, an 

antagonist of NR2B-containing receptors, abolished the late phase of the PREGS effect. 

Collectively, these findings indicate that postsynaptic NR2B-subunit containing NMDARs 

mediate the late phase of PREGS LTP. We are currently further characterizing these 

postsynaptic effects of PREGS. 

We next investigated the presynaptic mechanism of action of PREGS. First, we 

used BAPTA-AM to chelate Ca 2+ at both the presynaptic and postsynaptic levels. In the 

presence of this agent, both the early and late phases of PREGS LTP were abolished. 

Second, we assessed the effects of bath application of NMDAR antagonists. The nonselective 

antagonist, DL-APV, also blocked the two phases. The antagonist of the glycine 

co-agonist binding site, 7-chlorokynurenate, had a similar effect. Importantly, both phases 

of the PREGS effect were eliminated by low concentrations of PPDA, which selectively 

116



antagonizes receptors containing NR2D subunits [2]. These findings indicate that a 

presynaptic elevation in [Ca 2+ ] i is required for the early phase of the PREGS-induced LTP 

and that this rise is mediated by NR2D-containing presynaptic receptors. The presence of 

this receptor in presynaptic terminals was confirmed by bath application of the 

nonselective NMDA agonist homoquinolinic acid. Importantly, homoquinolinic acid did 

not increase mEPSC frequency in slices from P6-10 animals, indicating that functional 

expression of these presynaptic NMDARs is developmentally restricted. The role of 

presynaptic NR2D-containing NMDARs in the mechanism of action of PREGS is 

currently being further investigated. 

Studies have demonstrated that depolarization of immature neurons can increase the 

probability of glutamate release at presynaptic terminals. We investigated whether 

depolarization could trigger the release of endogenous PREGS. Depolarization (15 sec) 

produced a long-lasting increase in mEPSC frequency in slices from P3-4 but not P6-10 

rats. Importantly, pre-incubation of slices with PPDA or rabbit anti-PREGS polyclonal 

antibodies, but not rabbit IgG, blocked this effect. These findings suggest that a PREGSlike 

neurosteroid is an endogenous factor involved in synapse maturation, acting in a 

retrograde manner like other lipids such as endocannabinoids and arachidonic acid. 

On a related study, we examined the role of PREGS in the developmental actions of 

alcohol [4]. We previously showed that chronic prenatal ethanol exposure increases 

PREGS levels in the developing brain [1]. We discovered that 50 mM ethanol strengthens 

AMPAR-mediated transmission in the CA1 region in a PREGS-like manner. This effect of 

ethanol is age-dependent and blocked by application of an anti-PREGS antibody 

scavenger. These data suggest that the deleterious effects of ethanol on hippocampal 

development are mediated, in part, by alterations in the production and/or release of a 

PREGS-like neurosteroid, which might result in premature stabilization of excitatory 

synaptic connections. 

Supported by grants from the National Institute of Mental Health and the National Institute 

of Alcohol Abuse and Alcoholism. 


[1] J.C. Caldeira, Y. Wu, M. Mameli, R.H. Purdy, P.K. Li, Y. Akwa, D.D. Savage, J.R. 

Engen, C.F. Valenzuela, Fetal alcohol exposure alters neurosteroid levels in the 

developing rat brain, J Neurochem 90 (2004) 1530-1539. 

[2] B. Feng, H.W. Tse, D.A. Skifter, R. Morley, D.E. Jane, D.T. Monaghan, Structureactivity 

analysis of a novel NR2C/NR2D-preferring NMDA receptor antagonist: 1- 

(phenanthrene-2-carbonyl) piperazine-2,3-dicarboxylic acid, Br J Pharmacol 141 

(2004) 508-516. 

[3] M. Mameli, M. Carta, L.D. Partridge, C.F. Valenzuela, Neurosteroid-induced 

plasticity of immature synapses via retrograde modulation of presynaptic NMDA 

receptors, J Neurosci 25 (2005) 2285-2294. 

[4] M. Mameli, C.F. Valenzuela, Alcohol increases efficacy of immature synapses in a 

neurosteroid-dependent manner, Eur J Neurosci 23 (2006) 835-839. 

117


NEUROSTEROID RESPONSES AND GABA A RECEPTOR PLASTICITY 

DURING CHRONIC STRESS 

Biggio G. 1,2 , Mostallino M.C. 2 , Pisu M.G. 2 , Talani G. 1 , Carta M. 1 , Sanna E. 1 and 

Serra M. 1,2 

1 Department of Experimental Biology, Center of Excellence for Neurobiology of Drug 

Dependence, University of Cagliari, Cagliari, Italy. 2 C.N.R. Institute of Neuroscience, 

Italy 

Rats deprived of social contact with other rats at a young age experience a form of 

prolonged stress that leads to long-lasting alterations in their behavioral profile This 

chronic stress paradigm is thus thought to be anxiogenic for these normally gregarious 

animals and their abnormal reactivity to environmental stimuli, when reared under this 

condition, is thought to be a product of prolonged stress. 

We have previously demonstrated that social isolation of rats immediately after 

weaning is associated to a reduction in the cerebrocortical and plasma concentrations of 

progesterone and its metabolites 3α,5α-TH PROG and 3α,5α-THDOC (Serra et al., 2000). 

Moreover, although we found that the basal plasma concentration of adrenocorticotropic 

hormone in isolated rats was slightly decreased compared with that in group-housed 

animals, the functional response of the hypothalamic-pituitary-adrenal axis HPA axis to an 

acute stressful stimulus (foot shock), or to an acute injection of ethanol or isoniazid is 

markedly increased in isolated rats. We found that foot shock, used as a novel acute 

stressor, or acute intraperitoneally injection of isoniazid or ethanol, increased the 

cerebrocortical and plasma concentrations of progesterone, 3α,5α-TH PROG, and 3α,5α- 

TH DOC by a greater percentage in isolated rats than in group-housed animals (Serra et al., 

2000; 2003). Moreover, acute administration of ethanol in socially isolated rats increases 

the concentration of 3α,5α-TH PROG to a substantially greater degree in the cerebral 

cortex than in plasma. This finding is consistent with our observation that ethanol increases 

local neurosteroid synthesis in the brain independently of the HPA axis (Sanna et al., 

2004). Social isolation modified the effects of ethanol on the amounts of steroidogenic 

acute regulatory protein (StAR) mRNA and protein in the brain. The increased sensitivity 

of neuroactive steroid production to ethanol in socially isolated rats may thus be due in part 

to an increase in the expression of StAR in brain mitochondria. Ethanol also increased the 

amplitude of GABA A receptor–mediated miniature inhibitory postsynaptic currents 

(mIPSC) recorded from CA1 pyramidal neurons with a greater potency in hippocampal 

slices prepared from socially isolated rats than in those from group-housed. Indeed, 

whereas ethanol at a concentration of 50 mM significantly increased mIPSC amplitude in 

neurons from isolated animals, it proved ineffective in those from group-housed rats. The 

effect of ethanol on mIPSC amplitude was inhibited by finasteride supporting the idea that 

this action is mediated by an increased production of 3α,5α-TH PROG. This conclusion is 

supported by the lack of a difference in the effect of exogenous 3α,5α-TH PROG at 

concentrations of 1 and 3 µM, on mIPSC amplitude in CA1 pyramidal neurons between 

isolated and group-housed rats. Behavioral studies have also indicated that the ability of 

ethanol to inhibit isoniazid-induced convulsions is greater in isolated rats than in grouphoused 

animals and this effect of isolation is prevented by treatment with the 5α-reductase 

inhibitor finasteride. These observations and the finding that social isolation rats are more 

sensitive to the effects of ethanol on the brain concentrations of 3α,5α-TH PROG and 

3α,5α-TH DOC, both of which posses anxiolytic properties and potentiate the central 

118



actions of ethanol, suggest that chronic stress may induce plastic adaptation of neuronal 

systems that contributes to a vulnerability to alcohol abuse. 

The selective gene expression of GABA A receptor and its subsequent subunit 

composition is affected by chronic stress. In socially isolated rats the level of α 2 α 4 and δ 

subunits immunoreactivity was found to be increased, while the amounts of α 1 and γ 2 were 

decreased, throughout the hippocampus compared with that apparent in group-housed rats. 

Given that δ substitutes for γ 2 and that the latter subunit is essential for synaptic 

localization of GABA A receptors, those containing α 4 and δ would be expected to be 

extrasynaptic GABA A receptors responsible for tonic inhibition. Accordingly, we found 

that the amplitude of GABA A receptor–mediated tonic inhibitory currents in granule cells 

of the dentate gyrus was markedly greater in hippocampal slices from socially isolated rats 

than in those from group-housed animals. The reduction in tonic current noise induced by 

bath application of the GABA A receptor antagonist bicuculline (20 µM) was thus 

significantly greater in hippocampal slices than in those from group-housed animals. In 

addition, the enhancement of tonic current noise induced by 3α,5α-TH PROG (3 µM) was 

markedly greater in granule cells of the dentate gyrus from isolated rats than in those from 

group-housed animals. Since enhanced tonic inhibition is an effective means of reduced 

neuronal excitability, our data therefore suggest that the augmented tonic inhibitory current 

mediated by δ subunit–containing extrasynaptic GABA A receptors in the hippocampus of 

socially isolated rats might reflect a compensatory mechanism to counteract the increased 

seizure susceptibility due to the increased expression of the α4 subunit. 

Neurochemical, molecular and electrophysiological evidence demonstrate that 

social isolation is associated with alteration in the structure and function of GABA A 

receptors and suggest that endogenous levels of progesterone metabolites such as 3α,5α- 

TH PROG may be an important determinant in regulating brain excitability and sensitivity 

to stimuli and point out their possible role in psychiatric and neurological disorder. 


[1] Sanna, E., Talani, G., Busonero, F., Pisu, M.G., Purdy, R.H., Serra, M. and Biggio, 

G. (2004) Brain steroidogenesis mediates ethanol modulation of GABA A receptor 

activity in rat hippocampus. J. Neurosci. 24, 6521–6530. 

[2] Serra, M., Pisu, M.G., Floris, I., Cara, V., Purdy, R.H. and Biggio, G. (2003) Social 

isolation-induced increase in the sensitivity of rats to the steroidogenic effect of 

ethanol. J. Neurochem. 85, 257–263. 

[3] Serra, M., Pisu, M.G., Littera, M., Papi, G., Sanna, E., Tuveri, F., Usala, L., Purdy, 

R.H. and Biggio, G. (2000) Social isolation-induced decreases in both the 

abundance of neuroactive steroids and GABA A receptor function in rat brain. J. 

Neurochem. 75, 732–740. 

119


AXOGENIC EFFECT OF OESTROGEN IN MALE RAT HYPOTHALAMIC 

NEURONS INVOLVES Ca 2+ , PKC AND ERK SIGNALING 

Cambiasso M.J., and Gorosito S.V. 

Instituto de Investigación Médica M y M Ferreyra (INIMEC-CONICET), Friuli 2434 

(5016) Córdoba, Argentina. Fax: +54-351-4695163. E-mail: jcambiasso@immf.uncor.edu 

17-β-estradiol (E2) stimulates the growth of axons in male derived hypothalamic 

neurons in vitro [3,4]. This effect is not exerted through the classical intracellular 

oestrogen receptor (ER) but depends on a membrane mechanism [2] in which the tyrosine 

kinase receptor type B participates [1,5]. More recently, we showed that E2 rapidly 

induced phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK) mitogenactivated 

protein kinases (MAPK) [6]. In the present study we investigate the intracellular 

signaling cascades that mediate the axogenic effect of E2. Treatment with an intracellular 

Ca 2+ chelator, a Ca 2+ -dependent PKC inhibitor, or two specific inhibitors of MEK-ERK 

pathway completely inhibited the E2-induced axogenesis. E2 and the membrane 

impermeant construct E2BSA rapidly induced phosphorylation of ERK, which was 

blocked by the specific inhibitor of ERK pathway UO126 but not by the ER antagonist ICI 

182,780. Decrease of intracellular free Ca 2+ or disruption of PKC activation by Ro 32-0432 

attenuate ERK activation, indicating the confluence of signals in the MAPK pathway. Subcellular 

analysis of ERK demonstrated that the phospho-ERK signal is transduced to the 

nucleus by E2. We also have shown that E2 increased phoshorylation of CREB via ERK 

signaling. In summary, this study demonstrates that E2 induces axogenesis in malehypothalamic 

neurons through activation of the calcium-dependent PKC and ERK pathway 

leading to increased CREB phosphorylation, events required to induce axon elongation. 

Research is supported by CONICET (PIP6238) and FONCyT (PICT26331). 

IBRO Travel Grant is gratefully acknowledged. 


[1] Brito, V.I., Cambiasso, M.J., Carrer, H.F., 2004. Inhibition of TrkB synthesis blocks axogenic effect of 

estradiol on hypothalamic neurons in vitro. Eur. J. Neurosci. 20, 331-337. 

[2] Cambiasso, M.J., Carrer, H.F., 2001. Nongenomic mechanism mediates estradiol stimulation of axon 

growth in male rat hypothalamic neurons in vitro. J. Neurosci. Res. 66, 475-481. 

[3] Cambiasso, M.J., Colombo, J.A., Carrer, H.F., 2000. Differential effect of oestradiol and astrogliaconditioned 

media on the growth of hypothalamic neurons from male and female rat brains. Eur. J. 

Neurosci. 12, 2291-2298. 

[4] Cambiasso, M.J., Diaz, H., Caceres, A., Carrer, H.F., 1995. Neuritogenic effect of estradiol on rat 

ventromedial hypothalamic neurons co-cultured with homotopic or heterotopic glia. J. Neurosci. Res., 

42, 700-709. 

[5] Carrer, H.F., Cambiasso, M.J., Brito, V.I., Gorosito, S.V., 2003. Neurotrophic factors and estradiol 

interact to control axogenic growth in hypothalamic neurons. Ann. NY Acad. Sci. 1007, 306-316. 

[6] Carrer, H.F., Cambiasso, M.J., Gorosito, S., 2005. Effects of estrogen on neuronal growth and 

differentiation. J.Steroid Biochem. Mol. Biol., 93, 319-323. 

120



ALLOPREGNANOLONE DECREASE WITH SYMPTOM IMPROVEMENT 

DURING PLACEBO AND GnRH AGONIST TREATMENT IN WOMEN WITH 

PREMENSTRUAL DYSPHORIC DISORDER 

Nyberg S. 1 , Bäckström T. 1 , Zingmark E. 1 , and Sundström Poromaa I. 2 

1 Department of Clinical Science, Obstetrics and Gynecology, Umeå University Hospital, 

Umeå, Sweden. Fax +46-907852277 e-mail:Sigrid.nyberg@obgyn.umu.se 

2 Department of Women’s and Children’s Health, University Hospital, Uppsala, Sweden 

Background: Neurosteroids such as allopregnanolone and pregnanolone, are 

suggested to be of importance for the pathophysiology of premenstrual dysphoric disorder 

(PMDD). The aim of this study was to investigate if the luteal phase serum concentrations 

of these neurosteroids are associated with improvement of premenstrual symptoms in 12 

women with PMDD after treatment with low dose GnRH-agonist (100 µg buserelin) and 

placebo. 

Methods: Daily ratings for mood and physical symptoms were made prior to treatment and 

throughout the study and serum progesterone, allopregnanolone, and pregnanolone was 

assessed in the luteal phase (cycle day -1 to cycle day -9). Based on their symptom ratings 

prior and throughout the study, subjects were grouped as either buserelin responders (n = 

6) or placebo responders (n = 6). 

Results: Buserelin responders displayed decreased levels of allopregnanolone (p < 0.05) 

and progesterone (p < 0.05) in parallel with improvement of symptoms during buserelin 

treatment. During the placebo treatment, the placebo-responders had lower 

allopregnanolone serum concentrations compared to the serum concentrations in buserelin 

responders (p < 0.05). This was in parallel with improvement in symptoms compared to 

pre-treatment ratings. 

Conclusion: Treatment response, whether induced by buserelin or placebo, appears to be 

paralleled by a decrease in allopregnanolone concentration. 

121


12.00 – 13.00 

Plenary lecture: 

Swaab D.F. (Netherlands)



THE STRESS SYSTEM IN THE HUMAN BRAIN IN DEPRESSION AND 

NEURODEGENERATION 

Swaab D.F.*, Bao A-M* 

*Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The 

Netherlands, fax: 0031-20-6961006; 

e-mail: d.f.swaab@nin.knaw.nl, a.m.bao@nin.knaw.nl. 

The stress system 

The stress response is mediated by the hypothalamo-pituitary-adrenal (HPA) system. 

Activity of the corticotropin-releasing hormone (CRH) neurons in the hypothalamic 

paraventricular nucleus (PVN) forms the basis of the activity of the HPA axis. The CRH 

neurons co-express vasopression (AVP) that potentiates the CRH effects. CRH neurons 

project not only to the median eminence but also into brain area where they e.g. regulate 

the adrenal innervation of the autonomic system. The CRH neurons induce 

adrenocorticotropin (ACTH) release from the pituitary, which subsequently causes cortisol 

release from the adrenal cortex. In addition, the hypothalamo-neurohypophysial system is 

also involved in the stress response. It releases AVP from the PVN and the supraoptic 

nucleus (SON) and oxytocin (OXT) from the PVN via the neurohypophysis into the 

bloodstream. The suprachiasmatic nucleus (SCN), the hypothalamic clock, is responsible 

for the rhythmic changes of the stress system. The stress system is under the influence of 

sex hormones. Sustained hyperactivity of the stress system can cause prolonged 

overexposure of the brain to aberrant cortisol levels that are presumed to induce psychiatric 

disorders and neuropathology conditions such as hippocampal damage in depression and 

Alzheimer’s disease (AD) [1,2]. 

The stress system in depression 

Depression results from an interaction between environmental stress and a 

genetic/developmental predisposition that cause a permanent activation of the CRH 

neurons of the HPA-axis. Stressful life events such as child abuse and early maternal 

separation also form risk factors for later depression and anxiety disorder. 

Hypertrophy of the adrenals, decreased glucocorticoid receptor (GR) function, enhanced 

adrenal response to ACTH, as well as adrenal and pituitary enlargement is observed in 

depressed patients. Both centrally released CRH and increased levels of cortisol contribute 

to the signs and symptoms of depression. Symptoms such as decreased food intake, 

decreased sexual activity, disturbed sleep and motor behavior and increased anxiety can be 

induced in experimental animals by intracerebroventricular injection of CRH. Depression 

is also a frequent side effect of glucocorticoid treatment and of the syndrome of Cushing's 

disease. Inhibitors of cortisol production such as metyrapone can be clinically effective for 

treatment of depression, while the GR antagonist mifepristone (RU486) is effective in 

treating psychotic depression. The AVP neurons in the hypothalamic PVN and SON that 

are projecting to the neurohypophysis are also activated in depression, which contributes to 

the increased release of ACTH from the pituitary. Increased levels of circulating AVP are 

also associated with the risk for suicide. The increased activity of OXT neurons may be 

related to the eating disorders in depression, since OXT acts as a satiety peptide [2]. 

There is a clear sex difference in depression: the prevalence, incidence and morbidity risk 

is higher in females than in males, which is due to both organizing and activating effects of 

125


sex hormones on the HPA-axis. In particular the fluctuations of sex hormone levels are 

considered to be involved in the etiology of depression, e.g. in the premenstrual period, 

ante- and postpartum, during the transition phase to the menopause and by the use of oral 

contraceptives. About 40% of the activated CRH neurons in mood disorders co-express 

nuclear estrogen receptor (ER)-α in the PVN [3], while estrogen-responsive elements have 

been found in the CRH gene promoter region and estrogens stimulate CRH production. An 

androgen-responsive element in CRH gene promoter region initiates a suppressing effect 

on CRH expression [4]. 

The decreased activity of the SCN is the basis for the disturbances of circadian and 

circannual fluctuations in mood, sleep and hormonal rhythms found in depression. 

The stress system in AD 

CRH neurons are moderately activated in AD resulting in higher plasma cortisol levels. 

The SON and PVN remain largely intact , but the SCN is strongly affected from the 

earliest AD stages onwards[5]. Diminishment of sex hormones, i.e. of estrogens in 

menopause and of testosterone in aging men are considered to be risk factors for AD. 

Hippocampal damage in AD and depression 

The hippocampus is strongly affected in AD. Also the sensitivity for estrogens is changed 

in this disorder because of the changes in the ratio of the various splice variants and 

isoforms of the ER-α (see abstract T. Ishunina). Neuronal loss was also reported in the 

hippocampus of stressed or corticosteroid-treated rodents and primates. Because of the 

inhibitory control of the hippocampus on the HPA-axis, damage to this structure was 

expected to disinhibit the HPA-axis, and to cause a positive feedforward cascade of 

increasing glucocorticoid levels over time. This ‘glucocorticoid cascade concept of stress 

and hippocampal damage’ was hypothesized to be causally involved in an age-related 

accumulation of hippocampal damage in disorders like AD and major depression. 

However, in postmortem studies we could not find the presumed neuropathological 

consequences of steroid overexposure in either depressed patients or in patients treated 

with synthetic steroids[6,7] 


[1] Swaab DF, Bao AM, Lucassen PJ: The stress system in the human brain in depression and 

neurodegeneration. Ageing Res Rev 2005;4:141-194. 

[2] Swaab DF: The human hypothalamus. Basic and clinical aspects. Part II:. Handbook of clinical 

neurology. Amsterdam, Elsevier, 2004. 

[3] Bao AM, Hestiantoro A, Van Someren EJ, Swaab DF, Zhou JN: Colocalization of corticotropinreleasing 

hormone and oestrogen receptor-alpha in the paraventricular nucleus of the hypothalamus in 

mood disorders. Brain 2005;128:1301-1313. 

[4] Bao AM, Fischer DF, Wu YH, Hol EM, Balesar R, Unmehopa UA, Zhou JN, Swaab DF: A direct 

androgenic involvement in the expression of human corticotropin-releasing hormone. Mol Psychiatry 

2006; 11:567-576. 

[5] Wu YH, Fischer DF, Kalsbeek A, Garidou-Boof ML, van der Vliet J, van Heijningen C, Liu RY, Zhou 

JN, Swaab DF: Pineal clock gene oscillation is disturbed in alzheimer's disease, due to functional 

disconnection from the "master clock". FASEB J 2006;20:1874-1876. 

[6] Lucassen PJ, Muller MB, Holsboer F, Bauer J, Holtrop A, Wouda J, Hoogendijk WJ, De Kloet ER, 

Swaab DF: Hippocampal apoptosis in major depression is a minor event and absent from subareas at 

risk for glucocorticoid overexposure. Am J Pathol 2001;158:453-468. 

[7] Muller MB, Lucassen PJ, Yassouridis A, Hoogendijk WJ, Holsboer F, Swaab DF: Neither major 

depression nor glucocorticoid treatment affects the cellular integrity of the human hippocampus. Eur J 

Neurosci 2001; 14: 1603-1612. 

126


15.00 – 18.00 

Symposium: 

Corticosteroid effects and stress

Symposium: 

Corticosteroid effects and stress 

(Chairs: Riva M.A., Swaab D.F.) 

• Sousa N (Portugal) Corticosteroid receptors and neuroplasticity 

• Pryce CR (Switzerland) Postnatal ontogeny of hippocampal expression of the 

mineralocorticoid and glucocorticoid receptors in the common marmoset monkey 

• Gass P (Germany) Mice with compromised glucocorticoid receptor expression 

show behavioural and biochemical features of depression 

• Matthews SG (Canada) Maternal adversity, glucocorticoids and programming of 

neuroendocrine function and behaviour. 

• Darnaudéry M., Morley-Fletcher S. and Maccari S. (France) Long lasting effects 

of stress during pregnancy on HPA function and behaviour in mother and offspring 

rats



CORTICOSTEROID RECEPTORS AND NEUROPLASTICITY 

Sousa N. 

Life and Health Science Research Institute, University of Minho, Portugal 

The balance in actions mediated by mineralocorticoid (MR) and glucocorticoid 

(GR) receptors in certain regions of the brain, predominantly in the limbic system, appears 

critical for neuronal activity, stress responsiveness, and behavioral programming and 

adaptation. Altered MR/GR balance renders nervous tissue into a vulnerable status, with 

consequences for the regulation of stress response and enhances susceptibility to 

psychopathology, especially in predisposed individuals. The influence of corticosteroids on 

limbic functions is now indisputable. However, closer scrutiny of early studies together 

with interpretations from newer studies would suggest that the proposition that 

corticosteroid-induced neuronal death accounts fully for the associated corticosteroidinduced 

cognitive deficits is only partially correct. Firstly, it is now clear that specific subpopulations 

of neurons are more sensitive to changes in the corticosteroid environment. 

Secondly, from a critical analysis of the available data, the picture that emerges is that 

corticosteroids, by acting through two distinct receptors, influence not only cell birth and 

death, but probably mainly synaptic plasticity. MR occupation appears to be essential for 

the survival of existing and newly generated neurons. In contrast, while GR can induce loss 

of neurons in the absence of MR activation, it appears that their occupation usually results 

in less drastic effects involving only dendritic atrophy and loss of synaptic contacts. 

In this presentation we will build a revised scheme of corticosteroid actions on neuronal 

plasticity that ultimately determine brain structure and function. 

129


POSTNATAL ONTOGENY OF HIPPOCAMPAL EXPRESSION OF THE 

MINERALOCORTICOID AND GLUCOCORTICOID RECEPTORS IN THE 

COMMON MARMOSET MONKEY 

Pryce C.R. 

Department of Neuroscience, Novartis Institutes for Biomedical Research, Novartis 

Pharma AG, Basel, Switzerland e-mail: christopher.pryce@novartis.com 

The few primate studies to-date of the CNS distribution of corticosteroid receptor 

expression have been conducted in adults and have demonstrated some marked differences 

to the expression distribution in adult rodents, and some important homologies to that in 

adult humans. Developmental studies in primates were lacking until recently [1], but are 

important in order to understand the roles of the mineralocorticoid receptor (MR) and 

glucocorticoid receptor (GR) in CNS maturation, both in a homeostatic environment and as 

mediators of the short- and long-term effects of early-life stress. The common marmoset is 

a small South American monkey that is suitable for developmental study, e.g. gestation 

period of 5 months, infancy period of 3 months, and sexual maturation at 15 months. 

Marmosets live in extended family groups, and reproduction is characterized by twinning 

and biparental care. Based on the findings obtained in long-term descriptive and 

experimental studies conducted in family groups of common marmosets, the following will 

be presented: (1) the development profiles of basal and acute-stress ACTH and cortisol 

titres from birth until adulthood in undisturbed family groups; (2) the development profiles 

of hippocampal and hypothalamic MR and GR expression from birth until adulthood in 

family groups. 

(1) Basal plasma levels of ACTH were similar in neonates (day 1-2) and infants (week 4) 

and elevated at these life stages relative to juveniles (month 4-10) and adults (year 2-6). 

Basal plasma and CSF levels of cortisol were markedly (10-fold) elevated in neonates 

relative to infants and elevated (2-fold) in infants relative to juveniles and adults. The high 

cortisol levels in neonates were associated with large adrenal (fetal zone) glands. Using 

blood sampling as an acute challenge, infants (week 8) demonstrated similar ACTH and 

cortisol peak stress responses to juveniles and adults; ACTH post-stress recovery was 

similar in infants, juveniles and adults, whereas cortisol recovery was retarded, suggesting 

slower cortisol metabolism at younger life stages [2]. 

(2) For MR and GR, the marmoset cDNA sequence exhibited high homology (97%) with 

human MR and GR. Using marmoset-specific riboprobes and in situ hybridization, it was 

demonstrated that MR mRNA expression in the dentate gyrus and Ammon’s horn (CA1-4) 

was greater in marmoset infants (week 4-6) than in neonates (day 1-2), juveniles (month 4- 

5) and adults (year 3-6), with expression in the latter three stages being similar. In the same 

subjects and ontogenetic stages, GR mRNA expression was developmentally consistent in 

DG, CA1-4 and paraventricular nucleus of the hypothalamus (PVNh). Qualitative 

immunohistochemical analysis demonstrated that developmental profiles of MR and GR 

protein expression reflected those of their respective mRNA profiles [1]. 

Combining the findings summarized in (1) and (2), infancy is characterized by a 

combination of high ACTH and cortisol levels and high hippocampal MR expression, 

relative to subsequent life stages. 

130




[1] Pryce CR, Feldon J, Fuchs E, Knuesel I, Oertle T, Sengstag C, Spengler M, Weber E, 

Weston A, Jongen-Relo A. Postnatal ontogeny of hippocampal expression of the 

mineralocorticoid and glucocorticoid receptors in the common marmoset monkey. Eur 

J Neuroscience 2005;21:1521-1535. 

[2] Pryce CR, Palme R, Feldon J. Development of pituitary-adrenal endocrine function in 

the marmoset monkey: Infant hyper-cortisolism is the norm. J Clin Endocrinol Metab 

2002;87:691-699. 

131


MICE WITH COMPROMISED GLUCOCORTICOID RECEPTOR EXPRESSION 

SHOW BEHAVIOURAL AND BIOCHEMICAL FEATURES OF DEPRESSION 

Gass P. 

Central Institute of Mental Health Mannheim (ZI), University of Heidelberg, Germany 

Altered glucocorticoid receptor (GR) signalling is a postulated mechanism for the 

pathogenesis of major depression. To mimic the human situation of altered GR function 

claimed for depression we have generated mouse strains that under- or overexpress GR, 

but maintain the regulatory genetic context controlling the GR gene. We generated: i) GR 

heterozygous mutant mice (GR +/- ) with a 50% GR gene dose reduction; and ii) mice 

overexpressing GR by a yeast artificial chromosome resulting in a 2-fold gene dose 

elevation. These strains were subjected to a large battery of basal and stress-related 

behavioral tests. Furthermore, they were analysed for neuroendocrinological and 

neurochemical alterations. GR +/- mice exhibit normal baseline behaviors, but demonstrate 

increased helplessness after stress exposure, a behavioral correlate of depression in mice. 

Similar to depressed patients, GR +/- mice have a disinhibited HPA system and a 

pathological DEX/CRH test. Thus, they represent a murine depression model with good 

face and construct validity. Overexpression of GR in mice evokes reduced helplessness 

after stress exposure, and an enhanced HPA system feedback regulation. Therefore they 

may represent a model for a stress-resistant strain. These mouse models can now be used to 

study biological changes underlying the pathogenesis of depressive disorders. As a first 

potential molecular correlate for such changes we identified a downregulation of BDNF 

protein content in the hippocampus of GR +/- mice, which is in agreement with the so-called 

neurotrophin hypothesis of depression. Furthermore, these strains may represent a tool to 

detect pharmacological mechanisms or develop new psychopharmacological principles for 

the treatment of depression. 

132



MATERNAL ADVERSITY, GLUCOCORTICOIDS AND PROGRAMMING OF 

NEUROENDOCRINE FUNCTION AND BEHAVIOUR 

Matthews S.M. 

Departments of Physiology, Obstetrics and Gynecology and Medicine, Faculty of 

Medicine University of Toronto, 1 King’s College Circle, Toronto, Ontario, M5S 1A8, 

CANADA. 

The fetus may be exposed to increased endogenous glucocorticoid (GC) or synthetic 

glucocorticoid (sGC) in late gestation. Indeed, approximately 7% of pregnant women in 

Europe and North America are treated with sGC to promote lung maturation in fetuses at 

risk of pre-term delivery. It is now established that exposure of the fetal brain to excess GC 

can have life-long effects on neuroendocrine function and behaviour. Using the guinea pig, 

we have shown that both endogenous GC and sGC exposure has a number of rapid effects in 

the fetal brain in late gestation, including modification of neurotransmitter systems and 

transcriptional machinery. Such fetal exposure permanently alters hypothalamo-pituitaryadrenal 

(HPA) function and behaviour in prepubertal, post-pubertal and aging offspring, in a 

sex-dependent manner. However, these effects are dependant on the time in gestation at 

which GC exposure occurs as well as the age at which endocrine and behavioural outcomes 

are assessed. More recently, we have identified transgenerational effects of prenatal exposure 

to sGC on HPA function, behaviour and growth. Indeed, the magnitude of effects in the 

second generation is greater than that in the first generation. Defining the mechanisms 

involved in programming of HPA function and behaviour across generations will have 

considerable implications for clinical management of pregnancy. 

Supported by: Canadian Institutes of Health Research (CIHR) and Natural Sciences & 

Engineering Research Council (NSERC). 

133


LONG LASTING EFFECTS OF STRESS DURING PREGNANCY ON HPA 

FUNCTION AND BEHAVIOUR IN MOTHER AND OFFSPRING RATS 

Darnaudéry M., Morley-Fletcher S. and Maccari S. 

University of Lille 1, Laboratory of Neurosciences and Adaptive Physiology, Perinatal 

Stress Team, Bât. SN4.1, 59655 Villeneuve d’Ascq, France. 

E-mail:muriel.darnaudery@univ-lille1.fr; Fax: +33 3 20 43 46 02 

Life events occurring during the perinatal period have strong permanent long-term effects 

on the behavioural and neuroendocrine response to stressors. In rats, repeated restraint 

stress of the pregnant dam during the last week of pregnancy produces long lasting changes 

in the hypothalamic-pituitary-adrenocortical (HPA) axis function and behaviours in the 

offspring. These changes include a hyperactivity of HPA axis response associated with a 

reduction in the number of hippocampal corticosteroid receptors [8]. This can be evidenced 

by a more prolonged elevation of plasma ACTH and/or corticosterone after moderate 

stressors such as exposure to novelty, restraint stress or exposure to elevated plus 

maze[25,25,33,48,48,52,52]. The HPA dysfunctions have been reported in infant, young 

adult and aged animals, therefore suggesting a permanent effect of early stress [5, 7, 13, 

15]. Interestingly, after the confrontation to an intense inescapable footshock, prenatally 

stressed rats durably show a blunted corticosterone secretion after stress. Prenatal stress 

also induces a hyporeponse of the HPA axis when animals are exposed to an alcohol 

challenge [14]. These results suggest that HPA alterations associated to prenatal stress may 

vary according to the nature and/or the intensity of the stressor. 

Rats exposed to a prenatal stress also show behavioural disturbances known to be related to 

the HPA axis. Indeed, prenatal stress produces high anxiety levels and depressive-like 

behaviour during adulthood [10,12]. With ageing, these animals exhibit memory 

impairments in hippocampo-dependent tasks [13, 3]. Despite the permanent imprinting 

induced by stress in utero, the dysfunctions observed after prenatal stress can be reversed 

by environmental or pharmacological strategy. For example, early adoption [7] or 

environmental enrichment during adolescence [9], as well as a chronic treatment with 

Insulin-like growth factor 1 in aged animals [3] attenuated some HPA dysfunction’s 

produced by prenatal stress. 

Mechanisms underlying the prenatal stress effects on the offspring remain largely 

unknown. However, previous works demonstrated that maternal glucocorticoids during 

pregnancy may play an important role in the HPA disturbances reported. Thus, stressed 

mothers show high glucocorticoid levels during pregnancy [1, 6]. Furthermore, in the 

offspring of stressed mothers, the HPA response to stress is normalised by maternal 

adrenalectomy during pregnancy [1]. Recently, our group has reported that repeated 

restraint stress during pregnancy leads to a decrease of the placental 11β-HSD2 activity. 

Finally, gestational stress has long lasting effects on HPA axis and behaviour in female 

dams [2, 11]. Thus, during lactating period, stressed mothers show an impairment of 

maternal care and low aggressive behaviour against a male intruder. Moreover, females 

stressed during pregnancy show an increase of anxiety-like behaviour several weeks after 

the end of the stress period. Given that, several evidences suggest that changes in maternal 

care may durably program offspring’s HPA function and behaviours [4], it could be 

postulated that the alterations of the maternal behaviour during the early postnatal period 

may also strongly contribute to the long-term effect described after prenatal stress. 

134




1. A. Barbazanges, P.V.Piazza, M. Le Moal., and S.Maccari, Maternal glucocorticoid secretion mediates 

long-term effects of prenatal stress, J. Neurosci. 16 (1996) 3943-3949. 

2. M. Darnaudery, I.Dutriez, O.Viltart, S.Morley-Fletcher, and S.Maccari, Stress during gestation induces 

lasting effects on emotional reactivity of the dam rat, Behav. Brain Res. 153 (2004) 211-216. 

3. M. Darnaudery, M.Perez-Martin, G.Belizaire, S.Maccari, and L.M.Garcia-Segura, Insulin-like growth 

factor 1 reduces age-related disorders induced by prenatal stress in female rats, Neurobiol. Aging. 27 

(2006) 119-127. 

4. E.W. Fish, D.Shahrokh, R.Bagot, C.Caldji, T.Bredy, M.Szyf, and M.J.Meaney, Epigenetic Programming 

of Stress Responses through Variations in Maternal Care, Ann. N. Y. Acad. Sci. 1036:167-80. (2004) 

167-180. 

5. C. Henry, M.Kabbaj, H.Simon, M.Le Moal, and S.Maccari, Prenatal stress increases the hypothalamopituitary-adrenal 

axis response in young and adult rats, J. Neuroendocrinol. 6 (1994) 341-345. 

6. M. Koehl, M.Darnaudery, J.Dulluc, O.Van Reeth., M. Le Moal., and S.Maccari, Prenatal stress alters 

circadian activity of hypothalamo-pituitary-adrenal axis and hippocampal corticosteroid receptors in 

adult rats of both gender, J. Neurobiol. 40 (1999) 302-315. 

7. S. Maccari, P.V.Piazza, M.Kabbaj, A.Barbazanges, H.Simon, and M. Le Moal., Adoption reverses the 

long-term impairment in glucocorticoid feedback induced by prenatal stress, J. Neurosci. 15 (1995) 110- 

116. 

8. S. Maccari, M.Darnaudery, S.Morley-Fletcher, A.R.Zuena, C.Cinque, and.O.Van Reeth, Prenatal stress 

and long-term consequences: implications of glucocorticoid hormones, Neurosci. Biobehav. Rev. 27 

(2003) 119-127. 

9. S. Morley-Fletcher, M.Rea, S.Maccari, and G.Laviola, Environmental enrichment during adolescence 

reverses the effects of prenatal stress on play behaviour and HPA axis reactivity in rats, Eur. J. Neurosci. 

18 (2003) 3367-3374. 

10. S. Morley-Fletcher, M.Darnaudery, M.Koehl, P.Casolini, O.Van Reeth., and S.Maccari, Prenatal stress 

in rats predicts immobility behavior in the forced swim test. Effects of a chronic treatment with 

tianeptine, Brain Res. 989 (2003) 246-251. 

11. J.W. Smith, J.R.Seckl, A.T.Evans, B.Costall, and J.W.Smythe, Gestational stress induces post-partum 

depression-like behaviour and alters maternal care in rats, Psychoneuroendocrinology. 29 (2004) 227- 

244. 

12. M. Vallee, W.Mayo, F.Dellu, M. Le Moal., H.Simon, and S.Maccari, Prenatal stress induces high 

anxiety and postnatal handling induces low anxiety in adult offspring: correlation with stress-induced 

corticosterone secretion, J. Neurosci. 17 (1997) 2626-2636. 

13. M. Vallee, S.Maccari, F.Dellu, H.Simon, M.Le Moal, and W.Mayo, Long-term effects of prenatal stress 

and postnatal handling on age-related glucocorticoid secretion and cognitive performance: a longitudinal 

study in the rat, Eur. J. Neurosci. 11 (1999) 2906-2916. 

14. V. Van Waes, M.Enache, I.Dutriez, J.Lesage, S.Morley-Fletcher, E.Vinner, M.Lhermitte, D.Vieau, 

S.Maccari, and M.Darnaudery, Hypo-response of the hypothalamic-pituitary-adrenocortical axis after an 

ethanol challenge in prenatally stressed adolescent male rats, Eur. J. Neurosci. 24 (2006) 1193-1200. 

15. O. Viltart, J.Mairesse, M.Darnaudery, H.Louvart, C.Vanbesien-Mailliot, A.Catalani, and S.Maccari, 

Prenatal stress alters Fos protein expression in hippocampus and locus coeruleus stress-related brain 

structures, Psychoneuroendocrinology. 31 (2006) 769-780. 

135

Posters’ Exhibition

Posters’ Exhibition: 

Effects mediated by classical steroid receptors 

• Benedusi V., Pozzi S., Maggi A. and Vegeto E. (Italy) The anti-inflammatory 

activity of estrogenic compounds in microglia 

• Mattsson, A., Mura, E., Halldin, K., Panzica G.C., Brunström, B (Sweden) 

Embryonic exposure to an ERα agonist affects reproductive organ development but 

does not alter copulatory behavior or the parvocellular vasotocin system in male 

Japanese quail 

• Mayoral, S.R. and Penn, A.A. (USA) Brain estrogen receptor expression in 

perinatal mice 

• Pozzi S., Benedusi V., Vegeto E. and Maggi A. (Italy) Anti-inflammatory activity 

of estrogen in acute and chronic brain inflammation 

• Sanz A., Carrero P., Pernía O., Garcia-Segura L.M. (Spain) Both basal and 

estradiol-regulated activation of IGF-I receptor signaling in the rat brain are 

affected by the duration of previous ovarian hormonal deprivation 

• Walf, A.A., Frye, C.A (USA) Estradiol and selective estrogen receptor modulators 

with activity at estrogen receptor beta have dose-dependent effects to reduce 

anxiety and depressive behavior


THE ANTI-INFLAMMATORY ACTIVITY OF ESTROGENIC COMPOUNDS IN 

MICROGLIA 

Benedusi V., Pozzi S., Maggi A. and Vegeto E. 

Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological 

Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. 

Fax: 0039.02.50318284 

e-mail: elisabetta.vegeto@unimi.it 

The generation of proinflammatory and neurotoxic factors by microglia, the resident brain 

immune cells, plays a prominent role in mediating the progressive neurodegenerative 

process. Since there is an increasing evidence of beneficial effects of estrogen against 

neuroinflammation, we hypothesized that microglia could be a target for estrogen antiinflammatory 

activity in brain. 

Using primary cultures of microglia we initially found that estrogen blocks the LPSinduced 

conversion of microglia cells towards the activated phenoptype and inhibits the 

production of inflammatory mediators like MMP-9, iNOS and PGE2 associated with 

microglia reactivity. To elucidate the molecular mechanism of estrogen anti.-inflammatory 

activity we analysed whether estrogen could modify the activity of p65, a transcription 

factor which stimulates the espression of many genes involved in the inflammatory process 

(e.g. IL2, TNF, adhesion molecules and acute phase proteins). Using molecular biology 

techniques we demonstrated that estrogen prevents p65 translocation into the nucleus 

through a non genomic, ERalpha-mediated event which requires PI3K activation. Current 

studies are underway to identify which inflammatory genes are modulated by estrogen and 

whether isoform-specific or selective-estrogen receptor modulators mimic the antiinflammatory 

activity of the endogenous hormone in microglia cells. Recent preliminary 

data in our laboratory show that estrogen is able to reduce LPS-induced TNF-alpha 

production; TNF-alpha expression is regulated by NF-kB so this observation is in 

agreement with our previous findings. Interestingly, selective ERalpha agonists are able to 

reduce LPS-induced TNF-alpha production, whilst selective ERbeta agonists do not show 

any effect. These preliminary data are further confirming the key role of ERalpha, and not 

ERbeta, in the anti-inflammatory effect of estrogen in the brain; further recent results will 

also be discussed. 

138



EMBRYONIC EXPOSURE TO AN ERα AGONIST AFFECTS REPRODUCTIVE 

ORGAN DEVELOPMENT BUT DOES NOT ALTER COPULATORY BEHAVIOR 

OR THE PARVOCELLULAR VASOTOCIN SYSTEM IN MALE JAPANESE 

QUAIL 

Mattsson A. a , Mura E. b , Halldin K. c , Panzica G.C. b , Brunström B. a 

a Dept. of Environmental Toxicology, Uppsala University, Norbyvägen 18A, SE-75236 

Uppsala, Sweden. E-mail: Anna.Mattsson@ebc.uu.se, Fax: +46-18518843 

b Dept. of Anatomy, Pharmacology, and Forensic Medicine, Laboratory of 

Neuroendocrinology, University of Torino, Torino, Italy 

c Inst. of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. 

Estradiol plays an important role in female sex differentiation of the brain and the 

reproductive organs during embryonic development in birds. In Japanese quail, the 

copulatory behavior and the parvocellular vasotocin (VT) system are sexually dimorphic 

traits that are organized during embryonic development. The nucleus of the stria terminalis, 

pars medialis (BSTm), the lateral septum and the medial preoptic nucleus (POM) contain 

denser VT-immunoreactivity (VT-ir) in males than in females [4]. Xenoestrogen exposure 

can demasculinize the brain of male embryos and interfere with the differentiation of the 

reproductive organs in both males and females. This may result in long lasting effects that 

impair fertility and reproductive success. For instance, the copulatory behavior of male 

Japanese quail exposed to estradiol benzoate, diethylstilbestrol, or genistein is reduced or 

totally abolished, which is paralleled by a decreased VT-ir [3]. Ethinylestradiol (EE2) 

exposure has been shown to result in retention of oviducts, increased testicle weight 

asymmetry, and reduced copulatory behavior in males [1,2]. The respective roles of the 

two nuclear estrogen receptors, ERα and ERβ, in normal and disrupted sexual 

differentiation are not well known. The aim of this study was to elucidate whether ERα 

can mediate disrupted differentiation of the brain and the reproductive organs in male 

Japanese quail embryos. We injected fertilised Japanese quail eggs with the selective ERα 

agonist Propyl pyrazole triol (PPT) on incubation day 3, i.e. well before sexual 

differentiation of reproductive organs and copulatory behaviour is completed. A second 

group was injected with EE2, a potent agonist to both ERα and ERβ. The males were 

tested for copulatory behavior at sexual maturity, and the gross morphology of the 

reproductive organs was examined. The brains were processed for VT 

immunohistochemistry. The copulatory behaviour was significantly reduced in the EE2 

group, whereas PPT had no effect. There was no correlation between behavioral 

performance and plasma concentration of testosterone, which confirms that the behavioural 

changes were not due to changes in testosterone concentration, but were rather caused by 

an organizational effect established during embryonic life. The VT-ir in BSTm, the lateral 

septum and POM was not affected by EE2 or PPT treatment. The effects on the 

reproductive organs were similar in the PPT-exposed as in the EE2-exposed birds. Effects 

included presence of oviducts which frequently were malformed, testicle weight 

asymmetry and reduced cloacal gland area. Our results show that treatment with the 

selective ERα agonist PPT causes similar adverse effects on reproductive organ 

development as treatment with EE2, and hence these effects can be mediated via ERα. 

However, PPT exposure was not sufficient to demasculinize the copulatory behavior or the 

VT system in male Japanese quail. This indicates that the demasculinizing effect of 

estrogens on the male brain is not mediated by ERα alone. Ongoing studies in our 

139


laboratory aim to resolve whether selective activation of ERβ may result in disrupted sex 

differentiation in the quail embryo. 


[1] Berg, C., Halldin, K., Fridolfsson, A.K., Brandt, I. and Brunstrom, B., The avian egg as a test 

system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ 

development, Sci Total Environ, 233 (1999) 57-66. 

[2] Halldin, K., Berg, C., Brandt, I. and Brunstrom, B., Sexual behavior in Japanese quail as a test end 

point for endocrine disruption: Effects of in Ovo exposure to ethinylestradiol and diethylstilbestrol, 

Environmental Health Perspectives, 107 (1999) 861-866. 

[3] Panzica, G., Mura, E., Pessatti, M. and Viglietti-Panzica, C., Early embryonic administration of 

xenoestrogens alters vasotocin system and male sexual behavior of the Japanese quail, Domestic 

Animal Endocrinology, 29 (2005) 436-445. 

[4] Panzica, G.C., Balthazart, J., Pessatti, M. and Viglietti-Panzica, C., The parvocellular vasotocin 

system of Japanese quail: a developmental and adult model for the study of influences of gonadal 

hormones on sexually differentiated and behaviorally relevant neural circuits, Environ Health 

Perspect, 110 Suppl 3 (2002) 423-8. 

140



BRAIN ESTROGEN RECEPTOR EXPRESSION IN PERINATAL MICE 

Mayoral S.R. and Penn A.A. 

Department of Pediatrics, Stanford University, 300 Pasteur Drive, Stanford, CA 94305- 

5208, USA. Fax: 650-725-7724 e-mail: apenn@stanford.edu. 

Estrogen is important for neuronal growth and survival [1, 2]. Studies of preterm infants, 

who experience an early loss of placental estrogen, show that preterm males suffer worse 

brain abnormalities and cognitive outcomes than preterm females [3]. These observations 

suggest that estrogen is crucial for the proper development of the brain. However, the role 

and the expression of the two principal estrogen receptors, ERα and ERβ, in CNS 

development are still not well established. We examined perinatal ERα and ERβ mRNA 

expression in CD1 mouse cortex, hippocampus and cerebellum by quantitative real-time 

RT-PCR. We found significant gender-dependent differences in ERα expression in the 

developing cortex. In embryonic (E18) CD1 mice, ERα levels in males and females in the 

cortex were similar (P=.24; t-test). However, after birth (P0 and P7) these values diverge 

significantly (P


ANTI-INFLAMMATORY ACTIVITY OF ESTROGEN IN ACUTE AND 

CHRONIC BRAIN INFLAMMATION 

Pozzi S., Benedusi V., Vegeto E. and Maggi A. 

Center of Excellence on Neurodegenerative Diseases, Department of Pharmacological 

Sciences, University of Milan, Via Balzaretti 9, 20133 Milan, Italy. Fax: 

0039.02.50318284 

e-mail: adriana.maggi@unimi.it 

Activation of microglia cells, the resident macrophage cells of the brain, is the hallmark of 

acute and chronic neurodegenerative disorders, such as ischemia, Multiple Sclerosis (MS) 

and Alzheimer Disease (AD), characterized by inflammatory events. 

Recent studies reported that 17β-estradiol (E 2 ) acts as a neuroprotective agent in brain by 

both targeting neurons and inhibiting the brain inflammatory reactions. In our lab we 

recently developed an experimental model of brain inflammation, in which LPS 

(lipopolysaccharide), a potent inflammatory agent, is injected in the cerebral ventricles, 

resulting in a transient and localised acute neuroinflammatory reaction. We used this 

system to investigate the E 2 anti-inflammatory activity in the central nervous system and 

demonstrated that E 2 is a potent inhibitor of microglia reactivity in several regions of the 

brain, including cortex, hippocampus and noncortical areas and that hormone 

administration results in a significant reduction of the expression of inflammatory markers, 

such as TNF-α, MCP-1 and MIP-2 [1]. Our data thus show that estrogen is able to quench 

acute brain inflammation in vivo, in agreement with data published by other groups on 

neuroinflammatory processes such as ischemia or experimental allergic encephalomyelitis 

(EAE). On the other hand, we used the APP23 transgenic mice, expressing the human 

amyloid precursor protein (APP) with a mutation reported in familial AD, in order to 

understand the role of E 2 in chronic neurodegenerative diseases. In this animal model of 

AD microglia displays the characteristic activated morphology and immunoreactive 

phenotype induced by the chronic deposition of the amyloid peptide (Aβ). We observed 

that ovariectomy increases microglia activation at Aβ deposits whereas chronic 

replacement with E 2 in ovarectomized APP23 mice reduced neuroinflammation. Thus, 

chronic neuroinflammatory events, associated with neurodegeneration, are also targeted by 

hormone action. Future studies using synthetic estrogenic compounds will be discussed. 


[1] E. Vegeto, S. Belcredito , S. Ghisletti, C. Meda, S. Etteri and A. Maggi, The endogenous estrogen status 

regulates microglia reactivity in animal models of neuroinflammation, Endocrinology 147 (5) (2006), pp. 

2263-2272 

142



BOTH BASAL AND ESTRADIOL-REGULATED ACTIVATION OF IGF-I 

RECEPTOR SIGNALING IN THE RAT BRAIN ARE AFFECTED BY THE 

DURATION OF PREVIOUS OVARIAN HORMONAL DEPRIVATION 

Sanz A.*, Carrero P., Pernía O., Garcia-Segura L.M. 

Instituto Cajal, C.S.I.C., Avenida Doctor Arce, 37, E-28002, Madrid, Spain. *E-mail: 

sanz_amaya@hotmail.com FAX: +34-915854754 

The ovarian hormone 17 beta-estradiol (E2) is neuroprotective in many experimental 

models of neurodegeneration. This pro-survival effect of E2 is partially due to its ability to 

regulate the activity of some proteins of the signaling pathways of growth factors, such as 

the phosphoinositide 3-kinase (PI3K) pathway and the mitogen-activated protein kinase 

(MAPK) pathway. However, the neuroprotective effects of E2 have not been always 

confirmed in clinical studies. There is evidence that early initiation of hormone therapy, 

during the perimenopausal period, may provide cognitive benefits to postmenopausal 

women. In contrast, late initiation of hormone therapy, several years after menopause, may 

increase cognitive deficits. In addition to possible differences due to age, the discrepant 

results of hormone therapy may be explained if brain responsiveness to E2 is sensitive to 

the duration of previous ovarian hormonal deprivation. To test this hypothesis, we have 

assessed whether different lengths of ovarian hormonal deprivation before E2 

administration may affect the basal and E2-regulated phosphorylation of kinases associated 

to the IGF-I receptor (IGF-IR) in the brain. Wistar female rats were ovariectomized 10 

days (short-term hormonal deprivation) or 30 days (long-term hormonal deprivation) 

before the i.p. injection of 300 micrograms E2 and killed at the age of 3 months, 24 h after 

the administration of E2. The cerebral cortex, the hippocampus, the hypothalamus and the 

cerebellum were dissected out and the levels of phosphorylation of Akt, glycogen synthase 

kinase 3 beta (GSK3 beta) and extracellular signal-regulated kinases (ERK1/2), the levels 

of expression of the IGF-IR and the levels of expression of estrogen receptors (ER alfa and 

ER beta) were assessed by Western blotting. Basal levels of phosphorylation of Akt, GSK3 

beta and ERK1/2 showed significant differences depending on the time elapsed after 

ovarian removal and the brain region. Long-term hormonal deprivation resulted in 

increased basal phosphorylation levels of Akt and ERK-1 in the hippocampus, GSK3 beta, 

ERK1 and ERK2 in the hypothalamus and Akt, ERK-1 and ERK-2 in the cerebral cortex. 

