ABSTRACT OF DISSERTATION Deanna Lynn McCullers The ...

ABSTRACT OF DISSERTATION 

Deanna Lynn McCullers 

The Graduate School 

University of Kentucky 

2001

ADRENOCORTICOSTEROID RECEPTOR EFFECTS 

ON HIPPOCAMPAL NEURON VIABILITY 

____________________________________ 


____________________________________ 

A dissertation submitted in partial fulfillment of the 

requirements for the degree of Doctor of Philosophy in the 

College of Medicine at the University of Kentucky 

By 


Lexington, Kentucky 

Director: Dr. James P. Herman, Associate Professor of Anatomy and Neurobiology 


2001 

Copyright © Deanna Lynn McCullers 2001




Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), the 

mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), influences hippocampal 

neuron vulnerability to injury. Excessive activation of GR may compromise hippocampal 

neuron survival after several types of challenge including ischemic, metabolic, and excitotoxic 

insults. In contrast, MR prevents adrenalectomy-induced loss of granule neurons in the dentate 

gyrus. The present thesis addresses the respective roles of MR and GR in modulating neuronal 

survival following two forms of neuronal injury, excitotoxicity and traumatic brain injury. Male 

Sprague-Dawley rats were pretreated with MR antagonist spironolactone or GR antagonist 

mifepristone (RU486) and subsequently injected with kainic acid, an excitotoxic glutamate 

analog, or injured with a controlled cortical impact. Twenty-four hours following injury, 

hippocampal neuron survival was measured to test the hypotheses that MR blockade would 

endanger and GR blockade would protect hippocampal neurons following injury. Messenger 

RNA levels of viability-related genes including bcl-2, bax, p53, BDNF, and NT-3 were also 

measured to test the hypothesis that ACR regulation of these genes would

correlate with neuronal survival. In addition, ACR mRNA levels were measured following 

receptor blockade and injury to test the hypothesis that glucocorticoid signaling is altered 

following neuronal injury via regulation of ACR expression. 

Mineralocorticoid receptor blockade with spironolactone increased neuronal vulnerability to 

excitotoxic insult in hippocampal field CA3, and GR blockade with RU486 prevented neuronal 

loss after traumatic brain injury in field CA1. These results are consistent with the hypotheses 

that MR protects and GR endangers hippocampal neurons. Adrenocorticosteroid receptor 

blockade decreased mRNA levels of the anti-apoptotic gene bcl-2 in select regions of uninjured 

hippocampus, yet ACR regulation of bcl-2 did not consistently correspond with measures of 

neuronal survival after injury. Kainic acid decreased MR mRNA levels in CA1 and CA3, while 

both kainic acid and controlled cortical impact dramatically decreased GR mRNA levels in 

dentate gyrus. These data suggest that injury modulation of glucocorticoid signaling through 

regulation of ACR expression may influence hippocampal neuron viability following injury. 

KEYWORDS: Mineralocorticoid Receptor, Glucocorticoid Receptor, Messenger RNA, Kainic 

Acid, Traumatic Brain Injury 

Deanna L. McCullers 

December 6, 2001



By 


Dr. James P. Herman 

Director of Dissertation 

Dr. Brian MacPherson 

Director of Graduate Studies 

December 6, 2001

RULES FOR THE USE OF DISSERTATIONS 

Unpublished dissertations submitted for the Doctor’s degree and deposited in the University of 

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rights of the authors. Bibliographical references may be noted, but quotations or summaries of 

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acknowledgments. 

Extensive copying or publication of the dissertation in whole or in part also requires the consent 

of the Dean of the Graduate School of the University of Kentucky.

DISSERTATION 


The Graduate School 


2001



____________________________________ 

DISSERTATION 

____________________________________ 

A dissertation submitted in partial fulfillment of the 

requirements for the degree of Doctor of Philosophy in the 

College of Medicine at the University of Kentucky 

By 



Director: Dr. James P. Herman, Associate Professor of Anatomy and Neurobiology 


2001 

Copyright © Deanna Lynn McCullers 2001

ACKNOWLEDGMENTS 

I would like to acknowledge my advisor and Dissertation Chair, Dr. James Herman, for his 

instrumental role in my training and in the completion of this work. For many years Dr. Herman 

has been an excellent role model as a scientist, a teacher, and a person. 

I also thank the 

members of my committee, Dr. Brian Davis, Dr. Steve Estus, Dr. Kurt Hauser, Dr. Jim Geddes, 

and Dr. Joe Springer, and the outside examiner, Dr. Thomas Foster, for their comments, insight, 

and commitment to my academic development. The former and current Directors of Graduate 

Studies, Dr. Harold Traurig and Dr. Brian MacPherson, have also been of great assistance 

throughout the dissertation process. I would further like to acknowledge Dr. Stephen Scheff for 

providing assistance and resources necessary for performing the studies on traumatic brain 

injury. 

Expert technical assistance was provided by several members of the Herman lab including: 

Garrett Bowers, Mark Dolgas, Dr. Helmer Figueiredo, Erin Murphy, Megan Paskitti, Dr. Kim 

Sipe, and Dr. Dana Ziegler. Dr. Patrick Sullivan, Katie Kraft, and other members of the Scheff 

lab provided technical assistance essential for the completion of the studies on traumatic brain 

injury, and Cynthia Long assisted with several histological procedures. I would also like to 

express my appreciation to Kelly Bolte Svec, Susan Herman, Liz Jones, Betty Newsom, Kim 

Stroth, and Kim Wilkerson for their valuable administrative assistance. Finally, I am indebted to 

my parents, Lynda J. McCullers and L. Arnold McCullers, for their continuing support. 

The contents of Chapter Two have previously been published as an article in the journal 

Neuroreport titled, “Mineralocorticoid receptors regulate bcl-2 and p53 mRNA expression in 

hippocampus”, by Deanna L. McCullers and James P. Herman, Copyright © 1998 Lippincott 

iii

Williams & Wilkins. This work is reprinted with permission of Lippincott Williams & Wilkins. 

The contents of Chapter Three have been previously published as an article in the Journal of 

Neuroscience Research titled, “Adrenocorticosteriod receptor blockade and excitotoxic 

challenge regulate adrenocorticosteroid receptor mRNA levels in hippocampus”, by Deanna L. 

McCullers and James P. Herman, Copyright © 2001 Wiley-Liss Inc. This work is reprinted with 

permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc. The contents of Chapter 

Four are currently in press to be published as an article in the journal Neuroscience titled, 

“Mifepristone protects CA1 hippocampal neurons following traumatic brain injury in rat”, by 

D.L. McCullers, P.G. Sullivan, S.W. Scheff, and J.P. Herman, Copyright © 2001 IBRO. Work 

contained in the following dissertation was supported by AG12962 (J.P.H.), AG10836 (J.P.H), 

AG00242 (D.L.M), NS39828 (S.W.S.), and KSCHIRT (S.W.S.). 

iv

TABLE OF CONTENTS 

Acknowledgments.............................................................................................................. iii 

List of Tables .................................................................................................................... vii 

List of Figures.................................................................................................................. viii 

List of Files ..........................................................................................................................x 

Chapter One: Introduction ..................................................................................................1 

Adrenocorticosteroid receptors in brain.........................................................................1 

The hypothalamic-pituitary-adrenal axis .................................................................3 

Properties of adrenocorticosteroid receptors ...........................................................5 

Central nervous system distribution of adrenocorticosteroid receptors...................6 

Regulation of adrenocorticosteroid receptor expression..........................................7 

Adrenocorticosteroid receptor activation ................................................................8 

Adrenocorticosteroid receptor regulation of gene transcription ..............................9 

Adrenocorticosteriod receptor roles in neuronal viability ...........................................12 

Excessive glucocorticoid receptor activation compromises hippocampal 

neuron viability ...................................................................................................12 

Mechanisms for glucocorticoid receptor endangerment of hippocampal 

neurons................................................................................................................14 

Neurotrophic effects of mineralocorticoid receptor activation..............................16 

Kainic acid as a model of excitotoxicity......................................................................17 

Glutamate receptors ...............................................................................................17 

Kainic acid excitotoxicity ......................................................................................18 

Glucocorticoid modulation of kainic acid excitotoxicity.......................................21 

Regulators of cell survival and death.....................................................................22 

Controlled cortical impact as a model of traumatic brain injury .................................25 

Controlled cortical impact pathology.....................................................................26 

Glucocorticoid modulation of traumatic brain injury ............................................27 

Regulators of cell survival and death.....................................................................27 

Chapter Two: Mineralocorticoid receptors regulate bcl-2 and p53 mRNA expression 

in hippocampus ...............................................................................................................29 

Introduction..................................................................................................................29 

Materials and Methods.................................................................................................30 

Results..........................................................................................................................31 

Discussion....................................................................................................................38 

Chapter Three: Adrenocorticosteriod receptor blockade and excitotoxic challenge 

regulate adrenocorticosteroid receptor mRNA levels in hippocampus ..........................41 

Introduction..................................................................................................................41 


v

Results..........................................................................................................................45 

Discussion....................................................................................................................50 

Chapter Four: Mifepristone protects CA1 hippocampal neurons following traumatic 

brain injury in rat ............................................................................................................55 

Introduction..................................................................................................................55 


Results..........................................................................................................................61 

Discussion....................................................................................................................68 

Chapter Five: Traumatic brain injury regulates adrenocorticosteroid receptor mRNA 

levels in rat hippocampus................................................................................................75 

Introduction..................................................................................................................75 


Results..........................................................................................................................79 

Discussion....................................................................................................................86 

Chapter Six: Discussion.....................................................................................................91 

Adrenocorticosteriod receptor regulation of hippocampal neuron viability................91 

Adrenocorticosteriod receptor regulation of hippocampal neuron survival ..........91 

Adrenocorticosteriod receptor regulation of viability-related gene expression.....92 

Adrenocorticosteriod receptor regulation of hippocampal neuron survival – 

potential mechanisms..........................................................................................93 

Adrenocorticosteriod receptor modulation of calcium homeostasis................93 

Adrenocorticosteriod receptor suppression of inflammation...........................94 

Adrenocorticosteriod receptor regulation of hippocampal neurotrophic 

factor expression ...........................................................................................96 

Summary..........................................................................................................98 

Injury regulation of adrenocorticosteroid receptor mRNA levels .............................100 

Injury regulation of adrenocorticosteroid receptor mRNA levels – potential 

mechanisms.......................................................................................................100 

Glutamate regulation of adrenocorticosteriod receptor mRNA levels ..........100 

Calcium regulation of adrenocorticosteriod receptor mRNA levels..............101 

Injury regulation of adrenocorticosteroid receptor mRNA levels – potential 

influence on hippocampal neuron viability.......................................................101 

Potential effects of GR downregulation on hippocampal circuitry ...............101 

Potential effects of GR downregulation on neurogenesis..............................102 

Summary....................................................................................................................104 

Appendix..........................................................................................................................105 

References........................................................................................................................106 

Vita...................................................................................................................................139 

vi

LIST OF TABLES 

Table 3.1. Effects of adrenocorticosteroid receptor blockade and kainic acid on 

plasma corticosterone levels .........................................................................45 

Table 3.2. Effects of adenocorticosteroid receptor blockade and kainic acid on 

hippocampal neuron viability .......................................................................49 

Table 4.1. Mean coefficient of error .................................................................................59 

Table 4.2. Mean coefficient of variation...........................................................................59 

Table 5.1. Effects of adrenocorticosteroid receptor blockade and controlled cortical 

impact on plasma corticosterone levels ........................................................79 

vii

LIST OF FIGURES 

Figure 1.1. The hypothalamic-pituitary-adrenal axis..........................................................4 

Figure 1.2. Functional domains of the rat glucocorticoid receptor and 

mineralocorticoid receptor ..........................................................................5 

Figure 1.3. Microdistribution of mineralocorticoid receptor and glucocorticoid 

receptor mRNAs in rat hippocampus...........................................................7 

Figure 1.4. Models for transcriptional regulation by adrenocorticosteriod receptors.......11 

Figure 1.5. Schematic diagram of the intrinsic circuitry of the hippocampal formation..19 

Figure 1.6. Photomicrograph illustrating the effects of saline versus kainic acid on 

CA1 hippocampal pyramidal neurons .......................................................20 

Figure 2.1. Photomicrographs illustrating bcl-2, bax, and p53 mRNA distribution in 

hippocampus of saline- and kainic acid-treated rats..................................32 

Figure 2.2. Effects of mineralocorticoid receptor blockade and kainic acid on bcl-2 

mRNA expression in hippocampus ...........................................................33 

Figure 2.3. Effects of mineralocorticoid receptor blockade and kainic acid on bax 


Figure 2.4. Effects of mineralocorticoid receptor blockade and kainic acid on p53 


Figure 2.5. Effects of mineralocorticoid receptor blockade and kainic acid on 

brain-derived neurotrophic factor expression in hippocampus..................36 

Figure 2.6. Effects of mineralocorticoid receptor blockade and kainic acid on 

neurotrophin-3 mRNA expression in hippocampus ..................................37 

Figure 3.1. Photomicrographs illustrating the effects of saline and kainic acid on 

mineralocorticoid receptor and glucocorticoid receptor mRNA levels 

in hippocampus of vehicle-pretreated rats .................................................46 

Figure 3.2. Effects of adrenocorticosteroid receptor blockade and kainic acid 

treatments on mineralocorticoid receptor mRNA levels in rat 

hippocampus ..............................................................................................47 

Figure 3.3. Effects of adrenocorticosteroid receptor blockade and kainic acid 

treatments on glucocorticoid receptor mRNA levels in rat 

hippocampus ..............................................................................................48 

Figure 4.1. Effects of mineralocorticoid receptor blockade, glucocorticoid receptor 

blockade, and controlled cortical impact injury on hippocampal neuron 

viability as measured by the optical fractionator method ..........................62 

Figure 4.2. Photomicrographs illustrating bcl-2, bax, and p53 mRNA levels in 

hippocampus of vehicle-pretreated, injured rats at twenty-four hours ......63 


blockade, and controlled cortical impact injury on bcl-2 mRNA levels 

in hippocampus .........................................................................................65 


blockade, and controlled cortical impact injury on bax mRNA levels 

in hippocampus ..........................................................................................66 


blockade, and controlled cortical impact injury on p53 mRNA levels 

in hippocampus ..........................................................................................67 

viii

Figure 5.1. Twenty-four hour time course analysis of plasma adrenocorticotropin 

releasing hormone levels following controlled cortical impact.................81 

Figure 5.2. Twenty-four hour time course analysis of plasma corticosterone levels 

following controlled cortical impact..........................................................81 

Figure 5.3. Photomicrographs illustrating the effects of sham operation and 

controlled cortical impact on mineralocorticoid receptor and 

glucocorticoid receptor mRNA levels in hippocampus of vehicle 

pretreated rats.............................................................................................82 


blockade, and controlled cortical impact injury on mineralocorticoid 

receptor mRNA levels in hippocampus .....................................................84 


blockade, and controlled cortical impact injury on glucocorticoid 

receptor mRNA levels in hippocampus .....................................................85 

Figure 6. Potential mechanisms for glucocorticoid modulation of hippocampal 

neuron survival after injury........................................................................99 

ix

LIST OF FILES 

mcculler.pdf ............................................................................................................ 2.49 MB 

x

Chapter One: Introduction 

Adrenocorticosteroid receptors in brain 

Glucocorticoids are steroid hormones that play an essential role in adaptation to stress. 

Named for their profound effects on glucose metabolism, glucocorticoids stimulate 

gluconeogenesis, mobilize stored energy, and inhibit subsequent energy storage during the stress 

response. In addition to regulating metabolism, glucocorticoids also increase cardiovascular 

tone, inhibit growth and reproductive capabilities, restrain immune and inflammatory responses, 

and influence learning and memory (Munck et al., 1984; Sapolsky et al., 2000). Glucocorticoids 

are essential for surviving stress, but excessive exposure can have detrimental effects such as 

hypertension, steroid diabetes, immunosuppression, inhibition of growth, and infertility (Munck 

et al., 1984; Sapolsky et al., 1986; McEwen and Stellar, 1993). Elevated levels of the steroid 

may also contribute to the development of affective disorders such as depression (De Kloet et al., 

1998) and neuropathological processes including Alzheimer’s disease (Landfield et al., 1992) 

and age-related neurodegeneration (Sapolsky et al., 1986; Landfield and Eldridge, 1991; 

McEwen, 1992). Furthermore, many studies have demonstrated that excessive exposure to 

glucocorticoids increases neuronal vulnerability to many types of challenge including ischemia, 

excitotoxicity, and oxidative stress (see Sapolsky, 1994). 

At the cellular level, glucocorticoid effects are mediated by two types of ligand-activated 

receptors, the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). These 

adrenocorticosteroid receptors (ACRs) are differentially activated in different glucocorticoid 

contexts; MR is extensively activated at basal hormone levels, while GR is activated by high 

glucocorticoid concentrations such as those experienced during stress or circadian peak (Reul 

and de Kloet, 1985; Reul et al., 1987). This arrangement suggests that the MR mediates tonic 

influences of glucocorticoids, while GR drives glucocorticoid actions aimed at restoring 

homeostasis (De Kloet and Reul, 1987). The dichotomous activation of the receptors also 

implies that MR maintains normal neuronal function while GR mediates the deleterious effects 

of excessive glucocorticoids. Studies demonstrating rescue of dentate gyrus granule neurons 

from adrenalectomy-induced death provide some support for the notion that MR has 

neurotrophic properties (Woolley et al., 1991). In addition, numerous studies have demonstrated 

1

that chronic GR activation compromises neuronal survival in hippocampus (see Sapolsky, 1994), 

a structure particularly rich in ACR expression (Reul and de Kloet, 1985) and particularly 

vulnerable to neuronal injury and degeneration (see Sapolsky, 1994). 

Improved understanding of the mechanisms underlying pathogenic effects of glucocorticoids 

is of crucial importance to facilitate treatment of neurodegenerative and psychiatric illness. The 

negative effects of chronic exposure to supraphysiological glucocorticoid levels have been well 

established in animal models (Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; Sapolsky, 1986a, 

b; Elliott et al., 1993; Stein-Behrens et al., 1994a), and in vitro studies provide convincing 

evidence that deleterious glucocorticoid effects are mediated by GR (Packan and Sapolsky, 

1990; Behl et al., 1997b). Some studies demonstrate that GR blockade with antagonist drug 

mifepristone (RU486) can protect cultured cells against glucocorticoid toxicity (Packan and 

Sapolsky, 1990; Behl et al., 1997b; Behl et al., 1997a). However, the potential for attenuating 

physiological glucocorticoid neurotoxicity with GR blockade has not been thoroughly examined 

in vivo. One previous study has demonstrated transient RU486 protection against ischemiainduced 

hippocampal cell loss (Antonawich et al., 1999), but RU486 protection against other 

forms of neuronal injury has not been studied in animal models. Furthermore, the possible 

neuroprotective role of normal MR activation has not been previously addressed. 

The current thesis aims to evaluate the respective roles of MR and GR on hippocampal 

neuron survival in vivo under conditions of neuronal challenge. Adrenocorticosteroid receptor 

regulation of neuronal viability was assessed after administration of ACR antagonist drugs and 

injury with kainic acid, an excitotoxic glutamate analog, or controlled cortical impact, a model of 

experimental traumatic brain injury. Following antagonist pretreatments and injury, survival of 

challenged neurons, neuronal expression of viability-related genes, and neuronal expression of 

ACR mRNAs were measured to test the hypotheses that (1) MR blockade would decrease 

hippocampal neuron survival of injury and expression of genes associated with neuronal 

viability, (2) GR blockade would increase hippocampal neuron survival of injury and expression 

of genes associated with neuronal viability, and (3) glucocorticoid signaling is altered through 

regulation of ACR expression following injury. 

2

The hypothalamic-pituitary-adrenal axis 

Glucocorticoid secretion is regulated by the hypothalamic-pituitary-adrenal (HPA) axis 

(Figure 1.1). Perception of stress or circadian drive stimulates the paraventricular nucleus of the 

hypothalamus to release corticotropin-releasing hormone (CRH) and other adrenocorticotropinreleasing 

hormone (ACTH) secretogogues into the hypophysial portal system in the median 

eminence. The neuropeptides stimulate anterior pituitary release of ACTH, which travels though 

systemic circulation and promotes synthesis and release of glucocorticoids from the adrenal 

cortex [see (Antoni, 1986; Jones and Gillham, 1988)]. Circulating glucocorticoids subsequently 

inhibit their own release by acting at multiple levels of the HPA system including brain and 

pituitary (Keller-Wood and Dallman, 1984; Jones and Gillham, 1988). This negative feedback 

system participates in termination of the stress response and prevents overexposure to catabolic 

glucocorticoid effects that might threaten homeostasis (Munck et al., 1984). Hypothalamicpituitary-adrenal 

dysregulation and elevated basal cortisol levels have been associated with 

affective disease and cognitive decline in humans (Sapolsky et al., 1986; Martignoni et al., 1992; 

Axelson et al., 1993). 

3

Figure 1.1. The hypothalamic-pituitary-adrenal axis. Stress or circadian drive stimulate the 

paraventricular nucleus of the hypothalamus (PVN) to release corticotropin releasing hormone 

(CRH), which stimulates release of adrenocorticotropin-releasing hormone (ACTH) from the 

anterior pituitary. ACTH promotes glucocorticoid release from the adrenal cortex. 

Glucocorticoids act at multiple levels to inhibit further HPA activation. 

4

Properties of adrenocorticosteroid receptors 

As members of the nuclear hormone receptor superfamily, MR and GR share a characteristic 

three-domain structure consisting of an N-terminal transactivation domain, a central DNAbinding 

domain, and a carboxy-terminal ligand-binding domain (Evans, 1988) (Figure 1.2). The 

non-conserved N-terminal domain provides unique transactivation properties to each receptor 

(Evans, 1988). The highly conserved DNA-binding domain suggests that both receptor types 

bind to identical response elements on target genes (Arriza et al., 1987). In addition to 

interaction with hormone ligand, the carboxy-terminal domain also mediates binding to 

chaperone proteins, nuclear translocation, dimerization, and transactivation (Bertorelli et al., 

1998). Rat and human ACR sequences are highly homologous; comparisons indicate complete 

identity in MR and GR DNA-binding domains and high coincidence in steroid-binding domains 

(Hollenberg et al., 1985; Miesfeld et al., 1986; Patel et al., 1989). 

Figure 1.2. Functional domains of the rat glucocorticoid receptor (GR) and mineralocorticoid 

receptor (MR). Each protein consists of three main functional domains: an N-terminal 

transactivation domain, a central DNA binding domain, and a carboxy-terminal hormone-binding 

domain (Miesfeld et al., 1986; Picard and Yamamoto, 1987; Evans, 1988; Patel et al., 1989). 

Approximate percentages of amino acid identity within each domain are based on human MR 

and GR homology (Evans, 1988). 

5

Differences in ligand binding affinity underlie the differential activation of MR and GR with 

increasing plasma glucocorticoid levels. The MR, also termed the “type I” receptor, binds 

corticosterone with high affinity (Kd ∼ 0.5-1 nM), resulting in extensive occupation even at low 

circulating glucocorticoid levels (Reul and de Kloet, 1985). The GR, or “type II” receptor, has a 

lower affinity for corticosterone (Kd ∼ 2.5-5 nM) and is activated (in addition to MR) only at 

high CORT levels as seen at the circadian peak or during stress (Reul and de Kloet, 1985). In 

contrast to a simple dose-response relationship, this two-receptor system is capable of generating 

unique cellular responses based on the strength of glucocorticoid signal. 

Central nervous system distribution of adrenocorticosteroid receptors 

The GR is widely expressed in the brain, in both neurons and glial cells (Fuxe et al., 1985; 

van Eekelen et al., 1987; Ahima and Harlan, 1990). Particularly high levels of GR expression 

are found in the hippocampal formation; other regions expressing moderate to high levels of GR 

include the paraventricular nucleus of the hypothalamus, the lateral septum, brainstem 

monoaminergic neurons, thalamic nuclei, the central amygdala, striatum, and layers of cortex 

(Aronsson et al., 1988; Van Eekelen et al., 1988; Chao et al., 1989). Expression of MR is limited 

mainly to limbic areas, with highest expression in the hippocampal formation (Reul and de 

Kloet, 1985; Ahima and Harlan, 1990). Colocalization of both MR and GR has been reported in 

dorsal subiculum, septum, amygdala, and particularly in hippocampus (Reul and de Kloet, 1985; 

Van Eekelen et al., 1988). The distribution of ACRs varies by subregion in hippocampus 

(Herman et al., 1989a; Herman, 1993) (Figure 1.3). The rich expression of ACRs in 

hippocampal neurons may contribute to their unusual vulnerability to challenge and their 

extreme sensitivity to the neurotoxic effects of glucocorticoids. 

6

Figure 1.3. Microdistribution of mineralocorticoid receptor (MR) (panel A) and glucocorticoid 

receptor (GR) (panel B) mRNAs in rat hippocampus. MR and GR mRNAs are heterogeneously 

distributed within the hippocampal formation (Herman et al., 1989a; Herman, 1993). MR 

mRNA levels are highest in CA2 and comparable in CA1, CA3, and dentate gyrus (DG) (panel 

A). GR signal is highly expressed in CA1 and DG and low in CA3 (panel B). 

Regulation of adrenocorticosteroid receptor expression 

Adrenocorticosteroid receptor expression is subject to short-term and long-term regulation by 

many factors, including glucocorticoids. Autoregulation of hippocampal ACR expression has 

been demonstrated in adrenalectomized animals and in subjects treated with ACR antagonists 

(i.e. receptor expression is increased in the absence of receptor activation). Results from several 

studies are consistent with regulation of MR expression by MR (Chao et al., 1998a; McCullers 

and Herman, 2001) and with regulation of GR expression by both MR (O'Donnell and Meaney, 

1994; Chao et al., 1998a; McCullers and Herman, 2001) and GR (Herman et al., 1989a; Reul et 

al., 1989; Chao et al., 1998a; Herman and Spencer, 1998; McCullers and Herman, 2001) 

(Chapter Three). Furthermore, ACR expression is subject to regulation by glucocorticoid 

secretion during acute stress (Herman and Watson, 1995; Paskitti et al., 2000), chronic stress 

(Sapolsky et al., 1984; Gomez et al., 1996; Paskitti et al., 2000), social conflict (Johren et al., 

1994), and the circadian rising phase (Herman et al., 1993). It is important to note that ACR 

expression is differentially regulated by brain region, particularly across subfields of the 

hippocampus (Herman, 1993). 

