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THE ROLE OF THE ANDROGEN RECEPTOR IN ANXIETY-
RELATED BEHAVIORS, THE HYPOTHALAMIC PITUITARY
ADRENAL AXIS, AND SENSOFIIMOTOFI GATING: STUDIES IN
RODENTS WITH THE TESTICULAR FEMINIZATION MUTATION

presented by

Damian Zuloaga

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5108 K:lProj/Acc&Pres/ClRC/DateDue.indd

THE ROLE OF THE AN DROGEN RECEPTOR IN ANXIETY-RELATED
BEHAVIORS, THE HYPOTHALAMIC PITUITARY ADRENAL AXIS, AND
SENSORIMOTOR GATING: STUDIES IN RODENTS WITH THE TESTICULAR
FEMINIZATION MUTATION

By

Damian Zuloaga

A DISSERTATION

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

DOCTOR OF PHILOSOPY
Department of Psychology

2008

ABSTRACT
THE ROLE OF THE ANDROGEN RECEPTOR IN ANXIETY-RELATED
BEHAVIORS, THE HYPOTHALAMIC PITUITARY ADRENAL AXIS, AND
SENSORIMOTOR GATING: STUDIES IN RODENTS WITH THE TESTICULAR
FEMINIZATION MUTATION
By

Damian Zuloaga

Androgens such as testosterone play a role in the display of anxiety-related
behaviors and sensorimotor gating. However, the role of a speciﬁc hormone receptor, the
androgen receptor (AR) in the regulation of these behaviors is less clear, because
testosterone can be converted to estrogens that act on estrogen receptors. In this series of
experiments we investigated the role Of the AR in anxiety and sensorimotor gating by
comparing AR deﬁcient male mice and rats with the testicular feminization mutation
(Tfm) to their wild type siblings in rodent models of these behaviors. Since increased
anxiety-related behaviors are often correlated with an elevation of the hypothalamic-
pituitary-adrenal (HPA) axis we also measured the release of the adrenal stress hormone
corticosterone at baseline and at time points after exposure to an anxiety-provoking
situation. Results Of these studies indicate increased indices of anxiety and increased
activation of the HPA axis in both Tfm male rats and mice, while sensorimotor gating
appeared normal in the absence of ARS. An investigation into the role of androgens and
the AR prior to adulthood in the organization of anxiety-related behaviors and
sensorimotor gating revealed that neonatally gonadectomized Tfm and wt male rats
showed decreased anxiety-related behavior and increased sensorimotor gating compared

to neonatally sham Operated Tﬁn and wt males. These results suggest that androgens act

primarily via an AR-independent mechanism, perhaps through estrogen receptors, during
development to inﬂuence these behaviors. Together our ﬁndings in Tfm rodents indicate
a role of the AR and/or developmental androgens in the regulation of anxiety-related

behavior, the HPA axis, and sensorimotor gating.

This dissertation is dedicated to my mom and Kristen without whom this would never
have been accomplished

iv

ACKNOWLEDGMENTS

Many thanks to all my friends in the lab that helped me along with different aspects of
my dissertation and members of my committee: Tony Nunez, Joe Lonstein, and Lyn
Clemens for their valuable advice. Special thanks to Marc and Cindy for encouraging me
to pursue a project that was a little bit different and for mentoring and supporting me
during my years at MSU. It is because of this that I truly enjoyed my years here, despite

the miserable weather, and am sad to leave.

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
CHAPTER 1

CHAPTER 2

THE ROLE OF THE AN DROGEN RECEPTOR IN THE MASCULTNIZATION OF
THE BRAIN AND BEHAVIOR: WHAT WE’VE LEARNED FROM RODENTS WITH
THE TESTICULAR FEMINIZATION MUTATION ......................................... 8

CHAPTER 3

MICE WITH THE TESTICULAR FEMINIZATION MUTATION DEMONSTRATE A
ROLE FOR ANDROGEN RECEPTORS IN THE REGULATION OF ANXIETY-
RELATED BEHAVIORS AND THE HYPOTHALAMIC-PITUITARY-ADRENAL

AXIS .................................................................................................. 35
CHAPTER 4

ELEVATED ANXIETY-RELATED BEHAVIOR AND CORTICOSTERONE
RESPONSE IN RATS WITH THE TESTICULAR F EMINIZATION MUTATION ..... 58
CHAPTER 5

THE ORGANIZATIONAL ROLE OF ANDROGENS AND THE ANDROGEN
RECEPTOR IN ANXIETY-RELATED BEHAVIORS AND SENSORIMOTOR

GATING ............................................................................................. 82
CHAPTER 6

DISCUSSION ...................................................................................... 101
APPENDICIES .................................................................................... I 17
REFERENCES ..................................................................................... 145

vi

LIST OF TABLES

TABLE 1
MORPHOLOGICAL FINDINGS IN THE TFM RODENT CENTRAL NERVOUS
SYSTEM ............................................................................................ 117

TABLE 2
BEHAVIORAL FINDINGS IN TFM RODENTS ............................................ 1 18

TABLE 3
BODY WEIGHT AND TESTOSTERONE LEVELS IN WT AND TFM MALE
MICE ................................................................................................ 119

TABLE 4
OPEN FIELD, NOVEL OBJECT TEST, LIGHT DARK BOX, AND ELEVATED PLUS
MAZE ................................................................................................ 120

TABLE 5

THE NUMBER OF C-FOS POSITIVE CELLS PER MM2 IN WT AND TFM MALE
RATS AT BASELINE OR AFTER EXPOSURE TO AN OPEN FIELD WITH A
NOVEL OBJECT .................................................................................. 121

vii

LIST OF FIGURES

FIGURE 1
BRAIN AND BODY WEIGHTS OF WILDTYPE MALE, TFM MALE, AND
WILDTYPE FEMALE MICE AND RATS .................................................... 122

FIGURE 2
THE POSTERODORSAL MEDIAL AMYGDALA (MEPD) IN NISSL-STAINED
CORONAL SECTIONS FROM A WILD TYPE (WT) MALE, A TFM MALE WITH A

DYSFUNCTIONAL AN DROGEN RECEPTOR, AND A FEMALE RAT .............. 123
FIGURE 3

TIME SPENT VISITING A NOVEL OBJECT IN ADULT MALE WT AND TFM
MICE ................................................................................................ 125
FIGURE 4

PLASMA CORTICOSTERONE LEVELS 20 MINUTES AFTER INITIAL EXPOSURE
TO AN OPEN FIELD WITH A NOVEL OBJECT IN WT AND TFM MALE

MICE ................................................................................................. 126
FIGURE 5

PREPULSE INHIBITION IN GONADALLY INTACT WT AND TFM MALE MICE
IN TEST ORDER 1 AND TEST ORDER 2 ................................................... 127
FIGURE 6

ANXIETY-RELATED BEHAVIOR IN GONADALLY INTACT WT AND TFM
MALE MICE ....................................................................................... 128
FIGURE 7

ACOUSTIC STARTLE RESPONSE AND PREPULSE INHIBITION IN T AND B-
TREATED WT AND TF M MALE MICE ..................................................... 131
FIGURE 8

ANXIETY-RELATED BEHAVIOR IN T AND B-TREATED WT AND TF M MALE
MICE ................................................................................................ 132
FIGURE 9

(A) PLASMA CORTICOSTERONE LEVELS AT BASELINE AND 20 MINUTES
AFTER EXPOSURE TO AN OPEN FIELD WITH A NOVEL OBJECT WERE
ELEVATED IN TFM MALES COMPARED TO WT MALES. (B) PLASMA
CORTICOSTERONE LEVELS AT BASELINE AND 20, 40, 60, AND 120 MINUTES
AFTER EXPOSURE TO AN OPEN FIELD WITH A NOVEL OBJECT IN TFM AND
WT MALES ........................................................................................ 135

viii

FIGURE 10

PREPULSE INHIBITION (PPI; A), ACOUSTIC STARTLE RESPONSE (B), AND
ACOUSTIC STARTLE RESPONSE BY TRIAL IN GONADALLY INTACT WT
MALE, TFM MALE, AND WT FEMALE RATS ............................................. 137

FIGURE 11

THE AMOUNT OF TIME SPENT IN/WITH AND NUMBER OF VISITS TO: (A) THE
CENTER AREA OF THE OPEN FIELD OR A NOVEL OBJECT IN THE OPEN
FIELD ARENA IN WT MALE, TFM MALE, AND WT FEMALE RATS .............. 138

FIGURE 12

PLASMA CORTICOSTERONE LEVELS WERE ELEVATED AT BASELINE AND
20 MINUTES AFTER INITIAL EXPOSURE TO AN OPEN FIELD WITH A NOVEL
OBJECT WERE ELEVATED IN TF M AND WT FEMALES COMPARED TO WT
MALES ............................................................................................. 140

FIGURE 13

THE NUMBER OF C-FOS POSITIVE CELLS PER MM2 IN WT AND TFM MALE
RATS AT BASELINE OR AFTER EXPOSURE TO AN OPEN FIELD WITH A
NOVELOBJECT .................................................................................. 142

FIGURE 14

NEONATAL CASTRATION INCREASED THE AMOUNT OF TIME SPENT IN AND
THE NUMBER OF VISITS TO THE CENTER AREA OF THE OPEN FIELD IN
BOTH TFM AN WT MALES ................................................................... 144

FIGURE 15

THE AMOUNT OF TIME SPENT WITH AND THE NUMBER OF VISITS TO A
NOVEL OBJECT IN THE OPEN FIELD ARENA IN NEO-GDX OR NEO-SHAM

TF M AND WT MALES .......................................................................... 146

FIGURE 16
THE AMOUNT OF TIME SPENT IN THE LIGHT AREA, NUMBER OF VISITS TO
THE LIGHT AREA, AND REARIN GS IN THE LIGHT AREA OF THE LD BOX... 148

FIGURE 17

THE AMOUNT OF TIME SPENT ON AND THE NUMBER OF ENTRIES INTO THE
OPEN ARMS OF THE EPM IN NEO-GDX OR NEO-SHAM TF M AND WT

MALES ............................................................................................. 150

FIGURE 18

PREPULSE INHIBITION, ACOUSTIC STARTLE RESPONSE, AND ACOUSTIC
STARTLE RESPONSE BY TRIAL IN NEO-GDX AND NEO-SHAM WT AND TF M
MALE RATS ....................................................................................... 152

ix

FIGURE 19
PLASMA CORTICOSTERONE LEVELS AT BASELINE AND 20 MINUTES AFTER

, INITIAL EXPOSURE TO AN OPEN FIELD WITH A NOVEL OBJECT ............... 155

FIGURE 20
BRAIN AND BODY WEIGHT IN NEO-GDX AND NEO-SHAM WT AND TFM
MALE RATS ....................................................................................... 156

Chapter 1: Introduction
In humans, there are startling gender differences in neuropsychiatric disorders such as
dyslexia, autism, schizophrenia, and anxiety. For example, the onset of schizophrenia
generally occurs earlier in men (Hambrect et al., 1992) and the symptoms tend to be more
severe (Castle et al., 1995). Females, however, are twice as likely as men to suffer ﬁom
generalized anxiety disorder (DSMIV, 1994). Gonadal hormones, testosterone (T) and
estrogen (E) particularly through their activation of estrogen receptors (ERS), have been
implicated in the development and maintenance of sex differences in neuropsychiatric
disorders (Perlman et al., 2004: Ostlund et al., 2003). However, the role of the androgen
receptor (AR), and particularly its inﬂuence on the development these disorders in males,
is less understood.

An overwhelming body of evidence indicates that sex differences in mammalian
behavior are the result of (a) differences in early hormone exposure that permanently
“organize” the nervous system and/or (b) differences in exposure to hormones in
adulthood that “activate” the neural circuitry that was previously organized. T is an
important hormone in the organization and activation of male typical behavior. T can
facilitate changes in behavior by acting on both ARS and ERS, which then alter gene
expression and protein synthesis. In rodents, the AR is directly activated by T itself or a
metabolite of T such as 5a-dihydrotestosterone (DHT). ERS are indirectly activated by T
through the process of aromatization in which T is converted to estrogens such as
estradiol (E2) by the enzyme aromatase, and subsequently E2 binds to ERS. Since T can
act on both receptor types it can be difficult to discern whether AR, ER, or both receptors

are involved in the masculinization of a particular behavior. As a result, genetic models

such as testicular feminization mutant (Tfm) rodents, which lack ﬁmctional ARS, provide

a unique approach for examining the role of sex hormone receptors in behavior.

Hormonal Influences on Anxiety

In humans, anxiety disorders are estimated to affect about 1 in 5 people (Kessler et al.,
1994), and there are striking sex differences in the prevalence of these disorders. Women
suffer more ﬁ'om social anxiety disorder than do men (Weinstock et al., 1999) and tend to
receive the majority of diagnoses for speciﬁc phobias (i.e., arachnophobia; DSMIV,
1994). Differences in circulating sex hormones, E and T, are likely involved in the
occurrence of gender differences in anxiety. E replacement has positive effects on mood
and decreases anxiety in postmenopausal women (Yazici et al., 2003), while other studies
indicate that T also plays a role in the regulation of anxiety in humans. Chemical
castration, through androgen blockade therapy, increases anxiety and when treatment is
ceased, anxiety levels decrease (Almeida et al., 2004).

Sex differences in anxiety-related behavior have been demonstrated in rats, as
assessed through rodent models of anxiety, with females generally showing more activity
and reduced indices of anxiety compared to males (Frye et al., 2000; Lucion et al., 1996).
However, gender does not appear to play a major role in the display of these same
behaviors in mice (V oikar et al., 2001). Sex hormones T and E are involved in regulating
anxiety in both mice and rats, with increases in either hormone correlated with a decrease
in anxiety-related behaviors (Frye and Walf, 2004; Bing et al., 1998). Recent reports
indicate that anxiolytic actions of estrogens are mediated via activation of ERB type

receptors. ERB knockout female mice exhibit greater anxiety in the elevated plus maze

anxiety test compared to their wild type (wt) siblings (Irnwalle et al., 2002) and
pharmacological activation of ERB in male and female rats decreases anxiety-related
behaviors in this same test (Lund et al., 2004). On the other hand, pharmacological
activation of ERa increases indices of anxiety in some rodent tests Of anxiety (Lund et al.,
2004). These ﬁndings indicate that ERu and ERB subtypes may play opposing roles in
the regulation of anxiety-related behaviors. There is also evidence indicating a role Of
ARS in the regulation of anxiety-related behavior in rodents. In both mice and rats, DHT
treatment reduces anxiety behaviors in the elevated plus maze (Aikey et al., 2002;
Edinger and Frye, 2004; Frye and Edinger, 2004). However DHT can also interact with
other hormone (ERB) and non-hormone (GABA) receptors via further metabolism,

making it difﬁcult to decipher the speciﬁc receptors involved in reducing anxiety.

Hypothalamic-Pituitary-Adrenal (HPA) Axis

The hypothalamic-pituitary adrenal axis (HPA) plays a critical role in an animal’s
reaction to an anxiety-provoking situation and dysregulation of the HPA axis is related to
the incidence of anxiety disorders. When an individual is exposed to a physical or
psychological stressor, the paraventricular nucleus of the hypothalamus begins to secrete
corticotropin releasing hormone (CRH) and vasopressin, and these hormones, in turn,
activate the anterior lobe of the pituitary gland to secrete adrenocorticotropic hormone
(ACTH). ACTH then activates the adrenal cortex, which results in the release of
glucocorticoids (primarily cortisol in humans, and corticosterone in rodents).
Glucocorticoids can then activate tissues throughout the body, including the brain which

contains glucocorticoid receptors that when activated can play a role in the cessation of

CRH release through a negative feedback mechanism. The display of anxious behavior is
Often correlated with increased levels of blood ACTH and glucocorticoids with the
elevation of these stress hormones likely increasing anxiety in stressful situations.

There are sex differences in rats in stress-induced blood corticosterone levels with
females showing a greater elevation (Handa et al., 1994). These differences may, in part,
result from the ability of testosterone to inhibit the HPA axis while estrogen can enhance
its activation (Handa et al., 1994). There is also evidence that exposure to hormones
around the time of birth can affect basal as well as stress-induced corticosterone levels

(Seale et al., 2005a).

Hormonal Inﬂuences on Sensory motor gating

Prepulse Inhibition (PPI) is a common measure of sensorimotor gating in human and
rodent models. PPI involves measuring a fast muscle twitch response to aloud acoustic
stimulus that is preceded, by approximately 100ms, by a weaker acoustic stimulus or
“prepulse”. The prepulse should permit the subject to anticipate the loud pulse, and
consequently startle less severely. Sensorimotor gating is necessary to ﬁlter sensory,
motor, and cognitive information (Kodisi & Swerdlow, 1994) and is reduced in people
with schizophrenia (Braff et al., 1978). These deﬁcits in sensorimotor gating may be
linked to symptoms of schizophrenia such as hallucinations, delusions, and disruptions in
thought (Rigdon & Weatherspoon, 1992; Perry & Braff, 1994). Sex differences in PPI
have also been reported in humans and rodents, in which males exhibit greater PPI than
females (Swerdlow et al., 1993). Because reduced PPI has been associated with

schizophrenia, it is curious that males, who are more severely affected by the disorder,

Show greater PPI than females. This suggests that susceptible males may be those who
do not exhibit a fully masculinized PPI response before onset of the disorder.

E has been suggested to play an important role in the regulation of PPI, and may
contribute to the delay in onset and milder symptoms observed in women with
schizophrenia. In female rats, administration of E causes an increase in PPI (Van den
Buuse & Eikelis, 2001), and symptoms Of schizophrenia in women are generally less
severe when endogenous E levels are high (Hafner et al., 1993). Because sex hormones
seem to affect both PPI and schizophrenia, investigating the inﬂuence of sex hormones

and their receptors on PPI may shed light on the etiology of schizophrenia.

Organizational vs. Activational role of the AR

The role of the AR in sensorimotor gating, anxiety, and HPA axis regulation is not well
understood, but is likely important. If ARS do contribute to these behaviors it is also
essential to decipher whether ARS affect these behaviors during development and/or in
adulthood. Once we know when androgen acts to regulate these ﬁmctions, we can then
look during these same time periods for morphological and/or functional changes in the

brain that might critically mediate the change in behaviors.

Chapters
Chapter 1 (the present chapter): We introduce the role Of sex hormones and their
receptors in the regulation of anxiety-related behaviors, the HPA axis, and sensorimotor

gating.

Chapter 2: We review evidence for the role of ARS in the masculinization of the brain
and behavior with a focus on what has been learned from studies in rodents with the
testicular feminization mutation.

Chapter 3: In this chapter we explore the role of the AR in anxiety-related behaviors,
activation of the HPA axis, and sensorimotor gating by comparing Tfm and wt male mice
in rodent models designed to measure these behaviors. Since Tfm male mice also have
lower circulating T levels, behaviors were also compared in T-replaced Tfm and wt
males.

Chapter 4: Here we explore the role of the AR in anxiety-related behaviors, activation
of the HPA axis, and sensorimotor gating by comparing behavioral and physiological
(corticosterone) responses in Tfm male, wt male, and wt female rats, and neural (c-fos)
responses in Tfm and wild type males. Female rats were included in this experiment
because, unlike in mice, sex differences in anxiety-related behaviors in rats are well
documented, allowing us to observe whether Tﬁn male behavior better resembled that of
wt males or females.

Chapter 5: In this chapter we investigate the role of androgens and the AR prior to
adulthood in the organization of anxiety-related behaviors, sensorimotor gating, and HPA
axis activity. In this experiment we compared behaviors in Tfm and wt male rats that
were gonadectomized (N eO-de) or sham-operated (N eo-Sham) on the day of birth. In
adulthood, rats were either gonadectomized or sham-operated and implanted with T
capsules to equilibrate adult circulating T and compared in tests of anxiety and
sensorimotor gating. Corticosterone levels were also measured in these rats at baseline or

following exposure to an anxiety-provoking situation.

Chapter 6: Here we present an overarching discussion of our ﬁndings and their

implications.

Chapter 2: The Role of Androgen Receptors in the Masculinization of
Brain and Behavior: What we’ve learned from the Testicular

Feminization Mutation

Abstract

Many studies demonstrate that exposure to testicular steroids such as testosterone early in
life masculinizes the developing brain, leading to permanent changes in behavior.
Traditionally, masculinization of the rodent brain is believed to depend on estrogen
receptors (ERS) and not androgen receptors (ARS). According to the aromatization
hypothesis, circulating testosterone from the testes is converted locally in the brain by
aromatase to estrogens, which then activate ERS to masculinize the brain. However, an
emerging body of evidence indicates that the aromatization hypothesis cannot fully
account for sex differences in brain morphology and behavior, and that androgens acting
on ARS also play a role. The testicular feminization mutation (Tfm) in rodents, which
produces a nonfunctional AR protein, provides an excellent model to probe the role of
ARS in the development of brain and behavior. Tfm rodent models indicate that ARS are
normally involved in the masculinization of many sexually dimorphic brain regions and a
variety of behaviors, including sexual behaviors, stress response and cognitive
processing. We review the role of ARS in the development of the brain and behavior,
with an emphasis on what has been learned from Tfm rodents as well as from related

mutations in humans causing complete androgen insensitivity.

Introduction

Exposure to testicular steroids such as testosterone (T) early in life masculinizes the
developing brain, leading to permanent changes in behavior in a wide variety of animal
models (Morris et al., 2004). According to the aromatization hypothesis, T is converted
by aromatase into 17-[3 estradiol (E2), which then acts on estrogen receptors (ERS) to
masculinize the brain (N aftolin et al., 1975). Traditionally, aromatization is believed to
be the mechanism by which the rodent brain becomes masculinized and defeminized.
Some sexually dimorphic regions within the hypothalamus adhere well to this hypothesis,
including the sexually dimorphic nucleus of the preoptic area (SDN-POA) and
anteroventral periventricular nucleus (AVPV). T spares neurons from death in the SDN-
POA, while it promotes cell death in the AVPV, both through activation of ERs. The
aromatization hypothesis also seems to hold for the development of some sexual and
nonsexual rodent behaviors. Defeminization Of rat sexual behavior, as measured by the
proclivity to show lordosis (the female posture of sexual receptivity), and masculinization
of aggressive behavior in rodents are largely controlled by estrogenic metabolites of T
acting on ERS (Olsen, 1979; Vreeburg et al., 1977; Ogawa et al., 2000; Scordalakes and
Rissman, 2004).

However, an emerging body of evidence suggests that the aromatization
hypothesis cannot account for all sex differences in brain morphology and behavior. In
primates, including humans, brain masculinization may be accomplished primarily via
androgens acting directly on the androgen receptor (AR). For example, alpha-fetoprotein
binds estrogen and prevents it from entering and masculinizing the brain in rodents

(Bakker et al., 2006; Puts et al., 2006), but has very low affinity for estrogen in primates

(Swartz and Soloff, 1974). If the aromatization hypothesis were generally true in
primates, it would seem that ovarian estrogens would cross the blood-brain barrier and
masculinize the female brain. Moreover, people with complete androgen insensitivity
syndrome (CAIS) exhibit feminine behavior (see below) and morphology. CAIS
individuals have a 46,XY karyotype and develop testes that remain undescended in the
abdominal cavity. Despite producing normal-tO-high male levels of T, individuals with
CAIS have completely nonfunctional ARs, and so are phenotypically female (Imperato-
McGinley et al., 1982). Because individuals with CAIS regard themselves as females,
we refer to them hereafter as women. Their feminine behavior suggests that functional
ARS are required to masculinize the human brain. Conversely, human males with
mutations rendering the aromatase enzyme dysfunctional present as normal males,
despite the absence of aromatization in the brain or elsewhere (Grumbach and Auchus,
1999). Taken together, the feminine behavior of people with CAIS and the masculine
behavior of men with dysfunctional aromatase suggest that ER stimulation has a very
limited role, if any, in masculinizing the human brain.

In the meantime, there is growing evidence that even in rodents, androgens act on
ARS to shape the brain and behavior. Traditionally, exploration into the role of ARS in
brain morphology and behavior involved the administration of a nonaromatizable AR
agonist (usually dihydrotestosterone: DHT) or antagonist (e.g., ﬂutamide).
Unfortunately, DHT administration does not guarantee exclusive activation of ARS.
DHT can be metabolized to 3a-androstanediol (3u-diol) which has a low afﬁnity for ARS
but a high afﬁnity for GABA receptors, and 38-diol, an estrogenic compound which

binds ERS (Kuiper et al., 1998). Even if administration of DHT did act solely upon ARS,

10

it might not reveal a contribution of the AR in cases where T acts synergistically upon
both ERS and ARS. For example, T acts upon ARS to increase aromatase activity in many
brain regions (Roselli et al., 1987; Rosenfeld et al., 1977), which would provide more
estrogens to stimulate ERs. Therefore DHT treatment alone would not reveal such a
normal role for ARS because while it might boost aromatase activity, there would be no
aromatizable androgenic precursor to be converted by the enzyme to provide estrogens
for ERS. Furthermore, neonatal DHT administration may not masculinize some areas of
the brain because AR expression levels in those areas may depend on ERS (McAbee and
DonCarlos, 1999a). Neonatal gonadectomy in male rats decreases AR mRNA, and
replacement with T or E2, but not DHT, restores AR mRNA to levels similar to
gonadally intact male rats (McAbee and DonCarlos, 1999a; McAbee and DonCarlos,
1999b). These ﬁndings suggest that T normally acts through ERS to upregulate AR.
Because DHT treatment fails tO restore AR mRN A, DHT may fail to masculinize brain
areas Simply because AR levels are too low. These data also suggest reciprocal
interactions between ARS and ERS in the brain, a theme emphasized in the remainder of
this review.

The AR antagonist ﬂutamide is also an imperfect test Of AR contributions since
its administration, while blocking ARS, also alters T production from the testes, making it
difﬁcult to attribute changes in morphology and behavior solely to AR blockade (Clos et
al., 1988; Ayub and Levell, 1987).

Testicular feminization mutant (Tfm) rodents provide a Lurique model for
examining the role of the ARs in the brain and behavior, because this mutation in the AR

gene renders the protein nonfunctional. As a result of this mutation (the rodent analog of

11

CAIS in humans), genetically male Tfm rodents appear phenotypically female: they
possess nipples typical of female rodents, lack normal male genitalia, and are infertile.
Since this trait is X-linked, only genetically male (XY) carriers are wholly androgen
insensitive. Female Tﬁn carriers are heterozygous, carrying one T ﬁn and one wildtype
(wt) allele of the AR gene, thus allowing the mutation to be passed on to future
generations. Although Tfm rodents present a feminine exterior, we refer to them
hereafter as Tﬁn males because they are genetically male, possess testes and, unlike
women with CAIS, Show signs of at least partial masculinization Of many behaviors, as
discussed below. We also refer to them as Tfm males to distinguish them from females
carrying the Tfm allele.