In contrast, long-term hormonal deprivation reduced basal phosphorylation levels of GSK3 

beta, ERK-1 and ERK-2 in the cerebellum. In addition, E2 decreased the phosphorylation 

levels of ERK-1 and ERK-2 in the cerebral cortex at 10 days, but not at 30 days, after 

ovariectomy and decreased ERK-2 phosphorylation in the cerebellum at 30 days, but not at 

10 days, after ovariectomy. The levels of expression of ERs and IGF-IR were also affected 

by the duration of ovarian hormonal deprivation and E2 administration. IGF-IR expression 

was decreased in the cerebral cortex after long-term hormonal deprivation and by E2 

treatment after short-term hormonal deprivation. The expression of ER alpha was 

decreased after long-term hormonal deprivation in the hippocampus. In conclusion, our 

findings indicate that, within the same age group, the duration of previous ovarian 

hormone deprivation differentially affects basal levels of IGF-IR signaling and its 

responsiveness to estrogen and is therefore an important parameter to consider when 

assessing neuroprotective and cognitive effects of hormone therapy. 



143


ESTRADIOL AND SELECTIVE ESTROGEN RECEPTOR MODULATORS WITH 

ACTIVITY AT ESTROGEN RECEPTOR BETA HAVE DOSE-DEPENDENT EFFECTS 

TO REDUCE ANXIETY AND DEPRESSIVE BEHAVIOR 

Walf, A.A. 1 , Frye, C.A. 1-4 , 

Dept. of Psychology 1 , Biological Sciences 2 , and The Centers for Neuroscience 3 and Life Science 4 Research 

The University at Albany-SUNY, Life Sciences 01048, 1400 Washington Avenue, Albany, NY USA 12222; 

aawalf@yahoo.com, 518-591-8838 

Estradiol (E 2 ) alters anxiety and depressive behavior of female rodents. Female rats during 

proestrus, which have high physiological E 2 levels, have decreased anxiety and depressive behavior 

compared to females with lower E 2 levels and males [3,4,11]. Similarly, subcutaneous (SC) administration 

of E 2 that produces physiological E 2 concentrations anxiety and depressive behavior of ovariectomized (ovx) 

rats and mice [3-6]. However, E 2 dosing that produces more sustained and/or higher E 2 concentrations does 

not consistently decrease anxiety and depressive behavior [11-14]. Indeed, some of the variations that have 

been reported for the effects of E 2 on anxiety and depressive behavior among females may be related to E 2 

dosing regimen utilized [11-12]. 

Another factor that may modulate the functional response to E 2 may involve E 2 ’s estrogen receptor 

(ER) isoform-specific effects. Administration of ER antagonists systemically or directly to an area of the 

brain that is important for E 2 ’s effects on anxiety and depressive behavior, the hippocampus, attenuates E 2 ’s 

anti-anxiety and anti-depressive effects in ovx rats [11,13]. Although these data suggest that ERs are 

important for the functional effects of E 2 , the ER antagonists utilized in these studies were not ER isoform 

(ERα or ERβ)-specific. Indeed, recent studies using knockout mice, antisense oligonucleotides, and 

administration of selective estrogen receptor modulators (SERMs) suggest that E 2 ’s effects for anxiety and 

depression may be dependent upon ERβ, whereas E 2 ’s effects for reproductive behavior are dependent upon 

ERα [1,2,5,11-14]. Whether there are dose-dependent effects of ERα or ERβ agonists for these effects is of 

interest. 

These data suggesting that there are ERβ-specific effects of E 2 for anxiety and depressive behavior 

is intriguing given that estrogens can have robust proliferative effects on peripheral, E 2 -sensitive tissues, such 

as mammary glands and uterus, that involve actions of ERα. As such, it is important to further characterize 

ER-isoform-specific and dose-dependent effects of SERMs to be able to parse beneficial effects in brain from 

possible negative effects in these peripheral tissue. To begin to address this, the effects of SC administration 

of different dosages (0, 0.3, 0.6, 0.9, or 1.8 mg/kg) of 17β-E 2 , an ERα agonist (propyl pyrazole triol; PPT, or 

an ERβ agonist (diarylpropionitrile; DPN) for behavior in measures of anxiety (elevated plus maze), 

depression (forced swim test), and reproduction/sexual receptivity (lordosis) were compared in ovx rats. We 

hypothesized that there would be dose-dependent effects of E 2 and DPN for anxiety and depression behavior 

and a dose-dependent effects of E 2 and PPT for lordosis. 

Results of the present study supported our hypothesis of the dose-dependent, ER-isoform specific 

effects of SERMs for anxiety, depression, and reproductive behavior. Administration of 0.9 mg/kg, but not 

lower or higher dosages of E 2 , decreased anxiety (i.e. increased open arm time in the elevated plus maze), 

decreased depressive behavior (i.e. decreased immobility in the forced swim test), compared to vehicle, 

which replicated our previous findings [4]. All E 2 dosages increased lordosis. 0.3 mg/kg DPN, compared to 

vehicle, increased open arm time, decreased immobility, and did not alter lordosis. All doses of PPT 

enhanced lordosis compared to vehicle, but did not alter affective behavior. 

Together, these data suggest that there are dose-dependent effects of ERβ agonists for affect and 

ERα agonists for sexual receptivity. Studies are ongoing investigating the proliferative effects of these 

compounds concomitant with these behavioral effects. 


Supported by: NSF (IBN03-16083) U.S. Dept. of Defense (BC051001). 

[1] D.B. Imwalle, J.A. Gustafsson, E.F. Rissman, Lack of functional estrogen receptor β influences anxiety behavior 

and serotonin content in female mice, Physiol Behav. 84 (2005) 157-63. 

[2] W. Krezel, S. Dupont, A. Krust, P. Chambon, P.F. Chapman, Increased anxiety and synaptic plasticity in estrogen 

receptor β -deficient mice. PNAS. 98 (2001) pp. 12278-82. 

[3] C.A. Frye, S.M. Petralia, M.E. Rhodes, Estrous cycle and sex differences in performance on anxiety tasks coincide 

with increases in hippocampal progesterone and 3α,5α-THP, Pharmacol. Biochem. Behav. 67 (2000) pp. 587-596. 

[4] C.A. Frye, A.A. Walf, Changes in progesterone metabolites in the hippocampus can modulate open field and forced 

swim test behavior of proestrous rats. Horm. Behav. 41 (2002) pp. 306-15 

144

% of control- Open Arm Time 

% of control- Lordosis Quotient 

% of control- Immobility Time 

% of control- Open Arm Time 

% of control- Lordosis Quotient 

% of control- Immobility Time 



[5] T.D. Lund, T. Rovis, W.C. Chung, R.J. Handa, Novel actions of estrogen receptor-beta on anxiety-related behaviors. 

Endocrinology. 146 (2005) pp. 797-807. 

[6] S. Ogawa, J. Chan, A.E. Chester, J.A. Gustafsson, K.S. Korach, D.W. Pfaff, Survival of reproductive behaviors in 

estrogen receptor β gene-deficient male and female mice, PNAS 96 (1999) pp. 12887-92. 

[7] S. Ogawa, J. Chan, J.A. Gustafsson, K.S. Korach, D.W. Pfaff, Estrogen increases locomotor activity in mice through 

estrogen receptor α: specificity for the type of activity. Endocrinology 144 (2003) pp. 230-9. 

[8] B.A. Rocha, R. Fleischer, J.M. Schaeffer, S.P. Rohrer, G.J. Hickey, 17β-Estradiol-induced antidepressant-like effect 

in the Forced Swim Test is absent in estrogen receptor-β knockout (BERKO) mice. Psychopharmacology. 179 (2005) pp. 

637-43. 

[9] A.A. Walf , I. Ciriza, L.M. Garcia-Segura, C.A. Frye, , Antisense oligodeoxynucleotides for estrogen receptor beta and 

alpha attenuate estrogen’s modulation of affective and sexual behavior, respectively, Neuropsychopharmacology (in 

revision). 

[10] A.A. Walf, C.A., Frye, Administration of estrogen receptor beta-specific selective estrogen receptor modulators to 

the hippocampus decrease anxiety and depressive behavior of ovariectomized rats, Pharmacol Biochem Behav. (in 

press). 

[11] A.A. Walf, C.A., Frye, A review and update of mechanisms of estrogen in the hippocampus and amygdala for 

anxiety and depression behavior. Neuropsychopharmacology 31 (2006) pp. 1097-111. 

[12] A.A. Walf, C.A., Frye, Estradiol’s effects to reduce anxiety and depressive behavior may be mediated by estradiol 

dose and restraint stress. Neuropsychopharmacology 30 (2005) pp. 1288-301. 

[13] A.A. Walf, C.A., Frye, ERβ-selective estrogen receptor modulators produce antianxiety behavior when 

administered systemically to ovariectomized rats. Neuropsychopharmacology.30 (2005) pp. 1598-609. 

[14]A.A. Walf, M.E. Rhodes, C.A. Frye, Anti-depressant effects of ERβ selective estrogen receptor modulators in the 

forced swim test. Pharmacol Biochem Behav. 78 (2004) pp. 523-9. 

300 

300 

200 

* 

200 

100 

vehicle 

control 

100 

vehicle 

control 

0 

300 

0.3 0.6 0.9 1.8 

0 

300 

0.3 0.6 0.9 1.8 

200 

* 

200 

100 

vehicle 

control 

100 

vehicle 

control 

0 

300 

0.3 0.6 0.9 1.8 

0 

300 

0.3 0.6 0.9 1.8 

200 

200 

* * * * 

100 

vehicle 

control 

100 

vehicle 

control 

Figure 1: * vs. vehicle 

(p


Neuroactive steroids and neurogenesis 

• Alias A.G. (USA) Does 5α-reductase stimulation improve cognitive functions, 

while inhibition improve immunity? 

• Eser D., Schüle C., Romeo E., Uzunov DP., di Michele F., Baghai T.C., Pasini A., 

Schwarz M. and R Rupprecht (Germany) Influence of mirtazapine on plasma 

concentrations of neuroactive steroids in major depression and on 3αhydroxysteroid 

dehydrogenase activity 

• Hill M., Cibula D., Včelaková H, Kancheva L. and Pařízek A. (Czech Republic) 

Pregnanolone isomers and their polar conjugates in late pregnancy: A longitudinal 

study 

• Kancheva L , Včelaková H, Hill M., Vrbíková J. and Stárka L (Czech Republic) 

Neuroactive steroids in adult men 

• Bo E., Casella D., Martini M., Viglietti-Panzica C., Deviche P., Panzica G.C. 

(Italy) Photoperiod influences aromatase expression in junco hyemalis 

prosencephalon 

• Pawluski J.L., Walker C.A., Galea L.A.M. (Canada) Adult hippocampal 

neurogenesis is altered with maternal experience 

• Rahman M, Lindblad C., Johansson I-M, Bäckström T and Wang M-D (Sweden) 

Neurosteroid modulation of recombinant rat α 5 β 2 γ 2l and α 1 β 2 γ 2L GABA A receptors 

in xenopus oocyte



DOES 5α-REDUCTASE STIMULATION IMPROVE COGNITIVE FUNCTIONS, 

WHILE INHIBITION IMPROVE IMMUNITY? 

A.G. Alias 

Fulton State Hospital, Fulton, MO 65251, USA. Fax: +01- 573-441-9666 

e-mail: aliasag@yahoo.com 

The two hypotheses presented here may seem too good to be true. And it may well be so. If 

not, these hypotheses have substantial utility. In any case, these simplified versions need to 

be reformulated as more research results are accumulated, or existing ones unbeknownst to 

the author are incorporated. These hypotheses have been generated by the author’s 

empirical research of over 30 years. Those findings are summarized in two papers [1,2]. 

Steroid 5α-reductase (5αR) stimulates the conversion of steroids such as 

testosterone (T) and progesterone (P) to dihydrotestosterone (DHT) and 

dihydroprogesterone (DHP), respectively [11,13,23]. DHT and DHP are further reduced by 

3α-hydroxysteroid dehydrogenase (3αHSD) to 3α-androstanediol (A-diol), and 3α,5αtetrahydroprogesterone 

(THP/ “Allo”) [11,13]. Such metabolic activities take place in 

central and peripheral nervous system sites as well [11,13]. 

Frye et al [12] demonstrated that “DHT has cognitive enhancing effects, 

independent of estradiol, which are attenuated by a [3αHSD] inhibitor, indomethacin [by 

inhibiting 

A-diol formation from DHT].” In yet another animal model [5], T, but not nonaromatizable 

DHT, improved working memory, however. And Brinton et al [6] 

demonstrated that THP promoted “neurogenesis in vitro and in vivo in transgenic mouse 

model of Alzheimer's disease [AD].” And THP is decreased in the prefrontal cortex of AD 

patients; the “levels are inversely correlated with neuropathological disease stage” [21]. P 

has neuroprotective properties in experimental models of neurodegeneration [12,17,18]. 

“[P] increased the levels of DHP and THP in plasma and hippocampus and prevented 

kainic-acid-induced neuronal loss” [8]. By contrast, the synthetic progestin 

medroxyprogesterone acetate (Provera) failed to mimic P in this experiment [8]. 5αR 

inhibitor finasteride blocked the increase in DHP and THP in plasma and hippocampus, 

following P administration, and also abolished the neuroprotective effect of P [8]. Further, 

indomethacin blocked the neuroprotective effect of both DHP and THP [8]. Nevertheless, 

indomethacin, along with other non-steroidal antiinflammatory agents (NSAIAs), has been 

used in the treatment of AD [17], though many “long-term, placebo-controlled clinical 

trials … produced negative results” [17]. A mechanism for the beneficial effects of 

NSAIDs against AD is thought to be “an allosteric modulation of gamma-secretase 

activity, the enzyme responsible for the formation of amyloid-beta” which is considered to 

be “independent from the anti-cyclooxygenase activity [of NSAIDs] and is related to the 

chemical structure of the compounds, with some NSAIDs being active (ibuprofen, 

sulindac, flurbiprofen, indomethacin, diclofenac) and others not (naproxen, aspirin, 

celecoxib)” [17]. 

It has been known that substantial differences exist in innate immune functions 

between the sexes [3,7]. These differences have been (largely) attributed to sex hormonal 

influences [3,7]. Though estrogens are believed to potentiate immune functions and 

androgens to suppress them, the influences of sex hormones on immune functions are more 

complex, as are on cognitive functions. A well-known example to psychiatrists is the 

beneficial effects of female gender, as well as of estradiol [19], in the course and severity 

of schizophrenia, whereas, the prevalence of excess anxiety is nearly twice as common in 

147


women of menstruating age. And a single sublingual dose of T inhibits fear induced startle 

response in women [16]. 

Anabolic hormones have been used as immune enhancing agents. “Ghione [14] was 

the first to demonstrate an ‘anti-infective’ action of 4-chlorotestosterone in experimental 

infection by Nocardia asteroids in rabbits and mice, and in experimental staphylococcal 

infection of mice” [22]. Indeed, more recently, addition of an anabolic agent, in addition to 

good nutrition, has been advocated in wound healing [9]. This should follow that T, with 

its anabolic properties, should have immune enhancing properties. However, numerous 

studies have shown otherwise [3,7]. And yet, the same group [7] later identified, and 

confimed, DHT as the crucial immune depressing hormone after trauma-hemorrhage [7- 

see Fig. 3]. More specifically, DHT suppresses interleukin-4 and gamma-interferon [4]. 

And, the powerful immunosuppressant, cyclosporin A, stimulates 5αR [10]. Furthermore, 

Gilliver et al [15] demonstrated that “systemic [5αR inhibition mimicked] the effects of 

castration in a rat model of cutaneous wound healing” [15]. 

Thus it is reasonable to hypothesize that 5αR stimulation could enhance certain 

cognitive functions, and that.5αR inhibition could enhance certain immune functions. 


1. Alias AG. Schizotypy and leadership: a contrasting model for deficit symptoms, and a possible therapeutic 

role for sex hormones. Med Hypotheses 2000;54:537-552. 

2. Alias AG. A role for 5alpha-reductase activity in the development of male homosexuality? Ann N Y Acad 

Sci 2004;1032:237-44. 

3. Angele MK, Ayala A, Cioffi WG, Bland KI, Chaudry IH. Testosterone: the culprit for producing 

splenocyte immune depression after trauma-hemorrhage. Am J Physiol 1998;274:C1530-1536. 

4. Araneo BA, Dowell T, Diegel M, Daynes RA. [DHT] exerts a depressive influence on the production of 

interleukin-4 (IL-4), IL-5, and gamma-interferon, but not IL-2 by activated murine T cells. Blood 

1991;78:688-699. 

5. Bimonte-Nelson HA, Singleton RS, Nelson ME, et al. [T], but not nonaromatizable [DHT], improves 

working memory ... nerve growth factor levels in aged male rats.. Exp Neurol 2003;181:301-12. 

6. Brinton RD, Wang JM. ... therapeutic potential of allopregnanolone to promote neurogenesis in vitro and 

in vivo in transgenic mouse model of Alzheimer's disease. Curr Alzheimer Res 2006;3:11-17. 

7. Choudhry MA, Bland KI, Chaudry IH. Gender and susceptibility to sepsis following trauma. Endocr 

Metab Immune Disord Drug Targets 2006;6:127-35. 

8. Ciriza I, Carrero P, Frye CA, Garcia-Segura LM. J Neurobiol 2006;66:916-28. 

9. Collins, 2004. N. The right mix: using nutritional interventions and an anabolic agent to manage a stage IV 

ulcer. Adv Skin Wound Care 2004;17:36,38-39. 

10. Cutolo M, Giusti M, Villaggio B, et al. Testosterone metabolism and cyclosporin A treatment in 

rheumatoid arthritis. Br J Rheumatol 1997;36:433-439. 

11. Frye C.A. Some rewarding effects of androgens may be mediated by actions of its 5α-reduced metabolite 

3α-androstanediol. Pharm Biochem Behav (2006/07 – in press). 

12. Frye CA, Edi nger KL, Seliga AM, Wawrzycki JM. Psychoneuroendocrinology 2004;29:1019-1027. 

13. Garcia-Segura LM, Melcangi RC. Steroids and glial cell function. Glia 2006;54:485-498. 

14. Ghione M. Antinfective action of an anabolic steroid. Proc Soc Exp Biol Med 1958;97:773-775. 

15. Gilliver SC, Ashworth JJ, Mills SJ, Hardman MJ, Ashcroft GS. J Cell Science 2006;119:722-732. 

16. Hermans EJ, Putman P, Baas JM, et al. Biol Psychiatry 2006;59:872-874. 

17. Imbimbo BP. The potential role of non-steroidal anti-inflammatory drugs in treating Alzheimer's disease. 

Expert Opin Investig Drugs 2004;13:1469-8141. 

18. Koenig HL, Schumacher M, Ferzaz B, et al. Science 1995;268:1500-1503. 

19. Kulkarni J, Riedel A, de Castella AR, et al. Schizophr Research 2001;48:137-144. 

20. Marx CE, Trost WT, Shampine LJ, et al. The Neurosteroid Allopregnanolone Is Reduced in Prefrontal 

Cortex in Alzheimer’s Disease. Biol psychiatry 2006;60:1287-1294. 

21. Melcangi RC, Garcia-Segura LM. Therapeutic approaches to peripheral neuropathy based on neuroactive 

steroids. Expert Rev Neurotherapeutics 2006;6:1121-1125. 

22. Tolentino P. Androgens and antibody formation. Pharmacol and Therap 1975;1:209-216. 

23. Wilson JD, Griffin JE, Russell DW. Steroid 5α–reductase 2 deficiency. Endocr Rev 1993;14:577-593. 

148



INFLUENCE OF MIRTAZAPINE ON PLASMA CONCENTRATIONS OF 

NEUROACTIVE STEROIDS IN MAJOR DEPRESSION AND ON 3α- 

HYDROXYSTEROID DEHYDROGENASE ACTIVITY 

Eser D. 1 , Schüle C. 1 , Romeo E. 2 , Uzunov DP. 3 , di Michele F. 2 , Baghai T.C. 1 , Pasini A. 

2 , Schwarz M. 1 and R Rupprecht 1 

1 Department of Psychiatry and Psychotherapy, Ludwig-Maximilian-University, 

Nussbaumstr. 7, 80336 Munich, Germany 

2 IRCCS Santa Lucia, Tor Vergata University, Via Ardeatina 306, 00179 Rome, Italy 

3 Novartis Institutes for BioMedical Research, Neuroscience Research, Novartis Pharma 

AG, WSJ-386.3.26, CH-4002 Basel, Switzerland 

E-mail: Daniela.Eser@med.uni-muenchen.de, Tel.: ++49-89-5160-5876, Fax: ++49-89- 

5160-5391 

Concentrations of 3α-reduced neuroactive steroids are altered in depression and normalize 

after antidepressant pharmacotherapy with SSRIs. We investigated the impact of 

mirtazapine on the activity of a key neurosteroidogenic enzyme, the 3α-hydroxysteroid 

dehydrogenase (3α-HSD), and on the levels of neuroactive steroids in relation to clinical 

response. Twenty-three drug-free inpatients suffering from major depression (DSM-IV 

criteria) underwent 5-week treatment with mirtazapine (45 mg/day). Plasma samples were 

taken weekly at 8:00 AM and quantified for neuroactive steroids by means of combined 

gas chromatography/mass spectrometry analysis. Enzyme activity was determined by 

assessment of steroid conversion rates. Irrespectively of clinical outcome, there were 

significant increases in 3α-reduced neuroactive steroids after mirtazapine treatment, 

whereas 3β-reduced steroids were significantly decreased. In-vitro investigations 

demonstrated a dose-dependent inhibitory effect of mirtazapine on the activity of the 

microsomal 3α-HSD in the oxidative direction, which is compatible with an enhanced 

formation of 3α-reduced neuroactive steroids. However, the changes in neuroactive steroid 

concentrations more likely reflect direct pharmacological effects of this antidepressant 

rather than clinical improvement in general. 

149


PREGNANOLONE ISOMERS AND THEIR POLAR CONJUGATES IN LATE 

PREGNANCY: A LONGITUDINAL STUDY 

Hill M. 1 , Cibula D. 2 , Včelaková H, Kancheva L. 1 and Pařízek A. 2 

1 Institute of Endocrinology, Narodni triad 8, Prague 11694, Czech Republic 

2 Clinic of Gynecology and Obstetrics, General Faculty Hospital, Prague 

Pregnanolone isomers (PIs) are known as modulators of GABA A receptors (GABA A -r). 

The 3α-PIs operate as the positive modulators and the 3β-PIs are their inactive competitors 

for the receptors. The polar conjugates of PIs negatively modulate the GABA A -r and they 

are also active on NMDA-receptors depending on the position of C5 hydrogen. Free 5β-PIs 

may also operate on nuclear pregnane X receptors (PXr) influencing uterine contractility 

and on calcium channels of type T participating in pain transmission. This study addresses 

the question of whether changes in the biosynthesis and metabolism of neuroactive 

pregnanolone isomers (PIs) may be associated with the timing of human parturition. Using 

the GC-MS, the time profiles of unconjugated pregnenolone isomers (PIs) 

allopregnanolone (3α-hydroxy-5α-pregnan-20-one, P3α5α), pregnanolone (3α-hydroxy- 

5β-pregnan-20-one, P3α5β), isopregnanolone (3β-hydroxy-5α-pregnan-20-one, P3β5α) 

and epipregnanolone (3β-hydroxy-5β-pregnan-20-one, P3β5β), pregnenolone, their polar 

conjugates, progesterone, 5α-dihydroprogesterone (P5α), and 5β-dihydroprogesterone 

(P5β) were monitored in the circulation of 30 healthy women to obtain data from the 10 th 

week before parturition (WBP) up to labor at one-week intervals (Fig. 1). Changes in the 

steroid levels were evaluated by two-way ANOVA with WBP and subject as independent 

factors. The mean concentrations of free PIs ranged from 2–50 nmol/L (Fig. 1) but their 

polar conjugates were in 40–140 fold excess over free PIs (Fig. 2). The decelerating 

biosynthesis of the P5β was found from the 7 th WBP till parturition (Fig. 3) but their 

escalating sulfation was found in all PIs from the 10 th or 9 th WPB (Fig. 4). The conjugation 

capacity for P3α5β in pregnancy is limited. As we recently reported, the ratio of 

conjugated to free steroid (C/F) for P3β5α was higher in NPW (150:1) that in the pregnant 

women (PW) (60:1). However, the C/F of P3α5β rise in the third trimester (Fig. 2). In 

contrast, the C/F was higher in PW for both P3α5α and P3β5α. The ratio for P3α5α was 

about 65 in PW but only about 15 in NPW and the ratio for P3β5α being about 80 for PW 

dropped to about half value in NPW. The changes in sulfation capacity probably diminish 

the difference between P3α5α and P3α5β levels in pregnancy. The accelerating ratio of 

conjugated to free P3β5α points to potential importance of the steroid for the timing of 

parturition. This conjugate exerts a reverse modulating effect on the GABA A -r than the 

unconjugated 3αPIs. The modulation efficiencies of both substances are comparable in 

absolute values, however the circulating polar conjugates of P3β5α are in a great excess 

over the P3α5α. In contrast to the expectedly inferior physiological role of P3α5β in 

NPW, the steroid may participate in sustaining of pregnancy and conversely, its reduced 

biosynthesis at accelerating conjugation may contribute to inducement of human 

parturition given its effectiveness in uterine relaxation via the PXr-dependent mechanism, 

its relative abundance and potency (like P3α5α) to suppress the activity of oxytocin 

producing cells via positive modulation of GABA A -r. 

The study was supported by grants 1A/8649-5, NR/8991-3, NR/9055-4, of the Internal 

Grant Agency of the Czech Ministry of Health and GAČR 303/06/1817. 

150



P3!5! (nmol/L) 

60 

50 

40 

30 

20 

10 

0 

Week: F=8.58, p


NEUROACTIVE STEROIDS IN ADULT MEN 

Kancheva L , Včelaková H, Hill M., Vrbíková J. and Stárka L 

Institute of Endocrinology, Steroid Hormone Unit, Národní 8, Prague 11694, Czech 

Republic, fax +420224905325, email lkantcheva@endo.cz 

Pregnane steroids (PS), and androstane metabolites (AM) modulate ionotropic receptors. 

Women produce significant amounts of neuroactive progesterone metabolites. The steroid 

neuromodulators in men are mostly of testicular origin. The 3-oxo-4-ene androgens are 

converted to their 3α- and 3β-hydroxy-5α/5β-reduced metabolites (Fig. 1). The 

neuromodulating effects of AM and PS prompted us to follow their circulating levels to 

estimate metabolic pathways in periphery that may implicate brain concentrations of the 

steroids. Accordingly, the levels of 20 steroids and 16 steroid polar conjugates including 

17-oxo- and 17β-hydroxy-derivatives of 5α/β-androstane-3α/β-hydroxy-AM were 

quantified in 15 men (16-62 years) using GC-MS (Tab. 1). 

The levels of AM were in excess over the respective PS, which pointed to the potential 

physiological effect of AM in men. The levels of conjugated AM were by 2 or 3 orders of 

magnitude higher compared to free steroids (Tab. 1). The neuroinhibitory unconjugated 

3α-AM represent only a minute fragment from the pool of the circulating AM. 

OSO 2 O 

OSO 2 O 

OSO 2 O 

X 

X 

X 

OSO 2 O 

H 

H O 

H 

O 

H 

H O 

H 

OSO 2 O 

H 

X :...=O...Androsterone/3!-s ulf ate 

X:...-OH ...5!-Androstane-3!,17"-diol /3!-sulf ate 

17"-sulf ate /3!,17"-dis ulf ate 

X:...=O...5!-Androstane-3,17-dione 

X:...-OH...5!-D ihy drotestosterone/17"-s ulf ate 

X :...=O...Epiandrosterone/3"-sulf ate 

X:...-OH ...5!-Andros tane-3",17"-diol /3"-s ulf ate 

17"-s ulf ate /3",17"-disulf ate 

X 

OSO 2 O 

O 

X:...=O...Androstenedione 

X:...-OH ...Testosterone/17"-s ulf ate 

OSO 2 O OSO 2 O OSO 2 O 

X 

X 

X 

OSO 2 O 

H 

H O 

H 

O 

H 

H O 

H 

OSO 2 O 

H 

X :...=O...Etiocholanolone/3!-s ulf ate 

X:...-OH ...5"-Androstane-3!,17"-diol /3!-sulf ate 

17"-sulf ate /3!,17"-disulf ate 

X:...=O...5"-Androstane-3,17-dione 

X:...-OH...5"-D ihy drotestosterone/17"-s ulf ate 

X :...=O...Epietioc holanolone/3"-sulf ate 

X:...-OH ...5"-Andros tane-3",17"-diol /3"-s ulf ate 

17"-sulf ate /3",17"-disulf ate 

FIGURE 1. Biosynthesis of androstane metabolites 

152



Table 1. Levels of neuroactive androstane and pregnane steroids including their precursors and 

ratios of conjugates to free steroids in the serum of adult men (16-65 years old) 

Unconjugated 

steroids 

(n=15) 

[nmol/L] 

Steroid polar 

conjugates 

(n=10) 

[nmol/L] 

Conjugates/free 

steroids 

(n=10) 

[nmol/L] 

Median 

Lower 

quartile 

Upper 

quartile 

Median 

Lower 

quartile 

Upper 

quartile 

Median 

Lower 

quartile 

Upper 

quartile 

Steroid 

Pregnenolone 2.19 1.63 2.89 292 216 424 163 110 226 

17-Hydroxy-pregnenolone 2.98 2.54 8.74 --- --- --- --- --- --- 

Dehydroepiandrosterone 13.8 10.0 17.1 6848 3200 8981 593 280 1080 

5-Androstene-3 ! , 17 ! -diol 2.18 1.78 3.21 1840 1367 2300 1002 731 1722 

Testosterone 21.7 19.0 31.9 --- --- --- --- --- --- 

Androstenedione 5.43 4.32 7.74 --- --- --- --- --- --- 

5 " -Androstane-3, 17-dione (A5 " ) 0.366 0.195 0.700 --- --- --- --- --- --- 

Androsterone (A3 " 5 " ) 0.649 0.539 0.889 2535 1387 3293 4173 3185 6114 

Epiandrosterone (A3 ! 5 " ) 0.212 0.135 0.313 510 335 654 2941 2658 4198 

Etiocholanolone (A3 " 5 ! ) 0.510 0.189 0.971 166 76 242 346 158 475 

Epietiocholanolone (A3 ! 5 ! ) 0.017 0.011 0.067 65 45 140 3339 2383 5060 

5 " -Dihydrotestosterone 1.36 1.12 1.65 13.9 11.9 20.9 12.3 8.9 13.1 

5 " -Androstane-3 " , 17 ! -diol (A3 " 5 " 17 ! ) 0.475 0.352 0.548 154 133 199 347 201 521 

5 " -Androstane-3 ! , 17 ! -diol (A3 ! 5 " 17 ! ) 0.149 0.057 0.251 246 149 346 2199 747 5723 

5 ! -Androstane-3 " , 17 ! -diol (A3 " 5 ! 17 ! ) 0.067 0.045 0.120 70.7 17.8 96.1 803 630 1434 

5 ! -Androstane-3 ! , 17 ! -diol (A3 ! 5 ! 17 ! ) 0.085 0.062 0.171 10.8 8.7 13.7 120 78 347 

Allopregnanolone (P3 " 5 " ) 0.341 0.154 0.399 9.0 6.8 14.4 50.4 20.6 93.4 

Isopregnanolone (P3 ! 5 " ) 0.233 0.177 0.367 15.0 12.7 26.6 76.0 44.6 124.4 

Pregnanolone (P3 " 5 ! ) --- --- --- 38.4 24.6 51.6 --- --- --- 

Epipregnanolone (P3 ! 5 ! ) --- --- --- 6.94 3.39 8.89 --- --- --- 

The ratios of conjugates to free steroids in AM were by 1-2 orders of magnitude higher 

compared to values found for the corresponding PS. The neuroinhibiting reduced 3α-5α/β- 

AM prevailed over the inactive 3β-derivatives. Strong correlations were detected between 

3α-, 3-oxo- and 3β- derivatives in both AM and PS. A5α correlated with A3α5α (r=0.604, 

p


PHOTOPERIOD INFLUENCES AROMATASE EXPRESSION IN JUNCO 

HYEMALIS PROSENCEPHALON 

Bo E., Casella D., Martini M., Viglietti-Panzica C., Deviche P.*, Panzica G.C. 

Dep.Anatomy, Pharmacology and Forensic Medicine, University of Turin, Italy. 

*School of Life Sciences, Arizona State University, Phoenix, AZ, USA 

Aromatase (ARO) is produced by the CYP19 gene and it’s responsible of estradiol (E2) 

biosynthesis from testosterone (T). In the avian brain, this enzyme is localized in regions 

controlling sexual behavior, as the POM (preoptic medial nucleus), the BnST (bed nucleus 

of the stria terminalis), the VMN (ventromedial nucleus) and the medial amygdala. In 

passerine birds, ARO was found also in hippocampus and in HVC (High Vocal Center). 

ARO expression in male galliforms is related to circulating levels of T, it almost 

disappears in castrated quails and its expression is restored to the level of intact males 

when castrated birds receive exogenous T [1,2]. Gonadal hormones’ levels, in particular T, 

fluctuate in wild birds when exposed to different seasonal conditions, in particular they 

may change according to the lenght of the photoperiod, paralleling changes of gonadal 

functions. 

Studies on the effects of seasonal changes on ARO system of wild birds are rare [3,5], and 

never performed on a migratory species. The aim of the present study was to investigate 

brain ARO immunoreactivity distribution in a migratory songbird, Junko hyemalis. In 

addition, we have also investigated changes in limbic and hypothalamic distribution of 

ARO according to the exposure of these birds to different photoperiods. 

Birds were collected from a local population in Fairbanks, Alaska (65°N, 148°W), in 

different periods and were divided in two groups:the first one (not photostimulated, no- 

PHOT) includes three animals captured in September at the age of three months and 

exposed to a short photoperiod (8h of light and 16h of dark) for 8 months. The second one 

(photostimulated, PHOT) includes three one year old animals captured in May (natural 

photoperiod of 20h of light and 4h of dark) and sacrificed immediately after the capture. 

The animals were perfused and processed for the immunocitochemistry using the Harada 

antibody to detect ARO immunoreactivity. To identificate brain nuclei we used the Serinus 

Canaria atlas with modifications for the Junco hyemalis [4]. The sections were analyzed to 

count the cell number in selected regions and the results were statistically analyzed. 

In PHOT birds, ARO positive neurons were observed in POM, BnST, VMN, medial 

amygdala, hippocampus, nidopallium, nidopallium caudalis, and reticular formation of the 

mesencephalon. We have quantitatively analyzed hypothalamic and limbic nuclei that are 

related to reproductive behavior in birds (i.e., POM, BnST, and VMN). 

In all these structures we observed a strongly significant decrease of the number of AROimmunoreactive 

(ir) elements in no-PHOT animals, comparable to the decrease observed 

in castrated male quail. 

It seems therefore that, as demonstrated in quail [1], in Junko there is a small population of 

ARO-ir cells that are insensitive to the fluctuations of circulating T levels. We can 

hypothesizes that, when photoperiod increases and circulating T levels raise, these 

elements start to locally convert T into E2 stimulating at the same time the surrounding 

cells to produce ARO. 

Acknowledgements. This work was supported by grants from PRIN, Università di Torino, 

Fondazione CRT, and Regione Piemonte 

154




[1] N. Aste, G.C. Panzica, P. Aimar, C. Viglietti-Panzica, N. Harada, A. Foidart and J. 

Balthazart, Morphometric studies demonstrate that aromatase-immunoreactive cells 

are the main target of androgens and estrogens in the quail medial preoptic nucleus, 

Exp. Brain Research 101 (1994) 241-252. 

[2] N. Aste, G.C. Panzica, C. Viglietti-Panzica, N. Harada and J. Balthazart, 

Distribution and effects of testosterone on aromatase mRNA in the quail forebrain: 

a non-radioactive in situ hybridization study, Journal of Chemical Neuroanatomy 

14 (1998) 103-115. 

[3] A. Foidart, B. Silverin, M. Baillen, N. Harada and J. Balthazart, Neuroanatomical 

distribution and seasonal variations of aromatase activity and aromataseimmunoreactive 

cells in the Pied Flycatcher (Ficedula hypoleuca), Hormones and 

Behavior 33 (1998) 180-196. 

[4] G.C. Panzica, L. Plumari, E. Garcia-Ojeda and P. Deviche, Central vasotocinimmunoreactive 

system in a male passerine bird (Junco hyemalis), Journal of 

Comparative Neurology 409 (1999) 105-117. 

[5] L.V. Riters, M. Eens, R. Pinxten, D.L. Duffy, J. Balthazart and G.F. Ball, Seasonal 

changes in courtship song and the medial preoptic area in male European starlings 

(Sturnus vulgaris), Hormones and Behavior 38 (2000) 250-261. 

155


ADULT HIPPOCAMPAL NEUROGENESIS IS ALTERED WITH MATERNAL 

EXPERIENCE 

Pawluski J.L., Walker C.A., Galea L.A.M. 

Program in Neuroscience, Department of Psychology and Brain Research Centre, 

University of British Columbia, 2136 West Mall, Vancouver B.C., V6T 1Z4, CANADA,. 

Fax: 001-604-822- 6923, email: jodi@psych.ubc.ca 

Adult neurogenesis in the dentate gyrus of the hippocampus is influenced by steroid 

hormones (estradiol and corticosterone), which fluctuate during the estrous cycle, 

pregnancy and lactation. Pregnancy and lactation have been demonstrated to be a time of 

maximal neural and behavioural plasticity as a host of learning and memory processes are 

activated in the mother to ensure the acquisition of adequate maternal care and 

reproductive success. Recent work has shown that motherhood differentially affects 

hippocampus-dependent learning and memory performance [1] and hippocampal 

morphology [2] and these effects differ with reproductive experience (number of times 

pregnant and given birth). Thus, it is possible that hippocampal neurogenesis may also be 

affected by reproductive experience. However, very little research has investigated the role 

of motherhood on hippocampal neurogenesis. The present study aimed to thoroughly 

investigate the role of motherhood and/or pup exposure on hippocampal neurogenesis via 

cell proliferation and cell survival. Four groups of female Sprague-Dawley rats were used; 

multiparous, primiparous, nulliparous, and sensitized (pup-exposed nulliparous females). 

All rats were injected with BrdU (200 mg/kg) 24 hours after birth/pup-exposure with agematched 

controls. Rats were perfused either 24 hours (cell proliferation) or 21 days (cell 

survival) after injection. Parous/sensitized rats remained with pups until perfusion. Results 

show there is a significant decrease in BrdU-labeled cells in the dentate gyrus surviving 

throughout lactation in primiparous dams compared to multiparous, nulliparous and 

sensitized rats. Interestingly this effect appears to be independent of pup-exposure. In 

addition, multiparous rats have a greater percentage of cells surviving throughout lactation 

compared to all other groups. Future research aims to determine the hormonal mechanisms 

mediating these changes. 


1. Pawluski, J.L., Walker, S.K., and Galea, L.A.M (2006). Reproductive experience differentially 

affects spatial reference and working memory performance in the mother. Hormones and Behavior. 

49(2):143-9. 

2. Pawluski, J.L. and Galea L.A.M. (2006). Hippocampal morphology is differentially affected by 

reproductive experience. Journal of Neurobiology. 66(1):71-81. 

156



PRIMARY CELL CULTURES FROM FETAL BOVINE BRAIN: AN IN VITRO 

MODEL TO STUDY NEUROACTIVE STEROIDS 

Peruffo A., Buson G. Cozzi B. and Ballarin C. 

Department of Experimental Veterinary Science, University of Padova, 

Viale dell’Università 16, 35020 Legnaro – Agripolis (PD), ITALY 

Phone +39.049.8272626, Fax +39.049.8272669, e-mail: antonella.peruffo@unipd.it 

Primary cell cultures represent a simplified experimental model useful to control and 

modulate media’s composition and study the effects of neuroactive steroids on neural cells. 

We set up a procedure to obtain primary cell cultures from fetal bovine brain to study the 

mRNA expression and localization of P450 aromatase (P450 AROM ) at the cellular level in 

neurons and astrocytes. Aim of the study was to verify if primary cultures from fetal 

bovine brain may be a reliable model for the expression of P450 AROM . We chose the bovine 

as experimental species for studying the role of neural aromatization in the sexual 

differentiation of the central nervous system because of its large brain, extended duration 

of gestation and incidence of spontaneous intersex calves. 

Hypothalamus and frontal cortex areas were isolated from a series of bovine fetuses of 

different developmental stages. Previous published data from our laboratory [1] 

demonstrated that tissue fragments obtained from the bovine cerebral cortex and 

hypothalamus could be cryopreserved in liquid nitrogen and used for primary cell cultures. 

We compared mRNA expression of P450 AROM in both fetal brain tissue and primary cell 

cultures harvested from the same cerebral region. Furthermore, we detected the presence 

and localization of the enzyme by immunohistochemistry on fetal tissue and by 

immunocytochemistry on primary cell cultures. 

The mRNA expression of P450 AROM was confirmed using RT-PCR analysis. 

Immunohistochemistry performed with an anti P450 AROM antibody was used to identify 

immunoreactive neural cell in hypothalamic sections and to study the cellular localization 

of the enzyme in cultured neurons and astrocytes by confocal microscopy. Cellular lisates 

obtained from cell cultures were analysed by Western blot to detect the P450 AROM protein 

encoded by the aromatase transcripts. 

Neural cells from primary cultures were able to express P450 AROM mRNA. The enzyme 

encoded by transcripts was detected by Western blot and its localization in both neurons 

and astrocytes was confirmed by immunocytochemistry. The presence of P450 AROM in 

neuron and astrocytes was observed in rat cerebral cortex [2] and recently also in the 

human temporal cortex [3]. 

We conclude that primary cultures from fetal bovine brain could represent a good in vitro 

model for future investigations on P450 AROM expression, activity and regulation. 


1. Peruffo, A., Massimino, M.L., Ballarin, C., Carmignoto, G., Rota, A., Cozzi, B., 2004. Primary cultures 

from fetal bovine brain. Neuroreport 15, 1719-1722. 

2. Zwain, I.H. and Yen, S.S.C. 1999. Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of 

cerebral cortex of rat brain. Endocrinology 140, 3843-3852. 

3. Yague., J.G., Munoz, A., de Monasterio-Schrader, P., Defelipe, J., Garcia-Segura, L.M., Azcoitia, I. 

2006. Aromatase expression in the human temporal cortex. Neuroscience 138, 389-401. 

157


NEUROSTEROID MODULATION OF RECOMBINANT RAT α 5 β 2 γ 2L AND α 1 β 2 γ 2L 

GABA A RECEPTORS IN XENOPUS OOCYTE 

Rahman M., Lindblad C., Johansson I-M, Bäckström T. and Wang M-D 

Umeå Neurosteroid Research Center, Department of Clinical Science, Obstetrics and Gynecology, Umeå 

University, S-901 85 Umeå, Sweden. Phone: +46 90 785 3323 Fax: +46 90 77 60 06; 

E-mail: mingde.wang@obgyn.umu.se 

GABA A receptors containing α 5 -subunit have an important role in cognitive function. As 

the potentiating effect of 3α-hydroxy ring-A reduced steroids depends on subunit 

combinations of GABA A receptors, the antagonistic effect of 3β-hydroxypregnane steroids 

may vary between α 5 -subunit and α 1 -subunit containing receptors. We investigated the 

effect of steroid agonists and antagonists in recombinant α 1 β 2 γ 2L and α 5 β 2 γ 2L receptors 

expressed in Xenopus oocytes using a two electrodes voltage-clamp technique. We did not 

find any significant difference in potency and efficacy of GABA response between the 

α 1 β 2 γ 2L and α 5 β 2 γ 2L receptor. Compared to the α 1 β 2 γ 2L receptor, a significantly lower 

degree of desensitization was observed in the α 5 β 2 γ 2L receptor. In addition, the potency of 

3α-OH-5α-pregnan-20-one (3α5αP); 5α-pregnan-3α,21-diol-20-one (3α5αTHDOC) and 

5α-androstane-3α,17β-diol (3α5αADL) to enhance GABA response was significantly 

higher in the α 5 β 2 γ 2L receptor, whereas their efficacy remained unchanged between the 

receptors. In either receptor, the efficacy of the 3α5αTHDOC was significantly higher than 

that of 3α5αP and 3α5αADL. The efficacy of 5β-pregnan-3β,21-diol-20-one(UC1015) 

and 5α-pregnan-3β,20α-diol(UC1019) to inhibit GABA response and 5β-pregnan-3β, 20βdiol 

(UC1020) to inhibit 3α5αTHDOC enhanced GABA response were higher in the 

α 5 β 2 γ 2L receptor compared to α 1 β 2 γ 2L receptor. The potencies of 3β-hydroxypregnane 

steroids to inhibit GABA response or 3α5αTHDOC enhanced responses did not vary 

between the α 1 β 2 γ 2L and α 5 β 2 γ 2L receptors. Interestingly, the potencies and efficacies of 

3β-hydroxy-pregnane steroids to inhibit GABA response were positively correlated to 

potencies and efficacies inhibiting 3α5αTHDOC enhanced GABA response. Results from 

the current study revealed a different pattern of steroid modulation in the rat α 1 β 2 γ 2L and 

α 5 β 2 γ 2L receptors. 


Rahman, M., Lindblad, C., Johansson, I.M., Backstrom, T. and Wang, M.D., Neurosteroid modulation of 

recombinant rat alpha(5)beta(2)gamma(2L) and alpha(1)beta(2)gamma(2L) GABA(A) receptors in Xenopus 

oocyte, Eur J Pharmacol, 547 (2006) 37-44. 

158


Neuroprotective effects 

• Barreto G., Veiga S., Azcoitia I., Garcia-Segura L.M., Garcia-Ovejero D. (Spain) 

Testosterone decreases reactive astroglia and reactive microglia after brain injury 

in male rats: role of its metabolites estradiol and dihydrotestosterone 

• Berumen L.C., Tecozautla A., Sánchez-Ramos M.A., García-Servín M., García- 

Alcocer G. (México) Steroid hormone effects on 5-HT 5A serotonin receptor-like 

immunolabelling in the rat hippocampus 

• F.Biamonte, G.Assenza, R.Marino, D.Caruso, S.Crotti, R.C.Melcangi, R.Cesa, 

P.Strata, F.Keller. (Italy) Interaction between estrogens and reelin in purkinje cell 

development 

• Carroll J.C., Emily R. Rosario, Lilly Chang, Frank Z. Stanczyk, Salvatore Oddo, 

Frank M. LaFerla, Christian J. Pike (USA) Progesterone blocks estrogen 

regulation of alzheimer-like neuropathology in female 3xTG-AD mice 

• Danza G., Cecchi C., Pensalfini A., Formigli L., Nosi D., Stefani M, Liguri G, 

Rosati F., Dichiara F., Morello M.,Pieraccini G., Serio M., Peri A. (Italy) The 

estrogen-regulated gene Seladin-1/DHCR24 exerts its neuroprotective effects 

through membrane cholesterol modulation 

• Fargo K.N., Sengelaub D.R. (USA) Androgenic, but not estrogenic, protection of 

motoneurons from somal and dendritic atrophy induced by the death of neighboring 

motoneurons 

• Forsberg M. K., Hallberg M., Nyberg F., Svensson A-L (Sweden) Neuronal 

protection of dehydroepiandrosterone on PC12 cells pretreated with β-amyloid 

• S. Giatti, I. Roglio, M. Pesaresi, R. Bianchi, G. Cavaletti, L.M. Garcia-Segura, G. 

Lauria, R.C. Melcangi (Italy) Progesterone and its derivatives as protective agents 

in experimental diabetic neuropathy 

• Jarrahi M, Vafaei AA, Rashidy-Pour A (Iran) An evaluation of the effect of 

dexamethasone in preventing Tourniquet neurepathy in rats 

• Kibaly C., Meyer L., Patte-Mensah C. and Mensah-Nyagan A.G. (France) 

Involvement of endogenous dehydroepiandrosterone in the modulation of spinal 

nociceptive mechanisms

• Lee J.E., Kim H.J., Kang H.S., Ahn H.S., and Gye M.C. (Korea) Postnatal 

changes in the expression of aquaporin 1 and effect of estrogen on the expression 

in ovariectomized mouse brain 

• I. Roglio, S. Giatti, M. Pesaresi, R. Bianchi, G. Cavaletti, D. Caruso, S, Scurati, 

L.M. Garcia-Segura, G. Lauria, R.C. Melcangi (Italy) Testosterone derivatives are 

neuroprotective agents in experimental diabetic neuropathy 

• Rosario, E.R., Carroll, J.C., Pike, C.J. (USA) Estrogen and androgens regulate 

Alzheimer-like neuropathology in male 3xTG-AD mice 

• Schaeffer V., Patte-Mensah C., Eckert A., Mensah-Nyagan A.G. (France) Effects 

of beta amyloid peptide 1-42 and oxidative stress on neurosteroid formation in 

human neuroblastoma cells 

• Szegő É.M., Kékesi K.A., Juhász G., Ábrahám I.M. (Hungary) Effect of estrogen 

treatment on protein expression pattern in famale mice brain using fluorescent 

differential 2-D gel electrophoresis (dige) 

• Tapia González S., Diz-Chaves Y., Pernía O., Carrero P., Garcia-Segura L.M. 

(Spain) Selective estrogen receptor modulators decrease microglia activation in 

the cerebellum of male rats



TESTOSTERONE DECREASES REACTIVE ASTROGLIA AND REACTIVE 

MICROGLIA AFTER BRAIN INJURY IN MALE RATS: ROLE OF ITS 

METABOLITES ESTRADIOL AND DIHYDROTESTOSTERONE 

Barreto G. 1* , Veiga S. 1 , Azcoitia I. 2 , Garcia-Segura L.M. 1 , Garcia-Ovejero D. 3 

(1) Instituto Cajal, C.S.I.C., Avenida Doctor Arce 37, E-28002 Madrid, Spain.* Email: 

gemilio.sampaio@cajal.csic.es FAX: +34-915854754 

(2) Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 

Madrid, Spain 

(3) Laboratorio de Neuroinflamación, Hospital Nacional de Parapléjicos, Toledo, Spain 

Stab wound injury in the brain elicits a complex cascade of events involving glial cells. 

Astrocytes and microglia actively react to brain damage, participating in the repair of the 

disrupted blood-brain barrier and in the reorganization of the injured neuronal circuits. The 

response of astrocytes and microglia to brain injury is modulated by local factors produced 

in the injured tissue and also by substances transported by the systemic circulation. Among 

other peripheral molecules, the hormones secreted by the gonads exert a modulation of 

reactive gliosis. In this study we have assessed the effect of testosterone therapy on gliosis 

after a stab wound injury affecting the hippocampal formation. In addition, to determine 

whether the effects of testosterone were mediated by its metabolites, some animals were 

treated with estradiol or dihydrotestosterone (DHT). Wistar male rats were bilaterally 

orchidectomized at the age of 2 months to reduce circulating levels of testicular secretions. 

A penetrating injury affecting the hippocampal formation was performed one month after 

orchidectomy. The effects of early and late treatments after injury with testosterone or its 

metabolites estradiol and DHT were assessed. Therefore, a group of animals received one 

subcutaneous injection of testosterone (5 mg/Kg), estradiol (1 mg/Kg) or DHT (5 mg/Kg) 

on days 0, 1 and 2 after injury (early steroid administration). A second group of animals 

were injected with the same steroids at the same doses on days 5, 6 and 7 after injury 

(delayed steroid administration). One week after lesion, animals were killed by fixative 

perfusion and the brains processed for immunohistochemistry for vimentin, a marker of 

reactive astroglia or MHC-II, a marker of reactive microglia. The number of vimentin 

immunoreactive astrocytes was assessed in the hippocampus using the optical disector 

method. The volume fraction of MHC-II immunoreactive microglia was estimated 

according to the point-counting method of Weibel. Both early and delayed administration 

of testosterone resulted in a significant decrease in the number of vimentinimmunoreactive 

astrocytes in the studied area (0-345 micrometers from the lateral border 

of the wound). Early and delayed treatments with estradiol also resulted in a decrease in 

the number of vimentin-immnoreactive astrocytes compared to control values. DHT 

administration, either early or delayed, did not affect the number of vimentin 

immunoreactive astrocytes. The volume fraction of MHC-II immunoreactive microglia 

showed a significant decrease in the animals that received testosterone or estradiol in both 

early and delayed treatments. In contrast, the volume fraction of MHC-II immunoreactive 

cells was not affected by the delayed administration of DHT. However, early 

administration of DHT significantly reduced the volume fraction of MHC-II 

immunoreactive cells. These findings indicate that both early and delayed testosterone 

administration after a stab wound injury reduce astroglia and microglia reactivity in male 

rats. Testosterone may exert its effects on reactive gliosis by acting on androgen receptors 

or after local conversion to estradiol. Previous studies have shown that a stab wound injury 

induces the expression of aromatase, the enzyme that converts testosterone in estradiol, in 

161


reactive astrocytes [2]. Furthermore, a stab wound injury induces the expression of 

estrogen receptors in reactive astroglia [1]. Therefore, testosterone may be converted into 

estradiol in reactive astrocytes and then estradiol may act by a paracrine or autocrine 

mechanism on reactive astrocytes expressing estrogen receptors. In addition, estradiol may 

indirectly reduce reactive astrogliosis by preventing neuronal death. In agreement with this 

possibility, we have detected in the present study that the early or late administration of 

estradiol after injury to adult orchidectomized male rats reduces reactive astrogliosis in the 

borders of the wound. Therefore, it may be postulated that at least part of the early and late 

effects of testosterone on reactive astrogliosis are mediated by its local conversion to 

estradiol. Local conversion to estradiol may also in part mediate the effects of testosterone 

on microglia. Previous studies have shown that estradiol may reduce microglia activation 

in female rodents [3] and our present findings indicate that early or late administration of 

estradiol may also reduce microglia activation in male rats. Testosterone may also be 

converted in the brain to its reduced metabolite DHT, by the enzyme 5alpha-reductase, 

which is also expressed in glial cells. DHT is a potent agonist of androgen receptors and 

many of the biological effects of testosterone are mediated by this metabolite. However, 

since DHT did not affect the number of vimentin immunoreactive astrocytes, we may 

conclude that the effect of testosterone on reactive astrogliosis is not mediated by the 

conversion in DHT and the subsequent activation of androgen receptors. Furthermore, 

androgen receptors have not been detected in reactive astrocytes in the rat brain. In 

contrast, early expression of androgen receptors has been detected in reactive microglia 

after a stab wound [1]. Our present findings, indicating that early administration of DHT 

reduces the volume fraction of MHC-II immunoreactive cells in the hippocampus in the 

proximity of the wound, suggest that part of the early effect of testosterone on reactive 

microglia may be mediated by its conversion to DHT and the consecutive action on 

androgen receptors. Therefore, we may conclude that early and late effects of testosterone 

on reactive astroglia and reactive microglia may be at least in part mediated by estradiol, 

while DHT may mediate part of the early effects of testosterone on reactive microglia. 