Adrenocorticosteroid receptor expression is also regulated by numerous factors other than 

glucocorticoids, emphasizing the importance of ACR regulation as a means for modulating 

glucocorticoid signaling. Neurotransmitters appear to play a prominent role in regulating ACR 

7

levels; there is evidence for serotonergic (Seckl et al., 1990; Mitchell et al., 1992), noradrenergic 

(Yau and Seckl, 1992), and dopaminergic (Antakly et al., 1987) regulation of neuronal ACR 

mRNAs and/or receptor binding. Antidepressant drugs also affect ACR density, but whether 

antidepressants act directly to alter ACR expression or indirectly by increasing monoamine 

levels is unclear (Seckl and Fink, 1992). Disruption of hippocampal circuitry also influences 

ACR mRNA expression; lesions of the medial septum (Yau and Seckl, 1992), dentate gyrus 

(Brady et al., 1992), entorhinal cortex (O'Donnell et al., 1993), and central 5-HT lesions (Yau et 

al., 1994) can regulate hippocampal ACR mRNA levels. Excitatory amino acids (Lowy, 1992) 

and growth factors (Sarrieau et al., 1996) are among numerous other factors shown to regulate 

ACR expression. 

Expression of ACR mRNAs is regulated at multiple levels including transcription, 

polyadenylation, and/or alternative splicing (see De Kloet et al., 1998). Splicing at the 3’-end of 

human GR heteronuclear RNA results in at least two GR mRNA variants, GRα and GRβ 

(Hollenberg et al., 1985). Notably, GRβ is not expressed in rat (Otto et al., 1997). Rat GR and 

MR mRNAs exhibit heterogeneity in 5’-untranslated exon 1 (McCormick et al., 2000) (Kwak et 

al., 1993; Zennaro et al., 1995). Interestingly, expression of rat GR mRNA variants can be 

regulated by environmental manipulations (i.e. postnatal handling) (McCormick et al., 2000), 

and select MR transcripts are regulated by glucocorticoids (Kwak et al., 1993). Splice variants 

may exhibit different stabilities and/or translation efficiencies, influencing overall levels of ACR 

expression based on the differential expression of transcripts (Kwak et al., 1993; Zennaro et al., 

1995). 

Adrenocorticosteroid receptor activation 

Adrenocorticosteroid receptors are activated upon the binding of glucocorticoid ligand, 

specifically cortisol in human or corticosterone (CORT) in the rat. Unliganded ACRs reside in 

the cytoplasm as part of a multi-protein complex containing two molecules of heat shock protein 

(HSP) 90, a molecule each of hsp70, hsp56, p23, and an immunophilin (Pratt, 1993; Bruner et 

al., 1997; Bertorelli et al., 1998). The chaperone complex facilitates hormone binding to the 

receptor and participates in receptor trafficking (Pratt, 1993). Glucocorticoid binding induces a 

conformational change in the ACR molecule that favors dissociation from the chaperone 

complex (Pratt, 1993). Following dissociation, the liganded receptors become 

8

hyperphosphorylated (Orti et al., 1992) and translocate into the nucleus (Picard and Yamamoto, 

1987) where they act as transcription factors (Yamamoto, 1985). 

Adrenocorticosteroid receptor regulation of gene transcription 

Models proposed to explain ACR regulation of gene transcription identify chromatin 

remodeling and stabilization of the transcriptional preinitiation complex as essential mechanisms 

(Jenster et al., 1997). Adrenocorticosteroid receptor regulation of these processes is 

accomplished via binding to DNA response elements or through interactions with other 

transcription factors (Figure 1.4). In the case of the former, the activated receptors can bind as 

homo- or heterodimers (Luisi et al., 1991; Trapp et al., 1994) to specific DNA sequences called 

glucocorticoid response elements (GREs). These palindromic sequences are located in or near 

promoter regions of glucocorticoid-responsive genes and serve as inducible enhancer elements 

(see Yamamoto, 1985). Response element-bound ACRs recruit coactivator proteins capable of 

remodeling chromatin structure via histone acetyltransferase activity. By acetylating histone N- 

termini, coactivators loosen nucleosomal structure and increase accessibility for other 

transcription factors. The steroid receptors and coactivators may also stabilize assembly of the 

preinitiation complex, thereby increasing the rate of transcription initiation (Jenster et al., 1997). 

The MR and GR can repress gene transcription by recruiting corepressor proteins or by acting at 

negative GREs, which differ in sequence from positive GREs (Drouin et al., 1993). It is 

important to note that interactions with coactivator proteins will vary depending on dimer 

configuration, with distinct transactivation properties conferred by each type of homo- or 

heterodimer (Trapp et al., 1994). 

Ligand-activated ACRs may also influence transcription of genes via interactions with other 

transcription factors (Karin, 1998) (Figure 1.4). For instance, GR modulates activity of 

transcription factors including activator protein-1 (AP-1), cyclic AMP response element binding 

protein (CREB), and nuclear factor-κB (NF-κB), in the absence of DNA binding (Herrlich, 

2001). Steroid receptor cross-talk with transcription factors most frequently results in negative 

interference, but synergism is also possible under specific conditions (i.e. when AP-1 is a 

homodimer of c-Jun) (Diamond et al., 1990). The exact molecular mechanism of steroid 

receptor cross-talk has yet to be elucidated. Models for direct protein-protein interactions 

between GR and other transcription factors have been proposed (McEwan et al., 1997). 

9

Adrenocorticosteroid receptors may also repress gene transcription by inducing specific 

inhibitors of other transcription factors. For example, GR may repress NF-κB activity by 

inducing IκB, an inhibitor of NF-κB (Auphan et al., 1995; Scheinman et al., 1995). The 

evidence indicates that multiple mechanisms are likely to account for glucocorticoid-mediated 

gene repression (see De Bosscher et al., 2000). 

10

Figure 1.4. Models for transcriptional regulation by adrenocorticosteriod receptors. Activated 

mineralocorticoid receptors (MRs) or glucocorticoid receptors (GRs) bind DNA hormone 

response elements as dimers or interact with other transcription factors to influence transcription 

(McEwan et al., 1997; Karin, 1998; De Bosscher et al., 2000). Possible mechanisms for 

modulating transcription include: recruitment of transcription coactivators or repressors (A, B); 

binding to negative hormone response elements (nGREs)(C); interference with activity of other 

transcription factors (D); competive DNA binding (E, F); and induction of inhibitors of other 

transcription factors (G). 

11

Adrenocorticosteriod receptor roles in neuronal viability 

Differential ACR activation at varying glucocorticoid levels can elicit unique cellular 

responses due to the distinct regulatory effects of MR and GR. Collaborative binding of MR/GR 

heterodimers to GREs demonstrates the potential for ACR cooperation in regulating gene 

expression (Trapp et al., 1994). In some cases, however, MR and GR effects appear to be quite 

opposite. For instance, predominant MR activation at low glucocorticoid concentrations results 

in very small depolarization-induced calcium (Ca 2+ ) currents in CA1 pyramidal neurons (Karst et 

al., 1994), while the additional occupation of GR at high glucocorticoid levels results in 

relatively large Ca 2+ currents (Kerr et al., 1992). Further, MR activation by low glucocorticoid 

levels or aldosterone decreases accommodation and afterhyperpolarization amplitude in CA1, 

enhancing excitatory transmission (Joels and de Kloet, 1990); in contrast, high glucocorticoid 

levels and GR agonist RU28362 increase accommodation and the duration of 

afterhyperpolarization, suppressing CA1 excitability (Kerr et al., 1989). Differential activation 

of GR can also elicit structural effects on hippocampal neurons that do not occur at lower, MRactivating 

levels. For instance, chronic exposure to GR-saturating glucocorticoid levels achieved 

by 21-day corticosterone (CORT) administration (Woolley et al., 1990; Magarinos and McEwen, 

1995) or repeated stress (Magarinos and McEwen, 1995) reversibly decreases both numbers and 

length of apical dendritic branches in CA3 pyramidal neurons. 

The preferential activation of MR or GR can also influence the survival of hippocampal 

neurons. The vast majority of literature to date examines negative GR effects on hippocampal 

neuron viability, as GR activation is associated with the exacerbation of several forms of 

neuronal insult (Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; Sapolsky, 1986a, b; Sapolsky et 

al., 1988; Packan and Sapolsky, 1990; Elliott et al., 1993; Stein-Behrens et al., 1994a; Goodman 

et al., 1996; McIntosh and Sapolsky, 1996a; Behl et al., 1997b). In contrast, MR activation is not 

associated with reduced neuronal survival (Packan and Sapolsky, 1990), and there is some 

evidence to indicate that MR may have trophic or neuroprotective effects (Woolley et al., 1991). 

Excessive glucocorticoid receptor activation compromises hippocampal neuron viability 

Chronic exposure to high concentrations of glucocorticoids can be toxic to neurons, with 

preferential damage to the hippocampus (see Sapolsky, 1994). Studies describing hippocampal 

12

degeneration in aging male rats with elevated basal glucocorticoid levels provided the first 

evidence of a physiological role for glucocorticoid neurotoxicity. Landfield and colleagues 

demonstrated a correlation between the degree of glucocorticoid hypersecretion and the amount 

of hippocampal degeneration in mid-aged rats (Landfield et al., 1978). Removal of 

glucocorticoids via adrenalectomy was subsequently shown to prevent the hippocampal cell loss 

and glial reactivity (Landfield et al., 1981). These studies led to the formation of the 

“glucocorticoid hypothesis of brain aging and neurodegeneration”, which proposes that 

glucocorticoids contribute to age-related degeneration of the hippocampus (Landfield and 

Eldridge, 1991). Sapolsky and colleagues later demonstrated that chronic exposure to high 

levels of glucocorticoids, achieved by repeated injection of corticosterone, resulted in the death 

of hippocampal pyramidal neurons in rat (Sapolsky et al., 1985). It remains unclear whether 

glucocorticoids alone are sufficient to induce neuronal death in hippocampus. 

While glucocorticoids may not kill neurons directly, there is ample support for the notion that 

chronic exposure to high levels of glucocorticoids increases the vulnerability of hippocampal 

neurons to subsequent challenge (Sapolsky, 1985). In rat, administration of high physiologic 

concentrations of CORT potentiates ischemic injury to CA3 neurons produced by the four-vessel 

occlusion method of forebrain ischemia (Sapolsky and Pulsinelli, 1985). High CORT levels also 

enhance damage induced in CA3 by the excitotoxin kainic acid (Sapolsky, 1985, 1986a, b; 

Elliott et al., 1993; Stein-Behrens et al., 1994a) and in DG by the antimetabolite toxin 3- 

acetylpyridine (Sapolsky, 1985, 1986b). Glucocorticoid exacerbation of excitotoxic (Sapolsky et 

al., 1988; Packan and Sapolsky, 1990; Goodman et al., 1996) and metabolic (Sapolsky et al., 

1988) challenge is also seen in primary hippocampal cultures. Furthermore, in vitro experiments 

have shown that glucocorticoids increase the amount of hippocampal cell death induced by 

oxidative stress (Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a; 

Behl et al., 1997b) and amyloid β-peptide toxicity (Goodman et al., 1996). 

Despite the graded activation of GR with increasing CORT levels, it remains difficult to 

differentiate between specific MR and GR effects at high CORT concentrations, as both receptor 

types are activated. Likewise, both MR and GR are inactivated in adrenalectomized animals due 

to the removal of endogenous CORT. In order to dissect the individual effects of each receptor 

type, selective MR/GR agonists may be administered in adrenalectomized animals or in culture 

to study the selective activation of a receptor type. Conversely, MR/GR antagonists may be 

13

administered to intact animals or in culture to observe the effects of loss of function of a specific 

ACR type. A number of studies using ACR agonists and antagonists in vitro have provided 

convincing evidence for the hypothesis that the endangering effects of excessive glucocorticoid 

exposure are mediated by the GR. In primary hippocampal cultures, a specific GR agonist, 

dexamethasone, exacerbates kainic acid damage, where the MR agonist aldosterone has no effect 

(Packan and Sapolsky, 1990). In the same culture system, GR antagonist RU486 protects against 

glucocorticoid/kainic acid synergy, but MR antagonist RU28318 does not prevent the damaging 

glucocorticoid effects (Packan and Sapolsky, 1990). Other in vitro studies provide strong 

support for GR enhancement of amyloid β protein, hydrogen peroxide, and glutamate toxicity, as 

dexamethasone exacerbates (Behl et al., 1997b) and RU486 prevents (Behl et al., 1997b; Behl et 

al., 1997a) negative glucocorticoid effects with these types of insult. In vivo, RU486 transiently 

protects gerbil CA1 neurons from ischemia-induced cell loss, indicating that blockade of GR can 

prevent the detrimental effects of post-ischemia CORT elevations (Antonawich et al., 1999). 

Mechanisms for glucocorticoid receptor endangerment of hippocampal neurons 

Glucocorticoids can increase neuronal susceptibility to injury by inducing a state of 

metabolic vulnerability (Sapolsky, 1985). In the absence of additional challenge, temporary 

energy crisis induced by glucocorticoids is not likely to threaten neuronal survival. However, by 

compromising cellular energy supplies, glucocorticoids can greatly intensify the toxicity of a 

wide variety of insults that impair generation of energy (i.e. ischemia) or place pathogenic 

demands on the neuron for energy (i.e. excitotoxicity) (Sapolsky, 1990). Glucocorticoids impair 

glucose metabolism in regions including hippocampus, locus coeruleus, hypothalamic 

paraventricular nucleus, median eminence, and pituitary anterior lobe (Kadekaro et al., 1988). 

Activation of GR may compromise neuronal energy stores by impairing glucose transport, as 

demonstrated in cultured hippocampal neurons and glia in vitro (Horner et al., 1990). Chronic 

glucocorticoid exposure also markedly induces expression of ATP-requiring enzymes such as 

glutamine synthetase and glycerol-phosphate dehydrogenase, which may result in the further 

depletion of cellular ATP stores (Nichols and Finch, 1994). Energy supplementation with 

glucose or mannose has been shown to attenuate glucocorticoid neurotoxicity in vivo (Sapolsky, 

1986b) and in vitro (Sapolsky et al., 1988). 

14

Neuronal energy depletion following excessive glucocorticoid exposure has been proposed to 

exacerbate hippocampal insult by contributing to excitotoxic cascades (Sapolsky, 1990). Under 

normal conditions, glutamate interaction with postsynaptic receptors mobilizes free cytosolic 

Ca 2+ in the postsynaptic neuron in a highly regulated fashion. Glutamate is removed from the 

synapse by energy-dependent glutamate transporters (Kanai et al., 1993). Calcium in the 

postsynaptic neuron is effectively buffered by Ca 2+ binding proteins including calbindin and 

parvalbumin, sequestered by cellular organelles such as smooth endoplasmic reticulum and 

mitochondria, or extruded via ATP-dependent transporters including a Na + /Ca 2+ exchanger and 

the plasma membrane calcium pump (PMCA) (Blaustein, 1988). However, energy depletion and 

necrotic insult can lead to the pathologic accumulation of synaptic excitatory amino acids, 

leading to excessive influx of Ca 2+ into the postsynaptic neuron. Energy crisis compromises 

glutamate removal from the synapse and may even result in reversal of glutamate transporters 

(Takahashi et al., 1997), resulting in the further depolarization-induced opening of voltage-gated 

Ca 2+ channels and NMDA-gated Ca 2+ channels on the postsynaptic neuron. Energy deficits can 

also lead to the failure of ATP-driven Ca+ sequestration and extrusion systems, exacerbating 

increases in intracellular Ca 2+ levels (see Tymianski and Tator, 1996). Excessive Ca 2+ influx can 

set in motion many destructive processes including mitochondrial failure, the degradation of 

structural proteins by Ca 2+ -activated proteases (i.e. calpain)(Siman et al., 1989), and the 

generation of free radicals [see (Choi, 1992; Tymianski and Tator, 1996)]. 

Many experimental studies support the theory that glucocorticoids contribute to excitotoxic 

glutamate/Ca 2+ cascades. Chronic exposure to high levels of glucocorticoids has been shown to 

increase extracellular levels of excitory amino acids following kainic acid administration (Stein- 

Behrens et al., 1992; Stein-Behrens et al., 1994b). Glucocorticoids also prolong intracellular 

Ca 2+ 

elevation after kainic acid treatment (Elliott and Sapolsky, 1992) and increase basal 

intracellular Ca 2+ levels (Elliott and Sapolsky, 1993) in culture. Furthermore, glucocorticoids 

exacerbate Ca 2+ -mediated degenerative events in the hippocampus following intrahippocampal 

kainic acid infusion, as measured by spectrin proteolysis and altered tau immunoreactivity 

(Elliott et al., 1993; Stein-Behrens et al., 1994a). Glucocorticoid exacerbation of 3- 

acetylpyridine toxicity is eliminated with administration of an NMDA receptor antagonist, 

aminophosphonovaleric acid, providing evidence that negative glucocorticoid effects are 

dependent on activation of the NMDA receptor (Armanini et al., 1990). 

15

Glucocorticoids can also influence neuronal survival via genomic mechanisms. High 

glucocorticoid concentrations may modulate Ca 2+ homeostasis by increasing P/Q- and L- type 

Ca 2+ channel subunit mRNA levels, as demonstrated in isolated CA1 neurons (Nair et al., 1998). 

Glucocorticoids also decrease hippocampal mRNA levels of plasma membrane calcium pump 

isoform 1 (PMCA1) (Bhargava et al., 2000), which regulates intracellular Ca 2+ levels by 

coupling ATP hydrolysis with Ca 2+ extrusion. These changes in gene expression, if indicative of 

changes in protein expression, are consistent with increased intracellular Ca 2+ accumulation with 

GR activation. Furthermore, glucocorticoid regulation of neurotrophin expression may lead to 

decreased neurotrophic support and the exacerbation of neuronal insults. Immobilization stress 

(Smith et al., 1995a) and CORT administration (Chao and McEwen, 1994; Smith et al., 1995a) 

decrease hippocampal brain-derived neurotrophic factor (BDNF) mRNA levels and increase 

hippocampal neurotrophin-3 (NT-3) levels in hippocampus. 

Neurotrophic effects of mineralocorticoid receptor activation 

The potentially damaging effects of GR activation at high glucocorticoid concentrations are 

well documented. In contrast, the MR, which is persistently activated even at basal 

glucocorticoid levels (Reul and de Kloet, 1985), is not associated with neuronal energy depletion 

or exacerbation of neuronal insult (Packan and Sapolsky, 1990). On the contrary, MR may 

function in a neurotrophic manner, as activation of MR is required for the survival of dentate 

gyrus granule neurons. Removal of endogenous glucocorticoids by adrenalectomy causes 

granule cell loss within as little as three days (Gould et al., 1990), and nearly all granule cells are 

lost within three to four months after surgery (Sloviter et al., 1989). Administration of the 

selective MR agonist aldosterone prevents adrenalectomy-induced granule neuron loss, but 

treatment with GR agonist RU28362 provides only partial protection for DG granule neurons 

(Woolley et al., 1991). Thus, MR occupation is necessary for maintenance of DG neuron 

survival, but the role for GR is less clear (Woolley et al., 1991). MR activation also regulates 

granule cell neurogenesis in DG, maintaining a balance between granule cell birth and cell death 

(Gould et al., 1991). MR may regulate neurogenesis by enhancing excitatory input to the region 

(Gould, 1994) or indirectly through regulation of neurotrophin expression (Lindholm et al., 

1994; Qiao et al., 1996). 

16

Kainic acid as a model of excitotoxicity 

Kainic acid (KA) is a glutamate analog notable for its excitotoxic and epileptogenic effects. 

Systemic or intracerebral injection of KA induces epileptiform seizures and cell loss in 

hippocampus in a pattern similar to human temporal lobe epilepsy (Nadler et al., 1978; Ben-Ari 

and Cossart, 2000). The extensive literature demonstrating glucocorticoid modulation of KA 

excitotoxicity, the selective toxicity of KA in hippocampus, and the substantial but incomplete 

neuronal loss following seizure indicate KA as an ideal model for use in studying ACR effects 

on hippocampal neuron viability. 

Glutamate receptors 

Glutamate receptors are divided into two major categories - metabotropic and ionotropic 

receptors. Metabotropic glutamate receptors are coupled to G-protein-mediated second 

messenger pathways; ionotropic glutamate receptors allow the direct influx of cations upon 

activation. Ionotropic glutamate receptors are responsible for the majority of fast excitatory 

synaptic transmission in the central nervous system. Three types of ionotropic glutamate 

receptors have been identified based on their preferential activation by each compound – the 

AMPA, kainate, and NMDA receptors (Dingledine and McBain, 1999). 

Non-NMDA glutamate receptor subunits are approximately 900 amino acid residues in 

length, have a calculated molecular weight of approximately 100 kDa, and are predicted to have 

four transmembrane domains (Keinanen et al., 1990). Non-NMDA receptors are formed by 

homomeric or heteromeric associations of AMPA (GluR-1/-2/-3/-4) or kainate (GluR-5/-6/-7 and 

KA-1/-2) receptor subunits. Subunit composition may confer distinct functional and 

pharmacological properties (Hollmann et al., 1991; Bettler and Mulle, 1995). Kainate receptors 

exhibit significant structural homology to AMPA receptors and can be expressed in the same 

cells as AMPA receptors, yet KA receptor subunits and AMPA receptor subunits do not seem to 

coassemble (Frerking and Nicoll, 2000). Kainate receptors generate excitatory postsynaptic 

potentials (EPSPs) in response to glutamate release (Frerking and Nicoll, 2000), and kainate 

responses undergo rapid and total desensitization from which recovery is slow (Lerma et al., 

1997). As compared to AMPA receptor-mediated EPSPs, kainate-mediated EPSPs have smaller 

17

peak amplitude and slower decay kinetics. A controversial presynaptic role for kainate receptors 

has also been proposed (see Malva et al., 1998). 

NMDA receptors are believed to play a key role in glutamate neurotoxicity due to their high 

Ca 2+ permeability as compared to non-NMDA ionotropic glutamate receptors (Rothman, 1987). 

NMDA receptors are unique in that they require bound ligand and the voltage-dependent 

removal of a Mg2+-dependent block to initiate activation, and the presence of glycine is required 

for full activation (Malva et al., 1998). NMDA receptors are insensitive to AMPA and kainate 

(Hollmann and Heinemann, 1994) but may be activated by kainate-induced seizures or 

excitotoxic cascades (Ben-Ari and Gho, 1988). 

Kainic acid excitotoxicity 

The distribution of KA binding sites roughly corresponds to regional susceptibility to KA 

excitotoxicity. Regions displaying KA binding and sensitivity to KA toxicity include the 

hippocampus, amygdala, entorhinal cortex, piriform cortex and insular cortex (Kohler and 

Schwarcz, 1983). High-affinity kainate receptors are expressed at very high density in rat brain 

in the mossy fiber synaptic region, or stratum lucidum of CA3 (Foster et al., 1981; Represa et al., 

1987). The epileptogenic effects of KA are attributed to excitation of CA3 neurons by postsynaptic 

kainate receptors at mossy fiber synapses (Ben-Ari and Gho, 1988). It is important to 

note that degeneration of CA3 neurons is probably due to the sustained release of glutamate 

subsequent to recurrent seizures, not to the activation of KA receptors per se (Coyle et al., 1981; 

Ben-Ari, 1985; Ben-Ari and Cossart, 2000). Seizures generated in CA3 propagate to CA1 via a 

network of recurrent collateral glutamatergic axons that interconnect pyramidal neurons. 

Secondary activation of CA1, the major source of hippocampal output, leads to the spread of 

seizures to other limbic structures including entorhinal cortex (Figure 1.5) (Ben-Ari and Cossart, 

2000). 

18

Figure 1.5. Schematic diagram of the intrinsic circuitry of the hippocampal formation (adapted 

from Amaral and Witter, 1989). The hippocampal formation is composed of four basic regions: 

the entorhinal cortex (EC), the dentate gyrus (DG), the hippocampus proper (which may be 

divided into subfields CA1, CA2, and CA3), and the subicular complex (S). The major 

connections linking hippocampal subfields are glutamatergic and largely unidirectional. Input 

from EC reaches DG via the perforant path. Granule cells of the DG project to the CA3 field via 

mossy fibers. CA3 pyramidal neurons give rise to Schaffer collaterals, which terminate on CA1 

pyramidal neurons. CA1 pyramidal neurons project to S, which projects to regions including 

EC. 

The behavioral effects of systemic KA administration have been described in detail (Sperk et 

al., 1983). Approximately five minutes after injection, most animals assume a catatonic posture 

with staring behavior. Within approximately one hour, subjects develop generalized clonic-tonic 

convulsions accompanied by frequent rearing, falling over to one side, “wet dog shakes”, and 

hemorrhagic foam from the mouth. Convulsions often subside two hours after injection and are 

replaced by a phase of motor hyperactivity. In more severely affected animals, the convulsive 

phase may last up to 4-5 hours after KA injection. After twenty-four hours the animals appear 

physically exhausted. Rare convulsions may still occur spontaneously or in response to noise or 

handling (Sperk et al., 1983). 

Kainic acid lesion preferentially destroys hippocampal pyramidal cells, particularly those of 

region CA3 (Nadler et al., 1978). Neurons of CA1 are vulnerable to a lesser degree, and DG 

19

granule cells are relatively insensitive to KA excitotoxicity (Nadler et al., 1978; Kohler and 

Schwarcz, 1983). As early as one to three hours after exposure, damaged neurons appear 

shrunken, with darkly stained cytoplasm and pyknotic nuclei (Sperk et al., 1983) (Figure 1.6). 

Some neurons appear swollen, containing small vacuoles in mitochondria and in cytoplasm due 

to swelling of endoplasmic reticulum and Golgi apparatus (Sperk et al., 1983). Cell death 

induced by KA administration has been classified as necrotic (Sperk et al., 1983; Fujikawa et al., 

2000) yet exhibits some characteristics consistent with apoptosis such as TUNEL positivity and 

DNA laddering (Fujikawa et al., 2000). Damaged neurons appear to be partially removed by 

three days after injection but may be seen for at least two weeks (Nadler et al., 1978). Neuronal 

degeneration is accompanied by edema, perivenous hemorrhage, gliosis, and vacuolization of the 

neuropil (Nadler et al., 1978; Sperk et al., 1983). The entorhinal cortex, piriform cortex, frontal 

cortex, and medial thalamic nuclei are also subject to degrees of KA-induced degeneration 

(Nadler et al., 1978; Kohler and Schwarcz, 1983; Sperk et al., 1983). 

Figure 1.6. Photomicrograph illustrating the effects of (A) saline versus (B) kainic acid on CA1 

hippocampal pyramidal neurons (40X magnification). Twenty-four hours following systemic 

administration, saline-treated neurons are healthy and densely packed with rounded borders and 

prominent nucleoli. Neurons damaged by kainic acid appear pyknotic and shriveled with darkstaining 

somata and shrunken processes. 

20

Kainate-induced death of hippocampal neurons is likely due to the sustained release of 

glutamate during recurrent seizures (Coyle et al., 1981; Ben-Ari, 1985; Ben-Ari and Cossart, 

2000) and subsequent increases in intracellular Ca 2+ levels (Hori et al., 1985; Sun et al., 1992). 