Rat and mouse Tfm models differ in terms of the type of mutation in AR. In rats,
androgen insensitivity results from a mutation involving a single base pair replacement in
the AR gene (Yarbrough et al., 1990), while in the Tfm mouse the mutation results from a
single base deletion, which causes a frameshift mutation (Charest et al., 1991). Thus
there is only a single amino acid difference between wt and Tfm AR protein in rats
resulting in expression Of a normal sized but dysfunctional AR protein in Tfm rats. On
the other hand, the mutation in Tfm mice introduces a premature stop codon, resulting in
a shortened transcript and essentially no AR protein (He et al., 1991; Monks et al., 2007).
Due to differences in the nature of these mutations, Tﬁn rats have some residual
sensitivity to androgens through ARS, although it is greatly reduced (Yarbrough et al.,
1990), while Tfm mice have virtually no sensitivity to androgen through ARS, as in CAIS
women (Drews, 1998). In both models estrogen binding in the brain appears normal

(Attardi et al., 1976; Olsen and Whalen, 1982).

12

Rat and mouse Tfm models also differ in terms of circulating T levels. Tfm male
mice have signiﬁcantly less endogenous T compared to their wt siblings (Jones et al.,
2003), while Tfm male rats have circulating T levels in the high male range (Rosseli et
al., 1987). In this regard, the Tﬁn rat resembles untreated humans with CAIS, who also
have circulating T levels in the high-male range (Vague, 1983). In both rat and mouse
Tﬁn models, indirect evidence suggests that T levels are near the normal male range in
the perinatal period (Olsen, 1979; Goldstein and Wilson, 1972), but no one has actually
measured circulating androgens in perinatal Tfm rodents.

Aromatase activity is also decreased in several areas of the adult Tﬁn brain
compared to wt males (Roselli et al., 1987; Rosenfeld et al., 1977), so differences
between wt males and Tfm males in brain morphology and behaviors associated with
these regions may be caused by reduced ER activation. Therefore in the survey that
follows, as we ﬁnd evidence that the T ﬁn allele of the AR gene affects brain and behavior,
we must keep in mind that such ﬁndings do not disprove a role for ER, since AR may be

acting in part by affecting aromatase.

AR and Brain Morphology

Increasing evidence suggests that activation Of AR also normally mediates
masculinization of the nervous system and behavior. Some of the ﬁrst evidence for this
idea came from analysis of motoneurons of the spinal nucleus of the bulbocavernosus
(SNB), which mediate penile reﬂexes during copulation (see Sengelaub and Forger,
2008, in this issue). There is a sex difference in the number of SNB motoneurons

(males>females) that is dependent on AR activation, since the SNB system is feminized

l3

in Tﬁn rats (Breedlove and Arnold, 1980; Breedlove and Arnold, 1981). Recent research
utilizing Tfm rodent models suggests that the absence of functional ARS also alters
discrete areas Of the brain. This demasculinization of speciﬁc brain regions is not due to
an effect on the overall size of the brain. In both rats and mice, males have greater brain
weights than do females, which probably reﬂect the sex difference in overall body size.
We now report for the ﬁrst time that the brain weight Of Tfm males is fully masculine in
both species (Figure 1). Interestingly, body weight Of Tfm males is intermediate between
that Of wt males and females in both species, indicating that sexual differentiation of
overall brain weight and body weight are dissociable. These results suggest that this
global masculinization of brain weight may occur via ER activation or via some other
mechanism not dependent on ARs. However, they also set the stage for the following
results where brain regions that are typically larger in males than in females are partially
or wholly feminine in Tfm males, indicating that regional brain demasculinization in Tfm
animals is not a generalized effect on overall brain size, but a speciﬁc decrease in some

brain areas caused by lack of AR stimulation.

Posterodorsal medial amygdala (MePD)

Morphology Of the posterodorsal medial amygdala (MePD), which receives olfactory and
pheromonal information and is important for some aspects of male sexual behavior, is
highly dependent on adult hormones. MePD volume is 1.5 times greater in male rats than
in females, but this sex difference can be abolished by castration of adult males and/or
administration of T to adult females (Cooke et al., 1999). Structural plasticity in the adult

MePD appears to be mediated through activation of both ARS and ERs. Cooke et a1.

14

(2003) found that treating castrated adult male rats with E2 and the nonaromatizable
androgen DHT each masculinize aspects of MePD morphology. Treatment with E2
increases both volume and soma size Of MePD cells compared to untreated castrates,
while DHT treatment increased MePD soma size but did not affect volume. MePD
volume and soma size have also been examined in Tfm male rats (Morris et al., 2005)
and both were found to be partially demasculinized, signiﬁcantly different ﬁ'om both wt
males and females (Figure 2a-f). In contrast, the rostrocaudal extent of the MePD, which
is greater in wt males than females, is fully masculinized in Tfm males. Because T levels
are in the high male range and aromatase activity is normal in the medial amygdala of
Tfm male rats (Rosseli et al., 1987), adequate amounts of estrogens should be available
for ER activation. Given that both MePD soma size and volume are smaller in Tfm
males than in wt males, T activity through ERs appears insufficient to fully masculinize
these characteristics (Morris et al., 2005). On the other hand, masculinization of the
rostrocaudal extent Of the MePD appears to be independent of AR activation, with Tﬁn
and wt males equivalent (Morris et al., 2005). Similarly, Tfm and wt male rats also show
an asymmetry, with MePD volumes greater in the right hemisphere than the left. This
laterality in MePD volume is not found in females, indicating that its presence may
depend on some masculinizing factor unrelated to AR activation, the leading candidate

being ER activation (Morris et al., 2005).

Suprachiasmatic Nucleus
Sexual dimorphisms have been found in the brain’s biological clock, the suprachiasmatic

nucleus (SCN), in both humans and rodents. The vasoactive intestinal polypeptide (VIP)

15

containing subnucleus Of the SCN is twice as large in men as in women, and the
vasopressin containing subnucleus of the SCN has a different shape in men than in
women (Swaab et al., 1994; Swaab et al., 1985). Sexual orientation has also been
correlated with the size of the human SCN. Swaab and Hofrnan (1990) found the
vasopressin subnucleus of the SCN Of homosexual men to be 1.7 times as large and
contain twice as many cells as that of heterosexual men. Studies of sex differences in the
SCN Of rats have yielded somewhat conﬂicting ﬁndings in which males have a greater
SCN volume than females (Robinson et al., 1986; Gorski et al., 1978) or there were no
differences (Madeira et al., 1995; Bloch and Gorski, 1988). These discrepancies suggest
that sex differences in the rat SCN may either be small and/or strain dependent. In
gerbils, volume of the male SCN was twice that of the female SCN (Holman and
Hutchison, 1991). Furthermore, castration of gerbils on the day of birth reduced SCN
volume to female levels in adulthood, suggesting that the SCN is inﬂuenced by early
androgen exposure. Morris et al. (2005) analyzed the SCN in Tfm male rats and found
that volume and neuronal soma size was decreased compared to wt male rats, indicating
that functional ARS are essential for the full masculinization of this brain area. However,
given that aromatase activity is decreased in the SCN of Tfm male rats (Roselli et al.,
1987), it is possible that the main role the AR plays in masculinization of the SCN is to
regulate the level of aromatase and that ERS mediate downstream changes in SCN
structure. This scenario would still indicate an important role for ARS in normal
masculinization of the SCN, but it would also suggest that sufﬁcient stimulation of ERS,

such as with exogenous estrogen, can fully masculinize the nucleus.

16

Ventromedial Hypothalamus

The ventromedial hypothalamus (V MH) is another sexually differentiated region that is
involved in sexual and parental behaviors. VMH volume and soma size are greater in
males than in females, and males also have a higher concentration Of ARS in the VMH
than do females (Matsumoto and Arai, 1983; Madeira et al., 2001). The VMH contains
four morphologically distinct regions, including the anterior (V MHa), dorsomedial
(VMHdm), ventrolateral (V MHvl), and central (V MHc) subdivisions, which have distinct
connectivity patterns to other brain regions and may affect a variety of functions
(McClellan etal., 2006). In a recent study, volume and neuronal soma size of the entire
VMH and its four subdivisions were compared between wt males, females, and Tﬁn male
rats (Dugger et al., 2007). Conﬁrming earlier ﬁndings, overall VMH volume was greater
in males than in females. This difference was accounted for by a larger male VMHvl and
a marginally greater male volume in the VMHdm. In Tfm males, overall VMH volume
was intermediate between wt males and females, but did not signiﬁcantly differ from
either group. However, the VMHvl, which accounts for most of the sexual dimorphism
in VMH volume as a whole, was signiﬁcantly smaller in Tfm males than wt males and
similar to that Of females. Analysis of neuronal soma size revealed a sex difference in
which males had a greater soma size than females in three Of the four VMH subdivisions
(V MHvl, VMHc, and VMHdm). Tfm males had signiﬁcantly smaller somata than wt
males in the VMHdm and marginally smaller somata in the VMHvl and VMHc.
Together these data suggest that functional ARS normally play a role in the full

masculinization of the VMH. In fact, ARS may be the predominant steroid receptor

17

mediating masculinization of the VMH, since Tfm males did not signiﬁcantly differ from
females on any measure. However, differences in AR-induced aromatase activity, as
discussed for the SCN, may also inﬂuence VMH morphology. Finally, these results from
the VMH, along with those discussed for the MePD and SCN, emphasize the fact that
different morphological traits Of a given brain region may or may not be affected by ARS,
and that the effects of the AR on individual cells within a region may not affect overall

volume.

Bed Nucleus of the Stria Terminalis

The bed nucleus of the stria terminalis (BST) is a sexually dimorphic region within the
hypothalamus that plays a role in social and reproductive behaviors (De Vries and
Panzica, 2006). A portion of this region, the posteromedial nucleus of the bed nucleus of
the stria terminalis (BSTMPM) shows several sexual dimorphisms, including greater AR
density and regional volume in males compared to females (Hines et al., 1992; Lisciotto
and Morrell, 1994; Roselli, 1991). The sex difference in volume appears to result from
hormone exposure both perinatally and in adulthood. Gonadectomy Of newborn males
decreases BSTMPM volume, and androgen treatment of newborn females increases it
(Guillamon et al., 1988b), while gonadectomy of adult males also decreases regional
volume (Malsbury and McKay, 1994). Based on neuroanatomical similarities Of the
BSTMPM and the MePD (proximity and high AR content), and similar hormone
responsiveness between the two regions, it was hypothesized that ARS may also play a
role in the sexual differentiation of this region. Durazzo et a1. (2007) compared
characteristics of the BSTMPM in wt male, wt female, and Tfm male rats and found

group differences in regional volume, but not neuronal soma size. There was a sex

18

difference in BSTMPM volume in both the left and right hemisphere (males > females),
while volume was increased in wt males compared to Tfm males only in the left

hemisphere. These results suggest that, as in the MePD, ARS normally contribute to the

full masculinization of the BSTMPM in males.

Arginine vasopressin (AVP) innervation of the septal area (including the BST),
which plays a role in pair bonding, parental, and aggressive behavior (De Vries and
Panzica, 2006), is also sexually differentiated. AVP innervation of this area is greater in
males than in females and is dependent upon androgens in both early development and
adulthood. Castration of male rats in adulthood decreases AVP innervation, although not
as extensively as does castration neonatally (Wang et al., 1993). Androgens contribute to
the sex difference in AVP via aromatized metabolites of T acting on ERS, although ARS
also appear to contribute to the perinatal organization of this system to some degree. For
example, gonadectomized rat pups treated in the early postnatal period with DHT show a
partial masculinization of BST vasopressin, although this masculinization is less than that
found in rat pups treated with E2 either alone or combined with DHT (Han and De Vries,
2003). On the other hand, Tﬁn male mice are not different from wt males after adult
castration and treatment with E2 (Scordalakes and Rissman, 2004). These results suggest
that the role Of ARS in the masculinization of AVP innervation of the septa] area in mice

is minimal.

Sexually Dimorphic Nucleus of the Preoptic Area (SDN—POA)

19

Even the SDN-POA, in which abundant literature suggests that sex differences depend on
the metabolism of T into E2 and activation of ERS, may normally depend on ARS for ﬁrll
masculinization. Regional volume and soma size Of SDN-POA neurons are greater in
male than in female rats (Gorski et al., 1980; Madeira et al., 1995), and perinatal hormone
manipulations suggest that ERS, not ARS, are important in the masculinization of SDN-
POA volume (Dohler et al., 1986). However, Tfm male rats show only a partial
masculinization Of the SDN-POA: volume is fully masculine but neuronal soma size is
not, with Tfm males having smaller neurons in the SDN-POA than wt males (Morris et
al., 2005). Again, as in several other brain regions, aromatase activity is decreased in the
medial preoptic area of Tfm male rats (Roselli et al., 1987), so ARS may not be directly
necessary for the masculinization Of neuronal soma size in the SDN-POA, if sufﬁcient
estrogen is provided. Nevertheless, ARS appear to contribute normally to the full

masculinization Of the rat SDN-POA.

Locus Coeruleus

The locus coeruleus (LC), a brainstem nucleus implicated in the physiological response
to stress and panic, is an example of a sexual dimorphism in which females show a larger
volume and a greater number of neurons than do males (Guillamon et al., 1988a). This
dimorphism appears to be under the control of both testicular and ovarian hormones, with
testicular steroids decreasing and ovarian steroids increasing growth and survival of LC
cells. Both neonatal and postpubertal gonadectomy of females decrease the number of
neurons in the LC (De Blas etal., 1990; De Blas et al., 1995), while pre- and postnatal

androgen administration also decreases LC neuron number (Guillamon et al., 1988a). In

20

males, ARS rather than ERS appear to play a critical role in the masculinization of the LC.
Garcia-Falgueras et a1. (2005) found that, compared to wt male littermates, Tﬁn males
had a larger volume and greater number of neurons in the LC. Based on these results,
Guillamon’s group suggests that when a sexual dimorphism exists in which females show
more neurons and/or greater volume it may be at least partially mediated via AR

activation in males.

Hippocampus

The hippocampus is another sexually dimorphic area of the brain in which males show a
greater overall volume than females (Madeira et al., 1995; Nunez et al., 2000). One area
Of the hippocampus, the dentate gyrus, is sexually dimorphic in some strains of mice in
which males show a greater number, size, and density of cells within this region (Wimer
and Wimer, 1984; Wimer et al., 1988; Wimer and Wimer, 1989). Study of Tfm male
mice suggests that ARS may play some role in the development of the mouse dentate
gyrus. Although no sex difference was reported in volume of the granule cell layer of the
dentate gyrus (GCL) in C57BL6J mice, both wt males and females had a larger right than
left GCL, a laterality that was absent in Tfm males and partially androgen insensitive
Tfm carrier female mice (Tabibnia et al., 1999). These results suggest that ARS play a
role in establishing laterality in the mouse GCL regardless of whether a sexual
dimorphism exists. In the rat dentate gyrus, the presence of functional ARS also appears
to affect morphology, despite a lack of sexual dimorphism. Jones and Watson (2005)
found that Tﬁn male rats show a greater GCL volume than do females, although there

was no sex difference in GCL volume. Why GCL volume is greatest in Tfm rats is

21

unclear, but the authors suggest it may result from increased ER activation within the
GCL due to the higher levels of T in Tfm male rats.

Androgens also increase the pyramidal cell dendritic Spine density (PSSD) in
CA1 of the hippocampus in male and female rats (MacLusky et al., 2004; Leranth et al.,
2004; Hajszan et al., in this issue). In females, estrogens can also increase CA1 PSSD,
though they have no effect in males (Leranth et al., 2003; MacLusky et al., 2005). This
result indicates that the androgen-induced increase in PSSD in males may be mediated
through ARS. However, Tfm males with dysfunctional ARS showed a PSSD comparable
to wt males (MacLuscky et al., 2006). Interestingly, DHT administration increased CA1
PSSD in both wt and Tfm males, suggesting that androgens may exert their inﬂuence
through nonclassical pathways.

Our review of the literature indicates that, for every known sexual dimorphism in
the rat and mouse nervous system that has been examined in Tfm models so for, results
indicate that ARS normally contribute to masculinization of at least some aspects Of
morphology. In several cases, both regional volume and neuronal soma size are
signiﬁcantly demasculinized in Tﬁn males (SNB, MePD, VMH, SCN). Sometimes
volume is demasculinized while neuronal soma size is not, sometimes the reverse is true.
Our conclusion is that ARS are integral to masculine structural development throughout

the rodent brain. These studies are summarized in Table 1.

AR and Behavior
In line with the emerging role of the AR in shaping brain morphology, evidence also

suggests that the AR is important in shaping behavior, both during its early organization

22

and its later activation in adulthood. Although aromatization may account for many sex
differences in rodent behavior, research using Tfm models suggests that the AR normally

plays a critical role in a variety of behaviors ranging from sexual to cognitive.

Spatial Memory
Males outperform females in spatial memory performance in both rodents and humans
(Rizk-Jackson et al., 2006; Roof, 1993; Astur etal., 1998; Jonasson, 2005). The sex
differences in rodents have generally been attributed to the organizational effects of
androgens, since neonatal castration of males or administration of T to newborn females
eliminates such sex differences (Isgor and Sengelaub, 2003). The aromatization of
androgens to estrogens may be particularly important in the development Of the sex
difference, since neonatal administration of E2 masculinizes spatial ability in female rats
(Williams et al., 1990; Williams and Meck, 1991). However, other evidence suggests
that perinatal AR activation may also play a role in enhancing male spatial memory
performance. Isgor & Sengelaub (1998) found that prenatal E2 treatment did not
masculinize performance in the Morris Water Maze (MWM) in female Sprague-Dawley
rats, whereas treatment with either T or DHT did. In addition, males administered AR
antagonists prenatally show poorer MWM performance than do control males,
eliminating the sex difference in this behavior (Isgor and Sengelaub, 1998; Joseph et al.,
1978)

Hormonal inﬂuence on such behaviors, however, may not be solely
organizational. Adult circulating androgens and estrogens can also affect certain aspects

of spatial memory performance and these effects may, at least partially, be mediated

23

through activation of ARS in adulthood (Gibbs, 2005; Sandstrom et al., 2006; Naghdi et
al., 2001). To further explore the role of ARS in spatial memory, performance in the
MWM has been examined in both the rat and mouse Tfm models. Jones and Watson
(2005) compared MWM performance in wt male, female, and Tfm male rats and found
that Tfm males showed an intermediate pattern Of performance in which they took longer
to reach male-typical performance levels. Tfm male mice also show impaired MWM
performance compared to partially androgen-insensitive Tfm carrier females, although
sex differences in wt mice were not found (Rizk et al., 2005). Together, these ﬁndings
suggest that the AR is necessary for the full masculinization of spatial memory.

Other facets of memory also appear to be inﬂuenced by androgens, such as object
memory in rodents, which is strongly inﬂuenced by circulating androgens and estrogens
in adulthood (Frye and Lacey, 2001; Walf et al., 2006). Use of the Tﬁn models would
further elucidate the role of T and its mechanism of action in this and other memory
tasks.

Sex differences in human spatial ability also appear to be mediated by androgens
(Puts et al., 2007; Puts et al., in press), and evidence supports a role of ARs in this sex
difference. In the only published study of CAIS and Spatial ability to date, CAIS females
performed signiﬁcantly worse on spatial tasks than did their male relatives (Imperato-
McGinley et al., 1991). Although this ﬁnding suggests that androgens masculinize
spatial ability via ARS, it is also possible that CAIS women exhibit less masculine spatial
abilities because they were socialized in a manner concordant with their phenotypic
gender. A more powerful comparison is that between CAIS women and their unaffected

(46,XX) female relatives. If spatial ability is AR-mediated, then CAIS women should

24

perform signiﬁcantly worse on tests of spatial ability than their unaffected female
relatives, who are also socialized as females yet produce and receive some androgen
stimulation. In fact, this is what was found (ImperatO-McGinley et al., 1991). Even this
comparison must be interpreted cautiously however, as it is possible that ovarian
hormone production in unaffected females caused them to differ from their CAIS

relatives.

Anxiety-Related Behavior
In humans, anxiety disorders are estimated to affect about 1 in 5 people (Kessler et al.,
1994), and there are striking sex differences in the prevalence of these disorders. Women
suffer more ﬁom social anxiety disorder than do men (Weinstock, 1999) and tend to
receive the majority of diagnoses for speciﬁc phobias (e.g., arachnophobia; DSM-IV,
1994). Differences in circulating sex hormones, E2 and T, are likely involved in the
occurrence of gender differences in anxiety. Estrogen replacement has repeatedly been
shown to increase mood and decrease anxiety in postmenopausal women (Yazici et al.,
2003). Other studies indicate that androgens also play a role in the regulation of anxiety
in humans. Chemical castration through androgen blockade therapy in men with prostate
cancer was correlated with an increase in anxiety. When treatment ceased, anxiety levels
decreased (Almeida et al., 2004).

Gonadal hormones also play a role in the display of anxiety-related behavior in
rodents as assessed by open ﬁeld testing, exposure to a novel Object, the elevated plus
maze task, and light dark box (Edinger and Frye, 2004; Walf and Frye, 2005; Lund et al.,

2005; Bridges and Starkey, 2004; Frye and Lacey, 2001). In general, increases in either

25

T or E2 in both male and female rodents are correlated with a decrease in anxiety-related
behaviors (Frye and Walf, 2004; Bing et al., 1998; Frye et al. 2007). Recent reports have
shed some light on the hormone receptors that are activated to provide anxiolysis.
Imwalle et al. (2005) reported that estrogen receptor beta (ERB) knockout female mice
exhibit greater anxiety on an elevated plus maze anxiety test compared to their wt
littermates. Furthermore, pharmacological activation of ERB in rats decreased, while
estrogen receptor alpha (ERa) activation increased, anxiety-related behaviors in an Open
ﬁeld and when exposed to a novel Object (Lund etal., 2005). Together these studies
suggest that estrogen action via ERa and ERB subtypes differentially regulates anxiety-
related behaviors. I

ARS also appear to be involved in anxiety in rodents. In rats, DHT treatment
reduces anxiety behavior in the elevated plus maze (Edinger and Frye, 2004; Frye and
Edinger, 2004). However, as previously mentioned, DHT can also interact with other
hormone and non-hormone receptors via further metabolism. DHT can be metabolized to
3a-androstanediol (3u-diol) which has a low afﬁnity for ARS but a high affinity for
GABA receptors, and treatment with 3a-diol increases anxiolysis in gonadectomized rats
(Frye and Edinger, 2004). 3B-diol, which can also be derived from DHT, binds ERS,
with greater affinity for ERB than ERa (Kuiper et al., 1998; Lund et al., 2004). Given
what is known about the role of ERB in the display of anxiety, it is likely that increases in
3B-diol levels following DHT administration can also alter this behavior (see Handa et
al., in this issue). Consequently, Tﬁn male rodents with nonfunctional ARs provide a
useful and novel method for exploring the role of the AR in anxiety-related behaviors,

while avoiding some limitations of DHT treatment.

26

Studies using Tfm mice indeed suggest that ARS are involved in the anxiolytic
actions of androgens. Rizk et al. (2005) found that, as a group, mice carrying the Tfm
allele (Tfm males and Tfm carrier females) showed increased indices Of anxiety on the
elevated plus maze compared to wt male and female mice. Furthermore, in a novel
Object exposure test conducted in our laboratory, Tfm male mice tend to spend less time
exploring the novel object in an open ﬁeld compared to wt males, indicating that mice
without functional ARS show greater anxiety-related behavior (Fig 3a). This difference
does not appear to depend on inherent differences in circulating T levels between Tfm
and wt males, as androgen treatment, which increases the time spent with a novel Object
in castrated wt males, does not alter the behavior of Tfm males in this test (Fig 3b).

Androgens play a critical role in the regulation of the hypothalamic-pituitary-
adrenal (HPA) axis, with administration of T or DHT reducing the rise Of stress
hormones (adrenocorticotropic hormone (ACTH) and corticosterone) following exposure
to a stressful situation (Handa et al., 1994; Lund et al., 2004; Lund et al., 2005; Lund et
al., 2006). Increased HPA axis activation is Often correlated with increased anxiety
(Lund et al., 2005; Salome et al., 2004), which suggests that androgens may regulate the
display of anxiety-related behavior by depressing HPA axis activation. In a recent study
comparing activation Of the HPA axis in Tfrrr and wt male mice, we found that Tfm
males show an increased release of corticosterone following exposure to an Open ﬁeld
with a novel object (Fig 4), indicating that the increased anxiety-related behavior in Tﬁn

male mice described above may be related to hyperactivation of the HPA axis.

Play Fighting and Aggression

27

Play ﬁghting (which includes two major components, playful attack and playﬁil defense)
is a common juvenile behavior in many mammalian species and appears to be regulated
by gonadal hormones. In rats, play ﬁghting, also called rough-and-tumble play, peaks
between postnatal days 30-40 (Thor and Holloway, 1984), and occurs more frequently in
males than females (Olioff and Stewart, 1978; Pellis and Pellis, 1990). Play ﬁghting is
decreased in male rats castrated at birth and its frequency begins to decline at the onset Of
puberty (Beatty et al., 1981; Meaney, 1988; Pellis and Pellis, 1990). Analysis of Tfm
male rats suggests that ARS are involved in the development Of play ﬁghting behavior
since, as juveniles, Tfm males Show decreased play ﬁghting behavior compared to wt
males (Meaney et al., 1983; Meaney, 1988), although recent data suggest that this
difference may depend on the testing paradigm (Field et al., 2006). Tfm males also fail
to show the male-typical decline in playful attack with age, but like wt males they do
Show a decline in some aspects Of playful defense, indicating that ARS are involved in
some but not all aspects of play ﬁghting (Field et al., 2006).

Unlike juvenile play ﬁghting behavior, aggression in adulthood may rely more
heavily on the activation of ERS. In male mice, castration decreases aggressive behavior
while administration of T, E2, DHT, or a combination of E2 and DHT have been shown
to increase male aggressive behavior (Gandelman, 1980; Matochik et al., 1994; Burge
and Edwards, 1971; Finney and Erpino, 1976; Luttge and Hall, 1973). Gonadally intact
Tfm mice also Show decreased aggressive behavior compared to intact males, suggesting
that ARS play a role in mediating this behavior (Ohno et al., 1974). However, when adult
wt and Tfm male mice were castrated and supplemented with exogenous E2, they showed

similar levels of aggression, indicating that low levels of circulating testicular hormones,

28

and not dysfunctional ARS, underlies decreased aggression in gonadally intact Tﬁn male
mice (Scordalakes and Rissman, 2004). Studies using hormone receptor knockout mice
also indicate that aggression may be mostly, if not entirely, mediated through activation
Of estrogen receptors, with enhancement Of aggression via ERa activation and inhibition

via ERB activation (Scordalakes and Rissman, 2004; Nomura et al., 2002).

Sexual/Social Behavior
The masculinization and defeminization Of male sexual behavior in rodents are
traditionally believed to rely on the conversion of androgens to estrogens and the
subsequent activation Of ERS during the perinatal period, a hypothesis most recently
supported by data from mice deﬁcient in either aromatase or ERS (Ogawa et al., 2000;
Matsurnoto et al., 2003; Ogawa et al., 1997). However, newborn male rats administered
an aromatase inhibitor retain masculine sexual behaviors (Dominguez-Salazar et al.,
2002), while genetically produced AR knockout mice (ARKO) show less masculine
sexual behavior (Sato et al., 2004), suggesting that ARS are also involved in male sexual
behavior. Tﬁn male mice, similar to ARKO mice, also display impaired male sexual
behavior (Ohno et al., 1974), but this impairment in coital behavior (mounts and thrusts),
is not observed after E2 replacement in adulthood (Bodo and Rissman, 2007). This
ﬁnding indicates that AR activation during development is Of little importance for the
expression of coital male behavior in mice, or that sufﬁcient ER stimulation can
overcome a lack of AR activation.