Therefore, our findings suggest that metabolism of testosterone is an essential mechanism 

involved in its effects on reactive astroglia and reactive microglia. 




[1] D. Garcia-Ovejero, S. Veiga, L.M. Garcia-Segura and L.L. DonCarlos, Glial 

expression of estrogen and androgen receptors after rat brain injury, J Comp Neurol 450 

(2002) 256-271. 

[2] L.M. Garcia-Segura, S. Veiga, A. Sierra, R.C. Melcangi and I. Azcoitia, Aromatase: a 

neuroprotective enzyme, Prog Neurobiol 71 (2003) 31-41. 

[3] E. Vegeto, C. Bonincontro, G. Pollio, A. Sala , S. Viappiani, F. Nardi, A. Brusadelli, B. 

Viviani, P. Ciana and A. Maggi, Estrogen prevents the lipopolysaccharide-induced 

inflammatory response in microglia, J Neurosci 21 (2001) 1809-1818. 

162



STEROID HORMONE EFFECTS ON 5-HT 5A SEROTONIN RECEPTOR-LIKE 

IMMUNOLABELLING IN THE RAT HIPPOCAMPUS 

Berumen L.C. 1 , Tecozautla A. 1 , Sánchez-Ramos M.A. 2 , García-Servín M. 3 , García- 

Alcocer G. 1 

1 Universidad Autónoma de Querétaro, Facultad de Química, Centro Universitario S/N, 

Cerro de las Campanas, 76010, Querétaro, México, Fax+52-442-1921302, email: 

berumen@uaq.mx. 2 Universidad Autónoma de Querétaro, Facultad de Ciencias Naturales, 

Qro. México. 3 Instituto de Neurobiología, Campus UNAM-UAQ, Juriquilla, Qro. México. 

The activity of different neurotransmitter systems can be modulated by steroid hormones. 

The effect of steroids over serotonergic pathway has been widely studied, by binding of 

radioligands, selective receptor antibodies and hibridization probes, and the use of agonist 

and antagonist drugs [1, 2]. The partial agonism of classical drugs for receptors, where 5- 

hydroxitriptamine (5-HT, serotonin) is their principal agonist, and the finding of different 

types of serotonin receptors led us to evaluate the response of 5-HT 5A receptors to steroid 

hormones, particularly estradiol and progesterone, using specific antibodies to label the 

presence of this protein in the hippocampal CA1 region. 

In the present study, thirty rats were ovariectomized and injected with 50µg/Kg 17-betaestradiol 

benzoate (E2), 7.5 mg/Kg progesterone (P) or the combination of both steroids; 

corn oil was used for ovariectomized controls. The 5-HT 5A –like immunosignal in the 

hippocampal CA1 region decreased in the group treated with E2 compared to the 

ovariectomized control group, and also decreased in the E2+P supplemented group. In 

contrast, the expression of 5-HT 5A receptor in the hippocampus of P-treated rats was not 

significantly different from ovariectomized controls. We also examined 5-HT 2C receptor 

immunolabelling, and found the contrary tendence, that is, decreased signal in 

hippocampus of P-supplemented rats compared to E2- or E2+P-supplemented ones. 

Studies have been made in order to understand the discrete effects found over serotonergic 

pathways, but the existence of several receptor proteins coupled to different transduction 

signals sometimes overlapping or cross-linked makes it complex to analyze [3]. Here we 

found that 5-HT 5A receptor responded similar to 5-HT 1 receptor [4], although the latter 

receptor is coupled to Gi-protein while 5-HT 5A receptor has been reported to stimulate 

adenylate cyclase activity, not yet defined [2]. In conclusion 5-HT 5A receptors participate 

in the modulation of serotonin signaling exerted by sexual hormones estradiol and 

progesterone. 

Acknowledgements 

The authors appreciate the work of Berenice Flores and Karina Hernández. This work was 

supported by PROMEP/103.5/05/1798. 


[1] Hoyer, D., Hannon, J.P., Martin, G.R., 2002. Molecular, pharmacological and functional diversity of 5- 

HT receptors. Pharm Biochem Beh. 71, 533-554. 

[2] Kroeze, W.K., Roth, B.L., 2006. Molecular biology and genomic organization of G protein-coupled 

serotonin receptors. In The Serotonin Receptors: from molecular pharmacology to human therapeutics (Roth 

BL, ED) Humana Press New Jersey. 1, 1-38. 

[3] Mouillet-Richard, S., Pietri, M., Schneider, B., Vidal, C., Mutel, V., Launay, J.M., Kellermann, O., 2005. 

Modulation of serotonergic receptor signaling and cross-talk by prion protein. J Biol Chem. 280(6), 4592- 

601 

[4] Biegon, A., Reches, A., Snyder, L., McEwen, B.S., 1983. Serotonergic and noradrenergic receptors in the 

rat brain: modulation by chronic exposure to ovarian hormones. Life Sci. 32(17), 2015-21. 

163


INTERACTION BETWEEN ESTROGENS AND REELIN IN PURKINJE CELL 

DEVELOPMENT 

F.Biamonte 1 , G.Assenza 1 , R.Marino 1 , D.Caruso 2 , S.Crotti 2 , R.C.Melcangi 3 , R.Cesa 4 , 

P.Strata 4 , F.Keller 1 

1. Lab of Dev Neurosci, Univ Campus Bio-Medico, Roma, Italy. 2. Dept of Pharmacol Sci, Univ of 

Milan, Italy 3. Dpt of Endocrin and Ctr of Excellence on Neurodegen Dis, Univ of Milan, Italy. 4. 

Rita Levi Montalcini Ctr for Brain Res, Univ of Turin, Italy 

Mariani et al. have demonstrated that mutations like reeler and staggerer in the 

heterozygous state, leading to haploinsufficiency of the gene product, cause a loss of 

Purkinje cells (PC) in the adult mouse cerebellum. Furthermore, the loss of PC is more 

prominent in male than female heterozygous mice. In order to test whether the PC loss 

may start at earlier stages, we have assessed PC numbers in neonatal cerebella of 

heterozygous malea (rl/+ M ) and female (rl/+ F ) vs. wild-type (wt) littermates using 

stereological counting methods. At ages between P10 and P18, PC numbers are decreased 

in male rl/+ M . Of all cell populations of the cerebellar cortex and their input and output 

nuclei, PC appear to be selectively affected, since the total number of neurons in deep 

cerebellar nuclei and in the inferior olivary nucleus, as well as the volume of the cerebellar 

cortex, are not reduced rl/+ M . PC are well known to be sensitive to gonadal sex steroids, 

particularly estrogens, and express the aromatase enzyme converting androgens into 

estrogens. In principle, it could be that estrogens protect rl/+ F from reelin 

haploinsufficiency or, alternatively, that androgens increase the effect of reelin 

haploinsufficiency in rl/+ M . To investigate the interaction between estrogens and reelin in 

our animal model, we injected 4-OHtamoxifen (TMX), an estrogen receptor antagonist, 

into the cisterna magna of P4 mice of either sex and genotype, and then assessed PC 

numbers again at P10-P18. We found that TMX selectively reduces PC numbers in rl/+ F 

and wt F , but has no effect in rl/+ M or wt M . To confirm the interaction between reelin and 

estrogens, we did similar experiments with 17-β-Estradiol (17βΕ2): 17βΕ2 was found to 

increase the number of PC in rl/+ M but had no effect in rl/+ F and wt F . Furthermore, we 

started to assess tissue levels of gonadal steroids and their precursors in cerebella of either 

sex and genotype at various developmental times, using mass spectrometry. The 

preliminary results indicate decreased 17βΕ2 levels and increased testosterone levels in 

cerebella of rl/+ M , a finding that is consistent with an aromatase deficit in rl/+ M . 

Experiments aimed at testing the effect of androgens are in progress. Our current results 

converge toward a model where the same genetic mutation is more penetrant in males than 

in females because of the protective effect of estrogens. Our animal model could be 

relevant for genetically complex neurodevelopmental disorders, such as autism, where 

genetic and hormonal factors have been recently postulated to interact (see e.g. 

Knickmeyer et al., Horm Behav 49:282-292, 2006). 

This work was supported by grants from the Fondation Jerome Lejeune, from 

NAAR-AUTISM SPEAKS (Grant number 1391) 

164



PROGESTERONE BLOCKS ESTROGEN REGULATION OF ALZHEIMER- 

LIKE NEUROPATHOLOGY IN FEMALE 3XTG-AD MICE 

Carroll J.C. a,b , Rosario E.R. a,b , Chang L. c ,. Stanczyk F.Z. c , Oddo S. d , LaFerla F.M. d , 

and Pike C.J. b 

a Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, 

b Davis School of Gerontology, c Department of Obstetrics and Gynecology, d Department of 

Neurobiology and Behavior, University of California Irvine, Irvine, CA 

cjpike@usc.edu 

fax: 213-740-4787 

Estrogen depletion in post-menopausal women is a significant risk factor for the 

development of Alzheimer’s disease (AD) and estrogen-based hormone therapy may be 

capable of reducing this risk. However, the effects of progesterone both alone and in 

combination with estrogen on AD pathology remain unknown. In this study, we utilized 

the 3xTg-AD mouse model of AD to investigate the effects of estrogen and progesterone 

on beta-amyloid and tau pathology as well as hippocampal-dependent memory deficits. 

Female 3xTg-AD mice were ovariectomized at 3 mo of age and immediately replaced with 

a 90d, subcutaneous, slow-release pellet containing either 17-beta-estradiol, progesterone, 

placebo, or both 17-beta-estradiol and progesterone pellets. After 3 months, depletion of 

sex steroid hormones in female 3xTg-AD mice significantly increased beta-amyloid 

accumulation in the CA1 of hippocampus, subiculum, and frontal cortex and worsened 

memory performance. Continuous replacement with 17-beta-estradiol prevented these 

effects. Continuous progesterone replacement alone had no beneficial effect on betaamyloid 

pathology or cognitive deficits but did significantly decrease tau 

immunoreactivity. However, when both hormones were replaced in combination, 

progesterone attenuated the beneficial effect of 17-beta-estradiol on beta-amyloid 

pathology and memory deficits. These results support the hypothesis that estrogen 

treatment may be beneficial in reducing the risk of AD and suggest possible therapeutic 

strategies regarding estrogen and progesterone-based hormone therapy. 

This research was funded by NIH grant AG23739 (CJP) and AG026572 (R.Brinton/CJP). 

JCC was supported by NIH Grant AG00093 (C. Finch). ERR was supported by NIH Grant 

NS52143 (ERR). 

165


THE ESTROGEN-REGULATED GENE SELADIN-1/DHCR24 EXERTS ITS 

NEUROPROTECTIVE EFFECTS THROUGH MEMBRANE CHOLESTEROL 

MODULATION 

Danza G. 1 , Cecchi C. 2 , Pensalfini A. 2 , Formigli L. 3 , Nosi D. 3 , Stefani M 2 , Liguri G 2 , 

Rosati F. 1 , Dichiara F. 1 , Morello M. 1 ,Pieraccini G. 4 , Serio M. 1 , Peri A. 1 

1 Endocrine Unit, Department of Clinical Physiopathology, University of Florence, Center 

for Research, Transfer and High Education on Chronic, Inflammatory, Degenerative and 

Neoplastic Disorders for the Development of Novel Therapies (DENOThe), Viale 

Pieraccini, 6, 50139 Florence, Italy (g.danza@dfc.unifi.it Fax n. +39 055 4271371) 

2 Department of Biochemical Sciences and Interuniversity Centre for the Study of the 

Molecular Basis of Neurodegenerative Diseases, University of Florence, viale Morgagni 

50, 50134 Florence, Italy 

3 Department of Anatomy, Histology and Forensic Medicine, University of Florence. 

4 Interdepartmental Mass Spectrometry Center, University of Florence. 

Alzheimer’s disease (AD) is a progressive, degenerative disorder of the brain characterized 

by loss of neurons and synapses in selective brain regions. Neuronal damage is apparently 

due to the altered production of intracellular neurofibrillary tangles and by extra-cellular 

and perivascular deposit of amyloid beta (Abeta) peptides. The reason for selective brain 

vulnerability has not been fully explained yet. However, a few years ago low levels of 

expression of a novel gene named seladin-1 (for Selective Alzheimer disease indicator 1) 

have been demonstrated in brain areas involved in AD [1]. Seladin-1 confers protection 

against Abeta mediated toxicity and from oxidative stress in vitro. In addition, it inhibits 

caspase 3 activity, a key mediator of apoptosis, thus protecting from apoptotic death. We 

have demonstrated previously that the expression of seladin-1 is up-regulated by estrogen 

and the selective estrogen receptor modulators (SERMs) tamoxifen and raloxifene, in a 

long-term cell culture of human foetal neuroblasts from human olfactory epithelium (FNC) 

that express both alpha and beta estrogen receptors. In these cells estrogen and SERMs 

significantly increased cell resistance to Abeta toxicity. Furthermore, upon seladin-1 

silencing, the neuroprotective effects of estrogen were abolished, indicating that this 

protein is a mediator of estrogen-related neuroprotection. Remarkably, seladin-1 has been 

found to be identical to DHCR24, the enzyme that catalyzes the last step of cholesterol 

biosynthesis by reducing the Delta 24 double bond of desmosterol [2]. Therefore, seladin- 

1/DHCR24 can be depicted as a multi-faced protein, which appears to have either antiapoptotic 

as well as enzymatic properties as a key enzyme of cholesterol biosynthesis. 

Neuronal cell death in AD is partly induced by the interaction of the Abeta peptides with 

the plasma membrane of target cells. It has been proposed that Abeta acts forming specific 

channels in the plasma membrane that allow a toxic flux of Ca2+ ions into the cell. In 

PC12 and in GT1-7 neurons it has been demonstrated that the enrichment of the plasma 

membrane with cholesterol, by modifying membrane fluidity, prevents the incorporation 

and pore formation of Abeta into cell membranes. Moreover we have previously shown 

that the variable susceptibility of different cell types to amyloid toxicity significantly 

correlates to membrane cholesterol content. 

Because the synthesis de novo of cholesterol is essential for cholesterol supply in the 

central nervous system, in this study we established the role of seladin-1/DHCR24 in the 

regulation of membrane cholesterol and in the control of the cytotoxicity of Abeta 

peptides. 

166



We modulated the enzymatic activity of seladin-1/DHCR24 by transient overexpression of 

the gene or using the specific inhibitor 5,22E-cholestadien-3β-ol (Δ22) in the human 

neuroblastoma cell line SH-SY5Y. These treatments determined a 30-40% increase or a 

15-25% decrease, respectively, of the membrane cholesterol (measured by GC/MS 

analysis). Cell membranes were also enriched or depauperated in cholesterol using specific 

reagents (PEG-cholesterol and methyl-beta-cyclodextrin, respectively) and the effect of all 

these treatments on Abeta toxicity was evaluated. We found that both seladin-1 

overexpressing cells with high membrane cholesterol and cells enriched in PEGcholesterol 

showed a significantly lower susceptibility to Aβ1-42 aggregates compared to 

control neuroblastoma cells. Confocal microscopic analysis, using monoclonal anti-Aβ 

antibodies or Fluo3-AM as fluorescent calcium indicator, revealed that the difference in 

cell viability was related to the ability of high membrane cholesterol content to: (i) reduce 

the interaction of amyloid assemblies with the plasma membrane; (ii) prevent intracellular 

Ca 2+ spikes induced by oligomeric inclusion. Conversely, membrane cholesterol loss in 

neuroblastoma cells treated with Δ22 or with methyl-beta-cyclodextrin triggered a quicker 

amyloid accumulation on cell surfaces and a higher cytosolic Ca 2+ increase resulting in a 

reduced cell survival. These data demonstrate that one of the mechanisms of 

seladin1/DHCR24 neuroprotection is dependent on the modulation of membrane 

cholesterol. 


1. Greeve, I., Hermans-Borgmeyer, I., Brellinger, C., Kasper, D., Gomez-Isla, T., Behl, 

C.,Levkau, B., Nitsch. RM., 2000. The human DIMINUTO/DWARF1 homolog 

seladin-1/DHCR24 confers resistance to Alzheimer’s disease-associated 

neurodegeneration and oxidative stress. J Neurosci. 20, 7345-52. 

2. Waterham, HR., Koster, J., Romeijn, GJ., Hennekam, RC., Vreken, P., Andersson, 

HC., FitzPatrick, DR., Kelley, RI., Wanders, R.J, 2001. Mutations in the 3betahydroxysterol 

Delta-reductase gene cause desmosterolosis, an autosomal recessive 

disorder of cholesterol biosynthesis. Am J Hum Genet. 69, 685-94. 

167


ANDROGENIC, BUT NOT ESTROGENIC, PROTECTION OF MOTONEURONS 

FROM SOMAL AND DENDRITIC ATROPHY INDUCED BY THE DEATH OF 

NEIGHBORING MOTONEURONS 

Fargo K.N. * , and Sengelaub D.R. 

Program in Neuroscience and Department of Psychological and Brain Sciences, Indiana 

University, 1101 East 10th Street, Bloomington, Indiana 47405, USA; 

sengelau@indiana.edu, Fax (812) 855-4691 

* This author now at Loyola University Chicago, Stritch School of Medicine; 

kfargo@lumc.edu 

Motoneuron loss is a significant medical problem, capable of causing severe movement 

disorders or even death. We have been investigating the effects of motoneuron loss on 

surviving motoneurons in a lumbar motor nucleus, the spinal nucleus of the 

bulbocavernosus (SNB). SNB motoneurons undergo marked dendritic and somal atrophy 

following the experimentally-induced death of other nearby SNB motoneurons. However, 

treatment with testosterone at the time of lesioning completely prevents this atrophy [1, 

2]. Because testosterone can be metabolized into the estrogen estradiol (as well as other 

physiologically active steroid hormones), it was unknown whether the protective effect of 

testosterone was an androgen effect, an estrogen effect, or both. In the present experiment, 

we used a retrogradely-transported neurotoxin to kill the majority of SNB motoneurons on 

one side of the spinal cord only, in adult male rats. Some animals were also treated with 

either testosterone, the androgen dihydrotestosterone (which cannot be converted into 

estradiol), or the estrogen estradiol. As seen previously, partial motoneuron loss led to 

reductions in soma area and in dendritic length and extent. Testosterone and 

dihydrotestosterone prevented these reductions, but estradiol had no protective effect. 

These results indicate that the neuroprotective effect of testosterone on the morphology of 

SNB motoneurons following partial motoneuron depletion is an androgen effect rather than 

an estrogen effect. 

This work supported by NIH-NINDS NS047264 (D.R.S.) and T32 DC 00012 


1. Fargo, K.N., Sengelaub, D.R., 2004. Testosterone manipulation protects motoneurons from dendritic 

atrophy after contralateral motoneuron depletion. J Comp Neurol. 469, 96-106. 

2. Fargo, K.N., Sengelaub, D.R., 2004. Exogenous testosterone prevents motoneuron atrophy induced by 

contralateral motoneuron depletion. J Neurobiol. 60, 348-359. 

168



NEURONAL PROTECTION OF DEHYDROEPIANDROSTERONE ON PC12 

CELLS PRETREATED WITH β-AMYLOID 

Forsberg M. K 1 ., Hallberg M 2 ., Nyberg F 2 ., Svensson A-L 1 

Department of Pharmaceutical Biosciences, 1 Division of Pharmacology, 2 Division of 

Biological Research on Drug Dependence, Uppsala University, P.O. Box 591, SE-751 24 

Uppsala, Sweden, Fax +46-18501920, e-mail: marie.forsberg@farmbio.uu.se 

The neuroactive steroid dehydroepiandrosterone (DHEA) is the most abundant 

prohormone produced by the adrenal glands. It is synthesized from cholesterol and 

secreted to peripheral tissues where it can be converted into androgens and/or estrogens 

[Labrie et al., 2005]. It is an important source of sex steroids apart from the gonads, leaving 

the adrenals as the only source of testosterone and estrogens after menopause in women. 

Neurosteroids have been implicated in loss of memory and memory acquisition in rodents 

[Brown et al., 2000]. Some neurosteroids are able to enhance memory performance in rats 

[reviewed in Mellon and Griffin, 2002]. It has been suggested that DHEA and its sulfate ester 

may protect the brain from neurodegeneration [Compagnone and Mellon, 2000]. DHEA has 

been shown to increase neuronal survival and differentiation. Furthermore, DHEA has 

been reported to protect hippocampal neurons against NMDA-induced toxicity in vitro 

[Kimonides et al 1998, Cardounel et al., 1999]. Although DHEA have shown neuroprotective 

properties, the mechanism(s) for its neuroprotective effects is not known. 

A plausible link between DHEA, other neurosteroids and neurodegenerative disorders like 

Alzheimer´s disease (AD) has been discussed. In AD the levels of neurosteroids such as 

DHEA and allopregnanolone are reduced compared to controls [Weill-Engerer et al., 2002]. It 

has recently been suggested that beta-amyloid (Aβ) peptide can activate DHEA synthesis. 

DHEA are also suggested to feed back onto glial cells and protect them against Aβinduced 

toxicity [Brown et al. 2000]. Beta-amyloid (Aβ) is a peptide that is a byproduct of the 

transmembrane protein amyloid precursor protein (APP). AD are characterized 

pathologically by deposits of Aβ peptide in the brain. Aβ peptide has been implicated in 

cell death during the course of AD and exerts toxic effects on neurons both in vivo and in 

vitro [reviewed in Jellinger, 2006]. 

In the present study, the effect of DHEA on Aβ-induced toxicity was investigated in rat 

pheochromocytoma PC12 cells. PC12 cells were exposed to Aβ alone and in the presence 

of DHEA for 24 hours. Untreated cells were used as controls. The number of necrotic 

cells were determined by the tryptane blue exclusion assay. The apoptosis was evaluated 

by measurement of caspase-3 activity using immunocytochemistry [Östergren et al., 2005]. 

Treatment of PC12 cells with Aβ (10 -6 M) significantly increased the number of necrotic 

cells. When PC12 cells were treated with Aβ in the presence of DHEA (10 -9 and 10 -6 M) 

the number of necrotic cells were significantly reduced. 

Treatment of PC12 cells with Aβ (10 -6 M) caused a significant increased number of 

caspase-3-positive cells. When PC12 cells were treated with Aβ in the presence of DHEA 

(10 -9 and 10 -6 M) the number of caspase-3-positive cells were significantly reduced. 

These results demonstrated that DHEA can protect PC12 cells against Aβ-induced toxicity. 

The mechanism(s) for the effect of DHEA is not fully understood but some evidence 

points towards the sigma receptors as a possible site of action. To investigate whether the 

169


neuroprotective effect of DHEA was achived through sigma receptors, PC12 cells were 

treated with DHEA in the presence of BD-1047 (a sigma-1 antagonist and sigma-2 agonist 

[Matsumoto R.R., et al.1995]). BD-1047 (10 -6 M) was observed to partly attenuate the toxicity 

induced by Aβ (10 -6 M) in PC12 cells. However, BD-1047 (10 -6 M) was not able to 

prevent the neuroprotective effect of DHEA against Aβ-induced toxicity. 

These data demonstrate that DHEA exerts neuroprotective properties, but the mechanism 

behind is still unclear and needs to be investigated. Knowledge of the role of neurosteroids 

on processes that are ongoing in Alzheimer brains might lead to clinical important 

applications. 


Brown R.C., Cascio C. and Papadopoulos V.,2000. 

Pathways of neurosteroid biosynthesis in cell lines from human brain: regulation of 

dihydroepiandrosterone formation by oxidative stress and β-amyloid peptide. J Neurochem, 74:847-859. 

Cardounel A., Regelson W. and Kalimi M., 1999. 

Dihydroepiandrosterone protects hippocampal neurons against neurotoxin-induced cell death: mechanism 

of action. Proc Soc Exp Biol Med, 222:145-149. 

Compagnone N.A. and Mellon S.H., 2000. 

Neurosteroids: Biosynthesis and function of these novel neuromodulators. Frontiers in 

Neuroendocrinology 21:1-56. 

Jellinger K.A., 2006. 

Alzheimer 100 – highlights in the history of Alzheimer research. J Neural Transm. 113:1603-1623. 

Kimonides VG, Khatibi NH, Svendsen CN, Sofroniew MV, Herbert J., 1998. 

Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against 

excitatory amino acid-induced neurotoxicity. Neurobiology 95:1852-7. 

Labrie F, Luu-The V, Bélanger A, Lin X-S, Simard J, Pelletier G., 2005. 

Is dehydroepiandrosterone a hormone? Journal of Endocrinology 187:169-96. 

Matsumoto R.R., Bowen W.D., Tomm. A., Vo V.N., Truong D.D., De Costa B.R., 1995. 

Characterization of two novel σ receptor ligands: Antidystonic effects in rats suggest σ receptor 

antagonism. 

European Journal of Pharmacology 280:301-310. 

Mellon S.H. and Griffin L.D., 2002. 

Neurosteroids: biochemistry and clinical significance. Trends Endocrinology Metabolism 13:35-43. 

Weill-Engerer, S., David, J. P., Sazdovitch, V., Liere, P., Eychenne, B., Pianos, A., Schumacher, M., 

Delacourte, A., Baulieu, E. E., Akwa, Y., 2002. 

Neurosteroid quantification in human brain regions: comparison between Alzheimer's and nondemented 

patients. J. Clin. Endocrinol. Metab. 87:5138-43. 

Östergren, A., Svensson, A-L., Lindquist, N. G., & Brittebo, E. B., 2005. 

Dopamine melanin-loaded PC12 cells: a model for studies on pigmented neurons. Pigment Cell 

Research 18:306-314. 

170



PROGESTERONE AND ITS DERIVATIVES AS PROTECTIVE AGENTS IN 

EXPERIMENTAL DIABETIC NEUROPATHY 

S. Giatti #1 , I. Roglio 1 , M. Pesaresi 1 , R. Bianchi 2 , G. Cavaletti 3 , L.M. Garcia-Segura 4 , 

G. Lauria 5 , R.C. Melcangi 1 

1 Dept. of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 

Milano, Italy; 

2 Dept. of Molecular Biochemistry and Pharmacology, "Mario Negri" Institute for 

Pharmacological Research, Milano, Italy; 3 Dept. of Neurosciences and Biomedical Technologies, University 

of Milan"Bicocca", Monza, Italy; 4 Instituto Cajal, C.S.I.C., Madrid, Spain; 5 Neuromuscular Diseases Unit, 

National Neurological Institute “Carlo Besta”, Milano, Italy. 

# Presenting author: Dept. of Endocrinology and Center of Excellence on Neurodegenerative Diseases, 


silvia.giatti@guest.unimi.it 

Deleterious effects of diabetes on the nervous system are responsible for several disorders 

including damage to the peripheral nervous system. However, in spite of the number of 

studies on human and experimental diabetic neuropathy, the current therapeutic arsenal is 

meagre. Consequently, the search for substances to protect the nervous system from the 

degenerative effects of diabetes has high priority in biomedical research. 

Because of the importance of neuroactive steroids in the control of the nervous system 

functions and of their neuroprotective effects in several experimental models of 

neurodegenerative diseases, we have assessed whether chronic treatment with progesterone 

(P), and its derivatives, dihydroprogesterone (DHP) and tetrahydroprogesterone (THP), 

had protective effects against (STZ)-induced peripheral neuropathy at the 

neurophysiological, functional, biochemical and neuropathological levels. Data obtained 

have indicated that chronic treatment for 1 month with P, or with its derivatives, DHP and 

THP, counteracted the impairment of nerve conduction velocity (NCV) and thermal 

threshold, restored skin innervation density, and improved Na + ,K + -ATPase activity and 

mRNA levels of myelin proteins, such as glycoprotein zero and peripheral myelin protein 

22. Moreover, protective effects of these neuroactive steroids in STZ-rat were also evident 

on morphological degeneration of the sciatic nerve. Indeed, treatment with P or DHP 

induced a significant reduction in the number of fibers with myelin infoldings. Altogether 

these observations suggest that these neuroactive steroids might be useful protective agents 

in diabetic neuropathy. Interestingly, different receptors seem to be involved in these 

effects. Thus, while morphological integrity of myelin, the expression of myelin proteins 

and Na + ,K + -ATPase activity are only influenced by P and DHP, (i.e., two neuroactive 

steroids interacting with P receptor, PR), NCV, thermal nociceptive threshold and intraepidermal 

nerve fiber density are also affected by THP, which interacts with GABA-A 

receptor. Because, a therapeutic approach with specific synthetic receptor ligands could 

avoid the typical side effects of steroids, future experiments will be devoid to evaluate the 

role of PR and GABA-A receptor in these protective effects. 


171


AN EVALUATION OF THE EFFECT OF DEXAMETHASONE IN PREVENTING 

TOURNIQUET NEUREPATHY IN RATS 

Jarrahi M, Vafaei AA, Rashidy-Pour A 

Physiology Research center, Semnan University of Medical Sciences, Semnan, Iran 

E-mail: jarrahi44@yahoo.com 

INTRODUCTION 

Paralysis after the use of a tourniquet is a well recognized clinical phenomenon. Although ischemia 

to nerve causes fiber degeneration [1] Inflammation following IR injury has been shown to lead to 

the induction of the ca2+-independent (inducible) form of NOS (iNOS) [2]. Dexamethasone is a 

glucocorticoid that has been shown to inhibit iNOS production by repressing gene expression, 

inhibiting protein synthesis and decreasing iNOS protein stability [3,4]. The present experiment 

was designed to evaluate the effects of dexamethasone during post tourniquet IR injury. 

MATERIAL AND METHODS: 

36 male Wistar rats (200-250 gr) were chosen and divided randomly into 6 groups as control, 

vehicle, tourniquet-vehicle, Dex1, Dex 2 and Dex 3. Tourniquet was applied to the right hind limb 

of all Animals except of control and vehicle groups for 3 hours at the beginning of experiments. 

Animals of tourniquet-vehicle, Dex1, Dex 2 and Dex 3 groups were injected 30 min before 

reperfusion by 1 cc Normal saline containing 4% ethanol, 1mg/kg, 2mg/kg and 3mg/kg 

Dexamethasone dissolved in saline and Alcohol respectively and motor nerve conduction velocity 

(MNCV) was measured one week after releasing the tourniquet. MNCV of tibial nerve in 

response to nerve stimulation were measured at 1 week post-tourniquet release. 

The procedure was done under ketamine-xylocin (90mg/kg-100mg/kg,Ip) anesthesia. Ischemia of 

the lower hind limb was produced by placing a pneumatic tourniquet around the right tigh and 

inflating the cuff to115 to125 mmHg. After one week for evaluation of MNCV the animals were 

acclimated for at leas thirty minutes in a temperature controlled room maintained at 25± 0.5 Cº 

before measurements were reperoformed. The procedure was done under urethane anesthesia (1.5 

g/kg), with insitu preparation in a pool of liquid paraffin previously saturated with saline to avoid 

nerve dehydration. Rectal temperature was monitored throughout anesthesia for this procedure (2 – 

4 min). No hypothermia was observed and all rats remaining between 37 and 38 C. The right 

sciatic nerve was stimulated first at the sciatic notch and then at the Achilles tendon. Stimulation 

comprises single 0.1 ms pulses of amplitude 1– 4 V were delivered via fine bipolar needle 

electrodes. Consequent each stimulation an electromyogram (EMG) was recorded, again using fine 

needle electrodes, from the gastrocinemus muscle via a 250X gain AC preamplifier on a single 

beam storage oscilloscope. The temporal separation of the peaks of the EMGs, was induced by 

stimulation the two sites, was measured using dividers. The mean of six measurements was taken 

on each occasion. Nerve length, separating the two points (sciatic notch and Achilles tendon) was 

measured using vernier caliper. The MNCV was calculated by the difference between the two 

stimulating electrodes. 

RESULTS: 

Application of the tourniquet for 3 h decreased significantly (p



90 

* 

MNCV (M/S) 

60 

30 

* 

0 

Control Vehicle TOUR DEX1 DEX2 DEX3 

Experimental groups 

Fig 1. Effect of Matricaria chamomilla extract dissolved in olive oil with comparison of olive oil 

on the percentage of wound healing in days after beginning of experiments. *P


INVOLVEMENT OF ENDOGENOUS DEHYDROEPIANDROSTERONE IN THE 

MODULATION OF SPINAL NOCICEPTIVE MECHANISMS 

Kibaly C., Meyer L., Patte-Mensah C. and Mensah-Nyagan A.G. 

Institut des Neurosciences Cellulaires et Intégratives, UMR 7168/LC2-CNRS, Université Louis 

Pasteur, Equipe Stéroïdes et Système nociceptif, 21 rue René Descartes, 67084 Strasbourg Cedex, 

France. Fax: +33 388 613 347; e-mail: gmensah@neurochem.u-strasbg.fr 

The excessive advertisement of dehydroepiandrosterone (DHEA) as a miraculous 

anti-aging drug resulted in its widespread self-administration in Europe and the USA 

where DHEA is available without medical prescription. However, basic data supporting 

beneficial effects of DHEA are rare and the risks related to long-term supplementation of 

DHEA are almost completely unknown. Here, we used a multidisciplinary approach to 

show that endogenous DHEA locally synthesized in sensory networks of the spinal cord 

(SC) is a pro-nociceptive molecule the production of which is down-regulated by 

neuropathic rats to cope with their chronic pain state. Real-time polymerase chain reaction 

after reverse transcription revealed a down-regulation of the gene encoding cytochrome 

P450c17 (P450c17), the key DHEA-synthesizing enzyme, in the SC of rats submitted to 

neuropathic pain generated by sciatic nerve ligature. Pulse-chase experiments combined 

with high-performance liquid chromatography and flow scintillation detection showed 

decreased P450c17 enzymatic activity in the SC of neuropathic-pain rats. 

Radioimmunoassays demonstrated that, in vivo, the chronic pain dramatically reduced 

DHEA concentration in the SC. Moreover, in vivo blockade of DHEA production in the 

SC by intrathecal administration of ketoconazole, a pharmacological inhibitor of P450c17, 

induced analgesia in neuropathic-pain rats. Unlike ketoconazole, DHEA potentiated both 

thermal hyperalgesia and mechanical allodynia characterizing the neuropathic pain state. 

The results draw attention on the potential risks linked to abusive use of DHEA, 

particularly in victims of neuropathic pain. Perspectives are opened for analgesic strategies 

based on the selective modulation of DHEA or neurosteroid biosynthetic pathways in 

neural centers controlling pain sensation. 

174



POSTNATAL CHANGES IN THE EXPRESSION OF AQUAPORIN 1 AND EFFECT 

OF ESTROGEN ON THE EXPRESSION IN OVARIECTOMIZED MOUSE BRAIN 

Lee J.E., Kim H.J., Kang H.S., Ahn H.S., and Gye M.C.* 

Department of Life Science, Hanyang University, Seoul 133-791, Korea 

Fax: +82-2-2298-8646 

e-mail: mcgye@hanyang.ac.kr 

1. Introduction 

Brain aquaporins (AQP) play important roles in the dynamic regulation of brain water 

homeostasis and the production of cerebrospinal fluid (CSF) under normal, as well as 

pathological, conditions. In females, endogenous estrogen can affect cognitive functioning 

during the menstrual cycle [1], preserve certain aspects of cognitive function in 

postmenopausal women [2], act as a neuroprotective and neuroregenerative agent in stroke 

and traumatic brain injuries, and reduce the risk of developing Alzheimer's disease [3]. To 

elucidate the role estrogen in the maintenance of brain water homeostasis, changes in the 

expression of AQP1, an important structural element of choroid plexus following ovariectomy 

(OVX), as well as the effect of estrogen (17ß estradiol) on the expression of AQP1 in OVX 

brain, was examined in mice. 

2. Materials and Methods 

Normal cyclic female mice were subjected to oveaiectomy (OVX) under ketamine anesthesia 

(100 mg/kg); a sham operation was performed as a control. After recovery, to allow clearance 

of circulating estrogen following OVX or sham, mice were rested for two weeks, and then 

subjected to estrogen treatment. Additionally, some OVX mice received intraperitoneal 

injections of 17ß estradiol (E 2 ) at 20 µg/head (single injection) or 1 µg/head (for 7 days) 

dissolved in sesame oil (4 heads for each treatment). As a control, sesame oil was injected as a 

vehicleOVX mice were sacrificed 24 h after final dosing of E2. Sham operated mice were 

sacrificed at the estrous stage. Hippocampus were dissected and subjected to protein and PCR 

analysis. Primers for AQP1 were designated 5’-AGTATGACCTGG ATGCTGAC-3’ 

(forward) and 5'-ACTCCTCCATGATGTCAAAG-3' (reverse) according to the mouse AQP1 

cDNA sequence (GenBank Acc, NM_007472, product size: 360bp). To confirm the estrogen 

response in brain expression of transthyretin (TTR) synthesized in choroids plexus was 

verified (Tang et al., 2004). Primers for TTR were 5’-aga cgt ggc tgt aaa agt gt-3’ (forward) 

and 5'-ctg tag gag tat ggg ctg ag-3' (reverse) according to the mouse TTR cDNA sequence 

(GenBank Acc, NM_013697, product size: 273bp). GAPDH mRNA was amplified as an 

internal control. The primers for GAPDH were 5'-AGTGGAGATTGTTGCCATCAACGAC- 

3' (forward) and 5'-GGGAGTTGCTGTTGAAGTCGCAGGA-3' (reverse) (GenBank Acc, 

NM001001303, product size: 360bp). PCR products were analyzed on 2% agarose gels 

containing ethidium bromide. The relative abundance of AQP1 mRNA versus GAPDH was 

determined by performing densitometric analysis of the amplicons. For immunohistochemical 

analysis of AQP1, cryocuts (5 µm) of brain at estrous stage were made and processed for 

immunohistochemical analysis using AQP1 antibody (rabbit polyclonal, SantaCruz, CA, 

1:200 dilution) and HRP conjugated secondary antibody. As a negative control, primary 

antibody was omitted. Following counterstaining with Mayer hematoxylin, slides were 

mounted permanently. Observation and photography were performed with a microscope 

equipped digital camera (DFC320, Leica, Germany). 

175


3. Results 

Throughout brain development, the immunoreactivity of AQP1 was found in the choroid 

plexus. Ependyma, pia, and veins were also positive for AQP1 immunoreactivity. AQP1 

mRNA level showed gradual decrease until postnatal day 8 but reincreased at adulthood. Two 

forms of AQP1 polypeptides with Mr. 35 and 28 kDa in brain. Both forms were much higher 

in the fetal brain but only 35kDa form was detected in adult brain. AQP1 was significantly 

down regulated together with transthyretin in OVX brain compared with the sham control at 

the estrous stage. In OVX female, repeated dosage with E 2 (1 or 10 µg/head) for 7 days 

significantly augmented AQP1 mRNA level together with TTR mRNA in OVX female 

brains; however, single high dose of E 2 (20 µg/head) resulted in no significant changes in 

AQP1 expression in OVX brains. 

4. Conclusion 

Together, these results suggest that expression of AQP1 in female brain tissue is tightly 

regulated by estrogen. Alteration of AQP1 expression in brain should be considered as a 

likely candidate mechanism for the pathological changes in the CNS in estrogen deficiency. 


[1] E. Hogervorst, J. Williams, M. Budge, W. Riedel, and J. Jolles, The nature of the effect of 

female gonadal hormone replacement therapy on cognitive function in postmenopausal 

women: a meta-analysis, Neuroscience 101, (2000), pp. 485-512. 

[2] M.H. Birkhauser, J. Strnad, C. Kampf and M. Bahro, Oestrogens and Alzheimer's disease. 

Int. J. Geriatr. Psychiatry 15 (2000), pp. 600-609. 

[3] J.I. Koenig, Estrogen and brain function. Trends Endocrinol. Metab. 12 (2001), pp. 4-6. 

176



TESTOSTERONE DERIVATIVES ARE NEUROPROTECTIVE AGENTS IN 

EXPERIMENTAL DIABETIC NEUROPATHY 

I. Roglio #1 , S. Giatti 1 , M. Pesaresi 1 , R. Bianchi 2 , G. Cavaletti 3 , D. Caruso 4 , S, Scurati 4 , 

L.M. Garcia-Segura 5 , G. Lauria 6 , R.C. Melcangi 1 

1 Dept. of Endocrinology and Center of Excellence on Neurodegenerative Diseases, University of Milan, 

Milano, Italy; 

2 Dept. of Molecular Biochemistry and Pharmacology, "Mario Negri" Institute for 

Pharmacological Research, Milano, Italy; 3 Dept. of Neurosciences and Biomedical Technologies, University 

of Milan"Bicocca", Monza, Italy; 4 Dept. of Pharmacological Sciences, University of Milan, Milano, Italy; 

5 Instituto Cajal, C.S.I.C., Madrid, Spain; 6 Neuromuscular Diseases Unit, National Neurological Institute 

“Carlo Besta”, Milano, Italy. 

# Presenting author: Dept. of Endocrinology and Center of Excellence on Neurodegenerative Diseases, 


ilaria.roglio@unimi.it 

Our recent studies have shown that progesterone and its metabolites exert important 

protective effects on peripheral nerves against pathological alterations induced by diabetes. 

In the present study we have assessed whether other members of the neuroactive steroid 

family, such as testosterone (T) and its derivatives, dihydrotestosterone (DHT) and 5alpha 

-androstan-3alpha, 17beta-diol (3alpha -diol) also exert protection against diabetic 

neuropathy. Diabetes was induced in adult male rats by the injection of streptozotocin. 

After 3 months of diabetes, T plasma levels, evaluated by liquid chromatography/mass 

spectrometry, showed a decrease associated to an increase of 3alpha-diol levels. In 

contrast, in sciatic nerve of diabetic rats low levels of DHT were observed and the 

expression of the enzyme converting T into DHT (i.e., the 5alpha-reductase), evaluated by 

real time PCR, was also reduced. Chronic treatment for 1 month with DHT or 3alpha-diol 

increased tail nerve conduction velocity and partially counteracted the increase of thermal 

threshold induced by diabetes. Treatment with DHT increased tibial Na + ,K + -ATPase 

activity and the gene expression of myelin protein P0 (i.e., a protein which plays a crucial 

role in maintaining the multilamellar structure of peripheral nerve myelin). Not only DHT 

and 3alpha-diol, but also T treatment, were able to totally restore the reduction of 

intraepidermal nerve fiber density induced by diabetes. Altogether these observations 

indicate that T derivatives can reverse behavioral, neurophysiological, morphological and 

biochemical alterations induced by peripheral diabetic neuropathy. These findings further 

strengthen the evidence that different members of the neuroactive steroid family exert 

relevant protective effects at the level of peripheral nerves 


177


ESTROGEN AND ANDROGENS REGULATE ALZHEIMER-LIKE 

NEUROPATHOLOGY IN MALE 3XTG-AD MICE 

Rosario E.R., Carroll J.C., and Pike C.J. 

Davis School of Gerontology, University of Southern California 3715 McClintock Avenue 

Los Angeles, CA 90089-0191 USA Tel: 213-740-4205 Fax: 213-740-4787 

Email: cjpike@usc.edu 

Normal, age-related, androgen depletion in men is a recently identified risk factor 

for the development of Alzheimer’s disease (AD). Recently, using a triple-transgenic 

mouse model of AD (3xTg-AD), we found that androgen depletion in males results in 

robust increases in accumulation of β-amyloid (Aβ), the protein implicated as the primary 

causal factor in AD pathogenesis. Androgen treatment, in the form of the nonaromatizable 

androgen dihydrotestosterone (DHT), prevented increased neural 

accumulation of Aβ; however, the mechanism of androgen regulation of Aβ remains 

unclear. Although several androgen actions in the brain are mediated through androgen 

pathways involving androgen receptors (AR), androgen actions may also result from 

estrogen-mediated pathways. To examine whether androgen actions on Aβ accumulation 

involve estrogen and / or androgen pathways we examined the effects of testosterone (T), 

dihydrotestosterone (DHT), and 17β-estradiol (E2) in androgen depleted male mice. Male 

3xTg-AD mice were gonadectomized (GDX) to deplete endogenous levels of androgens at 

3 months of age, and exposed to subcutaneous, slow-release drug delivery pellets 

containing either vehicle, 10 mg DHT, 10mg T, 0.025mg E2, or 0.01mg E2. After 4 

months of hormone treatment (7 months of age), mice were evaluated for severity of ADlike 

neuropathology. Similar to our previous results, we observed a significant increase in 

Aβ pathology in GDX mice in the subiculum and CA1 of hippocampus and amygdala, an 

effect prevented by DHT treatment. Treatment with testosterone also prevented increased 

accumulation of Aβ pathology in both hippocampus and amygdala. Interestingly, estrogen 

prevented Aβ accumulation in the subiculum and CA1 of hippocampus but had no effect in 

amygdala. These findings suggest that androgen regulation of Aβ is mediated through 

both androgen and estrogen pathways depending on the brain region. 

Supported by the Alzheimer’s Association (IIRG-04-1274; CJP) and NIH grants AG23739 

(CJP), NS52143 (ERR), AG00093 (JCC). 

178



EFFECTS OF BETA AMYLOID PEPTIDE 1-42 AND OXIDATIVE STRESS ON 

NEUROSTEROID FORMATION IN HUMAN NEUROBLASTOMA CELLS 

Schaeffer V. + , Patte-Mensah C. + , Eckert A.°, Mensah-Nyagan A.G. +# 

+ 

Institut des Neurosciences Cellulaires et Intégratives, Equipe Stéroïdes et Système 

Nociceptif, UMR 7168/LC2, CNRS, Université Louis Pasteur, 21 rue René Descartes, 

67 084 Strasbourg cedex, France. Fax: +33 (0)3 88 61 33 47 

# 

e-mail: 

gmensah@neurochem.u-strasbg.fr. 

° Neurobiology Research Laboratory, Psychiatric University Clinic, Wilhelm Klein-Strasse 

27, CH-4025 Basel, Switzerland. 

Pharmacological and behavioral studies performed in animals suggest neurosteroid 

involvement in neuroprotection. However, the contribution of neurosteroidogenesis in the 

protection against degenerative processes in humans remains to be determined. In a recent 

work, we showed that the overexpression of key Alzheimer’s disease (AD) proteins (native 

tau, mutant P301L tau and amyloid peptide precursor) interferes with the process of 

neurosteroidogenesis in human neuroblastoma SH-SY5Y cells. In addition, we observed 

that extracellular treatment of SH-SY5Y cells with aggregated synthetic Beta-amyloid 

fragment 25-35 (AB 25-35 ) stimulated progesterone synthesis and had no effect on estradiol 

synthesis. Because the activity of AB 25-35 can be different from that of the full-length AB 1- 

42 which is the real pathogenic peptide involved in AD, we have now combined pulsechase 

experiments with HPLC and continuous flow scintillation detection to study the 

effect of AB 1-42 on neurosteroid production in SH-SY5Y cells. AB 1-42 mimicked the action 

of AB 25-35 on progesterone formation. In contrast, the biosynthesis of estradiol, which was 

totally unaffected by AB 25-35, was stimulated in SH-SY5Y cells by AB 1-42 . In addition to 

the comparative analysis of the effects of AB 1-42 and AB 25-35 , we have also investigated the 

action of oxidative stress (another pathological factor leading to AD) on neurosteroid 

synthesis in human neuroblastoma cells. A significant SH-SY5Y cell death was observed 

24h and 48h after treatment with the oxidative stressor H 2 O 2 . A decreased estradiol 

production was detected in SH-SY5Y cells 12h, 24h and 48h after treatment with H 2 O 2 . 

Our results show that the cellular components responsible for endogenous formation of 

estradiol in human neuroblastoma cells are selectively modified by various factors 

involved in the etiology of AD. Consequently, it appears that molecular and cellular 

elements contributing to estradiol production in nerve cells may potentially be interesting 

to decipher functional interactions between the process of neurosteroidogenesis and 

neurodegenerative or neuroprotective pathways. Elucidation of such interactions will 

certainly open new perspectives for the development of efficient therapies against 

neurodegenerative disorders. 

179


EFFECT OF ESTROGEN TREATMENT ON PROTEIN EXPRESSION PATTERN IN 

FAMALE MICE BRAIN USING FLUORESCENT DIFFERENTIAL 2-D GEL 

ELECTROPHORESIS (DIGE) 

Szegő É.M., Kékesi K.A., Juhász G., Ábrahám I.M. 

Research Group of Neurobiology at Eötvös Loránd University - Hungarian Academy of Sciences, Pázmány 

P. Stny 1/c, Budapest, Hungary 

e-mail: eva.szego@freemail.hu 

Steroid hormons can alter neuronal functions such as learning, behavior and they can 

effect the cell-viability as well. The main female gonadal hormone estrogen (E2) could 

affect neurons on two different ways: via direct DNA-binding and transcriptional activity 

of liganded ERs (classical effect); or via intracellular signaling system (non-classical 

effect) such as protein kinase A, calcium-calmodulin dependent protein kinase IV leading 

to activation of many transcription factors and consequent changes in gene transcription 

and protein expression in the brain. In this study we characterized the impact of estrogen 

on the brain proteome using proteomic approaches. Differential two-dimensional gel 

electrophoresis (DIGE) is one of the key tools for comparative proteomic research. This 

method enable us to separate complex protein mixtures with high resolution, DIGE is a 

technique commonly employed for protein profiling studies. In our experiemnts total 

protein was extracted from the brain of ovariectomized mice at 24 hrs following E2 or 

vehicle injection. Equal amounts of protein from individual animals were homogenized, 

labeled with Cy3 (absorbtion max: 553 nm, emission max: 572 nm) or Cy5 (A max: 648 

nm, E max: 669 nm) and were focused isoelectrically and run on the same analytical gel. A 

pool composed of equal aliquot of each sample in the study was labeled with Cy2 dye (A 

max: 491 nm, E max: 506 nm) and loaded on each gel as a between-gel reference to 

minimize the gel-to-gel variation. 800 microgram protein was loaded on preparative gels 

for MS analysis. Gels were scanned with Typhoon Trio+ confocal laser scanner and 

analyzed using DeCyder (spot detection, noise filtering) and BVA (gel matching, statistica) 

softwares (GE Healthcare). We identified approximately 3000 protein spots in our 2D-gels. 

After trypsin-digestion, proteins were analysed with ESI LC MS/MS method. Estradiol 

altered the abundance of 76 spots (p≤0.05), and 48 of these proteins were identified with 

MS. Proteins can be clustered into 7 groups according to their biological functions. Our 

result suggest that estrogen may alter the rate of protein synthesis in the brain. Following 

estrogen treatment the amount of enzymes of the TCA cycle and glycolisis decreased 

(cytrate synthase; glycerol phosphate dehydrogenase, phosphoglycerate kinase 1 etc), still 

the quantity of proteasomal subunit, calpain, aspartyl aminopeptidase, 

phosphoglucomutase increased. Moreover, the EGFR-binding protein, peroxiredoxin, 

glutathione-S-transferase also increased. The enhanced protein turnover and increased 

antioxidant mechanisms provide a new aspect to the known neuroprotective mechanism of 

estrogen in the brain. 

Using bioinformatics we also studied the promoter regions of the corresponding genes, and 

only three of them contain ERE, but all have at least 7 half EREs. Based on these 

experiments, estrogen may effect the protein expression pattern acting on half ERE and/or 

AP-1 sites rather than ERE. These data may reformulate our view about the function of 

classical estrogen effects and estrogen receptor–ERE interaction. Our results also indicate 

that we can sucsessfully utilize the DIGE methodology to evaluate the effect of estrogen 

and ovariectomy on the brain proteome. We suppose that the enhanced rate of protein 

turnover and antioxidant mechanisms can be an important aspect of estrogen induced 

neuroprotection. 

Research supported by: RET, MEDICHEM, OTKA, ETT 

180



SELECTIVE ESTROGEN RECEPTOR MODULATORS DECREASE 

MICROGLIA ACTIVATION IN THE CEREBELLUM OF MALE RATS 

Tapia González S.*, Diz-Chaves Y., Pernía O., Carrero P., Garcia-Segura L.M. 

Instituto Cajal, C.S.I.C., Avenida Doctor Arce, 37, E-28002, Madrid, Spain. 

