SEXUAL DIMORPHISMS AND ANDROGEN INFLUENCE IN MEDIAL
POSTERODORSAL AMYGDALA ASTROCYTES
By
Ryan T. Johnson

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Neuroscience
2011

ABSTRACT
SEXUAL DIMORPHISMS AND ANDROGEN INFLUENCE IN MEDIAL
POSTERODORSAL AMYGDALA ASTROCYTES
by
Ryan T. Johnson
The amygdala is a highly interconnected brain region involved in fear, anxiety,
social and reproductive behaviors. In humans and laboratory species the amygdala
exhibits sexual dimorphisms in neuroanatomy and function both in juveniles and adults.
In rodents, the medial posterodorsal amygdala (MePD) is particularly sexually dimorphic
and gonadal hormone sensitive, and while neurons have been examined in this region,
few reports have examined the potential influence of gonadal hormones on other
cellular components of the MePD. Astrocytes are a subtype of glia involved in synapse
formation and known to be plastic and dynamic cells sensitive to gonadal hormone
influence in several brain regions. My dissertation reveals sexual dimorphisms in the
number of astrocytes in the juvenile rat MePD and that this sexual dimorphism remains
present in adult animals. I also found sex differences in the arbor complexity of
astrocytes in adults that are not present prior to puberty. Astrocytes also respond to
changes in circulating hormone levels in adulthood. Furthermore, while the sex
difference in astrocyte numbers in juvenile animals is androgen receptor-independent,
the sex differences found in adult astrocyte numbers and arbor complexity are both
androgen receptor-dependent. Finally, I provide evidence that astrocytes in the MePD
contain androgen receptors, suggesting that androgens may act directly on these cells.
The influence of gonadal hormones on astrocytes in the MePD is likely an important

part of pubertal development and has implications for our understanding of the cellular
organization of the amygdala and its function.

DEDICATION
This dissertation is dedicated to Sarah L. Johnson

iv

ACKNOWLEDGEMENTS
Thank you to my advisors Drs. Cynthia Jordan and Marc Breedlove, and thank
you to my other committee members Drs. Cheryl Sisk and Joe Lonstein for their insight
and guidance. I am also thankful for technical assistance from Heather Malinowski,
Sandy Troxell, Diane Redenius and Kelsey Korabichk. I would also like to thank Tom
Merritt, Daptone Records and Frank Herbert for helping me through very long periods of
scope work. I am most thankful to Sarah Johnson for her support, patience and
devotion. This work was supported by a predoctoral NRSA award F31-MH084655-02
with additional NIH: NS028421, NS0450195 and NSF grant support: IOS0956831.

v

TABLE OF CONTENTS

List of Tables

viii

List of Figures

ix

Abbreviations

xiv

Introduction
The Amygdala
Gonadal Hormones and the Amygdala
A Review of Glia in the Amygdala
Amygdala Glia and Disease
Amygdala Glia and Hormones
Amygdala Glia: Conclusions
Overview
References

1
1
3
6
8
18
23
24
26

Chapter 1: Sex Differences and Laterality in Astrocyte Numbers and Complexity
in the Adult Rat Medial Amygdala
40
Abstract
40
Introduction
41
Methods
43
Results
48
Discussion
52
Appendix
60
References
66
Chapter 2: Astrocytes in the Rat Medial Amygdala Are Responsive to Adult
Androgens
74
Abstract
74
Introduction
74
Methods
76
Results
84
Discussion
88
Appendix
95
References
107
Chapter 3: Androgen Dependent Sex Differences in Astrocyte Number but not
Astrocyte Arbor Complexity in the Juvenile Medial Posterodorsal Amygdala 115
Abstract
115
Introduction
116
Methods
117
Results
122

vi

Discussion
Appendix
References

124
130
136

Chapter 4: Differential Hormone Action in the Left and Right Amygdalae 143
Abstract
143
Introduction
143
Hemispheric Sex Differences in the Human Amygdala
144
Hemispheric Sex Differences in the Rodent Amygdala
151
Combining the Human and Rodent Literature
159
Appendix
165
References
168
GENERAL DISCUSSION
180
Astrocytes are Sexually Dimorphic in the Juvenile and Adult MePD
181
Androgen Receptors Mediate Gliogenesis and Astrocyte Arbor Proliferation183
MePD Astrocytes are Plastic in Adults and Contain Androgen Receptors 184
Conclusions
186
Future Directions and Closing Remarks
190
Appendix
194
References
197

vii

LIST OF TABLES

Table 1 Percent of astrocytes within each of the four categories of complexity by
genotype and hemisphere.
61

Table 2 Lateralized sexual dimorphisms in the human amygdala from
experiments examining both men and women.