Excessive accumulation of glutamate in the synapse will activate post-synaptic glutamate 

receptors, resulting in Ca 2+ influx. Calcium accumulation is mediated predominantly by the 

NMDA receptor, but AMPA or kainate receptors may also contribute to rises in intracellular 

Ca 2+ (Choi, 1992). The pathologic energy demands of repeated seizures lead to impairment of 

Ca 2+ sequestration and extrusion systems, contributing to Ca 2+ elevations (see Tymianski and 

Tator, 1996). Calcium homeostasis may be further disrupted by the release of Ca 2+ from internal 

stores, activation of certain enzymes (such as calpains and phospholipases), and changes in 

immediate early gene expression. Glutamate efflux from injured neurons may further exacerbate 

excitotoxic cascades (Choi, 1992). 

Glucocorticoid modulation of kainic acid excitotoxicity 

Glucocorticoids exacerbate KA toxicity (Sapolsky, 1985, 1986a, b; Sapolsky et al., 1988; 

Packan and Sapolsky, 1990; Stein-Behrens et al., 1994a), making KA administration an excellent 

model for studying ACR effects on neuronal viability. Glucocorticoids intensify KA-induced 

accumulation of extracellular glutamate (Stein-Behrens et al., 1992; Stein-Behrens et al., 1994b) 

and KA-induced calcium elevations (Elliott and Sapolsky, 1992, 1993). Chronic exposure to 

glucocorticoids also impairs antioxidant defenses in response to KA (McIntosh et al., 1998a) and 

increases the electophysiological sensitivity to KA in hippocampal neurons (Talmi et al., 1995). 

In addition, glucocorticoids influence immediate early gene responses to KA (Unlap and Jope, 

1995a) and exacerbate KA-induced spectrin proteolysis (Elliott et al., 1993; Lowy et al., 1994). 

Glucocorticoids may also influence neuronal viability to KA via genomic mechanisms. 

Kainate administration results in the altered expression of a multitude of genes, including: cell 

survival/death genes (bcl-2, bax, p53, KROX-20, COX-2, cyclin D1, and GADD45) (Sakhi et 

al., 1994; Gillardon et al., 1995; Vreugdenhil et al., 1996; Zhu et al., 1997; Hashimoto et al., 

1998; Timsit et al., 1999), transcription factors (AP-1 and NFκB) (Kaminska et al., 1994; 

Pennypacker and Hong, 1995; Won et al., 1999), neurotrophic factors [BDNF and nerve growth 

factor (NGF)] (Ballarin et al., 1991; Gall et al., 1991; Hashimoto et al., 1998), neuropeptides 

(Pennypacker and Hong, 1995; Friedman, 1997), heat shock proteins (Zhang et al., 1997; 

21

Hashimoto et al., 1998), immediate early genes (Honkaniemi and Sharp, 1999), GABA-A 

receptor subunits (Tsunashima et al., 1997), and countless others. Altered gene expression is 

likely to influence cellular response to KA toxicity; accordingly, glucocorticoid modulation of 

gene expression may modulate cell survival. Experiments in the current thesis focus on 

glucocorticoid regulation of members of the Bcl-2 family, p53, and neurotrophins BDNF and 

NT-3, all of which have been implicated in KA-induced neuronal death (see below). 

Regulators of cell survival and death 

Members of the bcl-2 family of genes are critical regulators of cell viability. Interactions 

between pro-survival genes, including bcl-2, bcl-x L , and bcl-w, and pro-death genes, such as bax 

and bad, are thought to influence cellular susceptibility to necrotic and apoptotic death (Oltvai et 

al., 1993; Korsmeyer, 1995; Merry and Korsmeyer, 1997). Bcl-2 family members influence cell 

survival via multiple mechanisms including stabilization of mitochondria and interference with 

cell death pathways (see below). 

A key event associated with cell death is the opening of a large conductance channel called 

the mitochondrial permeability transition pore. Permeability transition can be induced by a 

number of stimuli, including sustained increases in intracellular Ca 2+ levels, oxidative stress, 

caspase activation (see below), uncoupling of the respiratory chain, and modification of the Bcl- 

2 complex (Green and Reed, 1998). Opening of the nonselective permeability transition pore 

allows molecules up to approximately 1.5 kD to pass through the mitochondrial inner membrane 

into the matrix. The loss of inner membrane selectivity causes collapse of the mitochondrial 

transmembrane potential and dissipation of the H + gradient, resulting in the uncoupling of the 

electron transport chain, a decrease in ATP production, and an increase in production of reactive 

oxygen species. In addition, permeability transition pore opening causes release of caspaseactivating 

proteins such as cytochrome c and apoptosis-inducing factor (AIF) into the cytoplasm, 

promoting apoptosis [see (Kroemer et al., 1997; Green and Reed, 1998)]. 

Mitochondrial release of cytochrome c promotes apoptosis by initiating protease cascades 

programmed to dismantle the cell (Eldadah and Faden, 2000). Cytochrome c facilitates 

interaction between apoptotic protease activating factor-1 (Apaf-1) and procaspase-9 (Li et al., 

1997; Zou et al., 1997). Subsequent processing yields caspase-9, which activates “executioner” 

caspases such as caspase-3 (Li et al., 1997; Pan et al., 1998b). Caspase-3 cleaves ICAD, the 

22

inhibitor of CAD, a caspase-activated deoxyribonuclease (Enari et al., 1998; Sakahira et al., 

1998). Once released from ICAD inhibition, CAD is responsible for internucleosomal DNA 

degradation characteristic of apoptosis (Enari et al., 1998; Sakahira et al., 1998). 

Conditions favoring apoptosis trigger bax translocation from the cytoplasm to mitochondrial 

membranes (Green and Reed, 1998), promoting the release of cytochrome c from the 

intramembranous compartment (Jurgensmeier et al., 1998). Anti-apoptotic proteins such as Bcl- 

2 and Bcl-xL may inhibit apoptosis by preventing molecules such as Apaf-1 from activating 

caspases (Pan et al., 1998a). Bcl-2 can also prevent mitochondrial permeability transition (Green 

and Reed, 1998), the release of cytochrome c (Kluck et al., 1997; Yang et al., 1997), and the 

release of AIF from mitochondria (Green and Reed, 1998), each of which could prevent caspase 

activation. Although the functional significance is not fully understood, Bcl-2 family members 

can also form cation-selective pores, which could speculatively influence ion transport, regulate 

volume of the matrix space, control pH in the intermembrane space, or induce permeability 

transition in mitochondria (Green and Reed, 1998). In addition, Bcl-2 acts at cell nuclei (Marin 

et al., 1996), endoplasmic reticula (He et al., 1997), and mitochondria (Green and Reed, 1998) to 

influence subcellular Ca 2+ distribution. Whether the many effects of Bcl-2 are separate functions 

or consequences of Bcl-2 regulation of a single cell death process remains uncertain. 

Accumulating evidence supports a role for bcl-2 family members in modulating neuronal 

viability after KA-induced seizure. Systemic KA administration decreases bcl-2 mRNA levels in 

rat hippocampus (McCullers and Herman, 1998) (Chapter Two) and Bcl-2 immunoreactivity in 

mouse hippocampus and cortex (Gillardon et al., 1995). In contrast, systemic KA increases bax 

mRNA levels in rat hippocampus (McCullers and Herman, 1998; Lopez et al., 1999) (Chapter 

Two) and mouse brain (Gillardon et al., 1995). Increased Bcl-w expression has also been 

observed in rat hippocampus and piriform cortex following KA microinjection into the amygdala 

(Henshall et al., 2001). Bcl-2 promotes neuronal survival of KA insult, as Bcl-2 overexpression 

with viral vectors protects hippocampal neurons against KA toxicity in culture and in vivo 

(Phillips et al., 2000). Furthermore, KA-induced degeneration is attenuated in Bax-deficient 

cultured motor neurons (Bar-Peled et al., 1999), demonstrating Bax participation in KA-induced 

cell death. 

The p53 tumor-suppressor gene has been described as a guardian of the genome. In response 

to DNA damage, the transcription factor induces either growth arrest or apoptosis depending 

23

upon the cell type, environment, and expression of other regulatory factors. In either scenario, 

the end result is the genetic death of the damaged cell, preventing potential neoplastic risk (Evan 

and Littlewood, 1998). The mechanisms by which p53 promote apoptosis are not fully 

delineated, but many studies indicate the involvement of repression or induction of specific 

target genes such as bcl-2 and bax (Miyashita et al., 1994; Miyashita and Reed, 1995; Evan and 

Littlewood, 1998). Transrepression of anti-apoptotic genes and non-transcriptional mechanisms 

may also be involved (Evan and Littlewood, 1998). The signaling pathways by which insults 

activate p53 are mostly unknown (Evan and Littlewood, 1998). 

Experimental evidence suggests that p53 influences hippocampal vulnerability to KAinduced 

neuronal degeneration. P53 mRNA is induced in KA-sensitive brain regions including 

hippocampus, amygdala, piriform cortex, and thalamus following systemic KA injection (Sakhi 

et al., 1994) (Chapter Two), and nuclear accumulation of p53 protein has been observed in 

degenerating neurons after KA administration (Sakhi et al., 1996). Furthermore, p53-deficient 

mice (“knock-out” mice) exhibit little or no hippocampal cell loss after KA treatment (Morrison 

et al., 1996; Xiang et al., 1996). 

Neurotrophic factors (i.e. nerve growth factor, brain-derived neurotrophic factor, 

neurotrophins-3 and 4/5, basic fibroblast growth factor, ciliary neurotrophic factor, and insulinlike 

growth factors) support neuronal differential and survival during development (Barde, 1989) 

and may protect against select forms of neuronal injury (Lindsay et al., 1994; Mattson and 

Scheff, 1994; Mocchetti and Wrathall, 1995). A variety of neurotrophic factors have been 

reported to protect against injury resulting from physical, ischemic, excitotoxic, metabolic, and 

oxidative insults (Mattson and Scheff, 1994; Mocchetti and Wrathall, 1995). The 

neuroprotective function of these factors is proposed to involve enhanced maintenance of Ca 2+ 

homeostasis and free radical metabolism (Mattson and Scheff, 1994). 

Several studies have examined the involvement of two members of the neurotrophin family, 

brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), in relation to KA 

excitotoxicity in hippocampus. Intrahippocampal (Ballarin et al., 1991) or systemic (Dugich- 

Djordjevic et al., 1992; Lee et al., 1997) KA administration dramatically increases hippocampal 

BDNF mRNA levels. Levels of BDNF mRNA remain elevated for as long as two weeks 

following intracranial ventricular injection of KA (Garcia et al., 1997a). Increased BDNF 

protein levels have also been demonstrated following systemic KA administration (Rudge et al., 

24

1998; Katoh-Semba et al., 1999). In contrast, NT-3 mRNA levels are decreased following 

systemic (Lee et al., 1997; Katoh-Semba et al., 1999) or intracranial ventricular (Garcia et al., 

1997a) KA administration, with decreases lasting up to two weeks post-injection in the latter 

model (Garcia et al., 1997a). NT-3 protein levels correspond with altered mRNA levels, 

decreasing following systemic KA injection (Katoh-Semba et al., 1999). Experiments indicate 

that BDNF and NT-3 protect cultured rat hippocampal, septal, and cortical neurons against 

damage induced by glucose deprivation or glutamate neurotoxicity by attenuating harmful rises 

in intracellular Ca 2+ levels (Cheng and Mattson, 1994). BDNF also protects cultured rat 

cerebellar granule neurons against glutamate neurotoxicity (Lindholm et al., 1993). In contrast, a 

study examining the effects of in vivo intrahippocampal neurotrophin administration reports no 

neuronal protection against KA toxicity with NT-3 infusion and exacerbation of KA-induced 

injury in CA3 with BDNF administration (Rudge et al., 1998). 

Controlled cortical impact as a model of traumatic brain injury 

Hypothalamic-pituitary-adrenal abnormalities are prevalent in human cases of head injury 

(King et al., 1970; Pentelenyi and Kammerer, 1984; Chiolero and Berger, 1994; Lieberman et al., 

2001), indicating the urgent need for research examining the physiological effects of 

glucocorticoid secretion after traumatic brain injury (TBI). Synthetic glucocorticoid steroids 

such as methylprednisolone have been extensively used in the clinical treatment of central 

nervous system trauma. In 1990, the American National Acute Spinal Cord Injury Study 

demonstrated significant benefits of high-dose methylprednisolone in the treatment of human 

spinal cord injury (Bracken et al., 1990). However, the utility of these compounds with respect 

to brain injury requires further evaluation (see McIntosh et al., 1998b). Not surprisingly, 

neuroprotection conferred by methylprednisolone is thought to be independent of hormonal 

(receptor-mediated) glucocorticoid activities; rather, the primary neuroprotective mechanism of 

methylprednisolone is hypothesized to be the inhibition of oxygen free-radical induced lipid 

peroxidation via direct membrane effects (Hall et al., 1992). While the nonhormonal effects of 

exogenous synthetic steroids have been researched extensively, the physiological role for 

endogenous glucocorticoids after TBI remains largely unexamined. Increased cortisol levels 

reported with human TBI (King et al., 1970; Pentelenyi and Kammerer, 1984) emphasize the 

25

potential importance of receptor-mediated glucocorticoid effects on neuronal viability after 

trauma. 

Many animal models of mechanical brain injury have been developed in an attempt to 

reproduce aspects of human closed-head injury. Of these, several commonly used techniques 

are: fluid percussion, which produces brain injury by the rapid injection of fluid into the closed 

cranial cavity (Dixon et al., 1987); closed head injury, in which a weight-drop device delivers a 

standard blow to the skull (Shapira et al., 1988); and the cortical impact model, which uses a 

pneumatic impactor to deliver a controlled impact to the exposed dura, with measurable velocity 

and cortical deformation (Dixon et al., 1991). Experiments in the present thesis employ the 

controlled cortical impact (CCI) model of TBI in conjunction with ACR antagonist treatments in 

order to study ACR effects on hippocampal neuron viability following TBI. 

Controlled cortical impact pathology 

Traumatic injury of the human brain causes neuronal loss in regions including cortex, 

hippocampus, thalamus, and cerebellum (Adams et al., 1985; Kotapka et al., 1992; Ross et al., 

1993). Neuronal degeneration caused by TBI is the product of direct mechanical insult (primary 

injury) and delayed pathological events including ion disequilibration, free radical formation, 

membrane damage, protease activation, and cytoskeletal disruption (secondary injury) (Faden, 

1993). The delayed component of TBI pathology is of great significance as it provides 

opportunity for therapeutic intervention after injury. The pattern of neuronal loss in 

experimentally injured rat brain mimics that of human TBI, with neuronal degeneration observed 

in cortical areas overlying the evolving contusion, in hippocampus, and in lateral thalamus 

(Sutton et al., 1993; Dietrich et al., 1994; Hicks et al., 1996). Dark, shrunken neurons and 

swollen astrocytes can be detected in the acute phase, as early as ten minutes following injury 

(Hicks et al., 1996). With the controlled cortical impact model at moderate severity, neuronal 

loss in CA3 is evident by one hour after injury (Baldwin et al., 1997). Furthermore, there is no 

significant difference in the percentage of surviving CA3 neurons estimated 24 hours or 30 days 

post-injury, indicating that neuronal loss is rapid and complete by 24 hours (Baldwin et al., 

1997). Moderate levels of experimental TBI result in neuronal death by both necrosis and 

apoptosis, with the greatest loss caused by necrosis (Raghupathi et al., 2000; Zipfel et al., 2000). 

26

Secondary neuronal degeneration following traumatic insult is driven by an array of 

physiological and biochemical changes. Excessive glutamate release (Faden et al., 1989; 

Bullock and Fujisawa, 1992), increased intracellular Ca 2+ levels (Young, 1992; Nilsson et al., 

1993; McIntosh et al., 1998c), mitochondrial dysfunction (Sullivan et al., 1998; Sullivan et al., 

1999), metabolic changes (Hovda et al., 1992), tissue edema (Wahl et al., 1988), lipid 

peroxidation (Braughler and Hall, 1992), free radical formation (Lewen et al., 2000), vascular 

disruption (Cortez et al., 1989), neurochemical alterations (Hayes et al., 1992), inflammation 

(Feuerstein et al., 1997), astroglial responses (Cortez et al., 1989), axonal injury (Povlishock and 

Christman, 1995), proteolysis (Kampfl et al., 1997), activation of cell death pathways (Beer et 

al., 2000; Raghupathi et al., 2000), and altered gene expression (Mattson and Scheff, 1994; 

Hayes et al., 1995; Raghupathi et al., 2000) are among the many factors identified as contributors 

to trauma-induced degeneration (see Faden, 1993). 

Glucocorticoid modulation of traumatic brain injury 

Many factors contributing to secondary neuronal degeneration following TBI are likely 

influenced by glucocorticoids. Glucocorticoids reduce edema (Xu et al., 1992), influence 

neurotransmitter systems (Meijer and de Kloet, 1998), reduce inflammation (De Bosscher et al., 

2000), and interact with cell death pathways (Reagan and McEwen, 1997). Steroid hormones 

also prevent lipid hydrolysis and the subsequent formation of oxygen free radicals, as noted 

above (Hall et al., 1992). Furthermore, glucocorticoids exacerbate cognitive deficits associated 

with experimental TBI (Hicks et al., 1993; White-Gbadebo and Hamm, 1993; Smith et al., 1994; 

Scheff et al., 1997). Experiments in the current thesis focus on glucocorticoid and traumainduced 

regulation of viability-related genes, specifically members of the bcl-2 family and 

tumor-suppressor p53. 

Regulators of cell survival and death 

Bcl-2 family members are critical regulators of cell viability, and an increasing body of 

evidence suggests that bcl-2 related proteins control neuronal survival following TBI. Bcl-2 

expression increases in injured human brain (Clark et al., 1999) and in rat hippocampal and 

cortical neurons surviving cortical impact with secondary hypoxia (Clark et al., 1997). In 

contrast, bcl-2 expression is reported to decrease in rat cortical neurons following closed head 

27

injury (Lu et al., 2000). A neuroprotective role for Bcl-2 after TBI has been supported by studies 

demonstrating reduced neuronal loss in injured cortex and hippocampus of Bcl-2 overexpressing 

mice (Raghupathi et al., 1998; Nakamura et al., 1999). Bax may also participate in the 

regulation of TBI-induced neuronal death, as bax translocation to nuclei of injured cortical and 

hippocampal neurons has been reported forty-eight hours after cortical contusion (Kaya et al., 

1999). 

P53, a regulator of bcl-2 and bax expression (Miyashita et al., 1994), is also associated with 

experimental TBI in the rat. Induction of p53 mRNA has been observed in cortex, hippocampus, 

and thalamus following lateral fluid-percussion injury (Napieralski et al., 1999), and 

translocation of p53 proteins to nuclei of injured neurons has been reported using the controlled 

cortical impact model (Kaya et al., 1999). Neuronal apoptosis in thalamus is attenuated in p53 - 

/- mice (Martin et al., 2001), but a study measuring cortical lesion volume after controlled 

cortical impact in p53 -/- mice reported no significant differences compared to wild-type 

(Tomasevic et al., 1999). 

Several studies suggest that neurotrophic factors may also influence hippocampal neuron 

survival following TBI. Nerve growth factor mRNA levels are increased in dentate gyrus after 

injury by lateral fluid percussion (Truettner et al., 1999), while NT-3 mRNA levels are decreased 

(Hicks et al., 1997). In addition, increased hippocampal BDNF mRNA levels are observed 

following injury with CCI (Yang et al., 1996) and lateral fluid percussion (Hicks et al., 1997; 

Truettner et al., 1999) models of TBI, suggesting the potential for enhanced neuronal survival 

due to altered neurotrophin expression. In support of a neuroprotective role for BDNF 

expression after fluid percussion injury, BDNF mRNA levels are decreased in cortical regions 

containing degenerating neurons, generally unchanged in adjacent areas, and increased in more 

remote regions such as piriform cortex (Hicks et al., 1999). However, in a recent study 

intrahippocampal infusion of BDNF did not improve measures of neuronal loss, neuromotor 

function, learning, or memory following lateral fluid percussion injury (Blaha et al., 2000). 

28

Chapter Two: Mineralocorticoid receptors regulate bcl-2 and p53 mRNA 

expression in hippocampus 

Introduction 

Glucocorticoids regulate gene expression in hippocampal neurons through mineralocorticoid 

receptor (MR) and glucocorticoid receptor (GR) proteins. MR binds glucocorticoids with high 

affinity but low capacity, whereas GR binds with low affinity and high capacity (Reul and de 

Kloet, 1985). These differences in binding efficacy allow MR and GR to regulate gene 

expression in a concentration-dependent manner; MR is predominantly active at low 

concentrations of glucocorticoids, while GR becomes increasingly bound and dominant at higher 

glucocorticoid concentrations (Reul and de Kloet, 1985). MR activity at low glucocorticoid 

levels is associated with increased cell excitability (Joels and de Kloet, 1990), long-term 

potentiation (Pavlides et al., 1995), and dentate gyrus (DG) granule cell survival (Woolley et al., 

1991). In contrast, high levels of glucocorticoids acting mainly through GR have been shown to 

reduce long-term potentiation (Pavlides et al., 1993), shrink hippocampal pyramidal cell 

dendrites (Woolley et al., 1990), and exacerbate damaging effects of toxic insults (Sapolsky et 

al., 1988; Packan and Sapolsky, 1990). Thus, MR and GR seem to promote differential cellular 

effects; MR appears to enhance and GR appears to compromise neuronal viability. 

The influence of MR and GR on survival of hippocampal neurons is likely exerted at the 

level of gene regulation. To address the role of the MR in this regulation, we tested the effect of 

spironolactone (SPIRO), a specific, competitive MR antagonist (Sutanto and de Kloet, 1991), on 

basal and kainic acid (KA) stimulated expression of mRNAs associated with apoptosis or 

neuronal survival. Messenger RNAs include those that encode: 1) bcl-2, an inhibitor of 

apoptosis which is down-regulated by KA (Gillardon et al., 1995); 2) bax, a death-inducing 

member of the bcl-2 gene family which is up-regulated by KA (Gillardon et al., 1995); 3) p53, a 

tumor-suppressor which is induced by KA-related DNA damage (Sakhi et al., 1994); 4) brainderived 

neurotrophic factor (BDNF), a neurotrophin which is induced by KA excitotoxicity 

(Ballarin et al., 1991); and 5) neurotrophin-3 (NT-3). To investigate the hypothesis that MR 

promotes neuroprotective gene activity, we evaluated the effects of MR blockade and KA 

29

treatment by in situ hybridization analysis of mRNA expression in CA1, CA3, and DG of the rat 

hippocampal formation. 

Materials and Methods 

Animal treatment and tissue preparation 

All animal handling and protocols were approved by the University of Kentucky IACUC. 

Young adult male Sprague-Dawley rats were subcutaneously injected twice daily, in morning 

and afternoon, with MR antagonist spironolactone (50 mg/kg) or sesame oil vehicle. Treatment 

continued for three days until a total of five injections were administered. On day three, two 

hours after the final morning injection, half of each group received intraperitoneal injection of 

kainic acid (12 mg/kg), while the remaining half received 0.9% saline vehicle. Rats were 

monitored for 8 hours following KA injections; only animals exhibiting behavioral changes 

indicative of KA seizures as described previously (Sperk et al., 1983) were included in the study. 

Animals were decapitated on day four, twenty-four hours after onset of kainic acid-induced 

seizure activity. Brains were rapidly removed and frozen in isopentane (Sigma) cooled to -30 o 

to -40 o C on dry ice. Tissue was sectioned (16 um) with a Bright-Hacker cryostat, thaw-mounted 

onto Ultrastick Slides (Becton Dickinson), and stored at -20 o C. 

In situ hybridization 

Assessment of mRNA expression was accomplished using probes specific for 1) bcl-2, 2) 

bax, 3) p53, 4) BDNF, and 5) NT-3. Probes were labeled by standard in vitro transcription, 

using 33 P-UTP, according to previously published methods (Seroogy and Herman, 1996). 

Frozen tissue sections removed from a -20 o C freezer were placed in 4% paraformaldehyde for 

10 min. Slides were rinsed with 5mM KPBS, 5mM KPBS with 0.2% glycine, and again with 

5mM KPBS. Following 10 min in 0.1 M TEA with 0.25% acetic anhydride and rinses in 0.2X 

SSC, sections were dehydrated. Labeled probes were added to a hybridization buffer containing 

50% formamide. 50 uL (1 x 10 6 cpm) of diluted probe were applied to each slide. Slides were 

coverslipped, placed in moistened chambers, and incubated overnight at 55 o C. Following 

hybridization, coverslips were removed in 2X SSC, rinsed once in fresh 2X SSC, rinsed 3X in 

30

0.2X SSC for 10 min. per wash, followed by a 1 hour wash in 0.2X SSC at 60 o C. After 

dehydration, slides were exposed for 14 - 21 days to Kodak BioMAX film. 

Data analysis 

Densitometric measurements were taken from subfields CA1, CA3, and dentate gyrus using 

NIH Image 1.55 software for Macintosh. Data were analyzed by two-way ANOVA, with 

planned comparisons used to distinguish significance of KA and SPIRO effects. The 

relationship between bax and p53 mRNAs was assessed by regression analysis. 

Results 

Figure 2.1 illustrates expression of bcl-2, bax, and p53 in hippocampus of saline- and KAtreated 

rats. Bcl-2 mRNA levels were significantly affected by SPIRO (F(1, 25) = 4.305; p < 

0.05) and KA (F(1, 25) = 5.845; p < 0.05) in CA1 and by KA in CA3 (F(1,25) = 6.468; p < 

0.05). Planned comparisons indicate that SPIRO decreased basal bcl-2 mRNA levels in CA1 and 

CA3 of saline-treated subjects (p < 0.05). Kainic acid significantly decreased bcl-2 mRNA 

levels in CA1 (p < 0.05) and CA3 (p < 0.01) of oil-treated subjects (Figure 2.2). 

Kainic acid treatment significantly influenced bax mRNA expression in CA1 (F(1, 12) = 

5.555; p < 0.05) and CA3 (F(1, 24) = 8.049; p < 0.01) (Figure 2.3). Planned comparisons 

revealed that following KA injection, bax mRNA expression significantly increased in CA1 (p = 

0.05) of SPIRO-treated animals and in CA3 (p < 0.05) of oil-treated animals. MR blockade had 

no significant effect on bax mRNA expression. 

Spironolactone (F(1,20) = 4.481; p < 0.05) and KA treatment (F(1, 20) = 15.819; p < 0.001) 

elicited significant main effects on p53 mRNA expression in CA3, the only hippocampal region 

in which p53 was observed in consistently quantifiable amounts (Figure 2.4). Kainic acid 

significantly increased p53 mRNA levels in CA3 of oil-treated animals (p < 0.001), while 

SPIRO pre-treatment significantly attenuated this increase (p < 0.05). 

31

Figure 2.1. Photomicrographs illustrating bcl-2 (A, B), bax (C, D), and p53 (E, F) mRNA 

distribution in hippocampus of saline- (A, C, E) and KA-treated (B, D, F) rats. KA decreased 

bcl-2 mRNA expression in CA1 and CA3, increased bax mRNA expression in CA3, and 

increased p53 mRNA expression in CA3. 