*E-mail: stapia75@cajal.csic.es FAX: +34-915854754 

Estradiol prevents neuronal loss in diverse experimental models of 

neurodegenerative diseases and enhances cognitive skills in animals and humans. Antiinflammatory 

actions of the hormone may represent an important component of its 

neuroprotective effects and previous studies have shown that estrogen therapy reduces the 

activation of microglia after acute brain lesions in female rodents [3,4]. Selective estrogen 

receptor modulators (SERMs) may represent an alternative to estrogen therapy for the 

treatment or the prevention of neurodegenerative disorders in humans. In the present study 

we have assessed the neuroprotective potency of several SERMs in male rats injected with 

lipopolysaccharide (LPS). Adult (3 months old) Wistar male rats were given two 

intraperitoneal injections of LPS, separated by an interval of 3 days [2]. One hour before 

the administration of LPS the animals were injected with vehicle or estrogenic compounds. 

Animals were killed 7 days after the first injection of LPS and a morphometric analysis of 

OX42 and major histocompatibility complex class II (MHCII) immunoreactive microglia 

in the central white matter of the cerebellum was performed using the optical disector 

method. OX42 was used as a marker of total microglia and MHC-II as a marker of reactive 

microglia. The estrogenic compounds assessed were: 17 beta-estradiol (50, 250, 500 and 

700 micrograms/Kg bw), tamoxifen (0.6, 1 and 2 mg/Kg bw), raloxifene (1 mg/Kg bw), 

lasofoxifene (1 mg/Kg bw) and bazedoxifene (2 mg/Kg bw). The selected doses of 

estrogenic compounds were based on previous studies that analyzed their neuroprotective 

properties [1]. LPS induced a significant increase in the number of MHC-II 

immunoreactive cells in the white matter of the cerebellum. The estrogenic compounds did 

not affect the basal number of MHC-II immunoreactive cells. However, estradiol, 

tamoxifen, raloxifene and bazedoxifene significantly reduced the number of MHC-II 

immunoreactive cells in LPS injected animals. The number of OX42 immunoreactive cells 

was neither affected by LPS administration nor by treatment with estrogenic compounds, 

suggesting that changes in MHC-II reflect differences in microglia activation and not 

differences in proliferation or survival. In summary, our data suggest that some SERMs 

may exert anti-inflammatory effects in the brain, reducing reactive microglia. 


[1] I. Ciriza, P. Carrero, I. Azcoitia, S.G. Lundeen and L.M. Garcia-Segura, Selective estrogen receptor 

modulators protect hippocampal neurons from kainic acid excitotoxicity: differences with the effect of 

estradiol, J Neurobiol 61 (2004) 209-221. 

[2] Y.K. Ng and E.A. Ling, Induction of major histocompatibility class II antigen on microglial cells in 

postnatal and adult rats following intraperitoneal injections of lipopolysaccharide, Neurosci Res 28 

(1997) 111-118. 

[3] E. Vegeto, S. Belcredito, S. Etteri, S. Ghisletti, A. Brusadelli, C. Meda, A. Krust, S. Dupont, P. Ciana, P. 

Chambon and A. Maggi, Estrogen receptor-alpha mediates the brain antiinflammatory activity of 

estradiol, Proc Natl Acad Sci U S A 100 (2003) 9614-9619. 

[4] E. Vegeto, S. Belcredito, S. Ghisletti, C. Meda, S. Etteri and A. Maggi, The endogenous estrogen status 

regulates microglia reactivity in animal models of neuroinflammation, Endocrinology 147 (2006) 2263- 

2272. 

181


ALLOPREGNANOLONE (ALLO) EXERTS A PROTECTIVE EFFECT AGAINST 

OXIDATIVE STRESS IN NIEMANN PICK C CELLS 

Zampieri S * , Mellon SH # , Pittis MG * , Nevyjel M*, Bembi B*, Dardis A * . 

* Unita di Malattie Metaboliche, IRCCS Burlo Garofolo, Via dell’Istria 65/1, 34137, 

Trieste, Italy. FAX 390403785500, email: andrea.dardis@area.trieste.it 

# Department of Obstetrics, Gynecology and Reproductive Sciences, The Center for 

Reproductive Sciences, University of California, San Francisco, USA. 

Niemann Pick C disease (NPC) is an autosomal recessive neurodegenerative disorder 

caused by different mutations in the NPC1 (95% of cases) and NPC2 (5% of cases) genes. 

The abnormal function of either of these proteins leads to an accumulation of unesterified 

cholesterol and other lipids, such as sphingolipids and glycosphingolipids (GSLs) in the 

lysosomal compartment of the cell [1]. The molecular mechanisms underlying the 

pathophysiology in NPC disease are not clear. However, oxidative damage has been 

implicated in the pathophysiology of different neurological disorders and 

neurodegenerative diseases [2], and the effect of GSL accumulation on the intracellular 

redox state has been well documented [3], suggesting a possible role of oxidative stress in 

the pathophysiology of this disease. The synthesis of neurosteroids is altered in a time- and 

region-specific fashion in the BALB\c Niemann Pick type C mouse, and neurons and 

neuroglia expressing the steroidogenic enzymes are lost in the NPC mouse. In particular, 

the synthesis of ALLO is substantially diminished at birth, and decreases further over time. 

These data suggest a pivotal role of neurosteroidogenesis abnormalities in the phenotypic 

expression of the disease. In addition, the treatment of NP-C mice with ALLO increased 

their lifespan and delayed the onset of neurological impairment [4]. However, the 

molecular mechanism by which ALLO exerts these neuroprotective effects is not fully 

understood.. 

In this study we determined whether the intracellular redox state might contribute to the 

pathophysiology of NPC disease, and analyzed the possible effects of (ALLO) on the 

oxidative damage in human NPC cells. 

We first analyzed the levels of reactive oxygen species (ROS) in cultured fibroblasts from 

NPC patients and healthy controls. ROS levels were approximately three times higher in 

NPC than in normal fibroblasts (p0.01). Higher 

concentration did not show a further benefitial effect. In addition, pretreatment of cells for 

24 h with 50 nM ALLO almost complete reverted peroxide-induced apoptosis (p



due, at least in part, to its antioxidant properties. In addition these results demonstrated that 

the effects of ALLO are quite pleiotropic and that probably other brain diseases, including, 

but not limited to congenital storage diseases, may benefit from similar treatments with 

neuro-active steroids. 


[1] Patterson, M.C., Vanier, M.T., Suzuki, K., Morris, J.A., Carstea, E.D., Neufeld, E..B., Blanchette- 

Mackie, E.J. and Pentchev, P.G., 2001, Niemann–Pick disease type C: a lipid trafficking disorder. In: 

C.R. Scriver, A.L. Beaudet, W.S. Sly, D. Valle, B. Childs, K.W. Kinzler and B. Vogelstein, Editors 

(eighth ed.), The Metabolic and Molecular Bases of Inherited Disease, McGraw Hill, New York, pp. 

3611–3634. 

[2] Culmsee, C., Landshamer, S., 2006. Molecular insights into mechanisms of the cell death program: role 

in the progression of neurodegenerative disorders. Curr Alzheimer Res. 3, 269-83 

[3] Garcia-Ruiz, C., Colell, A., Paris, R., and Fernandez-Checa J.C., 2000. Direct interaction of GD3 

ganglioside with mitochondria generates reactive oxygen species followed by mitochondrial permeability 

transition, cytochrome c release, and caspase activation. FASEB J. 14,847-858. 

[4] Griffin, L.D., Gong, W., Verot, L., Mellon, S.H., 2004. Niemann-Pick type C disease involves disrupted 

neurosteroidogenesis and responds to allopregnanolone. Nat Med. 10, 704-11. 

183


Xenoestrogens and brain circuitries 

• Martini M., Miceli D., Palanza P., Viglietti-Panzica C., Panzica G.C. (Italy) 

Effects of Bisphenol A on the hypothalamic nitrinergic system of CD1 mouse 

• Sica M., Håkansson H., Halldin K. (Sweden) Effects on estrogenic activity in 

pituitary gland and hypothalamus of male ERE reporter mice, after single oral 

TCDD exposure



EFFECTS OF BISPHENOL A ON THE HYPOTHALAMIC NITRINERGIC 

SYSTEM OF CD1 MOUSE 

Martini M.*, Miceli D.*, Palanza P.°, Viglietti-Panzica C.*, Panzica G.C.* 

*Laboratory of Neuroendocrinology, Dept Anatomy, Pharmacology and Forensic 

Medicine, C.so M. D’Azeglio, 52. 10126 Torino (Italy) 

e-mail: mariaangela.martini@unito.it 

°Dept Evolutive And Functional Biology, Parma (Italy) 

Bisphenol A (BPA) is a well-known pollutant derived from plastic used for aliments and is 

characterized by the ability of binding to estrogen receptors [1,8]. It is therefore considered 

an endocrine disrupting chemical of the category of xenoestrogens [3]. Several studies 

have been performed to elucidate its toxicological properties and its impact on different 

behaviors in rodents [4,9,10]. However, studies on the effects of BPA on neural circuits are 

at the moment limited to the catecholaminergic system [2,5-7]. In the present study we 

have investigated the effects of early exposure to BPA on the nitrinergic system of adult 

mice of both sexes. 

Pregnant mice (11th gestational day) were daily treated with different doses of BPA up to 

8 days after delivery. In this way, the pups of both sexes were exposed for 10 prenatal and 

8 postnatal days at BPA. Mice treated in this way were sacrificed at the age of 2 months by 

intracardiac perfusion and brains were dissected, frozen and sectioned. Serial sections 

taken each 100 µm were then treated for the immunohistochemical detection of nNOS 

(antibody from Diasorin, at a dilution of 1:12,000). Quantitative analysis of the number of 

nNOS-ir cell bodies was performed for 6 nuclei (medial preoptic nucleus, POM; nucleus of 

the stria terminalis, BST; paraventricular nucleus; ventromedial nucleus; arcuate nucleus, 

and caudate-putamen). Significant effects of BPA treatments were observed only in the 

POM and in the ventral subdivision of BST (BSTv), in a sex-oriented way. In the POM, 

where the nNOS-ir population is sexually dimorphic in controls, the higher doses of BPA 

(40 µg/kg/day) induced a decrease in the cell number in males, whereas the intermediate 

doses (20 µg/kg/day) induced an increase in the females. In the BSTv, where no sex 

dimorphism was observed in controls, the intermediate doses of BPA induced a significant 

decrease of the cell number only in males. 

These results indicate that BPA has a powerful effect on specific portions of the nNOS-ir 

system (i.e. POM and BSTv) that are particularly important for the control of male sexual 

behavior. In addition, they confirm that precocious exposure to xenoestrogens, in particular 

to BPA, may have a high impact on the organization of specific neural pathways that can 

later affect complex behaviors or functions as those related to reproduction. 


[1] Brotons, J.A., Olea-Serrano, M.F., Villalobos, M., Pedraza, V. and Olea, N., Xenoestrogens released 

from lacquer coatings in food cans, Environ Health Perspect, 103 (1995) 608-12. 

[2] Chu, H.P. and Etgen, A.M., A Potential Role of Cyclic GMP in the Regulation of Lordosis Behavior 

of Female Rats, Hormones and Behavior, 32 (1997) 125–132. 

[3] Fujimoto, T., Kubo, K. and Aou, S., Prenatal exposure to bisphenol A impairs sexual differentiation 

of exploratory behavior and increases depression-like behavior in rats, Brain Res, 1068 (2006) 49- 

55. 

[4] Henley, D.V. and Korach, K.S., Endocrine-disrupting chemicals use distinct mechanisms of action 

to modulate endocrine system function, Endocrinology, 147 (2006) S25-32. 

[5] Hull, E.M., Du, J., Lorrain, D.S. and Matuszewich, L., Testosterone, preoptic dopamine, and 

copulation in male rats. In: Hormones, Brain, and Behavior (G.C.Panzica and J.Balthazart eds), 

Brain Research Bullettin, 44 (1997) 327-333. 

185


[6] Hull, E.M., Lorrain, D.S., Du, J., Matuszewich, L., Lumley, L.A., Putnam, S.K. and Moses, J., 

Hormone-neurotransmitter interactions in the control of sexual behavior, Behavioural Brain 

Research, 105 (1999) 105-116. 

[7] Hull, K.L. and Harvey, S., GH as a co-gonadotropin: the relevance of correlative changes in GH 

secretion and reproductive state, Journal of Endocrinology, 172 (2002) 1-19. 

[8] Olea, N., Pulgar, R., Perez, P., Olea-Serrano, F., Rivas, A., Novillo-Fertrell, A., Pedraza, V., Soto, 

A.M. and Sonnenschein, C., Estrogenicity of resin-based composites and sealants used in dentistry, 

Environ Health Perspect, 104 (1996) 298-305. 

[9] vom Saal, F.S., Timms, B.G., Montano, M.M., Palanza, P., Thayer, K.A., S.C., N., Dhar, M.D., 

Parmigiani, S. and Welshons, W.V., Prostate enlargement in mice due to fetal exposure to low doses 

of estradiol or diethylstilbestrol and opposite effects at high doses, Proceedings of the National 

Academy of Sciences of the United States of America, 94 (1997) 2056–2061. 

[10] Welshons, W.V., Thayer, K.A., Judy, B.M., Taylor, J.A., Curran, E.M. and vom Saal, F.S., Large 

effects from small exposures. I. Mechanisms for endocrine-disrupting chemicals with estrogenic 

activity, Environ Health Perspect, 111 (2003) 994-1006. 

186



EFFECTS ON ESTROGENIC ACTIVITY IN PITUITARY GLAND AND 

HYPOTHALAMUS OF MALE ERE REPORTER MICE, AFTER SINGLE ORAL 

TCDD EXPOSURE 

Sica M., Håkansson H., Halldin K. 

Institute of Environmental Medicine, Karolinska Institute, nobels väg 13, 171 77 Stockholm, 

Sweden 

Many chemicals are known to interfere with endocrine system. 2,3,7,8-tetrachlorodibenzo-pdioxin 

(TCDD) is a potent endocrine disrupter that provokes disturbances in male [1,2,3,4] 

and female reproduction [5,6], and in the central nervous system (CNS). 

Neurodevelopmental delays and neurobehavioral effects on children [6, 7], impairment of 

seratonine metabolism [8] and alteration in brain sexual dimorphism [9, 10], have been 

observed. 

The toxic effects of TCDD are due to binding to a soluble, ligand –activated transcription 

factor (AhR). During the last few years, there has been growing evidence for molecular 

interactions between estrogen receptor (ER) and AhR signaling [11, 12]. 

The present study was designed to investigate rapid effects of TCDD on estrogen signaling 

in the pituitary gland and hypothalamus, with major focus on nuclei that control reproductive 

behavior. The main objective of our studies is to clarify whether disturbed estrogen 

signaling is involved in the observed effects of dioxin-like contaminants on reproductive 

function. 

For this purpose an in vivo model that detects activation of estrogen receptors (ERs) was 

used. 66 transgenic mice carrying a luciferase reporter gene under control of three ERE 

sequence (3X ERE reporter mice; 13) were dosed with 200µg/kg bw of TCDD or vehicle 

(corn oil) and sacrificed at several time points (6h, 24h, 3 days, 7 days, 14 days) after 

exposure. Brains and pituitary were removed from the skull and the pituitary were snap 

frozen in liquid nitrogen and used for luciferase assay. Brains were immersed in acrolein 

10%, frozen in isopentane, sectioned at cryostat and processed for anti-luciferase 

immunohistochemistry. 

Luciferase activity in brains has been determined by means anti-luciferase 

immunohistochemistry, in order to give a more detailed picture of the rapid effects of TCDD 

on estrogenic activity in the different nuclei of male mouse hypothalamus. 

Luciferase assay performed on pituitary did not show any clear change regarding luciferase 

activity in all the time point analyzed. 

Preliminary results on antiluciferase immunostaining show a wide distribution of luciferase 

immunoreactive cells on hypothalamus and limbic system. Luciferase immunoreactive 

elements was observed in lateral septum, medial preoptic area, amygdala, ventromedial 

nucleus and arcuate nucleus. The distribution of luciferase immunoreactive distribution is 

comparable to estrogen receptors alpha and beta distribution. 

At 7 days after TCDD exposure an increase in the number of luciferase immunoreactive cells 

was noticed in the medial preoptic nucleus and currently other time points and other nuclei 

of hypothalamus are under investigation. 

Our data show that TCDD may interfere on estrogen receptor signaling in a tissue-specific 

manner. 

187


Acknowledgments The authors wish to thank, Louise Lyrenäs, Christina Trossvik, Daniel Borg, 

Maria Herlin for excellent technical support. This work is supported from the EU-research 

projects BoneTox (QLRT-2002-02528) and CASCADE (FOOD-CT-2004-506319). Monica Sica is 

recipient of a grant from Blanceflor Boncompagni-Ludovisi nee Bildt Foundation. 


[1] Sommer R.J., Ippolito D.L., Peterson R.E., 1996. In utero and lactational exposure of the male 

Holtzman rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: decreased epididymal and ejaculated sperm 

numbers without alterations in sperm transit rate. Toxicol Appl Pharmacol. 140,146-539. 

[2] Bjerke D.L., Peterson R.E. 1994. Reproductive toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin 

inmale rats: different effects of in utero versus lactational exposure. Toxicology and Applied 

Pharmacology 127,241-9. 

[3] Kakeyama M., Sone H., Miyabara Y., Tohyama C., 2003. Perinatal exposure to 2,3,7,8,- 

tetrachlorodibenzo-p-dioxin alters activity-dependent expression of BDNF mRNA in the neocortex 

and male rat sexual behavior in adulthood. Neurotoxicology 24, 207-217. 

[4] Ikeda M., Tamura M., Yamashita J., Suzuki C., Tomita T.,2005. Repeated in utero and lactational 

2,3,7,8-tetrachlorodibenzo-p-dioxin exposure affects male gonads in offspring, leading to sex ratio 

changes in F2 progeny. Toxicology and Applied Pharmacology 133, 285-294 

[5] Gray L.E and Ostby J.S., 1995. In utero 2,3,7,8-Tetraclhorodibenzo-p-dioxin (TCDD) alters 

reproductive morphology and function in female rat offspring. Toxicology and Applied 

Pharmacology 133,285-294. 

[6] Vorderstrasse B.A., Fenton S.E., Bohn A.A., Cundiff J.A., Lawrence B.P., 2004. A novel effect of 

dioxin: exposure during pregnancy severely impairs mammary gland differentiation. Toxicology 

Science. 78, 248-57 

[7] Patandin S., Lanting C.I., Mulder P.G., Boersma E.R., Sauer P.J., Weisglas-Kuperus N., 1999. 

Effects of environmental exposure to polychlorinated biphenyls and dioxins on cognitive abilities in 

Dutch children at 42 months of age. J Pediatr. 134,33-41. 

[8] Kuchiiwa S., Cheng S.B., Nagatomo I., Akasaki Y., Uchida M., Tominaga M., Hashiguchi W., 

Kuchiiwa T., 2002. In utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin 

decreases serotonin immunoreactive neurons in raphe nuclei of male mouse offspring. Neurosci 

Lett. 317, 73-6 

[9] Zareba G., Hojo R., Zareba K.M., Watanabe C., Markowski V.P., Baggs R.B, Weiss B., 2002. 

Sexually dimorphic alterations of brain cortical dominance in rats prenatally exposed to TCDD. 

Journal of Applied Toxicology 129-37 

[10] Hojo R, Stern S., Zareba G, Markowski VP, Cox C., Weiss B. 2002. Sexually dimorphic behavioral 

response to prenatal dioxin exposure. Environmental Health Perspective 247-254. 

[11] Brunnberg S., Pettersson K., Rydin E., Matthews J., Hanberg A., Pongratz I.,2003. The basic helixloop-helix-PAS 

protein ARNT functions as a potent coactivator of estrogen receptor-dependent 

transcription. Proc Natl Acad Sci U S A. 27, 6517-22. 

[12] Chaffin C.L., Trewin A.L., Hutz R.J. 2000. Estrous cycle-dependent changes in the expression of 

aromatic hydrocarbon receptor (AHR) and AHR-nuclear translocator (ARNT) mRNAs in the rat 

ovary and liver Chem Biol Interact. 124, 205-1623 

[13] Lemmesn J.G., Arends R.J., Ven Boxtel A.L., Van der Saag P.T., Van Der Burg B., 2004. Tissue 

and time-ependent estrogen receptor activation in estrogen reporter mice Journal of Molecular 

Endocrinology 32,689-701. 

188


Effects mediated by membrane receptors 

• Dieni C.V., Tobin V., Menzies JRW., Dutia M.B. (Italy) Effects of THDOC and 

allopregnanolone on the gabaergic current evoked by muscimol in the neurons of 

the rat medial vestibular nuclei 

• Frondaroli A., Grassi S., Pettorossi V.E. (Italy) Long-term effects of THDOC on 

the neuronal activity of the rat medial vestibular nuclei 

• Hariri O.R., Micevych P.E. (USA) The nongenomic effects of estrogen regulates 

the effects of oxytocin in the hypothalamic astrocytes 

• Lindblad C., Bierzniece V., Turkmen S., Bäckström T. and Johansson I-M. 

(Sweden) Metabolism prevent the UC1010 antagonistic effect to allopregnanolone 

in the morris water maze 

• G. Ragagnin, M. Rahman, E. Zingmark, J. Strömberg, P. Lundgren, M. Wang, T. 

Bäckström (Sweden) Structure-activity relationship of GABA A -steroids antagonists


EFFECTS OF THDOC AND ALLOPREGNANOLONE ON THE GABAERGIC 

CURRENT EVOKED BY MUSCIMOL IN THE NEURONS OF THE RAT 

MEDIAL VESTIBULAR NUCLEI 

Dieni C.V.*, Tobin V.°, Menzies JRW.°, and Dutia M.B.° 

* Department of Internal Medicine, Section of Human Physiology, University of Perugia, 

Via del Giochetto, 06100, Perugia, Italy, Fax: +39-0755857371 

email: cristinavd@libero.it 

° Centre for Integrative Physiology, Biomedical Sciences, Hugh Robson Building, George 

Square, Edinburgh, EH8, 9XD, UK, email: m.b.dutia@ed.ac.uk 

Introduction: Plasticity in the medial vestibular nuclei (MVN) after unilateral 

labyrinthectomy and the resultant vestibular compensation involve time-dependent 

changes in MVN neuron intrinsic excitability and a re-balancing of the neuronal activity of 

the bilateral MVN [3], in part through changes in GABA receptor function [2, 5]. 

Neurosteroids are known to modulate GABA receptor function in many brain areas [1, 6] 

by primarily but not exclusively increasing the sensitivity of the receptor and thus 

increasing inhibitory drive. The enzymes responsible for the de novo synthesis of 

neurosteroids are present in the MVN [4] and previous work in our lab has shown that 

neurosteroid exposure can increase the inhibitory effect of the GABA A receptor agonist 

Muscimol on the spontaneous firing rate of MVN neurons (unpublished data). Thus, we 

hypothesised that the Muscimol-induced Cl - current in MVN neurons may be augmented 

by exposure to the neurosteroids Allopregnanolone (ALLO) and 3α, 5αtetrahydrodeoxycorticosterone 

(THDOC). 

Methods: Coronal brainstem slices including the rostral half of the MVN (200-250 µm) 

were prepared from Lister Hooded rats (P14-P21) and incubated (60 min, 35 o C) in 

oxygenated artificial cerebrospinal fluid (ACSF). The slices were then transferred to a 

recording chamber (35 o C) with constant oxygenated perfusion of ACSF or drugs dissolved 

in ACSF (2 ml/min). Neurons were identified using IR-DIC optics and generally had ovoid 

soma with 15 µm diameter with at least 2 processes visible. Patch pipettes (7-8 MΩ) were 

filled with (in mM): CsMeSO 4 145, HEPES 5, EGTA 0.1, MgATP 5, pH 7.2, 290-295 

mOsm). Clamping the membrane potential at -30 mV and dialysis of the cells with cesium 

blocked sodium and potassium currents respectively, allowing us to record the whole cell 

Cl - current elicited by bath application of Muscimol (3 µM, 1 min). Currents were acquired 

using Axon 200B amplifier and Clampex v9 software and filtered at 2 kHz. Both 

amplitude and area of current recording (integral of evoked current minus holding current) 

were measured using Clampfit v9. These were measured before (control) and 5, 15 and 25 

min after the start of neurosteroid application (ALLO 3 µM or THDOC 3 µM, 5 min) and 

compared using either paired t-test or one-way ANOVAs with Holm-Sidak post-hoc 

analysis. P values of less than 0.05 were accepted as significantly different. 

Results: In 20 cells, the amplitude of the GABA current immediately after ALLO was 

significantly increased (124.8 ± 23.9 pA vs. 90.13 ± 13.8 pA, P



the same intervals as above but in the absence of neurosteroid were unchanged compared 

to the first response. 

Conclusions: These results indicate that ALLO and to a lesser extent THDOC augment 

Muscimol-induced GABAergic currents in MVN neurons, consistent with previous studies 

in other brain regions that have shown neurosteroids can enhance the sensitivity of the 

GABA A receptor to its ligand. An interesting finding is the persistence of this enhancement 

after wash out of the neurosteroid. This may reflect either binding of neurosteroid to the 

GABA A receptor or a persistent change in the receptor function. Further studies are 

ongoing to clarify the biochemical mechanisms utilised by the neurosteroids. However, our 

results demonstrate that neurosteroids can modify the neuronal activity of MVN neurons 

by facilitating GABA currents and therefore inhibitory drive. As vestibular compensation 

is dependent on stress hormones [3], and stimulation of the stress axis has been shown to 

increase production of neurosteroids in different brain areas [6], we suggest that post-UL 

compensatory changes may involve the actions of neurosteroids in the vestibular nuclei. A 

possible mechanism may be an enhancement of GABAergic drive to induce long lasting 

depression of discharge of the MVN neurons in the intact side, while GABAergic receptor 

sensitivity of the MVN neurons on the lesioned side is down-regulated [5]. 


1. E.E. Baulieu, Neurosteroids: a novel function of the brain, Psychoneuroendocrinol. 23 (1998) 963-987. 

1998. 

2. S.A. Cameron, M.B. Dutia, Cellular basis of vestibular compensation: changes in intrinsic excitability of 

MVN neurons, Neuroreport 8 (1997) 2595-2599. 

3. S.A. Cameron, M.B. Dutia, Lesion-induced plasticity in rat vestibular nucleus neurones dependent on 

glucocorticoid receptor activation, J. Physiol. 518 (1999) 151-158. 

4. E. Dupont, J. Simard, V. Luu The, F. Labrie, G. Pelletier, Localization of 3 beta-hydroxysteroid 

dehydrogenase in rat brain as studied by in situ hybridization. Mol. Cell. Neurosci. 5 (1994) 119-123. 

5. T. Yamanaka, A. Him, S.A. Cameron, M.B. Dutia, Rapid compensatory changes in GABA receptor 

efficacy in rat vestibular neurones after unilateral labyrinthectomy, J. Physiol. 523 (2000) 413-424. 

6. R.H. Purdy, A.L. Morrow, P.H. Jr. Moore, S.M. Paul, Stress-induced elevations of γ-aminobutyric acid 

type A receptors-active steroids on the rat brain. Proc. Natl. Acad. Sci. USA 88 (1991) 4553-4557. 

191


LONG-TERM EFFECTS OF THDOC ON THE NEURONAL ACTIVITY OF THE 

RAT MEDIAL VESTIBULAR NUCLEI 

Frondaroli A., Grassi S., Pettorossi V.E. 

Department of Internal Medicine, Section of Human Physiology, University of Perugia, Via del Giochetto, 

06100, Perugia, Italy, Fax: +39-0755857371 email: sgrassi@unipg.it 

Aim: Stressfull events induce synthesis of neurosteroids (NS) in the brain, which rapidly 

influence the neuronal excitability by acting on different neurotransmitter receptors, 

including GABA A and ionotropic glutamate (NMDA and AMPA) receptors [1]. 

There is evidence that the medial vestibular nuclei (MVN) are susceptible to stress 

hormones (glucocorticoids, GC), as activation of GC receptors plays a role in vestibular 

compensation, which leads to the re-balance of neuronal activity in the medial vestibular 

nuclei (MVN) after unilateral labyrinthectomy (UL) [2]. Besides GC, it is also possible 

that NS are involved in the compensation, since NS could be locally synthesized in the 

MVN [3]. To evidence the role of NS in the vestibular plasticity phenomena, it is firstly 

necessary to demonstrate their influence on the spontaneous and synaptically driven 

activities of the MVN neurons and, secondly, to evidence possible long term effects. The 

NS used in this study was the tetrahydrodeoxycorticosterone (THDOC), since its secretion 

is correlated with that of GC [5]. 

Methods: The experiments were performed in rat transverse brainstem slices (350 µm), 

containing the medial vestibular nuclei (MVN), which were perfused with oxygenated 

artificial cerebrospinal fluid (2 ml/min) and maintained at 30-31 °C. The field potentials 

were recorded in the ventral part (Vp) of the MVN by stimulating (40-100 µA intensity, 

0.07 ms duration and 0.06 Hz frequency) the ipsilateral vestibular afferents at the point 

where they enter the MVN. Single unit potentials were also extracellularly recorded. The 

effect of THDOC (3 µM, applied for 15 min), was assayed on the amplitude of the field 

potential N1 wave (expressed as a percentage of the baseline) and on spontaneous firing 

rate of single neurons (spikes/sec). To recognise the receptors which could be involved in 

mediating the THDOC effects, we used the antagonists for the following receptors: 

GABA A (Bicuculline, 30 µM), NMDA (DL-AP5, 100 µM), group I metabotropic 

glutamate (mGlu-I) (AIDA, 200 µM) and AMPA/kainate (NBQX, 10µM). 

Results: THDOC induced long-lasting changes of the field potential amplitude consisting 

in both depressions and potentiations (Fig. 1A). In addition, THDOC induced two 

temporally distinct effects, which were not necessary combined: the early one, developing 

at 7-12 min after the start of drug application, and the late one at 20 - 30 min. Under block 

of GABA A receptors (Bicuculline), field potential depressions were annulled, while the 

occurrence of potentiation remained unchanged (Fig. 1A). Under this condition, the block 

of NMDA (AP-5) and mGlu-I (AIDA) receptors did not significantly change the 

occurrence of early and late potentiations (Fig. 1A and B), contrary to that normally 

observed in the vestibular synaptic long-term potentiation [4]. The same result was 

obtained by analysing the effect of THDOC on the neuron firing rate in normal condition, 

under Bicuculline and under block of NMDA and mGlu-I receptors (Fig. 1C and D). By 

contrast, the block of AMPA/kainate (NBQX) receptors significantly modified the 

occurrences of both early and late potentiations, which were respectively significantly 

reduced and increased, compared with those observed under Bicuculline alone (Fig. 1D). 

Conclusions: These results show that THDOC changes synaptic and spontaneous activity 

of the MVN neurons by inducing long-lasting effects. These effects consisted in depression 

and potentiation showing short or long induction time (early and late effects). The analysis 

of the possible action mechanism of THDOC indicates that 1) early and late depressions 

192



depend on a facilitatory influence of THDOC on GABA A receptors, which mediate the 

inhibitory transmission in the MVN; 2) the early THDOC potentiation depends on a 

facilitation of AMPA/kainate mediated glutamate transmission, which might be due to the 

enhancement of receptor sensitivity to glutamate and/or to the increase of glutamate 

release; 3) the late THDOC potentiation could depend on the enhancement of intrinsic 

neuronal excitability, probably through an effect on ionic channels, since it is not annulled 

by the whole glutamate receptor block. In conclusion, THDOC modifies the activity of 

MVN neuronal circuitry rapidly, by increasing the inhibitory and excitatory transmission, 

through potentiation of GABA A and AMPA/kainite receptor activation, and changes the 

neuronal intrinsic excitability, later. The presence of different influences of THDOC, on 

vestibular activity, in terms of sign and timing of effects, may explain opposite behaviour 

of vestibular neurons during UL compensation, which requires an increase of activity in 

the ipsilesional side and decrease in the contralateral side. 


1. E.E. Baulieu, Neurosteroids: a novel function of the brain, Psychoneuroendocrinol. 23 (1998) 963-987. 

1998. 

2. S.A. Cameron, M.B. Dutia, Lesion-induced plasticity in rat vestibular nucleus neurones dependent on 

glucocorticoid receptor activation, J. Physiol. 518 (1999) 151-158. 

3. E. Dupont, J. Simard, V. Luu The, F. Labrie, G. Pelletier, Localization of 3 beta-hydroxysteroid 

dehydrogenase in rat brain as studied by in situ hybridization. Mol. Cell. Neurosci. 5 (1994) 119-123. 

4. S. Grassi, V.E. Pettorossi, Synaptic plasticity in the medial vestibular nuclei: role of glutamate 

receptors and retrograde messengers in rat brainstem slices. Prog. Neurobiol. 64/6 (2001) 527-553. 

5. R.H. Purdy, A.L. Morrow, P.H. Jr. Moore, S.M. Paul, Stress-induced elevations of γ-aminobutyric acid 

type A receptors-active steroids on the rat brain. Proc. Natl. Acad. Sci. USA 88 (1991) 4553-4557. 

Event occurrence (%) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

A 

* 

* 

P otentiation (early + l ate) 

Depression 

Null effect 

P otentiation occurrence (%) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

B 

* 

Early potentiation 

Late potentiation 

0 

0 

Event occurrence (%) 

100 

80 

60 

40 

20 

C 

* 

* 

* 

Potentiation (early + late) 

Depression 

Null effect 

P otentiation occurrence (%) 

100 

80 

60 

40 

20 

D 

* 

* 

* 

* 

Early potentiation 

Late potenziation 

0 

0 

Fig. 1 Occurrence percentage (mean ± SD,* p < 0.05) of the effects induced by THDOC in the MVN on the 

field potential amplitude (A and B) and on neuronal firing rate (C and D) under normal condition (control), 

under block of GABA A receptors (Bicuculline) and under block of NMDA (AP-5), mGlu-I (AIDA) and/or 

AMPA (NBQX) glutamate receptors. 

193


THE NONGENOMIC EFFECTS OF ESTROGEN REGULATES THE EFFECTS OF 

OXYTOCIN IN THE HYPOTHALAMIC ASTROCYTES 

Hariri O.R. * , Micevych P.E. * 

* University of California, Los Angeles, Le Conte Ave, Los Angeles, CA, USA. Fax +310-825- 

2224 Email: pmicevych@mednet.ucla.edu 

Astrocytes have important roles in the development and physiology of the central nervous 

system. In the hypothalamus, astrocytes have been implicated in sexual differentiation, synthesis of 

neurosteroids and synaptic function. Astrocytes can regulate neuronal activity through physical 

integration of synaptic signals, regulate interneuronal contacts and have been recently implicated in 

estrogen positive feedback responses. It is clear that astrocyte activity has implications for the 

functional understanding of a variety of different circuits systems. Astrocyte activity can be 

tracked by observing variations in intracellular free cytoplasmic calcium levels ([Ca 2+ ] i ). Previous 

studies have demonstrated that hypothalamic astrocytes respond to both estradiol and oxytocin 

(OT). Studies have shown that a high proportion of ventromedial hypothalamic neurons express 

estrogen receptors (ER) mRNAs and oxytocin receptor (OTR) mRNA. Although estradiol 

influences the expression of oxytocin receptors (OTR) in neurons, there is a paucity of studies that 

examined the interaction ER and OTR inastrocytes. In non-neuronal cells of the corpus luteum, 

chronic estradiol treatment caused the down-regulation of OTR. Because both estradiol and OT 

affect astrocytes and because their interaction may have important consequences for astrocyte 

function, we tested whether estradiol could modulate oxytocin signaling in hypothalamic 

astrocytes. Separate studies have shown that both rapid actions of estradiol and oxytocin increase 

free cytoplasmic calcium levels [Ca 2+ ] i in astrocytes, but the proximal steps of their signaling 

mechanisms have not been examined. The present study examines the interaction of E2 and OT on 

cell signaling by monitoring ([Ca 2+ ] i transients in post-pubertal astrocytes. 

We used digital fluorescence microscopy with Fura-4 as the Ca 2+ indicator and tested the 

response of post-pubertal hypothalamic astrocytes to both OT and estradiol. Application of 

estradiol (4nM) to the astrocyte cultures rapidly increased the [Ca+2]i by 570.2 ± 70 (relative 

units). Also, application of OT (400nM) increased the [Ca 2+ ] i 285.2 ± 42. In another experiment, 

astrocytes were treated with 400nM OT inducing a significant increase in [Ca 2+ ] i levels . After a 

washout of three minutes, addition of 2nM estradiol produced a significant increase in [Ca 2+ ] i . But 

when astrocytes were treated with estradiol (2nM) first and then OT (400nM), OT did not 

significantly increase the [Ca 2+ ] i levels, demonstrating a estradiol mediated attenuation of the OTinduced 

[Ca 2+ ] i response. In another experiment, astrocytes were treated chronically with 10nM 

estradiol for 22 hours. Astrocytes did not have a [Ca 2+ ] i response to OT challenge after the 

estradiol. 

Previous studies demonstrate that estradiol and OT rapidly activate phospholipase C (PLC) 

to induce the release of intracellular stores of calcium through the inositol trisphosphate (IP3) 

receptor, suggesting that OT and estradiol signaling converge onto a common intracellular 

pathway. Rapid, estradiol signaling in neurons appears to require an interaction of membrane ER 

with metabotropic glutamate receptor (mGluR), and the activation of [Ca 2+ ] i flux requires the type 

1a receptor (mGluR1a). To test whether the same mechanism was present in astrocytes, 20nM of 

LY367385, an mGluR1a antagonist, was added to the media. The estradiol-induced [Ca 2+ ] i 

transient was significantly attenuated. Interestingly, the OT-induced [Ca 2+ ] i transient was also 

prevented by LY367385, indicating that the convergence of E2 and OT signaling occurs at the 

mGluR1a, which activates a G-protein to activate the PLC-IP3 pathway. 

Although estradiol or OT separately rapidly increases [Ca 2+ ] i , flux in hypothalamic 

astrocytes, an order of treatment effect was also observed: pre-incubation/treatment with estradiol 

prevents OT signaling in hypothalamic astrocytes. These results demonstrate a negative estradiol 

regulation of OT signaling. We hypothesize that the association of the ER with mGluR1a prevents 

interaction of the OTR with the ER/mGluR complex, and therefore preventing OT signaling 

through the PLC-IP3 pathway. These results suggest regulatory mechanism through which 

estradiol modulates OT responses during pregnancy and lactation. 

194



METABOLISM PREVENT THE UC1010 ANTAGONISTIC EFFECT TO 

ALLOPREGNANOLONE IN THE MORRIS WATER MAZE 

Lindblad C., Bierzniece V., Turkmen S., Bäckström T. and Johansson I-M. 

Department of Clinical Sciences, Obstetrics and Gynecology, Umea University Hospital, S-901 85, 

Umea, Sweden. Fax: +46-90-776006, e-mail: charlotte.lindblad@obgyn.umu.se 

Background: The progesterone metabolite allopregnanolone (allo, 3α-OH-5α-pregnan- 

20-one) is a neurosteroid which act as a positive modulator on the GABA-A receptor and 

enhance the GABA activity. It is produced at many different sites and occasions. In 

humans, allo from corpus luteum is fluctuating during the menstrual cycle with its peak 

during the luteal phase. During pregnancy placenta produces allopregnanolone which is 

rising to high levels. Allopregnanolone is also increased during stress, but in this case the 

source is the adrenal glands. It can also be synthesized in the brain. 

Allopregnanolone has been shown to have negative effects on learning and memory in 

rat [2]. A key area for learning and memory is the hippocampus and it has been shown that 

the Morris water maze is a good model for testing spatial learning in rat [5]. In humans it 

has been shown that short-term spatial working memory is affected by menstrual cycle 

changes, with more errors in the luteal phase [4], when allo is accumulated in the brain [1]. 

Since allo has these negative effects on cognition, mood and learning, it would be 

helpful to be able to block these negative effects. As an antagonist we have used UC1010 

(3β-OH-5α-pregnan-20-one) a 3β-isomer to allopregnanolone. UC1010 has been shown to 

work as an antagonist with several different in vitro systems. Wang et al. in 2000 showed 

that UC1010 antagonize the inhibitory effect of allopregnanolone on population spikes in 

the CA1 region of the rat hippocampus dose-dependently [6]. Lundgren et al. (2003) 

showed that allo-induced GABA-mediated chloride ion influx into rat cortical microsacs is 

inhibited by UC1010 [3]. 

Aim: To block the negative allopregnanolone effects on learning in the Morris Water 

Maze with the antagonist UC1010. 

Material and Methods: Adult male Wistar rats were injected i.v. daily with allo (2 mg/kg, 

n=7), UC1010 (32 mg/kg, n=5), allo:UC1010 (2:32 mg/kg, n=5) and vehicle (10,7 ml/kg, 

n=4) respectively. Rats started a trial session for place navigation in the Morris Water 

maze 8 minutes after injection. Each session consisted of 4 searches for the platform. 

Animals were decapitated eight minutes after injection at day 8 (allo and vehicle) or 7 

(UC1010 and allo:UC1010). Trunk blood and selected brain areas were taken care of for 

further analyses. 

Results: The negative effects of allopregnanolone on learning in the Morris water maze 

were not blocked by UC1010 (fig. 1A, filled squares). Instead UC1010 enhanced the 

negative allo effect, i.e. rats injected with both allo and UC1010 didn’t find the platform at 

all. Allopregnanolone (filled circles) inhibited learning as shown earlier. Animals injected 

with only UC1010 (open squares) had an allo-like learning curve, with slightly, but not 

significant lower latency to find the platform compared to allo group. Vehicle group (open 

circles) learned to find the platform already at day two of the experiment. 

The lack of learning in the treated groups was not due to loss of swimming skills or 

reduced motor activity. The only group that differed in swim speed was the UC1010 

group, and those animals had a higher speed compared to the vehicle and allo groups (fig. 

1B). 

195


We see is a changed search pattern in all treated groups compared to the vehicle group 

(fig. 1C). Allo and UC1010 groups decreased their time spent close to the wall 

(thigmotaxis) throughout the experiment. But in the group that received both allo and 

UC1010 there was instead a slight increase in thigmotaxis time. 

Preliminary data of allopregnanolone concentrations in hippocampus shows higher 

levels of allo in the group that was injected with only UC1010 (3300 nmol/kg) than in 

animals injected with allo (2100 nmol/kg). The highest levels were found in animals 

injected with both allo and UC1010 (4600 nmol/kg). All treated groups had allo levels 

) 

several thousand times higher than the vehicle group (4 nmol/kg). 

) 

s 

( 

y 

c 

n 

e 

t 

a 

L 

120 

100 

80 

60 

40 

20 

Latency 

0 

0 1 2 3 4 5 

Day 

) 

s 

/ 

m 

c 

( 

A B C 

) 

s 

/ 

m 

c 

( 

d 

e 

e 

p 

s 

Figure 1. Morris water maze results 

m 

i 

40 

30 

20 

Swim speed 

40 

dVehicle 

e 

Allo (2 mg/kg) 

e 30 

pUC1010 (32 mg/kg) 

sAllo:UC1010 (2:32 mg/kg) 

20 

m 

i 

10 

w 

S 

0 

0 1 2 3 4 5 

Swim speed 

Day 

Thigmotaxis time (%) 

s 100 

i Vehicle 

x Allo 80(2 mg/kg) 

a UC1010 (32 mg/kg) 

t 60 

o 

Allo:UC1010 (2:32 mg/kg) 

m 

g 

40 

i 

h 

20 

T 

0 

0 1 2 3 4 5 

Discussion: UC1010 didn’t 10 succeed to block the negative effect of allo on learning in the 

w 

Morris water maze even though S it had been shown to work as an antagonist to allo in vitro 

0 

[2,6]. This taken together with the allo like effects we had seen in the group injected with 

0 1 2 3 4 5 

UC1010 and the high concentration of allo Day in the UC1010 group that had not been injected 

with allo points towards a possible metabolism of UC1010 to allopregnanolone within the 

brain. 

% 

( 

e 

m 

i 

t 

Day 

Vehicle 


UC1010 (32 mg/kg) 


Vehicle 


UC1010 (32 mg/kg) 



[1] M. Bixo, A. Andersson, B. Winblad, R.H. Purdy, T. Bäckström, Progesterone, 5α-pregnane-3,20-dione 

and 3α-hydroxy-5α-pregnane-20-one in specific regions of the human female brain in different endocrine states, 

Brain Res. 764 (1997) 173-178 

[2] I-M. Johansson, V. Birzniece, C. Lindblad, T. Olsson, T. Bäckström, Allopregnanolone inhibits learning 

in the Morris water maze, Brain Res. 934 (2002) 125-131 

[3] P. Lundgren, J. Strömberg, T. Bäckström, M. Wang, Allopregnanolone-stimulated GABA-mediated 

chloride ion flux is inhibited by 3β-hydroxy-5α-pregnan-20-one (isoallopregnanolone), Brain Res. 982 (2003) 

45-53 

[4] M.S. Man, I. MacMillan, J. Scott, A.H. Young, Mood, neuropsychological function and cognitions in 

premenstrual dysphoric disorder, Psychol. Med. 29 (1999) 727-733 

[5] G. Riedel, J. Micheau, A.G. Lam, E. Roloff, S.J. Martin, H. Bridge, L. Hoz, B. Poeschel, J. McCulloch, 

R.G. Morris, Reversible neural inactivation reveals hippocampal participation in several memory processes, 

Nat. Neurosci. 2 (1999) 898-905 

[6] M.D. Wang, T. Bäckström, S. Landgren, The inhibitory effects of allopregnanolone and pregnanolone on 

the population spike, evoked in the rat hippocampal CA1 stratum pyramidale In vitro, can be blocked 

selectively by epiallopregnanolone, Acta Physiol. Scand. 169 (2000) 333-341 

196



STRUCTURE-ACTIVITY RELATIONSHIP OF GABA A -STEROIDS 

ANTAGONISTS 

G. Ragagnin*, M. Rahman, E. Zingmark, J. Strömberg, P. Lundgren, M. Wang, T. 

Bäckström 

*Umeå Neurosteroid Research Center, Department of Clinical science, Obstetrics and 

Gynecology, Norrland University Hospital by5B, tr5, 901 85 Umeå, Sweden 

Fax +46 90 776006; e-mail: gianna.ragagnin@obgyn.umu.se 

Neuroactive steroids have effects on serotonin, glutamate and GABA systems. 

Their importance is enormous since they are involved in the regulation of mood, memory, 

Alzheimer’s disease, stress-induced depression and burn-out syndrome, anxiety disorders, 

postnatal and major depression as well as premenstrual syndrome (PMS), premenstrual 

dysforic disorder (PMDD), and mood changes related to hormone replacement therapy,. 

They are 3α-hydroxy-5α/β metabolites of progesterone (allopregnanolone and 

pregnanolone), testosterone (3α-hydroxy-5α-androstan-diol), and deoxycorticosterone 

(tetra-hydro-deoxycorticosterone). 

Allopregnanolone acts via the GABA A receptor by changing receptor’s expression or 

sensitivity. It is involved in premenstrual mood changes and induces cognitive deficits, 

such as spatial-learning impairment. 

Me 

O 

Me 

HO 

H 

Allopregnanolone 

Our aim is to find steroids than could antagonize the negative effects of allopregnanolone. 

A X-ray analysis of the GABA A receptor is required in order to design an optimal structure 

of a possible agonist/inverse agonist/antagonist by simple computer-aided modelling. 

Such an analysis is still not available yet, because of the receptor’s complexity. It must also 

be noticed that there are at least 20 different subunit compositions in the brain and there 

are evidences that more than one active site for steroids are present in a single receptor. 

We have synthesized, in some steps followed by chromatographic purification, a number 

of new steroids with potential activity as partial inverse agonists against allopregnanolone 

and neutral antagonists against GABA. The compounds have been tested in vitro by 

voltage-clamp and patch-clamp methods. 

The systematic investigation of several isomer combinations of GABA A -active 

neurosteroid derivatives, led us to the finding that, as a general rule, the following features 

are of importance for an effective drug: 

- Geometry between rings A/B is crucial 

- Hydrogen-bond donator in position 3 

- Hydrogen-bond acceptor in position 20 

- Flexible bond at position 17 

In addition, the title compounds fitfull the Lipinski’s rule of five for effective oral 

administration, and display acceptable values for logBBB. 

197


Corticosteroid effects and stress 

• Berry A., Giorgio M., Martin-Padura I.; Pelicci P.G., de Kloet E.R., Alleva E., 

Minghetti L. and Cirulli F. (Italy) Mice carrying a deletion of the P66 Shc gene show 

reduced behavioural and neuroendocrine responses to stressful stimuli 

• Hirst JJ , Palliser HK, Yates DM, Walker DW (Australia) Role of 5αReductase 

enzymes in the fetal brain and placenta in neurosteroid production following 

intrauterine growth restriction 

• Macrì S., Cirulli F. , Pasquali P. , Bonsignore L.T. , Laviola G. (Italy) Neonatal 

exposure to low doses of corticosterone increases novelty seeking and resistance to 

bacteria infection in adult male mice 

• Maggio N. and Segal M (Israel) Corticosteroids modulation of long term 

potentiation along the septo_temporal axis of the hippocampus 

• Morley-Fletcher S, Mairesse J, Daszuta A, Soumier A, Banasr M, Zuena AR, 

Mocaer E, Matteucci P, Casolini P, Catalani A, Maccari S. (France) 

Neuroplasticity in the prenatal stress rat model of depression: effects of 

agomelatine treatment 

• Ognibene E., Adriani W., Macrì S., Laviola G (Italy) Neurobehavioural disorders 

in the infant reeler mouse model: interaction of genetic vulnerability and 

consequences of maternal separation 

• Vafaei AA, Taherian AA, Jarrahi M (Iran) Assessment of modulatory effects of 

corticosterone on anxiety related behavior in mice 

• Velickovic N.A., Djordjevic A.D., Horvat A.I., Demajo M.A. (Serbia) Late effects 

of ionizing irradiation on corticosteroid receptor expression in rat hippocampus: 

the role of hypothalamus-pituitary-adrenal axis



MICE CARRYING A DELETION OF THE P66 Shc GENE SHOW REDUCED 

BEHAVIOURAL AND NEUROENDOCRINE RESPONSES TO STRESSFUL 

STIMULI 

Berry A. (1, 2); Giorgio M. (3); Martin-Padura I. (3); Pelicci P. G. (3); de Kloet E.R. (2); 

Alleva E. (1); Minghetti L. (4), and Cirulli F. (1) 

(1) Section of Behavioral Neuroscience, Dept. BCN, ISS, Rome, Italy; (2) LACDR, Div. of Medical 

Pharmacology, Gorlaeus Labs., Leiden University, Leiden, The Netherlands, (3) Department of 

Experimental Oncology, IEO, Milan, Italy; (4) Section of Degenerative and Inflammatory 

Neurological Diseases, Dept. BCN, ISS, Rome, Italy. 

Targeted mutation of the p66 Shc gene in the 129Sv/Ev mouse strain has been shown to increase 

both resistance to oxidative stress (OS) and life span. A growing body of evidence suggests 

that there might be an interaction between stressful stimuli and OS. P66 mutant mice represent 

an ideal animal model to study such an interaction. P66-/- (KO) and p66+/+ (WT) mice (4- 

and 11-months-old subjects) were tested in the open field (OF) and in the elevated plus maze 

(EPM) to assess emotional reactivity. They also underwent a chronic restraint stress paradigm 

as well as an acute systemic LPS challenge. Data from the OF and the EPM give an overall 

indication that KO mice were characterized by a less anxious phenotype. Results from the 

chronic restraint did not reveal any difference in the HPA axis responsiveness. By contrast, 

results from the LPS challenge indicate a reduced HPA axis activation in adult KO subjects. 

These results indicate that behavioral responses to arousing stimuli, as well as neuroendocrine 

responses to an immunogenic stimulus, are reduced in KO mice in an age-dependent fashion. 

Supported by: Italian Ministry of Health (grant ex art. 56 to F. C. and L. M.) and by Marie 

Curie fellowship (The Genetic Basis of Disease) granted to Alessandra Berry. 

199


EFFECTS OF PRENATAL DEXAMETHASONE TREATMENT ON MOTOR 

DEXTERITY IN THE JUVENILE MARMOSET MONKEY 

Hauser J.*, Zuercher N.*, Feldon J.*, Diaz-Heijtz R.#, Dettling-Artho R.*, Knapman 

A.*, Pryce C.R.$ 

* Behavioural Neurobiology Laboratory, Swiss Federal Institute of Technology – Zurich, 

Schoerenstrasse 16, CH- 8603 Schwerzenbach, Switzerland, Fax: +41 (0)44 655 72 02, e-mail: 

jonas.hauser@behav.biol.ethz.ch 

# Behavioural Neuroscience Laboratory, Karolinska Institute, Stockholm, Sweden 

$ Novartis Institutes for BioMedical Research Basel, Novartis Pharma AG, Basel, Switzerland 

There is considerable evidence that prenatal stress has deleterious effects on development 

of the central nervous system (CNS) and those functions that it controls, including endocrine 

homeostasis and behaviour [7]. Prenatal stress results in increased maternal hypothalamicpituitary-adrenal 

(HPA) axis activity and thereby in higher plasma corticosteroids levels in 

maternal blood as well as, to some extent, fetoplacental unit. Corticosteroid binds specifically 

to mineralocorticoid receptor and glucocorticoid receptor (GR), this last being recruited 

primarily under stressful condition. GR are expressed in many organs, including brain, where 

they can, as transcription factors, regulate genes expression. GR undergo fetal programming: 

the setting up of adult expression level of a receptor by the presence of its ligand during early 

development, a mechanism hypothesized to mediate long term effects of prenatal stress. In 

rats, prenatal stress has been associated with delayed early life motor development [6] and 

increased HPA axis activity [4], this was also observed with the specific GR agonist 

dexamethasone (DEX) [3,11]. Similarly in rhesus monkey, prenatal stress or ACTH exposure 

resulted in impairement in an adaptation of the human Brazelton Newborn Behavioural 

Assessment Scale [8,9]. These associations between both prenatal stress and prenatal DEX 

exposure and impaired early motor development clearly point to the importance of increased 

understanding of any causal relationships between prenatal exposure to synthetic GC and 

development of motor dexterity beyond infancy. This long-term effect are furthermore 

important to study as synthetic specific GR agonists, such as DEX, are commonly prescribed 

in obstetric medicine for the prophylactic treatment of morbid symptoms associated with 

preterm birth, most importantly intra-ventricular haemorrhage and respiratory distress 

syndrome [5]. Such treatment is endorsed by the US National Institutes of Health (NIH) [1], 

with emphasis on the importance to study potentially harmful long-term effect especially in 

the case of repeated exposure [2]. 