Masculinization Of other sex-related behaviors, such as partner preference in

mice, does appear to rely on the presence Of a functional AR during development. For

29

example, while wt males prefer females, Tfm males act like wt females in showing no
partner preference, even when E2 is equated across groups (Bodo and Rissman, 2007).
Tfm males and females also prefer to explore male-soiled bedding, while males prefer
exploring female-soiled bedding (Bodo and Rissman, 2007). Expression of the
immediate early gene c-Fos in the MPOA and BST was also elevated in Tfm males and
females but not in wt males after exposure to male-soiled bedding (Bodo and Rissman,
2007). Together these data suggest that ARS are necessary for the masculinization of at
least some aspects of sexual and social behavior in mice. Similarly, people with CAIS do
not differ from genetic (XX) females in sexual orientation, are just as likely to be
married, and as children were involved in female typical play (Money et al., 1968; Hines
etaL,2003)

In rats, defeminization Of sexual behavior appears to be AR-independent. Olsen
(1979) found that neonatally castrated Tfm male rats, compared to intact Tfm males,
showed an increased display of lordosis, indicating that functional ARS are not necessary
for the defeminization of this behavior, but rather androgens normally act through
another mechanism (presmnably ERS) to defeminize lordosis. The role Of ARS in the
development of masculine sexual behavior (i.e., mounting behavior), however, is not as
clear. Tfm male rats have been reported to show variable, but consistently reduced
sexual behavior compared to wt males (Beach and Buehler, 1977; Hamson et al., 2005;
Olsen and Whalen, 1981; Olsen, 1992; Shapiro et al., 1976). Interestingly, c-FOS
expression following a sexual encounter was similar in relevant brain areas (such as the
medial preoptic area (MPOA) and medial amygdala (MeA)) of wt and Tfm males that

showed comparable levels Of sexual behavior (Hamson et al., 2005), suggesting that

30

activation of relevant neural circuitry may be normal in Tfm rats despite the lack of
functional ARS.

Diminished sexual behavior in Tﬁn male rats may actually have little to do with
their motivation, since partner preference is masculinized in Tfm male rats (Hamson et
al., 2005; unlike in Tfm male mice), and more to do with a female’s disinterest in them.
Tfm male rats make fewer ultrasonic vocalizations during sexual encounters compared to
wt males, a deﬁcit that may reduce the receptivity of estrous females (Hamson et al.,
2006). For Tfm animals in both species, we also cannot discount the inﬂuence of having
a female genital phenotype on male sexual behavior because, as Frank Beach was fond of
saying, “it’s hard to be a good carpenter without a hammer”.

In conclusion, just as Tfm models indicate that AR normally contributes to
masculine development of every neural region examined so far, Tfm models also indicate
that AR normally contributes to masculine development of a wide range of behaviors.

We summarize these ﬁndings in Table 2.

Structure/function relationships in Tfm rodents

Thus far, little evidence has been generated to suggest a strong correlation between
behavior and brain morphology in Tfm males. However, morphological differences have
been found in brain areas that have been associated with particular behaviors. One such
example includes differences in hippocampal morphology and spatial memory. Studies
in Tfm mice and rats suggest that functional ARS are beneﬁcial for optimal spatial
memory performance (Jones and Watson, 2005; Rizk et al., 2005), and differences in

GCL morphology have been reported in both species (Jones and Watson, 2005; Tabibnia

31

et al., 1999). However, morphological differences are not the same in the two Tfm
models and it is difﬁcult to interpret how an increased GCL volume in Tfm male rats, or
a lack of GCL laterality in Tfm male mice, could result in spatial memory impairment. It
is possible that morphological differences in unexamined hippocampal regions, such as
CA1 of Tfm males, contribute to this deﬁcit.

As reviewed above, several brain areas believed to regulate sexual behavior in
rodents (MePD, VMH, SDN-POA, BST) are demasculinized in Tfm male rats, and thus
may contribute to their reduced sexual behavior (Beach and Buehler, 1977; Shapiro et al.,
1976; Hamson et al., 2005). In this instance, it is easier to infer how demasculinization of
sex behavior-related circuitry could lead to a demasculinization of behavior. Ultrasonic
vocalizations, which are disrupted in Tfm males, appear to rely heavily on androgens
acting in the VMH (Harding and McGinnis, 2003; Harding and McGinnis, 2004), so the
demasculinization of this nucleus in Tfm males (Dugger et al., 2007) may contribute to
this deﬁcit in masculine behavior. Furthermore, along with its role in sexual behavior,
the BST is also an important component of the circuitry involved in the regulation of the
HPA axis and anxiety-related behavior. Therefore, altered BST morphology in Tfm
males could potentially underlie differences in HPA axis physiology and anxiety. Of
course, it is also possible that structural changes in the brains of Tfm male rats and mice

are unrelated to the differences observed in their behavior.

Conclusion
The Tfm models have provided overwhelming evidence that functional ARS are indeed

necessary for the full masculinization of rodent brain and behavior, and studies Of CAIS

32

strongly implicate a role of ARS in human behavior as well. Certain rodent brain sexual
dimorphisms rely heavily on the presence Of functional ARS (MePD, VMH, LC),
whereas others do to a lesser extent (SDN-POA), and yet others appear largely unaffected
by ARS (septal AVP innervation).

However, there is more to be learned, and there are many holes in the Tfm story.
In terms of differences in brain morphology, very little has been examined in the Tfm
mouse for at least two reasons: (1) sex differences in the rat brain are better documented,
and (2) Tfm male mice also have much lower T levels than wt males in adulthood,
making it more difficult to attribute morphological differences to the AR per se. Few
studies of Tﬁn mouse behavior have provided exogenous T to equilibrate this factor as
was done for Figure 3. It is also not known when during development T titers begin to
decline in Tfm male mice, although, as mentioned earlier, indirect evidence suggests that
T levels are normal during the pre- and early postnatal period (Goldstein and Wilson,
1972). One important beneﬁt of the Tﬁn mouse model is that, unlike in the Tfm rat, the
AR is completely nonfunctional, making it easier to detect contributions of ARS.
Furthermore, genetic tools and approaches are already available in mice, Offering more
opportunities to dissect the differing contributions of the AR in various tissues.

For the behaviors discussed in this review, analysis has Often been performed in
only one of the two models or in some cases experiments were performed using Tﬁn
mice that were not supplemented with hormones in adulthood to equate levels in wt and
Tfm males. The strength and generalizabilty of previous ﬁndings would beneﬁt from
addressing these issues. There are undoubtedly other differences in the brain and

behavior of Tfm and wt males that have yet to be discovered, some of which could

33

involve non-neuronal mechanisms. It is possible that AR deﬁciency could affect the
morphology and number of glia such as astrocytes, and in turn inﬂuence behavior.

In many ways, the Tfm rodent model can serve as a starting point upon which the
role Of ARS in brain morphology and behavior can be explored, but it has its limitations.
Standard use of these models does not resolve the issue of whether any differences are
the result of organizational or activational inﬂuences of hormones, since the Tfm defect is
present throughout ontogeny. We know that in the MePD, volume and soma size are
dependent on adult androgens, so in other cases in which brain morphology differs in
Tfm males, the notion that adult AR activation can alter morphology should not be
discounted. Another question concerns cellular targets that mediate a particular androgen
effect, and this is not readily answered with traditional Tfm models. For example, the
cell type(s) (neurons, astroctyes, oligodendrocytes) that are the site of action for ARS to
alter morphology or behavior are difﬁcult to determine using this model. The
development of conditional knockout models (in which the AR is deleted only in
neurons, or only in astrocytes, or in both at particular times in the lifespan) could help
answer such questions (see J untti et al., 2008, in this issue). First, however, it would be
beneﬁcial to learn more about the role of global AR deﬁciency using the Tfm rodent
models. Future research will undoubtedly add to what we have already learned from the
Tfm models which, simply stated, is that the AR does play an important role in the

masculinization of rodent brains and behaviors.

34

Chapter 3: Mice with the Testicular Feminization Mutation
Demonstrate a Role for Androgen Receptors in the Regulation of
Anxiety-Related Behaviors and the Hypothalamic-Pituitary-Adrenal

Axis

Abstract

Testosterone (T) appears to play a role in anxiety and sensorimotor gating in rodents, but
whether T acts through the androgen receptor (AR) to inﬂuence these behaviors is less
clear. We compared adult genetic male mice with the testicular feminization mutation
(Tfm), which lack functional ARS, to wild type male littermates (wt males) on an assay of
sensorimotor gating (prepulse inhibition Of the acoustic startle response; PPI) and several
tests thought to reﬂect anxiety: open ﬁeld exposure, novel object exposure, elevated plus
maze (EPM), and light/dark (LD) box. PPI was similar between groups, but indices of
anxiety in the novel object and LD box tests were increased in Tfm males with no
signiﬁcant differences found in the open ﬁeld or EPM. Since Tfm male mice have
decreased circulating T, the same tests were conducted in mice that were gonadectomized
(wt males) or sham-operated (Tfm males) as adults and supplemented with T or nothing
(B). Although no signiﬁcant group differences were found in PPI, both B and T-treated
Tfm males continued to Show increased anxiogenesis compared to T-treated wt males in
the novel object and LD box tests. Increased indices Of anxiety in Tfm males appear to
be related to hyper-activation of the hypothalamic-pituitary—adrenal axis since levels of

the stress hormone corticosterone were elevated in Tfm males compared to wt males at

35

baseline and at several time points after exposure to a novel object. These ﬁndings

demonstrate that ARS inﬂuence anxiety and stress response.

Introduction
In humans, gonadal hormones have been implicated in the development and maintenance
of several mental disorders including anxiety, depression, and schizophrenia. Women
suffer from anxiety disorders and depression more frequently than do men (Weinstock,
1999; Kornstein, 1997; Wilhelm et al., 1998), and variations in the ovarian hormone
estrogen appear to contribute to the etiology of these disorders in women (Arpels, 1996;
Yazici et al., 2003). Evidence in animal models supports a role of estrogen in mood
disorders. In both male and female rodents, estrogens have been shown to decrease
depressive and anxiety-related behavior (Frye and Lacey, 2001; Walf and Frye, 2005),
particularly through activation of the estrogen receptor (ER) isofonn ERB (Lund et al.,
2005; Imwalle et al., 2005).

Emerging evidence suggests that androgens also contribute to mood disorders.
Boys and girls with low T levels show increased indices of depression and anxiety
compared to those with high T (Granger etal., 2003). Furthermore, in hypogonadal and
aging men who have low levels of circulating T, mood disorders are increased
(Karninetsky, 2005; Lund et al., 1999; Amore, 2005; Eskelinen et al., 2007; Cooper and
Ritchie, 2000). T treatment of these individuals can decrease depressive symptoms and
increase mood (Kumano, 2007; Karninetsky, 2005; Cooper and Ritchie, 2000).

In rodents, androgens have also been shown to play a role in anxiety-related

behavior, with an increase in T generally correlated with decreased indices Of anxiety

36

(Frye et al., 2008; Bing et al., 1998; Bitran et al., 1993). Reducing androgen levels via
gonadectomy in male rodents increases anxiety and fear-related behaviors (Adler et al.,
1999; Bitran et al., 1993; Frye et al., 2008; Frye and Edinger, 2004). Supplementation
with T also decreases indices of anxiety in several rodent models (Frye et al., 2008;
Aikey et al., 2002; Edinger and Frye, 2005). As in hypogonadal men, diminished
testicular production of T in mice is associated with indices of decreased mood (U mehara
et al., 2006). In rodents, aging, which has also been correlated with increased anxiety-
related behaviors (Koprowska et al., 2004; Boguszewski and Zagrodzka, 2002), is
accompanied by a decline in T. A recent study suggests that anxiety-related behavior in
aged male mice can be reduced by administration of androgens (Frye et al., 2008).

One mechanism through which androgens may inﬂuence anxiety-related behavior
is through the regulation Of the hypothalamic-pituitary-adrenal (HPA) axis. While HPA
axis activation is generally adaptive, hyper-activation may be maladaptive and has been
associated with increased indices Of anxiety and depression in both humans and rodents
(Scott and Dinan, 1998; Landgraf et al., 1999; Lund et al., 2005). In rodents,
administration of T, and its metabolites dihydrotestosterone (DHT) and Salpha-
androstane, 3beta,17beta-diol (3 B-diol), can suppress the normal rise of stress hormones
(adrenocorticotropic hormone (ACTH) from the pituitary and corticosterone from the
adrenal cortex) following exposure to a stressful situation (Handa et al., 1994; Lund et al.,
2004; Lund et al., 2005; Lund et al., 2006), suggesting androgens can inﬂuence HPA axis
activity, and in turn, may potentially inﬂuence anxiety-related behaviors.

Gonadal hormones, particularly estrogens, have also been hypothesized to play a

neuroprotective role in schizophrenia, which may contribute to the later onset and milder

37

symptoms Of this disease in women compared to men (Hafner et al., 1998; Castle et al.,
1995). One common characteristic in people with schizophrenia is a deﬁcit in
sensorimotor gating, the capacity to ﬁlter sensory, motor, and cognitive information
(Kodisi and Swerdlow, 1994). This reduction in sensorimotor gating may be linked to
symptoms Of schizophrenia such as information processing deﬁcits, disorders of thought
and auditory hallucinations (Perry and Braff, 1994; Kumari et al., 2008). In an
operational measure of sensorimotor gating, prepulse inhibition Of the acoustic startle
response (PPI; Swerdlow et al., 1996), people with schizophrenia show reduced PPI
indicating a deﬁcit in sensorimotor gating (Braff et al., 1992). This deﬁcit is alleviated
by treatment with atypical antipsychotic medications (Kumari et al., 2002).

Animal models suggest that gonadal hormones can also inﬂuence sensorimotor
gating. In female rats, PPI varies depending on the phase of the estrus cycle (Koch,
1998) and administration Of estrogen to ovariectomized females increases PPI (van den
Buuse and Eikelis, 2001). T has also been shown to facilitate PPI in male and female rats
(Gogos and Van den Buuse, 2003), however, another recent study suggests that in male
rats PPI is unaffected by treatment with androgens or estrogens (Turvin et al., 2007). In
mice the role Of gonadal hormones in PPI has been less studied, and the roles Of speciﬁc
hormone receptors, particularly the AR, in modulating PPI are largely unknown, though
studies in aromatase knockout (ArKO) males suggest that estrogenic metabolites of
androgen acting through ERS can inﬂuence PPI (van den Buuse et al., 2003; Gogos et al.,
2006)

One model for exploring the role of the AR in the brain and behavior is testicular

feminization mutant (Tfm) mice (Zuloaga et al., 2008). Tfm mice have a mutation in the

38

AR gene that involves a single nucleotide deletion (Charest et al., 1991) which introduces
a reading ﬁ'arne shift and premature stop codon, resulting in a shortened transcript and
essentially no AR protein (He et al., 1991; Monks et al., 2007). Consequently these mice
have virtually no sensitivity to androgen through the AR (Drews, 1998). Since this trait
is X-linked, only genetic males (XY) are wholly androgen insensitive. TO ﬁirther
investigate the role of the AR in anxiety-related behavior, regulation of the HPA axis, and
sensorimotor gating in males, we compared behavioral and physiological responses in wt
and Tﬁn male mice. Because Tfm male mice have signiﬁcantly decreased circulating T
levels (Jones etal., 2003) behavior was also assessed in mice provided with exogenous T
in adulthood. These studies indicate that the AR regulates the HPA axis and plays a role
in many behaviors associated with anxiety in mice, but plays little if any role in

regulating PPI.

Method

Animals

C57BL6J mice bred in our Tfm colony at Michigan State University were group housed
with a 12/ 12 L/D cycle, lights on at 0600. The mice in this colony have been sired
exclusively by commercially purchased C57BL6J males for over 10 generations. Upon
weaning at 21 days old, mice were ear punched and genotyped using a modiﬁed
polymerase chain reaction (PCR) described elsewhere (Fernandez et al., 2003). Products
of this reaction differentiated between the Tﬁn and the wt allele for the AR, and male
versus female, based on the presence or absence of the Sry gene found only on the Y

chromosome.

39

Procedure
Experiment 1: To address the role of the AR in sensorimotor gating and anxiety-related
behavior, 90-120 day old wt male (N=9) and Tﬁn male mice (N =10) were tested for PPI

and anxiety-related behaviors as described in detail below.

Experiment 2: Since endogenous T levels differ in Tfm and wt male mice (Tfm<< wt)
and might explain differences in behavior, a second experiment was performed. Wt
males were gonadectomized at 70-90 days Of age and implanted with Silastic capsules
containing T (N =10) or nothing (blank, B; N=9), while Tﬁns were sham-operated and
also implanted with capsules containing T (N =10) or nothing (N =9). Tﬁn males were not
gonadectomized because (1) their testes produce negligible amounts of T (Jones et al.,
2003; present study), and (2) gonadectomy would be more intrusive in these animals
because the testes reside in the abdominal cavity. To better mimic gonadectomy in wt
males, Tfm males received an incision into the perineal cavity (procedure described in
detail below). Three weeks later, mice were assessed for sensorimotor gating and

anxiety-related behavior as described below.

Experiment 3: To assess whether differences in anxiety-related behavior might be

related to differences in HPA axis activation, plasma corticosterone was collected from
90-120 day old wt and Tfm male mice at baseline (wt male: N=10; Tfm male: N=10) or
at 20 min following the onset Of exposure to an Open ﬁeld with a novel object (wt male:

N=10; Tﬁn male: N=10).

40

Experiment 4: To assess whether the time course of HPA axis recovery differed in wt
and Tfm males following exposure to an open ﬁeld with a novel object, 90—120 day old
mice were assayed for corticosterone at baseline (wt male: N=7; Tfm male: N=6) as well
as at 20 (wt male: N=7; Tfm male: N=7), 40 (wt male: N=6; Tfm male: N=7), 60 (wt
male: N=7; Tfm male: N=7), and 120 (wt male: N=6; Tfm male: N=6) min after exposure

to a novel object in the Open ﬁeld testing arena.

Adult castration and hormone replacement

At 70-90 days, wt male mice were anesthetized with isoﬂurane and the testes extemalized
via bilateral incisions made in the scrotum. The vas deferens were tied off with silk
suture prior to cutting. For Tfm males, bilateral incisions were made in the perineal
cavity in a similar location as the scrotum in wt males. Animals also received a
subcutaneous Silastic capsule (1.57 mm inner diameter, 3.18 mm outer diameter: 6 mm
effective length) containing either free T (T) or nothing (blank (B)) via a 2 cm incision
dorsally at the nape of the neck. These Silastic capsules were designed to deliver normal
male physiological levels Of T to adult mice. Incisions were closed with wound clips and

the analgesic buprenorphine (0.1 mg/kg) was injected sc post operatively.

Behavior Testing
Animals in experiments 1 and 2 were tested for sensorimotor gating (PPI) and anxiety-
related behaviors. Testing took place in the following order: PPI, open ﬁeld/novel Object

test, elevated plus maze (EPM), and light/dark (LD) box, with each animal receiving a

41

minimum of 72 hours between tests. All tests were administered between 1000-1400
except for PPI, which was conducted during the dark cycle beginning at 1900. In order to
address the possibility that exposure to the most fear provoking test (PPI) might alter
behavior on the following tests, tests were also conducted in another cohort of intact wt
(n=10) and Tﬁn (n=10) male mice in order from those judged to be the least to most

anxiety-provoking (open ﬁeld/novel Object test, light dark box, elevated plus maze, PPI).

Prepulse Inhibition

Mice were tested for PPI 1 hour after lights off in a room illtuninated by dim red light.
PPI was measured in acoustic startle response chambers (SR Lab startle response system,
San Diego Instruments, San Diego, CA). Animals were placed into the chamber for 18
minutes, the ﬁrst 5 minutes of which is an acclimation period. For the remaining 13
minutes the fast twitch startle responses of mice were recorded via SR Lab software (San
Diego Instruments) to a 100 decibel (dB) tone alone (acoustic startle response), or the
same tone preceded by a 100 msec tone of 3, 8, 10, or 15 decibels. The prepulse should
permit the subject to anticipate the loud pulse, and consequently startle less severely. PPI
was calculated as the percentage decrease in startle response relative to responding to the
100 dB tone without a prepulse. Each of these trials was repeated 6 times at pseudo-
random intervals. After the test, animals were removed and the chamber was cleaned

with 70% ethanol.

Open F ield/Novel object test

42

Open ﬁeld/novel object testing for mice took place in a 16”x 16” Plexiglas chamber

(V ersamax animal activity monitor, Accuscan Instruments Inc, Columbus, OH) that was
illuminated from above. The chamber was equipped with a grid of invisible infrared
beams, and breaks in beams were used to measure movement characteristics such as
rearings (vertical movements that involve removal of the forelimbs from the ﬂoor) and
anxiety-related indices, including the number Of entries into and time spent in the center
area of the ﬁeld or visiting the novel Obj ect at the ﬁeld center. Animals were ﬁrst placed
into a corner Of the empty Open ﬁeld and data were collected for 5 minutes. After 5
minutes mice were removed, the chamber was cleaned with 70% ethanol, and a novel
object (a 2” d x 0.75” h Petri dish with red and blue tape) was placed in the center of the
chamber. Three minutes after ﬁrst removal from the open ﬁeld, the mouse was then
replaced into the chamber and data were collected for another 5 minutes. The chamber

was cleaned with 70% ethanol between each test.

Elevated Plus Maze (EPM)

The EPM (Phenome Technologies Inc.; Lincolnshire, IL) consisted Of two open and two
closed arms (30 x 5 cm) that extended from a center platform and was elevated 50 cm
above the ﬂoor. Testing took place in a dimly lit room with a video camera suspended
above the maze to record behavior for assessment at a later time by an Observer blind to
the experimental condition. During testing, animals were placed in the center area of the
EPM facing an open arm and allowed to freely explore for the 10 minute duration of the

test. The numbers Of entries into and the amount of time spent on the open and closed

43

arms were assessed as indices of anxiety-related behavior. The EPM was cleaned with

70% ethanol between each test.

Light/Dark (LD) Box

The LD box (Phenome Technologies Inc.; Lincolnshire, IL) consisted Of a rectangular
box divided into two regions, a dark (21 cm length x 24 cm width x 19 cm height) and a
light (21 cm length x 24 cm width x 19 cm height) area connected by a (10 cm height x 5
cm width) opening between the two. The dark region was constructed of black plastic
and was covered by a black plastic lid, while the light region was constructed Of clear
Plexiglas and was illuminated by a 60 watt light 3 feet directly above it. The small
opening connecting the two chambers allowed the animals to freely enter either area.
Animals were placed in the light side of the chamber facing the Opening to the dark
chamber and were allowed to move freely between the two compartments for 10 minutes
with behavior recorded via an overhead video camera. The number of entries into, time
spent in the two compartments, and the number of rearings in the light compartment were
scored at a later time as indices Of anxiety-related behavior by an Observer blind to the

experimental condition. The chamber was cleaned with 70% ethanol between each test.

Plasma Collection, Corticosterone and Testosterone Assay

In experiment 3, blood was collected from adult mice between 0900-1100 at baseline or
20 min after exposure to an open ﬁeld with a novel object (a 2” d x 0.75” h Petri dish
with red and blue tape). In experiment 4, blood was collected from adult mice between

0900-1100 at 20, 40, 60, and 120 min after exposure to an open ﬁeld with a novel Object

44

or from control mice that remained in their home cage. Exposure to a novel object in an
Open ﬁeld was for 10 minutes, after which they were returned to their home cage where
they remained until sacriﬁce. Control mice remained in their home cage until sacriﬁce.
Mice were deeply anesthetized with isoﬂurane and decapitated, with trunk blood
collected within 2 minutes of cage disturbance. Blood was also collected from mice in
experiment 2 to verify T levels after supplementation. These mice were deeply
anesthetized with soditun pentobarbital and blood was collected through cardiac
puncture. All blood was collected in 1.5 ml tubes containing 250ul of heparin and held
on crushed blue ice until centrifugation. Samples were centrifuged at 8° C for 20 minutes
and plasma was collected and stored frozen at —20° C. Plasma was assayed for
corticosterone and T at the Diagnostic Center for Population and Animal Health at
Michigan State University using Coat a Count Corticosterone and Coat a Count Total
Testosterone kits (Diagnostics Products Corporation, Los Angeles, CA, USA). All

plasma samples were run in duplicate and the results averaged.

Statistical Analysis

For PPI, a mixed design AN OVA was used to analyze data for all treatment groups
and/or genotypes as between group factors and the different prepulse trials as within
group (or repeated measures) factors. Anxiety-related behaviors were analyzed in
experiment 1 using Student’s t-tests, or in experiment 2 using a 2x2 analysis of variance
(AN OVA) with hormone treatment (T, no T) and genotype (wt male and Tfm male) as
independent factors. TO compare behavioral differences in mice exposed to the PPI test

prior to anxiety tests (test order 1) and mice tested in order from least to most anxiety

45

provoking (test order 2) a 2-way ANOVA was used with genotype and test order (test
order 1, test order 2) as independent factors. In experiment 3, T and corticosterone were
analyzed using a 2x2 analysis of variance (AN OVA) using testing condition (open
ﬁeld/novel object exposure, no manipulation) and genotype (wt male and Tfm male) as
independent factors. In experiment 4 differences in corticosterone were analyzed using a
two-way AN OVA (genotype x time). All signiﬁcant main effects or interactions were
further analyzed using Newman-Keuls multiple comparisons post hoc tests. Differences
were considered signiﬁcant when p<0.05 and all data are reported as means :1: standard

error of the mean (SEM).

Results
Experiment 1.
For PPI, a mixed design AN OVA (genotype as a between groups factors and prepulse
intensity as a within groups factor) revealed an expected effect of prepulse intensity
(F(3,42)= 87.679, p<.001) in which PPI increased as intensity of the prepulse increased.
There was also a signiﬁcant effect of genotype in which wt males showed an overall
greater PPI than did Tfm males (F(1,45)= 4.869, p<.05; Figure 1a). This difference
appeared to be independent of prepulse intensity as there was only an overall effect of
genotype and no signiﬁcant interaction. No signiﬁcant difference in startle response to
the loud acoustic pulse alone was found in this test (wt males: 63.9 a: 6.2; Tfm males:
50.3 i 8.4; in arbitrary units).

In the Open ﬁeld test, no signiﬁcant differences were found between wt and Tﬁn

males in the number of entries into or time spent in the center area of the chamber (Figure

46

2a). There was also no signiﬁcant difference in the total number of rearings, although
there was a trend in which wt males tended to rear more frequently than did Tfms (t(l 7)=
1.971, p=.065; Figure 2e, left-most bars). When mice were exposed to a novel Object,
independent t-tests revealed that wt males entered the center area of the Open ﬁeld
containing the object more frequently (t(l7)= 2.225, p<.05; Figure 2b) and spent more
time visiting the novel Object (t(l7)= 2.177, p<.05; Figure 2b) than did Tﬁn males. The
number of rearings was also greater in wt than Tfm males (t(l 7)= 3.279, p<.01; Figure
2e, middle bars).