32

Figure 2.2. Effects of MR blockade and kainic acid on bcl-2 mRNA expression in hippocampus. 

MR blockade with SPIRO significantly decreased basal bcl-2 mRNA expression in CA1 and 

CA3 of saline-treated animals (*p < 0.05). KA decreased bcl-2 mRNA expression in CA1 and 

CA3 of oil-treated animals ( ‡ p < 0.05). 

33

Figure 2.3. Effects of MR blockade and kainic acid on bax mRNA expression in hippocampus. 

Kainic acid (KA) significantly increased bax mRNA expression in CA1 of SPIRO-treated 

animals and in CA3 of oil-treated animals ( ‡ p < 0.05). 

34

Figure 2.4. Effects of MR blockade and kainic acid on p53 mRNA expression in hippocampus. 

Kainic acid significantly increased p53 mRNA levels in CA3 ( ‡ p < 0.05). MR blockade with 

SPIRO significantly attenuated the KA-induced increase (*p < 0.05). 

Kainic acid treatment significantly altered BDNF mRNA levels in CA1 (F(1, 23) = 26.952; p 

< 0.0001) and CA3 (F(1, 23) = 13.511; p < 0.01); expression increased significantly (p < 0.05) in 

CA1 and CA3 of both oil- and SPIRO-treated animals (Figure 2.5). NT-3 mRNA levels were 

affected by KA in DG (F(1, 24) = 107.00; p < 0.0001), showing significant decreases in 

expression in both oil- and SPIRO-treated animals (p < 0.0001) (Figure 2.6). MR blockade had 

no significant effect on BDNF or NT-3 mRNAs. 

35

Figure 2.5. Effects of MR blockade and kainic acid on brain derived neurotrophic factor 

(BDNF) expression in hippocampus. Kainic acid significantly increased BDNF mRNA 

expression in CA1 and CA3 ( ‡ p < 0.05). 

36

Figure 2.6. Effects of MR blockade and kainic acid on neurotrophin-3 (NT-3) expression in 

hippocampus. KA decreased NT-3 mRNA expression in DG ( ‡ p < 0.05). 

37

Discussion 

Within the bcl-2 gene family, dimerization among death-repressing members, such as Bcl-2, 

and death-inducing members, such as Bax, is thought to determine cellular response to apoptotic 

signals. The ratio of life-promoting to death-promoting proteins determines if dimerization 

favoring cell survival will occur (Oltvai et al., 1993). We report that MR blockade by SPIRO 

decreases basal bcl-2 mRNA expression in CA1 and CA3, demonstrating that MR activity is 

essential in maintaining normal levels of bcl-2 mRNA in these regions. This finding supports the 

hypothesis that MR functions as a neuroprotective factor, as decreases in bcl-2 mRNA could lead 

to the alteration of cellular Bcl-2/Bax ratios and predispose cells to apoptotic cell death. We also 

report a decrease in bcl-2 mRNA expression in CA1 and CA3 of oil-treated animals with KA 

administration, an effect previously demonstrated in mouse hippocampus (Gillardon et al., 

1995). Bcl-2 mRNA levels were similarly decreased in SPIRO and oil-treated animals after KAtreatment. 

Thus, whereas SPIRO pretreatment decreased bcl-2 mRNA levels in control rats, it 

did not exacerbate decreases in bcl-2 mRNA expression induced by KA treatment. Overall, the 

data suggest that MR modulates bcl-2 expression in hippocampus and may therefore play a role 

in pyramidal cell survival. 

The observed induction of bax mRNA expression in CA1 and CA3 following KA treatment 

is consistent with previous studies (Gillardon et al., 1995). Notably, levels of bax mRNA did not 

differ significantly between SPIRO or oil-treated groups prior to or following KA administration. 

In combination with decreased bcl-2 mRNA expression, these results suggest a possible reduced 

Bcl-2/Bax ratio, a scenario believed to favor the neuroendangering actions of Bax (Oltvai et al., 

1993). 

In agreement with previous studies (Sakhi et al., 1994), we demonstrated that KA treatment 

increases p53 mRNA levels in rat hippocampus. We additionally report that SPIRO attenuated 

this KA-induced increase in CA3, indicating MR involvement in p53 mRNA expression under 

these conditions. As accumulation of p53 is associated with induction of apoptosis (Yonish- 

Rouach et al., 1991; Sakhi et al., 1996), these data suggest that, contrary in part to our principal 

hypothesis, the MR may play a role in regulating both protective and endangering gene products. 

Bax mRNA responses parallel those of p53 mRNA in this study. SPIRO appeared to 

attenuate the KA-induced increase of bax mRNA in CA3 and also significantly reduced the KA 

38

induction of p53 mRNA in the same region. These changes in bax and p53 mRNA levels after 

KA treatment are significantly correlated (r = 0.74; p < 0.05), indicating that the expression of 

bax and p53 mRNAs are regulated in parallel. P53 is capable of inducing the human bax gene 

(Miyashita and Reed, 1995) and is thought to participate in a signaling pathway involving bax 

and bcl-2 that predisposes cells to apoptosis (Miyashita et al., 1994). For these reasons, the 

similar responses observed in this study invite speculation that increased bax mRNA levels may 

result from upregulation by p53. However, p53 overexposure has been shown to cause apoptosis 

in hippocampal pyramidal neurons without bax induction (Jordan et al., 1997), so the possibility 

remains that the increases in bax and p53 mRNA are not causally linked. 

Consistent with previous reports (Ballarin et al., 1991), this study demonstrated that BDNF 

mRNA levels increase in rat hippocampus following KA treatment. As elevated glucocorticoid 

levels have been shown to decrease the expression of BDNF mRNA (Schaaf et al., 1997), this 

induction is most likely a consequence of another aspect of KA excitotoxicity, although possibly 

influenced by glucocorticoid input. In addition, NT-3 mRNA expression was decreased in DG 

of animals receiving KA. While neurotrophins are clearly susceptible to glucocorticoid 

regulation, as demonstrated by previous studies (Chao and McEwen, 1994; Smith et al., 1995a), 

there does not appear to be any differential regulation of BDNF or NT-3 mRNA with MR 

blockade prior to or following excitotoxic insult with KA. 

It should be noted that blockade of the MR may alter mRNA expression via activity of an 

unopposed GR. Without the MR available to form MR-GR heterodimers and otherwise compete 

with GR homodimers at the GC response element, an increase in GR-mediated transactivation 

may underlie changes in transcriptional regulation. Overall, it is clear that MR blockade affects 

bcl-2 and p53 mRNA levels, but whether this change is due to a loss of MR or increase in GR 

activity remains to be determined. Another important consideration is the substantial 

hippocampal pyramidal cell loss that accompanies KA treatment after 24 hours. It is possible 

that changes observed in bcl-2, bax, and p53 mRNAs could represent non-specific actions of 

dying cells, rather than specific regulatory events, or possibly reflect changes in surviving cell 

numbers. 

Our current understanding of the function of GC receptors and cell viability is far from 

complete. The MR likely participates in apoptosis regulation, but the present evidence does not 

convincingly support an exclusively neuroprotective role. One problematic issue is the 

39

complexity of receptor interaction involved. It is unclear at this time whether changes in mRNA 

expression following MR blockade are attributable to an absence of MR activity or an 

exaggerated presence of GR. Based on the available data, it appears that MR promotes 

protective gene induction under normal conditions, which may have important implications for 

survival of pyramidal cells following injury. 

40

Chapter Three: Adrenocorticosteriod receptor blockade and excitotoxic 

challenge regulate adrenocorticosteroid receptor mRNA levels in 

hippocampus 

Introduction 

Glucocorticoids are steroid hormones that convey hypothalamic-pituitary-adrenal (HPA) axis 

information throughout the body. Two types of adrenocorticosteroid receptors (ACRs) mediate 

glucocorticoid action in brain: the mineralocorticoid receptor (MR) and the glucocorticoid 

receptor (GR). Once the receptors bind glucocorticoid ligand, cortisol in humans and 

corticosterone (CORT) in rodents, MR and GR act as transcription factors (Yamamoto, 1985) to 

modulate genes controlling many physiological processes including development, metabolism, 

memory, inflammation, immune function, and cell death (Munck et al., 1984; McEwen et al., 

1986). 

Because the MR has greater affinity for binding CORT and the GR greater capacity, the MR 

is mainly active at basal glucocorticoid concentrations while the GR is extensively bound during 

periods of elevated glucocorticoid levels (Reul and de Kloet, 1985; Reul et al., 1987). 

Predominant activation of the MR or GR at different CORT concentrations appears to have 

opposing effects on neuronal viability in hippocampus, an area of MR and GR colocalization 

(Reul and de Kloet, 1985; Herman et al., 1989a) that is particularly susceptible to glucocorticoidmediated 

effects. For example, activation of the MR protects hippocampal dentate gyrus (DG) 

granule cells from adrenalectomy (ADX) induced death (Woolley et al., 1991). In contrast, 

prolonged elevation of glucocorticoid levels, acting mainly through the GR, shrinks pyramidal 

cell dendrites (Woolley et al., 1990), impairs calcium regulation (Elliott and Sapolsky, 1993), 

and exacerbates neuronal insults (Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; Sapolsky, 

1986a; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a). Thus, 

evidence exists that the MR protects and the GR endangers hippocampal neurons. 

Considering the differential effects of MR and GR on hippocampal cell survival, ACR 

activation may be of critical importance in the cellular response to injury. To address this issue, 

the present study is designed to investigate the roles of the MR and the GR in regulating ACR 

expression and neuron viability under basal conditions and following excitotoxic insult. Subjects 

41

were pretreated with vehicle, the MR antagonist spironolactone (SPIRO), or the GR antagonist 

mifepristone (RU486) and subsequently challenged with systemic injection of kainic acid (KA). 

In situ hybridization was performed to assess the effects of ACR blockade and subsequent 

challenge with KA on relative MR and GR mRNA levels. In addition, hippocampal neuronal 

loss was evaluated following MR or GR blockade and KA insult to determine the role of ACRs 

in hippocampal vulnerability to injury in vivo. The results of this study provide further evidence 

for homo- and heterologous regulation of ACR expression and indicate that ACR mRNA 

regulation is altered by glutamate receptor stimulation. 


Subjects 

Young adult male Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) 

weighing between 200-250 gm were maintained on a 12 hr light/dark cycle in an environment 

with constant temperature, humidity, and ad libitum access to food and water. All animal 

procedures were performed in accordance with the National Institutes of Health guidelines and 

approved by the University of Kentucky Institutional Animal Care and Use Committee. 

In vivo protocols 

All rats were injected s.c. twice daily, in morning and afternoon, with propylene glycol 

vehicle, MR antagonist spironolactone (SPIRO) (50 mg/kg) (Herman and Spencer, 1998) 

(Sigma, St. Louis, MO), or GR antagonist RU486 (25 mg/kg) (Ratka et al., 1989) (Sigma). 

Subjects were administered a final pretreatment injection at 900h on day three, followed by an 

i.p. injection of either saline vehicle or kainic acid (KA) (12 mg/kg)(Sigma) at 1100h. Subjects 

were monitored for seizure activity for 6h following KA injections, and only those displaying 

behavioral changes indicative of KA intoxication (Sperk et al., 1983) were included in the study. 

All animals were sacrificed on day 4 at 1200h, approximately 24h after seizure onset. 

Animals processed for plasma CORT determination and in situ hybridization experiments 

were killed by rapid decapitation. Brains were rapidly removed, frozen in isopentane cooled to – 

40 o C on dry ice, then stored at –80 o C. Brains were later warmed to -20 o C, sectioned to 16 um 

with a Bright-Hacker cryostat, thaw-mounted onto Ultrastick Slides (Becton Dickinson, Franklin 

42

Lakes, NJ), and stored at -20 o C until processing for in situ hybridization. Trunk blood samples 

were collected into heparinized tubes on ice, and plasma was separated by centrifugation and 

stored at –20 o C. 

Animals processed for cell counts received a lethal dose of sodium pentobarbitol (Butler, 

Columbus, OH)(150mg/kg) before undergoing transcardial perfusion with 0.9% saline followed 

by 4% paraformaldehyde. Brains were removed, post-fixed in 4% paraformaldehyde overnight, 

then placed in 30% sucrose at 4 o C for 24h. Brains were then stored in 70% ethanol until 

embedded in paraffin. 

Radioimmunoassay 

Radioimmunoassay was performed to measure plasma CORT concentration. A doubleantibody 

rat corticosterone kit (ICN Biochemicals, Costa Mesa, CA) was used to determine 125 I- 

labeled CORT levels. Due to a skewed distribution of CORT values, we performed a log 

transformation of the data before analysis by two-way ANOVA. Data shown in Table 3.1 

represent actual CORT values listed as mean ± standard error. Two-way ANOVA was used to 

analyze log-transformed CORT values for main effects (of antagonist groups and KA treatment) 

and their interactions. Pairwise comparisons were made to determine significance of individual 

group effects within treatments and vice versa using the Student-Newman-Keuls test, with 

significance set at p ≤ 0.05. 


Antisense riboprobes specific for rat MR (550 bp, complementary to the coding region and 3’ 

untranslated region of rat MR mRNA) and rat GR (456 bp, complementary to the coding region 

and 3’ untranslated region of rat GR mRNA) were labeled according to previously published 

methods (Seroogy and Herman, 1996) by standard in vitro transcription using [ 35 S]UTP. Tissue 

sections removed from a –20 o C freezer were fixed in 4% paraformaldehyde for 10 min. Slides 

were then rinsed sequentially in the following solutions: 5 mM KPBS, 5 mM KPBS with 0.2% 

glycine, 5mM KPBS, 0.1 M triethanolamine with 0.25% acetic anhydride, 0.2X SSC, and graded 

alcohols. Labeled probes were added to a hybridization buffer containing 50% formamide, and 

diluted probe (50 ul containing 1x 10 6 c.p.m.) was applied to each slide. Slides were 


43

hybridization, slides were rinsed in 2X SSC and incubated in 0.2X SSC at 60 o C for 1 hr. Slides 

were then dehydrated and exposed for 14 days to Kodak BioMAX xray film. 

Autoradiographic images were digitized with NIH Image 1.62 software for Macintosh. Based 

on morphological criteria, regions CA1, CA3, and DG of the hippocampal formation were 

manually sampled for densitometric analysis. The mean gray level of an unhybridized 

(background) area from each section was subtracted from sampled regions to arrive at corrected 

gray level. The data were then expressed as a percentage of control (vehicle/saline) and analyzed 

by two-way ANOVA within each subregion. Pairwise comparisons were made using the 

Student-Neuman-Keuls test to determine significant effects of groups within treatments and vice 

versa, with significance set at p ≤ 0.05. Data in Figures 3.2 and 3.3 are presented as percentage 

of vehicle/saline control ± standard error. 

Cell counts 

Nissl-stained sections of perfused tissue were examined by light microscopy for undamaged 

neurons in the CA1 and CA3 pyramidal layers of dorsal hippocampus approximately 3mm 

posterior to bregma. Cell counts were not performed in dentate gyrus because no damaged 

granule cells were observed and because of the general insensitivity of this subregion to KA 

(Sperk et al., 1983). Neurons identified by round, healthy-looking somata and prominent 

nucleoli were counted by an observer blinded to the experimental design. In preparation for cell 

counts, perfused, paraffin-embedded, slide-mounted tissue was deparaffinized in xylene for 10 

min, hydrated through graded alcohols, and immersed in cresyl violet Nissl stain for 5 min. 

Slides were then rinsed briefly in dH 2 O, dehydrated through graded alcohols, cleared in xylene 

for 5 min, and coverslipped with DPX mountant. 

To obtain cell counts, numbers of neurons with identifiable nucleoli were recorded from a 

100 um 2 field. Counts were performed in three sections per subject, and within each section, 

three fields were counted from CA1 and from CA3 then averaged to calculate subregional means 

per section. Subregional means from each section were then averaged to determine mean 

numbers of live CA1 and CA3 neurons for each animal. Cell counts were analyzed for 

significant effects using two-way ANOVA. Post hoc comparisons were made using the Student- 

Newman-Keuls test, with significance set at p ≤ 0.05. Data in Table 3.2 are presented as mean 

cell number per 100 um 2 field ± standard error. 

44

Results 

Effects of ACR blockade and KA on CORT levels 

Two-way ANOVA indicates that 24h post-insult, KA significantly increased CORT levels 

[F(1,42) = 65.42; p ≤ 0.05) (Table 3.1). Post hoc comparisons indicate that KA-induced 

elevations in CORT levels were significant in all pretreatment groups. ACR antagonists also 

produced a significant main effect on CORT levels [F(2,42) = 5.96; p ≤ 0.05]. In saline-treated 

animals, MR blockade with SPIRO increased basal CORT levels, while GR antagonist RU486 

decreased basal CORT levels relative to vehicle-pretreated animals. 

Table 3.1. Effects of adrenocorticosteroid receptor blockade and kainic acid on plasma 

corticosterone levels (ng/ml) 

Group Treatment N Mean CORT Level 

VEHICLE SALINE 11 78.35 ± 20.89 

SPIRO SALINE 7 137.01 ± 23.24 a 

RU486 SALINE 6 23.93 ± 5.07 a 

VEHICLE KA 11 332.03 ± 59.42 b 

SPIRO KA 6 275.43 ± 53.35 b 

RU486 KA 7 325.85 ± 69.87 b 

SPIRO = MR antagonist spironolactone 

RU486 = GR antagonist mifepristone 

KA = excitotoxin kainic acid 

a p ≤ 0.05 as compared to vehicle/saline subjects (effect of antagonist) 

b p ≤ 0.05 as compared to saline subjects within the same pretreatment 

group (effect of kainic acid) 

45

Effects of ACR blockade and KA on MR and GR mRNA levels 

Relative changes in ACR mRNA levels were determined by film densitometry following in 

situ hybridization with MR and GR-specific riboprobes (see Methods, Figure 3.1). 

Figure 3.1. Photomicrographs illustrating the effects of saline (A,C) and kainic acid (B,D) on 

MR (A,B) and GR (C,D) mRNA levels in hippocampus of vehicle-pretreated rats. Panels A,B: 

Kainic acid decreases MR mRNA levels in CA1 and CA3. Panels C,D: Kainic acid decreases 

GR mRNA levels in dentate gyrus. 

46

Pretreatment with ACR antagonists significantly affected MR mRNA levels in hippocampal 

subregions CA1 [F(2,42) = 5.51; p ≤ 0.05], CA3 [F(2,42) = 6.58; p ≤ 0.05], and DG [F(2,42) = 

4.80; p ≤ 0.05] (Figure 3.2). MR antagonist SPIRO increased MR mRNA signal in CA1, CA3, 

and DG of saline-treated animals, while GR antagonist RU486 had no significant effects on basal 

MR mRNA levels. KA effects on MR mRNA levels were significant in CA1 [F(1,42) = 83.43; p 

≤ 0.05] and CA3 [F(1,42) = 60.62; p ≤ 0.05]. KA decreased MR mRNA levels in regions CA1 

and CA3 of all pretreatment groups. 

Figure 3.2. Effects of ACR blockade and KA treatments on MR mRNA levels in rat 

hippocampus. Semi-quantitative analysis expressed as percentage of control (vehicle/saline) 

corrected gray level. SPIRO significantly increased basal MR mRNA levels in CA1, CA3, and 

DG of saline-treated animals. KA significantly decreased MR mRNA levels in CA1 and CA3 of 

all pretreatment groups. [ a p ≤ 0.05 as compared to vehicle/saline animals (effect of antagonist 

group). b p ≤ 0.05 as compared to saline animals within same pretreatment group (effect of KA 

treatment).] 

47

Two-way ANOVA demonstrates a significant group/treatment interaction effect on GR 

mRNA levels in region CA3 [F(2,42) = 5.16; p ≤ 0.05] (Figure 3.3). SPIRO and RU486 

significantly increased basal GR mRNA in region CA3, but no significant interaction effects 

within post hoc comparisons were observed. Main effects of KA treatment on GR mRNA levels 

were significant in regions CA1 [F(1,42) = 7.27; p ≤ 0.05], CA3 [F(2,42) = 24.72; p ≤ 0.05], and 

DG [F(2,42) = 91.25; p ≤ 0.05]. KA induced significant decreases in GR mRNA signal in CA1 

of RU486 animals, in CA3 of SPIRO and RU486-treated animals, and in DG of all pretreatment 

groups. 

Figure 3.3. Effects of ACR blockade and KA treatments on GR mRNA levels in rat 

hippocampus. Semi-quantitative analysis expressed as percentage of control (vehicle/saline) 

corrected gray level. SPIRO and RU486 significantly increased basal GR mRNA levels in CA3 

of saline-treated animals. KA decreased GR mRNA levels in CA1 of the RU486 pretreatment 

group, in CA3 of SPIRO and RU486 pretreatment groups, and in DG of all groups. [ a p ≤ 0.05 as 

compared to vehicle/saline animals (effect of antagonist group). b p ≤ 0.05 as compared to saline 

animals within same pretreatment group (effect of KA treatment).] 

48

Effects of ACR blockade and KA on hippocampal neuron viability 

Neurons were counted to determine the effects of ACR antagonists on hippocampal neuron 

viability in CA1 and CA3 following KA challenge (see Methods) (Table 3.2). Neither ACR 

blockade nor KA significantly affected the number of CA1 pyramidal neurons. Kainic acid 

produced a significant main effect on the number of viable neurons in CA3 [F(1,22) = 6.35; p ≤ 

0.05]. The excitotoxin decreased live cell numbers in CA3 of SPIRO-pretreated subjects, but 

post hoc comparisons indicate no significant effects of antagonist pretreatment. 

Table 3.2. Effects of adrenocorticosteroid receptor blockade and kainic acid 

on hippocampal neuron viability 

Region Group Treatment N 

Mean LIVE neurons 

per 100 um 2 

CA1 VEHICLE SALINE 4 63.54 ± 3.06 

SPIRO SALINE 4 62.46 ± 2.51 

RU486 SALINE 3 65.00 ± 2.25 

VEHICLE KA 8 60.79 ± 2.16 

SPIRO KA 4 47.25 ± 14.81 

RU486 KA 5 48.90 ± 8.36 

CA3 VEHICLE SALINE 4 39.67 ± 1.69 

SPIRO SALINE 4 34.63 ± 1.52 

RU486 SALINE 3 31.39 ± 6.54 

VEHICLE KA 8 30.38 ± 2.82 

SPIRO KA 4 22.60 ± 3.92 a 

RU486 KA 5 28.10 ± 5.20 


RU486 = GR antagonist mifepristone 

KA = excitotoxin kainic acid 

a p ≤ 0.05 as compared to SPIRO/saline subjects (effect of kainic acid) 

49

Discussion 

The present study characterizes the effects of MR and GR antagonists on plasma CORT 

levels and hippocampal ACR mRNA regulation under basal conditions and after excitotoxic 

challenge with kainic acid. We report increases in basal MR mRNA levels in CA1, CA3, and 

DG following MR blockade and demonstrate increases in basal GR mRNA levels in CA3 

following both MR and GR blockade. Further, we report that administration of the ionotropic 

glutamate receptor agonist kainic acid reduces MR and GR mRNA levels in specific 

hippocampal subfields at 24h following insult, indicating differential regulation of ACR mRNAs 

during excitotoxic injury. Cell counts following KA administration provide evidence that the 

magnitude of ACR mRNA downregulation exceeds that of cell loss. 

Effects of ACR blockade and KA on CORT levels 

In this study, pretreatment with SPIRO significantly increased basal CORT levels (Table 

3.1). There is evidence that MR blockade with antagonist RU28318 increases CORT levels in 

rat (Ratka et al., 1989; van Haarst et al., 1997), suggesting that the SPIRO-induced CORT 

increase is due to MR blockade and a resultant disinhibition of adrenocortical secretion. 

However, other studies using SPIRO have reported no significant effects on plasma CORT levels 

(Chabert et al., 1984; Herman and Spencer, 1998). The reason for this disagreement may be 

related to differing treatment protocols and durations. 

Glucocorticoid receptor blockade with RU486 decreased basal CORT levels in this study. 

This effect concurs with data demonstrating RU486-mediated suppression of CORT induction 15 

minutes following introduction to a novel environment (Ratka et al., 1989). Considering the 

relatively high CORT levels observed in our saline/vehicle-treated (control) subjects, the 

observed decrease in CORT levels may reflect RU486-mediated reduction of CORT levels 

subsequent to stress associated with either the euthanasia procedure or intraperitoneal injections 

received the previous day. Despite its efficacy as a GR antagonist, RU486 can also act as a 

partial GR agonist (Nordeen et al., 1995), which might explain a reduced stress response in 

saline animals. However, CORT response to KA is not attenuated in RU486-treated animals, 

suggesting that effects of RU486 on delayed negative feedback are minimal. 

50

Kainic acid induced a massive increase in CORT secretion observable 24h after injection. 

Acute effects of KA include hyperactivity, “wet dog shakes”, and prolonged tonic-clonic 

convulsions. Dehydration, brain edema, and perivenous hemorrhages are also common 24-48h 

after KA injection (Sperk et al., 1983). It is therefore likely that the excitotoxin is a potent 

stressor on multiple levels and thus increases CORT secretion for an extended period after 

injection. 

ACR antagonists modulate MR and GR mRNA levels 

Treatment with the MR antagonist SPIRO increased basal MR mRNA levels in all observed 

hippocampal subregions by approximately 30-40% (Figures 3.1 and 3.2). These changes 

complement reports of MR mRNA upregulation by adrenalectomy (Herman et al., 1989a; Reul 

et al., 1989; Herman et al., 1993; Chao et al., 1998a) and provide additional evidence for 

homologous feedback regulation of MR. The MR also appears to regulate expression of GR. 

The present data demonstrate a significant SPIRO-induced increase in GR mRNA in CA3; GR 

mRNA levels are similarly elevated in CA1 and DG of SPIRO-pretreated subjects but do not 

reach our criteria for statistical significance. These data add to previous results from our 

laboratory showing significant increases in GR mRNA levels in CA1 and DG with SPIRO 

pretreatment (Herman and Spencer, 1998). In addition, the MR agonist aldosterone decreases 

GR binding in primary hippocampal cell cultures (O'Donnell and Meaney, 1994), and 

administration of aldosterone or low, MR-activating doses of CORT reverses ADX-induced 

increases in GR mRNA (Chao et al., 1998a). Together, these results and the current data suggest 

that MR functions as a tonic inhibitor of GR expression in hippocampus. 

Preceding evidence also suggests that the activated glucocorticoid receptor negatively 

regulates ACR expression. MR mRNA levels decrease during periods of high CORT secretion 

(Herman et al., 1993) when the GR is most extensively activated (Reul and de Kloet, 1985). 