166

Table 3 Lateralized sexual dimorphisms in the rodent medial posterodorsal
amygdala.
167

viii

LIST OF FIGURES

Figure 1 Photomicrographs depicting astrocytes of varying complexity. Panel A
depicts a Group 1 astrocyte, which has few and short processes. Panel
B depicts a Group 2 astrocyte, which has several processes but
branches remain unelaborated. Panel C depicts a Group 3 astrocyte,
which has multiple elaborated processes and Panel D depicts a Group
4 astrocyte, which is highly elaborated. Because of the single plane of
focus depicted, only a portion of the full extent of arbor complexity is
visible here. Complex astrocytes frequently demonstrated multiple
branches that emerged as the plane of focus was moved through the
tissue. Scale bar = 50 microns.
62

Figure 2 Estimated regional volume (A) and total astrocyte number (B) in the
posterodorsal medial amygdala (MePD) of adult wildtype male and
female rats and testicular feminized mutant (Tfm) male rats, which lack
functional AR. The MePD volume is significantly larger on the right
than the left in all three groups, but this laterality was most dramatic in
males. The right hemisphere is also significantly larger in males than in
females and Tfm males. The right MePD contained significantly more
astrocytes than the left in all three groups (B). This laterality was most
prominent in males, leading to a sex difference in astrocyte number
only on the right side. Note that Tfm males have fewer astrocytes than
wildtype males but no more than do females in the right MePD,
indicating that the sex difference in astrocyte number depends on AR.
In contrast, the left hemisphere showed no effect of genotype. Values
are means of N = 8 10 rats/group (+ standard error of the mean,
SEM).
63

Figure 3 Estimated number of primary branches (A), branch points (B), branch
endings (C), and average branch length (D) in the MePD of adult
wildtype male, Tfm male and female rats. In all three groups,
astrocytes in the left MePD had more primary branches, more branch
points, more branch endings and a larger average branch length than
those on the right, indicating that astrocytes in the left MePD have more
complex morphologies than astrocytes in the right. Moreover, there
was a significant sex difference in astrocyte complexity in the left
MePD, with all measures indicating that males have more complex
astrocytes than females on the left side. Tfm males are not significantly
different from females for any measure of astrocyte complexity. Data
from Tfm males demonstrates that masculinization of astrocyte

ix

complexity in the left MePD depends on AR. Values are means of N =
5 rats/ group (+ SEM).
64

Figure 4 Representative photomicrographs of coronal sections through the adult
MePD of normal (A) and AR-knockout (B) male mice immunostained for
androgen receptor (AR; Epitomics #1852-1 rabbit monoclonal anti-AR
antiserum). Note the distinct nuclear labeling in the MePD of the
normal adult male that is absent in the MePD of an AR knock-out male.
Scale bar = 100 microns.
96

Figure 5 Confocal image showing that preadsorbing the AR primary antibody
with the immunizing peptide (Epitomics P-1852, diluted 1:80 or 1/25
less than the 1:2000 dilution used for the AR primary) eliminated AR
labeling in the MePD. Image depicts the result of a sequential scan
using the 405 and 488 nm lasers. Scale bar = 10 microns.
97

Figure 6 Confocal image showing that omission of the AR primary antibody
eliminated AR labeling in the MePD and other AR-containing brain
regions of normal adult male rats. Panels A-C depict the results of
individual scans with 405, 488 and 633 nm lasers. Both DAPI (A) and
GFAP (B) labeling remained intact while Ar labeling (B) was eliminated.
Panel D depicts a sequential scan using all three lasers. Scale bar =
20 microns.
98

Figure 7 MePD volume is sexually dimorphic and maintained by androgens in
adulthood especially in the right hemisphere. (A) Volume of the adult
MePD in rats is both sexually differentiated and asymmetric, as males
have a larger MePD than females, and the right (R) MePD is larger than
the left (L) in both sexes. (B) T treatment increases MePD volume
overall in both the left and right hemispheres of rats gonadectomized in
adulthood (collapsed across sex). However, analyzing the
hemispheres separately reveals that T treatment significantly increases
the volume of the left MePD only in females (C) but increases the
volume of the right MePD in both sexes (D). Values are means of N =
9 rats/group (± standard error of the mean, SEM).
99

Figure 8 Astrocyte numbers are lateralized, sexually dimorphic and maintained
by adult androgens in female rats. (A) The number of astrocytes in the
adult MePD is both sexually differentiated and asymmetric, with males
having more MePD astrocytes overall than females, and with the right
x

(R) MePD containing more astrocytes than the left (L) in both sexes.
(B) T treatment increased the number of MePD astrocytes only in the
right hemisphere (collapsed across sex). Examining the effect of
androgens separately in each hemisphere revealed that in the left
hemisphere, males have more MePD astrocytes than females, adult
testosterone (T) treatment affects this measure only in females (C).
Males also have more astrocytes than females in the right MePD,
where T increases astrocyte number in both sexes (D; the difference in
males is marginally significant (p = 0.056). Values are means of N = 9
rats/group (±SEM). Note that astrocyte numbers in this figure closely
mirrors the differences in MePD volume in figure 7.
101

Figure 9 Astrocyte complexity is increased by androgens in adulthood,
predominantly in males. Number of primary branches (A), branch
points (B), branch endings (C) and average branch length (D) of
astrocytes were measured in the MePD of adult gonadectomized male
and female rats treated with testosterone (T) or blank capsules for 30
days. In males, astrocyte process complexity in both the left and right
MePD is increased by T treatment, suggesting that adult androgens
may normally maintain the elaborate processes of adult MePD
astrocytes. The exception to this pattern is average branch length of
MePD astrocytes (D), which was unaffected by androgens in males and
was the only measure of MePD astrocyte complexity affected by T
treatment in females, and that only in the right hemisphere. Values are
means of N = 5 rats/ group, (±SEM).
103