In the EPM there were no group differences in the number of entries into the
Open arm of the maze, though wt males did show a trend toward spending more time in
the open arms of the maze than did Tfm males (t(l 7)= 1.781, p=.09; Figure 20). In the
LD box, independent t-tests revealed that wt males showed a greater number of entries
into the light area of the box (t(l 7)= 2.148, p<.05; Figure 2d), spent more time in the light
area (t(l 7)= 2.178, p<.05; Figure 2d), and reared more frequently in the light area (t(l 7)=
2.372, p<.05; Figure 2e, right-most bars) than did Tfm males.

When behavior tests were conducted in another cohort of wt and Tfm male mice,
in order from least to most anxiety-provoking (test order 2), similar group differences
and/or similarities were found for all tests except PPI. Time spent visiting the novel
Object, in the light area of the LD box, and in the open arm of the EPM were greater (or
marginally greater) in wt than in Tfm males ((t(18)= 3.376, p<.01; (t(18)= 2.290, p<.05);
(t(18)= 2.067, p=.057) respectively; data not shown), and again there were no group
differences in the open ﬁeld test. In mice in test order 2 there was no signiﬁcant main

effect of genotype for PPI (Figure 1b) and again ASRs did not differ. As a result of the

47

discrepancy in group differences in PPI mice from test order 1 and 2 were pooled to
determine if there was an overall effect of genotype. This analysis did not indicate a
signiﬁcant main effect Of genotype for PPI. No signiﬁcant main effect Of test order was
found for behaviors in the Open ﬁeld, novel Object test, LD box, EPM, or PPI test
between mice in test order 1 and test order 2, indicating behaviors in Tfm and wt males

were not signiﬁcantly affected by test order.

Experiment 2.

There was a signiﬁcant main effect of T treatment on body weight with no main effect of
genotype or interaction. Speciﬁcally, body weight was greater in T-treated wt males than
all other groups (all p’s<.05; Table 1). Blood concentrations of T also revealed a
signiﬁcant main effect of treatment with no main effect of genotype or interaction.
Animals provided T capsules had circulating T levels that were in the physiological
range, slightly lower than in gonadally intact wt males (Table 1). Castrated wt males
given B capsules had circulating levels of T that approximated those of untreated Tfm
mice in both studies (Table 1).

A mixed design AN OVA using genotype and hormone treatment as between
groups factors and prepulse intensity as a within groups factor again revealed the
expected effect of prepulse intensity (F(3,96)= 48.293, p<.001; Figure 3b) in which PPI
increased as the intensity of the prepulse increased. However, no signiﬁcant main effects
or interactions were found between groups for PPI in this cohort. As in experiment 1,

there were no significant group differences for startle response to the 100 dB pulse alone

(Figure 3a).

48

In the Open ﬁeld test, a 2x2 ANOVA revealed no signiﬁcant main effects or
interactions between groups in the number Of entries into and time spent in the center
area of the chamber (Figure 4a), or in the number Of rearings (Figure 4e, left-most bars).
In the novel Object test there was a signiﬁcant main effect Of genotype (F (1 ,34)= 9.35,
p<.05), with no main effect of treatment or interaction, indicating that as a group Tfm
males spent less time visiting the novel object than did wt males, replicating the results of
experiment 1 in gonadally intact mice. Post hoc comparisons revealed significant
differences between T-treated wt males and all other groups (p’s<.05; Figure 4b, left),
indicating that T increases this measure, but only in wt males and not in Tfm males. A
signiﬁcant main effect Of genotype was also revealed for the number of rearings in the
novel Object test (F(1,34)= 6.714, p<.05), with no main effect of treatment or interaction,
indicating that wt males reared more than did Tfrns. Post hoc comparisons revealed
signiﬁcant differences between T-treated wt males and both Tfm groups (T or no T,
p’s<.05; Figure 4e, middle bars). NO signiﬁcant main effects or interactions were found
for the number of novel Obj ect visits, though there was a trend toward a main effect Of
treatment (F(1,34)= 3.323, p=.076; Figure 4b, right).

In the EPM no signiﬁcant main effects or interactions were found for time spent
on or number of visits to the Open arms (Figure 4c). In the LD box a signiﬁcant main
effect of treatment was found for time spent in the light area (F(1,34)= 4.209, p<.05),
with no signiﬁcant main effect for genotype or interaction. Post hoc tests revealed that T-
treated wt males spent more time in the light area than did either B-treated wt males or T-
treated Tfm males (p’s<.05; Figure 4d, left), though T-treated wt males did not

signiﬁcantly differ from B-treated Tfm males. A signiﬁcant main effect of genotype was

49

found for the number of rearings in the light area of the LD box (F(1,34)= 6.356, p<.05),
with no signiﬁcant main effect of treatment or interaction. Post hoc comparisons
revealed signiﬁcant differences between T-treated wt males and all other groups (p’s<.05;
Figure 4e, right bars), indicating that T-treated wt males reared more than any other
group in the light area of the LD box. No signiﬁcant main effects or interactions were

found for the number of light area visits, although there was a trend toward a main effect

Of treatment (F(l ,34)= 3.966, p=.054; Figure 4d, right).

Experiment 3.

Two-way ANOVA revealed a signiﬁcant main effect of exposure (F(1,38)= 908, p<.001)
in which mice exposed to an open ﬁeld with a novel object showed an increased
corticosterone response 20 min after exposure compared to mice in the basal condition
(left in their cage). There was also a signiﬁcant main effect of genotype (F(1,38)= 177.4,
p<.001) with Tfm males showing an increased corticosterone response compared to wt
males. Post hoc comparisons revealed that Tfm males had greater blood corticosterone
levels at baseline (p<.01) and after exposure to an open ﬁeld with a novel object
(p<.001). A Signiﬁcant interaction was also found between exposure and genotype on
corticosterone concentrations (F (3,36)= 69.1, p<.001), which indicated that the increase
in corticosterone from baseline to that induced by exposure to a novel object was greater
in Tfm males than in wt males (Fig 5a). Two-way AN OVA revealed a signiﬁcant main
effect of genotype on T levels (F(1,38)= 134, p<.001), with no main effect of exposure or

interaction between genotype and exposure. Speciﬁcally, compared to wt males, Tfm

50

males had lower levels of T (Table 1). As expected, body weight was also signiﬁcantly

greater in wt than in Tfrrr males (t(l7)= 3.382, p<.01; Table 1).

Experiment 4.

A 2-way ANOVA conﬁrmed the expected main effect of time on corticosterone levels
(F(4,52)= 55.58, p<.001) indicating that compared to basal corticosterone levels, Tfm and
wt males had greater corticosterone levels overall after exposure to a novel Object. A
main effect of genotype was also revealed in which Tfm males showed overall greater
blood corticosterone concentrations compared to wt males (F(1,55)= 33.67, p<.001).

Post hoc comparisons revealed that Tfrns had higher corticosterone concentrations

compared to wt males at 20, 40, and 60 min (p’s<.05; Fig 5b).

Discussion

Tfm male mice showed increased indices Of anxiety in several behavioral tests (novel
Object and LD box tests), but not others (open ﬁeld and EPM), indicating a task-speciﬁc
increase in anxiety-related behavior in Tfm males compared to wt males. In the novel
object and LD box tests, Tfm males showed decreased exploration of a novel object and
of the light area of the LD box as well as a generalized decrease in exploratory behavior
in these tests, as assessed by the number of rearings. T-treatment in gonadectomized wt
males decreased indices of anxiety in the novel Obj ect and LD box tests suggesting that
T-treatment in adult male mice can produce anxiolysis. However, these behaviors were
unaffected by T treatment in Tfm males, which indicates that T’s anxiolytic action in wt

males is likely mediated through the AR. These ﬁndings support previous research

51

indicating that the AR is involved in the regulation of anxiety-related behavior in rodents
(Edinger and Frye, 2006; Femandez-Guasti and Martinez-Mota, 2005; Rizk et a1, 2005).

Since the Tfm mutation is present from ontogeny, it is unclear whether
differences between wt and Tﬁn males in anxiety-related behavior are the result of
“organizational” or “activational” inﬂuences of ARS (Arnold and Breedlove, 1985).
Other ﬁndings in rodents indicate that activation Of ARS in adulthood plays a prominent
role in regulating anxiety-related behavior (Edinger and Frye, 2006; Femandez-Guasti
and Martinez-Mota, 2005). In our study, increased anxiety-related behavior in wt males
given T capsules versus B capsules conﬁrms that T can act in adulthood to affect this
behavior, although it does not directly implicate the AR. It is possible that decreased AR
activation during development in Tfm males resulted in their increased indices of anxiety.
Little is known about the organizational role of ARs in anxiety, although a study showed
that neonatal treatment of rats with the AR antagonist ﬂutamide did not affect indices of
anxiety in the elevated plus maze (Zimmerberg and Farley, 1993).

Corticosterone levels were also elevated in Tfm males compared to wt males at
both baseline and 20 min after initial exposure to an Open ﬁeld with a novel Object
(experiment 3). During recovery, the corticosterone response remains consistently
elevated in Tfm males compared to wt males at 40 minutes and 60 minutes (experiment
4). These ﬁndings indicate that the increased anxiety-related behavior in Tﬁn male mice
may be related to a hyper-activation of the HPA axis. Previous studies suggested a
relationship between trait anxiety and increased HPA axis activity after exposure to

rodent tests of anxiety. Rats bred for high compared to low anxiety behavior show

52

elevations in stress hormones ACTH and corticosterone that correlate with increased
indices of anxiety in the EPM and open ﬁeld (Landgraf et al., 1999; Salome et al., 2004).

Androgens have been shown to play a role in the regulation of the HPA axis
response to stress in other models. Gonadectomy of adult wt male rats increased the
release of corticosterone and ACTH following footshock stress or exposure to an open
ﬁeld (Handa et al., 1994). Furthermore, T or DHT replacement in gonadectomized rats
decreased the rise in stress hormones to levels similar to those of intact rats (Handa et al.,
1994). Androgen’s inﬂuence on the HPA axis does not appear to be solely activational,
however. Neonatal gonadectomy in male rats increased the corticosterone response to
restraint stress, an increase that is not reduced by adult testosterone propionate (TP)
replacement as it is in rats gonadectomized as adults (McCormick et al., 1998). Neonatal
androgen exposure can also alter the female corticosterone response: female rats
administered TP on the day of birth showed decreased basal and stress induced
corticosterone levels (Scale et al., 2005a). Other evidence supports the notion that the
AR is involved in the organization of the HPA axis (McCormick and Mahoney, 1999;
Seale et al., 2005b). Seale et al. (2005b) demonstrated that perinatal AR blockade, by
administration of the AR antagonist ﬂutarnide, increased both basal and stress induced
corticosterone levels in adulthood compared to rats given vehicle perinatally.
Furthermore, serum corticosterone and ACTH levels are elevated in AR knockout male
mice, in which AR deﬁciency is present from ontogeny (Miyamoto et al., 2007), as in
Tﬁn males.

Previous research therefore indicates that increased corticosterone levels found in

Tfm male mice could be related to organizational inﬂuences, activational inﬂuences, or

53

both, resulting from a lack of functional ARS. However, since Tﬁn males have decreased
levels of T compared to wt males, it is also possible that when provided ample T, Tfm
males would show corticosterone levels similar to wt males. Evidence suggests that T,
after metabolism to DHT, can be converted to 3B-diol which reduces HPA axis activity
via activation Of ERB (Lund et al., 2004; Lund et al., 2005; Lund et al., 2006). Therefore,
without additional experiments, we cannot conclude that the increased corticosterone
levels seen in Tﬁn male mice result directly from a lack Of functional ARs. However,
Tfm male rats, which have elevated T levels compared to wt males, also show an
increased corticosterone response compared to wt males 20 min following exposure to an
Open ﬁeld with a novel object (Zuloaga et al., in preparation). Therefore, even when
provided similar T, Tfm male mice may still continue to show increases in corticosterone
compared to wt males. This outcome would be consistent with our behavioral measures
of anxiety suggesting that Tfm male mice show increased anxiogenesis regardless of
endogenous T levels.

Indirect evidence suggests that in Tfm male mice, T levels are near the normal
male range during the perinatal period (Goldstein and Wilson, 1972) and decrease during
development largely due to a reduction in the testicular enzyme 17a-hydroxylase
(Murphy and O’Shaughnessy, 1991). Thus, Tﬁn male mice may have ample T substrate
to activate ERS perinatally, leading us to suspect that the increased HPA axis activity
seen in Tfm male mice may indeed reﬂect a lack of functional ARS, although when AR is
critical remains unclear.

Since plasma corticosterone binding globulin (CBG) levels were not measured in

this study, it is possible that CBG concentrations are elevated in Tﬁn males and could

54

contribute to differences in corticosterone levels. An increase in CBG levels would
indicate that more corticosterone would need to be released in order for similar
corticosterone receptor binding to occur, because more circulating corticosterone is being
bound by CBGs and is therefore rendered biologically inactive. Increased T has been
shown to decrease plasma CBG concentrations (V iau and Meaney, 2004). Therefore, if
indeed a decrease in circulating T (or a decrease in functional ARS independent of
circulating T) in Tfm males increases plasma CBGS, elevated plasma corticosterone
levels in Tfm males may reﬂect an attempt to achieve normal levels of arousal rather than
a hyper-arousal.

Intact Tﬁn male mice also showed an overall decrease in PPI compared to wt
male mice in test order 1, suggesting that the AR may be involved in sensorimotor gating.
However, in test order 2 there were no group differences in PPI and in experiment 2
neither T treatment nor genotype affected PPI. These ﬁndings suggest that the original
differences found between wt and Tfm male mice may be subtle and somewhat
unreliable. Alternatively, group differences in PPI may have been reduced by prior
exposure to tests of anxiety in intact animals. However, hormone manipulated mice in
experiment 2 were tested for PPI without prior exposure to tests Of anxiety and did not
show differences in PPI, indicating that test order did not contribute to group differences.
Together these data suggest that in mice the role of T and ARS in the regulation of PPI is
minimal. This corresponds with a report of normal PPI in mice with low circulating T
levels (Umehara et al., 2006).

Tfm males provide a useful model for examining the role of ARS in behavioral

and physiological responses, while avoiding some of the limitations of phararnacological

55

AR agonists (e.g., DHT) and antagonists (e.g., ﬂutamide). DHT, along with activating
ARs, can be metabolized to 3a-androstanediol (3a-diol) which has a low afﬁnity for ARS
but a high affinity for GABA receptors. 3a-diol also appears to play a role in anxiolysis
in rodents, as its administration decreases anxiety-related behavior (Frye and Edinger,
2004; Edinger and Frye, 2004; Edinger and Frye, 2005). An estrogenic metabolite, 3B-
diol, which binds ERs with greater afﬁnity for ERB than ERa, can also be derived from
DHT (Kuiper et al., 1998; Lund et al., 2004). 3B-diol administration has also been
demonstrated to reduce anxiety-related behavior and activation Of the HPA axis through
its actions on ERB (Lund et al., 2004; Lund et al., 2005; Lund et al., 2006). Thus effects
of DHT treatment on anxiety-related behaviors and HPA axis activity could be mediated
either through AR, ERB, or GABA receptors.

A limitation of the Tfm model is that Tfm male rats have been reported to show
decreased aromatase activity in several areas of the brain (Rosseli et al., 1987).
Therefore it is possible that T-treatment in Tfm males failed to affect anxiety-related
behavior because there was insufﬁcient ER activation due to a decrease in the conversion
of T to estrogens. However, Tfm male mice have been reported to show both similar and
slightly decreased levels of aromatase activity in the hypothalamus compared to wt males
(Naftolin et al., 1975; Rosenfeld et al., 1977). In the study that indicated a decrease in
Tfm aromatase activity, it was the conversion of T to the less potent estrogen, estrone,
which contributed to an overall decrease in aromatization, while the conversion of T to
E2 was actually elevated in Tfm male mice (Rosenfeld et al., 1977). Estrone, unlike E2,
has a much higher affinity for ERa than ERB, and evidence suggests that a decrease in

ERo. activation would not contribute to increased anxiogenesis (Lund et al., 2005; Krezel

56

et al., 2001). Furthermore, aromatase knockout (ArKO) male mice have been shown to
exhibit normal levels Of anxiety behavior (Dala et al., 2005), suggesting that
aromatization of T to estrogens plays a minimal role in the display of these behaviors in
male mice.

In conclusion, the present ﬁndings in Tfm mice indicate that the AR is involved in
the regulation Of anxiety-related behaviors in males, as demonstrated by differences
between wt and Tﬁn males in some tests of anxiety. Furthermore, these differences in
anxiety-related behavior may be related to an increased activation of the HPA axis in Tfm
males since they Show increased levels of blood corticosterone at baseline as well as at
several time points following exposure to an open ﬁeld with a novel object. Further work
will be needed to explore whether ARS act during development and/or adulthood to affect
anxiety-related behaviors. On the other hand, the role Of the AR in sensorimotor gating
in mice, as indicated by PPI, appears minimal, suggesting that any role of gonadal

steroids on sensorimotor gating may be mediated via estrogen receptors rather than ARS.

57

Chapter 4: Elevated Anxiety-Related Behavior and Corticosterone

Response in Rats with the Testicular Feminization Mutation

Abstract

Testosterone has been shown to inﬂuence anxiety and sensorimotor gating in rodents but
the role of a target receptor, the androgen receptor (AR), in mediating these inﬂuences is
unclear. In this study male rats with the testicular feminization mutation (Tfm), which
lack functional ARS, were compared to wild type (wt) male and female littermates on an
assay of sensorimotor gating (prepulse inhibition (PPI) Of the acoustic startle response
(ASR)) and tests of anxiety (open ﬁeld, novel object exposure, elevated plus maze
(EPM), and light/dark (LD) box). NO differences were found for PPI, although Tfm
males had an increased ASR compared to wt males and females. Tﬁn males also showed
increased indices Of anxiety compared to wt males and females in the open ﬁeld and
when exposed to a novel object, with no signiﬁcant differences found in the EPM or LD
box. When exposed tO a novel object, Tﬁn males showed a peak corticosterone response
similar to wt females and greater than wt males, although baseline and recovery levels
did not differ from wt males. Analysis of the immediate early gene c-Fos revealed
activation of several brain areas after exposure to an open ﬁeld with a novel object, with
greater activation in Tfm males compared to wt males in some regions (medial preoptic
area) and lesser activation in others (dentate gyrus, posterodorsal medial amygdala).
These ﬁndings demonstrate that ARS play a role in the regulation of some anxiety-related

behaviors in rats, but have little or no role in sensorimotor gating.

58

Introduction

In humans, gonadal hormones play an important role in the regulation of anxiety and
sensorimotor gating (the capacity to ﬁlter sensory, motor, and cognitive information).
Women are diagnosed with disorders of anxiety and unipolar depression more than men,
and decreases in mood often coincide with a decline in levels of the ovarian hormone
estrogen during menopause (Arpels, 1996). Furthermore, estrogen replacement therapy
has been reported to increase mood and decrease anxiety in postmenopausal women
(Yazici et al., 2003). In men a similar but less abrupt decline in androgen levels is also
often accompanied by increased symptoms of anxiety and depression (Kaminetsky, 2005;
Lund et al., 1999; Eskelinen et al., 2007; Cooper and Richie, 2000). Androgen treatment
of these men, or of younger men with decreased testicular production of testosterone (T)
alleviates some of these symptoms (Kaminetsky, 2005; Cooper and Richie, 2000;
Kumano, 2007). Similarly, boys and girls with low T levels Show greater indices of
depression and anxiety than those with high T (Granger et al., 2003).

There are also sex differences in sensorimotor gating (men > women; Swerdlow
et al., 1993) as assessed by an experimental model of sensorimotor gating, prepulse
inhibition of the acoustic startle response (PPI; Swerdlow et al., 1996). Furthermore, PPI
varies in women according to stage of the menstrual cycle suggesting that variations in
sex hormones can inﬂuence PPI (Swerdlow et al., 1997; Jovanovic et al., 2004).

As in humans, hormones also appear to play a role in the display of anxiety-
related behaviors and sensorimotor gating in rodents. In rats there are sex differences in
tests Of anxiety with females Often showing decreased anxiety-related behaviors and

greater activity compared to males (Archer, 1975; Masur et al., 1980; Slob et al., 1981;

59

Seliger, 1977; Lucion et al., 1996). Furthermore, administration of both estrogens and
androgens generally result in decreased indices Of anxiety in rodents (Frye and Lacey,
2001; Walf and Frye, 2005; Lund et al., 2005; Frye et al., 2008; Bing et al., 1998; Bitran
etal., 1993). Evidence suggests that the anxiolytic actions of estrogens are largely
mediated through activation of the estrogen receptor (ER) isoform ERB (Lund et al.,
2005; Imwalle et al., 2005). On the other hand, the actions of androgens may be
mediated through several mechanisms including androgen receptors (ARS), ERS, and/or
GABA receptors (Edinger and Frye, 2006; Lund et al., 2005; Edinger and Frye, 2005).

Along with inﬂuencing anxiety, sex hormones have also been shOwn to mediate
hypothalamic-pituitary-adrenal (HPA) axis activity (Handa et al., 1994; Lund et al.,
2005), which in turn may affect behavior in rodents exposed to an aversive situation. In
particular, T decreases while estrogen increases the release of stress hormones
adrenocorticotropic hormone (ACTH) from the pituitary gland, and corticosterone from
the adrenal cortex. Speciﬁc activation of sex hormone receptors ERa and ERB increase
and decrease HPA axis activity respectively, while AR activation also appears to
decrease activity, although its role is less clear (Handa et al., 1994; Lund et al., 2004;
Lund et al., 2005; Lund et al., 2006).

Sensorimotor gating in rodents, like humans, is inﬂuenced by sex hormones. Sex
differences in PPI have been reported in some strains of rats and mice (Lehman et al.,
1999; Ralph et al., 2001; Ison and Allen, 2007) and PPI is inﬂuenced by estrus cycle in
rats (Koch, 1998). Administration of estrogens and androgens has also been

demonstrated to affect PPI (van den Buuse and Eikelis, 2001; Gogos and Van den Buuse,

60

2003), though little is known about the role of speciﬁc hormone receptors, including the
AR, that may be mediating these changes.

To investigate the role Of ARS in anxiety-related behaviors, regulation of the HPA
axis, and PPI, we compared behavior and hormonal (corticosterone) responses in wild
type (wt) male, wt female, and testicular feminization mutant (Tfm) male rats with
dysfunctional ARS. To further probe the role of ARS in males, corticosterone recovery
and neural activation were also measured in Tfm and wt male rats at baseline and after
exposure to an anxiety-provoking situation. Tfm rats are a genetic model for exploring
the role Of the AR in the brain and behavior (Zuloaga et al., 2008a). In rats this mutation
results from a single base pair replacement in the AR gene (Yarbrough et al., 1990), and
thus there is only a single amino acid difference between wt and Tfm AR protein that
results in expression of a normal sized but largely dysfunctional AR protein in Tfm rats
(Yarbrough et al., 1990). Since this trait is X-linked, only genetic males (XY) are

androgen insensitive.

Materials and Methods

Animals

Long Evans rats, which were bred with commercially purchased Long Evans (Charles
River) sires for over 10 generations, were group housed in our Tfm colony at Michigan
State University with a 12/ 12 L/D cycle, lights on at 0600. Upon weaning at 21 days old,

car punches were performed to obtain DNA for determining whether rats were wt male,

61

wt female, Tfm male, or female Tfm carriers. Rats were genotyped using a modiﬁed
polymerase chain reaction (PCR) to detect the Tfm versus the wt allele for the AR, and to
detect the presence or absence of the Sry gene found only on the Y chromosome
(Fernandez et a1, 2003). Wt males, wt females, and Tfm males were used in the
following experiments. All animals received care that meets standards of the National

Institutes of Health and all experiments were approved by the MSU IACUC.

Experiment I : The role ofARs in sensorimotor gating and anxiety-related behavior.
One hundred twenty-150 day Old wt male (N=9), wt female (N=11), and Tfm male rats
(N =10) were tested for PPI and anxiety-related behaviors as described below.
Experiment 2: The role of ARs in HPA axis regulation. Plasma corticosterone was
collected from 120-180 day Old rats at baseline (wt male: N=10; wt female: N=10; Tfm
male: N=10) or at 20 minutes after initial exposure to an open ﬁeld with a novel object
(wt male: N=10; wt female: N=10; Tfm male: N=10).

Experiment 3: The role of ARs in HPA axis recovery. One hundred twenty-180 day Old
rats were assayed for corticosterone at baseline (wt male: N=7; Tfm male: N=6), 20 (wt
male: N=7; Tfm male: N=7), 40 (wt male: N=6; Tﬁn male: N=6), 60 (wt male: N=6; Tfm
male: N=6), and 120 (wt male: N=6; Tfm male: N=6) minutes after exposure to an Open
ﬁeld with a novel Object. For this experiment we chose to focus only on wt and Tfm
males in order to further probe the role of ARs in the male corticosterone response to
stress. Furthermore, sex differences in the rodent corticosterone response to stress are
already well documented (Handa et al., 1994b; reviewed in Kudielka and Kirschbaum,

1995).

62

Experiment 4: The role of ARs in immediate early gene activation. To investigate how
speciﬁc brain areas may be activated differently due to the presence or absence of
functional ARS in males, we examined activation of the immediate early gene c-F OS in
120-150 day Old Tﬁn and wt male rats at baseline (wt male: N=7; Tfm male: N=7) and

after exposure to an open ﬁeld with a novel Object (wt male: N=7; Tfm male: N=7).

Behavior Testing

Animals in Experiment 1 were tested for sensorimotor gating (PPI) and anxiety-related
behaviors. Testing took place in the following order: PPI, Open ﬁeld/novel object test,
elevated plus maze (EPM), and light dark (LD) box, with a minimum of 72 hours
between tests. All tests were administered between 1000-1400 except for PPI, which was
conducted during the dark cycle beginning at 1900. TO address the possibility that
exposure to the most fear provoking test (PPI) can alter behavior in tests that follow,
anxiety tests were also conducted on another cohort of intact wt male (n=11), wt female
(n=6) and Tfm rats (n=9) in an order that we deemed was least to most anxiety-provoking

(open field/novel object test, LD box, EPM) without testing for PPI.

Prepulse Inhibition

Rats were tested for PPI 1 hour after lights Off in a room illuminated by dim red light.
PPI was measured in acoustic startle response chambers (SR Lab startle response system,
San Diego Instruments, San Diego, CA). Animals were placed into the chamber for 18
minutes, the ﬁrst 5 minutes of which is an acclimation period. For the remaining 13

minutes the fast muscle twitch startle responses of animals were recorded to a 100 decibel

63

tone alone (acoustic startle response; ASR), or to that same tone preceded by tones Of 3,
8, 10, and 15 decibels via SR Lab software (San Diego Instruments). The prepulse
should permit the subject to anticipate the loud pulse, and consequently startle less
severely. Each of these trials was repeated 6 times at pseudo-random intervals. After the

test, animals were removed and the chamber was cleaned with 70% ethanol.