Further, glucocorticoid receptor agonist dexamethasone reverses ADX-induced increases in GR 

(Herman et al., 1989a; Reul et al., 1989) and MR (Herman et al., 1989a) mRNA levels and 

reduces GR mRNA expression in culture (Rosewicz et al., 1988). In the present experiment, 

RU486 significantly increased GR mRNA levels in CA3 (Figures 3.1 and 3.3), suggesting that 

glucocorticoid receptor blockade increases GR biosynthesis by homologous feedback 

mechanisms. However, RU486 did not increase MR mRNA levels, implying that glucocorticoid 

51

eceptor regulation of MR is limited to suppressing MR levels during periods of extensive GR 

activation. 

SPIRO and RU486 are commonly used to achieve ACR blockade and are among the most 

specific antagonists available, but like many other antagonist drugs they may produce some 

nonspecific effects. SPIRO is considered relatively specific for the MR but has been 

demonstrated a weak GR antagonist in a transformed murine fibroblast cell line (Couette et al., 

1992). RU486 is an extremely potent GR antagonist in vitro, binding the GR with approximately 

18 times the affinity of CORT, but also has strong anti-progestin activity (Baulieu, 1989). 

RU486 may also act as a GR agonist under certain circumstances (Nordeen et al., 1995) and may 

not be sufficient to fully block GR activation in vivo. However, it should be noted that the 

currently reported antagonist effects correspond very well with the effects of adrenalectomy 

(Herman et al., 1989a; Reul et al., 1989; Herman et al., 1993; Chao et al., 1998a); the antagonist 

drugs increase hippocampal ACR mRNA expression in saline animals for the duration of the 

experiment. 

Excitotoxic challenge with KA decreases MR and GR mRNA levels 

The present data show dramatic reductions in hippocampal MR and GR mRNA levels 

following kainic acid insult (Figures 3.1, 3.2, and 3.3). KA-induced decreases in MR and GR 

binding have previously been demonstrated using whole dissected hippocampus from 

adrenalectomized rats (Lowy, 1992). Reductions in MR and GR binding are similar in 

magnitude to the currently reported mRNA decreases, are evident as early as one hour after 

systemic KA administration, and persist for at least 24 hours (Lowy, 1992). Therefore, the 

current data provide further evidence that MR and GR availability is reduced by KA and indicate 

that this reduction is likely achieved through genomic downregulation, rather than by protein 

degradation or receptor inactivation. 

ACR downregulation by KA does not appear to be MR or GR-dependent. In the current 

study, KA reduces ACR mRNA expression in the presence of MR or GR blockade. In addition, 

a previous study has demonstrated that KA decreases ACR binding in adrenalectomized rats 

(Lowy, 1992); corticosterone depletion in adrenalectomized rats prevents ACR activation and 

their potential participation in the regulation of receptor expression. Furthermore, the pattern of 

ACR downregulation produced by KA is inconsistent with the pattern of regulation associated 

52

with high CORT levels. High CORT levels decrease MR and GR mRNA levels by equal 

proportions in CA1, CA3, and DG (Herman et al., 1993). In contrast, KA regulation of ACR 

expression differs substantially by hippocampal subfield. Mineralocorticoid receptor expression 

is downregulated in KA-sensitive regions CA1 and CA3, while GR expression is reduced in the 

relatively KA-insensitive DG. These results suggest that KA regulation of ACR expression is 

not mediated by KA induction of CORT, but that differential ACR regulation by KA is achieved 

through other mechanisms related to KA susceptibility, such as glutamate receptor activation or 

Ca 2+ influx. 

Interestingly, increases in MR and GR mRNA that occur with ACR antagonist pretreatment 

are reversed by KA insult. Although KA does not affect MR mRNA levels in DG of vehiclepretreated 

animals, the SPIRO-induced increase in this region is negated by KA treatment. 

Similarly, KA does not reduce GR mRNA levels in the pyramidal layers of vehicle-pretreated 

animals, but SPIRO and RU486-induced increases in CA3 are abolished with KA injection. 

These results suggest that homologous regulation of the receptors is suppressed or overridden by 

KA, functionally disconnecting autoregulatory feedback. 

Hippocampal pyramidal neurons are extremely sensitive to KA toxicity; CA3 neurons are 

particularly vulnerable, while CA1 neurons are slightly less susceptible, to KA-induced damage 

and cell degeneration (Nadler et al., 1978). The potential impact of cell loss on mRNA levels as 

measured by in situ hybridization is an important consideration that is presently undetermined. 

However, it is clear that in the hippocampal pyramidal layers, KA-induced decreases in MR 

mRNA levels exceed the proportion of neuronal loss indicated by cell counts – by approximately 

25 to 50% (see Figures 3.1, 3.2, and Table 3.2). Furthermore, DG granule cells are resistant to 

KA toxicity and exhibit no histological damage after systemic KA administration (Sperk et al., 

1983). Therefore, the approximately 40-55% reduction in GR mRNA levels observed in DG is 

not attributable to a decline in cell number. 

KA-induced repression of ACR mRNA levels might influence neuronal viability during 

injury. The receptors have potent cellular effects, some of which could endanger neurons under 

compromising conditions. For example, MR-modulated excitation of pyramidal neurons 

(Reiheld et al., 1984; Joels and de Kloet, 1990, 1992) could be permissive to excitotoxic damage. 

MR blockade with SPIRO has been shown to inhibit CORT-mediated increases in susceptibility 

to KA-induced convulsions in mice (Roberts and Keith, 1994a, b). Thus, downregulation of the 

53

MR in pyramidal cells may reduce excitability and vulnerability to excitotoxic challenge. 

Moreover, the GR has long been recognized as a potentially compromising influence on 

hippocampal neurons. GR activation has been shown to increase intracellular calcium levels in 

CA1 pyramidal cells (Kerr et al., 1992; Karst et al., 1994) and exacerbate the damaging effects of 

toxic insults including KA, oxidative stress, and ischemia (Sapolsky, 1985; Sapolsky and 

Pulsinelli, 1985; Sapolsky, 1986a; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and 

Sapolsky, 1996a). Downregulation of the GR in dentate gyrus might protect granule cells from 

effects of excitotoxicity such as extracellular glutamate accumulation, Ca 2+ influx, and oxidative 

stress. 

MR blockade increases CA3 vulnerability to KA 

Kainic acid decreased the number of live CA3 neurons counted in SPIRO-pretreated 

subjects; however, KA had no significant effect on numbers of viable neurons in any other 

region or group (Table 3.2). These data indicate that MR blockade increases susceptibility to 

KA-induced degeneration in CA3. We have previously demonstrated that MR blockade with 

SPIRO decreases basal mRNA levels of the anti-apoptotic gene bcl-2 in the hippocampal 

pyramidal layers (McCullers and Herman, 1998) (Chapter Two). Thus, MR appears to play a 

neuroprotective role during neuronal injury that may occur through maintenance of viabilityenhancing 

genes such as bcl-2. 

The results of this study demonstrate that MR and GR mRNA levels respond to stress as 

conveyed not only by differential receptor activation, but also by ACR-independent signals 

generated by excitotoxic stress. Further, kainic acid regulation of ACR mRNA levels appears to 

override ACR-dependent feedback mechanisms. The present evidence for modulation of ACR 

mRNA expression during injury emphasizes the importance of ACR function for maintenance of 

neuronal viability, particularly considering the prevalence of HPA dysregulation in 

neurodegenerative conditions such as Alzheimer’s disease and in normal aging. 

54

Chapter Four: Mifepristone protects CA1 hippocampal neurons following 

traumatic brain injury in rat 

Introduction 

Glucocorticoids modulate many cellular processes relevant to injury, including metabolism, 

ion balance, immune function, inflammation, neurotrophin expression, and cell death (Munck et 

al., 1984; McEwen et al., 1986; Reagan and McEwen, 1997; Joels and Vreugdenhil, 1998). Two 

steroid hormone receptors mediate glucocorticoid action: the mineralocorticoid receptor (MR) 

and the glucocorticoid receptor (GR). Glucocorticoids bind GR with lower affinity but higher 

capacity relative to MR; thus, MR is extensively bound at very low (e.g. circadian nadir) 

glucocorticoid levels, while GR mediates the effects of high glucocorticoid levels (Reul and de 

Kloet, 1985; Reul et al., 1987). The MR and GR are abundantly expressed in hippocampus 

(Reul and de Kloet, 1985; Aronsson et al., 1988; Arriza et al., 1988; Reul et al., 1989), making 

this region a prime target for glucocorticoid action. Excessive activation of GR has been shown 

to increase hippocampal neuronal vulnerability to several forms of neuronal insult, including 

excitotoxicity, oxidative stress, and ischemia (Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; 

Sapolsky, 1986a; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a). 

Moderate to severe traumatic brain injury (TBI) in rat produces neuronal damage and cell 

death in hippocampus (Cortez et al., 1989; Lowenstein et al., 1992; Goodman et al., 1994; Hicks 

et al., 1996). The significant cell death that occurs following TBI is believed to evolve in a 

biphasic sequence consisting of the primary mechanical insult coupled with a progressive 

secondary necrosis. Results of experimental TBI research have proposed several injury 

mechanisms including excitotoxicity (Choi, 1992; Hayes et al., 1992), ischemia (Okiyama et al., 

1994), free radical formation (Braughler and Hall, 1992), elevated intracellular calcium (Young, 

1992), edema (Wahl et al., 1988), proteolysis (Kampfl et al., 1997), and mitochondrial 

dysfunction (Sullivan et al., 1998; Sullivan et al., 1999). Glucocorticoids likely modulate some 

or all of the processes mediating secondary injury. One possible mechanism whereby 

glucocorticoids could influence neuronal vulnerability to injury is through regulation of the antiapoptotic 

B-cell leukemia/lymphoma-2 gene, bcl-2. Several studies indicate that bcl-2 is induced 

by TBI and has neuroprotective effects after traumatic injury (Clark et al., 1997; Raghupathi et 

55

al., 1998; Nakamura et al., 1999). Our laboratory has demonstrated that MR blockade with 

spironolactone decreases basal bcl-2 messenger RNA (mRNA) levels (McCullers and Herman, 

1998) (Chapter Two), suggesting that MR activation maintains neuroprotective bcl-2 expression 

and may participate in regulation of neuronal viability after TBI. 

The present study is designed to test the hypotheses that GR blockade will attenuate and MR 

blockade will exacerbate hippocampal cell loss following controlled cortical impact (CCI), an 

experimental model of TBI. Animals were pretreated with vehicle, the MR antagonist 

spironolactone (SPIRO), or the GR antagonist mifepristone (RU486) and subsequently subjected 

to either sham operation or injury by CCI. Using the optical fractionator method, CA1, CA3, 

and dentate gyrus (DG) hippocampal neurons were counted twenty-four hours after surgery to 

determine SPIRO and RU486 effects on cell survival after CCI. In addition, mRNA levels of the 

anti-apoptotic gene bcl-2, the pro-apoptotic gene bax, and the tumor-suppressor gene p53 (a 

transcriptional regulator of bcl-2 and bax) (Miyashita et al., 1994) were examined after MR or 

GR blockade and CCI to determine whether MR or GR regulation of these genes correlates with 

neuronal viability after traumatic brain injury. 


Subjects 

Young adult male Sprague Dawley rats (n=60) (Harlan Sprague Dawley, Inc., Indianapolis, 

IN) weighing 200-250g were maintained on a 12 hr light/dark cycle in an environment with 

constant temperature, humidity, and ad libitum access to food and water. All animal procedures 

were performed in accordance with the National Institutes of Health guidelines and approved by 

the University of Kentucky Institutional Animal Care and Use Committee. All efforts were 

made to minimize animal suffering and the number of animals used. 


Subjects received subcutaneous injections of propylene glycol vehicle, MR antagonist 

spironolactone (SPIRO) (50 mg/kg) (Herman and Spencer, 1998) (Sigma, St. Louis, MO), or GR 

antagonist RU486 (25 mg/kg) (Ratka et al., 1989) (Sigma) twice daily, in morning and afternoon. 

Pretreatment was conducted for forty-eight hours to ensure adequate time for the altered 

56

expression of ACR-regulated genes (Herman and Spencer, 1998). After a final injection was 

administered at 900h on day three, subjects were transported to a surgical procedure room. 

Between 1100h-1200h, animals were subjected to craniotomy only (sham operation) or a 

moderate unilateral cortical contusion (2mm). All animals were sacrificed on day four at 1200h, 

approximately twenty-four hours after surgery. 8-12 subjects were included in each 

pretreatment/surgery group. Half of the subjects in each condition were perfused for cell counts, 

and half were decapitated for in situ hybridization analysis, yielding n = 4-6 subjects per 

statistical group. Surgical procedures are briefly described below. 

The moderate CCI injury used in these experiments results in significant loss of cortical 

tissue, blood brain barrier disruption, and loss of hippocampal neurons as previously described 

(Baldwin et al., 1997; Scheff and Sullivan, 1999), thus mimicking aspects of the pathology of 

human closed-head injury. Animals were anesthetized with isoflurane (2%) and placed in a 

stereotaxic frame (Kopf Instr., Tujunga, CA) prior to CCI injury. Using sterile procedures, the 

skin was retracted and a 6mm unilateral craniotomy was centered between bregma and lambda. 

The skull cap was removed without disruption of the underlying dura. The exposed brain was 

injured using a pneumatically controlled impacting device with a 5mm beveled tip that 

compressed the cortex 3.5 m/sec to a depth of 2mm. Following injury, Surgicel (Johnson and 

Johnson, Arlington, TX) was laid upon the dura, and the skull cap was replaced. A thin coat of 

dental acrylic was then spread over the craniotomy site and allowed to dry before the wound was 

stapled closed. Body temperature was monitored continuously throughout the surgical/recovery 

procedures and maintained at 37 o C with a heating pad. 

Animals processed for in situ hybridization experiments were killed by decapitation. Brains 

were rapidly removed, frozen in isopentane cooled to –40 o C on dry ice, then stored at –80 o C 

until sectioned. Trunk blood samples were collected on ice, and plasma was stored at -20 o C 

until plasma corticosterone levels were measured using commercial radioimmunoassay kits per 

manufacturers’ instructions (ICN Biochemicals, Costa Mesa, CA). Animals processed for cell 

counts received a lethal dose of sodium pentobarbitol (Butler, Columbus, OH) (150mg/kg) 

before undergoing transcardial perfusion with 0.9% saline followed by 4% paraformaldehyde. 

Brains were removed and post-fixed in 4% paraformaldehyde overnight before further 

processing. 

57

Tissue processing 

Frozen brains were removed from a –80 o C freezer, warmed to –20 o C, and sectioned to 16 um 

with a Bright-Hacker cryostat. Sections were then thaw-mounted onto Ultrastick Slides (Becton 

Dickinson, Franklin Lakes, NJ) and stored at –20 o C until processing for in situ hybridization. 

Perfused brains were processed as previously described (Baldwin et al., 1997). Briefly, brains 

were placed in a graded series of celloidin (Mallinckrodt Baker, Inc., Paris. KY) dissolved in 

50:50 ETOH:ethyl ether. After removal from 12% celloidin, the brains were placed in Peel-A- 

Way (Polyscience, Warrington, PA) disposable histology embedding molds with fresh 12% 

celloidin and exposed to chloroform vapors for 18 hours. Once the celloidin hardened, molds 

were removed. The embedded tissue was then attached to wooden blocks and stored in 70% 

EtOH until sectioning. The brains were cut in coronal sections (50 um) with a Zeiss HM440E 

sliding microtome. Sections were collected from anterior to posterior beginning at the 

appearance of anterior hippocampus. In order to select a series in an unbiased manner such that 

every section had an equal probability of being selected, a die was thrown to determine the first 

section saved, after which every twelfth section was kept for staining with Giemsa. Six drops of 

1% glacial acetic acid were added per 100 ml of Giemsa stain (Sigma, St. Louis, MO) diluted 1:4 

with water. Following staining the sections were placed in cedar wood oil for 5 min to reduce 

background and subsequently washed in Hemo-D (Fisher Scientific, Pittsburgh, PA). The 

sections were then mounted on slides, covered with permount, and coverslipped. 

Optical fractionator method 

Bioquant Image Analysis software (R and M Biometrics, Memphis, TN) was used to 

estimate total cell number in regions of interest using the optical fractionator method of cell 

counting. The optical fractionator method involves the combining of optical dissector counting 

with fractionator sampling. This method of cell counting has several desirable attributes, such as 

being unaffected by tissue shrinkage and not requiring rigorous definitions of structural 

boundaries. For detailed explanation and discussion please see (West et al., 1991). The optical 

fractionator involves counting neurons with optical dissectors in a uniform sample that 

constitutes a known fraction of the structure being analyzed; the method systematically samples 

a known fraction of the section thickness, of a known fraction of sectional area, of a known 

58

fraction of the sections that contain the region of interest. The equation is as follows (our 

specific parameters): 

N = ∑ Q • t 1 1 

• • 

h asf ssf 

Where N is the total number of neurons, and Σ Q is the total number of neurons counted in each 

region of interest in each animal. t is the measured section thickness (42-45 um), h is the height 

of the dissector (20 um), 1/asf is the counting grid area (100 um X 100 um)/ the dissector area 

(20 um X 20 um), and 1/ssf is the sampling section fraction (12). Criteria for counting neuronal 

nuclei were observed as previously described (Baldwin et al., 1997). Briefly, the plane of focus 

was moved 5 um down from the top of the section to obviate the problem of an uneven counting 

surface. Neuronal nuclei in focus at level –5 um, the top level of the dissector, were not counted, 

but nuclei in focus at –25 um, the bottom level of the dissector, were counted. Through the 

dissector, nuclei completely within the counting frame or touching the upper or right side of the 

frame were counted. Nuclei touching the left or lower sides of the frame were not counted 

(Baldwin et al., 1997). Coefficient of error (CE) and coefficient of variation (CV) were 

calculated to determine intra-animal variation and inter-animal variation, respectively (Tables 4.1 

and 4.2). 

Table 4.1. Mean coefficient of error (CE) 

CA1 CA3 Dentate gyrus 

Vehicle 0.082 0.144 0.059 

Spironolactone 0.090 0.143 0.093 

RU486 0.071 0.122 0.080 

Table 4.2. Mean coefficient of variation (CV) 

CA1 CA3 Dentate gyrus 

Vehicle 0.241 0.365 0.156 

Spironolactone 0.162 0.304 0.094 

RU486 0.372 0.218 0.086 

Cell count data were analyzed by two-way ANOVA within each subregion, with hemisphere 

and drug designated as independent variables. Pairwise comparisons were made using the 

Student-Neuman-Keuls test with significance set at p ≤ 0.05. Data in Figure 4.1 are presented as 

neurons x 10 4 ± standard error. 

59


Antisense riboprobes complementary to coding regions for bcl-2 (550 bp) (courtesy of Dr. D. 

Vaux, The Walter and Eliza Hall Institute, The Royal Melbourne Hospital, Victoria, Australia), 

bax (100 bp) (courtesy of Dr. J. Springer, University of Kentucky, KY, USA), and p53 (950 bp) 

(courtesy of Dr. G. Lozano, MD Anderson Cancer Center, Houston, TX, USA) mRNAs were 

generated for in situ hybridization. Probes were labeled according to previously published 

methods (Seroogy and Herman, 1996) by standard in vitro transcription using [ 35 S]UTP. Tissue 

sections removed from a –20 o C freezer were fixed in 4% paraformaldehyde for 10 min. Slides 

were then rinsed sequentially in the following solutions: 5 mM KPBS, 5 mM KPBS with 0.2% 

glycine, 5mM KPBS, 0.1 M triethanolamine with 0.25% acetic anhydride, 0.2X SSC, and graded 

alcohols. Labeled probes were added to a hybridization buffer containing 50% formamide, and 

diluted probe (50 ul containing 1x 10 6 c.p.m.) was applied to each slide. Slides were 


hybridization, slides were rinsed in 2X SSC and incubated in 0.2X SSC at 60 o C for 1 hr. Slides 

were then dehydrated and exposed for 14-18 days to Kodak BioMAX xray film. 

Autoradiographic images were digitized with NIH Image 1.62 software for Macintosh. A 

standard curve was generated prior to image capture to ensure linearity of the signal. Based on 

morphological criteria, regions CA1, CA3, and DG of the hippocampal formation were manually 

sampled from both hemispheres for densitometric analysis. The mean gray level of an 

unhybridized (background) area from each hemisphere was subtracted from sampled regions to 

arrive at corrected gray level. Ipsilateral mRNA data were normalized as a percent of control 

(sham/vehicle) values from the ipsilateral hemisphere, and contralateral mRNA data were 

normalized as a percent of control (sham/vehicle) values from the contralateral hemisphere, with 

sham/vehicle levels expressed as one hundred percent. In situ hybridization data were then 

analyzed by two-way ANOVA within each hemisphere and subregion with drug and injury 

designated as independent variables. Post hoc comparisons were made using the Student- 

Newman-Keuls test, with significance set at p ≤ 0.05. 

60

Results 

Effects of adrenocorticosteroid receptor blockade and traumatic brain injury on hippocampal 

neuron viability 

The numbers of hippocampal neurons surviving CCI at 24 hours in hippocampal subregions 

CA1, CA3, and DG were assessed using the optical fractionator method of cell counting (see 

Methods). Because sham operation does not cause cell loss and damage is confined to the 

ipsilateral hemisphere of injured rats, the numbers of neurons in sham/ipsilateral, 

sham/contralateral, and injured/contralateral hemispheres were statistically indistinguishable (i.e. 

unchanged by surgery) within each subregion (data not shown). Therefore, cell counts from 

injured/contralateral hemispheres were used as control values against which to compare 

injured/ipsilateral hemisphere values to determine the effects of CCI on neuronal survival. 

Effects of antagonist pretreatments were assessed as compared to vehicle within the ipsilateral 

and contralateral hemispheres. 

Two-way ANOVA indicates a significant effect of hemisphere on numbers of neurons in 

CA1 [F(1,24) = 9.32; p ≤ 0.05], CA3 [F(1,24) = 38.17; p ≤ 0.05], and DG [F(1,24) = 5.12; p ≤ 

0.05] (Figure 4.1). In CA1 of RU486-pretreated animals, numbers of surviving neurons were 

statistically indistinguishable in the ipsilateral and contralateral hemispheres. In contrast, 

numbers of hippocampal neurons in CA1 of vehicle and SPIRO-pretreated animals were 

significantly decreased by injury in the ipsilateral as compared to contralateral hemisphere. In 

CA3, injury significantly reduced the number of viable pyramidal neurons in the ipsilateral 

hemisphere in all pretreatment groups. Injury also significantly decreased the number of 

ipsilateral DG granule neurons counted in SPIRO and RU486-pretreated animals. Post-injury 

corticosterone levels fell within the range of approximately 15-80 ng/ml in all drug pretreatment 

groups, indicating that excessive corticosterone levels were not likely to contribute to the 

differential neuronal survival between groups. 

61

Figure 4.1. Effects of MR blockade, GR blockade, and controlled cortical impact injury on 

hippocampal neuron viability as measured by the optical fractionator method (see Methods). 

Injury significantly decreased the number of viable neurons in CA1 and CA3 of vehiclepretreated 

animals (n = 6). Pretreatment with RU486 protected CA1 pyramidal neurons from 

injury-induced cell loss (n = 5). Injury also decreased the number of viable neurons in CA3 and 

dentate gyrus of spironolactone (n = 4) and RU486-pretreated animals. [ B p ≤ 0.05 as compared 

to contralateral hemisphere within same pretreatment group (effect of injury).] 

Effects of adrenocorticosteroid receptor blockade and traumatic brain injury on bcl-2, bax, and 

p53 mRNA levels 

The effects of MR or GR blockade and CCI on hippocampal bcl-2, bax, and p53 mRNA 

levels were determined using densitometric analysis following in situ hybridization with genespecific 

radiolabeled riboprobes (see Methods, Figure 4.2). Unlike measures of cell loss, mRNA 

levels may be affected by injury both in the contralateral (unoperated) and ipsilateral (shamoperated 

or injured) hemispheres. Therefore, all injury-induced changes were assessed as 

compared to sham values from the same hemisphere. To this end, separate two-way ANOVAs 

were conducted comparing sham/contralateral to injured/contralateral values and comparing 

sham/ipsilateral to injured/ipsilateral values, with drug and injury as independent variables. In 

each hemisphere, effects of drug were assessed as compared to vehicle within the sham or 

injured treatment groups. 

62

IPSI 

CONTRA 

Figure 4.2. Photomicrographs illustrating (A) bcl-2, (B) bax, and (C) p53 mRNA levels in 

hippocampus of vehicle-pretreated, injured rats at 24h. Injury decreases bcl-2 mRNA levels in 

ipsilateral dentate gyrus (panel A) but has no significant effect on bax or p53 mRNA levels in 

vehicle-pretreated rats (panels B,C). 

63

Two-way ANOVA demonstrates significant drug/injury interaction effects on contralateral 

bcl-2 mRNA levels in CA1 [F(2,27) = 3.47; p ≤ 0.05] and DG [F(2,27) = 5.67; p ≤ 0.05] (Figure 

4.3). Post hoc comparisons indicate that bcl-2 signal was significantly decreased by RU486 and 

by SPIRO in contralateral CA1 and DG of sham-operated animals. Injury increased contralateral 

bcl-2 mRNA levels in DG of RU486 animals. 

Drug/injury interaction effects on bcl-2 mRNA levels are observed in CA1 [F(2,27) = 5.01; p 

≤ 0.05], CA3 [F(2,27) = 3.94; p ≤ 0.05], and DG [F(2,27) = 6.92; p ≤ 0.05] of the ipsilateral 

hemisphere (Figure 4.3). In sham-operated animals, ipsilateral bcl-2 mRNA levels were 

significantly decreased by SPIRO in DG and by RU486 in CA1 and DG. CCI decreased 

ipsilateral bcl-2 signal in DG of vehicle animals and increased ipsilateral bcl-2 signal in DG of 

RU486 animals. 

Overall, bax mRNA levels were increased in DG [F(1,28) = 4.49; p ≤ 0.05] (Figure 4.4) 

contralateral to CCI. However, post hoc comparisons demonstrate no significant effects of drug 

or injury on bax mRNA levels, in any region or hemisphere. 

Significant effects of injury on p53 mRNA levels are detected in contralateral CA1 [F(1,29) 

= 6.97; p ≤ 0.05] and DG [F(1,29) = 4.57] (Figure 4.5). Post hoc comparisons indicate that CCI 

decreased contralateral p53 signal in CA1 and DG of SPIRO-pretreated animals. No significant 

effects of drug or injury on p53 mRNA levels were observed in the ipsilateral hemisphere. 