In this study, we administered 5mg/kg/d (per os) DEX to pregnant marmoset monkeys 

(Callithrix jacchus; gestation length 144 days) for 7 days either during early gestation (day 42- 

48, putative stage of maximal neurogenesis) or late gestation (day 90-96, equivalent time of 

clinical treatment in pregnant women). We assessed postnatal developmental effects relative to 

controls in offspring between 3 and 7 months of age. Physical and endocrine developments 

were monitored monthly by measuring body weight, knee-heel length and collecting a blood 

sample for radioimmuno assay analyses of basal ACTH and cortisol plasma titres. Home-cage 

behaviours were scored one hour weekly during months 3, 5 and 7, using an ethogram based 

on one already published for the marmoset [10], with focus on the following relationships 

(behaviour elements in parentheses): infant–parent or infant-infant (social grooming, social 

play) and infant alone (mobility, scratch with hand or foot, exploration, self-grooming, eat 

200



solid food, solitary play, tail hair piloerection). Skilled motor dexterity – reach-to-grasp 

movement – was assessed using a conditioned test that was adapted from the elegantlydesigned 

and well-validated skilled reaching task for rodents [12], consisting in training and 

testing subjects to reach through a small opening for a reward, and measuring the frequency of 

specific reaching and grasping behaviours. 

In the EDEX subjects there was a female-specific increase in body weight and in body 

weight:knee-heel ratio. There were no differences between any of the treatment groups or 

between sex in terms of basal plasma ACTH and cortisol titres. When assessing motor 

dexterity in the skilled reaching task, the LDEX treatment led to a deficit, which was not 

improved by training, whereas the EDEX treatment resulted in a transient impairment, which 

was overcome by the end of the testing. This study extends the aforementioned reports of 

neonatal delay in motor reflexes development following prenatal GR activation, and provides 

novel evidence that prenatal DEX exposure impairs skilled motor function in juvenile 

primates, which is important to our understanding of the role of glucocorticoids in the 

ontogeny of motor behaviour and of the negative side effects of clinical prenatal 

glucocorticoid treatment. 


[1] Consensus Development Panel on the Effect of Corticosteroids for Fetal Maturation on Perinatal 

Outcomes: Effect of corticosteroids for fetal maturation on perinatal outcomes., JAMA, 273 (1995) 413- 

8. 

[2] Consensus Development Conference Statement: Antenatal corticosteroids revisited: repeat courses, 

Obstet Gynecol, 98 (2001) 144-50. 

[3] Burlet, G., Fernette, B., Blanchard, S., Angel, E., Tankosic, P., Maccari, S. and Burlet, A., Antenatal 

glucocorticoids blunt the functioning of the hypothalamic-pituitary-adrenal axis of neonates and disturb 

some behaviors in juveniles, Neuroscience, 133 (2005) 221-30. 

[4] Fride, E., Dan, Y., Feldon, J., Halevy, G. and Weinstock, M., Effects of prenatal stress on vulnerability 

to stress in prepubertal and adult rats, Physiol Behav, 37 (1986) 681-7. 

[5] Jobe, A.H. and Soll, R.F., Choice and dose of corticosteroid for antenatal treatments, Am J Obstet 

Gynecol, 190 (2004) 878-81. 

[6] Patin, V., Vincent, A., Lordi, B. and Caston, J., Does prenatal stress affect the motoric development of 

rat pups? Brain Res Dev Brain Res, 149 (2004) 85-92. 

[7] Ruiz, R.J. and Avant, K.C., Effects of maternal prenatal stress on infant outcomes: a synthesis of the 

literature, ANS Adv Nurs Sci, 28 (2005) 345-55. 

[8] Schneider, M.L. and Coe, C.L., Repeated social stress during pregnancy impairs neuromotor 

development of the primate infant, J Dev Behav Pediatr, 14 (1993) 81-7. 

[9] Schneider, M.L., Coe, C.L. and Lubach, G.R., Endocrine activation mimics the adverse effects of 

prenatal stress on the neuromotor development of the infant primate, Dev Psychobiol, 25 (1992) 427-39. 

[10] Stevenson, M.F. and Poole, T.B., An ethogram of the common marmoset (Calithrix jacchus jacchus): 

general behavioural repertoire, Anim Behav, 24 (1976) 428-51. 

[11] Welberg, L.A., Seckl, J.R. and Holmes, M.C., Prenatal glucocorticoid programming of brain 

corticosteroid receptors and corticotrophin-releasing hormone: possible implications for behaviour, 

Neuroscience, 104 (2001) 71-9. 

[12] Whishaw, I.Q., O'Connor, W.T. and Dunnett, S.B., The contributions of motor cortex, nigrostriatal 

dopamine and caudate-putamen to skilled forelimb use in the rat, Brain, 109 (Pt 5) (1986) 805-43. 

201


ROLE OF 5ΑREDUCTASE ENZYMES IN THE FETAL BRAIN AND PLACENTA IN 

NEUROSTEROID PRODUCTION FOLLOWING INTRAUTERINE GROWTH 

RESTRICTIONHirst JJ 1,2 , Palliser HK 1 , Yates DM 1 , Walker DW 3 

1 Mothers and Babies Research Centre & 2 School of Biomedical Sciences, University of Newcastle, 

Callaghan NSW 2308, Australia; 

3 Department of Physiology, Monash University, Melbourne Vic 

3800, Australia 

Email: Jon.Hirst@newcastle.edu.au 

Allopregnanolone concentrations throughout the brain are remarkable high during fetal life and fall 

dramatically after birth [1]. This decline may result for the loss of a supply of 5α-reduced precursors 

by the placenta. We have shown that acute episodes of fetal hypoxia-ischemia, induced by occlusion 

of the umbilical cord during late gestation, lead to a rapid and marked rise in both allopregnanolone 

concentrations and 5α-reductase expression in the in the fetal brain [2]. In recent studies we have 

shown that the suppression of this neurosteroid response by finasteride treatment, potentiates hypoxiaischemia 

induced brain injury [3]. This suggests that the stress induced rise in allopregnanolone 

concentrations play an important neuroprotective role in the fetal brain and may be responsible to the 

relative resistance of the fetal brain to hypoxic ischemic insults. Placental insufficiency reduces 

progesterone production and may limit the supply of precursors for these stress-induced responses. 

Alternatively, the intrauterine growth restriction (IUGR) caused by placental insufficiency has been 

shown to alter steroidogenic pathways in the fetus. This may increase the neuroprotective steroid 

concentrations and/or the expression of their synthetic enzymes. These observations further suggest 

that neurosteroidogenic enzyme expression in the brain and placenta may act together to protect the 

fetal brain. 

The aim of this study was to compare the expression of 5α-reductase enzymes in the brain and placenta 

of fetuses from normal pregnancies and IUGR fetuses. 

A model of IUGR was produced in guinea pigs by the surgical ablation of a proportion of the branches 

of the uterine artery supplying each placenta at 31-35 days of gestation. Fetal brains and placentas were 

collected at term (65 days) and prepared for immunoblotting. 5α-Reductase type 1 and 5α-reductase 

type 2 expression in the cortex, hippocampal region and placentas of the IUGR fetuses (n=4) were 

compared to sham operated controls (n=4). Denistometry of the specific bands was normalized against 

actin. 

Birth weights of IUGR fetuses were significantly lower than those of the sham operated controls (44.9 

6.9g and 80.2 6.0g respectively; P=0.001). Fetal brain to body weight ratio was higher in the IUGR 

fetuses (170%; P=0.001) indicating brain sparing occurred in response to the chronic restriction of the 

placental blood supply. There was no difference in the expression of 5α-reductase 1 in the cortex, 

hippocampal region or placenta between sham and IUGR fetuses. However, the placenta was found to 

have the highest level of expression of 5α-reductase 1, followed by the cortex and then the 

hippocampal region (P



following preterm birth may raise the vulnerability of the preterm neonate to brain injury if 

neurosteroidogenic pathways in the brain are not adequately mature to take over the role of the 

placenta. 


1. Nguyen, P. N., Billiards, S.B., Walker, D. W., Hirst, J.J. 2003. Changes in 5α-pregnane steroids and 

neurosteroidogenic enzyme expression in the perinatal brain. Pediatr. Res. 53, 956-964 

2. Nguyen, P. N., Yan, E. B., Castillo-melendez, M., Walker, D. W., Hirst, J. J. 2004. Increased 

allopregnanolone concentrations in the fetal brain following umbilical cord occlusion. J. Physiol. 560, , 593- 

6021 

3. Yawno, T., Yan, E.B., Walker D.W., Hirst, J.J. 2006. Inhibition of neurosteroid synthesis increases asphyxiainduced 

brain injury in the late gestation fetal sheep. Soc Gynecol Invest 53, Abstr31. 

203


NEONATAL EXPOSURE TO LOW DOSES OF CORTICOSTERONE INCREASES 

NOVELTY SEEKING AND RESISTANCE TO BACTERIA INFECTION IN ADULT 

MALE MICE 

Macrì S.* ,1 , Cirulli F. 1 , Pasquali P. 2 , Bonsignore L.T. 1 , Laviola G 1 . 

1 Section of Behavioural Neuroscience, Department of Cell Biology and Neuroscience, 

2 Department of Food Safety and Veterinary Public Health 

Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Roma, Italy. Fax +39-06-4957821 

email: simone.macri@iss.it; laviola@iss.it 

Neonatal maternal separation studies in laboratory rodents suggest that variable levels of 

environmental demands may modify mother-offspring interaction and, in turn, regulate the 

development of individual stress and fear responses in the adult progeny [1]. However, there is 

no agreement on the view that maternal care is the unique mediator of this form of adaptive 

plasticity [1,2,3]. In contrast with this hypothesis, we recently demonstrated that long periods 

of maternal separation increase levels of maternal care compared to no separation, and that 

brief separations increase active maternal care and reduce HPA activation in adult offspring 

[2]. It has therefore been proposed that, among other mediators (e.g. maternal care, food 

intake) neonatal manipulations may modify circulating levels of corticosterone in dams and/or 

pups, and, in turn, offspring development [4]. Additionally, we proposed that the effects of 

neonatal corticosteroids may follow a U-shaped curve whereby, compared to intermediate 

concentrations, both low and high circulating levels of maternal corticosterone would upregulate 

offspring stress and fear responses. In the present study we investigated this 

hypothesis through supplementing mouse dams with corticosterone in the drinking water 

during the first week of lactation [5]. Here we used this experimental procedure to mimic the 

physiological HPA activation consequent to variable levels of environmental demands in 

mouse dams. 

Methods: lactating CD-1 mouse dams were given ad libitum access to water (vehicle) or water 

supplemented with corticosterone (low, L-CORT, 33µg/ml or high, H-CORT, 100µg/ml) 

during the first lactation week. In order to control for the effects of corticosterone treatment on 

dam-pup interaction, maternal behaviour was scored according to a detailed ethogram 

originally developed for rats [6,7]. Naïve male adult offspring were tested for levels of novelty 

seeking (PND > 65), and anxiety-related behaviours in the elevated plus maze. To address the 

development of the immune response, a separate group of adult mice were infected with 

Brucella, and residual colony forming units in the spleen were measured two weeks following 

infection. Finally, corticosterone plasma levels were addressed in an additional group of male 

mice under basal conditions, and 0, 35 and 95 min after the termination of 25-min restraint 

stress. 

Results: Compared to control and L-CORT, H-CORT dams showed a significant reduction in 

levels of active maternal care (p



(p


CORTICOSTEROIDS MODULATION OF LONG TERM POTENTIATION ALONG 

THE SEPTO_TEMPORAL AXIS OF THE HIPPOCAMPUS 

Maggio N. and Segal M. 

Department of Neurobiology, The Weizmann Institute of Science, Israel 

Behavioral stress is correlated with an increased release of corticosteroids. Within the 

hippocampus, a structure that is associated with memory processes, corticosteroids have been 

shown to modulate synaptic plasticity. However, in recent years, evidence has accumulated to 

indicate that spatial memory is associated primarily with the dorsal (septal) sector of the 

hippocampus, while the role of its ventral/temporal sector is not yet clearly defined. In 

addition, slices taken from the ventral hippocampus exhibit a much lower ability to produce 

LTP compared to those taken from the septal sector. 

We explored the ability to produce LTP in dorsal and ventral hippocampal slices taken from 

the acutely stressed rats. Following stress, LTP was impaired in dorsal hippocampus, as seen 

before. Surprisingly, LTP was enhanced in ventral hippocampal slices. Bath applied 1µM 

Corticosterone in control slices mimicked this effect. Furthermore, the glucocorticoid agonist 

(dexamethazone) also induced an LTP impairment in dorsal hippocampal slices while it did 

not have any effect in the ventral hippocampus. On the other hand, aldosterone, a selective 

mineralocorticosterone receptor (MR) agonist, selectively enhanced LTP in the ventral 

hippocampus. This effect was blocked by a calcium channel antagonist nifedipine, but not by 

APV, indicating an involvement of voltage gated calcium channels in the facilitating effect of 

steroids on LTP in the ventral hippocampus. We conclude that a differential distribution of 

MR and GR receptors along the septo-temporal axis of the hippocampus could account for this 

effect. 

206



NEUROPLASTICITY IN THE PRENATAL STRESS RAT MODEL OF 

DEPRESSION: EFFECTS OF AGOMELATINE TREATMENT 

1 Morley-Fletcher S, 1 Mairesse J, 2 Daszuta A, 2 Soumier A, 2 Banasr M, 3 Zuena AR, 

4 Mocaer E, 3 Matteucci P, 3 Casolini P, 3 Catalani A, Maccari 1,3 S. 

1 Lab. of Perinatal Stress, University of Lille 1 USTL, Villeneuve d'Ascq, FR sara.morleyfletcher@univ-lille1.fr, 

fax +33.320.434602 ; 2 IC2N, CNRS, Marseille, FR ; 3 Univ of Rome 

La Sapienza, IT; 4 I.R.I.S, Courbevoie, FR 

Hippocampal neurogenesis can be influenced by several environmental factors and stressful 

experiences diminish the formation of the hippocampal granule cells in a number of 

mammalian species [4]. In rats, chronic antidepressants treatment can oppose the action of 

stress on the morphology and proliferation of hippocampal neurons, inducing changes in 

neuroplasticity and increasing adult neurogenesis in animals [9]. Thus, behavioural effects of 

antidepressant may be mediated by the stimulation of neurogenesis in the hippocampus [12]. 

Among the stress-induced animal models of depression that have been developed, the prenatal 

restraint stress (PS) in the rat represents a promising model of early stress with high face and 

predictive validity [7,10,11]. PS rats present a life span reduction of hippocampal 

neurogenesis [6], an impairment of hypothalamus-pituitary adrenal axis feedback inhibition 

[8], a generalized disorganization of circadian rhythms [3] and increased anxiety [13]. We 

evaluated the effect of a chronic treatment (6 weeks, 40 mg/kg i.p.) with the new 

antidepressant agomelatine, a melatonin agonist with 5-HT 2C antagonist properties [1], on 

hippocampal neurogenesis and on PSA-NCAM and BDNF expression, markers of 

neuroplasticity, in PS male adult rats. To evidence neurogenesis and cell survival, the 

thymidine-analogue bromodeoxyuridine (BrdU, 75 mg/kg i.p. twice daily for 4 days) was 

injected after 3 weeks of the agomelatine treatment which was then continued for additional 3 

weeks. Prenatal stress reduced hippocampal neurogenesis whereas it increased PSA-NCAM 

and BDNF. The effects of PS were reversed by the chronic agomelatine treatment. 

Interestingly, agomelatine’s effect on neurons’ survival was selectively observed in the ventral 

part of the dentate gyrus, a brain region specifically involved in anxiety [5]. Also, based on the 

potential involvement of glutamatergic system in neurogenesis and anxiety [2], we studied the 

long-term effects of PS and antidepressant on the expression of hippocampal mGluR5 

subtype. Prenatal stress reduced mGluR5 expression, whereas chronic agomelatine increased 

it. Finally, to investigate the functional behavioural impact of neurogenesis, we tested animals 

in the elevated-plus maze test to assess their anxiety-like response. PS rats treated with 

agomelatine spent more time on the open arms of the elevated-plus maze, suggesting a 

possible causal link between increased hippocampal neurogenesis and glutamatergic 

transmission and, the attenuated anxiety-like behaviour. The results obtained with agomelatine 

provide further evidence of neuroplasticity as one of the targets of antidepressants and, further 

reinforce the high predictive validity of the PS rat as animal model of depression. 


1. Banasr M, et al. (2006). Agomelatine, a new antidepressant, induces regional changes in hippocampal 

neurogenesis. Biol Psychiatry 2006 1:59(11):1087-96. 

2. Di Giorgi Gerevini V. et al. (2004). The mGlu5 metabotropic glutamate receptor is expressed in zones of 

active neurogenesis of the embryonic and postnatal brain. Brain Res.Dev.Brain Res. 150: 17-22 

207


3. Dugovic C. et al. (1999). High corticosterone levels in prenatally stressed rats predict persistent paradoxical 

sleep alterations. J.Neurosci. 19: 8656-8664 

4. Kempermann G, et al. (1997). More hippocampal neurons in adult mice living in an enriched environment. 

Nature 386:493-495. 

5. Kjelstrup KG, et al. (2002). Reduced fear expression after lesions of the ventral hippocampus. Proc Natl 

Acad Sci U S A 99:10825-10830. 

6. Lemaire V, et al. (2000). Prenatal stress produces learning deficits associated with an inhibition of 

neurogenesis in the hippocampus. Proc Natl Acad Sci U S A 97:11032-11037. 

7. Maccari S, et al. (2003). Prenatal stress and long-term consequences: implications of glucocorticoid 

hormones. Neurosci Biobehav Rev 27:119-127. 

8. Maccari S, et al. (1995). Adoption reverses the long-term impairment in glucocorticoid feedback induced by 

prenatal stress. J Neurosci 15:110-116. 

9. Malberg JE, et al. (2000). Chronic antidepressant treatment increases neurogenesis in adult rat 

hippocampus. J Neurosci 20:9104-9110. 

10. Morley-Fletcher S, et al. (2003). Prenatal stress in rats predicts immobility behavior in the forced swim test. 

Effects of a chronic treatment with tianeptine. Brain Res 989:246-251. 

11. Morley-Fletcher S, et al. (2004). Chronic treatment with imipramine reverses immobility behaviour, 

hippocampal corticosteroid receptors and cortical 5-HT(1A) receptor mRNA in prenatally stressed rats. 

Neuropharmacology 47:841-847. 

12. Santarelli L, et al. (2003). Requirement of hippocampal neurogenesis for the behavioral effects of 

antidepressants. Science 301:805-809. 

13. Vallee M, Mayo W, Dellu F, Le MM, Simon H, Maccari S (1997). Prenatal stress induces high anxiety and 

postnatal handling induces low anxiety in adult offspring: correlation with stress-induced corticosterone 

secretion. J Neurosci 17:2626-2636. 

208



NEUROBEHAVIOURAL DISORDERS IN THE INFANT REELER MOUSE MODEL: 

INTERACTION OF GENETIC VULNERABILITY AND CONSEQUENCES OF 

MATERNAL SEPARATION 

Ognibene E. 1 , Adriani W. 1 , Macrì S. 1 , Laviola G 1, *. 

1 Section of Behavioural Neuroscience, Department of Cell Biology and Neuroscience, 

Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy. Fax +39-06-4957821 email: 

laviola@iss.it 

Studies on heterozygous (HZ) reeler mice suggest a relationship between reelin (a protein of extra 

cellular matrix) haploinsufficiency and the presence of altered neural networks and behaviour. 

Neonatal adverse and/or stimulating experiences might interfere with the emergence of this geneticdependent 

phenotype. Repeated episodes of maternal separation early in ontogeny result in enduring 

neuroendocrine, neurochemical and behavioural alterations in the offspring. Therefore, in order to 

investigate whether developmental indexes of neurobehavioural disorders can be studied in the infant 

reeler mouse model, and whether ontogenetic adverse experiences may question or improve its 

suitability, homozygous reeler (RL), heterozygous (HZ) and wild-type (WT) mouse pups underwent 

maternal separation (SEP, 5 h/day) or handling (H, 3 min/day) on PND 2–6. As expected, a sex 

difference appeared, for measure of emotional and communicative behaviour in infant mice. In 

particular, male H pups emitted a higher number of ultrasonic vocalizations (USV) compared to H 

females (p < 0,05). Interestingly, such sex difference was not observed in the SEP group. On PND 7, 

compared to other genotypes, RL mouse pups from the H control group, showed reduced levels USV 

production and of locomotion. Surprisingly, this deficit in RL mice was fully reverted by maternal 

separation. Maternal separation per se reduced social motivation in the homing test at PND 9 in WT 

mice, with no effects on HZ and RL ones. Additionally, female pups emitted much lower levels of 

ultrasound production than males within the H control group. The deficit in both emotional and 

communicative capabilities, however, apparently disappeared when reeler pups were faced, early in 

development, with the SEP condition. In fact, repeated episodes of maternal separation exerted a strong 

contrasting effect on homozygous RL mice, dramatically increasing, and perhaps activating their 

masked USV capacity production. This behavioural activation rendered these pups similarly able to 

respond to a stress condition, such as the repeated maternal separation, as the other two genotypes. 

Although hypothetical, it is tenable that the “beneficial” effects of repeated maternal separation, 

observed in RL pups, reflect a compensatory process of neural plasticity involving the activation of 

hormonal steroid pathways. The repeated 5-h long episodes of maternal separation are frequently 

associated with a substantial corticosterone release in newborn pups [1]. In turn, such a corticosteroid 

release might exert a stimulatory action in RL pups from SEP dam condition, early in development, 

compared to those from H-control group. In line with this interpretation, it has been recently reported 

that i.c.v. steroid hormones administration on PND 4 compensates reelin haploinsufficiency in terms of 

Purkinje cell loss during development [2]. 

The present results provide evidence that unusual stress and related hormonal stimulation early in 

development may (i) independently shape individual behavioural phenotype and (ii) interact with a 

genetic make-up to substantially modify its “natural” developmental trajectories. 


[1] M.V. Schmidt, S. Levine, S. Alam, D. Harbich, V. Sterlemann, K. Ganea, E.R. de Kloet, F. Holsboer, M.B. Muller. 

Metabolic signals modulate hypothalamic–pituitary–adrenal axis activation during maternal separation of the neonatal 

mouse. J. Neuroendocrinol. 18 (2006) 865–74. 

[2] G. Assenza, F. Biamonte, R. Cesa, P. Strata, F. Keller. Interaction between reelin and estrogens on Purkinje cells during 

development: a model of cerebellar pathology in autism and related disorders. In: Abstract national congress of the Italian 

Society for Neuroscience and joint Italian-Swedish neuroscience meeting Regina Isabella Congress Center Lacco Ameno. 

2006. 

209


ASSESSMENT OF MODULATORY EFFECTS OF CORTICOSTERONE ON ANXIETY 

RELATED BEHAVIOR IN MICE 

Vafaei AA, Taherian AA, Jarrahi M 

Physiology Research Center, Semnan University of Medical Sciences, Semnan, Iran 

E-mail: aavaf43@yahoo.com 

Previous studies indicated that glucocorticoid receptors probably involve on anxiety reactions. This 

study was designed to evaluate modulatory effects of corticosterone on anxiety in mice. In this study, 

seventy male albino mice (25 – 30 gr) were used. Also we used of Elevated plus Maze (EPM) model 

for assessment of anxiety. Corticosterone (CORT) as a glucocorticoid receptor agonist (0.1, 0.5, 1, 3 

and 10 mg/kg) or vehicle were injected IP, 30 min before of test. At the first time for increasing 

activity animals have put inside the black wall box for 5 min. Then animal transfer to the EPM and 

evaluation their anxiety reaction that including of number entrances and time spent in open arm. 

Results indicated that injection of CORT in doses of 1 and 3 mg/kg reduced of reaction anxiety and 

with compare to sham and control groups in the test group animals have more number of entrances and 

spent more time in open arm (P



Time spent in open arm (Sec) 

120 

100 

80 

60 

40 

20 

0 

Sham Cont CORT 

0.1 

CORT 

0.5 

* 

CORT 

1 

* 

CORT 

3 

CORT 

10 

Number of entrance to open 

arm 

7 

6 

5 

4 

3 

2 

1 

0 

Sham Cont CORT 

0.1 

CORT 

0.5 

* 

CORT 

1 

* 

CORT 

3 

CORT 

10 

Fig 1 A: The effect of CORT (0.1, 0.5, 1, 3 

and 10 mg/kg, SC) on anxiety related 

behavior in mice. (Time spent in open arm) 

*P


LATE EFFECTS OF IONIZING IRRADIATION ON CORTICOSTEROID 

RECEPTOR EXPRESSION IN RAT HIPPOCAMPUS: THE ROLE OF 

HYPOTHALAMUS-PITUITARY-ADRENAL AXIS 

Velickovic N. A., Djordjevic A. D., Horvat A. I., Demajo M. A. 

VINCA Institute for Nuclear Sciences, Laboratory for Molecular Biology and 

Endocrinology, PO Box 522, Belgrade 11000, Serbia, Fax: +381112455561 

e-mail: natasaxx@vin.bg.ac.yu 

Radiotherapy continues to be the primary treatment modality for malignant brain 

tumors. This treatment, however, inevitably involves the inclusion of normal CNS tissue 

in the radiation field. Experimental studies have shown that radiation exposure induces 

both short and long-term deleterious effects on the functional capacity of the 

hypothalamus-pituitary-adrenal (HPA) axis [2]. The primary element in HPA axis 

feedback regulatory mechanisms considered to be the hippocampus. The hippocampus is 

highly sensitive to corticosteroids (CS), exemplified by its enriched complement of CS 

receptors. Two types of CS receptors are found in the brain: mineralocorticoid (MR) and 

glucocorticoid (GR) receptors. Differential activation of this dual receptor system may 

account for the opposing actions of corticosteroids on neuronal proliferation, survival, 

and death in hippocampus [1]. 

The aim of the present study was to estimate the effect of ionizing irradiation on 

corticosteroid receptor expression in the rat hippocampus and to compare the expression 

of GR protein with changes in HPA axis functioning during the late radiation response 

phase. Infantile (18 days old) male Wistar rats received a single dose of 10 Gy (the dose 

was selected to be clinically relevant [3]) in the head region from a Co 60 -source. Late 

effects of gamma-irradiation on HPA axis have been studied at the age of 42 days in 

basal and after restrain stress, followed by assessment of GR and MR mRNA levels and 

protein expression by RT-PCR and Western blot, respectively. 

In our study, radiation exposure lead to a decrease of stress-induced but not basal 

plasma corticosterone levels (Fig. 1, C-3 vs. IR-3, p< 0.05). Treatment with 

dexamethasone (DEX) revealed the nonsuppression of stress-induced HPA axis function 

in irradiated rats, thus suggesting the dampened sensitivity of negative feedback of 

glucocorticoids as a long-term consequence of irradiation (Fig. 1, IR-4 vs. C-4, p< 0.01). 

Corticosterone (ng/ml) 

800 

700 

600 

500 

400 

300 

200 

100 

0 

Basal 

Stress 

* 

** 

C-1 IR-1 C-2 IR-2 C-3 IR-3 C-4 IR-4 

DEX: - - + + - - + + 

Groups 

Figure 1. Plasma corticosterone 

levels in control (C) and irradiated 

(IR) animals under different 

treatment. Groups 1 and 3 

received vehicle injection, groups 

2 and 4 received DEX injection 

(30 µg/kg). Groups 3 and 4 were 

subjected to acute stress, groups 1 

and 2 were not subjected to this 

stress. 

Values are means ± SEM (n=7-9); 

* p< 0.05, ** p< 0.01 vs. controls. 

212



Since GR (coordinately with MR) mediates the corticosteroid control of the 

hippocampus on the HPA axis, we assessed the level of GR and MR mRNA and protein. 

Western blot analysis showed a decreased translocation of the GR from the cytoplasm to 

the nucleus in the presence of DEX, without affecting the level of GR protein in whole 

cell extracts. Nuclear trafficking of the GR is regulated by the macromolecular hsp90 

heterocomplex. Additional Western analyses were performed to determine whether 

deficits in GR translocation were associated with decreased chaperone expression or 

trafficking. Expression of hsp90 and hsp70 was not affected by irradiation in both nuclear 

and cytoplasmic extracts, thus indicating to some other mechanism lying in this 

phenomenon. In contrast, no change in nuclear (or cytosolic) MR protein was observed in 

rat hippocampus after irradiation, which may denote that the corticosteroid receptor 

translocation deficit was limited to the GR. The obtained results are confirmed by RT- 

PCR, since GR down-regulation is accompanied by reduced GR mRNA, whereas the MR 

mRNA level did not show statistically significant differences. 

In conclusion, our data suggest that irradiation of the cranial region may attenuate 

the translocation and activation of the GR by circulating hormones, thereby diminishing 

negative feedback on the HPA axis solely by GR, and not by MR, in the hippocampus. 

This study was supported by Project grant No. 143044, the Ministry of Science and 

Environmental Protection, Republic of Serbia. 


[1] Almeida, O.F.X., Conde, G. L., Crochemore, C., Demeneix, B. A., Ficher, D., Hassan, A. H. S., Meyer, 

M., Holsboer, F., Michaelidis, T. M., 2000. Subtle shifts in the ratio between pro- and antiapoptotic 

molecules after activation of corticosteroid receptors decide neuronal fate. FASEB J 14, 779-790. 

[2] der Meeren, A. V., Monti, P., Lebaron-Jacobs, L., Marquette, C., Gourmelon, P., 2001. Characterization 

of the acute inflammatory response after radiation exposure in mice and its regulation by Il4. Radiat. 

Res. 155, 858-865. 

[3] Schunior, A., Mullenix, P. J., Zengel, A. E., Landy, H., Howes, A., Tarbell, N. J., 1994. Radiation 

effects on growth are altered in rats by prednisone and methotrexate. Pediatr. Res. 35, 416-423. 

213


Others 

• Allieri F., Hardin-Pouzet H. (Italy) Monoaminergic regulation of AVP system in 

limbic nuclei during estrous cycle, possible implication in axiety and depression 

• Amini H., Salimpour S., Mirzaei M., Sabetkasaei M., Ahmadiani A. (Iran) 

Involvement of the enzyme 5alpha-reductase in morphine-induced dopamine 

release in the nucleus accumbens: a microdialysis study in rats 

• Andrade T.G.C.S., Sergio T.O., Broiz A.C.G., Avanzi V. (Brazil) The effect of 

oestradiol benzoate in the median raphe nucleus on the exploratory behaviour in 

forced swimming test 

• Andrade T.G.C.S. , Almada R.F., Nakamuta J.S., Avanzi V. . (Brazil) Effect of 

oestrogenic action in the median raphe nucleus on the exploratory behaviour of 

previously immobilized female rats, in the elevated plus maze 

• Aste N., Shimada K., Watanabe Y. and Saito, N. (Japan) Neurosteroidogensis in 

the quail brain 

• Atwood C.S., Wilson A.C., Bowen R.L., Vadakkadath Meethal S. and Liu T. 

(USA) The potential role of a neuronal autocrine/paracrine mechanism in the 

regulation of neurosteroid production: luteinizing hormone receptor mediates 

neurosteroid production via upregulation of steroidogenic acute regulatory protein 

expression 

• Bramanti V., Bronzi D., Raciti G., Avitabile M., Avola R. (Italy) Neurosteroidsgrowth 

factors interaction induces up and down regulation of GFAP and vimentin 

expression in astroglial cells maintained under serum-free stressed culture 

conditions 

• Balog J., Szegő É.M., Erdei F., Szabó G., Juhász G. and Ábrahám I.M. (Hungary) 

Sex differences in rapid estrogen action on GABAergic neurons in vivo 

• Campbell B.C. (USA) DHEAS and the hominoid brain 

• Carrillo B, Pinos H., Guillamón A., Panzica G.C., Pérez-Izquierdo M.A., Collado 

P. (Spain) Nitric oxide effects on sexual and maternal behavior in the female rat 

• Ceccarelli I., De Padova A.M., Fiorenzani P., Massafra C. and Aloisi A.M. (Italy) 

Single opioid administration modifies gonadal steroids in both the CNS and plasma 

of male rats 

• Chalbot S., Lecanu L., Greeson J. and Papadopoulos V. (USA) Oxidationdependent 

plasma DHEA formation as a diagnostic tool for Alzheimer’s disease 

pathology: results from a trial 

• Csakvari E., Hoyk S., Szajli A., Kurunczi A., Gyenes A., Berger A., and Parducz 

A. (Hungary) Lesion-induced glial reaction in the rat olfactory bulb: effect of 

DHEA and DHEA derivatives

• Diz-Chaves Y., Pernía O., Carrero P., Garcia-Segura L.M. (Spain) Dose-response 

study of antidepressant effects of estrogenic compounds in ovariectomized mice in 

the forced swim test 

• Do Rego J.L., Tremblay Y., Luu-The V., Acharjee S., Repetto E., Galas L., Castel 

H., Vallarino M., Kwon H.B., Bélanger A., Seong J.Y., Pelletier G., Tonon M.C., 

Vaudry H. (France) Neuroanatomical and biochemical evidence for the 

occurrence of cytochrome P450 C17 in the frog brain. Regulation by vasotocin and 

mesotocin 

• Fester L., Zhou L., Bütow A., Huber C., von Lossow R., Jarry H., M. Rune G.M. 

(Germany) Synaptogenesis: promoted by cholesterol or estradiol? 

• Hiroi R., Neumaier J.F. (USA) Estrogen selectively increases tryptophan 

hydroxylase-2 and decreases 5HT1 B mRNA expressions in distinct subregions of 

rat dorsal raphe nucleus: association between gene expression and anxiety 

behavior in the open field 

• Kanematsu T. and Hirata M. (Japan) PRIP, a phospholipase C-related inactive 

protein, regulates GABA A receptor endocytosis 

• Löfgren M., Johansson I-, Meyerson B. and Bäckström T. (Sweden) Progesterone 

withdrawal sensitivity in female rats relates to differences in baseline behavior of 

risk taking and exploration 

• Longo D., Baldelli E., Zini I., Zoli M., Avoli M., Biagini G. (Italy) P450scc is 

induced in neuronal and glial cells after status epilepticus: modulatory effects of 

neurosteroids on epileptogenesis 

• Martín-García E., Darbra S., Pallarés M. (Spain) Alterations of neonatal levels of 

allopregnanolone and the novelty-directed behavioural response to 

intrahippocampal administration of allopregnanolone in adulthood 

• Milani P., Ginanneschi F., Biasella A., Bonifazi M., Rossi A., Mazzocchio R. 

(Italy) Heightened seizure susceptibility following the administration of human 

chorionic gonadotropin 

• Mizokami A., Kanematsu T. and Hirata M. (Japan) Roles of PRIP in trafficking of 

gamma2 subunit containing GABA A receptor 

• Nasir RH, Chen C, Bellinger D, Korrick SA (USA) Prenatal estrogens and the 

development of memory and learning 

• Nobahar M, Vafaei AA (Iran) Assessment of interaction between sex hormones 

and incidence of epilepsy crisis in female 

• Ohya T., Kodama M., Hayashi S. (Japan) Vasotocin/isotocin neurons are 

decreased after spawning in the female medaka fish (Oryzias latipes) brain: 

localization of aromatase and estrogen receptor homologue

• Parkash J. and Kaur G. (India) GnRH-Astrocytes interactions involved in GnRH 

neurosecretion: role of PSA-NCAM through changing activity and expression 

levels of polsialyltrasferase. 

• Prange-Kiel J, Jarry H, Kohlmann P, Schön M, Lohse C, Rune GM (Germany) Is 

there a link between the hypothalamo-pituitary-gonad axis and the hippocampus? 

• Romanò N., Jasoni C.L. and Herbison A.E. (New Zealand) Rapid actions of 

estrogen on adult GnRH neurons 

• Scurati S., Maschi O., Crotti S., De Angelis L., Melcangi R.C. and Caruso D. 

(Italy) Assessment of neuroactive steroid levels in plasma and nervous sistem by 

liquid chromatography-mass spectrometry 

• Timby E., Bäckström T., Nyberg S., Wihlbäck A.-C.N., Bixo M. (Sweden) 

Administration of allopregnanolone decreases secretion of gonadotropins in 

healthy women of fertile age 

• Venard C., Boujedaini N., Belon P., Mensah-Nyagan A.G. and Patte-Mensah C. 

(France) Pharmacological modulators of the glycinergic system regulate 

allopregnanolone biosynthesis in the rat spinal cord 

• Vlad A.G (Romania) It is possible that the unspecific steroids for gonadotropin 

system modulate neuronal pulsatility activity from POA–SCH of the hypothalamus 

for regulating LH–RH relase and ovulation trigger?



MONOAMINERGIC REGULATION OF AVP SYSTEM IN LIMBIC NUCLEI 

DURING ESTROUS CYCLE, POSSIBLE IMPLICATION IN AXIETY AND 

DEPRESSION 

F.Allieri1,2, H. Hardin-Pouzet2 

1 Lab: Neuroendocrinologia, Dip.Anatomia, Farmacologia e Med. Legale Univ Torino, 

Italy 

2 Lab. Neurobiologie des Signaux Intracellulaires, CNRS UMR 7101, Université Pierre et 

Marie Curie Paris, France 

Estrogen appear to influence the intensity of several psychiartric and neurological 

disorders, including depression. In particular, depression and anxiety are common health 

problems affecting women, particularly during reproductive years. The cerebral structures 

implicated in such mood disorders are principally located in the limbic areas, namely in the 

bed nucleus of the stria terminalis (BST), the anterior part of medial amygdala (MeA) and 

the posterior part of the medial amygdala (MeP). These nuclei contain parvocellular 

arginine-vasopressinergic (AVP) neurons, which are known to be sensitive to gonadal 

steroids and receive a dense innervation from the serotoninergic and dopaminergic 

systems. As well know serotoninergic and dopaminergic systems are strongly implicated in 

the modulation of some behavioural disorders, like depression and anxiety. To understand 

the possible role of estrogen on the monoamines system and the consequent influence of 

both of theme on the AVP parvocellular neurons present in the BST, the MeA and the 

MeP. Particularly we focused on the possible interaction of monoamines during the estrous 

cycle. We treated male and female, taken in the different phases of the estrous cycle, mice 

C 3 H with para-clorophenyl alanine (pCpA) and alpha-metylparatyrosine (α-MPT); these 

two pharmaco can inhibit whole patway of serotonin and dopamine. We observe the a 

prominent decrease of AVP protein quantity in the MeP alpha-MPT treated male mice and 

an increase of AVP protein quantity in the MeA and MeP of pCpA treated male mice, 

comparing to control animals. Normally in the female mice AVP circulating levels 

detected in blood change during the different phases of the estrous cycle; we observe that 

treatment with α-MPT and pCpA can affect the quantity of AVP protein detected in the 

BST, in the MeA and in the MeP, in comparison with control female and male mice. We 

detect that MeA and MeP respond in a different manner comparing the differnt phases of 

estrous cycle. These data suggest that the estrogen can lead monoamines regulation of 

AVP neurons. So we suggest a double control of estrogen and monoamines on the 

AVPergic system present in nuclei, as the BST, the MeA and the MeP, strongly related 

with the control of behaviour. Moreover this doble action can partecipate to the beginning 

of behavioural disorders like anxiety and depression. 

217


INVOLVEMENT OF THE ENZYME 5ALPHA-REDUCTASE IN MORPHINE- 

INDUCED DOPAMINE RELEASE IN THE NUCLEUS ACCUMBENS: A 

MICRODIALYSIS STUDY IN RATS 

Amini H.*, Salimpour S., Mirzaei M., Sabetkasaei M., Ahmadiani A. 

Department of Pharmacology, Neuroscience Research Center, Shaheed Beheshti Medical 

University, P.O. Box 19835-355, Tehran, Iran. E-mail: hamini@sbmu.ac.ir 

The enzyme 5alpha-reductase (5alpha-R) is one of the key enzymes in the biosynthesis of 

neurosteroids. We have already reported that morphine increases the 5alpha-R activity in 

the rat CNS following acute and chronic administration, but the question remains about the 

significance of this finding in morphine effects. It is well known that morphine and other 

addictive drugs increase dopamine release in the nucleus accumbens as well as dopamine 

metabolites, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In 

the present study, the effects of concomitant administration of morphine and finasteride (an 

inhibitor of 5alpha-R) on the extracellular levels of DOPAC and HVA in the nucleus 

accumbens of rats were studied using in vivo microdialysis and high performance liquid 

chromatography with electrochemical detection. Acute morphine (7 mg/kg, i.p.) 

treatment increased the levels of DOPAC and HVA in the nucleus accumbens to 

approximately two-fold of basal levels. Pretreatment with finasteride (5 mg/kg, i.p.), 2 h 

before morphine injection (7 mg/kg. i.p.) significantly changed the effects of morphine on 

the levels of DOPAC and HVA in the nucleus accumbens. These results suggest that the 

enzyme 5alpha-R involves in the mesolimbic dopaminergic pathway. 

218



THE EFFECT OF OESTRADIOL BENZOATE IN THE MEDIAN RAPHE 

NUCLEUS ON THE EXPLORATORY BEHAVIOUR IN FORCED SWIMMING 

TEST 

Andrade, T.G.C.S. 1 , Sergio, T.O., Broiz, A.C.G., Avanzi,V 1 . 

Laboratory of Physiology – University UNESP- Assis – São Paulo - Brazil 1 - Fax: 55 18 

3302-5849 – 

E-mail: raica@assis.unesp.br 

The median raphe nucleus (MRN) is located in the brain stem and constitutes one of the main 

systems of ascending serotonergic innervations. Based on clinical and experimental evidence, 

Deakin & Graeff [1] have hypothesized that the 5-HT MRN-hippocampal pathway underlies 

adaptation to chronic stress and the failure of this mechanism would impair the putative 5-HT 1A 

“resilience” system, slowing adaptation to stress and predisposing animals to depressive symptoms 

and humans to depression. Recently, oestrogenic receptors have been identified in the MRN [2]. In 

addition to that, oestrogen deficiencies have been related to the manifestation of anxiety and 

depression. Women in periods of low estrogenic concentration tend to present high level of 

anxiety, which might lead to occurrence of depression. It is known that oestrogen reposition 

increases the serotonergic neurotransmission with a direct influence on the sensibility of the 5-HT 

receptors in different areas of the brain, especially in the hippocampus. The main objective of the 

study presented here was to investigate the action of Oestradiol Benzoate (OB), microinjected in 

the MRN, on the behavioural responses in Forced Swimming Test (FST), an animal depression 

model [3]. With this objective we analysed ovariectomized female Wistar rats, 200g medium 

weight at the beginning of the experimental sessions, previously exposed to FST 24 hours before 

the cannulation. Seven days after the stereotaxic surgery for the insertion of the guiding cannula of 

access to the MRN, the animals were microinjected with OB, in doses of 600ng and 1200ng, in 

volume of 0,2ul, and were placed in FST. The control group received the same volume of saline or 

oil (diluent of OB). The results showed that the microinjection of OB in the MRN, in higher dose, 

led to an increase in the mobility in the test [F(3,38)=7.78; p=0.000] without causing an increase in 

motor activity in the arena [F(3,38)=0.26; p=0.851], similar to the effect of anti-depressive drugs in 

this test. These results are in accordance with other studies carried out in our laboratory in males 

and females with lesions in NMR or direct microinjection of 8-OH-DPAT, an agonistic of 5-HT 

receptors, in this structure. As conclusion, the beneficial effect of Oestradiol Benzoate may be 

measured by specific receptors in the MRN. Neuron located in this structure would also be 

modulated by endogen oestrogen, confirming studies, which point out that there are significant 

betterments in emotional symptoms related to depression, in periods of higher level of this 

hormone or due to oestrogen reposition. 

Support: FAPESP- Brazil 


1. DEAKIN, J.F.W., GRAEFF, F.G. 5-HT and mechanisms of defense. Journal of 

Psychopharmacology, 5(4):305-315, 1991. 

2. LERANTH, C. SHANABROUGH, M., UPHOUSEL, L. Oestrogen receptor-α in the 

serotonergic and supramammillary area calretinin-containing neurons of the female rat. 

Experimental Brain Research, 128:417-420, 1999. 

3. PORSOLT, R.D. et al. Behavioral despair in rats: a new model sensitive to antidepressant 

treatments. European Journal Pharmacology, 47: 379-391, 1978. 

219


EFFECT OF OESTROGENIC ACTION IN THE MEDIAN RAPHE NUCLEUS ON 

THE EXPLORATORY BEHAVIOUR OF PREVIOUSLY IMMOBILIZED 

FEMALE RATS, IN THE ELEVATED PLUS MAZE 

Andrade T.G.C.S. 1 , Almada 1 R.F., Nakamuta 1 J.S., Avanzi V 1 . 

Laboratory of Physiology – University UNESP- Assis – São Paulo - Brazil 1 - Fax: 55 18 

3302-5849 – E-mail: raica@assis.unesp.br 

Serotonin has been implicated in the aetiology of anxiety disorders. Gender differences in 

serotonergic functions have been observed in animal models of anxiety. Several studies 

have shown changes in the serotonergic activity correlated to the phases of the female 

hormonal cycle, and that ovarian hormones act on the synthesis, liberation, reuptake and 

catabolism of 5-HT. Clinical studies have reported marked emotional alterations with 

periods of increased anxiety, during low-oestrogen phases of the hormonal cycle. 

Oestrogen replacement improves those symptoms and it also increases serotonergic 

neurotransmission. In the same direction, experimental studies in rodents using animal 

models of anxiety have shown anxiety reduction during oestrogenic phases of the 

hormonal cycle as well as after oestrogen administration. It seems that oestrogen 

replacement increase serotonergic neurotransmission, influencing the sensitivity of 5-HT 1A 

receptors in several brain areas, especially in the hippocampus and raphe nuclei. The 

existence of oestrogen receptors (ER) in cellular bodies of the median raphe neurones has 

been demonstrated [1]. The median raphe nucleus (MRN) is located in the brain stem and 

constitutes one of the main systems of ascending serotonergic innervations and has been 

related to behavioural inhibitions and stress resistance mechanisms. The inefficiency of 

this structure could lead to the occurrence of emotional disturbances, such as anxiety and 

depression. Injection of oestradiol into the MRN impairs the 5-HT innervation of the 

hippocampus [2], which is critical for the process of behavioural inhibition that is 

supposed to underlie anxiety. Recently, the microinjection of the oestradiol benzoate into 

MRN occasioned anxiolytic effect in the elevated plus-maze [3]. This effect was abolished 

by microinjection into MRN of the Way100635, a 5-HT 1A receptors antagonist [3,4,5]. 

Like this, the study presented here had as its main objective to evaluate the effect of an 

acute stressor, immobilization, on behavioural responses of ovariectomized female rats in 

the elevated plus maze (EPM), an animal anxiety model [6] and to verify if the direct 

microinjection of Oestradiol Benzoate (OB) in the MRN would alter the anxiogenic effect 

caused by the previous exposition the acute stressor. Events, like restraint and social 

separation, have been described like powerfull stressors, unchained of inhibitory response 

in animal tests of anxiety [7]. Females Wistar rats, 200g medium weight at the beginning 

of the experimental sessions, kept in groups of five per box, under controlled temperature 

and illumination (12/12 hours light-dark cycles, 50 lux and 21º C ±1º) receiving food and 

water ad libitum were submitted to a bilateral ovariectomy and after fourteen days were 

submitted to a stereotaxic surgery for the implantation of an access cannula to the NMR. 

Seven days after, the animals were microinjected in the MRN with saline, Sesame Oil or 

OB, in doses 600 or 1200 ng (0,2 µ/min) and evaluated immediately after the 

administration of the drug in the EPM, for the period between 14 and 17 hours. 

Previously, on the sixth day were immobilized for two hours in a metal box and kept in 

individual box for 24 hours till the behavioural evaluation (social separation). The results 

showed that the exposition to previous stress (immobilization) led to an anxiogenic effect 

in the elevated plus maze (EPM). The ovariectomy produced effect only when the animals 

were previously immobilized. We verified that the microinjection of EB in the MRN 

220



neutralized the stressing effect of the immobilization in this animal model of anxiety, 

leading to pattern of exploratory behavioural responses similar to the non immobilized 

females: that is, a clear anxiolytic effect of this substance microinjected in the MRN; % of 

entrances in open arms/total [F(3,79)=14,35; p=0.000]; % of time in open arms/total 

[F(3,79)=11.90; p=0.000]. As conclusion, it seems that the anxiogenic effect due to the 

decreasing in the oestrogen levels may be evidenced when the females are exposed to 

previous stressors. Women in puerperium, in climacterium, or in premenstrual period, may 

present emotional symptoms when exposed to aversive events during these situations, as 

there is a low concentration of oestrogen. The direct microinjection of Oestradiol 

Benzoate in the MRN has led to behavioural desinhibition of the females. This may 

explain the salutary effect of endogen oestrogen and of the oestrogenic reposition in the 

prevention and/or treatment of anxiety disorders and affective disturbances. 

Support: FAPESP- Brazil 


1. LERANTH, C. SHANABROUGH, M., UPHOUSEL, L. Oestrogen receptor-α in the 

serotonergic and supramammillary area calretinin-containing neurons of the female rat. 

Experimental Brain Research, 128:417-420, 1999. 

2. PRANGE KIEL, J., RUNE, G., M., LERANTH, C., Median raphe mediates estrogenic 

effects to the hippocampus in female rats. European Journal Neuroscience, 249:341- 

351, 2004. 

3. ANDRADE, T.G.C.S.; NAKAMUTA, J.S., AVANZI, V., GRAEFF, F.G. Anxiolytic 

effect of oestradiol in the median raphe nucleus mediated by 5-HT 1A. Behavioural Brain 

Research,. 163: 18-25, 2005. 

4. ANDRADE, T.G.C.S., AVANZI, V. Effect of oestradiol benzoate microinjected into 

median raphe nucleus on anxiety in the conditioned fear test. Frontiers in 

Neuroendocrinology, 27: 134-138. 

5. ANDRADE, T.G.C.S., VIANA, R.A., NAKAMUTA, J.S., AVANZI, V. Different 

effects of the oestradiol benzoate microinjected into raphe nuclei on anxiety. Frontiers 

in Neuroendocrinology, 27: 134-138, 2006. 

6. PELLOW, S., CHOPIN, P., FILE, S. E., BRILEY, M., Validation of open-closed arm 

entries in an elevated plus-maze as a measure of anxiety in the rat. Journal 

Neuroscience Methods, 14: 149-167, 1985. 

7. KENETT, G.A., DOURISH, C.T., CURZON, G. Antidepressant-like action of 5-HT1A 

agonist and conventional antidepressants in an animal model of depression. European 

Journal Pharmacology, 134: 265-274, 1987. 

221


NEUROSTEROIDOGENSIS IN THE QUAIL BRAIN 

Aste N., Shimada K., Watanabe Y. and Saito N. 

Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya 

University,Chikusa, Nagoya 464-8601 Japan, Tel & Fax: +81-52-789-4067. 

aste@agr.nagoya-u.ac.jp 

The brain, besides being a target for gonadal and adrenal steroids, is also a site of steroids 

synthesis. Steroids of brain origin (neurosteroids), may work locally by modulating 

centrally controlled functions related to reproduction, learning and memory formation, 

which show sexual differences [1, 4]. Furthermore in situ formation of estradiol from 

cholesterol may play a role in brain sexual differentiation [3]. It is therefore of importance 

to understand whether the synthesis and the location of neurosteroidogenic enzymes show 

sexual dimorphism either in the adult or in the developing animals. 

Among birds, Japanese quail has been long employed as a model for studying the role of 

gonadal steroids on the activation of copulatory behavior and on brain sexual 

differentiation [2]. The activity and the presence of mRNA of cytochrome P450 side-chain 

cleavage enzyme (P450scc), 3β-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3β- 

HSD), cytochrome P450 17α-hydroxylase/c17, 20-lyase (P450c17), which are required to 

synthesize testosterone from cholesterol, have been demonstrated in quail brain [5]. 

However very little attention has been given to sexual differences in their content and 

distribution. 

In this study we assessed for the first time the presence of a sexual dimorphism for 

P450scc, 3β-HSD and P450c17 mRNA level in discrete regions of adult Japanese quail. 

Moreover the sexually dimorphic expression of these enzymes and of aromatase (which 

conerts testosterone into estradiol) was investigated in the prosencephalon of embryos at 

several ages spanning the critical period for brain sexual differentiation (E7, E9, E11, E15) 

using real-time PCR as a method. 

Expression of P450scc, 3β-HSD and P450c17 mRNAs was detected in all the investigated 

regions (telencephalon, diencephalon, optic lobes, cerebellum, brainstem) and showed 

regional differences. P450scc mRNA level did not show sexual differences. It was higher 

in the diencephalon and very low in the cerebellum, the other regions displaying 

intermediate values. The level of 3β-HSD and P450c17 mRNAs showed sexual 

dimorphism in the optic lobes, where higher mRNA level was detected in males than in 

females. 

P450c17 mRNA level showed large region-related variations in males being relatively 

higher in the optic lobes and in the brainstem than in the cerebellum and in the 

telencephalon. In females the region-related variations were comparatively of modest 

amplitude. 

3β-HSD mRNA level showed a more uniform distribution. This enzyme was more 

expressed in the diencephalon of females and in the optic lobes of males. 