Open F ield/Novel object test

Open ﬁeld/novel Object testing took place in a 122cm x 122cm white plastic box
illuminated from directly above by a 60 watt light. A grid was drawn in the box to
demarcate entries into the center area and activity (grid crossings). Rats were ﬁrst placed
into a corner of the empty open ﬁeld and behavior was recorded via an overhead video
camera for 5 minutes. After 5 minutes rats were removed, the box was cleaned with 70%
ethanol, and a novel object (a 4” diameter x 8” high cylindrical metal oxygen tank cap)
was placed in the center Of the chamber. Three minutes after removal from the Open
ﬁeld, the rat was replaced into the chamber which now contained the novel Object and
behavior was recorded for another 5 minutes. The box was again cleaned with 70%
ethanol following the novel object test. The number of entries into the center area, time
spent in the center area, visits to the novel object, and time spent visiting the novel Object
were assessed as indices of anxiety-related behavior. The number of grid crossings and
rearings were assessed as measures Of activity and all behaviors were coded at a later

time by a “blind” observer.

Elevated Plus Maze (EPM)

64

The EPM consisted of two open and two closed arms (50 x 10 cm) that extended from a
center platform and was elevated 50 cm above the ﬂoor. Testing took place in a dimly lit
room in which animals were placed in the center area of the EPM facing an Open arm and
allowed to move freely between the arms for the 10 minute duration of the test. Behavior
was recorded using an overhead video camera and was coded at a later time by a blind
observer. The number of entries into and the amount of time spent on the Open arms
were assessed as indices of anxiety-related behavior. The maze was cleaned with 70%

ethanol between each test.

Light/Dark (LD) Box

The LD box consisted of a rectangular box divided into two regions, one dark (40 cm
length x 38 cm width x 29 cm height) and one light (31 cm length x 28 cm width x 29 cm
height). The dark region was constructed of black plastic and was covered by a black lid,
while the light region was constructed of transparent Plexiglas and was illuminated by a
60 watt light that was 3 feet directly above it. The two chambers were connected by a
small Opening (7.5 height x 7.5 cm width) that allowed the animals to freely enter either
area. Animals were placed in the light side of the chamber facing the opening to the dark
chamber and were allowed to move freely between the two compartments for 10 minutes.
Behavior was recorded via an overhead video camera for coding at a later time by a blind
Observer. The number of entries into and time spent in the two compartments, as well as
the number of rearings in the light compartment, were assessed as indices of anxiety and

activity.

65

Plasma Collection and Hormone Assays

In Experiment 2 blood was collected from adult rats between 0900-1100 at baseline or 20
min after initial exposure to the open ﬁeld with a novel object. In Experiment 3 blood
was collected from adult rats between 0900-1100 at 20, 40, 60, and 120 minutes after
exposure to an Open ﬁeld with a novel object or at baseline. Novel object exposed rats
were placed in an Open ﬁeld with a novel object for 10 minutes after which they were
returned to their home cage where they remained until sacriﬁce. Baseline rats remained
in their home cage until sacriﬁce. Rats were deeply anaesthetized with isoﬂurane and
decapitated, with trunk blood collected within 2 minutes of cage disturbance. All blood
was collected in 1.5 ml tubes containing 250ul of heparin and held on crushed blue ice
until centrifugation. After centrifugation plasma was collected and frozen at —20° C until
the assay was performed. Plasma was assayed for corticosterone and T at the Diagnostic
Center for Population and Animal Health at Michigan State University using Coat- A-
Count Corticosterone and Coat-A-Count Total Testosterone kits according to the
manufacturer’s instructions (Diagnostics Products Corporation, Los Angeles, CA, USA).
All plasma samples were run in duplicate and results were averaged. Intraassay and
interassay coefﬁcients of variation were < 5 and < 7%, respectively for corticosterone and

< 12 and < 7%, respectively for testosterone.

Tissue Processing and c-Fos Immunocytochemistry
One hour after a 10 minute exposure to an Open ﬁeld with a novel Object, or at baseline,
rats were administered an intraperitoneal overdose of pentobarbital, and perfused through

the heart with 100 ml of 0.9% saline followed by 200 m1 of phosphate-buffered 4%

66

paraforrnaldehyde. Brains were removed, placed in 4% paraforrnaldehyde, and
reﬁigerated overnight. The following morning brains were transferred into a 20%
sucrose solution where they remained until sectioning. No later than one week after
collection, brains were sectioned at 40 um on a freezing microtome into three alternative
series and stored at -20° C in cryoprotectant. For c-Fos labeling, one series of sections
was rinsed in several changes of 0.05 M tris buffered saline (TBS) to clear cryoprotectant
and then incubated as free-ﬂoating sections in 0.1% sodium borohydride in TBS for 15
minutes to clear residual ﬁxative. Sections were then rinsed in TBS, incubated in 1%
hydrogen peroxide and 0.3% Triton-X in TBS (TBS-TX) for 10 minutes, again rinsed in
TBS, then incubated in 20% normal goat serum (N GS) in TBS-TX for 25 minutes. After
rinsing in TBS, tissue was incubated in primary antisera (c-Fos rabbit polyclonal:
1:10,000, Santa Cruz Biotechnology) in 2% normal goat serum and TBS-TX for 48 hours
at room temperature. Tissue was then rinsed in TBS and incubated for 1 hour in
biotinylated goat-anti rabbit antibody in TBS-TX (1:500, Vector Laboratories,
Burlingame, CA) followed by rinses in TBS and a 1 hour incubation in avidin-biotin
complex (ABC Elite kit, Vector Laboratories, Burlingame, CA). Following rinses in
TBS, tissue was developed for visualization of c-F OS positive cells in a hydrogen
peroxide, diaminobenzidine, and nickel solution for 5 minutes, after which sections were
rinsed in TBS and immediately mounted on slides. The following day sections were

counterstained with neutral red, dehydrated, defatted, and coverslipped with Permount.

Microscope analysis

67

Analysis of c-Fos positive cells was conducted on a Nikon light microscope equipped
with a video camera and Bioquant stereological software (Bioquant, Nashville, TN,
USA). Brieﬂy, anatomical position was determined using a stereotaxic atlas (Paxinos
and Watson, 1998). Discrete brain regions were outlined using a 20x objective and cells
considered c-Fos positive (containing black nuclear label) within this region were
counted. C-Fos positive cells were quantiﬁed in androgen and/or stress responsive brain
areas including the basolateral amygdala (BLA), medial amygdala (MBA), posterodorsal
medial amygdala (MePD), ventral lateral septum (VLS), posteromedial nucleus of the
bed nucleus of the stria terminalis (BSTMPM), paraventricular nucleus Of the
hypothalamus (PVN), ventromedial nucleus of the hypothalamus (VMH), medial
preoptic area (MPOA), dentate gyrus (DG), and CA1. The observer was blind to the

experimental condition of the tissue.

Statistical Analysis

For PPI, a mixed design repeated measures analysis of variance (ANOVA) was used to
analyze data with genotype as a between groups factor and prepulse intensity as within
(or repeated) groups factor. ASR and anxiety-related behaviors were analyzed using one-
way AN OVAs. Acoustic startle response by trial was analyzed using a repeated
measures 2-way ANOVA with ASR trial order (1-6) and genotype as factors. To
compare differences in anxiety-related behaviors in rats exposed to either the PPI test
prior to anxiety tests (test order 1) and not exposed to the PPI test (test order 2), 2-way
AN OVAS using genotype and test order as factors were performed. In experiment 2,

testosterone and corticosterone concentrations were analyzed using a 2-way AN OVA

68

with testing condition (novel object exposure, no manipulation) and genotype (wt male,
wt female, and Tfm male) as factors. In experiment 3 differences in corticosterone were
analyzed using a two-way AN OVA (genotype x time). Separate 2-way AN OVAs were
run for each brain region analyzed for C-Fos expression with testing condition and
genotype as factors. All signiﬁcant main effects or interactions were further analyzed
using Tukey’s post hoc tests. Differences were considered signiﬁcant when p<0.05 and
all data are reported as means :t standard error of the mean (SEM) with N= the number of

animals.

Results

Experiment 1: The role of ARs in sensorimotor gating and anxiety-related behavior

A mixed design repeated measures ANOVA for PPI revealed the expected effect of
prepulse intensity (F(3,108)= 3.793, p<.01; Figure la) in which PPI increased along with
an increase in prepulse decibel intensity, with no main effect of genotype or interaction
between genotype and prepulse intensity. Analysis of ASR revealed a signiﬁcant main
effect of genotype (F(2,27)= 4.668, p<.01). Post hoc comparisons revealed that Tfm
males showed an increased ASR compared to both wt males and females (ps<.05; Figure
1b). An analysis of ASR by trial revealed a signiﬁcant main effect of trial (F (5,162)=
4.789, p<.01) indicating that, as a whole, rats showed a decrease in ASR, particularly
after the initial ASR trial. There was also a signiﬁcant main effect of genotype
(F(2,162)= 14.72, p<.01) and a genotype x trial interaction (F(10,162)= 4.789, p<.01),
again because Tfm males showed an elevated ASR. Post hoc comparisons revealed that

ASR was elevated in Tfm males compared to wt males and females only in the initial trial

69

(ps<.001; Figure 1c), and this difference largely accounted for the overall increase in
ASR in Tﬁn males.

In the Open ﬁeld test there was a signiﬁcant effect Of genotype for time spent in
the center area of the chamber (F(2,27)= 9.738, p<.01), and the number of entries into the
center area of the chamber (F (2,27): 4.111, p<.05). Post hoc comparisons revealed that
Tfm males Spent less time in the center area of the open ﬁeld compared to both wt males '
and females (ps<.05 and .01 respectively; Figure 2a) and visited the center area less than
did wt females (p<.05; Figure 2a). There was also a signiﬁcant sex difference in the
number Of rearings in the open ﬁeld (F (2,27)= 4.790, p<.05), as wt females reared more
frequently than did wt males (ps<.05; Table 1, test order 1). No signiﬁcant effect of
genotype was found for the total number of grid crossings or time spent grooming (Table
1, test order 1).

In the novel Object test there was a signiﬁcant effect Of genotype for time spent
visiting the novel object (F(2,27)= 5.233, p<.05) and the number of novel object visits
(F(2,27)= 4.729, p<.05). Post hoc comparisons revealed that Tfm males spent less time
visiting the novel Object compared to both wt males (p<.05) and females (p<.01; Figure
2b) and visited the object less frequently than did wt females (p<.05; Figure 2b). There
were no signiﬁcant differences in the number of grid crossings, or time spent grooming
(Table 1, test order 1), although there was a marginal effect of genotype on rearings
(F (2,27)= 3.292, p=.053) suggesting that wt females rear more than wt or Tﬁrr males
(Table 1, test order 1).

In the EPM there were no group differences in the number Of entries into the

open arms or in time spent in the open arms, although there was a signiﬁcant difference

70

in total arm entries (F(2,27)= 4.627, p<.05), indicating that Tfm males made fewer total
arm entries than did females (p<.05; Table 1, test order 1). In the LD box there were no
signiﬁcant differences in the number of entries into the light area of the box, time spent in
the light area, or rearings in the light area (Table 1, test order 1). Body weight
signiﬁcantly differed between groups of rats used in these tests (F(2,27)= 44.54, p<.001)
with Tfm males (401.46 :t 14.05 g) having body weights intermediate between wt males
(539.43 A: 22.12g) and wt females (319.38 :1: 13.56g).

When anxiety-related behavior tests (open ﬁeld/novel object tests, LD box, and
EPM) were conducted in rats from least to most anxiety provoking and not preceded by
the PPI test (test order 2), no signiﬁcant differences were found for measures of anxiety
in the open ﬁeld test (time spent in the center area of the open ﬁeld or number of visits to
the center area) or grid crossings. However, Tfm males again tended to show fewer visits
and decreased time in the center area of the open ﬁeld compared to wt males and females
(See Table 1, test order 2). There was a signiﬁcant effect of genotype for the number of
rearings (F (2,21 )= 3.458, p<.05) indicating that wt females reared more than did Tfm
males (p<.05; Table 1, test order 2). A signiﬁcant main effect was also found for time
spent grooming in the open ﬁeld (F (2,21)= 3.747, p<.05) with Tﬁn males grooming more
than wt males (p<.05; Table 1, test order 2).

As in the novel object test in test order 1, there was a signiﬁcant effect for time
spent visiting the novel object (F(2,21)= 17.55, p<.001) and the number of novel object
visits (F(2,21)= 12.12, p<.001) in the test order 2 cohort. Post hoc comparisons revealed
that Tfm males spent less time visiting the novel object compared to both wt males

(p<.05) and females (p<.01) and visited the object less frequently than did wt females

71

(p<.01), exhibiting the same pattern of differences as seen in the earlier cohort of animals
in test order 1 (Table l). Wt males also spent less time visiting the novel object and had a
fewer number of visits to the object than did wt females (ps<.05). In this test, there was a
signiﬁcant effect for the number of grid crossings (F(2,21)= 5.833, p<.01) with females
showing a greater number of grid crossings than wt males (p<.05) and Tfm males (p<.01)
and a signiﬁcant difference in the number of rearings F(2,21)= 8.420, p<.01) with
females showing a greater number of rearings compared to wt and Tﬁn males (ps<.01).
No differences were found for time spent grooming in the novel object test. There were
also no signiﬁcant differences in indices of anxiety or activity in the EPM or LD box in
the test order 2 cohort (Table 1).

Signiﬁcant differences in anxiety-related behavior and activity were not found in
the open ﬁeld and the LD box between rats in test order 1 and test order 2 (Table 1).
However, in the novel object test there was a main effect of test order in the number of
rearings (F(l,49)= 15.91, p<.001), with no other signiﬁcant differences in anxiety-related
or activity behaviors. Speciﬁcally, females showed a greater number of rearings in test
order 1 than in test order 2 (p<.05). In the EPM there was also a main effect of test order
in which animals in test order 2 spent more time in the open arms (F(l ,49)= 4.868,
p<.05), had more visits to the open arms (F(l,49)= 12.49, p<.001), and more total arm
entries (F (1,49)= 35.61, p<.001) than did animals in test order 1. Post hoc comparisons
revealed that wt females from the test order 2 cohort spent more time in the open arms
and visited the open arms more frequently than females from the test order 1 cohort

(ps<.05). Furthermore, all groups showed an increase in total arm entries in test order 1

72

compared to test order 2 (ps<.05). See Table 1 for a comparison of behavior in anxiety-

related tests in rats tested in the two orders.

Experiment 2: The role of ARS in HPA axis regulation

Two-way ANOVA revealed a signiﬁcant main effect of exposure (F(1,54)= 585.8,
p<.001) in which rats exposed to an open ﬁeld with a novel object showed an increased
corticosterone response 20 minutes aﬁer exposure compared to rats in the basal
condition. There was also a signiﬁcant main effect of genotype (F (2,54)= 8.484, p<.001)
and a signiﬁcant interaction between exposure and genotype on corticosterone
concentrations (F(2,54)= 8.50, p<.001). Post hoc comparisons revealed that all groups
had similar blood corticosterone levels at baseline, but after exposure to an open ﬁeld
with a novel object Tfm males and wt females showed an increased corticosterone
response compared to wt males (ps<.001; Fig 3a). Two-way ANOVA revealed a
signiﬁcant main effect of genotype on T levels (F(2,54)= 11.40, p<.001), with no main
effect of exposure or genotype x exposure interaction. Speciﬁcally, T levels were greater
in Tﬁn males (16.3 i: 4.0 nmol/L) than in wt males (5.9 :t 1.3 nmol/L; p<.05), and both

groups had higher T levels than wt females (0.1 :t 0.03; ps<.01).

Experiment 3: The role of ARS in HPA axis recovery

Two-way ANOVA revealed a signiﬁcant main effect of time on corticosterone levels
(F(4,50)= 95.22, p<.001). Compared to basal corticosterone levels, corticosterone levels
were greater in wt and Tfm males at 20 and 40 minutes after the onset of novel object

exposure (ps< .01). A signiﬁcant genotype x time interaction was also revealed

73

(F(4,50)= 3.874, p<.01). Tfm males showed increased corticosterone levels compared to
wt males at 20 and 120 (ps<.05) minutes after novel object exposure, but similar
corticosterone levels compared to wt males at baseline, 40, and 60 minutes (Figure 3b).

A signiﬁcant main effect of genotype was not found in this test.

Experiment 4: The role of ARS in immediate early gene activation

Replicating our earlier ﬁndings (35), body weight was signiﬁcantly greater in wt males
(526.4 d: 24.4g) than Tfm males (379.5 i 18.6g), though brain weight did not
signiﬁcantly differ (wt male: 1.68 i 0.03g; Tfm male: 1.62 i 0.04g) in rats used in this
experiment. A signiﬁcant main effect of exposure on the number of c-Fos
immunopositive cells was found in every brain region examined (Table 2). These results
indicate that compared to the baseline condition, c-Fos irnmunoreactivity (ir) was
increased in these areas after exposure to an open ﬁeld with a novel object.

In the DG a main effect of genotype was revealed (F(l,24)= 14.85, p<.001) and
reﬂected that fact that wt males exposed to a novel object had greater c-Fos—ir compared
to novel object exposed Tfm males (p<.001; Table 2; Figure 4a). There was also a main
effect of genotype in the MPOA (F(l ,24)= 12.35, p<.01). Post hoc comparisons revealed
that c-Fos-ir in rats exposed to a novel object but not controls was increased in Tfm
compared to wt males (p<.001; Table 2; Figure 4b). A signiﬁcant interaction was also
found for c-Fos-ir in the MPOA (F(l,24)= 10.08, p<.01), indicating that the increase in c-
Fos expression from baseline to one hour after novel object exposure was also greater in
Tﬁn males. Only a signiﬁcant interaction was found in the MePD (F(l,24)= 5.630,

p<.05), due to a greater increase in c-Fos expression from baseline to one hour after novel

74

object exposure in wt compared to Tfm males (Table 2; Figure 4c). No other signiﬁcant

main effects of genotype or interactions were found for the brain regions examined.

Discussion

Tfrn male rats showed increased indices of anxiety in two tests (open ﬁeld and novel
object test), but not two others (LD box and EPM), indicating a task-speciﬁc or moderate
increase in anxiety-related behavior compared to wt males and females. In the open ﬁeld
and novel object tests, Tfm males showed decreased exploration of the center area of the
open ﬁeld and novel object compared to both wt males and females. The current ﬁndings
support previous research indicating involvement of the AR in the regulation of anxiety-
related behavior in rodents (Edinger and Frye, 2006; F emandez-Guasti and Martinez-
Mota, 2005), including our previous ﬁndings in Tﬁn mice (Zuloaga et al., 2008b).

Tfm males, like wt males, were also consistently less active in tests of anxiety
compared to wt females as assessed by the number of rearings and grid crossings in the
open ﬁeld and novel object tests, and the total number of arm entries in the EPM.
Differences, however, did not reach signiﬁcance in several cases (Table 1). These
ﬁndings are consistent with reports of sex differences in ambulation and rearing in the
open ﬁeld and activity in the EPM, with females more active (Archer, 1975; Masur et al.,
1980; Slob et al., 1981; Seliger, 1977; Lucion et al., 1996). Activity measures did not
differ between Tfm and wt males suggesting that sex differences in activity in rats in our
study and others may be mediated via differential activation of ERs.

Test order signiﬁcantly inﬂuenced anxiety-related and activity measures in the

EPM and rearings in the novel object test, with wt female rats most affected. In the EPM,

75

wt females showed an increase in the amount of time spent on and entries into the open
arms when tested in order from least to most anxiety-provoking (test order 2) compared
to animals ﬁrst tested for PPI (test order 1), while all groups showed greater activity in
the EPM in test order 2. In the novel object test, wt female rats also showed a greater
number of rearings in test order 2 compared to test order 1. These ﬁndings are consistent
with other studies showing that that prior exposure to a stressful or anxiety producing
situation can inﬂuence behavior in tests of anxiety (MacNeil et al., 1997; F ile, 1993;
Ballaz et al., 2007). Since estrus status was not controlled for, it is also possible that the
phase of the estrus cycle may have differed in females in test order 1 and 2 cohorts when
exposed to the different tests of anxiety, and this difference may have contributed to
changes in behavior between the two groups. Phase of estrus cycle has previously been
shown to inﬂuence behavior in tests of anxiety, most notably in the EPM (Zuluaga et al.,
2005; Marcondes et al., 2001; Mora et al., 1996; Vinogradova, 1999).

Consistent with other studies comparing sex differences in the corticosterone
response to a stressful situation (Handa et al., 1994a; Seale at al., 2004), wt female rats
showed an increased corticosterone response compared to wt male rats after exposure to
an open ﬁeld with a novel object. A novel ﬁnding in our study is that Tﬁn males also
showed an increased peak corticosterone response (20 min) compared to wt males,
though recovery levels at 40 and 60 minutes after novel object exposure did not
signiﬁcantly differ from wt males. Since Tﬁn males had elevated T levels compared to
wt males, and T generally has an inhibitory effect on corticosterone release (Handa et al.,
1994b; Seale et al., 2004; Mitsushima et al., 2008), this ﬁnding suggests that ARs play a

role in decreasing the corticosterone response to stress. There was also a difference in

76

corticosterone levels between wt and Tﬁn males at 120 minutes after exposure to a novel
object. Since corticosterone recovery levels were normal at 40 and 60 minutes this
difference does not appear to reﬂect a prolonged difference in recovery, rather it may
reﬂect a difference in basal corticosterone levels at this time of day. We recently found
that Tfm mice also show a heightened corticosterone response to a novel object compared
to wt males (Zuloaga et al., 2008a), supporting the notion that AR may play a role in
adrenal steroid regulation across mammals.

Although an increased corticosterone response to stress in females compared to
males is not associated with increased indices of anxiety, increased corticosterone
responses within a gender have been associated with elevated anxiety. In female rats
administration of the ERB agonist diarylpropionitrile (DPN) reduces anxiety as well as
corticosterone levels in the EPM (Lund et al., 2005). Similarly, in male rats
pharmacological activation of ERB decreases the corticosterone response to stress (Lund
et al., 2004; Lund et al., 2005; Lund et al., 2006) and decreases anxiety-related behaviors
in the EPM (Lund et al., 2005). Furthermore, male rats bred for high anxiety also show
increases in corticosterone compared to rats showing normal anxiety behaviors (Landgraf
et al., 1999). Therefore, elevated anxiety-related behaviors and corticosterone responses
in Tﬁn males compared to wt males are likely correlated. However, since plasma
corticosterone binding globulins (CBGs) were not quantiﬁed, it remains possible that
differences in CBGs may have contributed to differences corticosterone levels found
between wt and Tﬁn males.

PPI did not differ between groups, indicating that the AR plays a minimal role in

this measure of sensorimotor gating in rats. These ﬁndings are in line with other studies

77

showing that administration of DHT does not affect PPI in male and female rats
gonadectomized in adulthood (Turvin et al., 2007; van den Buuse and Eikelis, 2001),
though the role of developmental AR activation on PPI had not previously been studied.
Since AR deﬁciency is present from ontogeny in Tfm male rats, these ﬁndings further
suggest that activation of ARs during development have a negligible effect on PPI. In
rodents, PPI enhancing effects of T and estrogen are more likely mediated by the
activation of ERs (van den Buuse and Eikelis, 2001).

Although PPI did not differ across groups, ASR was enhanced in Tfm males
compared to wt males and females in this test suggesting a role for ARS in this behavior.
This difference was most notable in the ﬁrst ASR trial, with only a moderate elevation in
the following trials. This result suggests that emotional responses in Tfm males are
increased during the ﬁrst exposure to a loud acoustic stimulus, but are greatly diminished
after the initial exposure to the pulse. Elevated ASR in Tfm males is in line with studies
showing administration of T (Turvin et al., 2007; Toufexis et al., 2005) and DHT
(Turvin etal., 2007) can attenuate ASR. As for PPI, the inﬂuence of AR stimulation in
development on ASR has not been previously examined, therefore increased ASR in Tfm
rats may result from reduced AR activation in adulthood, development, or both.
Increased ASR in Tﬁn male rats also appears to relate to our ﬁndings of increased
anxiety-related behaviors in Tfm males since elevations in ASR have been suggested to
reﬂect anxiety in mice and rats (Walker and Davis, 1997; Crawley et al., 1997; Belzung
etal., 2000; Hode et al., 2000). Therefore, increased ASR in Tﬁn male rats likely also

reﬂects increased anxiety-related behavior.

78

As might be expected, there was an increase in c-Fos activation in all brain
regions examined in rats exposed to the novel object compared to rats sacriﬁced at
baseline. However, c-Fos-ir differed between wt and Tfm males in some brain areas
(DG, MPOA, MePD) but not others (LSV, PVN, BSTMPM, VMH, BLA, MEA, CA1)
following exposure to an open ﬁeld with a novel object. In brain regions in which c-Fos
activation differed between wt and Tfm males there was both increased (MPOA) and
decreased (DG, MePD) immunoreactivity in Tfm males, suggesting that the AR seems to
act speciﬁcally, modulating neural reactivity differently in different brain regions.

Decreased activation of the DG was found after novel object exposure in Tfm
compared to wt males. This is similar to ﬁndings in another study in which decreased 0-
Fos activation in the DG was observed in rats bred for high versus low anxiety (58). The
hippocampus, including the DG, is an androgen responsive brain region, with abundant
ARs particularly in CA1 (Xiao and Jordan, 2002) and lower levels in the DG (Brannvall
et al., 2005; Tabori et al., 2005), which plays a role in anxiety-related behavior (Edinger
and Frye, 2004, Edinger and Frye, 2005, Edinger and Frye, 2006). The hippocampus also
regulates HPA axis feedback via inhibitory projections to the PVN, where increased c-
Fos-ir has been correlated with increased HPA axis activity and anxiety (Lun et al., 2006;
Salome et al., 2004). While this could be a pathway through which androgens inhibit
anxiety and HPA axis activity we found no differences in c-Fos activation of the PVN,
although it remains possible that there was a greater excitation of neurosecretory neurons
in the PVN of Tfm males not revealed in our analysis of c-Fos.

C-Fos-ir was increased in the MPOA of novel object-exposed Tfm males

compared to wt males. This increase is consistent with previous ﬁndings demonstrating a

79

correlation between elevated c-Fos activation of the MPOA and increased anxiety-related
behavior in an open ﬁeld and the open arm of the EPM (Salome et al., 2004). The
MPOA contains abundant ARs (Handa et al., 1987; Handa et al., 1996; Simerly etal.,
1990) and has been identiﬁed as an androgen responsive brain region involved in sexual
behaviors as well as the regulation of the HPA axis (McCormick et al., 2002; Viau and
Meaney, 1996). Therefore, these data may reﬂect an alteration in HPA axis activity of
Tfm males.

A decrease in c-Fos-ir was found in the MePD of Tfm male rats, an area that has
also been found to have decreased volume in Tfm rats compared to wt males (Morris et
al., 2005). The MePD is particularly involved in receiving olfactory and pheromonal
information and is important for some aspects of male sexual behavior (Lehman et al.,
1983; Swarm et al., 2001). However, the MePD projects to two other hormone and stress
responsive regions (BNST, MPOA) and through these projections may regulate anxiety-
related behaviors (Coolen and Wood, 1998; Gomez and Neuman, 1992). In areas where
we detected no change in c-Fos activation between Tfm and wt males, it remains possible
that there were actual differences in the activation of speciﬁc cell types within these
regions, yet net cell activation was the same. Altered activity in areas outside of the brain
may have also contributed to differences in behavioral and physiological responses.
Differences between Tfm and wt males, particularly in HPA axis activation, may result
from AR mediated changes in pituitary activity. AR knockout male mice show
elevations in ACTH and corticosterone that appear to be caused by an impairment of
negative feedback resulting from a decrease in pituitary glucocorticoid receptors

(Miyamoto et al., 2007).