64

Figure 4.3. Effects of MR blockade, GR blockade, and controlled cortical impact injury on bcl-2 

mRNA levels in hippocampus. Semi-quantitative analysis expressed as percentage of control 

(sham/vehicle; see methods) corrected gray level; n = 5-6 per group. Contralateral bcl-2 mRNA 

levels were decreased by spironolactone and RU486 in CA1 and dentate gyrus of sham-operated 

animals. Ipsilateral bcl-2 levels were decreased by spironolactone in dentate gyrus and by 

RU486 in CA1 and dentate gyrus of sham-operated animals. Injury increased contralateral bcl-2 

mRNA levels in dentate gyrus of RU486 animals. CCI decreased ipsilateral bcl-2 signal in 

dentate gyrus of vehicle animals and increased ipsilateral bcl-2 signal in dentate gyrus of RU486 

animals. [ A p ≤ 0.05 as compared to vehicle animals within the same surgery condition (effect of 

drug). B p ≤ 0.05 as compared to sham-operated animals within same drug pretreatment group 

(effect of injury).] 

65

Figure 4.4. Effects of MR blockade, GR blockade, and controlled cortical impact injury on bax 


(sham/vehicle; see methods) corrected gray level; n = 4-6 per group. Post hoc comparisons 

indicate no significant effects of antagonist pretreatment or injury on bax mRNA levels in any 

region or hemisphere. 

66

Figure 4.5. Effects of MR blockade, GR blockade, and controlled cortical impact injury on p53 


(sham/vehicle; see methods) corrected gray level; n = 5-6 per group. Injury decreased 

contralateral p53 mRNA levels in CA1 and dentate gyrus of SPIRO-pretreated animals. Post hoc 

comparisons indicate no significant effects of antagonist pretreatments. [ B p ≤ 0.05 as compared 

to sham-operated animals within same drug pretreatment group (effect of injury).] 

67

Discussion 

The present study demonstrates that GR blockade with RU486 prevents CA1 neuron loss 24h 

after CCI injury. The data do not suggest that regulation of viability-related genes bcl-2, bax, or 

p53 is involved in the selective preservation of CA1 neurons by RU486. Furthermore, MR 

blockade with SPIRO did not exacerbate CCI-induced neuron loss in hippocampus. 

Hippocampal neuron loss 24 hours after controlled cortical impact injury 

The present study uses the optical fractionator method of cell counting, a procedure that 

yields an unbiased estimation of total cell number, to evaluate survival of hippocampal neurons 

24h after CCI injury (Figure 4.1). Counts revealed that CCI significantly decreased the number 

of ipsilateral CA1 pyramidal neurons in vehicle and SPIRO-pretreated rats by approximately 

35%. In contrast, no significant cell loss occurred in RU486-pretreated animals, demonstrating 

protection of CA1 neurons by RU486. Approximately 40-55% of CA3 neurons were lost at 24h. 

We have previously reported a similar amount of cell loss in CA3 after the same level of injury 

using the optical dissector method of cell counting. The same report demonstrated that no 

significant cell loss occurs in CA3 beyond 24 hours post-injury, indicating that neuronal cell 

death in this region is rapid following TBI (Baldwin et al., 1997). In the current study, CCI also 

reduced the number of granule cells in DG by approximately 20-30%. While injury did not 

significantly decrease the number of DG neurons in the vehicle-pretreated group, post-hoc 

comparisons indicate that the amount of cell loss in vehicle-pretreated subjects was not 

statistically different from that observed in SPIRO and RU486-pretreated subjects. The 

appearance of the data suggests that the actual extent of cell loss in vehicle-pretreated animals 

may have been obscured by high variability in the number of contralateral cells counted in this 

region. 

RU486 protection is specific to CA1 

The selective defense of CA1 neurons suggests either that RU486 preferentially protects this 

neuronal population or that CCI injury in CA1 is more responsive to treatment. Differential 

action of RU486 might be achieved through the heterogeneous distribution of GR across 

subfields (Herman, 1993). The high density of GR expressed in CA1 relative to other 

68

hippocampal regions might explain, in part, the efficacy of RU486. Relatively little GR is 

expressed in CA3, perhaps lessening the impact of RU486 on this subregion. However, GR 

levels are substantial in DG, a region not protected by RU486, suggesting that additional factors 

are involved in determining neuronal susceptibility to injury. 

Previous studies predict that CCI may affect neurons differently according to subregion, 

allowing for selective intervention. Subpopulations of hippocampal neurons are selectively 

vulnerable to different forms of insult; CA1 neurons are most severely damaged by ischemia 

(Kirino, 1982; Pulsinelli et al., 1982; Schmidt-Kastner and Freund, 1991), while CA3 neurons 

are most vulnerable, and DG granule cells least vulnerable, to kainate excitotoxicity (Nadler et 

al., 1978; Kohler and Schwarcz, 1983). Ischemia (Okiyama et al., 1994) and excitotoxicity 

(Choi, 1992; Hayes et al., 1992) are among the many components of TBI which can lead to 

neuronal death. Differential vulnerability in hippocampal neuron populations could therefore 

govern the severity of CCI injury or the length of time over which cell death occurs, allowing for 

more successful treatment in specific regions. CCI results in very rapid cell loss in CA3 

(Baldwin et al., 1997). Death of CA1 neurons as induced by ischemia is slower in comparison, 

occuring over a period of hours to days (Kirino, 1982; Pulsinelli et al., 1982). It is possible that 

CCI injury is either delayed or less severe in CA1 as compared to CA3, such that intervention 

with RU486 is able to increase or prolong neuronal survival. Clarification of this issue will 

require further elucidation of the mechanisms governing CCI-induced cell death and a time 

course characterization of CA1 neuron survival after CCI. 

A variety of factors could account for differential vulnerability to TBI by region. For 

example, CA3 neurons do not express neuronal calcium-binding proteins calbindin-D 28k or 

parvalbumin (Sloviter, 1989), which may reduce CA3 Ca 2+ buffering capacity as compared to 

CA1. In addition, ionotropic glutamate receptors are heterogeneously distributed in 

hippocampus; NMDA and AMPA subunits are preferentially expressed in CA1 and DG, while 

kainate receptors are mainly found in CA3 (Greenamyre et al., 1985; Tarazi et al., 1996). 

Glutamate receptor configuration may dictate sensitivity to the large amounts of glutamate 

released during injury. Subregional differences in afferent input, intracellular signaling, control 

of ion homeostasis, antioxidant defenses, and neurotrophic factor expression are among other 

factors that could also differentially influence cell viability. 

69

Glucocorticoid receptor blockade and neuronal viability 

GR-activating concentrations of glucocorticoids exacerbate many types of neuronal insult 

including kainic acid toxicity, oxidative stress, amyloid-beta peptide toxicity, and ischemia 

(Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; Sapolsky, 1986a; Sapolsky et al., 1988; 

Goodman et al., 1996; McIntosh and Sapolsky, 1996a). The GR may heighten neuronal 

sensitivity to insult through several mechanisms. Glucocorticoids can inhibit glucose transport 

(Horner et al., 1990) and may increase neuronal vulnerability to insult through energy depletion 

(Sapolsky, 1986b). Elevated glucocorticoid concentrations can also result in excitatory amino 

acid accumulation (Stein-Behrens et al., 1992; Stein-Behrens et al., 1994b), increased 

intracellular Ca 2+ concentrations (Kerr et al., 1992; Elliott and Sapolsky, 1993; Karst et al., 

1994), and free radical formation (McIntosh and Sapolsky, 1996a, b), all of which may 

compromise cells by triggering Ca 2+ -dependent proteolysis, lipid peroxidation, oxidative stress, 

and mitochondrial dysfunction. 

Inactivation of GR by glucocorticoid removal or GR blockade has been shown to improve 

survival of rodent hippocampal neurons following several types of neurotoxic challenge. 

Metyrapone, an inhibitor of adrenal glucocorticoid synthesis, reduces loss of CA1 neurons after 

ischemia (Smith-Swintosky et al., 1996) and loss of CA1 and CA3 neurons after insult with 

kainic acid, an excitotoxic glutamate analog (Stein and Sapolsky, 1988; Smith-Swintosky et al., 

1996). RU486 reverses glucocorticoid-mediated vulnerability to glutamate in a cultured murine 

hippocampal cell line (Behl et al., 1997b). Further, RU486 (Antonawich et al., 1999) and 

removal of endogenous glucocorticoids by adrenalectomy (Sapolsky and Pulsinelli, 1985; Morse 

and Davis, 1990) attenuate loss of CA1 neurons following ischemia. However, studies have 

indicated that while adrenalectomy and RU486 improve CA1 viability in gerbil at 4 days 

following ischemia, the protection is lost at 7 days (Morse and Davis, 1990; Antonawich et al., 

1999). Thus it is possible that RU486 protection of CA1 neurons after CCI is a transient effect. 

Additional time points will be required in order to determine if RU486 completely prevents CA1 

cell loss or merely delays neuronal loss after injury. 

Metyrapone and adrenalectomy studies suggest that RU486 may exert its neuroprotective 

effects by blocking the deleterious effects of GR. RU486 is a potent inhibitor of GR activation 

that both impedes and competes with glucocorticoid binding to the GR. Complexes of RU486 

bound to the GR can bind glucocorticoid response elements found in target gene promoter 

70

egions but do not facilitate transcription, c.f.(Cadepond et al., 1997). While RU486 generally 

acts as an anti-glucocorticoid, it may also act as a GR agonist under certain circumstances 

(Nordeen et al., 1995). It is therefore possible that RU486 influences GR activation in an 

unpredicted manner after CCI. However, we have consistently observed in the current subjects 

(McCullers, 2001a) (Chapter Five) and in a previous study (McCullers and Herman, 2001) 

(Chapter Three) that in vivo pretreatment with RU486 increases GR mRNA levels. This effect 

concurs with GR mRNA upregulation after adrenalectomy (Herman et al., 1989a; Reul et al., 

1989) and provides evidence for the functional blockade of GR in this study. In addition to its 

anti-glucocorticoid effects, RU486 is also a potent progesterone receptor antagonist. However, 

modest hippocampal expression of progesterone receptor (Kato et al., 1994) and evidence that 

progesterone is neuroprotective after TBI (Roof and Hall, 2000) suggest that progesterone 

receptor blockade is unlikely to underlie RU486 protection of CA1 neurons after CCI. 

Potential mechanisms of RU486 neuroprotection 

Subfield-specific modulation of Ca 2+ homeostasis may influence neuronal viability after TBI. 

Studies of CA1 neurons in hippocampal slices demonstrate enhanced afterhyperpolarization 

(Joels and de Kloet, 1989; Kerr et al., 1989), Ca 2+ action potentials (Kerr et al., 1989), and Ca 2+ 

currents (Kerr et al., 1992; Karst et al., 1994) with increased GR activation. These reports 

indicate that GR influences Ca 2+ influx in CA1 neurons and suggest that excessive GR activation 

could lead to Ca 2+ neurotoxicity. In agreement with this hypothesis, prolonged exposure to high 

glucocorticoid concentrations increases basal intracellular Ca 2+ levels (Elliott and Sapolsky, 

1993) and prolongs intracellular Ca 2+ elevation after kainic acid treatment (Elliott and Sapolsky, 

1992) in hippocampal neuron cultures. Characterizations of Ca 2+ channel subunit mRNA 

expression at low and high glucocorticoid levels are also consistent with the hypothesis that 

predominant MR occupation leads to restricted Ca 2+ influx, while additional GR occupation 

enhances Ca 2+ influx (Nair et al., 1998). In the present study, RU486 blockade of GR should 

result in predominant MR activation. RU486 may therefore shift expression of Ca 2+ channel 

subunits and other Ca 2+ regulatory genes to a neuroprotective configuration that could prevent 

excessive intracellular Ca 2+ accumulation in CA1 neurons. 

RU486 could also improve CA1 neuronal viability via non-genomic action. RU486 is 

reported to have antioxidant effects that may mediate neuroprotection in CA1 pyramidal cells. 

71

Previous studies have shown that RU486 prevents oxidation of the low-density lipoprotein 

(Parthasarathy et al., 1994) and inhibits oxidative modification of proteins by pre-existing lipid 

peroxides in cell-free systems (Carpenter et al., 1996). Furthermore, Behl, et al. (1997a) have 

demonstrated neuroprotective antioxidant effects of RU486 in cellular and organotypic slice 

cultures. Pretreatment with 10 -5 M RU486 protects rat primary hippocampal cultures from beta 

amyloid toxicity, mouse clonal hippocampal HT22 cells from beta amyloid, H 2 O 2 , and glutamate 

insults, and organotypic rat hippocampal slices from H 2 O 2 -induced cell death (Behl et al., 

1997a). Intracellular peroxide accumulation and DNA degradation were also prevented by 

RU486 in HT22 cells. Experiments in monkey kidney CV 1 cells suggest that the protective 

effects of RU486 are independent of GR or PR antagonism or activation (Behl et al., 1997a). 

Structural similarity to other antioxidant compounds suggests that the dimethylaminophenyl side 

chain moiety of RU486 is likely responsible for the antioxidant properties of RU486 

(Parthasarathy et al., 1994; Carpenter et al., 1996; Behl et al., 1997a). 

Expression of apoptosis-related genes after corticosteroid receptor blockade and injury 

The present data demonstrate decreased hippocampal bcl-2 mRNA levels in SPIRO and 

RU486-pretreated rats 24h after sham-operation, as compared to vehicle-pretreated subjects 

(Figure 4.3). These results extend our previous finding that SPIRO decreases bcl-2 mRNA 

levels in hippocampus (McCullers and Herman, 1998) (Chapter Two). This evidence for 

reduction of bcl-2 mRNA levels by MR and GR antagonists suggests that both receptor types, 

perhaps functioning as heterodimers (Trapp et al., 1994), are required for optimal maintenance of 

bcl-2 expression. However, the antagonist-induced decreases in bcl-2 mRNA levels observed in 

sham animals do not appear to predict increased hippocampal neuron vulnerability to injury. 

Injury-induced cell loss is not increased in antagonist-injected subjects in any region (as 

compared to vehicle-injected controls); on the contrary, RU486 treated subjects clearly show 

protection despite decreased bcl-2 mRNA levels observed in sham animals. These data fail to 

support the hypothesis that decreased bcl-2 expression subsequent to ACR blockade will increase 

neuronal vulnerability to CCI. It should be noted, however, that the effects of ACR blockade on 

bcl-2 mRNA levels are very different in sham versus injured animals. Among injured animals, 

ACR blockade did not reduce bcl-2 mRNA levels (as compared to vehicle-injected controls) 24 

hours post-injury in any hippocampal region. Thus, CCI injury abrogates the differential 

72

decrease of bcl-2 mRNA levels in antagonist-pretreated groups, perhaps accounting for the lack 

of correlation between sham bcl-2 levels and injury induced cell loss. Nonetheless, it is possible 

that a shorter period of antagonist pretreatment may be sufficient for RU486 protection of CA1 

neurons while avoiding the potential undesired effect of bcl-2 mRNA downregulation. 

The present study demonstrates decreased ipsilateral bcl-2 mRNA levels in DG of vehiclepretreated 

subjects following CCI injury. Decreased bcl-2 expression has also been 

demonstrated in fluid percussion (Dong et al., 2001) and in vitro (Morrison et al., 2000) models 

of TBI. In contrast, Clark et al. have reported increases in hippocampal bcl-2 mRNA levels 24h 

following CCI with the addition of 30 minutes moderate hypoxemia following injury (Clark et 

al., 1997). Increased bcl-2 mRNA levels appear to be associated with neuroprotection after CCI 

with hypoxia (Clark et al., 1997), and Bcl-2 overexpression in transgenic mice decreases 

hippocampal tissue loss after CCI (Nakamura et al., 1999). In addition, decreased bcl-2 mRNA 

levels have been associated with increased kainate-induced hippocampal cell loss (Gillardon et 

al., 1995; McCullers and Herman, 2001) (Chapter Three). However, decreased bcl-2 mRNA 

levels observed in DG of injured animals in the current study were not associated with 

significant neuronal loss following CCI injury. As previously noted, a high degree of variability 

in contralateral DG cell counts may have obscured significant effects of CCI on DG neuron loss. 

Even so, the process of secondary neuronal degeneration is very complex and involves numerous 

factors, and decreased bcl-2 expression, though perhaps an endangering factor, need not dictate 

neuronal loss following injury. 

Hippocampal bax mRNA levels were unchanged by MR/GR antagonist pretreatments or CCI 

(Figure 4.4). These findings agree with results obtained using an in vitro model of TBI 

(Morrison et al., 2000). The ratio of anti-apoptotic proteins such as Bcl-2 to pro-apoptotic 

proteins such as Bax is believed to determine cell sensitivity to apoptotic signals (Oltvai et al., 

1993). The current data suggest that transcriptional regulation of this ratio would be 

accomplished by modulation of bcl-2, and not bax, following traumatic brain injury. 

Levels of p53 mRNA in the present study were not significantly elevated in vehiclepretreated 

animals at the 24 hour time point; however, our data do not preclude changes earlier in 

time. For instance, another group has demonstrated increases in p53 mRNA levels 6 hours 

following fluid percussion brain injury which return to sham levels by 24 hours (Napieralski et 

al., 1999). The current report also demonstrates that injury reduced contralateral p53 signal in 

73

CA1 and DG of SPIRO animals (Figure 4.5). We have previously shown that SPIRO attenuates 

kainic acid-induced increases in p53 mRNA levels (McCullers and Herman, 1998) (Chapter 

Two), but the effects of p53 downregulation on neuronal survival are presently unclear. 

Hippocampal cell death is associated with functional (Lowenstein et al., 1992) and 

neurobehavioral (Hicks et al., 1993; Smith et al., 1994) deficits following TBI. By preventing 

CA1 neuron loss after CCI, RU486 could potentially improve behavioral outcome after injury. 

RU486 may be particularly effective in promoting neuron survival due to its combined antiglucocorticoid 

effects and antioxidant properties. While glucocorticoids have been used 

clinically to control edema after TBI, the value of this treatment is unclear. Some evidence 

indicates that glucocorticoids can improve recovery after central nervous system injury (Hall et 

al., 1992), but many studies show no improvement with glucocorticoid administration (Tornheim 

and McLaurin, 1978; Cooper et al., 1979; Giannotta et al., 1984; Brain, 1996). In contrast, many 

studies have demonstrated exacerbation of neuronal insult by glucocorticoids (Sapolsky, 1985; 

Sapolsky and Pulsinelli, 1985; Sapolsky, 1986a; Sapolsky et al., 1988; Goodman et al., 1996; 

McIntosh and Sapolsky, 1996a) and improved neuronal viability with antiglucocorticoid 

treatments (Sapolsky and Pulsinelli, 1985; Stein and Sapolsky, 1988; Morse and Davis, 1990; 

Smith-Swintosky et al., 1996; Behl et al., 1997b; Antonawich et al., 1999). Considering the 

benefits of glucocorticoid removal and blockade in other models of neuronal injury, the 

antioxidant properties of RU486, and the current data demonstrating CA1 neuron protection, 

RU486 appears to have potential as a useful treatment for head injury. 

74

Chapter Five: Traumatic brain injury regulates adrenocorticosteroid 

receptor mRNA levels in rat hippocampus 

Introduction 

The mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), collectively 

referred to as adrenocorticosteroid receptors (ACRs), are ligand-activated transcription factors 

that mediate glucocorticoid action in brain. The MR binds glucocorticoids with high affinity (kD 

~ 0.5-1 nM) and is extensively occupied at basal glucocorticoid levels (Reul and de Kloet, 1985). 

The GR binds glucocorticoids with a five-fold lower affinity, resulting in greater GR activation 

with increasing glucocorticoid levels (i.e. during stress and at the peak of circadian hormone 

secretion)(Reul and de Kloet, 1985; Spencer et al., 1990). This two-receptor system allows for 

differential regulation of MR and GR-coordinated gene expression in response to changes in 

circulating glucocorticoid concentration. The binding of glucocorticoid ligand allows receptor 

translocation to the cell nucleus, where the MR and GR can regulate transcription either by direct 

DNA binding to hormone response elements in gene promoter regions or by protein-protein 

interactions with other transcription factors [see (Yamamoto, 1985; De Kloet et al., 1998)]. 

Considerable evidence suggests that excessive GR activation by CORT increases 

hippocampal vulnerability to several forms of neuronal insult including excitotoxicity, oxidative 

stress, and ischemia (Sapolsky, 1985; Sapolsky and Pulsinelli, 1985; Sapolsky, 1986a; Sapolsky 

et al., 1988; Goodman et al., 1996; McIntosh and Sapolsky, 1996a). Furthermore, blockade of 

GR reduces hippocampal neuron vulnerability to traumatic brain injury (TBI) (McCullers et al., 

2001b) (Chapter Four), indicating the potential deleterious effects of GR activation with this type 

of injury. The degree of GR activation experienced by cells is largely determined by circulating 

glucocorticoid concentration and the amount of available GR; therefore, regulation of GR 

expression and the magnitude of HPA response are crucial determinants of glucocorticoid 

influence on neuronal viability after injury. 

To examine the respective roles of MR and GR in regulating ACR expression and 

glucocorticoid secretion following TBI, adult male Sprague Dawley rats were pretreated with 

vehicle, the MR antagonist spironolactone (SPIRO), or the GR antagonist mifepristone (RU486). 

Subjects were subsequently sham-operated or injured using the controlled cortical impact (CCI) 

75

model of TBI. Twenty-four hours post-injury, ACR mRNA levels were measured to determine 

if glucocorticoid signaling is altered through regulation of ACR expression following injury. As 

the acute hypothalamic-pituitary-adrenal (HPA) response to CCI has not been characterized, 

additional studies were performed to assess CORT and ACTH secretion over the 24-hour period 

following injury. 


Subjects 

Young adult male Sprague Dawley rats (Harlan Sprague Dawley, Inc., Indianapolis, IN) 

weighing 200-250g were maintained in an environment with constant temperature, humidity, ad 

libitum access to food and water, and a 12-hour light/dark cycle, with lights on between 600h- 

1800h. All measures were taken to minimize animal pain or discomfort. All animal procedures 

were performed in accordance with the National Institutes of Health guidelines and approved by 

the University of Kentucky Institutional Animal Care and Use Committee. 


Subjects (n = 5-7 per design group) received subcutaneous injections of propylene glycol 

vehicle, MR antagonist spironolactone (SPIRO) (50 mg/kg) (Herman and Spencer, 1998) 

(Sigma, St. Louis, MO), or GR antagonist RU486 (25 mg/kg) (Ratka et al., 1989) (Sigma) in 

morning (900h) and afternoon (1600h) for two days. On day three, animals were administered a 

final pretreatment injection at 900h and transported to a surgical procedure room. Between 

1100h-1200h, animals were subjected to craniotomy (sham operation) or a moderate unilateral 

cortical contusion, described below. Subjects were killed on day 4 at 1200h, 24h after surgery. 

A separate set of animals (n = 6 per group) was used for analysis of plasma ACTH and 

CORT levels during the 24 hours following surgery. Baseline blood samples were drawn by tail 

nick between 900-1000 hours, after which animals were transported to a surgical procedure 

room. Subjects then underwent sham operation or moderate unilateral cortical contusion 

between 1200-1300 hours. Sham and CCI surgeries were performed under identical conditions 

on separate days. Blood samples were collected at 1.5, 6, 12, 18, and 24 hours after the start of 

surgery. All samples were collected by tail nick, with the exception of the 24-hour point, which 

76

was obtained by collection of trunk blood at the time of sacrifice. Blood sampling was initiated 

1.5 hours after the start of surgery to allow all subjects to wake from anesthesia, and six hour 

intervals were observed thereafter to assess circadian hormone levels at times including lights off 

(1800 h) and lights on (600h). 

Surgical procedures were performed as previously described (Baldwin et al., 1997). Briefly, 

animals were anesthetized with isoflurane (2%) and placed in a stereotaxic frame (Kopf Instr., 

Tujunga, CA). Sterile procedures were used to retract the skin and perform a 6mm unilateral 

craniotomy midway between bregma and lambda. The underlying dura was not disrupted during 

removal of the portion of skull. A pneumatically controlled impacting device with a 5mm 

beveled tip was used to injure the exposed brain, compressing the cortex at 3.5m/sec to a depth 

of 2mm. Following injury, Surgicel (Johnson and Johnson, Arlington, TX) was laid upon the 

dura, and the skull cap was replaced. After a thin coat of dental acrylic was spread over the 

craniotomy site and allowed to dry, the wound was stapled closed. Body temperature was 

monitored continuously throughout the surgical/recovery procedures and maintained at 37 o C 

with a heating pad. 

Animals processed for these experiments were killed by decapitation. Brains were rapidly 

removed, frozen in isopentane cooled to –40 o C on dry ice, then stored at –80 o C until sectioned. 

Trunk blood samples were collected on ice, and plasma was stored at –20 o C. Frozen brains were 

removed from a –80 o C freezer, warmed to –20 o C, and sectioned to 16 um with a Bright-Hacker 

cryostat. Sections were then thaw-mounted onto Ultrastick Slides (Becton Dickinson, Franklin 

Lakes, NJ) and stored at –20 o C until processing for in situ hybridization. 


Antisense riboprobes complementary to coding and 3’ untranslated regions for rat MR 

mRNA (550 bp, courtesy of P. Patel and S. Watson, Univ. Michigan) and rat GR mRNA (456 

bp, courtesy of K. Yamamoto, UCSF) were labeled according to previously published methods 

(Seroogy and Herman, 1996) by standard in vitro transcription using [ 35 S]UTP. Slide-mounted 

tissue sections were removed from a -20 o C freezer and fixed in 4% paraformaldehyde for 10 

min. Slides were then rinsed in 5 mM KPBS, 5 mM KPBS with 0.2% glycine, 5mM KPBS, 0.1 

M triethanolamine with 0.25% acetic anhydride, and 0.2X SSC, then dehydrated in graded 

alcohols. Labeled probes were diluted in hybridization buffer containing 50% formamide, and 

77

50 ul of diluted probe (containing 1x 10 6 c.p.m.) was applied to each slide. Slides were then 


hybridization, slides were rinsed in 2X SSC and incubated in 0.2X SSC at 60 o C for 1 hr. After 

dehydration in graded alcohols, slides were exposed for 14-18 days to Kodak BioMAX xray 

film. 

Densitometric analysis was performed using NIH Image 1.62 software for Macintosh. A 

standard curve was generated using 14 C standards prior to image capture to ensure signal 

linearity. Based on morphological criteria, regions CA1, CA3, and DG of the hippocampal 

formation were manually sampled from the ipsilateral (operated) and contralateral (unoperated) 

hemispheres. The mean gray level of an unhybridized area (background) from each hemisphere 

was subtracted from sampled regions to compute corrected gray level. Ipsilateral mRNA data 

were normalized as a percent of control (sham/vehicle) values from the ipsilateral hemisphere, 

and contralateral mRNA data were normalized as a percent of control (sham/vehicle) values 

from the contralateral hemisphere, with sham/vehicle levels expressed as one hundred percent. 

Each region of each hemisphere was then analyzed by two-way analysis of variance with drug 

and injury designated as independent variables. Post hoc comparisons were made using the 

Student-Newman-Keuls test, with significance set at p ≤ 0.05. 