Figure 10 Confocal images showing androgen receptor (AR) immunoreactivity
within nuclei of MePD astrocytes. In panel (A), DAPI, AR and GFAP
reactivity is shown for the same field of view in addition to a combined
sequential scan for these three labels and indicates colocalization of
DAPI and AR staining within the same nuclei (arrows) of GFAP+
astrocytes. The intensity of AR labeling in astrocytes varied and was
often less intense than what was seen in presumptive neuronal nuclei.
In panel (B), an astrocyte nucleus with AR immunoreactivity is shown
with orthogonal views (along the yellow and pink lines) demonstrating
that AR immunoreactivity is within the same Z-plane as the astrocytic
nucleus. Scale bars = 10 microns.
105

Figure 11 Confocal image showing nuclear AR immunoreactivity within
astrocytes of the arcuate nucleus (A) and CA1 hippocampus (B).
Arrows point to AR+ immunoreactivity (red) in nuclei of GFAP
expressing cells (green). Scale bars = 10 microns.

xi

106

Figure 12 Photomicrographs depicting astrocytes of varying complexity in the
juvenile MePD. Panel A depicts a simple astrocyte, which has few and
short processes. Panel B depicts a more complex astrocyte that has
multiple, elaborated processes. Scale bar = 10 microns.
131

Figure 13 Photomicrograph depicting a complex astrocyte in the juvenile MePD
(A) and the resulting tracing (B). Because of the narrow plane of focus,
only a portion of the full extent of arbor complexity is visible at a time.
Complex astrocytes frequently demonstrated multiple branches that
emerged as the plane of focus was moved through the tissue. Scale
bar = 10 microns.
132

Figure 14 Regional volume and astrocyte number are sexually dimorphic in the
posterodorsal medial amygdala (MePD) of juvenile wildtype male,
female and testicular feminized mutant (Tfm) male rats. MePD volume
is larger on the right than the left in juvenile wildtype and Tfm males but
not in females (A). In the right hemisphere, Tfm males had greater
regional volume than wildtype males and females. Males had greater
regional volume than females in this hemisphere as well. In the left
hemisphere, Tfms had greater volume compared to females but
wildtype males and females did not significantly differ. The right MePD
contained significantly more astrocytes than the left in all three groups
(B). In the right hemisphere both wildtype and Tfm males had greater
numbers of astrocytes than females. In the left hemisphere, only
wildtype males differed significantly from females. Values are means of
N = 9 rats/group (+ standard error of the mean, SEM).
133

Figure 15 Estimated number of primary branches in astrocytes of the MePD in
juvenile wildtype male, Tfm male and female rats. No hemisphere or
sex differences were seen in any group. All other measures of
astrocyte complexity exhibited a similar pattern. Values are means of N
= 5 rats/ group (+ SEM).
135

Figure 16 MePD astrocytes increase in number during puberty in both the left
and right hemispheres. In the left hemisphere Tfm males do not differ
from wt males before or after puberty and astrogenesis does not
depend on functional androgen receptors. Astrogenesis in the right
hemisphere is greater compared to the left and is at least partially
dependent upon functional androgen receptors. Tfm males are
masculine before puberty but feminine in adulthood. The right
hemisphere displays a more dramatic decline in astrocyte numbers
when adult circulating hormone levels are reduced and this effect is
xii

especially prominent in females. Values are means of N = 8-10 rats/
group (+ SEM).
195

Figure 17 Astrocytes exhibit an androgen-dependent increase in the number of
branch endings during puberty in the left MePD. Other measures of
astrocyte complexity such as number of primary branches, branch
points, and average branch length exhibit similar patterns. Eliminating
adult circulating hormone through gonadectomy reduces astrocyte
complexity in the left hemisphere. Values are means of N = 5 rats/
group (+ SEM).
196

xiii

ABBREVIATIONS
AD AR

androgen receptor

BrDU - Bromodeoxyuridine
Cy2 - Cyanine 2
Cy5 - Cyanine 5
DAPI - 4',6-diamidino-2-phenylindol
DHT - dihyrotestosterone
ER- estrogen receptor
GFAP - glial fibrillary acidic protein
GFAP-ir - glial fibrillary acidic protein immunoreactive density
LTP - Long-term potentiation
MeA - medial amygdala
MeAV - medial anteroventral amygdala
MePD - medial posterodorsal amygdala
MePV - medial posteroventral amygdala
PND - postnatal day
SNP - single nucleotide polymorphism
SRC - steroid response coactivator

xiv

T - testosterone
Tfm

testicular feminization mutation

VEGF - vascular endothelial growth factor
VMH- ventromedial hypothalamus
WKY - Wister Kyoto rats
Wt

wildtype

xv

INTRODUCTION
Although it focuses on astrocytes, this dissertation is primarily a continuation of