80

Aromatase activity is also decreased in several areas of the brain in adult Tfm
compared to wt males (Rosseli et al., 1987), so we cannot discount the possibility that
differences between these groups result from decreased activation of ERS in Tﬁn males.
However, anxiety-related behaviors are also increased in Tﬁn male mice compared to wt
males where it is not clear whether aromatase levels are signiﬁcantly altered (N aftolin et
al., 1975; Rosenfeld et al., 1977), and other studies have also implicated an involvement
of ARs in the regulation of the HPA axis and anxiety-related behaviors (Miyamoto et al.,
2007 ; Edinger and Frye, 2006; Femandez-Guasti and Martinez-Mota, 2005).

In conclusion, Tfm male rats showed increased anxiety-related behavior
compared to wt males and females in tests thought to reﬂect anxiety (open ﬁeld, novel
object test, ASR), with no differences in PPI. Compared to wt males, Tfm males also
showed increased peak corticosterone responses and differences in immediate-early gene
activation in select brain regions (DG, MPOA, and MePD) following exposure to an open
ﬁeld with a novel object that are consistent with greater anxiety in Tfm males than wt
males. Together these data indicate a role of the AR in the regulation of anxiety, the

HPA axis, and activation of discrete brain areas in rats that may underlie anxiogenesis.

81

Chapter 5: The Organizational Role of Testicular Hormones and the
Androgen Receptor in Anxiety-Related Behaviors and Sensorimotor

Gating in rats

Abstract

Exposure to testosterone (T) early in life, which can act upon both androgen receptors
(ARS) and, via aromatization of T into estrogens, upon estrogen receptors, organizes
adult behaviors in rodents. We compared behaviors in wild type (wt) male rats and AR-
deﬁcient rats with the testicular feminization mutation (Tﬁn), that were either
gonadectomized (Neo-de) or sham-operated (N eo-Sham) on the day of birth. In
adulthood, all rats were either gonadectomized or sham-operated and implanted with T
capsules to equilibrate adult circulating T. In each of four tests of anxiety (open ﬁeld,
novel object exposure, light dark box, elevated plus maze), Neo-de rats showed
decreased indices of anxiety compared to Neo-Sham rats, with no differences between wt
or Tfm males within treatment groups. These results indicate that testicular hormones act
perinatally to increase adult indices of anxiety, and functional ARs are not required for
this effect. Acoustic startle response (ASR) was also reduced by Neo-de, but only in
Tfm males, suggesting that perinatal stimulation of ERS can increase ASR, but this effect
is counteracted in normal males by stimulation of ARs. Corticosterone levels were
elevated in Neo—de rats at baseline with no differences in hormone levels after exposure
to a novel object in an open ﬁeld. Sensorimotor gating, as measured by prepulse
inhibition (PPI) of the ASR, was increased by neonatal castration, in both wt and Tfm

rats. These ﬁndings indicate a role of T prior to adulthood in the organization of anxiety-

82

related behaviors and sensorimotor gating in rats, which appears to be primarily AR-

independent.

Introduction

In rodents, exposure to hormones around the time of birth can “organize” adult sex
differences in both the central nervous system and behavior (Phoenix et al., 1959;
Jacobson et al., 1981; Breedlove et al., 1982; Arnold and Breedlove; 1985).
Traditionally, sex differences in behavior and brain morphology are believed to result
from testicular production of testosterone (T) in males in early development. According
to the aromatization hypothesis, T is converted locally in the brain by the enzyme
aromatase to estrogens, which then activate estrogen receptors (ERS) to masculinize the
rodent brain (MacLusky and Naftolin, 1981). However, emerging evidence suggests that
activation of androgen receptors (ARs) also contributes to the masculinization of brain
and behavior in rodents (Zuloaga et al., 2008; Durazzo et al., 2007; Dugger et al., 2007;
Morris et al., 2005), so the issue of the relative contribution of ER and AR to
masculinization of rodent behavior remains open.

Sex differences exist in many different rodent behaviors and the most apparent are
related to sexual behavior. Neonatal administration of T to guinea pigs masculinizes
sexual behaviors in both males gonadectomized on the day of birth and females (Phoenix
et al., 1959) indicating a role of T in the organization of this sex difference. Early
hormone exposure also contributes to sex differences in rodent spatial memory
performance in the Morris water maze (males perform better than females). Neonatal

castration of males or administration of T to newborn females eliminates such sex

83

differences (Isgor and Sengelaub, 2003), an effect that appears to be mediated through
activation of both ERS and ARS (Williams et al., 1990; Jones and Watson, 2005; Isgor
and Sengelaub, 1998).

In rats there are also sex differences in tests of anxiety with females typically
showing decreased indices of anxiety and greater activity compared to males (Powell et
al., 2002; Archer, 1975; Masur et al., 1980; Slob et al., 1981; Seliger, 1977; Lucion et al.,
1996; Zimmerberg and Farley, 1993). These differences appear to depend on both
organizational and activational effects of hormones as well as both ERS and ARs.
Neonatal gonadectomy in male rats eliminates sex differences in anxiety-related
behaviors in the elevated plus maze (Lucion et al., 1996). Furthermore, female rats
receiving either neonatal treatment with an ER antagonist (tamoxifen) or prepubertal
ovariectomy show greater indices of anxiety in the EPM compared to control females
(Zimmerberg and Farley, 1993). In adulthood, administration of both estrogens and
androgens generally result in decreased anxiety-related behavior in rodents (Frye and
Lacey, 2001; Walf and Frye, 2005; Lund et al., 2005; Frye et al., 2008; Bing et al., 1998;
Bitran et al., 1993) apparently by acting on both ERS and ARs (Lund et al., 2005;
Imwalle et al., 2005; Edinger and Frye, 2006; Fernandez-Guasti and Martinez-Mota,
2005; Zuloaga et al., 2008). Sex differences in anxiety-related behaviors are also
associated with sex differences in HPA axis activation in which female rats show a
greater release of stress hormones from the pituitary (adrenocorticotropin hormone) and
adrenal glands (corticosterone) following exposure to an anxiety-provoking or stressful

situation (Handa et al., 1994a; Kudielka and Kirshbaum, 1999). Androgens and

84

estrogens acting in development and adulthood appear to contribute to these differences
(Handa et al., 1994b; Seale et al., 2004; Seale et al., 2005; Bingharn and Viau, 2008).

Sex differences in sensorimotor gating have also been reported in humans and
some strains of mice and rats (Swerdlow et al., 1993; Lehman et al., 1999; Ralph et al.,
2001; Ison and Allen, 2007) as assessed by an experimental model of sensorimotor
gating, prepulse inhibition (PPI) of the acoustic startle response (ASR; Geyer et al.,
1990). In adult rodents, circulating levels of both androgens and estrogens can inﬂuence
aspects of PPI (Jovanovic et al., 2004; van den Buuse and Eikelis, 2001; Gogos and Van
den Buuse, 2003), and these effects appear to be largely mediated through activation of
ERs rather than ARs (van den Buuse and Eikelis, 2001; Zuloaga et al., submitted).

In order to further investigate the role of developmental androgens and ARs in
anxiety-related behaviors, sensorimotor gating, and activation of the HPA axis we
compared behavioral and hormonal responses in wild type (wt) male rats and AR
deﬁcient rats with the testicular feminization mutation (Tfm). Tﬁn rats represent a
genetic model for exploring the role of the AR in brain and behavior (Zuloaga et al.,
2008). In rats this mutation results from a single base pair replacement in the AR gene
resulting in the expression of a normal sized but dysfunctional AR protein and
consequently sensitivity to androgens through ARS is greatly reduced (Yarbrough et al.,
1990; Naess et al., 1976). Wt and Tﬁn male rats were either gonadectomized (N eo-de)
or sham-operated (N eo-Sham) on the day of birth, then treated in adulthood to provide
equivalent T before assessing anxiety, ASR, PPI and adrenal responses. We conﬁrm
reports that neonatal testicular hormones lead to greater indices of anxiety in adulthood,

and because we ﬁnd almost all effects of neonatal castration are equivalent in wt and Tﬁn

85

males, conclude that most of these neonatal effects of T increasing adult anxiety are
primarily due to stimulation of ERs. However, we also ﬁnd evidence that neonatal

stimulation of AR normally results in reduced ASR in adulthood.

Materials and Methods

Animals

Long Evans rats were bred at Michigan State University and group housed in a Vivarium
with a 12/12 L/D cycle, lights on at 0600, with food and water ad lib. On the day of
birth, Tfm and wt male pups were either gonadectomized (Neo-de Tfrns; N=11: Neo-
de males; N=9) or sham-operated (Neo-Sham Tfrns; N=10; Neo-Sharn males; N=9:
procedure described in detail below). All rats were weaned at 21 days old, at which point
ear punches were taken and rats were genotyped using a modiﬁed polymerase chain
reaction (PCR) described elsewhere (Fernandez et al., 2003). Products of this reaction
differentiated between the Tfm and the wt allele for the AR, and male versus female,
based on the presence or absence of the Sry gene found only on the Y chromosome. In
adulthood, animals were either gonadectomized (for those that were sham-operated at
birth) or sham-operated and implanted with T capsules to equilibrate adult circulating T

(procedure described in detail below).

Neonatal castration: On the day of birth, pups were removed from their home cage and
anesthetized by hypothermia. After pups were deeply anesthetized, incisions were made
on either side of the lower abdominal cavity and the gonads were visualized and removed

or leﬁ intact (Sham-operated). Incisions were closed with surgical glue and pups were

86

placed under a lamp until they were warm and mobile before returning to their home

cage.

Adult castration and hormone replacement: At 120 days, rats were anesthetized with
isoﬂurane and either gonadectomized (for those that were sham-operated at birth) or
sham-operated (for those that were gonadectomized at birth). In rats receiving
gonadectomy, testes were extemalized via bilateral incisions made in the scrotum (wt
males) or in the lower abdominal area (Tfm males) and testes were tied off with silk
suture and removed. In sham-operated rats bilateral incisions were also made into either
the scrotum (wt males) or lower abdominal area (Tfm males). Incisions in the abdominal
muscles were closed with silk suture and the overlying skin was closed with wound clips,
while scrotal incisions were closed with wound clips. All rats also received two
subcutaneous Silastic capsules (1.57 mm inner diameter, 3.18 mm outer diameter: 20 mm
effective release length) containing either free T (T) or nothing (blank (B)) via a 2 cm
incision dorsally at the nape of the neck. We recently demonstrated that these Silastic
capsules deliver normal male physiological levels of T to adult rats (Morris et al., 2008).

The analgesic buprenorphine (0.05 mg/kg) was injected sc post operatively.

Behavior Testing

Two weeks after adult gonadectomy or sham-operation, animals were tested for anxiety
related behavior, sensorimotor gating, and blood corticosterone levels. Testing took
place in an order we judged was least to most anxiety-provoking: open ﬁeld/novel object

test, light dark (LD) box, elevated plus maze (EPM), PPI, and blood corticosterone

87

collection (all described in detail below), with a minimum of 72 hrs between tests. All
tests were administered between 1000-1400 except for PPI, which was conducted during

the dark cycle beginning at 1900.

Open F ieId/Novel object test

Open ﬁeld/novel object testing took place in a 48”x 48” white plastic box illuminated
from directly above by a 60 watt light. A grid was drawn in the box to demarcate entries
into the center area and activity (grid crossings). Rats were ﬁrst placed into a corner of
the empty open ﬁeld and behavior was recorded via an overhead video camera for 5 min.
Aﬁer 5 min, rats were removed, the box was cleaned with 70% ethanol, and a novel
object (a 4” diameter x 8” high cylindrical metal oxygen tank cap) was placed in the
center of the chamber. Three minutes aﬁer removal from the open ﬁeld, the rat was
replaced into the chamber which now contained the novel object and behavior was
recorded for another 5 min. The number of entries into the center area, time spent in the
center area, visits to the novel object, and time spent visiting the novel object were
assessed as indices of anxiety-related behavior. The number of grid crossings and
rearings were assessed as measures of activity and all behaviors were coded at a later
time by a blind observer. The box was again cleaned with 70% ethanol before testing the

next subject.

Light/Dark (LD) Box
The LD box (Phenome Technologies Inc.; Lincolnshire, IL, USA) consisted of a

rectangular box that was divided into two regions, one dark (40 cm length x 38 cm width

88

x 29 cm height) and one light (31 cm length x 28 cm width x 29 cm height). The dark
region was constructed of black plastic and was covered by a black lid, while the light
region was constructed of transparent Plexiglas and was illuminated by a 60 watt light
that was 3 feet directly above it. The two chambers were connected by a small opening
(7.5 cm height x 7.5 cm width) that allowed the animals to freely enter either area.
Animals were placed in the light side of the chamber facing the opening to the dark
chamber and were allowed to move freely between the two compartments for 10 min.
Behavior was recorded via an overhead video camera for coding at a later time by a blind
observer. The number of entries into and time spent in the two compartments, as well as
the number of rearings in the light compartment were assessed as indices of anxiety and

activity. The box was cleaned with 70% ethanol between subjects.

Elevated Plus Maze (EPM)

The EPM consisted of two open and two closed arms (50 x 10 cm) that extended from a
center platform and was elevated 50 cm above the ﬂoor. Testing took place in a dimly lit
room in which rats were placed in the center area of the EPM facing an open arm and
allowed to move freely between the arms for 10 min. Behavior was recorded using an
overhead video camera and was coded at a later time by a blind observer. The number of
entries into and the amount of time spent on the open arms were assessed as indices of
anxiety-related behavior. The maze was cleaned with 70% ethanol between each test.

Total arm entries (open and closed) were assessed as a measure of activity.

Prepulse Inhibition (PPI) and Acoustic Startle Response (ASR)

89

Rats were tested for PPI 1 hour aﬁer lights off in a room illuminated by dim red light.
PPI was measured in acoustic startle response chambers (SR Lab startle response system,
San Diego Instruments, San Diego, CA). Animals were placed into the chamber for 18
min, the ﬁrst 5 min of which is an acclimation period. For the remaining 13 min the fast
muscle twitch startle responses of animals to a 100 decibel tone alone (acoustic startle
response; ASR), or preceded by 100 msec by tones of 3, 8, 10, or 15 decibels were
recorded via SR Lab software (San Diego Instruments). The prepulse should permit the
subject to anticipate the loud pulse, and consequently startle less severely. Each of these
trials was repeated 6 times at pseudo-random intervals. After the test, animals were

removed and the chamber was cleaned with 70% ethanol.

Corticosterone Collection and Assay: In the ﬁnal test, blood was collected from rats
between 0900-1100 at baseline or 20 min after initial exposure to an open ﬁeld with a
different novel object (black tape dispenser; 7” length x 3” width x 3” height) located in
the center. Novel obj ect-exposed rats were placed in the open ﬁeld with a novel object
for 10 min, after which they were returned to their home cage where they remained until
sacriﬁce. Baseline rats remained in their home cage until sacriﬁce. Rats were deeply
anaesthetized with isoﬂurane and decapitated, with trunk blood collected within 2
minutes of cage disturbance. All blood was collected in 1.5 ml tubes containing 250ul of
heparin and held on crushed blue ice until centrifugation. After centrifugation, plasma
was collected and frozen at —20° C until the assay was performed. Plasma was assayed
for corticosterone at the Diagnostic Center for Population and Animal Health at Michigan

State University using a Coat-A-Count Corticosterone kit (Diagnostics Products

90

Corporation, Los Angeles, CA, USA). All plasma samples were run in duplicate and
results were averaged. Brain weights were also measured in these animals immediately

following sacriﬁce.

Statistical Analysis: For PPI, a mixed design repeated measures analysis of variance
(ANOVA) was used to analyze data with genotype and neonatal treatment as a between
groups factors and prepulse intensity as within (or repeated) groups factor. Anxiety-
related behaviors and ASR were each analyzed separately using 2-way AN OVA. ASR
by trial was analyzed using a mixed design repeated measures ANOVA with genotype
and neonatal treatment as a between groups factors ASR trial order (1-6) as a within
groups factor. Corticosterone levels were analyzed using 3 and 2-way AN OVA. All
signiﬁcant main effects or interactions were further analyzed using Tukey’s multiple
comparisons tests. Differences were considered signiﬁcant when p<0.05 and all data are
reported as means :t standard error of the mean (SEM) with N = number of animals in

each group.

Results

In the open ﬁeld test, a 2x2 AN OVA revealed a signiﬁcant main effect of neonatal
treatment for time spent in the center area of the chamber (F(1,35)= 6.191, p<.05; Figure
1a) and the number of entries to the center area (F (1,35)= 4.763, p<.05; Figure lb)
indicating that Neo-de rats spent more time in and visited the center area more than
Neo-Sham rats, with no main effects of genotype nor any interactions. There was also a

signiﬁcant main effect of neonatal treatment for the number of grid crossings in the open

91

ﬁeld test (F (1,35)= 12.79, p<.01), indicating a greater number of crossings in Neo-de
compared to Neo-Sham males (Figure 1c, left), but no signiﬁcant effect on number of
rearings (Figure 1c, right). Post hoc comparisons revealed that Neo-Sham males had
signiﬁcantly fewer grid crossings than did either Neo-de male or Neo-de Tfm groups
(ps<.05 and .01 respectively). No signiﬁcant main effects of genotype or interactions
were found for any measures in the open ﬁeld test.

In the novel object test there was a signiﬁcant main effect of neonatal treatment
for the number of novel object visits (F (1,35)= 7.872, p<.01; Figure 2b) and the number
of grid crossings (F (l ,35)= 4.660, p<.05; Figure 2c, left) with no main effects of
genotype or interactions. These results represent an elevation in both measures in Neo-
de rats compared to Neo-Sham rats. Post hoc comparisons did not reveal any other
signiﬁcant group differences, indicating only an overall effect of neonatal treatment in
these measures. No signiﬁcant main effects or interaction was found for time spent
visiting the novel object (Figure 2a) or the number of rearings (Figure 2c, right).

In the LD box a signiﬁcant main effect of neonatal treatment was found for time
spent in the light area (F(1,34)= 9.663, p<.01), the number of entries into the light area
(F(1,34)= 9.663, p<.01), and the number of rearings in the light area (F(1,34)= 9.894,
p<.01), with no signiﬁcant main effect of genotype or interaction (Figure 3a—c).
Speciﬁcally, Neo-de rats showed an elevation in all measures compared to Neo-Sharn
rats. There were no signiﬁcant main effects of genotype or any interactions.

Again a signiﬁcant main effect of neonatal treatment was found in the EPM for
the number of entries into the open arms (F (1,34)= 9.894, p<.01), indicating that Neo-

de rats had more open arm entries than Neo-Sham rats (Figure 4b). Signiﬁcant effects

92

of neonatal treatment were not found for time spent on the open arms (F(l,34)= 3.682,
p=.063; Figure 4a) or total number of arm entries (F(l,34)= 3.707, p=.062; Figure 4c),
although there was a trend for neonatally gonadectomized rats to show elevations in both
measures. No signiﬁcant main effects of genotype or interactions were found for any
measures in the EPM.

A mixed design ANOVA using genotype and neonatal treatment as between
groups factors and prepulse intensity as a within groups factor revealed the expected
effect of prepulse intensity (F (3,140)= 22.86, p<.001) in which PPI increased as intensity
of the prepulse increased. There was also a signiﬁcant main effect of neonatal treatment
(F(l,35)= 9.970, p<.01) in which Neo-de rats showed overall increased PPI compared
to Neo-Sham rats (Figure 5a). There was no signiﬁcant effect of genotype and no
interaction.

A signiﬁcant main effect of neonatal treatment was also found for ASR(F(1,35)=
9.760, p<.01), indicating that Neo-de rats showed a decreased ASR compared to Neo-
Sham rats (Figure 5b), which is consistent with the above ﬁndings that Neo-de reduces
measures of adult anxiety. Speciﬁcally, Neo-Sham Tfrns showed an increased ASR
compared to Neo-de Tfm and Neo-de male groups (ps<.05 and .01 respectively). No
signiﬁcant main effect of genotype or interaction was found for average ASR. An
analysis of ASR by trial revealed the expected signiﬁcant effect of trial (F(5,175)= 7.24,
p<.01) indicating that, as a whole, rats showed a decrease in ASR particularly aﬁer the
initial ASR trial. However, there was also a signiﬁcant main effect of neonatal treatment
(F (1,35): 9.760, p<.01), again indicating that Neo-Sham rats show greater ASR than

Neo-de rats. This difference was largely accounted for by an increased ASR in Neo-

93

Sham Tfm males compared to all other groups in trials 1 (ps<.05) and 2 (ps<.05), and
compared to Neo-de males in trial 5 (p<.05; Figure 5c).

A 3-way ANOVA (exposure x neonatal treatment x genotype) revealed a
signiﬁcant main effect of exposure (F(1,54)= 585.8, p<.001) in which rats exposed to an
open ﬁeld with a novel object showed an increased corticosterone response compared to
rats in the basal condition (Figure 6a and 6b). Separate 2-way AN OVAs (neonatal
treatment x genotype) were also performed to compare corticosterone levels at either
baseline or at 20 minutes after exposure to an open ﬁeld with a novel object. At baseline
there was a signiﬁcant main effect of treatment (F (1,34)= 8.546, p<.05), with no main
effect of genotype or interaction, indicating that compared to Neo-Sham rats, Neo-de
rats had higher corticosterone levels (Figure 6a). Post hoc comparisons did not reveal
any further signiﬁcant group differences, indicating only an overall effect of neonatal
treatment in this measure. Analysis of corticosterone levels following novel object
exposure revealed no signiﬁcant main effects or interaction (Figure 6b).

There was a signiﬁcant main effect of genotype (F(l,35)= 30.53, p<.001),
neonatal treatment (F(l ,35)= 25.66, p<.001), and an interaction between genotype and
neonatal treatment (F (1 ,3 5)= 7.60, p<.01) on body weight. Speciﬁcally, body weight was
greater in Neo-Sham males compared to all other groups (all ps<.001; Figure 7b). A 2-
way AN OVA for brain weight also revealed a signiﬁcant main effect of neonatal
treatment (F(l,34)= 8.546, p<.05), with no main effect of genotype or interaction. Post
hoc comparisons revealed reduced brain weights in Neo-de Tﬁn and Neo-de male

groups compared to Neo-Sham males (ps <.01; Figure 7a).

94

Discussion
Compared to Neo-Sharn rats, Neo-de rats showed decreased anxiety-related behavior in
all our tests of anxiety, displaying a greater number of visits to the center area of the open
ﬁeld, to a novel object in the open ﬁeld, to the light area of the LD box, and to the open
arms of the EPM, as well as a decreased ASR. Neo—de rats also spent more time in the
center area of the open ﬁeld and light area of the LD box, and tended to spend more time
in the open arms of the EPM. These ﬁndings are in accord with a previous report of
decreased anxiety-related behavior in the EPM in male rats that were gonadectomized at
birth compared to sham-operated rats (Lucion et al., 1996). Unlike that previous study, in
our study, T levels were equilibrated in adulthood, strengthening the idea that differences
in T during development, rather than differences in circulating levels of T in adulthood,
increase adult expression of anxiety. These ﬁndings also indicate that sex differences in
anxiety behaviors that have been reported in rats (females < males: Lucion et al., 1996;
Zimmerberg and Farley, 1993, Irnhof et al., 1993) may, at least partially, be related to
organizational effects of T. However, female gonadal hormones, acting both in
development and adulthood also appear to contribute to these sex differences
(Zimmerberg and Farley, 1993; Frye and Walf, 2004).

For all of these effects of neonatal castration on anxiety-related behaviors, wt and
Tfm males were equivalent, indicating that the organizational inﬂuence of T on anxiety-
related behaviors is primarily AR-independent, and rather, may be mediated through
activation of ERs. We previously found that gonadally intact Tfm male rats showed
increased indices of anxiety in the open ﬁeld and novel object tests compared to wt male

rats (Zuloaga et al., 2008), but in the present study we did not see this difference. It is

95

possible that exposure to a surgical procedure (gonadectomy) two weeks prior to
behavior testing or neonatal anesthesia may have affected anxiety-related behaviors in
such a way as to eliminate differences between these groups. Neonatal anesthesia has
previously been demonstrated to affect hippocampal morphology and cognitive behavior
(Rothstein et al., 2008; Nunez et al., 2000) though its effects on anxiety-related behavior
have not been studied. Adult gonadectomy and T replacement also eliminated normal
differences between Tfm males and wt males in circulating T (Tﬁn males > wt males:
Roselli et al., 1987; Zuloaga et al., 2008) introducing the possibility that increased T
levels in Tﬁn males, or differences in other gonadal secretions may have contributed to
differences we previously found between these groups.

Several activity measures (grid crossings in the open ﬁeld and novel object tests,
transitions between compartments and rearings in the light area of the light dark box, and
total arm entries in the EPM) were elevated in Neo-de compared to Neo-Sham rats.
These data are also in accord with other reports in which neonatally gonadectomized rats
have shown increased ambulation in the open ﬁeld and more total arm entries in the EPM
(Slob et al., 1986; Lucion et al., 1996). Therefore, data from this study and others
indicate that the presence of T during development contributes to sex differences in
activity in adult rodents (females generally more active than males). Since there were no
differences in activity measures between Neo-Sham Tfm and Neo-Sham wt males, our
data also indicate that T does not act through the AR to alter this behavior. Presumably
T’s organizational inﬂuence on activity is ER—mediated.

There was only a single exception to the above pattern that AR is irrelevant to

neonatal T effects on adult anxiety, and that was in the ASR, which has also been

96

suggested to reﬂect anxiety in mice and rats (Walker and Davis, 1997; Crawley et al.,
1997; Belzung et al., 2000; Hode et al., 2000). Again, neo-Sham rats showed a greater
ASR, but this difference was largely due to effects in Tfm rats. We previously found an
increased ASR in gonadally intact Tfm males compared to wt males and females
(Zuloaga et al., 2008), and in this study Neo-Sham Tfm males showed an increased
startle response compared to Neo-Sham males as well as Neo-de males of either group.
While in this study the increased mean ASR in Tfm males compared to wt males among
Neo-Sham animals did not reach signiﬁcance (Figure 5b, leﬁ), the trend was clearly
consistent with our previous ﬁnding, and analysis of ASR by trial indicates Neo-Sham
Tfrns display greater ASR than their wt counterparts (Figure 5c). Thus depriving Tfm
males of testicular secretions during development eliminates the effect of the mutation on
ASR in adulthood. This result suggests an interaction of AR and ER stimulation for this
behavior. One possible explanation is that neonatal ER stimulation increases startle (as it
appears to do for all other measures of anxiety we tested), but neonatal stimulation of
ARs can counteract that effect for this particular behavior. This mechanism would
explain why Neo-Sharn Tfms show a greater startle than Neo-Sham males: they receive
ER stimulation that increases startle, but do not receive the AR stimulation that would
ameliorate this effect of ER. In this scenario, wt males fail to show a signiﬁcant
reduction in ASR following neonatal castration because the loss of ER stimulation is
counterbalanced by the loss of AR stimulation.