Radioimmunoassay 

Plasma ACTH and corticosterone concentrations were determined using ( 125 I-labeled) rat 

ACTH (Diasorin, Stillwater, MN, USA) and corticosterone (ICN Biochemicals, Costa Mesa, 

CA) kits per manufacturers’ instructions. The limits of detection for each kit are 15 pg/ml and 8 

ng/ml, respectively. 

A skewed distribution of CORT levels observed in subjects pretreated with antagonist drugs 

was normalized by log transformation of the data prior to analysis for significant main effects by 

two-way analysis of variance. Post hoc comparisons were made using the Student-Newman- 

Keuls test, with significance set at p ≤ 0.05. Data presented in Table 5.1 are actual CORT values 

listed as mean concentration ± standard error. Time course values for ACTH and CORT were 

analyzed for significant main effects using a repeated measures analysis of variance. Post hoc 

comparisons were made using the Student-Newman-Keuls test, with significance set at p ≤ 0.05. 

Data graphed in Figures 5.1 and 5.2 represent mean hormone concentration ± standard error. 

78

Results 

Effects of ACR blockade and CCI on plasma ACTH and corticosterone levels 

ACTH and CORT levels were measured 24 hours following surgery to assess the effects of 

ACR blockade and CCI on HPA activation. Two-way ANOVA demonstrates a significant 

drug/injury interaction effect on plasma CORT levels at 24h [F(2,29) = 8.02; p ≤ 0.05] (Table 

5.1). Post hoc comparisons indicate that SPIRO significantly increased CORT secretion in 

sham-operated animals. Controlled cortical impact significantly decreased CORT levels in 

SPIRO and RU486-pretreated animals as compared to sham-operated animals within the same 

drug pretreatment groups. RU486 animals also had significantly lower CORT levels after injury 

as compared to vehicle/injured animals. 

Table 5.1. Effects of adrenocorticosteroid receptor blockade and controlled cortical impact on 

plasma corticosterone levels (ng/ml) 

Group Treatment n Mean CORT Level 

VEHICLE SHAM 7 80.5 ± 29.9 

SPIRO SHAM 6 324.5 ± 77.2 a 

RU486 SHAM 6 121.0 ± 16.2 

VEHICLE INJURED 5 81.7 ± 33.6 

SPIRO INJURED 6 51.9 ± 26.0 b 

RU486 INJURED 5 14.7 ± 6.8 a,b 


RU486 = GR antagonist (mifepristone) 

a p < 0.05 compared to vehicle-pretreated subjects in same surgery group (effect of antagonist) 

b p < 0.05 compared to sham-operated subjects in same pretreatment group (effect of injury) 

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Plasma ACTH and corticosterone levels: time course after injury 

Blood samples taken 24 hours after surgery suggest the dynamic regulation of CORT 

secretion over the post-injury period. To examine the kinetics of HPA activation following CCI, 

additional animals were subjected to sham operation or injury, after which sequential blood 

samples were drawn by tail nick over the 24-hour post-surgery period. Plasma ACTH levels 

exhibited a significant injury/time interaction as determined by repeated measures analysis of 

variance [F(5,41) = 7.89; p ≤ 0.05] (Figure 5.1). Plasma ACTH concentrations were increased to 

approximately 200 pg/ml at 1.5 hours and at 18 hours post-surgery in injured animals. ACTH 

concentrations in sham-operated animals were indistinguishable from baseline at all time points. 

Plasma CORT concentrations also demonstrate a significant injury/time interaction as 

indicated by repeated measures analysis of variance [F(5,50) = 5.29; p ≤ 0.05] (Figure 5.2). 

Corticosterone levels are significantly increased in injured rats at 1.5 hours (109 ± 12 ng/ml) and 

at 6 hours (53 ± 4 ng/ml) following surgery, but returned to baseline by 12 hours and remained 

unchanged for the rest of the 24-hour period. Corticosterone concentrations in sham-operated 

animals were indistinguishable from baseline at all time points. 

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Figure 5.1. Twenty-four hour time course analysis of plasma ACTH levels (pg/ml) following 

controlled cortical impact. Injury increased ACTH levels at 1.5 and 18 hours after surgery (p ≤ 

0.05). ACTH levels in sham-operated animals were indistinguishable from baseline at all time 

points. 

Figure 5.2. Twenty-four hour time course analysis of plasma corticosterone levels (ng/ml) 

following controlled cortical impact. Injury increased corticosterone secretion at 1.5 and 6 hours 

after surgery (p ≤ 0.05); levels returned to baseline by 12 hours after injury. Corticosterone 

levels in sham-operated animals were indistinguishable from baseline at all time points. 

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Effects of ACR blockade and CCI on ACR mRNA levels 

Relative changes in mRNA levels were measured by film densitometry following in situ 

hybridization with specific complementary antisense riboprobes (see Methods) (Figure 5.3). 

Figure 5.3. Photomicrographs illustrating the effects of sham operation (A,C) and controlled 

cortical impact (B,D) on MR (A,B) and GR (C,D) mRNA levels in hippocampus of vehiclepretreated 

rats. Panels A,B: Controlled cortical impact has no significant effects on MR mRNA 

levels in vehicle-pretreated animals. Panels C,D: Controlled cortical impact decreases ipsilateral 

GR mRNA levels in dentate gyrus and increases contralateral GR mRNA levels in CA1 and DG 

of vehicle-pretreated subjects. 

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In the hemisphere contralateral to injury, two-way ANOVA demonstrates significant main 

effects of injury on MR mRNA levels in CA1 [F(1,28) = 13.10; p ≤ 0.05], CA3 [F(1,28) = 32.39; 

p ≤ 0.05], and DG [F(1,28) = 4.31; p ≤ 0.05] (Figure 5.4). Post hoc comparisons indicate that 

injury induces significant contralateral increases in MR mRNA levels in CA1 of SPIROpretreated 

animals and in CA3 of SPIRO and RU486 animals; comparisons in DG did not reach 

statistical significance. No significant effects of antagonist pretreatment were observed in 

contralateral MR mRNA levels. 

In the injured hemisphere, two-way ANOVA demonstrates significant main effects of injury 

on MR mRNA levels in CA1 [F(1,28) = 6.55; p ≤ 0.05] and CA3 [F(1,28) = 42.18; p ≤ 0.05] 

(Figure 5.4). Post hoc comparisons indicate that cortical impact significantly increased 

ipsilateral MR mRNA levels in CA1 of RU486-pretreated animals and in CA3 of SPIRO and 

RU486 animals. A significant main effect of drug on MR mRNA levels is observed in ipsilateral 

CA1 [F(2,28) = 3.68; p ≤ 0.05], but post hoc comparisons did not distinguish between individual 

pretreatment groups. 

Contralateral GR mRNA levels exhibited significant drug/injury interactions in CA1 [F(2,28) 

= 7.75; p ≤ 0.05] and DG [F(2,28) = 5.70; p ≤ 0.05] (Figure 5.5). In sham-operated animals, 

SPIRO and RU486 increased contralateral GR signal in CA1 and DG. Cortical injury increased 

contralateral GR mRNA levels in CA1 and DG of vehicle-pretreated animals and decreased 

contralateral GR mRNA levels in CA1 of RU486 animals. 

Main effects of drug/injury interaction were observed in ipsilateral GR mRNA levels in 

subregions CA1 [F(2,28) = 6.53; p ≤ 0.05], CA3 [F(2,28) = 3.71; p ≤ 0.05], and DG [F(2,28) = 

4.29; p ≤ 0.05] (Figure 5.5). RU486 significantly increased ipsilateral GR signal in CA1, CA3, 

and DG of sham-operated animals, and SPIRO increased ipsilateral GR signal in CA1. Cortical 

impact significantly decreased ipsilateral GR mRNA levels in CA1 and CA3 of SPIRO and 

RU486-pretreated animals and in DG of all antagonist pretreatment groups. 

83

Figure 5.4. Effects of MR blockade (SPIRO), GR blockade (RU486), and controlled cortical 

impact (CCI) injury on MR mRNA levels in hippocampus. Semi-quantitative analysis expressed 

as percentage of control (sham/vehicle) corrected gray level. Injury and antagonist pretreatments 

interact to increase contralateral and ipsilateral MR mRNA levels in CA1 and CA3. [ B p ≤ 0.05 

as compared to sham-operated animals within same pretreatment group (effect of CCI).] 

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Figure 5.5. Effects of MR blockade, GR blockade, and controlled cortical impact (CCI) injury 

on GR mRNA levels in hippocampus. Semi-quantitative analysis expressed as percentage of 

control (sham/vehicle) corrected gray level. Antagonist pretreatment increases GR mRNA levels 

in sham-operated animals, but antagonist-induced increases in GR mRNA levels are prevented 

by injury. Contralateral GR mRNA levels are increased in CA1 and DG of vehicle-pretreated 

animals, and ipsilateral GR mRNA levels are decreased in DG of all pretreatment groups after 

injury. [ A p ≤ 0.05 as compared to vehicle-pretreated animals within same surgical treatment 

(effect of antagonist pretreatment). B p ≤ 0.05 as compared to sham-operated animals within 

same pretreatment group (effect of CCI).] 

85

Discussion 

CCI modulates HPA activation 

The extent of HPA activation after TBI may influence neuronal survival, given evidence for 

glucocorticoid exacerbation of several forms of neuronal injury (Sapolsky, 1985; Sapolsky and 

Pulsinelli, 1985; Sapolsky, 1986a; Sapolsky et al., 1988; Goodman et al., 1996; McIntosh and 

Sapolsky, 1996a). In the first experiment of this study, vehicle-pretreated animals demonstrated 

equivalent plasma CORT levels 24 hours after sham operation and injury. The levels were 

slightly higher than would be expected in unoperated rats but were not of a magnitude to saturate 

GR binding at the 24-hour time point. Another study assessing ACTH and CORT levels in rats 

following closed head injury reported similar results at 24 hours (Shohami et al., 1995). 

Clinical studies have reported increased HPA activation in humans after head injury (King et 

al., 1970; Pentelenyi and Kammerer, 1984). To determine if differential activation of the HPA 

axis occurs during the first 24 hours after experimental CCI, a time course analysis of plasma 

CORT and ACTH levels was performed in sham-operated versus injured rats. Blood sampling 

over the 24-hour period following surgeries confirmed the differential activation of the HPA axis 

in animals injured by CCI. Increased levels of ACTH and CORT were observed 1.5 hours 

following injury, after which a prolonged, low level of CORT secretion was observed for at least 

6 hours after injury. 

Plasma ACTH and CORT levels six hours after surgery (time of day = 1800h) indicate a 

disruption of normal circadian rhythms. Diurnal peaks in rodent ACTH and CORT secretion 

normally occur near the onset of darkness (Ruhmann-Wennhold and Nelson, 1977; D'Agostino 

et al., 1982; Kwak et al., 1992). ACTH levels in CCI-injured animals were indistinguishable 

from baseline at 1800h, the time of lights-off, and plasma CORT concentrations, although 

slightly elevated at 1800h, did not approach typical peak levels of 150-200 ng/ml (D'Agostino et 

al., 1982; Kwak et al., 1992). Sham-operated animals also lacked ACTH and CORT rhythms, 

exhibiting unchanged plasma hormone levels across the 24-hour period after surgery. Alteration 

of circadian rhythms has been reported after surgery in rats (Levine et al., 1980) and in humans 

(King et al., 1970; McIntosh et al., 1981; Pentelenyi and Kammerer, 1984), but the exact cause 

for disruption in these subjects, whether by anesthesia or the trauma of craniotomy, remains to be 

determined. 

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Injured animals demonstrated an unusual peak in ACTH levels 18h after the onset of surgery 

(time of day = 600h). While the significance of this increase is unclear, we speculate that 

increased ACTH levels could result from decreased sensitivity to glucocorticoid-mediated 

feedback. Decreased sensitivity to feedback normally occurs at the peak of the circadian rhythm 

and after stress (Dallman, 1992) and could be precipitated by the stress of injury or alteration of 

circadian rhythms. 

ACR antagonists modulate plasma corticosterone levels 

Blockade of MR-mediated feedback inhibition of the HPA axis may account for increased 

plasma CORT levels observed in SPIRO/sham subjects (Table 5.1). Our laboratory has 

previously demonstrated increased basal CORT secretion with SPIRO administration (McCullers 

and Herman, 2001) (Chapter Three), although some studies report no effect of SPIRO on 

morning CORT levels (Chabert et al., 1984; Herman and Spencer, 1998; Yau et al., 1999). 

Activation of MR is required to maintain CORT secretion at the low basal levels normally 

observed in rats in the morning (Spencer et al., 1998). Furthermore, studies using the MR 

antagonist RU28318 have consistently demonstrated that MR blockade increases adrenocortical 

secretion (Ratka et al., 1989; van Haarst et al., 1997; Spencer et al., 1998). The mechanism 

whereby CCI interacts with ACR antagonists to reduce CORT secretion is presently unknown. 

The inhibitory influence of the hippocampus over HPA activation (Herman et al., 1989b; 

Jacobson and Sapolsky, 1991) invites speculation that this structure may be involved. It is 

possible that aberrant hippocampal activation subsequent to injury in combination with altered 

ACR signaling might culminate in decreased HPA secretion, but support for this hypothesis will 

require further study. 

ACR antagonists regulate GR mRNA levels 

SPIRO pretreatment did not alter MR mRNA levels in sham-operated animals (Figure 5.4). 

Previously we have observed that SPIRO pretreatment (followed by i.p. saline injection) 

increases MR mRNA levels by approximately 30-40% in CA1, CA3, and DG (McCullers and 

Herman, 2001) (Chapter Three), and many preceding studies suggest that MR is subject to autoregulation 

(Herman et al., 1989a; Reul et al., 1989; Herman et al., 1993; Chao et al., 1998a). The 

lack of MR mRNA upregulation observed in the SPIRO/sham-operated group may be 

87

attributable to significantly increased CORT secretion in these subjects (Table 5.1). MR mRNA 

levels are negatively regulated by GR activation at high glucocorticoid levels (Herman et al., 

1989a; Herman et al., 1993). Thus, enhanced GR activation due to elevated CORT levels in 

SPIRO/sham animals may attenuate the transcriptional effects of MR blockade. 

Both MR antagonist SPIRO and GR antagonist RU486 increased GR mRNA levels in shamoperated 

animals (Figure 5.5). We have observed similar effects with administration of these 

antagonists in other experiments (Herman and Spencer, 1998; McCullers and Herman, 2001) 

(Chapter Three). These data, together with reports that GR agonist dexamethasone (Herman et 

al., 1989a; Reul et al., 1989), MR agonist aldosterone, or low, MR binding doses of CORT 

(Chao et al., 1998a) each reverse ADX-induced increases in GR mRNA provide compelling 

evidence for regulation of hippocampal GR mRNA expression by both MR and GR. 

CCI increases MR mRNA and decreases GR mRNA levels 

Injury increased MR mRNA levels in CA1 and CA3 of MR and GR antagonist-pretreated 

animals (Figure 5.4). Increased MR mRNA levels in SPIRO-pretreated subjects may be 

attributable to MR auto-regulation (Herman et al., 1989a; Reul et al., 1989; Herman et al., 1993; 

Chao et al., 1998a); however, previous experiments in our laboratory have not indicated MR 

mRNA upregulation with RU486 pretreatment (McCullers and Herman, 2001) (Chapter Three). 

These results suggest that TBI influences ACR-mediated regulation of MR expression. It is 

interesting to note that CORT levels are significantly lower in these groups as compared to 

sham-operated animals (Table 5.1). These data raise the possibility that injury-induced CORT 

secretion could influence MR mRNA levels through auto-regulatory processes. 

Injury did not alter ipsilateral GR mRNA expression in CA1 and CA3 of vehicle-pretreated 

animals (Figure 5.5). However, injury abolished autoregulatory effects of SPIRO and RU486, 

sharply decreasing ipsilateral GR mRNA levels (as compared to sham) by approximately 50- 

80% in antagonist-pretreated subjects. These results indicate that CCI suppresses or interferes 

with GR autoregulatory processes in hippocampus. Our laboratory has reported similar effects 

of excitotoxic insult with kainic acid (McCullers and Herman, 2001) (Chapter Three). 

While it is clear that GR auto-regulation (Herman et al., 1989a; Reul et al., 1989) is disrupted 

after injury, the extent to which GR mRNA levels may be further decreased due to cell loss is 

presently ambiguous. Nonetheless, decreases in GR signal in the ipsilateral DG of all 

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pretreatment groups surpass the magnitude of cell loss experienced in this region (McCullers et 

al., 2001b) (Chapter Four). We have observed similar GR mRNA decreases of approximately 

40-55% in DG of vehicle, SPIRO, and RU486 pretreated animals 24h after excitotoxic insult 

with kainic acid, at a dose which does not cause cell loss in DG (McCullers and Herman, 2001) 

(Chapter Three). Thus, multiple types of neuronal injury result in the significant downregulation 

of GR mRNA levels in DG. 

The extent to which cell loss affects measurement of mRNA levels by in situ hybridization is 

an important consideration that is presently undefined. In parallel subjects, CCI resulted in the 

loss of approximately 40% of CA1 and CA3 pyramidal neurons and 10% of DG granule neurons 

24 hours after injury, while no cell loss was observed in the contralateral hemisphere of injured 

rats or in sham-operated subjects (McCullers et al., 2001b) (Chapter Four). If comparable loss is 

assumed in this study, the lack of demonstrable GR mRNA decrement in injured, vehicle-treated 

groups indicates that the in situ hybridization technique underestimates cell loss. The lack of 

correlation between cell loss and mRNA level is also seen for MR, where levels are significantly 

higher on the injured side. It is possible that GR mRNA is upregulated in CA1 and CA3 to 

compensate for signal that would otherwise be decreased by cell loss; however, the increases in 

MR mRNA argue against this explanation, as greater induction of MR mRNA would have to be 

assumed. In conjunction with the differential effects of antagonist pretreatment, it is likely that 

the observed levels of GR and MR are due to injury-related gene regulation rather than simple 

cell loss. This notion is supported by the large and disproportionate loss of GR mRNA in DG, 

the region showing least cell death following CCI. 

Traumatic brain injury initiates a multitude of cellular and molecular cascades that influence 

gene expression. Although it is difficult to isolate specific factors of the many that are likely to 

influence neuronal GR expression, results from the current study and others (Lowy, 1990, 1992; 

McCullers and Herman, 2001) (Chapter Three) suggest a role for glutamate in the regulation of 

hippocampal GRs. Extracellular glutamate levels increase in hippocampus after CCI injury 

(Faden et al., 1989) and kainic acid injection (Liu et al., 1997), and similar patterns of GR 

mRNA downregulation are observed in hippocampus after both types of injury (McCullers and 

Herman, 2001) (Chapter Three). Thus, excessive glutamate receptor activation may lead to 

interference with antagonist-mediated autoregulatory effects and to the preferential 

downregulation of GR mRNA in DG. Glutamate receptor activation and alterations in calcium 

89

signaling that accompany CCI (McIntosh et al., 1998c) could potentially regulate GR mRNA 

levels via activation of transcription factors (Cabral et al., 2001), expression of neurotrophic 

factors (Sarrieau et al., 1996), or altered neurotransmitter release (Seckl et al., 1990). The results 

of this study also indicate that glucocorticoid secretion is increased immediately following CCI, 

potentially exposing hippocampal neurons to endangering levels of hormone. Considering the 

negative effects of excessive GR activation on injured hippocampal neurons, injury modulation 

of HPA activation and ACR expression may influence hippocampal neuron viability after 

traumatic injury. 

90

Chapter Six: Discussion 

Adrenocorticosteriod receptor regulation of hippocampal neuron viability 

The present thesis examines the respective roles of MR and GR on hippocampal neuron 

vulnerability to injury. To isolate individual receptor effects on neuronal survival, MR and GR 

antagonists were administered in subjects prior to excitotoxic challenge with KA or traumatic 

injury by CCI. Cell counts performed twenty-four hours following insult tested the hypotheses 

that MR blockade would increase and GR blockade would decrease hippocampal neuron 

vulnerability to each form of injury. In addition, expression of bcl-2 and other viability-related 

genes was examined following injury to determine if ACR antagonist effects on viability 

correlate with regulation of the selected genes. Finally, MR and GR mRNAs were measured 

after injury to test the hypothesis that glucocorticoid signaling is altered following injury via 

regulation of hippocampal ACR expression. 

Adrenocorticosteriod receptor regulation of hippocampal neuron survival 

The results of these experiments support the hypotheses that MR protects and GR endangers 

challenged hippocampal neurons. In support of the latter, GR antagonist RU486 prevented the 

loss of CA1 neurons twenty-four hours following CCI injury (Chapter Four). The selective 

protection of CA1 neurons may be due to regional differences in CCI injury or density of GR 

expression (see Chapter Four). The potential for RU486 neuroprotection against KA 

excitotoxicity could not be evaluated due to the absence of measurable KA-induced injury in 

vehicle and RU486-pretreated subjects (Chapter Three). The current work predicts that RU486 

would protect hippocampal neurons subjected to a more severe KA lesion. 

Pretreatment with SPIRO, an MR antagonist, increased vulnerability to KA excitotoxicity in 

hippocampal field CA3 (Chapter Three). While these results may suggest selective MR 

protection of this region, the comparable distribution of MR in CA1 and CA3 suggests that 

differential cell loss is more likely attributable to the selective vulnerability of CA3 neurons to 

KA insult (Nadler et al., 1978). Whether MR blockade increases susceptibility to KA by 

increasing injury severity or by lowering the neuronal threshold for cell death remains unclear. 

MR blockade might exacerbate neuronal injury by disrupting cellular Ca2+ homeostasis. 

91

Adrenalectomy, which prevents endogenous CORT production and subsequent MR activation, 

increases Ca 2+ currents in CA1 neurons, while selective activation of MR results in very small 

Ca 2+ currents (Karst et al., 1994). Predominant MR activation may therefore reduce harmful 

Ca 2+ influx (Karst et al., 1994), whereas MR blockade may contribute to pathologic rises in 

intracellular Ca 2+ . Alternatively, MR blockade may lower the neuronal “set point” for cell death 

by modulating expression of pro- or anti- apoptotic factors, such as members of the bcl-2 family. 

Following TBI, SPIRO did not exacerbate neuronal loss in CA1 and CA3. However, these 

regions suffered substantial (35-40%) injury-induced neuronal loss in vehicle and SPIROpretreated 

subjects. Because CCI alone was sufficient to destroy vulnerable cells, compromising 

effects of MR blockade may not have been evident at this level of injury. 

Adrenocorticosteriod receptor regulation of viability-related gene expression 

The current data indicate potential for ACR influence on neuronal survival via regulation of 

the anti-apoptotic gene bcl-2. MR blockade with SPIRO decreased basal mRNA levels of the 

survival-promoting gene bcl-2 in CA1 (Chapter Two), suggesting that SPIRO would increase 

hippocampal vulnerability to insult in this region. However, subjects receiving KA injection 

following SPIRO pretreatment exhibited no differential cell loss in CA1 (Chapter Three). In 

addition, RU486 decreased bcl-2 levels in CA1 and DG of animals subjected to sham-CCI, yet 

RU486 had a dramatic neuroprotective effect on CA1 neurons after CCI injury (Chapter Four). 

Thus, ACR-induced decreases in bcl-2 mRNA expression observed in uninjured (saline-injected 

or sham-operated) animals did not consistently predict increased vulnerability following injury. 

Experiments in the present thesis do not provide support for bax, p53, BDNF, or NT-3 

involvement in ACR modulation of hippocampal survival. Adrenocorticosteriod regulation of 

hippocampal bax, BDNF, or NT-3 mRNA levels was not observed in any of the current 

experiments (note that BDNF and NT-3 mRNA levels were not assessed in the CCI model). In 

addition, ACR blockade had no significant effects on hippocampal p53 mRNA levels in CCIinjured 

hemispheres (Chapter Four). Kainic acid-induced increases in p53 mRNA levels were 

attenuated by MR blockade in CA3, suggesting the potential for enhanced resistance to apoptosis 

in this region, yet MR blockade increased CA3 vulnerability to KA excitotoxicity (Chapter 

Three). Therefore, ACR-mediated effects on p53 expression do not appear to have significant 

influence on hippocampal neuron survival. 

92

Adrenocorticosteriod receptor regulation of hippocampal neuron survival - potential mechanisms 

Adrenocorticosteriod receptor modulation of calcium homeostasis 

Altered Ca 2+ homeostasis is proposed to be a key cellular mechanism underlying 

glucocorticoid neurotoxicity. Voltage-dependent Ca 2+ currents are strongly regulated by 

corticosteroids with U-shaped dose dependency; in CA1 of hippocampus, Ca 2+ current amplitude 

is large in the absence of corticosteroids, small under conditions of predominant MR activation, 

and large with the additional occupation of GR at higher glucocorticoid levels (Kerr et al., 1992; 

Karst et al., 1994; Werkman et al., 1997). Evidence suggests that neuronal Ca 2+ influx is 

enhanced by glucocorticoid modulation of L-type Ca 2+ currents (Landfield and Eldridge, 1994; 

Joels, 2001). In combination with depolarizing stimuli such as KA-induced seizures or excessive 

levels of extracellular glutamate, GR-mediated increases in intracellular Ca 2+ levels may enhance 

neuronal vulnerability to Ca 2+ -related injury mechanisms (Landfield and Eldridge, 1991; Joels, 

2001). 

In addition to increasing Ca 2+ influx, GR activation reduces Ca 2+ extrusion in hippocampal 

neurons (Elliott and Sapolsky, 1993; Joels, 2001). Consistent with these findings, high CORT 

levels reduce expression of PMCA1 (Bhargava et al., 2000), a plasma membrane Ca 2+ ATPase 

responsible for translocating calcium from the cytosol to the extracellular space (Garcia and 

Strehler, 1999). Overexpression of PMCA isoform 4 in pheochromocytoma 12 cells reduces 

vulnerability to Ca 2+ -mediated cell death, suggesting that regulation of PMCA expression may 

influence cellular survival of insults involving pathological increases in intracellular calcium 

(Garcia et al., 2001). In agreement with this hypothesis, KA administration decreases 

hippocampal expression of PMCA isoforms 1 and 2 (Garcia et al., 1997b). Further reduction in 

PMCA levels prompted by GR activation would be expected to intensify injury-induced rises in 

intracellular Ca 2+ levels and exacerbate neuronal degeneration. 

Adrenocorticosteriod receptor blockade with MR or GR antagonists may influence 

hippocampal neuronal viability by altering Ca 2+ conductances or extrusion mechanisms. 

Blocking MR activation with SPIRO should result in the predominant activation of GR. It is 

well established that voltage-dependent Ca 2+ currents increase with GR activation at high CORT 

levels (Kerr et al., 1992; Karst et al., 1994; Werkman et al., 1997). However, it is currently 

93

unclear whether exclusive GR activation is sufficient to induce large Ca 2+ currents in 

hippocampal neurons. Electrophysiological studies in hippocampal slices have reported mixed 

results; one study indicates that GR agonist RU28362 increases Ca 2+ currents in CA1 neurons 

(Kerr et al., 1992) while another indicates that it does not (Karst et al., 1994). If exclusive GR 

activation is indeed sufficient to increase Ca 2+ currents, then SPIRO blockade of MR may 

endanger hippocampal neurons by enhancing Ca 2+ influx, particularly under conditions of GRsaturating 

CORT levels. In terms of the current experiments, this scenario is consistent with the 

increased vulnerability of CA3 neurons to KA toxicity following SPIRO pretreatment (Chapter 

Three). 