In quail embryos 3β-HSD mRNA level showed a strong sexual dimorphism at E7, E9 and 

E15. At E7, females showed higher 3β-HSD mRNA level than males whereas males had 

more 3β-HSD mRNA than females at E9 and E15. In females, 3β-HSD mRNA was at its 

highest level at E7. In males, the relative level of 3β-HSD mRNA was significantly higher 

at E9 and E15 than at E7 and at E11. 

222



Conversely, the level of P450scc and of P450c17 mRNAs did not show sexual dimorphism 

nor development-dependent regulation. The expression of aromatase varied as a function 

of the embryonic age being significantly elevated at E9 and at E15 when compared to E7 

and E11 in both the sexes. 

In conclusion our study showed for the first time a sexual dimorphism in mRNA level of 

neurosteroidogenic enzymes in the adult and in the embryonic quail brain; provided the 

first description for P450scc gene expression in the optic lobes and confirmed the 

widespread capacity of quail brain to synthesize steroids starting from cholesterol. 

Further investigation is needed to understand the exact anatomical location of the 

described dimorphisms. 


1. Ball GF, and Balthazart J. Hormonal regulation of brain circuits mediating male sexual behavior in 

birds. Physiol Behav. 83 (2004) 329-46. 

2. Balthazart J., Baillien M., Cornil CA., and Ball GF. Preoptic aromatase modulates male sexual 

behavior: slow and fast mechanisms of action. Physiol Behav. 83 (2004) 247-70. 

3. Holloway CC, and Clayton DF. Estrogen synthesis in the male brain triggers development of the avian 

song control pathway in vitro. Nat Neurosci. 4 (2001) 170-5. 

4. Kawato, S., Yamada, M. and Kimoto, T. Brain neurosteroids are 4 th generation neuromessengers in the 

brain: Cell biophysical analysis of steroid signal transduction. Adv. in Biophys. (2003) 1-48. 

5 Tsutsui, K., Matsunaga, M., Miysbara, H., and Ukena, K. Neurosteroids biosynthesis in the quail brain: a 

review.Journal of Exp. Zool. 305A (2006) 733-742. 

223


THE POTENTIAL ROLE OF A NEURONAL AUTOCRINE/PARACRINE 

MECHANISM IN THE REGULATION OF NEUROSTEROID PRODUCTION: 

LUTEINIZING HORMONE RECEPTOR MEDIATES NEUROSTEROID 

PRODUCTION VIA UPREGULATION OF STEROIDOGENIC ACUTE 

REGULATORY PROTEIN EXPRESSION 

Atwood C.S.*, Wilson A.C.*, Bowen R.L.°, Vadakkadath Meethal S.* and Liu T.* 

*Department of Medicine, University of Wisconsin and the Geriatrics Research, Education 

and Clinical Center, VA Hospital, 2500 Overlook Terrace., Madison, WI 53705, U.S.A. 

Fax +608-2807291 e-mail: csa@medicine.wisc.edu 

°OTB Research, Raleigh, North Carolina 27615, U.S.A. 

Neurosteroids are synthesized by both neurons and glia, however the hormonal 

regulation of neurosteroid synthesis is unknown. In the gonads, steroid production is 

induced by luteinizing hormone (LH) and its signaling is mediated via luteinizing 

hormone/human chorionic gonadotropin (LH/hCG) receptors. Importantly, LH/hCG 

receptors have been shown to be expressed by neuronal cells throughout the brain 

(reviewed in [1, 2]). Since LH/hCG can cross the blood-brain barrier [3]; are present in 

cerebrospinal fluid [4]; and are expressed by neuronal cells [5, 6], we tested whether LH 

also might signal via neuronal LH/hCG receptors to modulate neurosteroid synthesis. 

Treatment of differentiated rat primary hippocampal neurons and human M17 

neuroblastoma cells with LH (100 mIU/ml) resulted in a 2-fold increase in pregnenolone 

secretion in both cell types, suggesting an increase in P450scc mediated cleavage of 

cholesterol to pregnenolone and its secretion from neurons [7]. To explore how LH might 

regulate the synthesis of pregnenolone, the precursor for steroid synthesis, we treated rat 

primary hippocampal neurons with LH (0, 10 and 100 mIU/ml) and measured changes in 

the expression of LH receptor and steroidogenic acute regulatory protein (StAR). LH 

induced a rapid (within 30 min.) increase in the expression of StAR, but induced a dosedependent 

decrease in LH receptor expression. Consistent with these results, the 

suppression of serum LH in young rats treated with leuprolide acetate for 4 months 

downregulated StAR expression but increased LH receptor expression in the brain. Taken 

together, these results indicated that LH induces neuronal pregnenolone production by 

modulating the expression of the LH receptor, increasing mitochondrial cholesterol 

transport and increasing P450scc mediated cleavage of cholesterol for pregnenolone 

synthesis and secretion. 

The source of LH (and hCG in humans) to signal for neurosteroid production is unclear; 

neuronal LH may be from pituitary LH secreted into the blood stream that crosses the 

blood-brain barrier [3]. Alternatively, LH has been localized to the cytoplasm of neurons 

in the cerebral cortex and hippocampus of human brain (e.g. [5]), raising the possibility 

that neurons synthesize LH de novo. Since gonadotropin-releasing hormone receptor 1 

(GnRHR1) has been localized to the limbic system of the brain, we tested whether 

GnRH1-induced neuronal LH expression by treating cultured human M17 neuroblastoma 

cells with GnRH1 for 6 h. M17 neuroblastoma cells expressed LHbeta mRNA while 

immunoblot analyses indicated the presence of 3 LH variants (~30, 47 and 60 kDa) that 

were upregulated by low concentrations of GnRH1, but downregulated at higher GnRH1 

concentrations [6]. LH expression also increased in differentiating embryonic rat primary 

cortical neurons. Our results demonstrate that neurons expressing GnRHR1 respond to 

GnRH1 by upregulating LH production. Post-reproductive surges in GnRH1 secretion 

may explain the accumulation of LH in pyramidal neurons of the aged human. 

224



The contribution of steroids produced in the brain, versus those produced in the 

periphery, to normal neuronal function is unknown. Given the importance of steroids to 

brain function, it is possible that neurosteroid production may be a mechanism to fine-tune 

the level of sex steroids in the brain. The neuronal production of sex steroids may be more 

crucial to brain function when gonadal sex steroid production decreases with menopause 

and andropause. This is supported by a recent study in mice showing that despite the 

decrease in serum sex steroids following ovariectomy, brain estrogen levels remain as high 

as control mice [8], possibly due to an increased production of neurosteroids as a result of 

the ovariectomy-induced increases in serum LH [9]. In humans however, brain 

neurosteroid levels have been shown to decrease when serum LH levels are increasing with 

age [10]. Neurosteroid concentrations also are decreased in individuals with AD versus 

age-matched controls even though serum 17β-estradiol levels are unchanged [8] and serum 

LH levels are elevated [11]. Further studies are required to determine the contribution of 

neuronal versus peripheral LH in mediating neurosteroid production. 

Taken together, our results suggest that the production of neurosteroids may be 

regulated in an autocrine/paracrine manner, whereby GnRH neurons secrete GnRH1 which 

binds to GnRHR1 in the limbic system for the local production of LH. Secreted neuronal 

LH then binds to LH receptors to elicit neurosteroid synthesis and secretion. It might be 

predicted that like the hypothalamic-pituitary-gonadal (HPG) axis, neurosteroids 

negatively feedback on GnRH neurons to regulate neuronal hormone synthesis. Thus, we 

propose that neurosteroid levels may be modulated by neuronal autocrine/paracrine 

mechanisms in addition to peripherally produced steroids. 


1. Lei Z.M., Rao C.V., Neural actions of luteinizing hormone and human chorionic gonadotropin. Semin. 

Reprod. Med. 19 (2001) 103-109. 

2. Vadakkadath Meethal S., Atwood C.S., The role of hypothalamic-pituitary-gonadal hormones in the 

normal structure and functioning of the brain. Cell Mol. Life Sci. 62 (2005) 257-270. 

3. Lukacs H., Hiatt E.S., Lei Z.M., Rao C.V., Peripheral and intracerebroventricular administration of 

human chorionic gonadotropin alters several hippocampus-associated behaviors in cycling female rats. 

Horm. Behav. 29 (1995) 42-58. 

4. Bagshawe K.D., Orr A.H. & Rushworth A.G., Relationship between concentrations of human 

chorionic gonadotrophin in plasma and cerebrospinal fluid. Nature 217 (1968) 950-951. 

5. Bowen R.L., Smith M.A., Harris P.L., Kubat Z., Martins R.N., Castellani R.J., Perry G., Atwood C.S., 

Elevated luteinizing hormone expression colocalizes with neurons vulnerable to Alzheimer's disease 

pathology. J. Neurosci. Res. 70 (2002) 514-518. 

6. Wilson, A.C., Salamat, M.S., Haasl, R.J., Roche, K.M., Karande, A., Vadakkadath Meethal, S., 

Terasawa, E., Bowen, R.L. and Atwood, C.S., Human neurons express Type I GnRH receptor and 

respond to GnRH I by increasing luteinizing hormone expression. J Endocrinol. 191 (2006) in press. 

7. Liu T., Wimalasena J., Bowen R.L. & Atwood C.S., Luteinizing hormone receptor mediates neuronal 

pregnenolone production via upregulation of steroidogenic acute regulatory protein expression. J. 

Neurochem. in press. 

8. Yue X., Lu M., Lancaster T., Cao P., Honda S., Staufenbiel M., Harada N., Zhong Z., Shen Y., Li R., 

Brain estrogen deficiency accelerates Abeta plaque formation in an Alzheimer's disease animal model. 

Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 19 198-19 203. 

9. Parlow A. F., Effect of ovariectomy on pituitary and serum gonadotrophins in the mouse. 

Endocrinology. 74 (1964) 102-107. 

10. Rosario E.R., Chang L., Stanczyk F.Z., Pike C.J., Age-related testosterone depletion and the 

development of Alzheimer disease. JAMA. 292 (2004) 1431-1432. 

11. Short R.A., Bowen R.L., O'Brien P.C. & Graff-Radford N.R., Elevated gonadotropin levels in patients 

with Alzheimer disease. Mayo Clin. Proc. 76 (2001) 906-909 

225


NEUROSTEROIDS-GROWTH FACTORS INTERACTION INDUCES UP AND 

DOWN REGULATION OF GFAP AND VIMENTIN EXPRESSION IN 

ASTROGLIAL CELLS MAINTAINED UNDER SERUM-FREE STRESSED 

CULTURE CONDITIONS 

V. Bramanti 1 , D. Bronzi 2 , G. Raciti 3 , M. Avitabile 3 , R. Avola 1 

1 

Department of Chemical Sciences, Section of Biochemistry and Molecular Biology, via 

A. Doria 6, 95125 University of Catania Italy Fax 0039-0957384220 – e-mail: 

ravola@unict.it 

2 Department of Physiological Sciences, University of Catania, Italy. 

3 

Department of Biological Chemistry, Medical Chemistry and Molecular Biology, 

University of Catania, Italy. 

The glucocorticoid dexametasone (DEX) influences astroglial cells regulating glial 

fibrillary acidic protein (GFAP) expression (1, 2) and play also a pivotal role in neuronal 

differentiation in culture. 

Interesting evidences (3) demonstrated that DEX can differentially regulate the expression 

of growth factors [bFGF, Nerve Growth Factor (NGF) and S-100 beta], in hippocampal 

astrocytes in vitro, suggesting that one of the mechanisms through which glucocorticoids 

(GCs) affect hippocampal functions may be regulated by the expression of astrocytederived 

growth factors. 

Neuron – glia cross-talk is modulated by mitogenic growth factors (GFs) EGF, IGF-I, 

Insulin (INS) and bFGF able to induce astroglial and neuronal cell proliferation and 

differentiation under different culture conditions. 

Serum deprivation is one exogenous stimulus, like glucocorticoids and cAMP, capable to 

enhance spontaneous or growth factor-induced astroglial differentiation (4). 

The aim of present investigation was to study the interactions between GFs and DEX on 

cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental 

conditions. 

Condition 1: 24h pre-treatment with bFGF, subsequent 72h switching in serum-free 

medium (SFM) and final addition of GFs alone or by two in the last 24h, after a prolonged 

(60h) DEX treatment. 

Condition 2: 36h pre-treatment with DEX and bFGF in the last 24h followed by SFM for 

60h and final GFs addition for 24h alone or by two. 

Western blot analysis data showed a marked GFAP expression in cultures submitted to 

condition 1 comparing results to untreated or treated controls. In particular, the maximum 

level of GFAP expression was observed when EGF or INS or both together were added in 

a prolonged 60h DEX treatment, comparing the results to both untreated and pretreated 

control cultures. This finding well correlates with differentiative role played by 

glucocorticoids interacting with the “competence” factor bFGF and demonstrates as 

increased GFAP expression mostly depends on maturation, rather than proliferating status 

of astroglial cells in culture. 

Under the same culture conditions (condition 1), VIM expression was instead significantly 

reduced after GFs addition in the last 24h of 60h DEX treatment, respect to control DEXpretreated 

ones. On the contrary, referring data to untreated controls, VIM expression was 

significantly enhanced after GFs addition. 

GFAP showed also a significant increase in EGF or INS or IGF-I or both EGF+INS 

treated astrocytes submitted to condition 2 respect to both control untreated or treated 

cultures. 

226



VIM expression was up an down regulated under condition 2. In particular, the expression 

of vimentin was significantly increased in cultures pretreated for 36h with DEX and bFGF 

in the last 24h, switching under serum free conditions (SFC) for 60h and final treatment for 

24h with INS or EGF+IGF-I or EGF+INS, comparing the results to both untreated and 

pretreated control ones. 

On the contrary, vimentin expression was markedly decreased after IGF-I addition, when 

data were compared to both untreated and pretreated controls. 

Our data demonstrate that the pre-treatment with “competence” factor bFGF, the 

subsequent switching for a long period under SFC and final treatment with DEX for 60h 

induces an up and down regulation of cytoskeletal protein expression depending on 

mitogenic synergistic effect evoked by some “progression” growth factors , like EGF o 

INS or both together. 

Collectively, our results indicate that progression growth factors addition can regulate 

GFAP and VIM expression, depending on pre-treatment with DEX and/or bFGF in 

cultures switched in SFC. This suggests an interactive dialogue between these two class of 

neuroactive molecules and confirm the complex role played by glucocorticoids and both 

‘‘competence’’ and ‘‘progression’’ growth factors, regulating astrocytic cytosckeletal 

network under stressed and adversed environmental conditions. 

Reference list: 

1. Avola R, et al., Clin Exp. Hypertension 2004 May;26(4):323-33 

2. Avola R, et al. Clin Exp. Hypertension 2002 Oct-Nov;24(7-8):753-67 

3. Niu H, et al., Brain Res Mol Brain Res 1997 51: 97-105. 

4. Loo T. et al., J Neuroscience Research 1995, 42(2): 184-91. 

227


SEX DIFFERENCES IN RAPID ESTROGEN ACTION ON GABAERGIC 

NEURONS IN VIVO 

Balog J. 1 , Szegő É.M. 1 , Erdei F. 2 , Szabó G. 2 , Juhász G. 1 and Ábrahám I.M. 1 

1 

Research Group of Neurobiology of Hungarian Academy of Sciences, Eötvös Loránd 

University, Pázmány Péter sétány 1/c, Budapest, H-1117, Hungary 

2 

Laboratory of Molecular Biology and Genetics, Institute of Experimental Medicine of 

Hungarian Academy of Sciences, Szigony utca 43, Budapest, H-1083, Hungary 

email: turboka@inf.elte.hu 

GABA is the most important inhibitory transmitter in the brain. GABAergic neurons 

play pivotal role in all brain function including cognition, perception, regulation of stress 

response or fertility. Previous findings showed that there is clear sex difference in function 

of GABAergic neurons such as the GABA release or rate limiting enzyme of GABA 

production. The mechanism of this sexually dimorphic GABAergic functions is not clear. 

One possibility that estrogen induces sexually dimorphic actions on these neurons. 

Besides the well-established estrogen receptor (ER)-mediated direct genomic effect, 

estrogen also exerts rapid nonclassical actions via different signaling pathways. Previously 

we have demonstrated that estrogen can induce a rapid activation of signaling pathways 

regulating transcription factors such as cAMP responsive element binding protein (CREB) 

via ER in different brain regions and in cholinergic and gonadotropin relasing hormon 

(GnRH) neurons in vivo. In our present study, using the CREB phosphorylation as an 

indicator of rapid estrogen action, we examined the estrogen’s effect on CREB 

phosphorylation in the GABAergic neurons. In addition we also determined the ER 

expression of GABAergic neurons in different brain areas. 

In our experiments we used GAD65-GFP transgenic mice expressing green 

fluorescent protein (GFP) under the control of the GAD65 gene’s regulatory domain. 99% 

of all GABAergic neurons exhibited GFP fluorescence and there were no GFP 

fluorescence without GAD expression in the GAD-GFP transgenic mice. In order to 

eliminate endogenous estrogens, all experiments were performed on gonadectomized adult 

female or male mice to which exogenous estrogen was then administered. 

In our first experiment we have examined the rapid effect of estrogen on CREB 

phosphorylation in GAD-GFP neurons in different brain areas by means of quantitative 

fluorescent immunohistochemistry. Whereas estradiol had no effect on the numbers of 

GAD-GFP neurons expressing CREB, an increase in pCREB expression was detected in 

GAD-GFP neurons in medial preoptic area (mPOA), median preoptic area (MnPO) and 

subparaventricular area (SpA) in female mice 15 min following estrogen administration. 

pCREB in females 

pCREB in males 

100 

100 

% in GAD-GFP neurons 

80 

60 

40 

20 

* 

* * 

% in GAD-GFP neurons 

80 

60 

40 

20 

0 

LS MnPO BNST mPOA SI SpA mAMY STR 

0 

LS MnPO mPOA BNST SI SpA mAMY STR 

Fig. 1. Effect of estrogen on CREB phosphorylation in GAD-GFP neurons in specific brain areas in 

gonadectomized GAD-GFP female and male transgenic mice. *p



By contrast, the estrogen did not alter the pCREB expression of GAD-GFP neurons 

in any of the brain regions analyzed in males (fig 1). 

In the next experiment ER-expression of GAD-GFP neurons was determined in the 

brain using quantitative fluorescent immunohistochemistry. Our results showed that GAD- 

GFP neurons exclusively expressed ERα in lateral septum (LS), MnPO while GAD-GFP 

positive neurons were labelled for ERα and ERβ in bed nucleus of stria terminalis (BNST), 

mPOA, substantia innominata (SI), SpA, medial amygdala (mAMY). By contrast, GAD- 

GFP neurons do not express ERs in striatum (STR). There were significant difference in 

ERα levels of GAD-GFP neurons of female and male mice in mAMY, SI and SpA. 

Regarding the ERβ expression of GAD-GFP neurons, there were significant differences 

between sexes in BNST, and mAMY. 

In summary, the present study reveals that clear sex differences exists in the ability of 

estrogen to phosphorylate CREB within GABAergic neurons and the ERs expression in 

specific brain regions in vivo. Our findings also demonstrate that the anatomical difference 

of estrogen-induced CREB phosphorylation in GABAergic neurons in female and male 

mice does not correspond to the sexdimorphic expression of ERs in GABAergic neurons. 

229


DHEAS AND THE HOMINOID BRAIN 

Campbell B.C. 

Anthropology Department, Harvard Unversity, 11 Divinity Ave., Cambridge MA 02138 

USA Fax: 617-496-8041 email: bccampb@fas.harvard.edu 

Dehydroepiandrosterone (DHEA) and its sulfate (DHEAS), the most common steroids in 

the human body, have has been demonstrated to have a wide variety of physiological 

effects on the skeletal, immunological, vascular, and nervous systems. Furthermore, 

adrenarche, the prepubertal rise in adrenal production of DHEAS, is a distinctive feature of 

hominoid life history, documented for humans and the African apes, but lacking among 

other primates. Yet, the evolution of adrenarche remain a mystery. [1]. 

This lack of understanding may reflect three major sources of confusion in the current 

literature: 1) the wide variety of effects exhibited by DHEA makes it difficult to establish 

its main function; 2) DHEA can be converted into a variety of other steroids, including 

estrogen and testosterone making it unclear if DHEA itself has specific effects or acts 

primarily as a source of estrogen and testosterone; 3) evidence for a well-defined receptor 

for DHEA, physiologist’s hallmark of functionality, has been lacking, further obscuring 

attempts to delineate the specific actions (if any) of DHEAS. 

However, the characterization of DHEA as a neurosteroid [2], and recent evidence of a 

specific DHEA receptor [3] provide new insights into the role of adrenarche. As a 

neurosteroid, the original function of DHEAS may have been neurological, focusing 

attention on a possible role in brain expansion during human evolution. The existence of a 

DHEA receptor would suggest that DHEA represents a meaningful signal that can be 

altered by selection. The fact that DHEA represents one step in the biosynthetic steroid 

pathway makes DHEA a likely candidate for an evolvable system. Furthermore, the fact 

that increases in DHEA continue into the early 20s mean that DHEA production could 

coordinate life history changes in both brain and body throughout development. 

I argue that adrenarche emerged in the great apes to support the development of continued 

brain maturation associated with a long life span, extended juvenile period and a fissionfusion 

social organization. I base this argument on two lines of evidence; 1) the role of 

DHEA in primate fetal brain development; 2) anatomical and genetic features specific to 

the hominoid brain which suggest increasing complexity relative to other primates. 

Together these lines of evidence suggest that adrenarche may be a marker of an extended 

period of juvenile development that leads to slow somatic growth while promoting 

increased neuroplasticity and enhanced social learning. 

Among primates, DHEA appears to be particular important during prenatal development 

[4]. DHEAS is produced by the fetal adrenal gland in larger amounts as precursor to 

placental estradiol production. Both DHEA and DHEAS have been shown to effect the 

development of fetal neural cells [5], suggesting that the two hormones may play a special 

role in promoting primate fetal brain development. Thus the development of a distinct zona 

reticularis and DHEA production by the adrenal gland starting around the age of three and 

continuing through adolescence [6] suggests a similar neurological function throughout 

childhood development. 

230



While brain maturation undoubtedly involves many cellular processes DHEA may play a 

particular role in shaping the development of neural conntections. DHEAS is known to 

have a variety of neuronal effects, including acting at both the GABA [7], and NMDA [8] 

receptor, as well as promoting glutamate release [9]. The effect of DHEA on glutamate 

receptors is of special interest since humans and great apes share a hominoid specific gene 

thought to promote glutamate turnover [10]. Thus increasing levels of DHEA at adrenarche 

would potentiate the excitotoxic effects of glutamate in shaping neural connections. 

NDMA receptors are present at high density in the striatum, amygdala and prefrontal 

cortex, parts of the mesolimbic and corticolimbic systems important in emotion and the 

control of behavior. 

Taken together the available literature suggests that adrenarche may represent an 

adaptation among humans and the great apes to increase levels of DHEA in support of 

brain development in response to an extend period of development and complex 

environment. 


1. Campbell, B.C. 2006. Adrenarche and the evolution of human life history. Am J Hum Biol. 18,569-589 

2. Baulieu, E.E. 1998. Neurosteroids: a novel function of the brain. Pyschoneuroendocrinol 23,963-987. 

3. Chen, F., Knecht, K., Birzin, E., Fisher, J., Wilkinson, H., Mojena, M., Moreno, C.T., Schimdt, A., 

Harada, S., Freedman, L.P., Reszka, A.A. 2005. Direct agonist/antagonist functions of 

dehydroepiandrosterone. Endocrinology 4568-4576. 

4. Mesiano, S., Jaffe, R.B. 1997. Developmental and functional biology of the primate fetal 

adrenal cortex. Endocrine Rev. 18,378-403. 

5. Compagnone, N.A., Mellon, S.H. 1998. Dehydroepiandrosterone: a potential signaling molecule for 

neocortical organization during development. PNAS (USA). 95,4678-4683. 

6. Remer, T., Boye, K.R., Hartmann, M.F., Wudy, S.A. 2005. Urinary markers of adrenarche: reference 

values in health subjects, aged 3-18 years. J Clin Endocrinol Metabol. 90,2015-2021. 

7. Majewska, M., Demigoren, S., Spivak, C.E., London, E.D. 1990. The neurosteroid 

dehydroepiandrosterone sulfate is an allosteric antagonist of the GABAa receptor. Brain Res. 

526,143-146. 

8. Monnet, F.P., Maurice, T. 2006. The sigma1 protein as a target for the non-genomic effects of neuro 

(active) steroids: molecular, physiological, and behavioral aspects. J Pharmacol Sci. 100,93-118. 

9. Lhullier, F.L.R., Nicolaidis, R., Riera, N.G., Ciprirna, F., Junqueira, D., Dahm, K.C.S., Brusque, A.M., 

Souza, D.O. 2004. Dehydroepiandrosterone increases synaptosomal glutamate release and improves 

the performance in inhibitory avoidance task. Pharmacol Biochem Behav. 77,606-610. 

10. Birke, F., Kaessman, H. 2004 Birth and adaptive evolution of a hominoid gene that supports high 

neurotransmitter flux. Nature Genetics 36,1061-1063. 

231


NITRIC OXIDE EFFECTS ON SEXUAL AND MATERNAL BEHAVIOR IN THE 

FEMALE RAT 

Carrillo B, Pinos H., Guillamón A., Panzica G.C. 1 , Pérez-Izquierdo M.A., Collado P. 

Departamento de Psicobiología, Universidad Nacional de Educación a Distancia. 

C/ Juan del Rosal, 10. P.O Box 60148 28040-Madrid, Spain. 

1 Rita Levi Montalcini, Dipartimento di Anatomia, Farmacologia e Medicina Legale, Laboratorio di 

Neuroendocrinología, Università di Torino, Torino, Italy. 

Nitric oxide (NO) is a gas that represents a new form of neurotransmission. NO plays an 

important role in the regulation of reproductive behaviors such as maternal and sexual 

behavior [1-4] and its production is regulated by estradiol in several mammalian species 

[5-8]. Sexual behavior in female rats is complex and it is constituted by proceptive and 

receptive behaviors, all of them can be measured [9]. In previous studies, it has been 

shown that NO affects sexual behavior in female rats throughout its influence in hormonal 

secretion and in the activity of some cerebral structures related to this behavior [3]. 

Although, the possible NO role in the maternal behavior is controversial [10, 11]. 

In the present study, we investigate the role of NO in sexual and maternal behaviors in 

Wistar female rat. For this purpose, we have administered intraperitoneally (i.p.) a NO 

precursor (L- Arginina) and a NO inhibitor (L-NAME). 

Sexual behavior: In order to test sexual bahavior 22 female Wistar rats three month old 

were studied. Before the test Ss. were ovariectomized and primed with estradiol benzoate 

and progesterone to induce sexual behavior. Animals were randomly assigned to one of 

three groups: control (i.p. saline injected), precursor (i.p injected. with a dose of 25mg/kg 

of L-Arginina) and inhibitor (i.p. injected with a dose of 25 mg/kg of L-NAME). All 

experimental treatment was administered before testing sexual behavior. Data from this 

experiment have shown a significant difference between control and L-NAME groups, and 

between L-NAME and L-Arginina groups. It can be concluded that proceptive behaviors 

were not affected either by L-Arginina or L-NAME but lordotic behaviour decresed when 

NO inhibitor is inyected. 

Natural maternal behaviour: To test maternal behavior 28 primiparous Wistar female rats 

weere studied inmediately after delivery. The primiparous were distributed in three groups: 

control (i.p. saline injected), NO precursor (i.p. injected with a dose of 25mg/kg of L- 

Arginina) and NO inhibitor (i.p. injected with a dose of 25 mg/kg of L-NAME). Five 

components of maternal behavior were recorded, and only three were significantly affected 

by the treatment: nest building, grooming and licking behavior. In all of them results 

indicate that L-Arginina interferes with these maternal behavior patterns. 

In conclusion, these data suggest different NO effects depending on the reproductive 

behavior we are studing: NO inhibitor decreases female sexual behavior and NO precursor 

seems to deteriorate some aspects of maternal behavior. 

232




1. Hull, E.M., Lumley, L.A., Matuszewich, L., Dominguez, J., Moses, J., Lorrain, 

D.S., 1994. The roles of nitric oxide in sexual function of male rats. 

Neuropharmacology. Nov, 33 (11), 1499-504. 

2. Hull, E.M., Lorrain, D.S., Du, J.F., Matuszewich, L., Lumley, L.A., Putnam, S.K., 

Moses, J., 1999. Hormone-neurotransmitter interactions in the control of sexual 

behavior. Behv. Brain Res., 105, 105-116. 

3. Mani, S.K., Allen, J.M.C, Rettori, V., McCann, S.M., O’Malley, B.W., Clark, J.H., 

1994. Nitric oxide mediates sexual behavior in female rats. Proc. Natl. Acad. Sci. 

91, 6468-6472. 

4. Benelli, A., Bertolini, A., Poggioli, R., Cavazzuti, E., Calza, L., Giardino, L., 

Arletti, R., 1995 Nitric oxide is involved in male sexual behaviour of rats. Eur. J. 

Pharmacol. 294, 505-510. 

5. Collado, P., Guillamón, A., Pinos, H., Pérez-Izquierdo, M.A., García-Falgueras, A., 

Carrillo, B., Rodríguez, C., Panzica, G.C., 2003a. NADPH-diaphorase activity 

increases during estrous phase in the bed nucleus of the accessory olfactory tract in 

the female rat. Brain Res. 983, 223-229. 

6. Ceccatelli, S., Grandison, L., Scott, R.E.M., Pfaff, D.W., Kow, L.M., 1996. 

Estradiol regulation of nitric oxide synthase mRNAs in rat hypothalamus. 

Neuroendocrinology 64, 357-363. 

7. Okamura, H., Yokosuka, M., Hayashi, S., 1994. Estrogenic induction of NADPHdiaphorase 

activity in the preoptic neurons containing estrogen receptor 

immunoreactivity in the female rat. J. Neuroendocrinol. 6, 597-601. 

8. Rachman, I.M., Unnerstall, J.R., Pfaff, D.W., Cohen, R.S., 1998. Regulation of 

neuronal nitric oxide synthase mRNA in lordosis-relevant neurons of the 

ventromedial hypothalamus following short-term estrogen treatment. Mol. Brain 

Res. 59,105-108. 

9. Beach, F.A., 1976. Sexual attractivity, proceptivity, and receptivity in female 

mammals. Horm Behav. Mar, 7 (1), 105-38. 

10. Popeski, N., Woodside, B., 2004. Central nitric oxide synthase inhibition disrupts 

maternal behavior in the rat. Behav Neurosci. Dec, 118 (6), 1305-16. 

11. Numan, M., 2004. Maternal behaviors: central integration or independent parallel 

circuits? Theoretical comment on Popeski and Woodside (2004). Behav Neurosci., 

Dec, 118 (6), 1469-72. 

Sources of support MCyT: BSO2003-02526 (Paloma Collado) and BSO2003-08962 

(Antonio Guillamón). 

233


SINGLE OPIOID ADMINISTRATION MODIFIES GONADAL STEROIDS IN 

BOTH THE CNS AND PLASMA OF MALE RATS 

Ceccarelli I., De Padova A.M., Fiorenzani P., Massafra C. and Aloisi A.M. 

Pain and Stress Neurophysiology Lab., Neuroscience and Applied Physiology Section, 

Department of Physiology, University of Siena, Via Aldo Moro, 2, 53100 Siena, Italy 

While morphine remains one of the most widely used opioid agonists for the treatment of 

painful conditions, other opioid agonists are also commonly employed. Because of the 

interactions between opioids and gonadal hormones, in particular the opioid-induced 

hypogonadism, this study investigated the effects of widely used opioids on plasma 

testosterone and estradiol levels and brain testosterone levels in male rats. Animals were 

subcutaneously injected with two concentrations of morphine (5 or 10 mg/kg), fentanyl 

(0.05 or 0.1 mg/kg), tramadol (10 or 40 mg/kg), buprenorphine (0.05 or 0.1 mg/kg) or 

saline (0.7 ml/kg). Four or 24 hours after treatment, the rats were deeply anesthetized to 

collect blood samples from the abdominal aorta and to perfuse the brains with saline. 

Plasma and brain hormone levels were measured by radioimmunoassay. In rats studied 4 

hours after treatment, all opioids, but tramadol 10 mg/kg, decreased plasma testosterone in 

comparison with saline administration. At the same time, plasma estradiol levels were 

lower than control in the groups treated with the low doses of morphine, tramadol and 

buprenorphine, while estradiol remained at control levels in the other groups. Twenty-four 

hours after treatment, plasma testosterone levels were different (higher) than control only 

in the animals treated with the low doses of morphine, fentanyl and buprenorphine. 

Estradiol was lower than control in the low dose groups, while the high doses did not 

produce any changes with respect to control. Four hours after treatment, brain testosterone 

was drastically decreased in all groups treated with the higher dose, except buprenorphine, 

in which it remained at control levels. All groups returned to control levels at 24 hours 

after treatment. The different magnitude and time-course of the effects of the different 

opiate agonists on testosterone and estradiol levels are likely due to their different 

mechanism of action. 

234



OXIDATION-DEPENDENT PLASMA DHEA FORMATION AS A DIAGNOSTIC 

TOOL FOR ALZHEIMER’S DISEASE PATHOLOGY: RESULTS FROM A 

TRIAL 

Chalbot S. *† , Lecanu L. *† , Greeson J. ‡ and Papadopoulos V *† . 

* Georgetown University Medical Center, Department of Biochemistry, Molecular and 

Cellular Biology, 3900 Reservoir Rd NW, Washington DC, 20057, USA. † Samaritan 

Pharmaceuticals Research Laboratories, Georgetown University Medical Center. ‡ 

Samaritan Pharmaceuticals Inc., 101 Convention Drive Center, Las Vegas NV, 89109, 

USA 

Sonia Chalbot, snc9@georgetown.edu, Fax (202)687-2354 

Alzheimer’s disease (AD) is a progressive, irreversible neurodegenerative disorder 

characterized by loss of memory and other cognitive functions leading to dementia. 

Although powerful neuropsychological tests are available for the diagnosis and monitoring 

of the evolution of the disease, conclusive diagnosis of AD may only be made after postmortem 

examination of brain tissue for the presence of large numbers of plaques and 

tangles. Thus, the need for an Alzheimer’s disease biochemical marker (biomarker). The 

ideal AD biomarker must reflect a fundamental feature of AD neuropathology and should 

be reliable, reproducible, and non-invasive. Accordingly, such biomarker may improve our 

understanding of AD origin and diagnosis, and help monitor AD progression and the 

efficacy of AD therapeutic interventions. 

In search of a biomarker directed at the fundamental CNS pathophysiology of AD, 

we proposed to apply our recent findings on the presence of an alternative, oxidative 

stress-mediated, pathway of neurosteroid biosynthesis in the brain [1]. Brain cells can 

convert cholesterol to pregnenolone, which is the precursor for a number of steroid 

modulators of neuronal functions, including DHEA. We identified a novel, brain- and cellspecific 

mechanism for DHEA biosynthesis present in the rat, bovine and human species. 

In this scheme, DHEA biosynthesis is mediated by a cytochrome P450 17α-hydroxylase 

(CYP17)-independent mechanism involving a yet unidentified hydroperoxide precursor. 

This alternative pathway is regulated by agents, such as Fe ++ and β-amyloid (Aβ) peptide, 

both pro-oxidant agents abundant in AD brain. Using brain tissue specimens from control 

and AD patients we subsequently provided evidence that DHEA levels are elevated in AD 

brain tissue specimens and DHEA is formed in the AD brain by the oxidative stressmediated 

metabolism of an hydroxyperoxy-steroid precursor, thus depleting the levels of 

the precursor present in plasma. In the present study, we proposed to test for the presence 

of this DHEA precursor in human plasma using a simple Fe ++ -based reaction and 

determine the amounts of DHEA formed. 

A total of 40 patients were included in this study, 12 age-matched control, 10 AD mild, 3 

AD moderate, 8 AD severe and 7 MCI (mild cognitive impairment). Blood samples were 

collected on heparin to prevent any interaction between EDTA and our experimental 

protocol. Deuterated DHEA as the internal standard was added to human plasma and 

20mM FeSO4 solution. Tubes were shaken for 60 minutes at 37 °C. The incubations were 

stopped with the addition of ethyl acetate. After decantation and centrifugation, the organic 

phase was extracted and dry under nitrogen. The extraction process was repeated three 

times. Then the dry residue was partitioned between methanol/water (8:2 (v/v) and 

petroleum ether to eliminate the lipids and sterols. After decantation the methanol/water 

phase containing steroids for analysis was evaporated under nitrogen. The dry residues 

235


were derivatized with using BSTFA in pyridine, evaporated under a stream of nitrogen and 

then dissolved in toluene. Samples were injected into GC–MS (GC17A/QP5050A, 

Shimadzu). DHEA was detected in selected ion monitoring (SIM) mode using the 

fragments (m/z=304; deuterated-DHEA, m/z=272). Statistical analyses were performed the 

software JMP 5.1 (SAS Institute, Palo Alto CA). The test used included ANOVA, 

Dunnett’s method and correlation. 

Plasma oxidation induced an increase of DHEA levels measured in control patient sera 

whereas no dramatic change was observed in patients diagnosed with severe AD. This 

significant difference of the biochemical pattern between both these groups (pF = 0.035). The merged group displayed significant 

difference compared to the control group (p=0.012) and a very important difference versus 

the AD mild group (p=0.066). These results suggest that comparing DHEA levels in 

plasma before and after oxidation by FeSO4 permits to differentiate between healthy and 

AD mild patients on one side, and AD moderate and AD severe patients on the other side. 

The % of DHEA variation after oxidation correlates with the Mini Mental State Evaluation 

(MMSE) of the patients. The lower the MMSE, the lower the DHEA increase (Correlation 

Coefficient = 0.44, R 2 =0.196). The slope of the linear regression curve was determined to 

not be due to a random effect but to a statistically significant percent Diff DHEA/MMSE 

relationship (F ratio = 7.54, Prob>F = 0.009). The changes seen were independent of age 

and sex of the patients. 

These preliminary results suggest that the comparison of DHEA levels in patient 

plasma before and after oxidation by FeSO4 could provide a useful tool to diagnose 

Alzheimer’s disease. These results also suggest that the proposed assay will not 

misdiagnose MCI patients as Alzheimer’s patients. In addition, since a significant 

correlation was observed between DHEA variation (%) and patient MMSE, the developed 

methodology might be useful in the prediction/diagnosis of severity of the disease as well 

as to follow up on effects of various therapies used. It is evident that the validity of the 

proposed methodology and interpretation of these data will be significantly refined with 

the inclusion of more patients to this trial, in particular in the AD moderate group, as well 

as non-AD demented patients like the ones with fronto-temporal dementia. The correlation 

of the data obtained with clinical information on treatments taken by the patients, drugs 

used, doses, and duration of treatments would further define the utility of this test for 

Alzheimer’s disease pathology. 


1. Brown R.C., Han Z., Cascio C. and Papadopoulos V. Oxidative stress-mediated 

DHEA formation in Alzheimer’s disease pathology. Neurobiol. Aging 2003, 24(1): 

57-65. 

236



LESION-INDUCED GLIAL REACTION IN THE RAT OLFACTORY BULB: 

EFFECT OF DHEA AND DHEA DERIVATIVES 

Csakvari E., Hoyk S., Szajli A. * , Kurunczi A., Gyenes A., Berger A., and Parducz A. 

Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research 

Center, Temesvari krt. 62., H-6701 Szeged, Hungary, 

E-mail: parducz@brc.hu; Fax: +36-62-433-133 

* Department of Organic Chemistry, University of Szeged, Hungary 

The neuroactive steroid, dehydroepiandrosterone (DHEA) was shown to influence 

the glial reactions of the peripherally denervated olfactory bulb in adult male rats. GFAP 

and vimentin immunostaining revealed that the deafferentation-induced reactive gliosis in 

the glomerular layer of the olfactory bulb was significantly diminished by DHEA. Western 

blot experiments have also shown that both chronic and acute DHEA treatment resulted in 

a significant decrease of GFAP expression levels. These findings indicate that DHEA 

attenuates glial reaction to denervation and may regulate glial plasticity in the olfactory 

glomeruli. 

To reveal the possible molecular mechanism of DHEA effect we selected different 

DHEA derivatives and studied their effects on the glial reactions. 

Denervation was achieved by destroying the olfactory mucosa with ZnSO 4 (0.17 

M) irrigation of the nasal cavities. The neurosteroids were applied in different doses during 

7 days. Rats were killed on day 7 after chemical denervation and reactive gliosis was 

monitored in the olfactory bulb using GFAP and vimentin immunohistochemistry. 

Qualitative changes in GFAP expression were analyzed by western blot. 

This work was supported by grants OTKA T 043436, M36252 and RET 08/2004 

237


DOSE-RESPONSE STUDY OF ANTIDEPRESSANT EFFECTS OF ESTROGENIC 

COMPOUNDS IN OVARIECTOMIZED MICE IN THE FORCED SWIM TEST 

Diz-Chaves Y*., Pernía O., Carrero P., Garcia-Segura L.M. 

Instituto Cajal, C.S.I.C., Avenida Doctor Arce, 37, E-28002, Madrid, Spain. 

*E-mail: ydiz@cajal.csic.es. FAX: 34-915854754 

Estradiol (E2) may influence depressive symptoms of women and decrease 

depressive behavior among rodents. Removal of the primary source of E2 (ovariectomy, 

ovx) increases depressive behavior of female rats and mice and E2 replacement reverses 

this effect. Previous reports have shown the ability of E2 to reduce depressive behavior in 

rats and mice in the forced swim test (FST), a well-established behavioral paradigm 

typically used to test the efficacy of antidepressants. In mice, the doses of estradiol used 

were 100 or 200 micrograms/Kg, and it has been described that E2 decreased duration of 

immobility in the FST in a dose dependent manner in different ovx mouse strains, after a 

minimum of three consecutive daily doses. In addition, previous studies have shown that 

selective estrogen receptor modulators (SERMs) with actions at estrogen receptor (ER) 

beta reduced depressive behavior in rats, but little is know about mice. The aim of this 

study was to determine the effective dose of different estrogenic compounds: E2, the ER 

alpha-specific SERM, PPT and the ER beta-specifc SERM, DPN, to induce an 

antidepressant effect on ovariectomized female mice in the FST. Female C57BL6 mice 

(n=104) of two months of age, were ovariectomized bilaterally under Rompun (2%) and 

ketamin (50 mg/Kg) anesthesia. Mice were housed in groups of six and maintained on a 

12h light-dark cycle in a temperature-controlled room with free access to food and water. 

After two weeks, animals were randomly assigned to an experimental group and 

behavioral studies performed. Mice were administered sesame oil vehicle, 17 beta-E2 

(Sigma), PPT or DPN (Tocris) in single doses of 50, 100 or 200 micrograms/Kg. As a 

positive control, some mice were administered a single injection of a tricyclic 

antidepressant, desipramine hydrochloride (DMI; Sigma) in a dose of 10 mg/Kg, or saline 

vehicle. Mice were tested on two occasions. First, in order to discard a possible influence 

of drug treatments on locomotor activity, the effect of the estrogenic compounds and 

antidepressant drug test was tested in the VersaMax activity monitor (Accuscan 

Instruments). Mice were placed in one of the four squares of a cage of 42 cm that 

mechanically recorded the number of beam breaks that occurred during a 5 minutes period. 

Mice were habituated during three consecutive days during 1 hour period and immediately 

after the last day of habituation, mice were administered the vehicle, the estrogenic 

compounds or DMI, and different locomotor activities (horizontal and vertical activity, 

total distance, number of movements, stereotype and rearing activity) were recorded 24- 

hours later. After two weeks, mice were tested in the forced swim test, according to the 

method of Porsolt. Swimming sessions were conducted by placing mice into an individual 

Plexiglas cylinder (29 cm height x 12 cm diameter) filled with water at 25ºC. Two 

swimming sessions were conducted: an initial 5-minutes pretest, one hour after drugs 

administration, followed by a 6-minutes test, 24 hours later. Test sessions were run 

between 11:00 and 15:00 hours and videotaped for later scoring. Two distinct types of 

behavior were assessed: mobility and immobility. One-Way analyses of variance 

(ANOVA) were utilized to determine if there were differences among the effects of 

estrogenic compounds on the basal activity or the forced swim test. In the present study we 

observed that none of the doses of estrogenic compounds analyzed produced changes in 

the different locomotor activities studied: horizontal and vertical activity, total distance, 

238



and number of movements, stereotype and rearing behavior. By contrast, DMI reduced the 

horizontal locomotor activities studied. Furthermore, a single acute injection of the 

different studied doses of 17 beta-E2, were unable to decrease immobility in the FST. 

However, at the higher dose tested (200 micrograms/Kg) we observed a tendency to 

increased immobility, but statically differences were not found. The lack of effectiveness 

of E2 to reduce depressive behavior observed in this study may be due in part to the 

duration of treatment and the strain of mice studied. It has been described that baseline 

immobility and sensitivity to antidepressants in the FST are strain dependent in mice. Also, 

it is known that high supraphysiological doses of E2 do not decrease immobility in the FST 

test. As observed for E2, a single acute injection of the ER alpha-specific SERM, PPT, did 

not affect immobility at any of the doses studied. However, the ER beta-specific SERM, 

DPN, significantly increased the duration of immobility at the dose of 100 micrograms/Kg. 

The lower dose (50 micrograms/Kg), did not have a significant effect. This finding seems 

to be in apparent contradiction to previous reports suggesting that E2 antidepressive effects 

are mediated by ER beta. However, the data presented here did not rule out the importance 

of ER beta, as important differences in the methodology could explain the discrepant result 

obtained. In most of the published studies, the FST procedure was performed in a single 

day, where the aspect of novelty and stress had great importance. Studying the 

antidepressive behavior in a two swimming sessions, eliminate the stress induced by 

novelty and the increased immobility observed in mice treated with DPN at 100 

micrograms/Kg, may be due to a less anxious behavior. As was expected, DMI, used as a 

positive control drug, significantly reduced duration of immobility when compared to the 

respective saline control. The diminution in locomotor activity did not interfere with the 

expression of active behaviors indicating that the anti-immobility effect of DMI is specific 

in the FST. In summary, in contrast to what it has been observed in ovx rats, our findings 

indicate that the acute administration of the studied estrogenic compounds does not have 

an antidepressive action in ovx mice. Although this may represent a species difference, 

further studies should determine whether chronic treatments with estrogenic compounds 

might have antidepressive actions in mice. 



239


NEUROANATOMICAL AND BIOCHEMICAL EVIDENCE FOR THE OCCURRENCE 

OF CYTOCHROME P450 C17 IN THE FROG BRAIN. REGULATION BY VASOTOCIN 

AND MESOTOCIN 

Do Rego J.L.*, Tremblay Y. † , Luu-The V. † , Acharjee S. ‡ , Repetto E. § , Galas L.*, Castel H.*, 

Vallarino M. § , Kwon H.B. ‡ , Bélanger A. † , Seong J.Y.°, Pelletier G. † , Tonon M.C.*, Vaudry 

H.* 

*INSERM U413, Lab. Cell. Mol. Neuroendocrinol., Eur. Inst. Pept. Res. (IFRMP23), Univ. Rouen, 76821 

Mont-Saint-Aignan, France. Fax +33 235 14 6946. e-mail: jean-luc.do-rego@univ-rouen.fr 

† Lab. Ontog. Reprod., and MRC Group Mol. Endocrinol. Oncol., CHU Laval, Québec G1V 4G2, Canada 

‡ Horm. Res. Center, Chonnam National Univ., Gwangju 500-757, Korea 

§ Dept. Exp. Biol., Univ. Genova, 16132 Genova, Italy 

°Lab. G Prot. Coupled Recept., Korea Univ. Coll. Med., Seoul 136-705, Korea 

It is now clearly established that the brain has the capability of synthesizing various 

biologically active steroids including 17-hydroxypregnenolone (17OH-PREG), 

17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA) and androstenedione 

(AD) [5]. However, the presence, distribution and activity of cytochrome P450 17alphahydroxylase 

/ C17, 20-lyase (P450 C17 ), a key enzyme required for the biosynthesis of these 

steroids in the central nervous system (CNS), are poorly documented. We took advantage 

of the availability of an antiserum raised against bovine testicular P450 C17 to determine the 

distribution of P450 C17 in the frog CNS. Immunohistochemical studies showed that 

P450 C17 -like immunoreactivity is widely distributed in the frog brain and pituitary. 

Prominent populations of P450 C17 -containing cells were observed in a number of nuclei of 

the telencephalon, diencephalon, mesencephalon and metencephalon as well as in the pars 

distalis and pars intermedia of the pituitary. The expression of P450 C17 in the brain and 

pituitary was also confirmed by Western blot analysis. In the brain, P450 C17 -like 

immunoreactivity was predominantly located in neurons. However, a small proportion of 

P450 C17 -positive cells, notably large cells in the optic tectum, in the tectal lamina six and in 

the pretectal grey of the mesencephalon, were identified as glial cells [2]. Labeling of 

consecutive sections of frog diencephalon with the antiserum against P450 C17 and the 

antiserum against 3beta-hydroxysteroid dehydrogenase / delta 5 -delta 4 isomerase (3beta- 

HSD) revealed that, in several hypothalamic nuclei, P450 C17 -positive cell bodies also 

contain 3beta-HSD-like immunoreactivity [2]. Incubation of telencephalon, diencephalon, 

mesencephalon, metencephalon or pituitary explants with tritiated pregnenolone (Preg) 

resulted in the formation of several tritiated steroids including 17OH-PREG, 17OH-P, 

DHEA and AD. De novo synthesis of C 21 17-hydroxy-steroids and C 19 ketosteroids was 

reduced in a concentration-dependent manner by ketoconazole, a P450 C17 inhibitor, 

demonstrating the presence of authentic P450 C17 activity in the frog brain [2]. 

The areas where P450 C17 - and 3beta-HSD-positive cell bodies are located are richely 

innervated by vatocin (VT)- and mesotocin (MT)-immunoreactive fibers [9]. In 

amphibians, VT and MT, that are orthologues of mammalian vasopressin (VP) and 

oxytocin (OT), respectively, play a crucial role in the control of sexual behaviors [4, 6, 10]. 

Since several neurosteroids also regulate reproduction-related behaviors [7], we have 

investigated the possible effect of VT and MT in the control of neurosteroid production. 

Double immunohistochemical labeling of frog brain sections with polyclonal antibodies 

against 3beta-HSD or P450 C17 and a monoclonal antibody against VP/VT [8] revealed the 

presence of VT/MT-positive fibers in close proximity of neurons expressing the 

steroidogenic enzymes 3beta-HSD and P450 C17 [3]. High concentrations of VT and MT 

receptor mRNAs were observed in diencephalic nuclei containing the 3beta-HSD and 

P450 C17 neuronal cell bodies [1, 3]. Exposure of frog hypothalamic explants to graded 

240



concentrations of VT or MT produced a dose-dependent increase in the formation of 

progesterone (P), 17OH-PREG, 17OH-P and DHEA. The stimulatory effect of VT and MT 

on neurosteroid production was suppressed by the 3beta-HSD inhibitor trilostane and the 

P450 C17 inhibitor ketoconazole. Time-course experiments revealed that a 30-min 

incubation of hypothalamic explants with VT or MT was sufficient to induce a robust 

increase in neurosteroid production and that the maximum effect was observed after a 2-h 

exposure to VT or MT, suggesting that VT and MT activate steroidogenic enzymes at a 

posttranslational level [3]. The stimulatory effect of VT and MT on neurosteroid 

biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, 

while a V2 receptor agonist and an OT receptor agonist had no effect. VT-induced 

neurosteroid production was completely suppressed by selective V1a receptor antagonists, 

and was not affected by a V2 receptor antagonist or an OT receptor antagonist. 

Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by a 

selective OT receptor antagonist and a V1a receptor antagonist but not by a V2 receptor 

antagonist [3]. 

In conclusion, the present study provides the first detailed immunohistochemical 

mapping of the steroidogenic enzyme P450 C17 in the brain and pituitary of any vertebrate. 

These data provide additional evidence that CNS neurons and pituitary cells can synthesize 

androgens. Our data also demonstrate for the first time the existence of a regulatory effect 

of VT and MT on neurosteroid biosynthesis, suggesting that some of the behavioral effects 

of VT and MT may be mediated through modulation of the activity of P450 C17 - and 3beta- 

HSD-expressing neurons. 

Supported by INSERM (U413), a France-Québec exchange program (INSERM-FRSQ), France-Korean 

exchange programs (INSERM-KOSEF and STAR), the Regional Platform in Cell Imaging, and the Conseil 

Régional de Haute-Normandie. 


[1] Acharjee, S., Do Rego, J.L., Oh, D.Y., Ahn, R.S., Lee, K., Vaudry, H., Kwon, H.B., Seong, J.Y., 2004. 

Molecular cloning, pharmacological characterization, and histochemical distribution of frog vasotocin 

and mesotocin receptors. J. Mol. Endocrinol. 33, 293–313. 

[2] Do Rego, J.L., Tremblay, Y., Luu-The, V., Repetto, E., Castel, H., Vallarino, M., Bélanger, A., Pelletier, 

G., Vaudry, H., 2006. Immunohistochemical localization and biological activity of the steroidogenic 

enzyme cytochrome P450 17alpha-hydroxylase/C17, 20-lyase (P450C17) in the frog brain and 

pituitary. J. Neurochem. (in press). 

[3] Do Rego, J.L., Acharjee, S., Seong, J.Y., Galas, L., Alexandre, D., Bizet, P., Burlet, A., Kwon, H.B., 

Luu-The, V., Pelletier, G., Vaudry, H., 2006. Vasotocin and mesotocin stimulate the biosynthesis of 

neurosteroids in the frog brain. J. Neurosci. 26, 67496760. 

[4] Iwata, T., Toyoda, F., Yamamoto, K., Kikuyama, S., 2000. Hormonal control of urodele reproductive 

behavior. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 126, 221–229. 