Our ﬁnding of elevated corticosterone levels in Neo-de rats is in accord with a
recent report of increased basal corticosterone in neonatally gonadectomized rats

(Bingham and Viau, 1998). Furthermore, prenatal administration of ﬂutamide (an AR

97

antagonist) or 1,4,6-androstatriene-3,17-dione (ATD: an aromatase inhibitor) also results
in increased basal corticosterone levels in rats, indicating that androgens act through both
ARs and ERS around the time of birth to organize basal corticosterone levels (Seale et al.,
2005). However, supporting our previous ﬁnding in gonadally intact Tfm and wt males,
there were no signiﬁcant differences in basal corticosterone levels between Neo-Sham
Tfm and wt males in the current study, which suggests that AR deﬁciency during
ontogeny does not affect this measure (Zuloaga et al., 2008).

As opposed to the basal condition, levels of corticosterone following exposure to
a novel object did not differ between groups. We have previously found increased
corticosterone levels in gonadally intact Tﬁn compared to wt males after novel object
exposure (Zuloaga et al., 2008) and others have demonstrated that neonatal gonadectomy
increases the corticosterone responses to stress in adult rats (Bingham and Viau, 2008).
However rats in the current study had been subjected to repeated stress prior to
corticosterone analysis, while rats in other studies, including our previous study, were
not. Exposure to repeated stress alters the corticosterone response to stress (Stamp and
Herbert, 1999; Melia et al., 1994; Retana-Marquez, 2003), and as a result, may have
reduced differences between groups. Basal levels of corticosterone can also be altered by
prior exposure to stress (Ottenweller et al., 1994; Servatius et al., 1994) suggesting that
basal corticosterone levels may have been elevated in rats in the current study.

PPI was also increased in Neo-de rats compared to Neo-Sham rats, indicating an
organizational effect of T in sensorimotor gating. Gonadal hormone administration in
adulthood was previously shown to inﬂuence PPI in rats (Eikelis et al., 2001 , Gogos and

van den Buuse et al., 2003), but to our knowledge this is the ﬁrst evidence of an

98

organizational effect of hormones on PPI. Again this effect appears to be primarily
dependent on ER rather than AR activation since there were no differences between Neo-
Sham Tﬁn and Neo-Sham wt males.

Brain weight analysis also revealed an effect of neonatal treatment on brain
weight with Neo-Sham rats having heavier brains than Neo-de rats. This difference
was largely accounted for by heavier brain weights in Neo-Sham wt males compared to
both Neo—de groups, though Neo-Sham Tfm males also tended to have heavier brains
than did Neo-de rats. Decreased brain weight in Neo-de rats compared to Neo-Sham
wt males resembles generalized sex differences in brain weight (males > females; Peiffer
et al., 2003; Zuloaga et al., 2008a), and indicate that brain weight is increased by
testicular secretions during development in male rats. Conﬁrming previous ﬁndings in
intact rats (Zuloaga et al., 2008a, Zuloaga et al., 2008b) brain weight did not signiﬁcantly
differ between Tfm and wt males indicating that T increases brain weight via an AR-
independent mechanism. Because the Tﬁn mutation decreases body weight, but has no
effect on brain weight, masculinization of these two parameters are at least partially
independent in rodents (Zuloaga et al., 2008).

In conclusion, neonatally gonadectomized rats showed reduced anxiety-related
behavior compared to sham-operated rats in tests thought to reﬂect anxiety (open ﬁeld,
novel object test, LD box, EPM, ASR), indicating that testicular secretions during the
neonatal period normally result in increased anxiety in adult male rats. Virtually all of
these effects of neonatal testicular secretions are equivalent in wt and Tﬁn males,
indicating that AR does not mediate these effects. Only in ASR did we ﬁnd evidence that

neonatal AR stimulation affects adult anxiety, and even this effect suggests a contributing

99

role of ERs as well. Compared to Neo-Sham rats, Neo-de rats also showed increased
PPI and basal corticosterone levels as well as decreased brain weights, and these effects
are also independent of AR. Together, these ﬁndings indicate a role of T prior to
adulthood in the organization of anxiety-related behaviors, the HPA axis, sensorimotor
gating, and brain weight, and suggest that ERs mediate these masculinizing effects of

neonatal T.

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Chapter 6: Discussion

Together our studies in Tﬁn rodents indicate a role of the AR and/or developmental
androgens in the regulation of anxiety-related behavior, the HPA axis, and sensorimotor
gating. The contribution of ARs and developmental androgens to these behavioral and

physiological responses are discussed in this chapter.

Anxiety-Related Behaviors and Activity
In Chapters 3 and 4 we demonstrated that Tﬁn males in both mice and rats show
increased indices of anxiety compared to wt males in several behavioral tests. These
ﬁndings indicate that in male rodents ARs normally act to reduce anxiety-related
behaviors. Tﬁn male rats were unlike wt males or females, displaying elevated anxiety
compared to both groups, suggesting that AR normally plays this role in females as well.
In this study there were not robust sex differences in tests of anxiety that have previously
been reported (females less anxious than males; Lucion et al., 1996; Zimmerberg and
Farley, 1993). However, females tended to show decreased indices of anxiety compared
to males, particularly when tests were performed from least to most anxiety-provoking.
Together these results indicate that Tfm male rats were neither masculinized nor
feminized in this behavior—rather it suggests that functional ARs are needed for the
display of “normal” emotional responses in both sexes.

Differences in anxiety-related behaviors appear to be task speciﬁc and to vary
between the two rodent species. In both Tﬁn mice and rats, anxiety-related behaviors are
increased upon exposure to a novel object. However, both species showed increased

indices of anxiety in one other test: mice in the LD box and rats in the open ﬁeld. Why

101

Tfm male mice and rats differ in terms of the speciﬁc tests in which they display elevated
anxiety is not readily apparent but it suggests that the absence of ARs results in a
moderate rather than robust increase in anxiety-related behaviors. If the role of the AR
was more robust we would likely see increased anxiety-related behavior in Tfm males in
all tests of anxiety.

Alternatively, rodents may have differed in anxiety-related behaviors in only
speciﬁc tests because neural mechanisms underlying the behaviors may differ between
tasks. Compared to other traditional tests of anxiety, exposure to an open ﬁeld in rats has
been demonstrated to involve speciﬁcally the basloateral amygdala (BLA) and inputs
from other areas (particularly the hippocampus) into this region (Hale et al., 2008). In
contrast, a single exposure to the elevated plus maze in rats has been shown to activate
areas of the hypothalamus including the paraventricular nucleus and dorsomedial
hypothalamus as well as the cingulate cortex, inferior colliculus, and median raphe
nucleus (Albrecht-Souza et al., 2008; Andrade and Graeff, 2001). A role of the
hippocampus and median raphe nucleus has also been demonstrated in the regulation of
behavior in the light/dark box (Andrade and Graeff, 2001; Bannerman et al., 2003).
While other studies indicate that brain activation patterns are grossly similar between
traditional tests of anxiety (Salome et al., 2004) as are the neurotransmitters involved
(Ludwig and Schwarting, 2007; Steiner et al., 1997; Schmitt and Hiemke, 1999), it is
always possible that there are subtle variations in the relative contributions of different
brain regions to each test, and that AR has differing levels of involvement in the different
brain regions. As a result, further studies are needed to clarify differences underlying

behaviors in these tests of anxiety.

102

Our investigation into the role of androgens and the AR prior to adulthood in the
organization of anxiety-related behaviors (Chapter 5) did not replicate some of the
differences we had previously seen between gonadally intact Tfm and wt males.
Speciﬁcally, neonatally sham—operated Tﬁn and wt male rats did not show behavioral
differences in the open ﬁeld and novel object test, although ASR was again increased in
Tfm rats. This discrepancy (discussed in chapter 5) may have resulted from effects of
neonatal anesthesia or adult gonadectomy in rats used in the study in chapter 5. Since we
found increased anxiety-related behavior in Tfm male rats in the open ﬁeld and novel
object tests in two different cohorts of gonadally intact rats these differences in chapter 3
do not appear to be transient.

Ambulation did not differ between wt and Tfm male mice (data not reported) or
rats (see chapter 4) indicating that ambulatory behavior is not AR-dependent. However,
another measure of activity, rearing, was greater in wt than in Tﬁn male mice with no
differences found between wt and Tfm male rats. Differences between these Tfm models
could result from differences in (a) the role of the AR in the regulation of rearing in mice
and rats, (b) T during development between the rat and mouse models, or (c) the nature of
the mutation between the two models that results in residual AR functionality in rats
compared to virtually none in mice (Yarbrough et al., 1990; Charest et al., 1991).
Whatever the nature of the species difference, this outcome suggests that in mice the
absence of functional ARS decreases the display of rearing. In male rats, activity
measures are more likely mediated through activation of the ER during development
based on our evidence from neonatally gonadectomized and sham-operated wt and Tﬁn

males in Chapter 5. In this study, neonatal gonadectomy signiﬁcantly increased

103

ambulation and moderately increased rearings in tests of anxiety while Tfm and wt male
groups did not differ in these measures, indicating an organizational effect of androgens
that is AR-independent.

In humans there is abundant evidence that androgens are involved in anxiety and
depression. A decrease in T with aging and low levels of T in younger men is often
associated with elevated anxious and depressive symptomology (Karninetsky, 2005;
Lund et al., 1999; Amore, 2005; Eskelinen et al., 2007; Cooper and Ritchie, 2000). There
is also emerging evidence that the AR is related to the etiology of mood disorders,
particularly from comparisons of AR polymorphisms in which the number of CAG
repeats in the AR gene is negatively correlated with levels of AR mediated transcription
(Mhatre et al., 1993; Kazemi-Esfarjani et al., 1995). A greater number of CAG repeats in
the AR gene is associated with symptoms of anxiety and depression in adolescents (Su et
al., 2007) as well as aging men (Harkonen et al., 2003). Another study suggests that a 5-
alpha reductase inhibitor (ﬁnasteride) is also linked to decreases in mood. Individuals
taking ﬁnasteride to treat male pattern hair loss reported increased symptoms of
depression and anxiety after 2 months of treatment with the drug (Rahimi-Ardabili,
2007). Since 5-alpha reductase inhibition causes a decrease in DHT, which subsequently
leads to decreased AR activation, this result again suggests a role of the AR in mood
disorders. Recent evidence also indicates that the AR may mediate mood via regulation
of the HPA axis. The brains of depressed individuals, compared to non-depressed
controls, contained lower levels of AR mRN A in the paraventricular nucleus of the
hypothalamus (PVN), a key brain region involved in the regulation of the HPA axis

(Lund et al., 2006). Since AR activation in the PVN is believed to inhibit HPA axis

104

activation, a decrease in AR mRN A levels suggests a mechanism through which the HPA
axis could become hyper-activated and consequently exacerbate symptoms of depression.
This mechanism may contribute to the greater adrenal steroid response of Tfm male
rodents in our studies.

Together, our evidence and that of others suggests a potential therapeutic role of
AR stimulation in the treatment of anxiety and depression. AR agonists have been shown
to be beneﬁcial in disorders involving muscle wasting, osteoporosis, and sexual behavior.
However side effects, including enlargement of the prostate in males and virilization in
females, can out-weight the beneﬁts (Gao and Dalton, 2007). The development of
selective androgen receptor modulators (SARMs) may offer a solution to this problem
(Gao and Dalton, 2007). SARMs can act on speciﬁc tissues in the body, thus avoiding
the ill effects of AR stimulation in non-target tissues. As a result, it appears feasible to
generate a SARM that could activate AR in the brain and muscle (producing positive
effects) but not in the prostate (reducing negative effects). To date, only a few SARMs
have reached clinical trials.

In Chapter 5 we also present evidence that the removal of androgens during
development can lead to a decrease in anxiety-related behavior; essentially resulting in
male rats that act like females (less anxious than control males). However, in humans
there is an opposite gender difference in anxiety (females > males; DSM-IV, 1994) and
decreased T during development is more likely to contribute to increased anxiety
(Granger et al., 2003; Evardone and Alexander, 2008). For example, Evardone and
Alexander (2008) recently demonstrated that larger 2nd to 4th ﬁnger length ratios

(indicative of lesser prenatal androgen exposure) were correlated with greater levels of

105

anxiety in men. Given the conﬂicting ﬁndings between rats and humans in anxiety, it
isn’t realistic to extend our ﬁndings in neonatally gonadectomized rats to humans. It has
also been suggested that sex differences reported in rat models of anxiety may not
actually reﬂect differences in anxiety per se. A factor analysis of behavior in male and
female rats in the EPM revealed that anxiety primarily contributed to behavior in males
while activity was the primary contributor to female behavior (F emandes et al., 1999).
This interpretation indicates that there are sex differences in motivating factors that
underlie behavior in tests of anxiety and raises the question of whether male rats are truly

more anxious than female rats.

Hypothalamic-Pituitary-Adrenal Axis

Compared to wt males, Tfm mice and rats showed an increased peak corticosterone
response following exposure to an open ﬁeld with a novel object, indicating an
involvement of the AR in HPA axis regulation. Tfm male mice demonstrate a
particularly hyperactive corticosterone response with elevations at' baseline, peak, and
during recovery compared to wt males, whereas only peak corticosterone levels are
elevated in Tfm male rats. This species difference likely reﬂects differences in T levels
between the two Tfm models in which Tﬁn mice show decreased T, while Tﬁn rats show
increased T compared to wt male siblings. Therefore, although T’s actions through the
AR are greatly reduced in Tﬁn male rats, there is ample T to be converted to estrogens
and activate ERB to decrease the corticosterone response to stress. Since T is reduced in
Tﬁn male mice, activation of both AR and ERB is probably also reduced, resulting in

persistently increased corticosterone levels. It remains possible that residual AR function

106

in Tﬁn male rats (Yarbrough et al., 1990), compared to virtually none in Tfm male mice
(He et al., 1991; Monks etal., 2007), may also have accounted for their lesser elevation
in corticosterone levels. However, recent demonstrations of the powerful inhibitory role
of ERB on HPA axis activity (Lund et al., 2005; Lund et al., 2006) suggests that
differences in ERB activation may represent the greatest contribution to this species
difference.

In both Tfm models, increased HPA axis activation is reﬂected by increased
anxiety-related behavior compared to wt males. Similar elevations in corticosterone
levels have been demonstrated in male mice and rats that display trait anxiety behaviors
(J ankoveski et al., 2008; Landgraph et al., 1999). This ﬁnding indicates that the absence
of functional ARs in Tfm males predisposes them to HPA axis hyperactivation, which
contributes to increased anxiety-related behavior in these rodents. In humans,
hyperactivity of the HPA axis is correlated with disorders of anxiety and depression
(Swaab et al., 2005). Common symptoms of Cushing’s syndrome, a disorder
characterized by high levels of cortisol in the blood, are anxiety and depression.
Furthermore, the combined dexamethasone/CRH test of HPA axis activity can identify,
with near 80% sensitivity, individuals that are depressed (Heuser et al., 1994). In non-
depressed individuals, pretreatment with dexarnethasone (a corticosteroid) suppresses
pituitary—adrenal responses to corticotropin releasing hormone (CRH); however, in
depressed individuals the same procedure enhances hormonal responses to CRH
indicating a hyperactivation of the HPA axis. As mentioned above, depressed individuals

also show decreased AR mRN A in the PVN, suggesting a mechanism through which

107

ARs can alter activation of the HPA axis, and in turn contribute to the occurrence of
mood disorders.

However, unlike depressed individuals, people with disorders of anxiety do not
show increases in HPA axis hyperactivity, indicating that in humans the link between
anxiety and hyper-activation of the HPA axis is less clear. Furthermore, in rodents,
subjects that show increased indices of anxiety also commonly show increased
depressive-like behavior, therefore it is difﬁcult to decipher whether increased release of
stress hormones in these rodents is speciﬁcally associated with anxiety-related behaviors.
In our experiments, we did not test Tfm males in rodent models of depression, such as the
forced swim test, so along with being more anxious it remains possible that these rodents
may also show increased indices of depression, and it may be that increased depressive-
like behavior (not anxiety-related behavior) is particularly related to HPA axis hyper-
activation. As a result, further research is needed to explore the speciﬁc link between
HPA axis hyper-activation and anxiety.

Our investigation into the role of androgen and ARs in the development of adult
HPA axis activation yielded inconclusive results given that these rats had been exposed
to a form of chronic stress prior to testing for corticosterone. As discussed in chapter 4,
prior exposure to stress can alter both basal (Ottenweller et al., 1994; Servatius et al.,
1994) and stress-induced increases in stress hormone levels (Stamp and Herbert, 1999;
Melia et al., 1994). Therefore analysis of corticosterone levels in new cohorts of rats,
that were not previously exposed to stress, would help to elucidate the role of androgens

and the AR in the development of adult HPA axis function.

108

It remains possible that differences in anxiety-related behaviors and the HPA axis
in all our experiments may, at least partially, be attributed to epigenetic inﬂuences.
Maternal licking and grooming behaviors have been shown to alter glucocorticoid
receptor expression in the hippocampus of offspring, a modiﬁcation that can inﬂuence
the HPA axis as well as stress responses (Weaver et al., 2004; Weaver, 2007). In
neonatally gonadectomized and sham-operated rats, exposure to either cryo-anesthesia or
neonatal surgery may have resulted in differences in maternal care. Furthermore, the
removal of androgens through neonatal gonadectomy may also decrease maternal licking
and grooming since the presence of androgens in neonates elicits greater maternal care
(Moore, 1982). Likewise, maternal care may have also differed for neonatal Tfm males
compared to wt males because sex hormone secretions likely differ between these groups
during this time period. Some or all of these epigenetic inﬂuences may have contributed
to differences in anxiety-related behaviors and corticosterone responses presented in this

dissertation.

Immediate Early Gene (c-Fos) Activation

In chapter 4 we reported differences in c-Fos immunoreactivity between wt and Tﬁn
male rats in 3 brain areas (MPOA, DG, and MePD) after exposure to an open ﬁeld with a
novel object. However, we do not know whether differences in activation of these brain
areas are speciﬁcally related to either differences in anxiety-related behaviors,
corticosterone responses, both, or neither. It is quite possible that elevated anxiety and
HPA axis activation in Tfm males involve independent mechanisms and distinct

pathways. Although the dentate gyrus and the MPOA have been indicated in playing a

109

role in regulating both responses, it may be that alterations in activity within these
regions reﬂect only differences in anxiety-related behaviors. Differences in
corticosterone responses may not be reﬂect differences in brain activation between Tfm
and wt males, rather alterations in pituitary activity may primarily contribute to differing
corticosterone responses. Alternatively, group differences in HPA axis activity may
reﬂect alterations in brain activity reported in Chapter 4 and differences in anxiety-related
behaviors may be entirely unrelated. In either case, we do not know whether behavioral
or physiological differences contribute to, or are a consequence of, alterations in neural
activity.

In any case we did ﬁnd signiﬁcant differences in immediate early gene activation
in 3 brain regions between wt and Tfm males. As previously mentioned, differential
activation of the DG and MPOA may reﬂect differences in either anxiety-related
behavior or HPA axis activity. The MPOA has been identiﬁed as an area within which
androgens suppress activation of the HPA axis (V iau and Meaney, 1996). Therefore
increased activation within this area in Tfm males may reﬂect an absence of androgen
suppression, due to a lack of functional ARs, which results in elevated excitatory input to
other regions involved in the HPA axis, such as the PVN. Alternatively, increased
activation of the MPOA could reﬂect elevated anxiety-related behavior. C-Fos
immunoreactivity is increased in the MPOA of rats bred for high anxiety (HA) compared
to low anxiety rats (LA) after exposure to an open ﬁeld (Salome et al., 2004). However,
HA rats also show HPA axis hyper-activation, so whether elevated c-Fos in the MPOA is
related speciﬁcally to anxiety-related related behavior, HPA axis activity, or both

mechanisms, remains unclear.

110

C-Fos-ir within the DG was decreased in Tﬁn males compared to wt males after
exposure to an open ﬁeld with a novel object. The dentate gyrus plays a role in the
regulation of anxiety-related behaviors in rodents (Chen at al., 2007) and androgens have
been shown to act in the hippocampus to decrease anxiety, though it is unclear whether
androgens act on cells within the dentate gyrus or other regions of the hippocampus to
regulate this behavior. The hippocampus, including the dentate gyrus, contains abundant
adrenal hormone receptors and is involved in the regulation of the HPA axis. Therefore
differences in activation of the dentate gyrus could represent differences in anxiety-
related behavior, the HPA axis, or both. It is also possible that a decrease in c-Fos-ir in
the dentate gyrus could be a consequence of increased cell death within this region
resulting from chronically elevated corticosterone levels, although I did not quantify cell
number within this region.

Differences I found in c-Fos activation within the MePD (Tfm males < wt males)
are the most difﬁcult to relate to differences in anxiety-related behavior and the HPA axis
because although the MePD has abundant AR in wt males, this region is not generally
believed to directly inﬂuence anxiety behaviors. The MePD is particularly involved in
receiving olfactory and pheromonal information and is important for some aspects of
male sexual behavior (Lehman et al., 1983; Swann et al., 2001). Nevertheless, the MePD
projects to two other hormone and stress responsive regions (BNST, MPOA) and through
these projections may regulate anxiety-related behaviors and the HPA axis (Coolen and
Wood; 1998; Gomez and Newman, 1992). It may be that androgens act through ARs in
the MePD to inhibit activation of cells in these other brain areas, resulting in decreases in

anxiety and/or HPA activation. Alternatively, decreased c-Fos-ir in Tfm males may

111

reﬂect a more traditional role of the MePD; such as a disruption in the processing of
olfactory and pheromonal cues.

Even in areas where no change in c-Fos activation was detected between Tfm
and wt males, it remains possible that there were actual differences in the activation of
speciﬁc cell types within these regions, yet net cell activation was the same. Such are the
limitations of c-Fos. Dual immunocytochemisz of c-Fos combined with glutamate
decarboxylase (GAD‘ a marker for GABAergic cells), corticotropic releasing factor
(CRF), or arginine vasopressin (AVP), would be useﬁrl to better characterize the cell

types that are activated following exposure to a novel object.

Sensorimotor Gating

The role of the AR in sensorimotor gating appears minimal based on our experiments in
Tfm rodents. In mice our initial test for PPI indicated a moderate, but signiﬁcant
decrease in PPI in Tfm male mice compared to wt males. However, in our follow-up
studies in intact and T-replaced mice we did not ﬁnd any differences between Tfm and wt
males, suggesting that differences we had seen initially were transient and did not
represent group norms in this behavior. In comparisons of PPI between wt and Tfm male
rats (chapters 4 and 5), we did not ﬁnd any differences, again indicating little or no role
for the AR in the regulation of sensorimotor gating. Furthermore, PPI did not differ
between gonadectomized wt male mice treated with T or nothing. This result suggests
that in mice, adult circulating T levels also appear to have little or no inﬂuence on

sensorimotor gating.

112

Interestingly, in chapter 5, we demonstrated that neonatal gonadectomy
signiﬁcantly increased PPI, but that this is unrelated to AR since the results were
identical in wt and Tﬁn male rats. These results suggest that the T during postnatal
development in males normally increases sensorimotor gating by acting upon ERs. As
we are aware of no other studies that have explored the organizational role of androgens
in sensorimotor gating, this appears to be the ﬁrst demonstration of this kind. In humans,
sex differences have been reported in PPI, but this difference appears to be largely the
result of differences in circulating estrogen, not T levels, that result in ﬂuctuations in PPI
in women (Swerdlow et al., 1997). Since deﬁcits in sensorimotor gating are associated
with schizophrenia, this could mean that exposure to androgens during development in
males predisposes them to schizophrenia. There is not a sex difference in overall
incidence of schizophrenia, but the development and severity of this disease do show a
marked gender difference. In men the onset of schizophrenia typically occurs during
development (early adulthood and adolescence; Hambrect et al., 1992), a period during
which T levels are elevated. Furthermore, in adolescent male rats, but not female rats,
there is an overproduction of dopamine receptors, which are subsequently eliminated in
early adulthood (Andersen et al., 1997), and T replacement in gonadectomized male and
female rats increases D1 receptor binding (Andersen et al., 2002). Overproduction of
dopamine has been linked to schizophrenia (Lieberman et al., 1987) therefore this could
be a mechanism through which developmental T could predispose an individual to this

disorder.

Future Directions

113

Our ﬁndings in Tfm rodents indicate an increase in anxiety-related behavior and HPA
axis activation that appears to be AR dependent. However, further experiments would be
useful in verifying the role of ARs in these responses. AR deﬁciency in Tfm males might
also alter the expression of estrogen receptor subtypes ERa and ERB and through this
mechanism may inﬂuence the display of anxiety-related behaviors and the HPA axis. An
immunocytochemical comparison of ERa and ERB in the brain of Tfm and wt male
rodents would help to answer this question. In order to differentiate the respective
contributions of AR and ERB, one could generate double-knockout AR/ERBko mice and
compare their anxiety-related behaviors and corticosterone responses with ARko, ERBko,
and wild type mice. This experiment would be helpful in clarifying the individual roles
of these steroid hormone receptors in the regulation of these responses and could also be
used to compare relative contributions of these hormone receptors in both males and
females.

Our ﬁndings in chapter 4 also suggest that ARs may also play a role in decreasing
anxiety-related behaviors in female rats. Administration of DHT to female rats decreases
anxiety-related behaviors (Frye and Lacey, 2001), further indicating a role of the AR in
regulating this behavior in female rats. However, as mentioned before, DHT can act on
other receptor types in the brain to decrease anxiety-related behaviors (Lund et al., 2004),
so the role of ARs in the regulation of anxiety-related behaviors in female rats remains
somewhat inconclusive. In order to further explore the ARs role in females one could
compare groups of female rats that were (1) administered DHT, (2) administered both
DHT and ﬂutamide, or (3) treated with nothing. If ARs are important in regulating

anxiety-related behaviors we would expect DHT treated females to show decreased

114

anxiety compared to both DHT/ﬂutamide treated and untreated female rats. It is also
possible that DHT/ ﬂutamide treated rats would show decreased anxiety-related
behaviors compared to untreated females if metabolites of DHT, acting through other
receptor types (GABA/ERB), play a role in regulating this behavior.

Another issue that remains largely unanswered is the role of the AR at different
developmental time-points in the regulation of anxiety-related behaviors and the HPA
axis, particularly in mice. One way to investigate the developmental role of ARs is
through the generation of conditional AR knockout mice in which we could eliminate AR
function at several different points in the lifespan. This approach would help decipher
whether it is the lack of prenatal, early postnatal, pubertal, or adult AR stimulation that
contributes to the elevated anxiety-related behaviors and corticosterone responses we
observed in Tfm male mice. It is also possible that activation of ARs at several

developmental time-points contribute to differences we report in Tfm males.

Conclusion

The ﬁndings presented in this dissertation add to emerging evidence that ARs contribute
to the masculinization of the rodent brain and behavior (see Chapter 2). Speciﬁcally,
these data support a role of the AR in the regulation of anxiety-related behaviors and the
HPA axis. Furthermore, supporting the organizational hypothesis (Phoenix et al. 1959),
we present evidence of an organizational role of androgens in behavior. Extending
previous ﬁndings, we demonstrated that anxiety-related behaviors were affected by the
absence of androgens in development, and present a novel role of organizational

androgens, probably acting though ERs, in the development of sensorimotor gating.

115

Together, research presented in this dissertation represents a contribution to the ﬁeld of
behavioral neuroendocrinology and may potentially contribute to the development of

novel therapies for the treatment of psychiatric disorders in humans.