neuroanatomy. Therefore, I begin with an introduction to the amygdala and an
examination of previous work from our lab and others in this brain region. I then shift to
the cell of interest in my dissertation, astrocytes, and provide a review of astrocytes in
the amygdala. I then describe my work examining sex differences and plasticity of
these cells in juvenile and adult animals and discuss my findings including a review of
laterality in the amygdala.
The Amygdala
Located in the temporal lobes, the cluster of nuclei comprising the
amygdala is involved in several important behaviors including social behavior, emotion,
fear and anxiety (reviewed in LeDoux 1998; Bishop et al. 2004; Adolphs et al. 2005;
Mah et al. 2007; Spezio et al. 2007; Truitt et al. 2007). In common laboratory species
and in humans, the amygdala is divided into central, medial, cortical, lateral, basal and
accessory basal divisions which are strongly interconnected, although the direction and
strength of these connections does not appear uniform among the subregions (Price et
al. 1987; Krettek and Price 1978a,b, Grove 1988a,b; Alheid et al.1995; Canteras et al.
1995; Shammah-Lagnado et al. 2000). Tract tracing work and resting-state fMRI has
demonstrated that the amygdala is reciprocally connected through direct and indirect
connections to important homeostatic and cognitive centers such as the hypothalamus,
hippocampus, and much of the sensory cortex (Canteras et al. 1995, Stefanaci and
Amaral 2002, Kilpatrick et al. 2006). Given these extensive connections, it is not

1

surprising that the amygdala has been implicated in many behavioral disorders and
diseases including schizophrenia, drug addiction, anxiety disorders, depression, and
autism.
Much of the knowledge regarding amygdala function stems from its role in fear
and anxiety. A long history of conditioning experiments identified the region as crucial
to the fear response (reviewed in LaDoux 2003) and additional methodologies mapped

1995; Cahill 2000; reviewed in Cardinal et al. 2002). Within these domains, it is widely
acknowledged that cellular plasticity within the amygdala plays a vital role in shaping
behavior (Rogan and LeDoux 1995; Ling et al. 2011; Tan et al. 2010).
Plasticity within the various amygdala sub-regions and in the amygdala as a
whole has been examined through many paradigms and has been covered in numerous
reviews (Sah et al. 2008; Samson et al. 2005; Cooke 2006). The human literature has
examined plasticity in amygdala volume and functionality and the rodent literature has
repeatedly demonstrated plasticity of amygdala neurons based on fear-related longterm potentiation (LTP) and by manipulation of circulating hormone levels (human
plasticity: Killgore and Yurgelun-Todd 2004; Giedd et al. 1997; long term potentiation:
reviewed in Sigurdsson et al. 2007, see also Sah et al. 2008; hormone manipulation:
Cooke et al. 1999; Morris et al. 2008). While LTP and hormone mediated plasticity
certainly interact within the amygdala (Kavushansky and Richter-Levin 2006;
Kavushansky et al. 2006) the complex and extensive influence of gonadal hormones in
the region is intriguing in itself.

2

Gonadal Hormones and the Amygdala

involvement in social/sexual behavior (Kluver and Bucy 1938). Since then
neuroendocrine research has identified numerous functional and morphological sex
differences in the amygdala, which may be related to the has dense concentrations of
gonadal hormone receptors within this brain region (Cahill et al. 2004; Kilpatrick et al.
2006; Hines et al. 1992; Rubinow and Juraska 2009; Simerly et al. 1990; Li et al. 1993).
The medial subregion of the amygdala appears to be especially hormone
sensitive and sexually dimorphic. Bubenik and Brown (1973) found a sex difference in
average neuronal soma size in the medial amygdala of ovariectomized and estradiol
treated female squirrel monkeys compared to normal males. Another sex difference
was identified in cholinergic binding within the medial nucleus of mice (Arimatsu et al.
1981). This was followed by a string of findings from a single group demonstrating sex
differences in the volume and synaptic organization of the rat medial amygdala (males
greater than females) that could be reversed by treating females with estrogen
(Nishizuka and Arai 1982, 1983; Mizukami et al. 1983). Another group reported sex
differences in substance-P immunoreactivity in the medial amygdala of rats (Malsbury
and McKay 1989), and von Ziegler and Lichtensteiger (1992) demonstrated that
aromatase activity, which is responsible for the conversion of testosterone into estradiol,
undergoes a pattern of lateralized sex differences in the medial amygdala during
development, noting the potential such activity may have in shaping neuroanatomy (von
Ziegler and Lichtensteiger 1992).

3

Continued investigation of the medial amygdala revealed that the medial
posterodorsal portion (MePD) exhibited particularly striking sex differences (Malsbury
and McKay 1989). The sex difference in regional volume reported by Mizukami and
colleagues (1983) for the medial amygdala was also found for the MePD as was the
ability of this sex difference to be eliminated by hormone treatment. Specifically, the
greater regional volume of the MePD in males compared to females could be eliminated
by castration of adult males or androgen treatment of females (Cooke et al. 1999). A
sex difference in average neuronal soma size was also found in the MePD with males
having larger somata compared to females and this too could be eliminated by hormone
manipulation of adult animals (Cooke et al. 1999). Thus, many of the sex differences in
the MePD can be reversed or eliminated by adult hormone manipulations, suggesting a
profound amount of hormone-mediated plasticity in the region. Evidence demonstrating
that MePD regional volume and soma size were responsive to both social cues and
photoperiod in some species further emphasized the plasticity of the region (Cooke et
al. 2002).
Both androgen receptors (ARs) and estrogen receptors (ERs) contribute to
maintaining a fully masculinized MePD and full expression of male mating behaviors.
For example, the treatment with high affinity AR ligand dihydrotestosterone (DHT)
maintained penile erections, ultrasonic vocalizations, and ejaculatory behavior along
with neuronal soma size in castrated male rats. In contrast, the ER ligand estradiol
maintained intromission patterns, ejaculatory behavior, neuronal soma size and regional
volume in castrated males, demonstrating the complexity of the hormone interactions
involved in MePD neuroanatomy and associated behaviors (Cooke et al. 2003). One