[5] Mellon, S., Vaudry, H., 2001. Biosynthesis of neurosteroids and regulation of their synthesis. Int. Rev. 

Neurobiol. 46, 33–78. 

[6] Moore, F.L., Miller, L.J., 1983. Arginine vasotocin induces sexual behavior of newts by acting on cells 

in the brain. Peptides 4, 97-102. 

[7] Moore, F.L., Boyd, S.K., Kelley, D.B., 2005. Historical perspective: hormonal regulation of behaviors 

in amphibians. Horm. Behav. 48, 373383. 

[8] Robert, F.R., Léon-Henri, B.P., Chapleur-Château, M.M., Girr, M.N., Burlet, A.J., 1985. Comparison of 

three immunoassays in the screening and characterization of monoclonal antibodies against argininevasopressin. 

J. Neuroimmunol. 9, 205-220. 

[9] Smeets, W.J.A.J., González, A., 2001. Vasotocin and mesotocin in the brains of amphibians: state of the 

art. Microsc. Res. Tech. 54, 125–136. 

[10] Woolley, S.C., Sakata, J.T., Crews, D., 2004. Evolutionary insights into the regulation of courtship 

behavior in male amphibians and reptiles. Physiol. Behav. 83, 347–360. 

241


SYNAPTOGENESIS: PROMOTED BY CHOLESTEROL OR ESTRADIOL? 

Fester L. 1 , Zhou L. 1 , Bütow A. 1 , Huber C. 1 , von Lossow R. 1 , Jarry H. 2 , Rune G.M. 1 * 

Institute of Anatomy I: Cellular Neurobiology, University Medical Center Hamburg 

Eppendorf, Martinistr. 52, 20246 Hamburg, Germany; Department of Experimental 

Endocrinology, University of Göttingen, Robert Koch-Str. 40, 30707 Göttingen, Germany 

Cholesterol of glial origin has been demonstrated to promote CNS synaptogenesis (1). As 

neuron-derived estradiol also regulates synapse density, we questioned whether cholesterol 

promotes synapse formation directly or indirectly by providing elevated substrate levels for 

neuronal estrogen synthesis. In this study, we provide evidence that cholesterol-induced 

synaptogenesis results from its metabolization to estradiol. Estradiol release from the 

cultures into the medium was 8-fold higher, stimulated by cholesterol. Cholesterolpromoted 

synaptogenesis, as demonstrated by spine synapse counting and by quantitative 

evaluation of pre- and postsynaptic protein expression, is abolished when cholesterol and 

letrozole, a potent aromatase inhibitor, are simultaneously applied to hippocampal cultures. 

Most importantly, downregulation of synapse formation after knock-down of StAR is only 

rescued by estradiol but not by cholesterol. 

Acknowledgement: This study was supported by the DFG (Ru 436/4-1) 


1. Mauch et al., Science 2001 Nov 9; 294(5545):1354-7 

242



ESTROGEN SELECTIVELY INCREASES TRYPTOPHAN HYDROXYLASE-2 

AND DECREASES 5HT1 B mRNA EXPRESSIONS IN DISTINCT SUBREGIONS 

OF RAT DORSAL RAPHE NUCLEUS: ASSOCIATION BETWEEN GENE 

EXPRESSION AND ANXIETY BEHAVIOR IN THE OPEN FIELD 

Hiroi R. * , and Neumaier J.F. o 

* Department of Psychology, University of Washington, USA. 

o Department of Psychiatry and Behavioral Sciences, Neumaier Lab, University of 

Washington, Box 35125, Seattle, Washington, USA. Fax +1-206-341-5804 

e-mail: neumaier@u.washington.edu 

Mounting evidence suggests that estrogen has anxiolytic effects [1,6,7]. We recently found 

that estrogen decreased anxiety behavior in rats in the open field test and progesterone 

reversed this effect [4]. Ovarian steroids also regulate the serotonergic neurons in the 

dorsal raphe nucleus (DRN), which are implicated in the etiology of affective disorders. 

The effects of estrogen on serotonin synthesis, release and reuptake may affect the overall 

availability of serotonin in the forebrain, and in turn affect behavior. Therefore, we 

examined the effects of ovarian steroids on mRNA expression of a brain specific isoform 

of tryptophan hydroxylase (TPH2), the rate-limiting enzyme for serotonin synthesis, and 

on inhibitory serotonin autoreceptors, 5-HT 1A and 5-HT 1B [2,5]. In addition, we examined 

whether these mRNA levels in discrete subregions of DRN correlated with anxiety 

behavior. Ovariectomized rats were treated for two weeks with placebo, estrogen, or 

estrogen plus progesterone, exposed to the open field test, and subsequently processed for 

TPH2, 5-HT 1A , and 5-HT 1B in situ hybridization histochemistry. Estrogen significantly and 

selectively increased TPH2 mRNA optical density in the mid ventromedial and caudal 

subregions of the DRN (by 31-41%); there were no changes in median raphe nucleus. 

Combined estrogen and progesterone treatment had no effect on the TPH2 mRNA in any 

of the DRN subregions, suggesting that progesterone reversed the effects of estrogen with 

no further effect on gene expression. Furthermore, TPH2 mRNA in caudal DRN was 

associated with lower anxiety-like behavior whereas TPH2 mRNA in rostral dorsomedial 

DRN was associated with increased anxiety-like behavior. On the other hand, estrogen had 

no effect on 5-HT 1A in any of the subregions of the DRN, while selectively decreased 

5-HT 1B mRNA in the mid-ventromedial subregion. This decrease in 5-HT 1B mRNA was 

associated with higher TPH2 mRNA and with higher anxiety-like behavior. These results 

suggest that estrogen may increase TPH2 synthesis and reduce 5-HT 1B autoreceptor in a 

coordinated fashion, thereby increasing the capacity for serotonin synthesis and release in 

distinct forebrain regions that modulate specific components of anxiety behavior. To test 

this idea, we are currently working on manipulating gene expression by knockdown and 

overexpression to block or mimic the effects of estrogen on anxiety behavior. Thus far, we 

have successfully achieved selective knockdown of TPH2 mRNA in the rat midrostral 

DRN, as measured by immunohistochemistry and western blot assays, with preliminary 

behavioral results that support our hypothesis [3]. 


[1] Frye, C.A. and Walf, A.A., Estrogen and/or progesterone administered systemically 

or to the amygdala can have anxiety-, fear-, and pain-reducing effects in 

ovariectomized rats. Behav Neurosci 118 (2004) 306-13. 

[2] Hiroi, R., McDevitt, R.A. and Neumaier, J.F., Estrogen selectively increases 

tryptophan hydroxylase-2 mRNA expression in distinct subregions of rat dorsal 

243


raphe nucleus: association between gene expression and anxiety behavior in the 

open field, Biol Psychiatry, 60 (2006) 288-95. 

[3] Hiroi, R. and Neumaier, J.F., Morpholino antisense oligonucleotide-mediated 

knockdown of tryptophan hydroxylase-2 in a discrete subregion of rat dorsal raphe 

nucleus. Abstract of the 36th Annual Meeting of the Society for Neuroscience, 

p199.16. 

[4] Hiroi, R. and Neumaier, J.F., Differential effects of ovarian steroids on anxiety 

versus fear as measured by open field test and fear-potentiated startle, Behav Brain 

Res, 166 (2006) 93-100. 

[5] Hiroi, R. and Neumaier, J.F., Estrogen selectively decreases 5-HT 1B mRNA in 

distinct subregions of rat dorsal raphe nucleus: Inverse association between gene 

expression and anxiety behavior in the open field. Annual Northwest Chapter 

Meeting of the Society for Neuroscience, 2006. 

[6] Walf A.A. and Frye C.A., ERbeta-selective estrogen receptor modulators produce 

antianxiety behavior when administered systemically to ovariectomized rats. 

Neuropsychopharmacology, 30 (2005) 1598-609. 

[7] Walf A.A. and Frye C.A., A review and update of mechanisms of estrogen in the 

hippocampus and amygdala for anxiety and depression behavior. 

Neuropsychopharmacology, 31 (2006) 1097-111. 

244



PRIP, A PHOSPHOLIPASE C-RELATED INACTIVE PROTEIN, REGULATES 

GABA A RECEPTOR ENDOCYTOSIS 

Kanematsu T. and Hirata M. 

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu 

University, 3-1-1, Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan. 

e-mail: inositol@dent.kyushu-u.ac.jp Fax: +81-92-642-6322. 

Efficacy of synaptic inhibition depends on number of cell surface expressed 

GABA A receptors. In spite of growing number of reports, the detailed molecular 

mechanisms involved in regulation of the receptor number still remain unclear. Our recent 

studies revealed that PRIP (phospholipase C-related but catalytically inactive protein) 

regulates GABA A receptor signaling by analyzing PRIP knockout (KO) mouse [1]. In the 

present study, we studied the involvement of PRIP in the modulation of postsynaptic 

GABA A receptor number by brain-derived neurotrophic factor (BDNF), which rapidly 

down-regulates GABA A receptor surface number. The exposure to BDNF reduced the 

GABA-evoked inhibitory current (I GABA ) in cultured hippocampal neuron of wild type 

mice, whereas a little potentiation was observed in the PRIP-KO mice, corresponding to 

the surface expression of GABA A receptor number. As PRIP bound to beta subunits of 

GABA A receptor, we mapped the region in PRIP responsible for the interaction with the 

beta-subunits, and the peptide mimicking that region blocked the attenuation of I GABA in 

wild type hippocanpal neurons in response to BDNF application [2]. GABA A receptor 

endocytosis is mediated by clathrin/AP2 protein complex. Since clathrin/AP2 protein 

complex was co-immunoprecipitated with PRIP, PRIP might be involved in the 

clathrin/AP2-mediated GABA A receptor endocytosis [3]. These results indicate that PRIP 

plays an importnat role in the process of GABA A receptors endocytosis by the direct 

interaction with GABA A receptor beta-subunits. 


1. Kanematsu T., Jang I. S., Yamaguchi T., Nagahama H., Yoshimura K., Hidaka K., Matsuda M., Takeuchi 

H., Misumi Y., Nakayama K., Yamamoto T., Akaike N., Hirata M. and Nakayama K. (2002) Role of the 

PLC-related, catalytically inactive protein p130 in GABA A receptor function. EMBO J. 21, 1004-1011. 

2. Kanematsu T., Yasunaga A., Mizoguchi Y., Kuratani A., Kittler J. T., Jovanovic J. N., Takenaka K., 

Nakayama K. I., Fukami K., Takenawa T., Moss S. J., Nabekura J. and Hirata M. (2006) Modulation of 

GABA A receptor phosphorylation and membrane trafficking by phospholipase C-related inactive 

protein/protein phosphatase 1 and 2A signaling complex underlying BDNF-dependent regulation of 

GABAergic inhibition. J. Biol. Chem. 281, 22180-22189. 

3. Kanematsu T., Fujii M., Mizokami A., Kittler J. T., Nabekura J., Moss S. J. and Hirata M. Phospholipase 

C-related inactive protein is implicated in the constitutive internalization of GABA A receptors mediated by 

clathrin and AP2 adaptor complex. J. Neurochem. In press. 

245


PROGESTERONE WITHDRAWAL SENSITIVITY IN FEMALE RATS RELATES 

TO DIFFERENCES IN BASELINE BEHAVIOR OF RISK TAKING AND 

EXPLORATION 

Löfgren M., Johansson I-M, Meyerson B. a and Bäckström T. 

Department of Clinical Science, Obstetrics and Gynecology, Umeå Neurosteroid Research 

Center, Building 5B, 5 th floor, Umeå University Hospital, SE-901 85 Umeå, Sweden. 

a Department of Neuroscience, Division of Pharmacology. Box 593, BMC SE-751 24 

Uppsala, Sweden. Fax: +46-90-776006 

Email: Magnus.Lofgren@obgyn.umu.se 

Background: Progesterone effects and the subsequent withdrawal properties are 

similar to those of GABA A receptor acting drugs. The effects of progesterone are most 

likely caused by allopregnanolone (3alpha-hydroxy-5alpha-pregnane-20-one). This 

progesterone metabolite is produced during the luteal phase of the menstrual cycle, during 

pregnancy and by stressful events. In women elevated levels of allopregnanolone are 

correlated with negative mood symptoms during the luteal phase. Interestingly normal 

plasma concentrations of gonadal steroids trigger PMS symptoms more readily in 

susceptible women. In male rats the stable baseline behavior of risk taking and exploration 

has been shown to influence the severity of progesterone withdrawal (PWD). 

Method: 32 female Wistar rats were tested in their diestrus phase in the Open Field 

(OF) for baseline behavior of risk taking and exploration. From the OF data the rats were 

divided into high and low responders (HR/LR) and further assigned to either placebo or 

treatment. Vaginal lavage was performed daily. Injections were given i.p. twice daily for 

six days, either 5 mg/kg progesterone in conjunction with 10 µg/kg 17β estradiol, or vehicle 

(sesame oil). Blood samples for corticosterone (CORT) analysis were collected after the 

behavioral tests. At WD (24h) the animals were tested in the Elevated Plus Maze (EPM). 

Results: The high risk taking and exploring rats showed greater aversion of the 

open arms in the EPM and had lower CORT levels at PWD. The low risk taking rats did 

not show an adverse reaction at PWD. Within the control group the time in the inner parts 

of the baseline OF test correlated with time spent on the open arms of the EPM WD test. 

The CORT concentrations in plasma collected after the tests were correlated in the control 

group. 

Conclusions: Baseline exploration and risk taking behavior measured in the OF 

predicted the PWD reaction in female rats, with greater sensitivity found in high 

responders. The stability of the plasma CORT indicated a consistent individual stress 

response. 

246



P450scc IS INDUCED IN NEURONAL AND GLIAL CELLS AFTER STATUS 

EPILEPTICUS: MODULATORY EFFECTS OF NEUROSTEROIDS ON 

EPILEPTOGENESIS 

Longo D.*, Baldelli E.*, Zini I.*, Zoli M.*, Avoli M. # , Biagini G.* 

*Dipartimento di Scienze Biomediche, Università di Modena e Reggio Emilia, via Campi 

287, 41100 Modena; # Montreal Neurological Institute, McGill University, Montreal, 

Quebec, Canada. 

P. I.: Longo Daniela, Dipartimento di Scienze Biomediche, Sezione di Fisiologia, 

Università di Modena e Reggio Emilia, via Campi 287, 41100 Modena; phone +39 059 

2055358; fax +39 059 2055363; e-mail: danylong@virgilio.it 

The conversion of cholesterol into pregnenolone by the rate-limiting enzyme 

cholesterol side-chain cleavage cytochrome P450 (P450scc) is a critical step in the 

synthesis of brain-derived steroids (neurosteroids). Pregnenolone is subsequently 

metabolized through various enzymatic steps into main final products such as 

allopregnanolone and allotetrahydrodeoxycorticosterone. Steroids may play several roles 

in the nervous systems, as they can regulate the axonal growth, synaptogenesis and, in 

general, neuronal trophism. In addition, steroids display modulatory properties on 

glutamate and gamma-amino butyric acid (GABA) receptors. It is noteworthy that 

allopregnanolone and allotetrahydrodeoxycorticosterone function as GABA A 

receptor 

agonists, so enhancing GABA inhibitory effects on neuronal targets that could be critical 

in modulating seizure susceptibility. 

Although neuronal cells possess the molecular machinery to produce neurosteroids, 

these molecules are mainly synthesized in glial cells, particularly in oligodendrocytes and 

astrocytes. This last glial cell type is highly activated by neuronal damage, but it is 

presently unclear whether this activation leads to a enhanced neurosteroid synthesis. 

Temporal lobe epilepsy (TLE) associated with hippocampal sclerosis is characterized by 

reactive gliosis. In animal models mimicking this human disease, astrocytes show 

hypertrophy and, consequently, increased staining for the marker glial fibrillary acidic 

protein (GFAP). This phenomenon, defined as “glial reactivity”, is particularly pronounced 

in the early period that follows status epilepticus (SE), but no information is still available 

on the possible changes in neurosteroid levels after SE or whether such a modification 

could affect the normal course of epileptogenesis. 

In this work, we induced SE by injecting pilocarpine (380 mg/kg i.p.) in Sprague- 

Dawley rats (270-300 gr body weight). Seizures were blocked after 3 hours from the 

beginning of SE and subsequently the animals were sacrificed at 5 different time intervals 

(1, 3, 7, 21 days after SE) and analyzed with immunohistochemical procedures. To this 

aim, we used polyclonal antibodies against GFAP (1:500, DAKO, Glostrup, Denmark) for 

astroglial and against heme oxygenase-1 (HO-1, 1:500, Stressgen, Victoria, BC, Canada) 

for microglial cells, while oligodendrocytes were identified with a monoclonal antibody 

against human 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase, 1:100, Sigma- 

Aldrich, Milan, Italy). A polyclonal anti-P450scc antibody (1:200, Chemicon, Tamecula, 

CA, USA) was used to evaluate the putative source of neurosteroid production. Neuronal 

cell bodies were identified with a monoclonal antibody against NeuN (1:100; Chemicon). 

A triple immunolabelling was obtained by incubating first the neuronal marker along with 

an antibody for glial cells, then by developing the reaction for P450scc. 

In another set of experiments, the animals were treated with daily s.c. injections of 

100 mg/kg finasteride (Ivy Chiral Chemicals, NJ, USA) or with 30% hydroxypropyl-β- 

247


cyclodextrin in water (vehicle-treated, n = 3 for NECs and n = 13 for the pilocarpine 

group), starting 3 days after SE and continuing for 18 days, in order to block neurosteroid 

synthesis and to evaluate the latency period prior to the onset of spontaneous recurrent 

seizures. The animals were videorecorded 6 h/day 7 days/week to monitor time of 

appearance and frequency of spontaneous seizures. Only stage 5 (generalized tonic-clonic 

convulsions) seizures were scored. Results were analyzed with one-way analysis of 

variance followed by the Games–Howell test for multiple comparisons. The Kaplan–Meier 

method was used to estimate the rate of onset of stage 5 spontaneous seizures after SE. 

These curves were compared by the log rank test. 

We found that P450scc is upregulated in several areas of the hippocampal 

formation (CA1, CA3, dentate gyrus, subiculum, entorhinal cortex) as well as in 

extrahippocampal areas (amygdala, neocortex) few days after SE. In particular, we 

observed a remarkable increase of P450scc in the stratum lacunosum-moleculare as well as 

in the pyramidal cell layer of the CA3 hippocampal subfield. The triple immunolabelling 

evidenced that most P450scc-positive elements co-stained with the GFAP antibody. In 

addition, anti-CNPase and anti-P450scc co-stained cells were also identified. Interestingly, 

we found also that putative microglial cells stained with the anti-HO-1 antibody were 

positive to P450scc: some of them were clearly localized in the vessel wall of markedly 

dilated blood vessels. The CA3 pyramidal cell layer was also characterized by doublelabelled 

positive neurons. 

Then, we analyzed the intensity of immunolabelling with semiquantitative 

microdensitometric methods as function of the different time intervals considered. In 

particular, we observed that positive cell counts as well as the intensity of the 

immunostaining increased progressively from day 1 to 3, both in the pyramidal and 

lacunosum-molecular layer of CA3. However, in the pyramidal layer both values 

decreased from day 7 reaching the basal levels at day 14, while in the stratum lacunosummoleculare 

cell counts and the intensity of immunostaining were still significantly 

(p



ALTERATIONS OF NEONATAL LEVELS OF ALLOPREGNANOLONE AND 

THE NOVELTY-DIRECTED BEHAVIOURAL RESPONSE TO 

INTRAHIPPOCAMPAL ADMINISTRATION OF ALLOPREGNANOLONE IN 

ADULTHOOD 

Martín-García E., Darbra S., Pallarés M. 

Departament de Psicobiologia i Metodologia en Ciències de la Salut, 

Institut de Neurociències, Universitat Autònoma de Barcelona, 08193 

Bellaterra, Barcelona, Spain. Fax: +34 93 581 20 01. 

E-mail: marc.pallares@uab.es. 

Recent findings indicate that neurosteroids could act as important keys during the brain 

development. Neonatal allopregnanolone (AlloP) administration produces behavioural 

changes observable into adulthood. Moreover, fluctuations in neonatal AlloP could result 

in altered pharmacological properties of the GABA A receptor system in adulthood. The 

aim of the present work is to screen whether developmentally altered neurosteroid levels 

influence the behavioural response to intrahippocampal administration of AlloP, a GABA A 

positive modulating neurosteroid, in adulthood. For this purpose, pups received AlloP (10 

mg/kg, s.c.) or the 5alpha-reductase inhibitor (finasteride, 50 mg/kg, s.c.) or vehicle since 

the fifth to the tenth postnatal day. At maturity (i.e. 90 old-days) a bilateral cannulae was 

implanted into the hippocampus (AP, -3.6 mm; L, ±1.8 mm; V, 2.8 mm). After recovery 

from surgery, animals received an administration of AlloP (0.2 µg) or vehicle 5 min before 

they were tested in the open field test, a paradigm of novelty-directed exploration and 

neophobia. The evaluation of habituation in a new environment, a primitive form of nonassociative 

learning, was also evaluated. Results showed that the habituation of activity in 

an open field test in adulthood was affected by alterations of neonatal levels of AlloP. 

Furthermore, animals that received perinatal administration of finasteride showed 

anxiolityc-like behaviour in adulthood. Thus, fluctuations in neonatal AlloP affect the 

novelty-directed behaviour. This effect seems to be mediated by alterations of the mature 

functions of the hippocampus, possibly via the GABA A receptor. 

249


HEIGHTENED SEIZURE SUSCEPTIBILITY FOLLOWING THE 

ADMINISTRATION OF HUMAN CHORIONIC GONADOTROPIN 

Milani P., Ginanneschi F., Biasella A., Bonifazi* M., Rossi A., Mazzocchio R. 

Department of Neurological and Behavioral Sciences, Section of Clinical 

Neurophysiology, University of Siena, Italy. 

Fax +39057740327 e-mail: dr.milani@yahoo.com 

*Department of Physiology, University of Siena, Siena, Italy 

It is known that the intramuscular injection of hCG lowers the threshold for motor 

evoked responses (MEPs) in the first dorsal interosseous (FDI) muscle to transcranial 

magnetic stimulation (TMS) in humans [1]. We describe the case of a patient with a 

clinically silent left-sided nasofrontal dermoid cyst who, while being treated with hCG/LH 

for hypogonadotropic hypogonadism, presented with simple partial seizures, ipsilateral to 

the cyst, with secondary generalization. Motor cortex excitability was studied by single and 

paired TMS and MEPs were recorded from FDI. Resting motor threshold (RMT), active 

motor threshold (AMT), MEP size, intracortical inhibition (ICI) and intracortical 

facilitation (ICF) were tested during and after suspension of hormonal therapy. RMT and 

AMT were lower, MEP size was larger, ICI was decreased while ICF was unchanged 

during treatment. This indicated an increased intracortical excitability during hormonal 

therapy. It is concluded that treatment with hCG/LH may favour seizure onset in the 

presence of potentially epileptogenic lesions such as an intracranial dermoid cyst. 


1. Bonifazi M, Ginanneschi F, Della Volpe R, Rossi A. Effects of gonadal steroids on the input-output 

relationship of the corticospinal pathway in humans. Brain Res 2004;1011:187-94. 

250



ROLES OF PRIP IN TRAFFICKING OF GAMMA2 SUBUNIT CONTAINING 

GABA A RECEPTOR 

Mizokami A., Kanematsu T. and Hirata M. 

Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu 

University, 3-1-1, Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan. 

e-mail: akiko-k @dent.kyushu-u.ac.jp Fax: +81-92-642-6322. 

GABA A receptors are a family of ligand-gated ion channels that are pentamer 

composed predominantly of alpha, beta, and gamma subunits. They are the major target of 

the endogenous inhibitory neurotransmitter (GABA) and have been implicated in a variety 

of brain functions including sedation, hypnosis, anxiety, learning and memory. The 

heterologous subunit composition of the GABA A receptor is known to be associated with 

the distinct pharmacological and physiological properties. In the present study, we have 

elucidated that PRIP (phospholipase C-related, but catalytically inactive protein) regulates 

the GABA signaling via the receptors by analyzing PRIP knockout (KO) mice; the 

sensitivity to diazepam was reduced as assessed by biochemical, electrophysiological and 

behavioral analyses of PRIP KO mice, suggesting the dysfunction of the gamma2 subunitcontaining 

GABA A receptors, a target of diazepam, a typical benzodiazepine type drug. 

We then examined the mechanisms by which PRIP molecule regulates cell-surface 

expression of gamma2 subunit-containing GABA A receptor. Disruption of the direct 

interaction between PRIP and the beta subunit of GABA A receptors by PRIP-binding 

peptide inhibited cell-surface expression of gamma2 subunit-containing GABA A receptors, 

while the expression of alpha and beta subunits were not altered by the peptide in GH3 and 

HEK293 cells. Collectively, PRIP molecules are involved in trafficking of gamma2 

subunit-containing GABA A receptors to cell-surface membrane, probably by facilitating 

the function of GABARAP, GABA A receptor associated protein. 

251


PRENATAL ESTROGENS AND THE DEVELOPMENT OF MEMORY AND 

LEARNING 

Nasir RH 1 , Chen C 2 , Bellinger D 1,3,4 , Korrick SA 2,3,4 . 

1 Children’s Hospital, Boston, 2 Channing Laboratory, Brigham and Women’s Hospital, 3 Harvard Medical 

School, 4 Harvard School of Public Health. 

Developmental Medicine Center, Fegan 10, Children’s Hospital Boston,300 Longwood Ave, Boston MA 

02115, USA. Ramzi.nasir@childrens.harvard.edu. Fax: 1-617-738-0252. 

Background: In animal studies experimental alterations of estrogen levels early in 

development result in alteration of memory and learning patterns later in life [5]. It is not 

known what role prenatal estrogens play in the development of memory systems in humans 

or if variability in estrogen levels between individuals during fetal development contributes 

to differences in memory function later in life. 

Objective: Evaluate the relationship between umbilical cord serum estradiol (E2) level and 

memory and learning at age eight years. 

Design/Methods: This study utilizes data from an ongoing birth cohort study examining 

relations between in utero exposures to environmental pollutants and neurodevelopmental 

outcomes among 788 children residing near a polychlorinated biphenyl (PCB) 

contaminated Superfund site in New Bedford, MA. We examined data from 326 children 

enrolled in this parent study for whom data are available on umbilical cord serum E2 level 

and 8-year neurodevelopmental and behavioral assessments. Memory and learning were 

assessed using the Wide Range Assessment of Memory and Learning (WRAML) which is 

a standardized instrument used clinically to evaluate three distinct memory functions: 

visual memory, verbal memory, and learning. Scores are standardized to a mean (standard 

deviation) of 100 (15). In this preliminary analysis, non linearities were noted in the 

relationship between E2 and WRAML indices. Therefore, nonparametric smoothing was 

used to describe the dose-response relationship of E2 with each WRAML index (SAS 

PROC GAM)[1]. Models were adjusted for age at exam and the examiner who 

administered the test. Potential sex specific differences in the impact of E2 on memory and 

learning were assessed by stratifying by sex. 

Results: Selected population characteristics are presented in Table 1. Children were 

generally full term and healthy at birth. Mean E2 levels were higher in male infants than in 

females as has been described in other populations [4] 

Table 1: Selected population characteristics (n=326) 

Characteristic Mean (sd) Characteristic N(%) 

Estradiol (pg/ml) - Girls 162 (50) 

Male 8174.7 (5457) Low income 90 (32) 

household 

Female 6879.5 (4849.9) Child breastfed 107 (39) 

Birth Weight (g) 3383 (435.9) Maternal smoking 93 (30) 

during pregnancy 

Gestational age 39.7 (1.3) Married parents 182 (62) 

(weeks) 

Age at exam (years) 7.9 (0.4) White mother 238 (81) 

The mean WRAML score (standard deviation) for visual memory was 90 (12), verbal 

memory 88 (13), and learning index 97 (13). Thus, values were slightly lower than the 

standardization population. Nonparametric smooths describing the relation between E2 

and WRAML verbal memory and learning indices are shown in Figure 1. In this figure 

there is a suggestion of a biphasic dose response: at moderate cord serum E2 levels, 

252



increasing E2 levels are generally associated with improved verbal memory and learning 

whereas at higher cord serum E2 levels, the inverse relationship holds. However, the 

paucity of data at higher E2 levels makes the latter association less certain. Additionally, 

the impact of E2 on WRAML indices appears to be different among boys and girls, 

particularly at low E2 levels where girls have better, and boys poorer, performance with 

increasing cord serum E2 levels. 

Figure 1 

Smoothing plot of the adjusted relationship between cord serum Estradiol and WRAML 

Verbal Memory Index (right), and Learning Index (left), adjusted for age at exam and 

examiner. 

Discussion/Conclusion: The key findings in this preliminary analysis are that prenatal 

estrogen exposures (measured as cord serum E2 levels) may impact subsequent memory 

and learning skills at age 8, and that this impact potentially differs by both E2 level and 

sex. Our observed non-linear dose-response relationship may be related to complex 

interactions with other environmental or biological covariates, including other sex steroids. 

The potential differential impact of E2 on memory and learning in boys compared with 

girls is intriguing but consistent with previously published evidence of sexual dimorphism 

of the brain and differential impact of neurosteroids according to sex [2,3]. The next steps 

in the analysis include further model development by adjustment for a wide range of 

covariates and potential confounders, using the GAM model findings to develop possible 

parametric representations of the dose-response relationship, assess the possible sex 

difference in E2 effects, and fuller assessment for interaction including hypothesized 

interactions between E2 levels and other environmental exposures with likely estrogenic 

activity such as PCBs. 


[1] SAS/STAT user’s guide. Version 9.1., SAS Institute, Inc Cary, NC, 2002-2003. 

[2] C.N. Jacklin, K.T. Wilcox and E.E. Maccoby, Neonatal sex-steroid hormones and cognitive abilities 

at six years, Dev Psychobiol 21 (1988) 567-574. 

[3] T.J. Shors and G. Miesegaes, Testosterone in utero and at birth dictates how stressful experience 

will affect learning in adulthood, Proc Natl Acad Sci U S A 99 (2002) 13955-13960. 

[4] R. Troisi, N. Potischman, J.M. Roberts, G. Harger, N. Markovic, B. Cole, D. Lykins, P. Siiteri and 

R.N. Hoover, Correlation of serum hormone concentrations in maternal and umbilical cord samples, 

Cancer Epidemiol Biomarkers Prev 12 (2003) 452-456. 

[5] C.L. Williams, A.M. Barnett and W.H. Meck, Organizational effects of early gonadal secretions on 

sexual differentiation in spatial memory, Behav Neurosci 104 (1990) 84-97. 

253


ASSESSMENT OF INTERACTION BETWEEN SEX HORMONES AND 

INCIDENCE OF EPILEPSY CRISIS IN FEMALE 

Nobahar M, Vafaei AA 

Faculty of Nursing and Paramedical, Semnan University of Medical Sciences, Semnan, Iran 

E-mail: Nobahar43@yahoo.com 

INTRODUCTION: 

Epilepsy is one of the disorders with chronic, recurrent and sudden changes in neurological function 

due to an electrical abnormality of cerebrum [1]. Previous studies have shown that around forty five 

million through out the world are suffering from epilepsy and is estimated that between 0.5 and 2 

percent of the population could acquire this disorder at any age [2]. Also the steroid hormones 

estradiol and progesterone not only regulate the reproductive system but have other central nervous 

system effects that can directly affect a variety of behaviors. Generally, estradiol has been shown to 

have activating effects, including the ability to increase seizure activity, while progesterone has 

been shown to have depressant effects, including anticonvulsant properties. Because levels of these 

hormones fluctuate across the menstrual cycle, it is important to understand how changes in these 

hormone levels may influence levels of excitability in the brain, especially in women who have 

seizure patterns that are related to their menstrual cycle, a phenomenon known as catamenial 

epilepsy. Seizures are generally random events and thus most women with epilepsy will have had a 

seizure near their menstrual cycle at some point in time [3]. Ovarian steroid hormones alter 

excitability of neurons of the central nervous system. Estrogen reduces inhibition at the GABA A 

receptor, enhances excitation at the glutamate receptor, and increases the number of excitatory 

neuronal synapses. Progesterone enhances GABA mediated inhibition, increases GABA synthesis, 

and increases the number of GABA A receptors. In animal models of epilepsy, estrogen increases and 

progesterone decreases the likelihood that a seizure will occur. Women with epilepsy may 

experience changes in seizures at puberty, during the menstrual cycle. These seizure patterns are 

believed to be associated with changes in estrogen and progesterone levels. Seizure control may also 

change during perimenopause because of fluctuations in estrogen and progesterone. In female 

patients with epilepsy manifestation of complex partial seizures and generalized tonic-clonic 

seizures may be influenced by sexual steroid hormones and progesterone. The term catamential 

seizure refers to a seizure manifestation in relation to the menstrual cycle during the few days before 

menstruation the first days of menstruation and near the middle of the cycle before ovulation [4]. 

The aim of this study was evaluation of interaction between of changes of sex hormone level and 

incidence of epileptically crisis in female patients. 

METHODS: This study has been done as a clinical trial study that during one year we investigate 

of all female that conflicted of epilepsy. At the first time we record of demographic data include of 

age, sex and so on. Then we collected data regarding of situation of epilepsy crisis especially during 

of menstrual cycle by interview and questioner. 

RESULTS: The results indicated that mean of age was 17 years old, 27% of them had family 

history and also there is significantly correlation between of sex hormone and incidence of epilepsy 

crisis (P



VASOTOCIN/ISOTOCIN NEURONS ARE DECREASED AFTER SPAWNING IN 

THE FEMALE MEDAKA FISH (ORYZIAS LATIPES) BRAIN: LOCALIZATION 

OF AROMATASE AND ESTROGEN RECEPTOR HOMOLOGUE 

Ohya T., Kodama M., and Hayashi S. 

Laboratory of Endocrinology, Graduate School of Integrated Science, Yokohama City 

University, 22-2 Seto, Kanazawa-ku, Yokohama, 236-0027, Japan. Fax +81-45-787-2380 

e-mail: tamaki037@ybb.ne.jp. 

In teleosts, the distribution of neurons in the preoptic-hypothalamic region and their 

associated neurohypophysial hormones, such as vasotocin (VT), appears to be different 

among species. This differential distribution is thought to reflect the social and/or sexual 

status of individuals within a species. In the previous study, we analyzed the number, size 

and the distribution of vasotocin/isotocin (VT/IT) neurons in the brains of both male and 

female medaka using immunohistochemistry. VT/IT neurons were similarly located in an 

inverted L-shape in the nucleus preopticus (NPO) in both sexes, as has been already 

reported in salmonids [1]. However, computer-assisted image analysis revealed sexual 

dimorphism in the number of VT/IT-immunoreactive (ir) neurons, with greater numbers 

found in males as compared to females. Further, in the female brain, the number of VT/ITir 

neurons decreased significantly after spawning. In pre-spawning compared to postspawning 

females, the small-sized VT/IT-ir neurons dominated [2]. Sexual differentiation 

of the medaka is fully dependent upon the steroid status during the early developmental 

stages and steroids are also known to trigger sex-specific behavior in the adult medaka [3]. 

In addition, we have already reported that aromatase-ir cells ware localized in the nucleus 

preopticus parvocelluralis posterioris [4], where the VT/IT-ir fibers which have originated 

from the magnocellulalis of the NPO extended facing to the third ventricle [2]. 

Furthermore, mRNA of estrogen receptor homologue seems to be localized in the preoptic 

area of the medaka brain [5]. Although, roles of the activational effects and/or 

organizational effects of the steroids via aromatase and estrogen receptor to the VT/IT 

neurons is not clarified yet, our findings strongly suggest that VT and/or IT neurons may 

be functionally related to ovulation and/or the reproductive axes through connections to 

their steroidal status in the medaka. 


1. Ohya, T., Ando, H., Ueda, H., Urano, A., 1998. Subnuclei of the nucleus preopticus in sockeye salmon: 

presence of sexual dimorphism. Prog. Jpn. Soc. Comp. Endocrinol. 13, 16 

2. Ohya, T., Hayashi, S., 2006. Vasotocin/Isotocin-immunoreactive neurons in the Medaka fish brain are 

sexually dimorphic and their numbers decrease after spawning in the female. Zool. Sci. 23, 23-29 

3. Yamamoto, T., 1959 The effects of estrone dosage level upon the percentage of sex-reversals in genetic 

male (XY) of the medaka (Oryzias latipes). J. Exp. Zool. 141, 133-153 

4. Hayashi, S., Kodama, M., 2005 Two kinds of immunohistochemical distribution of aromatase in the 

medaka brain. Neurosci. Res. 52, 205. 

5. Kawahara, T., Okada, H., Yamashita, I., 2000. Cloning and expression of genomic and complementary 

DNAs encoding an estrogen receptor in the medaka fish, Oryzias latipes. Zool. Sci. 17, 643-649 

255


GNRH-ASTROCYTES INTERACTIONS INVOLVED IN GNRH 

NEUROSECRETION: ROLE OF PSA-NCAM THROUGH CHANGING 

ACTIVITY AND EXPRESSION LEVELS OF POLSIALYLTRASFERASE 

Parkash J. and Kaur G. 

Department of Biotechnology, Guru Nanak Dev University, Amritsar, Punjab, India. 

Abstract: In recent years compelling evidence has been provided that cell–cell interactions 

involving non-neuronal cells, such as glial and endothelial cells, are important in 

regulating the secretion of GnRH, the neuro-peptide that control the sexual development 

and adult reproductive function. We have shown previously that morphological changes 

occur in the external zone of the median eminence (ME) allowing certain GnRH nerve 

terminals to contact the perivascular space on the day of proestrous. Using dual label 

immunofluorescence in conjunction with confocal microscopy, we determined that 

terminals and perikarya of GnRH neurons in adult cycling female rats are intimately 

associated with polysialylated form of neural cell adhesion molecule (PSA-NCAM). In the 

preoptic area (POA) the intense PSA-NCAM immunoreactivity was evident around the 

periphery of GnRH cell bodies. This morphological remodeling includes a reduction in 

astrocytic coverage of GnRH neurons during proestrous phase of cycling rats. Due to this 

neuronal glial interaction GnRH neurons and glial cells continue to express PSA-NCAM in 

cyclic fashion indicating that PSA plays important role in the neurosecretory activity of the 

hypothalamus in adult brain. Our data addressed new communication pathway between 

glia cells and GnRH neurons in the POA as well as ME regions of the hypothalamus in the 

central of GnRH release. The second goal of study was to determine the functional 

significance of PSA-NCAM molecule to both structural remodeling and neurosecretory 

activity of hypothalamus in adult brain, we have studied the expression of PSA-NCAM on 

GnRH axon terminals and on the glial cells in the POA as well as ME-ARC regions of 

hypothalamus in the proestrous and diestrous phase of cycling rats by using confocal 

microscope. Both GnRH and PSA-NCAM immunostaining was much higher in the POA 

as well as in the ME regions from proestrous phase rats, whereas, in diestrous phase rats 

their expression significantly reduced. The co-localization of PSA-NCAM was studied 

with GFAP in the POA as well as in the ME-ARC regions of hypothalamus using dual 

immunohistofluorescent staining. Taken together, our observations add to the growing 

evidence that PSA-NCAM play permissive role for neuronal-glial remodeling and further 

suggest a functional role of PSA-NCAM in the GnRH release mechanism. 

256



IS THERE A LINK BETWEEN THE HYPOTHALAMO-PITUITARY-GONAD AXIS 

AND THE HIPPOCAMPUS? 

Prange-Kiel J 1 , Jarry H 2 , Kohlmann P 1 , Schön M 1 , Lohse C 1 , Rune GM 1 

1 Institute of Anatomy I: Cellular Neurobiology, Martinistrasse 52, University of Hamburg, 

20246 Hamburg, Germany; prange-kiel@uke.uni-hamburg.de, fax: ++49-40-42803-4966 

2 Experimental Endocrinology, University of Göttingen, Göttingen, Germany 

Spine density in the hippocampus varies with fluctuating estradiol serum 

concentrations during the estrous cycle in rats, although application of exogenous estradiol to 

hippocampal slice cultures does not increase spine number. Since we recently demonstrated 

that hippocampal neurons of the rat synthesize estradiol (E 2 ) de novo, which in turn regulates 

spine density, we are now looking for potential regulators of estrogen synthesis in the 

hippocampus. 

First, we showed by unbiased electron microscopic stereological calculation that cyclic 

changes in spine synapse density occur specifically in the hippocampus, but not in the neocortex. 

In accordance, we demonstrated by real-time RT-PCR gonadotrophin-releasing 

hormone receptor (GnRH-R)-mRNA expression to be specific in the hippocampus. 

Immunohistochemistry showed that a subpopulation of hippocampal cells expresses 

gonadotrophin-releasing hormone receptor (GnRH-R). This expression is differentially 

regulated by E 2 , as shown by treatment of hippocampal dispersion cultures with E 2 and the 

aromatase-inhibitor letrozole, immunohistochemistry and subsequent image analysis. GnRH 

treatment of hippocampal slice and dispersion cultures resulted in a dose-dependent regulation 

of hippocampal E 2 synthesis (measured by RIA) and synaptic proteins. GnRH effects could be 

abolished by using the GnRH antagonist antide. If GnRH-induced estrogen synthesis was 

suppressed with the specific aromatase inhibitor letrozole, the GnRH effect on synaptic 

plasticity was also abolished. 

We conclude that GnRH specifically mediates cyclic changes in hippocampal E 2 synthesis and 

via this mechanism synaptic plasticity. Therefore, GnRH may link the hypothalamo-pituitarygonad 

axis to the hippocampus. 

257


RAPID ACTIONS OF ESTROGEN ON ADULT GnRH NEURONS 

Romanò N.*, Jasoni C.L.* and Herbison A.E.* 

*Department of Physiology, University of Otago, New Zealand 

Gonadotropin-releasing hormone (GnRH) neurons are the principal regulators of 

reproductive function and fertility. Estrogen feedback on the GnRH neuronal network is 

essential for its correct functioning and regulation. In addition to the possibility of 

classical genomic actions of estrogen, recent studies have suggested that rapid estrogen 

actions also occur at the GnRH neurons [1,2]. To evaluate this, we have been 

undertaking experiments using a new transgenic mouse line, in which the geneticallyencoded 

calcium indicator ratiometric-pericam is expressed in GnRH neurons. We show 

that 17-β estradiol can rapidly influence intracellular calcium levels in GnRH neurons 

from adult female mice by either promoting or suppressing calcium transients in a dosedependent 

rapid manner. Whereas the inhibitory effect is reproducible using the 

membrane-insoluble estradiol conjugate E 2 -6-BSA, the stimulatory effect is not. This 

suggests the presence of at least two distinct pathways of rapid estrogen actions, one of 

which is mediated by a membrane receptor. As GnRH neurons have been reported to 

express estrogen receptor β (ER-β) [3], we repeated the experiments on a GnRHpericam/ER-β 

knock-out mouse line. Both the stimulatory and the inhibitory responses 

to estrogen were seen on GnRH neurons from these animals, suggesting that ER-β is not 

essential for the rapid effects on calcium levels. Similar results were obtained in the 

presence of TTX, suggesting a direct effect of estrogen on GnRH neurons. These 

studies identify multiple pathways and mechanisms of rapid estrogen action upon the 

GnRH neuronal phenotype. 


1. Temple JL, Laing E, Sunder A, Wray S – J Neurosci. 2004 24:6326-33 

2. Abraham IM, Han SK, Todman MG, Korach KS, Herbison AE – J Neurosci. 2003 23:5771-7 

3. Herbison AE, Pape JR – Front. Neuroendocrinol. 2001 22:292-308 

258



ASSESSMENT OF NEUROACTIVE STEROID LEVELS IN PLASMA AND NERVOUS 

SISTEM BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY 

Scurati S.# 1 , Maschi O. 1 , Crotti S. 1 , De Angelis L. 1 , Melcangi R.C. 2 and Caruso D. 1 

1 Dept. of Pharmacological Sciences and 2 Dept. of Endocrinology, University of Milano, Milano, Italy. 

#Presenting author: Dept. of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133, 

Milano Italy, samuele.scurati@unimi.it 

Several methods for the quantification of steroids in plasma and tissue have been so far 

developed. However, even if these methods have allowed significant progress in endocrine 

research, they have manifest limits for analysis in the nervous system, where low levels of several 

steroids in small amount of tissue samples are present. For instance, immunometric techniques 

present low specificity due to cross reactivity [1], while gaschromatography connected to mass 

spectrometry, which has been used for its high specificity, requires a very critical derivatization 

steps to enhance sensitivity, but only few steroids in a single analysis are detected. Liquid 

chromatography coupled with mass spectrometry (LC-MS) might be considered as a good 

alternative. Indeed, this analytical technique shows high specificity and since generally it does not 

require any derivatization step, permits to detect different steroids in a single analysis. 

On this basis, the aim of our study was to develop a LC-tandem mass spectrometry (LC-MS/MS) 

method for the simultaneous quantification of the main neuroactive steroids present in plasma and 

in different nervous tissues, namely cerebral cortex, cerebellum, lombar portion of spinal cord, 

sciatic and brachial nerves. Plasma (0.5ml) and nervous tissues (100mg) were obtained from 5- 

month old male Sprague Dawley rats, extracted and purified according to Vallée et al. with minor 

modifications [1]. LC-MS/MS was performed with a linear ion trap-mass spectrometer (LTQ- 

ThermoElectron Co) in positive MS/MS mode using an atmospheric pressure chemical ionization 

(APCI) source. Each steroid was identified on the basis of the mass spectra of reference the 

compounds and the quantitative analysis was obtained by means of calibration curves using three 

deuterium labeled internal standards (D4-E, D4-PREG, D9-PROG). For each steroid we obtained a 

good linearity, as shown by linear regression coefficient (R 2 ≥ 0.99), and good reproducibility, as 

demonstarted by the analysis of five different calibration curves. This LC-MS/MS method allows, 

in the same analysis, the detection and quantification of eight different neuroactive steroids: 

pregnenolone (PREG), progesterone (PROG) and its derivatives, dihydroprogesterone (DHP) and 

tetrahydroprogesterone (THP), testosterone (T) and its derivative, dihydrotestosterone (DHT) and 

5alpha-androstan-3alpha, 17beta-diol (3alpha-diol), and 17beta-estradiol (17beta-E 2 ). 

Neuroactive steroid levels were assessed as pg/µl in plasma and as pg/mg in nervous structures. 

The results have indicated that PREG, the first steroid formed by cholesterol and precursor for the 

further steroids, is present not only in plasma but also in all the nervous tissues here considered. 

The same pattern is evident in case of PROG and T. However, some differences occur in case of 

their metabolites. Indeed, in case of PROG metabolites (i.e., DHP and THP), DHP is detected in 

plasma and in nervous structure, such as cerebellum and brachial nerve, but it is under the detection 

limit in cerebral cortex, spinal cord and sciatic nerve. In addition, the metabolite of DHP, i.e. THP, 

is present in cerebellum, spinal cord, sciatic and brachial nerves but it is under the detection limit in 

plasma and cerebral cortex. In case of T metabolites (DHT and 3alpha-diol), DHT is present in all 

nervous structures here considered, but under the detection limit in plasma while its metabolite, i.e. 

3alpha-diol, is detected both in plasma and nervous structures. Finally, 17beta-E 2 is identified in 

plasma and in all nervous structures tested with the exception of cerebral cortex and spinal cord. 

In conclusion, we here demonstrate that LC-MS/MS method allows the assessment of 

neuroactive steroids in plasma and in structures of central and peripheral nervous system with high 

sensitivity and specificity. 


1) Anal. Biochem. 2000; 287, 153-166 

259


ADMINISTRATION OF ALLOPREGNANOLONE DECREASES SECRETION OF 

GONADOTROPINS IN HEALTHY WOMEN OF FERTILE AGE 

Timby E., Bäckström T., Nyberg S., Wihlbäck A.-C.N., and Bixo M. 

Department of Clinical Science, Obstetrics and Gynecology, University Hospital of Umeå, 

Umeå University, S-901 85 Umeå, Sweden 

Fax +46-90137540, e-mail: erika.timby@vll.se 

OBJECTIVE 

Allopregnanolone is an endogenous neuroactive steroid secreted by the ovarian and 

adrenal glands. It is also synthesized de novo in the central nervous system.Through 

binding to the GABA-A receptor it enhances inhibitory neurotransmission. Altered levels 

of allopregnanolone have been reported in major depression and premenstrual dysphoric 

disorder, although results are contradictory. In animals, memory and learning are impaired 

by allopregnanolone.The concept of neuroactive steroids does not include endocrine action 

per se [1]. 

The aim of this study was to determine any effect of allopregnanolone on part of the 

pituitary-ovarian axis in healthy volunteers. 

MATERIAL AND METHODS 

10 healthy women with regular menstrual cycles and without hormonal treatment were 

challenged with allopregnanolone administered intravenously in a cumulative dose of 0.09 

mg/kg. The doses were increasing over time starting with 0.015 mg/kg at 0 min and then 

0.03 and 0.045 mg/kg at 30 and 60 min respectively. The study was performed during the 

follicular phase of the menstrual cycle. All subjects had the allopregnanolone injections 

during the same time (morning.). Baseline levels of allopregnanolone (ALLO), folliclestimulating 

hormone (FSH), luteinizing hormone (LH), estradiol (E2) and progesterone 

(PG) were drawn from serum before the first allopregnanolone injection. Following 

allopregnanolone injections blood samples for ALLO, FSH, LH, E2 and PG were collected 

at 5,13, 21, 35, 43, 61, 65, 73, 81, 95, 105, 115, 150, 330, 600, 780 min. Saccadic eye 

movement parameters and subjective ratings of sedation were also measured during the 

study day. Levels of FSH, LH, E2 and PG were measured with an Immulite system. 

Changes of serum levels of each hormone were analyzed by one-way ANOVA (analysis of 

variance) with repeated measures using time-point as within-subjects factor. The SPSS 

statistical package was used for the analyses. P values less than 0.05 were considered 

statistically significant. 

RESULTS 

Intravenously administered allopregnanolone in a cumulative dose of 0.09 mg/kg reduces 

SEV significantly and increases subjective report of sedation which were correlated to 

serum levels of allopregnanolone [2]. Exogenous allopregnanolone in a cumulative 

intravenous dose of 0.09 mg/kg significantly reduces serum levels of FSH 

(F(16,144)=2,184, p=0,008). LH was reduced significantly (F(16,144)=2,633, p=0,001). 

Allopregnanolone as an intravenous injection of a cumulative dose of 0.09 mg/kg does not 

affect the serum levels of estradiol. Serum levels of progesterone did not increase, but a 

significant lower level was seen at 5 min (p=0.033) and 780 min (p=0.023) 

(F(16,144)=6,153, p< 0,001). 

260



CONCLUSION 

Our results suggest that allopregnanolone affects the secretion of FSH and LH while E2 

and PG are not increased, thus the effect is not mediated by negative feed-back by the 

ovarian hormones. Our hypothesis is that this could be an action mediated by the GABA- 

A receptor. 


1. Baulieu EE. Neurosteroids: a new function in the brain. Biol Cell 1991; 71:3-10. 

2. Timby E, Balgård M, Nyberg S, Spigset O, Andersson A, Porankiewicz-Asplund J, Purdy R, 

Zhu D, Bäckström T, Sundström Poromaa I. Pharmacokinetic and behavioral effects of 

allopregnanolone in healthy women. Psychopharmacology (berl) 1-11, 2005. 

261


PHARMACOLOGICAL MODULATORS OF THE GLYCINERGIC SYSTEM 

REGULATE ALLOPREGNANOLONE BIOSYNTHESIS IN THE RAT SPINAL 

CORD 

Venard C.*, Boujedaini N. + , Belon P. + , Mensah-Nyagan A.G.* # and Patte-Mensah C.* 

*Institut des Neurosciences Cellulaires et Intégratives, Equipe Stéroïdes et Système Nociceptif, UMR 

7168/LC2 CNRS – 21, rue René Descartes – Université Louis Pasteur, 67084, Strasbourg, France. Fax: +33 

(0)3 88 61 33 47 # e-mail: gmensah@neurochem.u-strasbg.fr. 

+ Laboratoires BOIRON, Sainte-Foy-lès-Lyon, France. 

The neurosteroid allopregnanolone, also called 5alpha,3alpha-tetrahydroprogesterone 

(5a,3a-THP), controls several important neurobiological processes including anxiety, stress, pain, 

neuroprotection, depression and motor activity. It is well demonstrated that 5a,3a-THP regulates 

the nervous system activity through a potent allosteric stimulation of GABA A receptors. 5a,3a-THP 

is also a modulator of T-type calcium channels. 

The biosynthesis of 5a,3a-THP requires enzymatic activities of 5a-reductase (5a-R) and 3ahydroxysteroid 

oxidoreductase (3a-HSOR) which convert progesterone (PROG) into 5alphadihydroprogesterone 

(5a-DHP) and 5a,3a-THP, respectively. We have recently observed that the 

spinal cord (SC), which controls several neurophysiological mechanisms, contains active forms of 

5a-R and 3a-HSOR. Biochemical analyses revealed that SC slices are capable of converting 

[ 3 H]PROG into [ 3 H]5a-DHP and [ 3 H]5a,3a-THP indicating that the spinal tissue is an active 

producing site of 5a,3a-THP. Moreover, we demonstrated that, owing to its ability to potentiate the 

GABAergic inhibitory transmission, 5a,3a-THP synthesized in the SC modulates nociceptive 

mechanisms controlling pain sensitivity. 