116

APPENDIX

Table 1. Key morphological ﬁndings in the Tﬁn rodent brain. MePD: posterodorsal
medial amygdala; SCN: suprachiasmatic nucleus; VMHdm: dorsomedial aspect of the
ventromedial hypothalamus; VMHvl: ventrolateral aspect of the ventromedial
hypothalamus; BSTMPM: posteromedial nucleus of the bed nucleus of the stria
terminalis; SDN-POA: sexually dimorphic nucleus of the preoptic area; LC: locus
coeruleus; DG: dentate gyrus; SNB: spinal nucleus of the bulbocavernosus; LS: lateral
septum; AVP: arginine vasopressin.

 

 

 

 

 

 

 

 

 

 

 

 

Re 'on Species Morphological Characteristics Tfm 6’s are Refs
ePD Volume: wt 6 > Tfm 6 > wt S2. Em Bdorris et a1,
Soma Size: wt 6 > Tfm 6 > wt 9. nt 005
SCN Volume: wt 6 > Tfm 6 ~ wt 9. cm orris et a1,
Soma Size: wt 6 > Tﬁn 6 ~ wt 9, but wt 6 ~ cm? 005
wt S2.
VMHdm Volume: No signiﬁcant group differences. -- ugger et a1,
Soma Sizezwt6>Tfm6~wt Q. 007
cm
olume: wt 6‘ > Tﬁn 6 ~ wt 9. em ugger et a1,
WMHvl IEoma Size: wt 6 > wt S2, but Tfm 6 not t? 007
_igniﬁcantly different from either sex.
LSeTMPM Rat Volume: wt 6 > Tfm 6‘ ~ wt 52. F::; urazzo et a1,
Soma Size: No signiﬁcant group differences. - 007
emisphere)
DN-POA Volume: wt 6 ~ Tfm 6‘ > wt S2. Morris et a1,
Soma Size: wt6>Tfm6~wt S2,butwt6~ 2005

thB.

 

 

Nolumezwt6<Tfm6~wt Q.

 

 

Garcia-Falgueras

 

 

 

 

 

 

[Neuron Number: wt 6 < Tﬁn 6 ~ wt 32. et a1, 2005
IDG olume: wt 6 ~ Tﬁn 6‘ > wt 92, but wt 6 not '12:? Jones and
i iﬁcantly different from either group. Watson, 2005
ISNB euron Number: wt 6‘ > Tﬁn 6 ~ wt 9. Eem medlove and
oma Size: wt 6 > Tfm 6‘ old, 1981
[LS ﬂMouse IAVP Density: wt 6 ~ Tfm 6 > wt 9. IMasc Ecordalakes and
'ssman, 2004

 

ll7

 

Table 2. Key Behavioral Findings in Tfm Rodents

 

6’s displaying superior performance.

Tﬁn 6’s show decreased spatial memory
performance compared to Tfm carrier 92’s,

S atial Memo
orris Water fm 6’s show an intermediate spatial memory
erforrnance between wt 6’s and 32’s, with wt
ouse

Jones and Watson, 2002

 

Jank et a1 2005
though there was no sex difference in wt mice.

 

 

 

 

 

 

 

nxietv-related
behavior
ovel Object ouse Tfm 6’s show greater anxiety-related behavior esent Study
xposure han wt 6’s.
levated Plus ouse fm mice (Tfm 6 ’s and carrier Q’s) show izk etal., 2005
increased indices of anxiety compared to wt
mice (6’s anﬂ’s).
la Fi htin
uvenile play at Tfm 6’s show decreased play ﬁghting eaney et a1, 1983
rghting compared to wt 6’s.
at Tﬁn 6’s fail to show wt 6 typical decline in ield et a1, 2006
playful attack and one form of playful defense
(complete rotations).
Aggression
esident-intruder Mouse Similar indices of aggression in Tfm and wt Scordalakes and Rissman,
list 6’s; both more aggressive than 9’5. 2004

 

 

 

 

at Tfm 6’s show wt 6 typical defeminization of
exual behavior.
at Tﬁn 6’s show reduced masculine sexual
behavior behavior compared to wt 6’s.
ouse Ll‘fm 6’s show reduced masculine sexual
ehavior compared to wt 6’s, a difference that
is eliminated in E2 treated mice.
[Partner Preference at fm 6’s show a masculinized partner
reference.
ouse ﬁn 6’s show a demasculinized partner
reference.
Olfactory

 

reference test
reference.

Olsen, 1979

 

each and Buehler, 1977;
1sen and Whalen, 1981

0 et a1, 1974; Bodo and
'ssman, 2007

amson et a1, 2005

odo and Rissman, 2007

ouse frn 6’s show a demasculinized soiled bedding odo and Rissman, 2007

 

118

 

Table 3. Body weight and testosterone levels in wt and Tfm male mice. In T and B
treated mice, body weight was greater in T-treated wt males than all other groups
(p’s<.05), and T levels were elevated in T compared to B-treated mice (p<.001). In intact
untreated mice, wt males had elevated body weights and T levels compared to Tfm males
(p<.01). T: testosterone, B: blank.

 

Mice Weight T (nmoI/L)
T or B Treated
WT Male (T) 9.38 :t .60 14.22 :t 2.95

WT Male (B) 25.93 d: .96 263 :t 1.52
TFM Male (T) 26.49 :1: .56 16.81 i 2.17
TFM Male (B) 25.78 d: 1.38 4.43 3: 2.28

 

 

 

 

 

ntact
T Male 28.38 3: .70 19.75 :t 4.39
7 FM Male 24.92 i .74 3.26 :1: .52

 

119

Table 4. Behavior in tests of anxiety in wt male, Tfm male, and wt female rats in test
order 1 (Open Field/Novel Object Test, EPM, and LD box, preceded by the PPI test) and
test order 2 (Open Field/Novel Object Test, LD box, and EPM, not preceded by the PPI
test). * indicates p<.05 compared to wt males, A indicates p<.05 compared to wt females,
00 indicates p<.01 compared to wt females, # indicates p<.05 compared to the same
genotype in test order 1.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Open Field Center Time (s) lCenter Entries earings rid Crossings—Frooming (s) ‘
Test order 1
Wt Males 16.1 i 2.2 6.0 :t 1.3 23.6 :h 3.6" 113.1 :1: 14.7 6.3 :1: 2.5
Tﬁn Males 8.5 i 2.1“ 00 3.7 i 0.9" 26.1 i 4.3 96.6 :1: 16.3 6.5 :1: 2.7
Wt Females 21.4 d: 2.0 7.6 :t 0.7 37.2 i 2.0 134.7 i 7.2 2.8 i 0.8
Test order 2
Wt Males 18.5 i 4.3 5.2 i 1.0 32.4 :1: 3.4 108.0 :h 7.5 2.4 :1: 0.1
Tfm Males 10.9 i 3.2 3.3 :t 0.9 28.4 :t 2.6" 99.3 i 9.6 5.7 i 1.6"“
Wt Females 24.2 i 4.9 5.8 :t 0.9 41 :t 2.0 126.4 i 2.1 3.3 d: 0.7
ovel Object Test bject Time (5) Object Visits earings IGrid Crossings Grooming (5)
Test order 1
Wt Males 38.1 i 5.5 5.9 i 0.8 12.2 i 2.5 68.1 i 9.7 9.8 i 2.7
Tfm Males 15.2 :t 4.4'M 3.3 :h 0.9A 15.6 :t 3.2 63.2 :I: 12.0 27.1 i 6.7
Wt Females 42.3 i 8.0 7.6 d: 1.3 24.4 :1: 4.2 86.2 i 11.5 14.1 d: 6.3
Test order 2
Wt Males 43.8 :t 9.5A 4.7 :h 0.9" 23.4 :t 2.700 79.8 d: 12.0" 8.3 :1: 2.0
Tfm Males 11.7 :t 4.5* 00 1.9 i 0.7 00 20.5 d: 4.100 66.5 i 11.000 18.7 :h 6.4
Wt Females 80.8 :1: 12.0 9.0 i 1.1 41.6 i 4.3# 125.8 i 7.8 3.8 :t 1.2
lElevated Plus Maze Open arm Time (8) Open Arm Entries otal Arm
ntries
Test order 1
Wt Males 41.1 i 5.8 2.8 :1: 0.4 13.6 i 1.6
Tﬁn Males 23.2 i 6.4 1.6 :t 0.4 11.1 :t 13"
Wt Females 45.4 i 11.0 3.1 d: 0.6 17.2 i 1.5
Ll‘est order 2
Wt Males 41.6 i 10.0 3.8 :t 0.9 20.7 :t 1.8#
Tfm Males 46.2 :h 15.2 3.5 i 0.9 20.5 :t 2.7#
Wt Females 79.3 i 13.6# 6.8 :1: 1.5# 27.2 i 1.7#
[Light Dark Box Light Area Time (3) Light Area Entries ight Area
earings ‘
Test order 1
Wt Males 206.7 i 46.8 6.9 i 1.0 21.5 :1: 4.9
Tfm Males 84.5 :1: 28.4 6.3 d: 0.8 7.9 :1: 2.9
Wt Females 134.0 1 39.9 5.4 :t 0.7 13.3 :1: 5.9
Test order 2
Wt Males 114.5 i312 5.4:t1.3 10.8:lz3.l
Tfm Males 86.8 i 46.0 4.6 :1: 1.7 6.5 d: 3.2
Wt Females 109.3 i 30.4 6.6 :t 1.5 15.4 :1: 4.9

 

 

120

 

 

 

 

 

 

 

 

 

 

 

 

 

     
 

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121

Figure 1. (A) Brain and (B) body weights of adult wildtype (wt) male, Tfm male, and wt
female mice and rats. All animals were between 120-150 days old at the time of
sacriﬁce. Brain weight measurements did not include the olfactory bulb and were taken
after prolonged post-ﬁxation in 10% formalin. Wt males show an increased brain weight
compared to females, and Tﬁn males resemble wt males, in both mice and rats. Tﬁn
males show a body weight intermediate between wt males and females. * indicates p< .05
compared to wt males.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A
— wr MALES , 25

E MICE Ea TFM MALES RATS -
A 0.6 f I: WT FEMALES A
E ' 1'53 * E
E a- . , E
g 0.5 : x + § —E_‘ 20 z.
e \ § . s
.2 0.4: \ k :1'5 Z

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B — wr MALES
MICE E3 TFM MALES RATS

=l WT FEMALES J

30 i
* 1 A
g 25 s r; :\ * i: g
g 20 : 2
8 15 x \ E 1300 ‘8
E \ \ i r
g .0 § \ 5 200 i
or 5 x \ ;_100 m

: o

 

 

 

 

 

 

 

 

 

 

 

122

Figure 2. The posterodorsal medial amygdala (MePD) in Nissl-stained coronal sections
from a wildtype (wt) male (top), a Tfm male with a dysfunctional androgen receptor
(middle), and a female rat (bottom). The panels on the leﬁ are from the caudalmost
appearance of the MePD, which served as an anchor point to assess changes in the
nucleus across the rostrocaudal dimension. The appearance of the MePD, as well as the
optic tract (ot), the stria terminalis (st), the anterolateral part of the amygdalohippocampal
transition area (AHiAL), and the lateral ventricle (v) are equivalent in wt males (3), Tfm
males (b), and females (c), indicating that the caudal termination of the MePD occurs in
the homologous region of the brain across groups. The panels on the right are from the
approximate middle of the rostrocaudal extent of the MePD where the nucleus is larger in
wt males (d) than in females (f) and is intermediate in size in Tfm males (e). The MePD
also extends farther rostrally in wt males and Tfm males than in females (Morris et al.,
2005). Scale bar = 250 “m in a (applies to a-c), d (applies to d-f).

123

Figure 2.

 

 

   
  

Caudal-Most Extent . Middle

 

124

Figure 3. Time spent visiting a novel object in adult male wt and Tfm mice that were
placed ﬁrst in an empty open ﬁeld and after ﬁve minutes were brieﬂy removed and
placed back into the open ﬁeld that contained a small object (a 2”x 2” Petri dish with red
and blue tape) in the center. (a) Upon exposure to a novel object, wt males spent more
time exploring the novel object than did Tfm males.

(b) Wt male mice castrated as adults spent less time visiting the object, an effect that was
averted if they were treated with testosterone (T) rather than blank capsules. T had no
effect in Tﬁn males, indicating that androgen receptors mediate this effect. * indicates
p< .05 compared to (a) Tfm males or (b) T-treated Tfm males.

A

_ Novel Object Test
30 " *

 

- Wl' MALES

- m TFM MALES
20 -

10~

Time Visiting Object (sec)

l I l I

 

 

 

 

TT

30 Castrates

_ WI" MALES
El TFM MALES

 

Time Visiting Object (sec)
- * %

 

Blank Testosterone

125

Figure 4. Plasma corticosterone levels 20 minutes aﬁer initial exposure to the 10 minute
open ﬁeld/novel object test were signiﬁcantly greater in Tfm male mice compared to wt
male mice. * indicates p< .01 compared to Tfm males.

 

 

 

: 50° ‘: — Wl' Males T\
g, : E1 TFM Males %
‘c’ C * \
g 300 -_ \
2 .

3 ; \
.2 200 -_ \
t : \
O .

o 100 -_ 1- \

 

 

 

 

 

 

 

Control Open/Object

126

Figure 5. Prepulse inhibition in gonadally intact wt and Tﬁn male mice in (A) test order
1 (PPI, open ﬁeld/novel object test, EPM, LD box) and (B) test order 2 (open ﬁeld/novel
object test, LD box, EPM, PPI). There is an overall decrease in prepulse inhibition in
Tfm compared to wt males (p <.05) in test order 1, but no signiﬁcant difference in test
order 2. When test order 1 and 2 are pooled together, no signiﬁcant difference between
Tfm and wt males in PPI is found.

 

 

A Prepulse Inhibition
Test Order 1
30- - WT Male IZZI TFM
60—
3'. 40-
.\°
20—
B
o-
PP3 PP8 PP10 PP15
80- Test Order 2
60-
% 4o-
°\°
20- L |
0..
PP3 PP8 PP10 PP15

Prepulse Decibel

127

Figure 6. Anxiety-related behavior in gonadally intact wt and Tfm male mice. The
amount of time spent in/with and number of visits to: (A) the center area of the open
ﬁeld, (B) a novel object in the open ﬁeld arena, (C) the open arms of the elevated plus
maze, and (D) the light area of the light dark box. (E) depicts the number of rearings in
the open ﬁeld, novel object, and LD box tests. These tests indicate greater anxiety-
related behaviors in Tfm mice compared to wt mice in the novel object test and light
dark box, but not the other two tests. * indicates p<.05 compared to wt males.

130

Figure 6.

Center Time(s)

an

Object Tim 9 (s)

0

Open Arm Time (s)

 

 

 

 

 

 

 

 

 

 

- WT Male IZZ TFM
40- Open Field - 40
30q l- 30
20- - 20
10- a L 10
0" r 0
Duration Visits
Novel Object Test
40— - 40
L 30
L 20
- 1o
- 0
Duration Visits
Elevated Plus Maze
200- - 25
150- " 2°
— 15
100-
— 10
50-
o-

 

Duration Visits

129

 

s1! SM iaaiqo susm mu 60

susm uuv uado

Figure 6 cont.

U

Light Area Tim e(s)

m

# Rearings

Light Dark Box

 

 

Duration Visits

50- Rearings

 

 

Open Novel Light Dark
Field Object Box

130

 

saw 291v N5!‘l

Figure 7. (A) Acoustic startle response and (B) Prepulse inhibition in T and B-treated wt
and Tfm male mice. No signiﬁcant group differences were found in acoustic startle
response or prepulse inhibition. T: testosterone, B: blank.

A

Acoustic Startle Response

4°“ -WTMalezszM

 

Startle Respone (Arbitrary Units)

 

Testosterone Blank

80 Prepulse Inhibition

% PPI

““‘\\““‘

7
r a
I
v 5
I a
f v
f I
f a
I I.
s a
I A

 

T B T B r B T B
PP3 PP8 PP10 PP15

Prepulse Decibel

131

Figure 8. Anxiety-related behavior in T and B-treated wt and Tfm male mice. The
amount of time spent in/with and number of visits to: (A) the center area of the open
ﬁeld, (B) a novel object, (C) the open arms of the elevated plus maze, and (D) the light
area of the light dark box. (E) depicts the number of rearings in the open ﬁeld, novel
object, and LD box tests. These results conﬁrmed the previously found differences
between wt males and Tfm males for the novel object and LD box tests (see Figure 2)
and suggest that T amelioration of anxiety-related behaviors are mediated by AR, since
Tfm animals showed no behavioral response to T treatment in any test. * indicates p<.05
compared to T—treated wt males. T: testosterone, B: blank.

132

T= Testosterone B= Blank

- WT Male

aTFM

Open Field

0033—. (EU

 

 

-30

Conan» 5&3

 

Novel Object Test

 

40-

E «:5 .330

 

Visits

Duration

C

-30

One: >3: 5&8

 

Elevated Plus Maze

 

 

E 25... E..< :30

B T

T

Duration

133

Figure 8 cont.

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Light Dark Box

Novel Object

Open Field

134

Figure 9. (A) Plasma corticosterone levels at baseline and 20 min after initial exposure
to an open ﬁeld with a novel object were elevated in Tfm males compared to wt males.
*p< .01 compared to wt males. (B) Plasma corticosterone levels at baseline and 20, 40,
60, and 120 min aﬁer initial exposure to an open ﬁeld with a novel object in Tﬁn and wt
males. *indicates p< .05 compared to the same time point in wt males.

A

600-
- WT Male

400 n TFM %

200

A

Corticoste rone (nglm I)

 

  

 

 

 

l
Baseline OpenIObJect

w

500 a

400 -

300-

200-

100-

Corticoste rone (nglm I)

 

 

0..
Baseline 20 40 60 120
Minutes

135

Figure 10. Prepulse inhibition (PPI; A), acoustic startle response (B), and acoustic startle
response by trial in gonadally intact wt male, Tfm male, and wt female rats. There were
no group differences in PPI, but ASR was greater in Tfm males than either wt males or
females, primarily due to differences in startle response to the ﬁrst pulse. * indicates
p<.05 compared to wt males and females. # indicates p<.001 compared to same trial wt
males and females.

136

Figure 10.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A Prepulse Inhibition
1°°' - Nhle a Tfm = Female
80- l
I
‘L I
.\° 404 ’
5
20a I
l 5
0 l l ’l
PP3 PP8 PP10 PP15
B .
Acoustic Startle Response
‘5‘ _
a
2
g 200—
i
2
a 100-
0
g i
2 ; I M
E o— ' + e
«‘5 Male Tfm ' Female
C

Startle By Trial

-O— Male
#
600- + 11""

 

 

Startle Respone (Arbitrary Units)
é

 

137

Figure 11. The amount of time spent in/with and number of visits to: (A) the center area
of the open ﬁeld or (B) a novel object in the open ﬁeld arena in wt male, Tfm male, and
wt female rats. We found no signiﬁcant differences between wt males and females in
these measures, but Tfm males displayed signs of greater anxiety than other groups. *
indicates p<.05 compared to Tﬁn males. # indicates p<.01 compared to Tfm males.

138

Figure
A

30-

N
O
1

Center Time(s)
A
o
I

11.

Open Field

- Male a TTm :3 Female

#
.1:

 

(bject Time (s)

 

 

 

la

*
J: .

 

la

 

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P10

 

 

Visits

Novel Object Test

*

* l

 

 

 

   

 

Duration

Visits

139

 

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Salem micro

Figure 12. (A) Plasma corticosterone levels at baseline and 20 minutes after initial
exposure to an open ﬁeld with a novel object were elevated in Tfm males and wt females
compared to wt males. * indicates p< .001 compared to the same treatment Tfm male
and wt female groups. (B) Plasma corticosterone levels from a second cohort of rats at
baseline and 20, 40, 60, and 120 minutes after initial exposure to an open ﬁeld with a
novel object in Tfm and wt males. * indicates p< .05 compared to the same time point
Tfm male group.

140

 

 

 

 

Minutes

 

 

 

 

141

A Corticosterone
: 800- - Male :2
g :22: Tfm
5 600' a Female
gm.
3 200-
8 ._j% In
Control OpenIObject
B
A 800-
E
5 600-
% 400-
g
*
8 0_
Baseline 20 40 60 120

Figure 13. The number of c-Fos positive cells per mm2 in wt and Tfm male rats at
baseline or aﬁer exposure to an open ﬁeld with a novel object. There were no differences
between wt and Tfm males at baseline, but following exposure to a novel object Tﬁn
males displayed fewer c-Fos positive cells in the dentate gyrus and MePD, and more c-
Fos positive cells in the MPOA, than did wt males. * indicates p< .05 compared to the
same treatment Tfm male group. # indicates a signiﬁcantly greater increase from control
treatment compared to Tfm males, p<.05.

142

Figure 13.

 

 

 

 

   

 

 

 

 

A
De ntate Gyrus
3°°' - Male
IZZ Tfm
'35 200‘ ,
2 ¢
3 /
o
”r 100- /
° 7/ /
,_ é . A
Control Open/Object
B
MPOA
4oo~
NE 300—
*
‘3 200-
ll,
0
Control Open/Object
C
MePD
eo- #
is 40¢
E
Ta
0
"r 20-
0
o-

 

 

 

 

 

Control

143

Figure 14. Neonatal castration increased (A) the amount of time spent in and (B) the
number of visits to the center area of the open ﬁeld in both Tfm and wt male rats
(ps<.05). (C) Neo-de rats also showed a greater number of grid crossings (p<.01) than
did Neo-Sham rats (left), but the number of rearings did not differ between groups (right).
These data indicate decreased anxiety-related behavior and greater open ﬁeld activity in
Neo-de rats compared to Neo-Sham controls. Mean :t SEM.

144

Open Field

Figure 14.

A

a Tfm

- Male

p<.05

 

 

 

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5

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B

p<.05

 

 

 

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Sh am

GDX
Neonatal Treatment

Sham

145

Figure 15. (A) The amount of time spent with and (B) number of visits to a novel object
in the open ﬁeld arena in Neo-de or Neo-Sham Tfm and wt males. (C) The number of
grid crossing (left) and rearings (right) in the novel object test. Neo-de rats visited the
novel object more frequently than Neo-de rats (p<.05) and showed a greater number of
grid crossings than did Neo-Sham rats (p<.05). Time spent visiting the object and the
number of rearings did not signiﬁcantly differ between groups. These data indicate that
neonatal castration decreases some indices of anxiety and increases activity in male rats,
and that an intact AR is not required for these effects. Mean :t SEM.

146

-Malequm

Novel Object Test

Figure 15.

A

23133 A3

0 w o 0 o
5 3 2 1 o
_ _ _ _ _ _

    

 

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Sham

p<.01

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10-

—
o
8

147

Neonatal Treatment

Figure 16. (A) The amount of time spent in the light area, (B) number of visits to the
light area, and (C) rearings in the light area of the LD Box. As a group, Neo-de rats
spent more time in (p<.01), showed more visits to (p<.01), and reared more frequently in
the light area (p<.01) than did Neo-Sham rats. These data indicate decreased anxiety-
related behavior and greater activity following neonatal castration in rats. Mean :1: SEM.

148

Figure 16.
A

LightIDa rk Box
- Male a Tfm

r—L—I

350_ p<.01

 

8
?

 

J

Time in Light Area (3)
o 3 § "s‘ § §

 

 

 

 

 

Sham GDX

w

p<.01

.3
0|
1

 

 

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O
1

Light Area Bitries (N)
0|
L

 

 

 

 

 

 

   

Sham

O

50- p<.01

 

 

Rearings (N)

 

 

 

 

 

 

x\\\\\\\N-4

 

l
Sham GD
Neonatal Treatment

1

A

9

Figure 17. (A) The amount of time spent on and (B) number of entries into the open
arms of the EPM in Neo-de or Neo-Sham Tfm and wt males. (C) The number of total
(open and closed) arm entries. Neo-de rats had greater number of open arm visits than
Neo-Sham rats (p<.01). They also tended to spend more time on the open arms (p=.063)
and show a greater number of total arm entries (p=.062) than Neo-Sham males. These
data indicate that neonatal castration decreases some indices of anxiety and may increase
activity in adult rats in the EPM. Mean i SEM.

150

Figure 17.
Elevated Plus Maze

A - Male a Tfm
p=.063

 

33‘

 

 

 

i

 

'13

 

Open Arm Time (s)

 

 

 

O
I

w
‘i"

 

 

 

Open Arm Entries (n)
h
i

N
I

 

 

 

 

O
l

 

 

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N
on

u

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C

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L i

Total Arm Entries (n)
0|
1

 

  

 

 

  

 

 

 

 

O
i

l l
Sham GDX
Neonatal Treatment

H
Ll!
#

Figure 18. Prepulse inhibition (PPI; A), acoustic startle response (B), and acoustic startle
response by trial (C) in Neo-de and Neo-Sham wt and Tfm male rats. PPI was
increased in Neo-de compared to Neo-Sham rats (p<.01), an effect that was equivalent
in wt and Tfm males. ASR was decreased in Neo-de compared to Neo-Sham rats
(p<.01), a difference largely accounted for by an increased ASR in Neo-Sham Tfm males.
Because neonatal testicular secretions increase adult ASR only in Tfm males, there seem
to be both AR- and ER-mediated developmental inﬂuences on this behavior. * indicates
p<.05 compared to all other groups in the same trial. # indicates p<.05 compared to same
trial Neo-de males. Mean at SEM.

152

Figure 18.

 

 

 

A Prepulse inhibition
1oo— - Male Sham - Male Cast
a TFMSham - TFMCast
80—
_ so—
a
n.
=2 40-
204
o—
PP3 PP8 PP10 PP15
Prepulse Decibel
B
Acoustic Startle Response
- Male a TFM
E 500_ p<.01
E
:3
E 400-
2
.n
i 3004
o i__i
c 200d
0
Q.
m
,2 100-
2
'5 0—
% Sham GDX

Neonatal Treatment

153

Figure 18 cont.

0

Sta rtie By Trial
800 + Male Sham "A" Male Cast
7 «e- Tfm Sham -----v Tfm Cast

6004

 

 

Startle Respone (Arbitrary Units)
é

 

154

Figure 19. Plasma corticosterone levels at (A) baseline and (B) 20 minutes after initial
exposure to an open ﬁeld with a novel object. Baseline corticosterone levels were
elevated in Neo-de rats (p<.05) with no differences after exposure to a novel object.
Mean i SEM.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

A Basal Corticosterone

- Male I22! Tfm

p<.05
400-
: i'_'_i
E
O
5 ,
O
C
2
8 2
0
O
.2
t
O
o
GDX
B
Novel Object Corticoste rone
800-

E
O
5
O
c
2
8
0
O
.2
t
O
o

Sham GDX

Neonatal Treatment

155

Figure 20. (A) Brain and (B) body weight in Neo-de and Neo-Sham wt and Tfm male
rats. Brain weight was greater in Neo-Sham compared to Neo-de rats (p<.05), and was
equivalent in wt and Tfm males. Thus neonatal stimulation of ER may be responsible for
the larger brain weight in male versus female rats. In contrast, body weight was greater
in Neo-Sham wt males compared to all other groups, suggesting that neonatal AR
stimulation may normally contribute to masculinization of adult body weight. * indicates
p< .001 compared to all other groups. Mean i SEM.

 

 

 

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- Male 22: Tfm
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B 1.8

 

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Sham GDX
Neonatal Treatment

156

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