4

caveat in these studies is that hormones were delivered systemically, so their effects on
neurons in the MePD may or may not be mediated by AR and/or ER in these same
neurons. Moreover, such hormone-induced changes in MePD neurons may or may not
critically mediate the behavioral changes induced by these same hormones.
Testicular feminized mutant (Tfm) rats have been an important tool in isolating
the contribution of the AR and androgens in the brain. Tfm rats carry a spontaneous
mutation in the AR gene that causes XY males to have in many ways feminine traits
(e.g., nipples and a short, feminine anogenital distance), despite having testes that
produce adequate amounts of circulating testosterone (Roseli et al. 1987). Tfm rats
have a single base-pair replacement mutation in the steroid binding domain of their AR
gene which renders the receptor largely dysfunctional (Yarbrough et al. 1990). Because
androgens and estrogens are present but only ER are functional (Olsen and Whalen
1982) research using Tfm animals has confirmed that functional AR are necessary for
complete masculinization of several aspects of brain morphology and function (reviewed
in Zuloaga et al. 2008). This evidence has been vital in revising the aromatization
hypothesis, which attributed brain masculinization to ER activity alone (Naftolin et al.
1975).
In Tfm males, while rostral-caudal extent of the MePD was unaltered compared
to wt males, the regional volume and average neuronal soma size were intermediate
between wt male and female animals, suggesting functional ARs are necessary for
complete masculinization of these MePD components (Morris et al. 2005). Similar
results were found in a portion of the extended amygdala, the posteromedial bed
nucleus of the stria terminalis (BSTMPM; Durazzo et al. 2007). Thus research using

5

Tfm animals provides further evidence that androgens acting via AR influence MePD
structure and presumably function.
Subsequent investigations attempted to determine which cellular components of
the MePD respond to androgens. Using gonadectomized male and female animals in
conjunction with androgen supplementation, Morris et al. (2008) reported that males
had greater numbers of MePD neurons and glia than females. However, adult
androgens did not influence the number of neurons in the MePD. In contrast, glial cell
numbers were affected by androgens with high androgen-treated male and female rats
having more glia in the right MePD than control-treated rats (Morris et al. 2008). This
result suggested that androgens may act on cells other than neurons to influence
amygdala function and opens new avenues of exploration. There are several types of
glia in the amygdala and I will provide background regarding the role of one type,
astrocytes.

A Review of Glia in the Amygdala
Edited from: Johnson, R. T., Breedlove, S. M., & Jordan, C. L. (2010). Astrocytes in the
amygdala in Vitamins and Hormones, 82, 23-45. Hormones of the Limbic System.
As in most of the central nervous system, several types of glia are present in the
amygdala, including astrocytes, oligodendrocytes, and micro-glia. However, much of the
exploration of glia in the limbic system has focused either on astrocytes specifically or
on glia in general. Thus, reports examining microglia or oligodendrocytes in the
amygdala are rare, but these cells are likely included in counts of total glial within the
amygdala. These circumstances make attributing effects or phenomenon to specific

6

glial subtypes sometimes difficult, but hopefully future investigations will alleviate this
confusion.
Astrocytes are perhaps the most thoroughly investigated glia subtype in the
central nervous system and certainly in the amygdala. Astrocytes were first described
in detail in 1895 when Cajal named them based upon their extensive star-like arbors.
Cajal (1909) later suggested that astrocytes were functioning to insulate neural
connections while Golgi (1885), who was also investigating glia, proposed that
astrocytes provide a link between neurons and the blood supply based upon their
predisposition to contact blood vessels. Unfortunately, after these initial descriptions
and hypotheses, astrocytes were mostly ignored. It was not until the identification of the
astrocyte inflammatory response that these cells again received attention.
Numerous reports demonstrate that astrocytes rapidly respond to all manner of
brain trauma, including injury, disease, and genetic disorders with a rapid synthesis of
glial fibrillary acidic protein (GFAP) at the site of injury. This reliable, complex response
has been carefully examined and reviewed in detail (Eddleston and Mucke, 1993; Eng
and Ghirnikar, 1994). The information derived from studies of reactive gliosis has been
vital in understanding brain injury and should not be minimized, but as Cotter et al.

Fortunately, the past two decades have seen an increase in attention to glia.
Much of the interest stems from findings that astrocytes exhibit rapidly propagating
calcium waves, suggesting that they may form a second path of communication working
in conjunction with the neuronal system (Guthrie et al., 1999). More recent work