Because the glycinergic system is also important for inhibitory neurotransmission, we 

combined pulse-chase experiments with HPLC analysis and flow scintillation characterization to 

investigate the effects of glycine and strychnine (a major antagonist of glycinergic receptors) on 

5a,3a-THP biosynthesis. Glycine at 10 -10 M strongly enhanced [ 3 H]PROG conversion into 

[ 3 H]5a,3a-THP in the SC. The amount of [ 3 H]5a,3a-THP newly synthesized from [ 3 H]PROG in the 

presence of glycine (10 -10 M) was 117% higher than the control. Strychnine (10 -5 M), which 

antagonizes glycinergic actions, did not modify by its own 5a,3a-THP synthesis in the SC. 

Strychnine is an alkaloid exhibiting structural analogy with gelsemine, the major active 

chemical agent of Gelsemium sempervirens (G. sempervirens) described as an anxiolytic and 

analgesic substance. Therefore, we have also assessed the actions of purified gelsemine and G. 

sempervirens on 5a,3a-THP formation in SC slices. Gelsemine (10 -10 M) and G. sempervirens (10 - 

10 M) were both capable of increasing the amount of [ 3 H]5a,3a-THP synthesized from [ 3 H]PROG. 

The stimulatory effects of gelsemine (10 -10 M) and G. sempervirens (10 -10 M) were respectively 

185% and 600% higher than the controls. 

Taken together, our results suggest that gelsemine, which exerts similar effects as glycine 

on neurosteroidogenesis, may be an activator of the central inhibition via the potentiation of 

glycinergic transmission. Since gelsemine enhances the production of 5a,3a-THP, a highly active 

stimulator of GABA A receptors, gelsemine may reinforce significantly inhibitory signalling in the 

central nervous system through simultaneous activation of both GABAergic and glycinergic 

systems. Consequently, anxiolytic and analgesic actions of G. sempervirens might be explained by 

the presence of gelsemine in this medical drug. However, as the stimulatory effect of G. 

sempervirens (10 -10 M) on 5a,3a-THP formation is higher than that of gelsemine (10 -10 M), the data 

suggest that additional ingredients present in G. sempervirens composition may potentiate the 

stimulatory action of gelsemine on neurosteroid biosynthesis. 

The work was supported by the contrat CIFRE n° 2005/659 between CNRS-ULP and Laboratories 

BOIRON. 

262



IT IS POSSIBLE THAT THE UNSPECIFIC STEROIDS FOR GONADOTROPIN 

SYSTEM MODULATE NEURONAL PULSATILITY ACTIVITY FROM POA– 

SCH OF THE HYPOTHALAMUS FOR REGULATING LH–RH RELASE AND 

OVULATION TRIGGER? 

Vlad A.G. 

Dept. of Endocrinology.Spitalul Clinic Judetean.1900- Timisoara. ROMANIA 

Email aurvlad@yahoo.com 

We describe in 1971 the pulsatile rhythmic activity of the neurons from POA of 

Hypothalamus also and rhythmic activity of some neurons from CA1, CA2 of 

Hippocampus. This fact was followed by revealance that LH-RH was pulsatile released by 

neuronal system from hypothalamus. We demonstrate that this pulsatile activity of the 

neuronal system behave as a, pacemaker, mechanism for ovulation triggering as a basic 

mechanism of the gonadotropin system which start during puberty by a genetic program. 

In same time by clinical study on the puberty troubles of the younger girls and mature 

woman with menstrual disorders, treatment with very small dose of some steroids can 

regulate these troubles. Our study was performed on a lot of girls and mature womans, 

2500 cases, with amenorheea, pspaniomenorheea with infertility. We assayed blood 

samples for LH, FSH, PROL, ESTRADIOL, PROGESTERON and TESTOSTERONE. 

We monitorized menstrual cycles during 1 – 3 years. We give some small quantity of 

chorionoc gonadotropin 500 ei weekly and small amount of Medoxyprogesteron, 

Orgametril and anabolic steroids Nandrolone. We do not give Estradiol or Estradiol 

combinations. From this kind of therapeutic strategy we was very surprised that menstrual 

troubles disappeared and obtain a regularized menses and establish the fertility probed by 

frequent pregnancy with normal baby. These effects put a very interesting problem about 

specificity of active site receptors from the neuronal membrane from POA – SCH neurons 

which behave as, pacemaker, triggering of LH- RH pulsatile release. In a previous paper 

recently we demonstrate that the pulsatile activity of neuronal units from POA – SCH from 

anterior hypothalamus start this automaticity by a genetic program and this kind of activity 

is modulated probably by neuroactive steroids and exogenous (adrenal and gonadal) 

steroids. Our data after applied unspecific steroids evoke a very interesting problem about 

functionality of neuronal units from gonadotropin system first is automaticity and second 

is possible that the functionality of this automatic mechanism can have some reflex 

troubles which can be regulated by modulatory effects of very different steroids. 

263


RTI: Steroid hormones and sexually dimorphic brain circuits 

• Bao A-M, Swaab D. F. (Netherlands) Gender difference in age-related number of 

corticotropin-releasing hormone expressing neurons in the human hypothalamic 

paraventricular nuclears and the role of sex hormones 

• Cannizzaro C., Plescia F., Barrile V., Diliberto I., La Barbera M., Noto G., 

Mantia G. (Italy) Neurosteroid pregs differently affects learning and memory 

performance by altering emotionality in a gender-related manner 

• Gotti S., Martini M., Pradotto M., Viglietti-Panzica C., Panzica G.C. (Italy) Sexual 

dimorphism and estrous cycle effects on nitrinergic system in mouse hippocampus 

• Maccari S, Mairesse J, Zuena AR, Morley-Fletcher S, Matteucci P, Cinque C, 

Catalani A, Nicoletti F, Casolini P. (Italy) Prenatal stress has long-term influence 

on neuroplasticity: sex differences 

• Mura E., Furnari P., Plumari L., Viglietti-Panzica C., Panzica G.C. (Italy) Role of 

apoptosis in the sexual differentiation of the bed nucleus of stria terminalis of 

japanese quail 

• Oboti L., Peretto P., Fasolo A., Panzica G.C. (Italy) The accessory olfactory bulb 

of the adult mouse: neurogenesis and morphological analysis in the two sexes 

• Pinos H, Carrillo B, Pérez-Izquierdo M, Ortega E., Collado P. (Spain) 

Undernurishment and food rehabilitation effects on plasma leptin levels in Wistar 

rat



GENDER DIFFERENCE IN AGE-RELATED NUMBER OF CORTICOTROPIN- 

RELEASING HORMONE EXPRESSING NEURONS IN THE HUMAN 

HYPOTHALAMIC PARAVENTRICULAR NUCLEARS AND THE ROLE OF SEX 

HORMONES 

Bao A-M*, Swaab D.F.* 

*Netherlands Institute for Neuroscience, Meibergdreef 47, 1105 BA Amsterdam, The 

Netherlands, fax: 0031-20-6961006; e-mail: a.m.bao@nin.knaw.nl 

Previous studies showed that the activity of the corticotropin-releasing hormone 

(CRH) neurons in the hypothalamic paraventricular nucleus (PVN) forms the basis of the 

activity of the hypothalamo-pituitary-adrenal (HPA)-axis, which is increased at times of 

stress and in specific neurological and psychiatric disorders [1, 2]. Changes in the total 

number of CRH expressing neurons in the PVN reflect the change in activity of CRH 

neurons [1, 3-5], which was confirmed by in situ hybridization of CRH-mRNA analyzed in 

the same patients [6-8]. The total number of CRH neurons in the human hypothalamic 

PVN increases with aging [3, 4]. To determine whether such an age-related change 

depends on gender and whether circulating sex hormones play a role, we analyzed the total 

number of CRH immunoreactive neurons by immunocytochemistry and image analysis in 

postmortem hypothalamic PVN of 22 control subjects (11 males and 11 females) between 

the ages of 22 and 89 year, and 10 subjects with abnormal sex hormone status. Our data 

shows that there are gender differences in the number of CRH neurons in the hypothalamic 

PVN, in that (i) there is a significant age-related increase of CRH neurons in men (p = 

0.032), but not in women (p = 0.733); and (ii) men have significantly more CRH neurons 

than women (p=0.004). Male subjects with low testosterone levels due to castration 

showed significantly fewer CRH neurons than well-matched intact males (p = 0.008), 

while castrated male-to-female transsexuals with estrogen replacement showed normal 

numbers of CRH neurons. One male case, which had high estrogen levels due to an 

estrogen-producing tumor, showed a large number of CRH neurons. Thus, although both 

circulating androgens and estrogens seem to play a stimulatory role with respect to CRH 

neurons, the age-dependent increase in the number of CRH neurons in the PVN of men, 

which has been interpreted as a sign of activation of the CRH neurons with age, seems to 

result from factors other than age-related changes of circulating sex hormone levels. The 

authors favor the idea that a number of factors seem to be involved in the changes of CRH 

neuron activity, alterations in circulating sex hormone levels being only one of them [9]. 


1. Swaab DF, Bao AM, Lucassen PJ. The stress system in the human brain in depression 

and neurodegeneration. Ageing Res Rev 2005;4(2):141-194. 

2. Sapolsky RM, Finch CE. Alzheimer's disease and some speculations about the 

evolution of its modifiers. Ann N Y Acad Sci 2000;924:99-103. 

3. Raadsheer FC, Hoogendijk WJ, Stam FC, Tilders FJ, Swaab DF. Increased numbers of 

corticotropin-releasing hormone expressing neurons in the hypothalamic 

paraventricular nucleus of depressed patients. Neuroendocrinology 1994;60(4):436- 

444. 

4. Raadsheer FC, Oorschot DE, Verwer RW, Tilders FJ, Swaab DF. Age-related increase 

in the total number of corticotropin-releasing hormone neurons in the human 

265


paraventricular nucleus in controls and Alzheimer's disease: comparison of the 

disector with an unfolding method. J Comp Neurol 1994;339(3):447-457. 

5. Bao AM, Hestiantoro A, Van Someren EJ, Swaab DF, Zhou JN. Colocalization of 

corticotropin-releasing hormone and oestrogen receptor-alpha in the paraventricular 

nucleus of the hypothalamus in mood disorders. Brain 2005;128(Pt 6):1301-1313. 

6. Huitinga I, Erkut ZA, van Beurden D, Swaab DF. Impaired hypothalamus-pituitaryadrenal 

axis activity and more severe multiple sclerosis with hypothalamic lesions. 

Ann Neurol 2004;55(1):37-45. 

7. Goncharuk VD, Van Heerikhuize J, Swaab DF, Buijs RM. Paraventricular nucleus of 

the human hypothalamus in primary hypertension: activation of corticotropinreleasing 

hormone neurons. J Comp Neurol 2002;443(4):321-331. 

8. Raadsheer FC, van Heerikhuize JJ, Lucassen PJ, Hoogendijk WJ, Tilders FJ, Swaab 

DF. Corticotropin-releasing hormone mRNA levels in the paraventricular nucleus of 

patients with Alzheimer's disease and depression. Am J Psychiatry 1995;152(9):1372- 

1376. 

9. Bao AM, Fischer DF, Wu YH, Hol EM, Balesar R, Unmehopa UA, et al. A direct 

androgenic involvement in the expression of human corticotropin-releasing hormone. 

Mol Psychiatry 2006;11(6):567-576. 

266



NEUROSTEROID PREGS DIFFERENTLY AFFECTS LEARNING AND 

MEMORY PERFORMANCE BY ALTERING EMOTIONALITY IN A GENDER- 

RELATED MANNER 

Cannizzaro C., Plescia F., Barrile V., Diliberto I., La Barbera M., Noto G., Mantia G. 

Dept. of Pharmacological Sciences, Laboratory of Neuropsychopharmacology, University 

of Palermo Via del Vespro 129, 90127 Palermo, Italy, Fax +39 0916553212 

carla.cannizzaro@katamail.com 

Neurosteroids exert an important role as modulators of inhibitory and excitatory 

neurotransmission by interacting with different receptors [1] .In particular, pregnenolone 

sulphate (PREGS) rapidly alters neuronal excitability via non-genomic mechanism [4]. 

This neurosteroid is a negative modulator of GABA A receptor , positive modulator of 

NMDA receptor [3] and agonists at sigma 1 receptor, influencing cognition as well as 

emotionality [2]. Indeed altered levels of neurosteroids together with alterations in 

acetylcholine transmission have been reported in human neurodegenerative pathologies 

like Alzheimer’s disease [6]. 

In this study we investigated, in adult male and female rats, the effects of PREGS 

(10mg/kg s.c.) administrated 60 min before the test sessions, on: learning and memory 

performance, using a motivated, non-aversive, spatial and tactile/visual learning task, the 

“Can test”; emotionality, using the Elevated Plus Maze test (EPM). The Can Test protocol, 

following an habituation period, consisted in two separate parts: the baseline training (BT), 

i.e. four-day sessions consisting in ten trials each, and the longitudinal evaluation (LE), i.e 

four ten-trial sessions every two weeks, according to Popovich [5]. Rats, following their 

performance in the baseline training, were divided in non active (NA), and active (A) 

animals, according to the quality of their performance. Our results showed that in A-male 

rats PREGS induced an increase in the number of correct responses (p



1) Baulieu E.E., Robel P., Neurosteroids: a new brain function? J. Steroid Biochem. Mol. Biol. 37 

(1990) 395-403 

2) Darnaudéry M., Koehl M., Piazza P.V., Le Moal M., Mayo W., Pregnenolone sulfate increase 

hippocampal acetylcholine release and spatial recognition, Brain Res 852 (2000) 173-179 

3) Irwin R.P., Maragaskis N.J., Rogawski M.A., Purdy R.H., Farb D.H., Paul S.M., Pregnonolone 

sulphate augments NMDA receptor mediated increases in intracellular Ca 2+ in cultured rat 

hippocampal neurons, Neurosci. Lett. 141 (1992) 30-34 

4) Paul S.M., Purdy R.H., Neuroactive steroids, FASEB J. 6 (1992) 2311-2322 

5) Popoviç M, Biessels G-J, Isaacson RL, Gispen WH. Learning and memory in a streptozotocininduced 

diabetic rats in a novel spatial/object discrimination task. Behav Brain Res, 2001; 122: 201- 

207 

6) Vallée M., Mayo W., Darnaudéry M., Corpéchot C., Young J., Koel M., M. Moal Le, Baulieu E.E., 

Robel P., Simon H., Neurosteroids: deficient cognitive performance in aged rats depends on low 

pregnenolone sulphate levels in the hippocampus, Proc. Natl. Acad. Sci. USA 94 (1997) 14865- 

14870 

268



SEXUAL DIMORPHISM AND ESTROUS CYCLE EFFECTS ON NITRINERGIC 

SYSTEM IN MOUSE HIPPOCAMPUS 

Gotti S., Martini M., Pradotto M., Viglietti-Panzica C., Panzica G.C. 

Laboratory of Neuroendocrinology, Dept Anatomy, Pharmacology and Forensic Medicine, 

C.so M. D’Azeglio, 52. 10126 Torino (Italy) 

e-mail: stefano.gotti@unito.it 

Fluctuating levels of estradiol and progesterone during the estrous cycle may 

induce structural changes in several brain nuclei including the hippocampus, where some 

neurons express estrogens receptors [1-3]. Nitric oxide plays a wide range of functions in 

the nervous system generally by acting as a neurotransmitter-like molecule that can 

contribute to both anterograde and retrograde signalling at the synapse. It has been 

demonstrated that long-term treatments with estradiol in ovariectomized females and with 

testosterone in castrated males induce neuronal nitric oxide synthase (nNOS) expression in 

rat hypothalamus [4-5], whereas changes in nNOS immunoreactivity or in associated 

NADPH-diaphorase activity were observed both in hypothalamus [6] and in the amygdala 

[7-8] during different phases of estrous cycle [for a review see 9]. Estradiol could induce 

nNOS expression in several brain regions in rodents. Therefore, to clarify if the 

hippocampal NO producing system is a target for gonadal hormones in physiological 

conditions, we have performed a comparison between two months old intact female and 

male mice in the expression on nNOS in the hippocampus. Moreover, we have investigated 

the effects of estrous cycle in the expression of nNOS immunoreactivity on two months 

old intact female mice. 

Immunoreactive cells were observed in all the hippocampal subregions: the higher number 

was detected in the pyramidal layer of CA1 region and in polymorph layer of dentate 

gyrus. Comparing proestrus female mice with male mice we observed a significantly 

dimorphism in the number of nNOS positive cells: female mice show a higher number of 

nNOS-immunoreactive elements in all hippocampal subregions. In the female, the number 

of nNOS positive neurons fluctuates during the estrous cycle, reaching its peak during 

proestrus and metaestrus, but these variations were statistically significant only in CA2 

region, that is a hippocampal region with fewer cells. 

In conclusions, our data demonstrate the presence of a sexually dimorphic population of 

nNOS positive neurons all over the hippocampus. This population is larger in females than 

in males, and the estrous cycle is playing a non-significant role for the expression of nNOS 

in the hippocampus. Sexually dimorphic structures or systems that are larger in females 

than in males are a minority within the rodent brain (i.e. locus coeruleus, AVPV) and the 

development of these structures is probably under the control of androgens and androgen 

receptors. Further studies should clarify the mechanisms that are influencing this sex 

dimorphism in the mouse hippocampus. 

Acknowledgements. This work was supported by Regione Piemone, University of Torino, 

Fondazione CRT, PRIN. 

269



[1] R.C. Melcangi and G.C. Panzica, Neuroactive steroids: old players in a new game, 

Neuroscience 138 (2006) 733-739. 

[2] E. Gould, C.S. Wooley, M. Frankfurt and B.S. McEwen, Gonadal steroids regulate 

dendritic spine density in hippocampal pyramidal cells in adulthood, Neuroscience 

10 (1990) 1286-1291. 

[3] C.S. Woolley and B.S. McEwen, Estradiol mediates fluctuation in hippocampal 

synapse density during the estrous cycle in the adult rat, J Neurosci 12 (1992) 

2549-2554. 

[4] S. Ceccatelli, L. Grandison, R.E. Scott, D.W. Pfaff and L.M. Kow, Estradiol 

regulation of nitric oxide synthase mRNAs in rat hypothalamus, 

Neuroendocrinology 64 (1996) 357-363. 

[5] J. Du and E.M. Hull, Effects of testosterone on neuronal nitric oxide synthase and 

tyrosine hydroxylase, Brain Research 836 (1999) 90-98. 

[6] M. Martini, M. Sica, C. Eva, C. Viglietti Panzica and G.C. Panzica, Dimorphism 

and effects of estrous cycle on the nitrinergic system in mouse hypothalamus, 

Hormones and Behavior 46 (2004) 95-96. 

[7] P. Collado, A. Guillamon, H. Pinos, M.A. Perez-Izquierdo, A. Garcıa-Falgueras, B. 

Carrillo, C. Rodrıguez and G.C. Panzica, NADPH-diaphorase activity increases 

during estrous phase in the bed nucleus of the accessory olfactory tract in the 

female rat, Brain Research 983 (2003) 223–229. 

[8] B. Carrillo, H. Pinos, G.C. Panzica, A. Guillamon and P. Collado, Nitrergic 

expression during estrous phase and morphologic sexual dimorphism in the medial 

amygdala anteroventral subdivision in rat, Hormones and Behavior 46 (2004) 85- 

86. 

[9] G.C. Panzica, C. Viglietti-Panzica, M. Sica, S. Gotti, M. Martini, H. Pinos, B. 

Carrillo and P. Collado, Effects of gonadal hormones on central nitric oxide 

producing systems, Neuroscience 138 (2006) 987-995. 

270



PRENATAL STRESS HAS LONG-TERM INFLUENCE ON 

NEUROPLASTICITY: SEX DIFFERENCES 

1,2 Maccari S, 1,2 Mairesse J, 2 Zuena AR, 1 Morley-Fletcher S, 2 Matteucci P, 2 Cinque C, 

2 Catalani A, 2,3 Nicoletti F, 2 Casolini P. 

1 Lab. of Perinatal Stress, University of Lille 1 USTL, Villeneuve d'Ascq, FR 

maccari@univ-lille1.fr, fax +33.320.434602 or +39.0649912524; 2 University of Rome La 

Sapienza, IT; 3 Neuromed, Pozzilli, IT. 

Prenatal Stress (PS) in rats is a well-documented model of early stress known to 

induce long-lasting neurobiological and behavioural alterations (i.e., altered circadian 

rhythms, high levels of “anxiety”, altered feedback mechanisms of the HPA axis, 

decreased hippocampal glucocorticoid receptors, increased hippocampal BDNF and basal 

Fos expression) (2,4,6). These results together suggest that PS affects the ability to cope 

with environmental challenges and alters neuroplasticity. In particular, we have found that 

PS rats prefer an active coping strategy when exposed to an anxiogenic environment, 

whereas they show less interest towards a more reassuring environment (7). This is 

reminiscent of what occurs in depression and anxiety, in which allostatic changes in 

neuroplasticity in limbic regions hamper the emotional response to the environment. (5). 

In the last two decades, however, effects of PS on neuroplasticity and on response to stress 

has been extensively studied, but most studies focus on male rats. One example is the 

effect of PS on hippocampal neurogenesis in male rats (1). Among the different factors that 

can be involved in the regulation of neurogenesis, metabotropic glutamate receptors 

(mGluR) seem to play a significant role. Indeed, we have recently shown that in male rats 

PS induced a decrease in neurogenesis, an attenuated activity of hippocampal group-I 

mGlu receptors associated with increased anxiety-like behaviors. Interestingly, in female 

rats, PS induced opposite long-term effects on group-I mGlu receptors and anxiety (2). 

Here we studied, in male and female rats, the long-lasting effects of PS on neurogenesis in 

two different hippocampal regions, the ventral part, mainly involved in the modulation of 

anxiety-like behaviours, and the dorsal part, more implicated in learning processes. In 

order to evidence cell survival, rats were injected with the thymidine-analog 

bromodeoxyuridine (BrdU 75 mg/kg i.p. twice daily for 4 days) 3 weeks before 

immunohistochemistry analysis. Two important results came out from this study: first, in 

female rats a higher percentage of cells differentiated in astroglia compared to males, even 

if the percentage of hippocampal neuronal differentiation was similar in the two sexes; 

second, PS differentially affect neurogenesis in male and female rats, in fact, in terms of 

cell survival, PS reduced neurogenesis in male rats both in the dorsal and in the ventral part 

of the hippocampus, but no effect was observed in females. These results show that the 

hippocampus of female rats seems to be less vulnerable to PS compared to males. This 

decreased vulnerability could be explained, at least in part, by the protective effect yielded 

by the increased activity of group-I mGlu receptors present in females, while the activity of 

the same receptors was decreased in the more vulnerable male rats. Furthermore, an higher 

percentage of astroglia in females could also play a role. 

271



1 Lemaire V, Koehl M, Le Moal M, Abrous DN. Prenatal stress produces learning deficits 

associated with an inhibition of neurogenesis in the hippocampus. Proc Natl Acad Sci U S 

A. 2000 Sep 26;97(20):11032-7 

2 Maccari S, et al. (2003). Prenatal stress and long-term consequences: implications of 

glucocorticoid hormones. Neurosci Biobehav Rev 27:119-127. 

3 Maccari S, Zuena AR, Cinque C, Morley-Fletcher S, Catalani A, Nicoletti F, Casolini P. 

Long term consequences of a restraint prenatal stress and hippocampal metabotropic 

receptors. Program No. 662.15.- (2004) San Diego, USA : Society for Neuroscience. 

4 Maccari S. Morley-Fletcher S, Mairesse J., Viltart O., Daszuta A., Soumier A., Hery M., 

Gabriel C, Mocaer E, Zuena A., Matteucci P., Cinque C. Catalani A. Casolini P. Chronic 

treatment with agomelatine reversed the decrease in hippocampal cells neurogenesis and 

survival in prenatally stressed adult rats Program no. 566.8. (2005) Washington, DC: 

Society for Neuroscience, 2005. 

5 McEwen BS, Wingfield JC (2003) The concept of allostasis in biology and biomedicine. 

Horm Behav. 43(1):2-15Viltart O, Mairesse J, et al. (2006). Prenatal stress alters Fos 

protein expression in hippocampus and locus coeruleus stress-related brain structures. 

Psychoneuroendocrinology 31:769-780. 

6 Viltart O, Mairesse J, Darnaudery M, Louvart H, Vanbesien-Mailliot C, Catalani A, 

Maccari S. Prenatal stress alters Fos protein expression in hippocampus and locus 

coeruleus stress-related brain structures. Psychoneuroendocrinology. 2006 Jul;31(6):769-80 

7 Mairesse et al., Prenatal stress disrupts the negative correlation between neuronal activation 

in limbic regions and behavioral responses in rats exposed to high and low anxiogenic 

environments. Submitted. 

272



ROLE OF APOPTOSIS IN THE SEXUAL DIFFERENTIATION OF THE BED 

NUCLEUS OF STRIA TERMINALIS OF JAPANESE QUAIL 

Mura E., Furnari P., Plumari L., Viglietti-Panzica C., Panzica G.C. 

Laboratory of Neuroendocrinology, Dept Anatomy, Pharmacology and Forensic Medicine, C.so M. 

D’Azeglio, 52. 10126 Torino (Italy) 

e-mail: elena.mura@unito.it 

Programmed cell death (or apoptosis) is an essential process during brain 

development that plays an important role in the differentiation of areas involved in 

sexually dimorphic functions. In fact, in rodents a different incidence of apoptosis between 

males and females during the first weeks of life establishes the sexual dimorphism of some 

brain nuclei [2,5,10]. 

In galliform birds, the parvocellular vasotocin (VT) system of the limbic-preoptic region 

shows a strong dimorphism [4,6] probably dependent by the exposure to estradiol during 

embryonic development [8]. In adult males VT-immunoreactive (VT-ir) cells and fibers 

are largely present in the bed nucleus of the stria terminalis (BST), the medial preoptic 

nucleus (POM), and the lateral septum (SL). In females very low levels of VT-ir fibers are 

present, whereas cell bodies are totally absent. This strong dimorphism is also confirmed 

by the detection of mRNA for VT by means of in situ hybridization [4,7]. The BST 

represents the major source of VT-ir fibers for the innervation of both POM and SL [1,9]. 

In the present study we investigated the possible sexually dimorphic incidence of apoptosis 

during the first ten postnatal days within the Bed Nucleus of Stria Terminalis (BST) of the 

Japanese quail. Quail BST shows, in fact, a strong sexual dimorphism of the parvocellular 

vasotocin (VT) system in adult, while at birth males and females have a comparable 

amount of vasotocinergic elements [3]. 

Male and female quail chicks were sacrificed by intracardiac perfusion and according to 

their age they were divided in the following groups: P1, P2, P3, P4, P5, P8 and P10. Brains 

were taken out of the skull, frozen with dry ice and cut with a cryostat. Coronal sections 

were stained with cresyl violet or immunostained for VT detection. Sections stained with 

cresyl violet were analyzed with a camera lucida in order to distinguish and count 

apoptotic cells (cells with picnotic nucleus and cells with fragmented nuclear material). 

Adjacent sections immunostained for VT were semiquantitative analyzed to estimate the 

extension of the VT system. 

The semiquantitative analysis of VT-immunoreactivity has confirmed the presence of the 

VT-ir system and birth in both sexes with a similar extension. VT-ir cells diminish in a 

progressive way during postnatal development up to day 10, whereas fibers have a 

moderate immunoreactivity in both sexes. 

The two-way ANOVA (age and sex as indipendent variables) for the total number of 

apoptotic cells demonstrated a strong effect for both factors. Two by two comparisons with 

student t-test demonstrated a statistically significant difference at day P3, with females 

showing a higher number of apoptotic cells than males. 

We can therefore conclude that, in Japanese quail, apoptosis in the BST is a sexually 

differentiated process that could be one of the mechanisms inducing the sexual 

dimorphism of VT-ir system of the BST. 

273



1. P. Absil, M. Papello, C. Viglietti Panzica, J. Balthazart and G.C. Panzica, The medial 

preoptic nucleus receives vasotocinergic inputs in male quail: a tract-tracing and 

immunocytochemical study, J Chem Neuroanat 24 (2002) 27-39. 

2. Y. Arai, Y. Sekine and S. Murakami, Estrogen and Apoptosis in the Developing 

Sexually Dimorphic Preoptic Area in Female Rats, Neurosci Res 25 (1996) 403-407. 

3. N. Aste, G. Baiamonte, R. Grossmann and G.C. Panzica, Postnatal development and 

sexual differentiation of quail vasotocin system, Italian J Anat Embriol 102, Suppl. 

(1997) 82. 

4. N. Aste, J. Balthazart, P. Absil, R. Grossmann, E. Mühlbauer, C. Viglietti-Panzica and 

G.C. Panzica, Anatomical and neurochemical definition of the nucleus of the stria 

terminalis in Japanese quail (Coturnix japonica), J Comp Neurol 396 (1998) 141-157. 

5. E.C. Davis, P. Popper and R.A. Gorski, The Role of Apoptosis in Sexual 

Differentiation of the Rat Sexually Dimorphic Nucleus of the Preoptic Area, Brain 

Res. 734 (1996) 10-18. 

6. A. Jurkevich, S.W. Barth, N. Aste, G.C. Panzica and R. Grossmann, Intracerebral sex 

differences in the vasotocin system in birds: possible implication on behavioral and 

autonomic functions, Horm Behav 30 (1996) 673-681. 

7. A. Jurkevich, S.W. Barth and R. Grossmann, Sexual dimorphism of arg-vasotocin 

gene expressing neurons in the telencephalon and dorsal diencephalon of the domestic 

fowl. An immunocytochemical and in situ hybridization study, Cell Tissue Res 287 

(1997) 69-77. 

8. G.C. Panzica, C. Castagna, C. Viglietti-Panzica, C. Russo, O. Tlemçani and J. 

Balthazart, Organizational effects of estrogens on brain vasotocin and sexual behavior 

in quail, J Neurobiol 37 (1998) 684-699. 

9. G.C. Panzica, E. Mura, M. Pessatti and C. Viglietti Panzica, Early embryonic 

administration of xenoestrogens alters vasotocin system and male sexual behavior of 

the Japanese quail, Dom An Endo 29 (2005) 436-445. 

10. M. Yoshida, K. Yuri, Z. Kizaki, T. Sawada and M. Kawata, The distributions of 

apoptotic cells in the medial preoptic areas of male and female neonatal rats, Neuros 

Res 36 (2000) 1-7. 

274



THE ACCESSORY OLFACTORY BULB OF THE ADULT MOUSE: 

NEUROGENESIS AND MORPHOLOGICAL ANALYSIS IN THE TWO SEXES 

Oboti L.*°, Peretto P.*, Fasolo A.*, Panzica G.C.° 

*Department of Animal and Human Biology, University of Turin, via Accademia 

Albertina 13, 10123 Turin, Italy 

e-mail: livio.oboti@unito.it 

Fax: +39-011-6704692 

°Department of Anatomy, Pharmacology and Forensic Medicine, University of Turin, Via 

Accademia Albertina 13, 10123, Italy 

The accessory olfactory bulb (AOB) is a sexually dimorphic structure [1] of the 

vomeronasal system whose development and function is regulated by steroid hormones [2]. 

The AOB is part of the accessory olfactory pathway that is devoted to the processing of 

information concerned with reproduction and social behaviours. Evidences in rats indicate 

that the persistence of neurogenesis occurring in the adult subventricular zone (SVZ) also 

involves the AOB [3]. In the rat the amount of the SVZ newborn neurons is sexually 

dimorphic in the anterior part of the granular layer (GrL) of the AOB [4]. Recent 

comparative analyses have shown significant differences in the organization and activity of 

the adult neurogenic regions in several mammalian species [5]. Specific aim of our work 

was to analyze the neurogenic activity in the adult mouse AOB. In particular we have 

studied the distribution of newborn cells in the AOB different layers and functional 

subdivisions (anterior/posterior) of both sexes in the CD1 mice strain. Systemic injections 

of the exogenous marker of cell proliferation BrdU, and markers of immature (DCX) and 

mature (Neu-N, CR, GABA, NOS) neurons were used to evaluate neurogenic activity 1 

month after the BrdU treatment. Quantitative analyses were used to establish number, 

distribution, and differentiating phenotypes of newly formed neurons in the AOB of both 

sexes. Moreover, by using G0α immunoreactivity we studied the AOB volumes 

considering each single layer in its own functional subdivision in males and females. 

Our results indicate the occurrence of newly formed neurons in the AOB of adult CD1 

mice in both sexes. No significant differences were found between sexes in the distribution 

and phenotype of newly formed neurons. Conversely, in both males and females we have 

found that newly formed neurons are preferentially located (p=0,0002) in the anterior part 

of the AOB. Finally, the volumetric study did not show any sexual difference. 

These overall results validate the hypothesis that the occurrence of such neurogenic 

phenomenon along the accessory olfactory pathway can be similar in phylogenetically 

related species. Nevertheless, the striking result concerning the absence of a morphological 

sexual dimorphism in the CD1mice AOB reinforce the importance of detailed comparative 

studies to understand the basis of sexual dimorphism in the mammalian brain. 

This work is supported by Compagnia di San Paolo (Neurotransplant Project 2004.2019) 


1. Guillamon A. & Segovia S., 1997. “Sex differences in the vomeronasal system”. In: 

Hormones, Brain, and Behavior (G.C.Panzica and J.Balthazart eds). Brain Res Bull 

44: 377-382. 

2. Segovia S. & Guillamon A., 1984. “Effects of sex steroids on the development of the 

accessory olfactory bulb in the rat: a volumetric study”. Dev. Brain Res. 16:312-314. 

275


3. Bonfanti L., Peretto P., Merighi A., Fasolo A., 1997. “Newly generated cells from the 

rostral migratory stream in the accessory olfactory bulb of the adult rat”. Neuroscience 

81:489-502. 

4. Peretto P., Giachino C., Panzica G.C., Fasolo A., 2001. ”Sexually dimorphic 

neurogenesis is topographically matched with the anterior accessory olfactory bulb of 

the adult rat”. Cell Tissue Res. 306:385-389. 

5. Rakic P. 2004. ”Neuroscience: immigration denied”. Nature 427:685-686. 

276



UNDERNURISHMENT AND FOOD REHABILITATION EFFECTS ON PLASMA 

LEPTIN LEVELS IN WISTAR RAT 

Pinos H, Carrillo B, Pérez-Izquierdo M, Ortega E (1), Collado P. 

Dpto Psicobiología, Fac. Psicología UNED, C/ Juan del Rosal nº 10, 28040 Madrid, Spain, 

pcollado@psi.uned.es (1) Dpto de Bioquímica y Biología Molecular, Fac Medicina, 

Universidad de Granada, Spain. 

Leptin is a peptide hormone secreted by adipose tissue which is crucial in the regulation of 

feeding behaviour and has an important role in the regulation of metabolim, energy 

balance and reproduction (Campfield et al, 1995; Pelleymounter et al, 1995; Ahima and 

Flier, 2000; Ahima et al, 2000; Ahima and Osei, 2004).. Recent studies have shown that 

during the development, this hormone can play a role like neurotrophic factor (Bouret and 

Simerly, 2004; Bouret et al, 2004; Steppan and Swick, 1999). In well nourished rats, 

plasma leptin levels are different between males and females, showing males higher levels 

than females of the same age. 

In a previous study, we have studied the effect of undernutrition from gestational period to 

weaning day, in the postnatal day 21, and food rehabilitation, on the morphology of the 

locus coeuruleus (LC) in male and female Wistar rats. Data showed that pre and postnatal 

food deprovation until 60-days of age results in a significant decrease in the number of 

neurons in the LC in rats of both sexes. However, food rehabilitation induces some 

recovery in the LC neuronal population in males, but not in females. 

Data from the present study has shown that a food restriction period from embryonic day 

6 until postnatal day 60, has a consequence a significant decrease of serum leptin levels in 

both, male and female rats during development at postnatal day 12 but only seems to have 

a permanent effect in males when the food restricted diet extents until adulthood. When a 

food rehabilitation (water and food ad libitum) is implemented early enough in males, 

serum leptin levels recover in some extent levels showed by adult control males. 

Undernutrition also has a long term effect on body weight in both sexes, but nutritional 

rehabilitation leads to some degree of body weight recovery depending on sex and age at 

which that rehabilitation was implemented. It could be suggested that undernutrition and a 

posterior nutritional rehabilitation during the first stages of life is affecting different 

developmental processes in male and female rats and therefore the response of body 

weight and serum leptin levels in each sex to these different nutritional conditions varies, 

being males more vulnerable than females. 


1. Ahima RS, Flier JS (2000) Leptin. Annu. Rev. Physiol. 62:413-437 

2. Ahima RS, Saper CB, Flier JS et al (2000) Leptin regulation of neuroendocrine 

systems. Front. Neuroendocrinol. 21:263-307. 

3. Ahima RS, Osei YO (2004) Leptin signalling. Physiol. Behav. 81:223-241. 

4. Bouret SG, Simerly RB (2004) Minireview: Leptin and development of 

hypothalamic feeding circuits. Endocrinology. 145:2621-2626. 

5. Bouret SG, Draper SJ, Simerly RB (2004) Trophic action of leptin on hypothalamic 

neurons that regulate feeding. Science. 304:108-110. 

277


6. Campfield LA, Smith FJ, Guisez Y et al (1995) Recombinant mouse OB protein: 

evidence for a peripheral signal linking adiposity and central neural networks. 

Science. 269:546-549. 

7. Pelleymounter MA, Cullen MJ, Baker MB et al (1995) Effects of the obese gene 

producto on body weight regulation in ob/ob mice. Science. 269:540-543. 

8. Pinos H, Collado P, Salas M et al (2004) Undernutrition and food rehabilitation 

effects on the locus coeruleus in the rat. Neuroreport. 15:1417-1420. 

9. Steppan CM, Swick AG (1999) A role for leptin in brain development. Biochem. 

Biophys. Res. Comm. 256:600-602. 

Sources od support:MCYT grant BSO2003-02526 (PC) 

278

AUTHOR INDEX 

A 

Abdelnabi M.......................89 

Ábrahám I.M. ......... 180, 228 

Abrous D.N.........................66 

Acharjee S........................ 240 

Adriani W. ....................... 209 

Ahmadiani A. .................. 218 

Ahn H.S............................ 175 

Alias A.G.......................... 147 

Alleva E...................... 97, 199 

Allieri F. ........................... 217 

Almada R.F. .................... 220 

Aloisi A.M........................ 234 

Amini H............................ 218 

Andrade T.G.C.S.... 219, 220 

Arcieri P..............................13 

Assenza G ........................ 164 

Aste N. .............................. 222 

Atwood C.S...................... 224 

Avanzi V................... 219, 220 

Avitabile M ...................... 226 

Avola R............................. 226 

Avoli M............................. 247 

Azcoitia I.......................... 161 

B 

Bäckström T...121, 158, 195, 

197, 246, 260, 

Baghai T.C....................... 149 

Bakker J..................... 57, 104 

Baldelli E.......................... 247 

Ballarin C. ....................... 157 

Balog J.............................. 228 

Balthazart J............... 59, 104 

Banasr M ......................... 207 

Bao A-M................... 125, 265 

Barha C ...............................63 

Barker J.M. ........................63 

Barreto G......................... 161 

Barrile V. ......................... 267 

Barton M.............................89 

Bass A.H................. 39, 56, 99 

Baulieu E.E.........................19 

Bélanger A. ...................... 240 

Belcher S.M. .................... 113 

Bellinger D....................... 252 

Belloni V..............................97 

Belon P. ............................ 262 

Bembi B............................ 182 

Benedusi V............... 138, 142 

Berger A........................... 237 

Bernardi G..........................28 

Berry A............................. 199 

Berumen L.C................... 163 

Biagini G...........................247 

Biamonte F .......................164 

Bianchi R ................. 171, 177 

Biasella A. .........................250 

Bierzniece V. ....................195 

Biggio G. ...........................118 

Bixo M...............................260 

Bo E ...................................154 

Bodo C.................................42 

Bonifazi M. .......................250 

Bonsignore L.T. ...............204 

Boujedaini N.....................262 

Bourque M............................8 

Bowen R.L. .......................224 

Bramanti V.......................226 

Brinton R.D. ..................7, 65 

Broiz A.C.G......................219 

Bronzi D ............................226 

Brunström B.....................139 

Buson G.............................157 

Bütow A. ...........................242 

C 

Cambiasso M.J. ...............120 

Campbell B.C...................230 

Campbell R.E.....................33 

Caniglia S............................13 

Cannizzaro C. ..................267 

Carrero P...10, 143, 181, 238 

Carrillo B................. 232, 277 

Carroll J.C.........78, 165, 178 

Carta M.................... 116, 118 

Caruso D....25, 164, 177, 259 

Casella D...........................154 

Casolini P................. 207, 271 

Castel H.............................240 

Catalani A................ 207, 271 

Cavaletti G............... 171, 177 

Ceccarelli I........................234 

Cecchi C............................166 

Cesa R ...............................164 

Chalbot S. .........................235 

Chang L. ...........................165 

Chen C...............................252 

Chen S. ...............................65 

Chung E. ............................65 

Cibula D............................150 

Cinque C ...........................271 

Ciriza I. ...............................10 

Cirulli F.................... 199, 204 

Clarkson J. .........................33 

Collado P.................. 232, 277 

Corrieri L. ..........................93 

Cozzi B. .............................157 

Crotti S...............25, 164, 259 

Csakvari E........................237 

D 

Danza G. ...........................166 

Darbra S. ..........................249 

Dardis A ............................182 

Darnaudéry M. ................134 

Daszuta A..........................207 

De Angelis L .....................259 

de Kloet E.R. ....................199 

De Nicola A.F. ........... 19, 115 

De Padova A.M................234 

Delitala G............................13 

Della Seta D. .......................93 

Demajo M.A. ....................212 

Desole M.S. .........................13 

Dessì-Fulgheri F.......... 93, 97 

Dettling-Artho R..............200 

Deviche P. .........................154 

Dewing P. ............................55 

Di Michele F. ....................149 

Di Paolo T. ............................8 

Diaz-Heijtz R....................200 

Dichiara F.........................166 

Dieni C.V. .........................190 

Diliberto I. ........................267 

Diz-Chaves Y.....10, 181, 238 

Djordjevic A.D.,...............212 

Dluzen D.E. ...........................8 

Do Rego J.L......................240 

Doodipala S.R.....................27 

Dutia M.B. ........................190 

Dykens J.A..........................77 

E 

Eckert A............................179 

Erdei F. .............................228 

Eser D................................149 

F 

Farabollini F.......................93 

Fargo K.N.........................168 

Fasolo A. ...........................275 

Feldon J.............................200 

Fester L. ............................242 

Fiorenzani P. ....................234 

Forlano P.M. ......................99 

Formigli L.........................166 

Forsberg M. K..................169 

Fowler C.D. ........................64 

Frondaroli A.....................192 

Frye C.A.............11, 106, 144

Furnari P. .........................273 

Fusani L. .............................93 

G 

Galas L. .............................240 

Galea L.A.M.............. 63, 156 

García-Alcocer G. ...........163 

Garcia-Ovejero D. ...........161 

Garcia-Segura L.M. 10, 143, 

161, 171, 177, 181, 238 

García-Servín M..............163 

Gass P................................132 

Gennuso F...........................13 

Giaquinta G........................13 

Giatti S ..................... 171, 177 

Ginanneschi F. .................250 

Giorgio M. ........................199 

Gong W ...............................73 

Gonzalez Deniselle M.C. 115 

Gonzalez S.L. ...................115 

Gorosito S.V. ....................120 

Gotti S. ..............................269 

Grassi S. ............................192 

Greeson J. .................. 68, 235 

Griffiths W.J. .....................21 

Grossmann R. ....................45 

Guennoun R. ............. 19, 115 

Guillamon A.............. 51, 232 

Gye M.C............................175 

Gyenes A. ..........................237 

H 

Håkansson H. ...................187 

Hallberg M .......................169 

Halldin K. ................ 139, 187 

Handa R.J...........................41 

Hardin-Pouzet H .............217 

Hariri O. .................. 111, 194 

Hauser J............................200 

Hausmann O. .....................84 

Hayashi S. .........................255 

Herbert J.............................67 

Herbison A.E............. 33, 258 

Higashi T.............................23 

Hill M. ...................... 150, 152 

Hirata M. ................. 245, 251 

Hiroi R. .............................243 

Hirst JJ..............................202 

Horvat A.I.........................212 

Hoyk S...............................237 

Huber C. ...........................242 

I 

Ibrahim A. ..........................68 

Imaizumi K.........................86 

Irwin R................................65 

Ishunina T.A. .....................43 

J 

Jarrahi M................. 172, 210 

Jarry H..................... 242, 257 

Jasoni C.L.........................258 

Johansson I-M 158, 195, 246 

Juhász G. ................. 180, 228 

K 

Kancheva L. ............ 150, 152 

Kanematsu T........... 245, 251 

Kang H.S...........................175 

Kaur G. .............................256 

Kékesi K.A........................180 

Keller F .............................164 

Keller M............................104 

Kibaly C..................... 81, 174 

Kim H.J.............................175 

Klein S.................................45 

Knapman A. .....................200 

Kodama M........................255 

Kohlmann P .....................257 

Kondo S...............................86 

Korrick SA .......................252 

Kurunczi A.......................237 

Kwon H.B .........................240 

L 

L’Episcopo F......................13 

La Barbera M. .................267 

Labombarda F.......... 19, 115 

LaFerla F.M .............. 78, 165 

Lauria G .................. 171, 177 

Laviola G ................. 204, 209 

Lavoie E. .............................89 

Le H.H...............................113 

Lecanu L.................... 68, 235 

Lee J.E. .............................175 

Liere P.................................19 

Liguri G ............................166 

Lindblad C. ............. 158, 195 

Liu B. .....................................8 

Liu T..................................224 

Löfgren M.........................246 

Lohse C .............................257 

Longo D.............................247 

Longone P...........................28 

Lundgren P.......................197 

Luu-The V. .......................240 

M 

Maccari S.........134, 207, 271 

Macrì S..................... 204, 209 

Maggi A.............. 91, 138, 142 

Maggio N. .........................206 

Mairesse J................ 207, 271 

Mameli M. ........................116 

Manieri G. ..........................28 

Mantia G...........................267 

Marchetti B. .......................13 

Marino R ..........................164 

Martín-García E..............249 

Martini M. .47, 154, 185, 269 

Martin-Padura I..............199 

Maruniak J.........................94 

Maschi O.................... 25, 259 

Massafra C. ......................234 

Materazzi P. .......................93 

Matteucci P.............. 207, 271 

Matthews S.M. .................133 

Mattsson A. ......................139 

Mayoral S.R. ....................141 

Mazzocchio R...................250 

McCourty A. ......................68 

McEwen B.S. ....................102 

Meffre D............................115 

Melcangi R.C. .... 25, 83, 164, 

171, 177, 259 

Mellon S.H................. 73, 182 

Mensah-Nyagan A.G. ......81, 

101, 174, 179, 262 

Menzies JRW...................190 

Meyer L.............. 81, 101, 174 

Meyerson B.......................246 

Miceli D.............................185 

Micevych P. ....... 55, 111, 194 

Miele E. ...............................13 

Milani P.............................250 

Minghetti L.......................199 

Mirzaei M. ........................218 

Mizokami A......................251 

Mocaer E ..........................207 

Morale M.C........................13 

Morello M.........................166 

Morissette M. .......................8 

Morley-Fletcher S. 134, 207, 

271 

Mostallino M.C................118 

Mura E..................... 139, 273 

N 

Nagahama A.......................23 

Nakamuta J.S...................220 

Nasir RH...........................252 

Neumaier J.F....................243 

Nevyjel M..........................182 

Nicoletti F .........................271 

Ninomiya Y.........................23 

Nobahar M .......................254 

Nosi D................................166 

Noto G. ..............................267 

Nyberg F ...........................169 

Nyberg S. ................. 121, 260

O 

Oboti L. ............................ 275 

Oddo S........................ 78, 165 

Ognibene E. ..................... 209 

Ohya T.............................. 255 

Ortega E........................... 277 

Ottinger M.A......................89 

P 

Palanza P. .................. 94, 185 

Pallarés M........................ 249 

Palliser HK ...................... 202 

Panzica G.C......47, 139, 154, 

185, 232, 269, 273, 275 

Papadopoulos V. ....... 68, 235 

Parducz A. ....................... 237 

Paris, J.J........................... 106 

Pařízek A. ........................ 150 

Parkash J ......................... 256 

Parmigiani S.......................94 

Pasini A. ..................... 28, 149 

Pasquali P. ....................... 204 

Patisaul H.B................. 54, 90 

Patte-Mensah C. ...... 81, 101, 

174, 179, 262 

Pawluski J.L.............. 63, 156 

Pelicci P. G....................... 199 

Pelletier G. ....................... 240 

Penn A.A.......................... 141 

Pensalfini A. .................... 166 

Peretto P........................... 275 

Pérez-Izquierdo M.A .... 232, 

277 

Peri A................................ 166 

Pernía O. ....10, 143, 181, 238 

Peruffo A.......................... 157 

Pesaresi M................ 171, 177 

Pettorossi V.E.................. 192 

Pieraccini G. .................... 166 

Pike C.J. .............78, 165, 178 

Pinos H. .................... 232, 277 

Pisu M.G. ......................... 118 

Pittis MG.......................... 182 

Plescia F. .......................... 267 

Plumari L......................... 273 

Polston E.K.........................90 

Ponzi D. ...............................94 

Porteous R. .........................33 

Pozzi S. ..................... 138, 142 

Pradotto M. ..................... 269 

Prange-Kiel J................... 257 

Pryce C.R................. 130, 200 

Q 

Quinn M. Jr........................89 

R 

Raciti C .............................226 

Ragagnin G.......................197 

Rahman M............... 158, 197 

Rashidy-Pour A ...............172 

Repetto E. .........................240 

Rhodes, M.E.....................106 

Rissman E.F. ......................42 

Ritz M.-F.............................84 

Roglio I..................... 171, 177 

Romano N...........................33 

Romanò N.........................258 

Romeo E..................... 28, 149 

Romeo R.D. ......................102 

Rosario E.R. ......78, 165, 178 

Rosati F. ............................166 

Rossi A. .............................250 

Rune G.M. ............... 242, 257 

Rupprecht R.............. 28, 149 

S 

Sabetkasaei M. .................218 

Saito N...............................222 

Salimpour S......................218 

Sánchez-Ramos M.A.......163 

Sanna E. ............................118 

Santucci D...........................97 

Sanz A. ..............................143 

Schaeffer V. ............... 81, 179 

Schön M ............................257 

Schonemann M. .................73 

Schüle C. ...........................149 

Schumacher M.......... 19, 115 

Schwarz M........................149 

Scurati S.............25, 177, 259 

Segal M..............................206 

Segovia S. ............................51 

Sengelaub D.R.................168 

Seong J.Y. .........................240 

Sergio T.O.........................219 

Serio M..............................166 

Serra M. ............................118 

Serra P.-A. ..........................13 

Shimada K. ................ 23, 222 

Sica M......................... 47, 187 

Simpkins J.W. ....................77 

Sinchak K. ........................111 

Smith J.T. ...........................37 

Soma K..............................111 

Soumier A .........................207 

Sousa N..............................129 

Spritzer M.D. .....................63 

Spurling L.........................113 

Stanczyk F.Z. ...................165 

Stárka L ............................152 

Stefani M...........................166 

Stein D.G.................... 79, 115 

Strata P .............................164 

Strömberg J......................197 

Sundström Poromaa I. ...121 

Svensson A-L....................169 

Swaab D.F....43, 53, 125, 265 

Szabó G. ............................228 

Szajli A..............................237 

Szegő É.M................. 180, 228 

T 

Taherian AA.....................210 

Talani G. ...........................118 

Tapia González S...... 10, 181 

Taziaux M.........................104 

Tecozautla A. ...................163 

Tena-Sempere M. ..............15 

Testa N. ...............................13 

Thompson N.......................89 

Timby E. ...........................260 

Tirolo C...............................13 

Tobin V. ............................190 

Tonon M.C. ......................240 

Tremblay Y. .....................240 

Turkmen S........................195 

U 

Uzunov DP........................149 

V 

Vadakkadath Meethal S.224 

Vafaei AA ........172, 210, 254 

Valenzuela C.F.................116 

Vallarino M. .....................240 

Vaudry H. .........................240 

Včelaková H ............ 150, 152 

Vegeto E................... 138, 142 

Veiga S. .............................161 

Velickovic N.A .................212 

Venard C...........................262 

Verzè L................................47 

Viglietti-Panzica C. . 47, 154, 

185, 269, 273 

Vlad A.G. ..........................263 

Vom Saal F.S......................94 

von Lossow R. ..................242 

Vrbíková J........................152 

W 

Walf A.A. ................... 11, 144 

Walker C.A. .....................156 

Walker DW ......................202 

Wang J.M...........................65 

Wang M-D ............... 158, 197 

Wang Y. ..............................21 

Wang Z.X. ..........................64 

Watanabe Y......................222

Whitehouse K.....................89 

Wihlbäck A.-C.N. ............260 

Wilson A.C. ......................224 

Y 

Yao W..................................68 

Yates DM ..........................202 

Z 

Zaccaroni M.......................97 

Zampieri S ........................182 

Zamudio, P.A. ..................116 

Zhao L...................................7 

Zhou L...............................242 

Zingmark E. ............ 121, 197 

Zini I..................................247 

Zoli M................................247 

Zsarnovszky A. ................113 

Zuena AR................. 207, 271 

Zuercher N. ......................200

Conference organized with the support of 

Università degli Studi di Torino 

Università degli Studi di Milano 

Facoltà di Scienze MFN, Torino 

Dipartimento di Anatomia, Farmacologia e Medicina Legale 

Fondazione Oasi, Troina, Italy 

Centro Rita Levi Montalcini, Torino 

Center of Excellence on Neurodegenerative diseases, Milano 

International Brain Research Organization (IBRO) 

Società Italiana di Neuroscienze 

National Science Foundation 

Regione Piemonte 

Provincia di Torino 

Comune di Torino 

Applied Biosystems 

Thermofisher 

BIORAD 

CELBIO 

DBA, Italy 

Nikon, Italy 

Vinci Biochem, Italy 

Elsevier Publisher 

Karger Publisher

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