Glucocorticoid receptor blockade with RU486 should result in the predominant activation of 

MR. Exclusive MR occupation yields relatively small Ca 2+ currents (Karst et al., 1994) and may 

thus protect hippocampal neurons from exposure to excessive Ca 2+ influx. In addition, GR 

blockade should prevent GR-mediated repression of PMCA isoforms, improving Ca 2+ extrusion 

mechanisms. These beneficial effects of GR blockade on neuronal Ca 2+ homeostasis are 

consistent with the neuroprotective effects of RU486 on CA1 neurons following TBI (Chapter 

Four). 

Adrenocorticosteriod receptor suppression of inflammation 

Glucocorticoids inhibit synthesis, release, and/or function of many factors involved in the 

inflammatory response, including: cytokines, such as interleukin (IL)-1β, IL-2, IL-3, IL-5, IL-6, 

IL-12, and tumor necrosis factor alpha (TNF-α); chemokines such as IL-8; and inflammatory 

mediators and enzymes such as histamine, bradykinin, and nitric oxide (Cato and Wade, 1996; 

Steer et al., 1997; Sapolsky et al., 2000). Glucocorticoids also repress expression of 

cyclooxygenase 2 (COX-2) (Cato and Wade, 1996; Sapolsky et al., 2000), an enzyme that 

catalyzes prostaglandin formation (Rivest, 1999). Further, glucocorticoids inhibit expression of 

adhesion molecules, alter lymphocyte trafficking, and reduce activation and proliferation of B 

and T cells (Cato and Wade, 1996; Sapolsky et al., 2000). Glucocorticoid inhibition of KAinduced 

AP-1 (Unlap and Jope, 1995a) and NF-κB (Unlap and Jope, 1995b) DNA binding has 

been demonstrated, suggesting that the potent anti-inflammatory and immunomodulatory action 

of glucocorticoids may be mediated in part through repression of AP-1 and NF-κB activity (De 

Bosscher et al., 2000). 

94

Inflammation has been proposed to influence neuronal survival following several forms of 

insult including KA-mediated excitotoxicity and traumatic brain injury. Kainic acid increases 

glial expression of several cytokines in hippocampus and cortex including IL-1β (Minami et al., 

1991; Yabuuchi et al., 1993; Eriksson et al., 2000). Administration of IL-1ra, an endogenous 

antagonist of IL-1, protects neurons against KA-induced degeneration in CA1 and CA3, 

suggesting a deleterious role for IL-1β in excitotoxic neuronal death (Panegyres and Hughes, 

1998). Hippocampal and cortical mRNA levels of IL-1β also increase following experimental 

TBI (Fan et al., 1995). Administration of IL-1 receptor antagonist IL-1ra has been shown to 

reduce cortical lesion volume (Toulmond and Rothwell, 1995), attenuate hippocampal neuron 

loss, and improve cognitive function following lateral fluid percussion (Sanderson et al., 1999), 

providing further evidence that IL-1β may contribute to injury-mediated neurodegeneration. As 

glucocorticoids decrease IL-1 production (McEwen et al., 1997), ACR regulation of this 

cytokine should enhance neuronal survival after injury and would not appear to explain the 

endangering effects of GR. 

Other studies raise the possibility that mediators of inflammation may play a neuroprotective 

role after neuronal injury. Mice lacking IL-6 demonstrate increased susceptibility to KAinduced 

damage in hippocampus, suggesting that IL-6 can improve survival after excitotoxic 

injury (Penkowa et al., 2001). In addition, selective inhibitors of COX-2 exacerbate KA-induced 

neuronal degeneration in hippocampus (Baik et al., 1999) and worsen motor performance 

following TBI (Dash et al., 2000), indicating a protective role for COX-2. Glucocorticoids 

decrease IL-6 production (McEwen et al., 1997) and inhibit COX-2 expression (Masferrer et al., 

1992), raising the possibility that the anti-inflammatory effects of glucocorticoids may contribute 

to neuronal vulnerability to injury. 

Glucocorticoids may also increase neuronal vulnerability to injury via suppression of TNF-α, 

a pro-inflammatory cytokine that is rapidly induced after several types of brain injury including 

KA excitotoxicity (Minami et al., 1991; de Bock et al., 1996) and TBI (Taupin et al., 1993; Fan 

et al., 1996; Knoblach et al., 1999). The role for TNF-α in mediating neuronal survival is 

controversial; several studies indicate that inhibition of TNF-α reduces neurological deficits and 

hippocampal damage following TBI (Shohami et al., 1996; Shohami et al., 1997; Knoblach et al., 

1999). However, increasing evidence suggests that TNFα may have neuroprotective effects 

under certain circumstances following central nervous system insult. Pretreatment with TNF-α 

95

protects cultured hippocampal neurons against oxidative insults (Barger et al., 1995; Mattson et 

al., 1997), excitatory amino acid toxicity (Cheng et al., 1994), and injury induced by glucose 

deprivation (Cheng et al., 1994). In addition, TNF-deficient mice exhibit long-lasting motor 

deficits following CCI brain injury as compared to wild-type (despite increased memory and 

motor deficits within the acute posttraumatic period), suggesting that TNF may facilitate longterm 

recovery after brain injury (Scherbel et al., 1999). Further, mice lacking TNF receptors are 

more susceptible to neuronal loss induced by focal cerebral ischemia and KA administration 

(Bruce et al., 1996). TNF-α appears to protect neurons by stabilizing cellular Ca 2+ homeostasis 

and stimulating antioxidant pathways via activation of NF-κB (Cheng et al., 1994; Barger et al., 

1995; Bruce et al., 1996; Mattson et al., 1997; Furukawa and Mattson, 1998). Glucocorticoids 

may counter these potentially neuroprotective effects through suppression of TNF-α release from 

monocytes and macrophages (Joyce et al., 1997; Steer et al., 1997) and inhibition of NF-κB 

activation (De Bosscher et al., 2000). 

Glucocorticoids have been proposed to protect the organism by limiting inflammatory 

responses that, left unchecked, might threaten cellular homeostasis (Munck et al., 1984). In this 

view, it is possible that glucocorticoids prevent excessive activation of mediators of 

inflammation that would otherwise contribute to neuronal degeneration after injury. However, it 

is also possible that glucocorticoid suppression of the beneficial effects of inflammation, such as 

Ca 2+ regulation and the expression of antioxidants (Wong and Goeddel, 1988; Cheng et al., 

1994), may contribute to neuronal degeneration. Administration of RU486 would be expected to 

attenuate the anti-inflammatory effects of GR activation, as RU486 has been shown to reverse 

GR suppression of TNFα release from monocytes (Steer et al., 1997). Thus, RU486 repression 

of anti-inflammatory glucocorticoid effects may contribute to the improved survival of 

hippocampal neurons after injury. 

Adrenocorticosteriod receptor regulation of hippocampal neurotrophic factor expression 

Despite negative findings reported in the current work, the possibility for glucocorticoid 

modulation of neuronal viability via regulation of neurotrophic factors can not be dismissed. 

Due to the twenty-six hour time period between antagonist injection and decapitation, ACR 

antagonist effects on BDNF or NT-3 mRNA levels may not have been detected. Glucocorticoid 

regulation of BDNF expression is rapid and transient; hippocampal BDNF mRNAs are reduced 

96

within one hour after the onset of stress, recover substantially by four hours, and return towards 

baseline levels within twenty-four hours (Smith et al., 1995b). Similarly, hippocampal nerve 

growth factor mRNA levels peak approximately three hours after CORT injection, fall below 

baseline levels by six hours, and return close to control levels by 24 hours (Lindholm et al., 

1994), emphasizing the transitory nature of growth factor induction. In addition, neurotrophin 

levels were not assessed in sham and CCI-injured animals in the current work, and ACR effects 

on numerous other trophic factors remain to be investigated. 

Several previous studies validate the possibility that ACR regulation of neurotrophic factors 

may influence hippocampal vulnerability to injury. Although reports are not entirely consistent, 

there is general agreement that in specific hippocampal subfields, CORT and stress decrease 

hippocampal BDNF mRNA levels, while adrenalectomy increases BDNF mRNAs and decreases 

NT-3 mRNAs in hippocampus (Chao and McEwen, 1994; Smith et al., 1995a, b; Schaaf et al., 

1997; Chao et al., 1998b). Futher, CORT and dexamethasone have been shown to increase nerve 

growth factor mRNA levels (Lindholm et al., 1994; Scully and Otten, 1995). There is also 

evidence to indicate that glucocorticoids regulate neurotrophic factor expression following 

injury. Adrenalectomy attenuates BDNF mRNA induction and nearly abrogates NGF mRNA 

induction by KA, and GR agonist dexamethasone enhances NGF mRNA induction by KA 

(Barbany and Persson, 1993). In cultured hippocampal neurons, KA-induced increases in BDNF 

mRNAs are negated by dexamethasone (Cosi et al., 1993). Furthermore, serum deprivationinduced 

death of cultured hippocampal neurons is accelerated by CORT and associated with 

decreases in BDNF mRNA levels and protein (Nitta et al., 1999). Addition of BDNF to serumdeprived 

cultures attenuates negative CORT effects, supporting the hypothesis that reduced 

synthesis of BDNF contributes to CORT-mediated endangerment of hippocampal neurons (Nitta 

et al., 1999). A recent study has also demonstrated that glucocorticoids influence neurotrophic 

factor expression following TBI. Adrenalectomy enhances fluid percussion injury-induced 

increases in BDNF mRNA levels, an effect that is reversed with administration of CORT 

(Grundy et al., 2000). Adrenalectomy also prevents and CORT replacement permits the 

induction of NGF mRNA levels following fluid-percussion injury, indicating the essential role of 

CORT in the NGF response (Grundy et al., 2001). 

Despite evidence for glucocorticoid regulation of neurotrophic factor expression following 

injury, the potential impact of this regulation on hippocampal neuronal viability is presently 

97

unclear. High glucocorticoid levels decrease BDNF mRNAs and increase pyramidal neuron 

vulnerability to injury (Sapolsky, 1994; Smith et al., 1995b), suggesting reduced BDNF 

expression as a potential mechanism for glucocorticoid toxicity. Reports demonstrating an 

association between decreased BDNF mRNA levels and increased neuronal degeneration support 

this hypothesis (Hicks et al., 1999; Nitta et al., 1999). In contrast, the attenuation of KA-induced 

increases in BDNF expression by adrenalectomy is not consistent with a protective role for 

glucocorticoid removal (Barbany and Persson, 1993). The neuroprotective value of BDNF is 

also uncertain, as exogenous administration of BDNF has been shown to exacerbate CA3 

neuronal loss following KA administration (Rudge et al., 1998). Furthermore, glucocorticoids 

potentiate NGF expression and maintain NT-3 mRNA levels, in seeming disagreement with the 

profile for neuronal endangerment by glucocorticoids. The literature suggests that blockade of 

GR with RU486 should enhance KA- and TBI-mediated induction of BDNF expression, while 

decreasing injury-induced NGF expression. However, the potential effects MR blockade on 

injury-induced neurotrophin expression are less clear because it has not been established whether 

GR activation alone is sufficient for neurotrophin regulation. Clarification of these issues will 

require further study. 

Summary 

The present thesis provides evidence supporting the hypotheses that MR protects and GR 

endangers hippocampal neurons following specific types of neuronal injury. The current 

experiments did not support the hypotheses that ACR effects on neuronal survival involve the 

regulation of bcl-2, bax, or p53 expression. Rather, the widespread effects of glucocorticoids on 

several other systems crucial to neuronal survival suggest that glucocorticoids influence 

hippocampal neuron viability via more global mechanisms. Glucocorticoids exert direct effects 

on cellular metabolism, Ca 2+ homeostasis, inflammatory processes, and neurotrophic factor 

expression; in addition, these targets are largely interrelated and capable of altering one another 

(Figure 6). For instance, glucocorticoid effects on neurotrophin expression, cytokine production, 

and cellular metabolism can influence intracellular Ca 2+ levels (Sapolsky, 1990; Mattson and 

Scheff, 1994; Tymianski and Tator, 1996; Jiang and Guroff, 1997). In turn, Ca 2+ influx can 

modulate neurotrophin expression (Finkbeiner, 2000), regulate cytokine production (Hotchkiss 

and Karl, 1996), and disrupt mitochondrial production of ATP (Green and Reed, 1998). It 

98

ecomes clear that glucocorticoid effects on hippocampal neuron viability are not likely carried 

out via isolated signaling pathways, but rather they result from the integration of many factors 

associated with the injury response. 

Figure 6. Potential mechanisms for glucocorticoid modulation of hippocampal neuron survival 

after injury. Excessive activation of the glucocorticoid receptor may impair antioxidant 

defenses, suppress neuroprotective inflammatory responses, decrease neuronal energy stores, 

increase voltage-dependent Ca 2+ currents, and compromise ATP-dependent processes 

responsible for maintaining membrane potential. All of these factors may culminate to increase 

oxidative stress and intracellular Ca 2+ levels, potentially leading to neuronal death. 

99

Injury regulation of adrenocorticosteroid receptor mRNA levels 

Experiments in the current thesis reveal that hippocampal ACR mRNA levels are 

dramatically regulated by excitotoxic and traumatic injury (Chapters 6 and 8). Kainic acid 

decreased MR mRNA levels by approximately 40% in CA1 and CA3 and decreased GR mRNA 

levels by approximately 40% in DG (Chapter Three). Traumatic injury did not influence MR 

mRNA levels but had powerful effects on GR levels; CCI decreased GR levels by approximately 

30% in DG of the injured hemisphere and increased GR levels by approximately 20% in CA1 

and DG of the contralateral hemisphere (Chapter Five). These data support the hypothesis that 

glucocorticoid signaling is altered through regulation of ACR expression following injury. 

Injury regulation of adrenocorticosteroid receptor mRNA levels – potential mechanisms 

As discussed in Chapters Three and Five, altered ACR expression following KA 

administration and CCI is not attributable to CORT-mediated auto-regulatory mechanisms. Still, 

the downregulation of dentate gyrus GR mRNA levels by both types of injury suggests the 

possibility of a common regulatory mechanism. Potential candidates for this role include 

glutamate receptor activation and Ca 2+ signaling, as both KA-induced seizures and TBI lead to 

the accumulation of extracellular glutamate and increased intracellular Ca 2+ levels (Ben-Ari, 

1985; Faden et al., 1989; Sun et al., 1992; McIntosh et al., 1998c). 

Glutamate regulation of adrenocorticosteriod receptor mRNA levels 

Receptor expression patterns provide no evidence for differential action of ionotropic 

glutamate receptors in DG, as NMDA and AMPA glutamate receptor subtypes are preferentially 

expressed in CA1, and KA receptors are mainly located in stratum lucidum of CA3 (Foster et al., 

1981; Represa et al., 1987). However, metabotropic glutamate receptor subtype 1 (mGluR1) is 

strongly expressed in granule neuron cell bodies and the molecular layer of dentate gyrus 

(Shigemoto et al., 1997), and mGluR1 expression is increased in dentate gyrus following 

systemic KA injection (Blumcke et al., 2000). The literature provides support for mGluR 

activation as a viable candidate mechanism for regulation of GR expression after injury. Group 

1 mGluRs, which include mGluR1 and mGluR5, stimulate formation of cyclic adenosine 

monophosphate (cAMP) (Aramori and Nakanishi, 1992), which has been shown to decrease GR 

100

expression in multiple cell lines. The cAMP analog 8-bromo-cAMP decreases GR binding in 

mouse AtT20 anterior pituitary corticotrope tumor cells, and forskolin, a drug that stimulates 

adenylate cyclase, decreases GR binding and mRNA levels (Sheppard et al., 1991). In addition, 

treatment with dibutyryl cAMP, another cAMP analog, decreases GR protein levels in rat C6 

glioma cells, B104 neuroblastoma cells, and in rat primary hippocampal and cortical cultures 

(Sipe, 2000). Thus, activation of mGluR1 represents a possible mechanism whereby injury may 

decrease GR expression in DG via cAMP-dependent pathways. 

Calcium regulation of adrenocorticosteriod receptor mRNA levels 

Intracellular Ca 2+ -dependent signaling may also mediate injury-induced changes in ACR 

expression. Calcium influx (Young, 1992; Fineman et al., 1993; Tymianski and Tator, 1996) has 

been hypothesized to regulate changes in gene expression induced by both kainate (Pennypacker 

and Hong, 1995) and TBI (McIntosh et al., 1997; Morrison et al., 2000). Furthermore, Ca 2+ 

regulation of GR expression has been demonstrated in vitro in AtT-20 cells (Sheppard, 1994). 

However, the lack of Ca 2+ accumulation in DG following KA injection (in contrast to significant 

Ca 2+ accumulation in CA3) (Friedman, 1997) and the high density of Ca 2+ -binding proteins in 

DG (Sloviter, 1989) indicate that GR downregulation does not simply correspond to overall Ca 2+ 

load. Rather, Ca 2+ -mediated regulation of GR expression might occur via distinct Ca 2+ signaling 

pathways linked to specific routes of Ca 2+ influx, such as L-type voltage-dependent Ca 2+ 

channels (Bading et al., 1993). In support of this hypothesis, treatment with BAY K8644, which 

selectively promotes Ca 2+ influx through L-type Ca 2+ channels, decreases GR binding in AtT-20 

cells (Sheppard, 1994). 

Injury regulation of adrenocorticosteroid receptor mRNA levels – potential influence on 

hippocampal neuron viability 

Potential effects of GR downregulation on hippocampal circuitry 

Ample evidence supports the hypothesis that GR compromises survival of CA1 and CA3 

pyramidal neurons after injury, yet GR activation does not appear to compromise DG granule 

neuron survival after administration of KA or TBI. Nonetheless, alterations in dentate neuron 

excitability may influence downstream synaptic transmission and the survival of pyramidal 

101

neurons, as DG is the major recipient of perforant path projections from the entorhinal cortex and 

delivers the majority of excitatory input into CA3 (Amaral and Witter, 1989) (see Figure 1.5). 

Electrophysiological studies demonstrating GR attenuation of CA1 neuron excitability (Kerr et 

al., 1989) raise the possibility that decreased GR expression in DG may enhance KA and TBIinduced 

granule neuron hyperexcitability (Lowenstein et al., 1992; Sloviter, 1992; Santhakumar 

et al., 2000), potentially contributing to excitotoxic stress in CA3 and CA1. However, a recent 

report demonstrates that in contrast to CA1, GR activation does not suppress synaptically evoked 

field potentials in dentate gyrus (Stienstra and Joels, 2000), indicating that decreased GR 

expression may not influence excitability in this region. In addition, GR blockade, which should 

have a maximal effect if any on glucocorticoid-modulated synaptic transmission, does not 

compromise CA3 or CA1 survival after TBI (Chapter Four). It therefore does not appear likely 

that altered GR expression influences pyramidal neuron survival via modulation of DG synaptic 

transmission. 

Alternatively, downregulation of GR expression may facilitate rearrangement of DG circuitry 

after excitotoxic and traumatic brain injury. This hypothesis is supported by studies 

investigating reactive synaptogenesis in response to entorhinal cortex lesion in the rat. 

Glucocorticoids inhibit axon sprouting (Scheff and Cotman, 1982) and influence synaptic 

replacement (Scheff et al., 1986) in DG following delivery of entorhinal cortex lesion. In 

addition, an approximately 33% bilateral decrease in GR mRNA levels has been observed in DG 

one day following unilateral entorhinal cortex lesion (O'Donnell et al., 1993). The authors of 

this study proposed that decreased GR expression might serve to attenuate glucocorticoid 

inhibition of lesion-induced synaptogenesis (O'Donnell et al., 1993). Strikingly, the patterns of 

GR downregulation following entorhinal cortex lesion, kainic acid insult, and TBI are very 

similar (Chapters Three and Five), raising speculation that decreased expression of GR in dentate 

gyrus may have a permissive effect on restructuring of DG circuitry following multiple types of 

injury. Kainic acid is known to induce mossy fiber sprouting (Davenport et al., 1990), but the 

extent of DG remodeling induced by TBI remains to be investigated. 

Potential effects of GR downregulation on neurogenesis 

In addition to facilitating rearrangement of residual DG circuitry, downregulation of GR 

expression may influence DG neurogenesis after injury. Neurogenesis of DG granule cells 

102

continues well into adulthood in the rat and is inhibited by corticosterone in a concentrationdependent 

manner (Cameron and Gould, 1994). Kainic acid (Gray and Sundstrom, 1998) and 

TBI (Dash et al., 2001) have both been shown to enhance DG neurogenesis. GR downregulation 

in dentate gyrus after KA and traumatic brain injury may therefore facilitate injury-induced 

neurogenesis and the subsequent restoration of functional DG circuitry. 

103

Summary 

The present thesis supports the hypotheses that MR activation enhances and GR activation 

endangers neuronal survival in specific hippocampal subregions following excitotoxic or 

traumatic brain injury. Experiments in the current work demonstrate a neuroprotective effect of 

GR blockade after experimental TBI in vivo, as pretreatment with RU486 prevented CCIinduced 

neuronal loss in hippocampal subregion CA1 (Chapter Four). The present data also 

provide evidence that MR protects hippocampal neurons following excitotoxic injury, as MR 

blockade with SPIRO increased CA3 cell loss following systemic KA injection (Chapter Three). 

In addition, this work demonstrates KA and TBI-induced downregulation of GR mRNA levels in 

dentate gyrus, which may facilitate the recovery of functional DG circuitry after injury (Chapters 

Three and Five). Adrenocorticosteroid receptors clearly influence hippocampal neuron survival 

after injury, and future study of ACR regulation under challenging conditions could contribute to 

the improved treatment of seizure, stroke, traumatic injury, and neurodegenerative diseases. 

104

List of Abbreviations 

Appendix 

mineralocorticoid receptor (MR), glucocorticoid receptor (GR), adrenocorticosteroid receptor 

(ACR), mifepristone (RU486), hypothalamic-pituitary-adrenal (HPA), corticotropin-releasing 

hormone (CRH), adrenocorticotropin-releasing hormone (ACTH), paraventricular nucleus of the 

hypothalamus (PVN), heat shock protein (HSP), glucocorticoid response element (GRE), 

activator protein-1 (AP-1), cyclic AMP response element binding protein (CREB), nuclear 

factor-κB (NF-κB), negative glucocorticoid response element (nGRE), calcium (Ca 2+ ), 

corticosterone (CORT), plasma membrane calcium pump isoform 1 (PMCA1), brain-derived 

neurotrophic factor (BDNF), neurotrophin-3 (NT-3), excitatory postsynaptic potential (EPSP), 

entorhinal cortex (EC), subicular complex (S), nerve growth factor (NGF), apoptosis-inducing 

factor (AIF), apoptotic protease activating factor-1 (Apaf-1), inhibitor of CAD (ICAD), caspaseactivated 

deoxyribonuclease (CAD), traumatic brain injury (TBI), controlled cortical impact 

(CCI), anti-apoptotic B-cell leukemia/lymphoma-2 gene (bcl-2), messenger RNA (mRNA), 

interleukin (IL), tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (COX-2), metabotropic 

glutamate receptor subtype 1 (mGluR1), cyclic adenosine monophosphate (cAMP) 

105

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Date of Birth: September 24, 1974 

Place of Birth: Newport News, Virginia 

Education: B.S. Psychology, 1995 

Virginia Polytechnic Institute and State University 

Research and Professional Experience: 

1995 - 1996 Undergraduate research assistant 

Virginia Polytechnic Institute and State University 

1996 - 2001 Graduate student 


Department of Anatomy and Neurobiology 

1998 - 1999 Teaching assistant, MD 817, Neurosciences 

University of Kentucky College of Medicine 

1999 Teaching assistant and guest lecturer 

ANA 636, Advanced Neuroanatomy 



2000 – 2001 Guest lecturer, ANA 206/209, Principles of Human Anatomy 



Awards: 

1996-1997 Hughes Predoctoral Fellowship 



1998-1999 Kentucky Research Challenge Trust Fund Fellowship 

1999-2001 NIH Training Grant: 

Molecular and Cellular Aspects of Brain Aging 

139

Publications and Abstracts 

McCullers, D.L., Sullivan, P.G., Scheff, S.W., and Herman, J.P. (2001) RU486 protects CA1 

hippocampal neurons following traumatic brain injury in rat. In press, Neuroscience. 

McCullers, D.L., Sullivan, P.G., Scheff, S.W., and Herman, J.P. (2001) Traumatic brain injury 

regulates adrenocorticosteroid receptor mRNA levels in rat hippocampus. Submitted. 

McCullers D. L. and Herman, J.P. (2001) Adrenocorticosteroid receptor blockade and 

excitotoxic challenge regulate adrenocorticosteroid receptor mRNA levels in hippocampus. 

Journal of Neuroscience Research 64: 277-283. 

Pedersen, W.A., McCullers, D.L., Culmsee, C., Haughey, N.J., Herman, J.P., and Mattson, M.P. 

(2001) Corticotropin-releasing hormone protects neurons against insults relevant to the 

pathogenesis of Alzheimer’s disease. Neurobiology of Disease 8(3): 492-503. 

McCullers, D.L., and Herman, J.P. (1998) Mineralocorticoid receptors regulate bcl-2 and p53 

mRNA expression in hippocampus. Neuroreport 9(13), 3085-3089. 

McCullers, D.L. and Herman, J.P. (2001) Adrenalectomy with corticosterone replacement 

prevents restraint stress-induced downregulation of glucocorticoid receptor mRNA levels in 

CA1 of rat hippocampus. Society for Neuroscience Abstracts 27. 

McCullers, D.L., Sullivan, P.G., Scheff, S.W., and Herman, J.P. (2000) Effects of 

adrenocorticosteroid receptor blockade and traumatic brain injury on hippocampal neuron 

viability. Society for Neuroscience Abstracts 26: 419. 

McCullers, D.L. and Herman, J.P. (1999) Mineralocorticoid receptor blockade 

and kainic acid-induced changes in viability-related gene expression. Society for 

Neuroscience Abstracts 25: 615. 

140

McCullers, D.L., and Herman, J.P. (1998) Blockade of mineralocorticoid receptors increases 

hippocampal cell death following kainic acid treatment. Society for Neuroscience Abstracts 

24: 1380. 

McCullers, D.L., Watson, R.E., and Herman, J.P. (1997) Blockade of mineralocorticoid 

receptors alters transcription of the apoptosis-related genes bcl-2 and bax. Society For 

Neuroscience Abstracts 23: 1242. 

Deanna L. McCullers 

141

ABSTRACT OF DISSERTATION Deanna Lynn McCullers The ...

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