7

suggests that propagating calcium waves are extremely rare in vivo (Wang et al., 2006),
but new evidence reaffirms that astrocytes are active and dynamic cells.
Mature astrocytes generally have large arbors extending from their cell bodies,
and in the cortex an average astrocyte connects to approximately four neurons, 300
600 dendrites, and 1,000,000 synapses (Halassa et al., 2007). Astrocyte processes are
closely involved in the formation of new synapses and these processes are highly
mobile and extend or retract to modulate contact between neurons (Hatton, 2002;
Nishida and Okabe, 2007; Ullian et al., 2001). Interestingly, increased branch
complexity in astrocytes has been associated with an increase in functional synapses in
some regions (Elmariah et al., 2005; Pfrieger and Barres, 1997) but has also been
associated with decreases in dendritic spines (Mong et al., 2001). Thus, it may be that
astrocytes have opposing roles under different conditions, promoting and maintaining
synapses in some cases, while promoting their elimination in others. In many ways, a
sensitivity to varying conditions has become the hallmark of glia, and their
responsiveness to various disease states, which I review next, illustrates this capacity.
Amygdala Glia and Disease
Glia are known to be important metabolic factories for normal brain functioning,
responding quickly and topographically to stimuli with rapid increases in metabolic
activity (Schummers et al., 2008). The nature of the glial response to disease has been
puzzling, with long-standing questions about whether this response is beneficial and
neuroprotective or is detrimental t
, 2008). Furthermore, in several diseases
and disorders it is not at all certain if the effects seen in glia are merely a response to

8

disease or a contributing cause. As in most cases, that dichotomy may be an illusion,
with glia likely playing both roles at various times and in various instances. What is
clear is that the extreme sensitivity of glia to brain dysfunction makes them powerful
indicators of abnormal brain function and has guided the examination of amygdala glia
in several disease models.
Epileptic states
Investigation of patients with temporal lobe epilepsy demonstrates glial
satellitosis, the accumulation of glia around neurons, in the lateral amygdala (Faberzuschratter et al., 2009) and interesting glial phenomena have been detailed in the
mouse amygdala as well. Kainic acid, an excitotoxin regularly used to investigate the
-like conditions, induces several responses in rodent glia.
After kainic acid injection, microglia become immunopositive for cyclin-dependent
kinase 4 and cyclin D1, important regulators of apoptotic cell death (Ino and Chiba,
2001). In amygdala astrocytes, kainic acid injection results in prolonged expression of
metallothioneins, a family of proteins involved in neuroprotection (Kim et al., 2003).
The apolipoproteins, a family including apolipoprotein E, are primarily expressed
in astrocytes under normal conditions. However, in cases when damage induced by
kainic acid extended beyond the injection site into regions such as the amygdala,
apolipoprotein E expression was also found in neurons. In cases of mild damage, this
effect was not seen, but increases in GFAP-immunoreactivity and microgliosis were
seen. The authors conclude that in severe cases, damage to the glial network is large
enough to allow accumulation of apolipoproteins from damaged glia in the surrounding

9

neurons, while in mild cases the reactive glia response may ameliorate this outcome
(Grootendorst et al. 2000).
Amygdala-kindled seizures also promote astrocyte proliferation in the piriform
cortex of rats (Vessal et al. 2004), and kindling of the olfactory bulb, a major source of
input for the amygdala, results in astrocyte proliferation in the basolateral amygdala and
the piriform cortex (Woldbye et al. 1996). These two studies together suggest that over
stimulation of the amygdala, be it through direct kindling or though kindling of a primary
input source, can have long-reaching effects on other parts of the brain. The authors
suggest that astrocytes become activated in response to the formation of a new
synaptic pathway brought on by the hyper-excitability of the epileptic discharge and
conclude that astrocytes may support the formation of the kindling pathway (Vessal et
al. 2004, 2005). Jung et al. (2009) also found amygdala astrocyte proliferation in a
lithium pilocarpine injection model, and surgical specimens from patients suffering from
temporal lobe epilepsy demonstrate an inverse correlation between the extent of
astrocytic-reactive gliosis and inhibitory synapses on GAD-positive projection neurons
(Yilmazer-Hanke et al., 2007). Again, these results suggest that astrocyte activity may
ure sensitive regions.
Astrocytes in the piriform cortex become immunoreactive for nestin, an
embryonic intermediate neurofilament protein, after amygdaloid kindling, providing
further evidence that astrocyte morphology is affected by seizures (Umeoka et al.,
2001). Relatedly, while astrocyte reactivity in the hippocampus and amygdala after
amygdala-kindled seizure was equivalent in S-100b knock outs (a type of calcium
binding protein found in some astrocytes) compared to controls, the knockout animals

10

kindled more rapidly and exhibited more severe seizures, suggesting changes in
astrocytic structural proteins are related to seizure progression (Dyck et al., 2002).
However, since S-100b is expressed only in a subtype of adult astrocytes around blood
vessels, only select astrocytes would be involved (Hachem et al., 2005).
Investigators have also begun to explore whether astrocytes help to preserve
neurons after seizures. Vascular endothelial growth factor (VEGF) protects neurons
from cell death and astrocytes in the amygdala up-regulate VEGF protein expression
approximately 24 h after seizure induction. This VEGF up-regulation in astrocytes may
benefit surrounding neurons (Newton et al., 2003; Nicoletti et al., 2008).
Finally, seizure-like states are also examined for their beneficial effects.
Electroconvulsive seizures are used for the treatment of major depression, and
Wennstrom et al. (2004) examined Wistar rats given five electroconvulsive treatments
and injected with bromodeoxyuridine (BrdU) to monitor cell proliferation. As the seizure
literature would predict, the authors report a proliferation of glial cells in the amygdala
after electroconvulsive treatment. Specifically, the most dramatic increase was in
oligodendrocytes, a proliferation that lasted 3 weeks after treatment and led to the
establishment of new mature oligodendrocytes. Interestingly, this effect was region and
cell-type specific with oligodendrocyte proliferation being found in the central, lateral,
and basal subregions and microglia proliferation found in the medial subregion. The
authors speculate that the formation of these new cells may play a role in regulating
synaptic function (Wennstrom et al., 2004).

11

Depression
As the previous report suggests, glia are also implicated in major depressive and
bipolar disorders. Social exploratory behavior is sometimes used as a model of
depression, and Lee et al. (2007) found that blocking glutamate uptake in the amygdala
resulted in a dose-dependent reduction in social exploratory behavior and altered
circadian activity patterns, reminiscent of depressive states in humans. Importantly,
selectively blocking glutamate uptake by astrocytes with dihydrokainic acid in the
basolateral amygdala also reduced social exploratory behavior, an effect that could be
reversed by simultaneous injection of the NMDA receptor antagonist AP5. These
results led the authors to conclude that impaired uptake and metabolism of glutamate
by astrocytes may be critically involved in depression (Lee et al., 2007).
Glial numbers in the amygdala are also affected in humans with major
depressive disorder. Bowley et al. (2002) examined the amygdala from patients with
major depressive and bipolar disorder postmortem and found that glia density and
glia/neuron ratio were greatly reduced compared to controls. Interestingly, this
reduction was mostly seen in the left hemisphere and was not due to a change in the
number of neurons. This reduction in glia was also found in untreated bipolar cases but
not in cases treated with either lithium or valproate, suggesting these medications may
reduce the loss of glia in the amygdala (Bowley et al., 2002). Relatedly, a 4-week
lithium treatment of adult males rats reduced oligodendrocyte proliferation compared to
controls, but the authors note did not look for effects of lithium on the survival of cells
(Orre et al., 2009).

12

Since the identification of reduced glial numbers in the amygdala in major
depressive disorder, other reports have examined this phenomenon in more detail. One
study examined the expression of glial markers in the basolateral amygdala in Wister
Kyoto (WKY) rats, often used as a model for anxiety and depression due to their
exaggerated responses to stressors and depressive-like performance in behavioral
tests (De La Garza and Mahoney, 2004). Gosselin et al. (2009) found a significant
deficit in the number of GFAP immunopositive cells (a common marker for astrocytes) in
the basolateral amygdala of WKY rats compared with Sprague-Dawley control animals,
a result confirmed by Western analysis of GFAP expression levels. However, the use of
a second astrocyte marker, S-100b, revealed no differences in expression between
controls and WKY rats, a result one might expect given that only a subpopulation of
astrocytes express S-100b (Hachem et al. 2005). However, the second marker did
provide an interesting finding; in control animals GFAP and S-100b were colocalized
while in WKY rats a population of S-100b-positive cells was found that were devoid of
GFAP immunoreactivity. This led the authors to suggest that in the WKY model GFAP
expression by astrocytes is altered not the number or density of total astrocytes
(Gosselin et al., 2009).
Another report found similar results using a maternal deprivation paradigm, a
model of early life stress, which is a major risk factor in the etiology of depressive
disorders. The maternally deprived animals exhibited a significant reduction in GFAPimmunoreactive astrocyte density in the basolateral amygdala and several other brain
regions. This reduction in immunoreactivity was greater than the overall reduction in
cell density, which suggests, as in the previous report, that GFAP expression, rather

13

than the number of astrocytes, was altered in this animal model (Leventopoulos et al.,
2007).
However, it is also clear that early stress can alter cell numbers in the amygdala.
Using a prenatal stress paradigm, Kraszpulski et al. (2006) found altered glial numbers
in different amygdala subregions at different developmental time points. For example,
the number of glia in the lateral amygdala nucleus was reduced in prenatally stressed
animals at postnatal day (PND) 7 only, whereas glia numbers were reduced in the
basolateral and central nuclei at PND 25. Neuron number exhibited the same pattern
and the authors speculate that these reductions in cell number may underlie the
enhanced anxiety seen in prenatally stressed animals in adulthood (Kraszpulski et al.,
2006). Unfortunately, how reductions of GFAP expression and/or reduced glia number
in the amygdala relate to depression is still unclear.
Despite several reports that astrocytes are affected in the amygdala of both
human cases and in animal models of major depressive disorder, other evidence
suggests that astrocytes may not be altered during depression. Hamidi et al. (2004)
labeled tissue from patients diagnosed with major depressive disorder for S-100b,
human leukocyte antigen, and stained for Nissl. Using S-100b, the group reported no
significant difference in astrocyte density in depressed patients compared to controls.
Moreover, human leukocyte antigen was used to identify microglia and revealed no
difference. However, oligodendrocyte density (identified by their compact deeply
stained nuclei in Nissl staining) was significantly lower in this population of depressed
patients. The authors concluded that the reduction of glia previously reported in the
amygdala of patients suffering from major depressive disorder was due to a reduction in

14

oligodendrocytes. These results are difficult to interpret for two reasons. First, as
previously mentioned, S-100b is expressed only in a subset of astrocytes (Hachem et
al. 2005). Furthermore, Gosselin et al. (2009) found a dissociation of S-100b and
GFAP-labeled cells in their WKY rat model of depression. Perhaps, if Hamidi and
colleagues had also used GFAP immunoreactivity as their astrocytic marker, they would
have seen a reduction in the number of astrocytes as well. However, it is certainly
possible that both astrocytes and oligodendrocytes are reduced in the amygdala of
depressive disorder cases.
Adding to the complexity is an interesting report examining S-100A1, a protein
closely related to S-100b that also colocalizes with GFAP. S-100A1 knockout mice
appear to develop normally and show no obvious differences in brain development.
However, S-100A1 knockout males exhibit reduced anxiety based on an approachavoidance paradigm (Ackermann et al., 2006). Reduced anxiety in knockouts suggests
S-100A1 may somehow facilitate fear and anxiety, two behaviors often elevated in
models of major depressive disorder. Regardless of what changes occur to amygdala
glia, a clear cause effect relationship remains elusive, and the question of whether
changes in glial numbers in the amygdala are a response to or a cause of depression
remains unanswered (see Hercher et al., 2009).
Proteinopathies
-synuclein
synuclein-immunoreactive inclusions in neurons has been an important marker in
research on this disease (Mukaetova-

-

15

synuclein-immunoreactive inclusions have also been described in the human amygdala
(Terada et al., 2003). These astrocytic inclusions appear in the amygdala at stage 3 of
the disease, after their initial appearance in neurons, and their immunoreactivity
increases in the latter stages of the disease (Braak et al., 2007).
-synuclein from afflicted neurons, likely explaining why glia are
-synucleinimmunoreactive glia influence the progression of disease symptoms is unclear, but
based upon how astrocytes respond to trauma in the healthy brain, afflicted astrocytes

the amygdala from other brain regions in the latter stages of the disease (Braak et al.,
-synuclein-immunoreactive astrocytes and astrocytes positive for
other disease markers (tau and ubiquitin inclusion bodies) have been found in the
amygdala of nontangles with calcification dementia, this disease may share pathophysiological

Yokota et al., 2002).
-gliosis in
the amygdala has been reported in many AD cases (Corsellis, 1970). Scott et al.
(1992) investigated the cortical and basal amygdala subregions in brains of AD patients
postmortem. They counted glia and divided them into two categories: large, which the
authors presumed to be astrocytes, and small, presumed to be oligodendrocytes and
microglia, based on size. When examining glial cell density (cells per mm 2), they found

16

that the overall glia density was greater in AD cases in both the basal and cortical
regions. However, this was attributable mostly to increased density of large glia. When
the data were corrected for structural atrophy, total numbers of glia were reduced in AD
cases in both size categories in the basal amygdala. In the cortical region, only the
number of small glia was reduced (Scott et al., 1992).
Additional reports found an increase in the number of astrocytes expressing
peroxiredoxin 6, an antioxidant enzyme, in the amygdala of AD cases compared to
controls. Peroxiredoxin 6 staining, which is not found in oligodendrocytes or microglia
and only at very low levels in neurons, was found mostly in astrocytes associated with
amyloidastrocytes (Power et al., 2008). Furthermore, Kaku et al. (2003) found that a mutant
mouse (osteopetrotic mice), known to have reduced number of microglia, also have
greater fibrillary plaques in the amygdala and other regions compared to controls,
suggesting microglia regulate plaque formation and/or clearance in the region.
Additional reports confirm that activated microglia follow plaque formation in the
amygdala, and activated astrocytes form later around these plaques (Dudal et al.,
2004).
One difficulty in measuring astrocyte proliferation in any age-related disease
such as AD is the well-documented increase in GFAP production as the brain ages
(Kohama et al., 1995; Linnemann and Skarsfelt, 1994). Interestingly, this increase in
GFAP can be attenuated in several brain regions, including the amygdala, by systemic
administration of the steroid pregnenolone in aging animals (Legrand and Alonso,
1998). This implies that reduced hormone levels during aging may cause widespread

17

increases in GFAP production and may contribute to the increased incidence of many
neural disorders with age.
Other pathologies
Along with their roles in epilepsy, depression, and various proteinopathies, glia
also figure into other areas of disease and disorder research. For example, myoinositol, a glial marker and second messenger in intracellular calcium regulation, is
reduced in the amygdala of narcoleptic patients compared to controls, suggesting glia
involvement in sleep disorders (Poryazova et al., 2009). Additionally, Yokota et al.
(2008) found several interesting glial abnormalities in the amygdala during their
investigation of cases of neurofibromatosis type 1, a disease in which tumors develop
from nervous system tissues. They conclude that the glial clusters and satellitosis seen
in the disease may be due to altered astrocyte growth regulation.
Although currently isolated in their fields, these two reports suggest that glia in
the amygdala may be involved in, or at least be an indicator of, a broad range diseases
and disorders. Due to their sensitivity and prominent role in maintaining brain
homeostasis, the use of glia as indicators of neurological trauma has great potential for
disease research. Furthermore, given their role in the formation of synapses and
facilitation of neuronal communication, exploration of glia in disease models may lead to
novel treatments focusing on restoring damaged communication pathways.
Amygdala Glia and Hormones
As described above, the amygdala contains a number of sexual dimorphisms
(reviewed in Hamann, 2005; Stefanova and Ovtscharoff, 2000). These sex differences
have important implications beyond sexual behavior as most of the diseases associated

18

with the amygdala such as depression, anxiety, schizophrenia and autism exhibit strong
sex biases in the population (American Psychiatric Association, 2000). While it is clear
that gonadal hormones are key in the early establishment of sex differences and in CNS
plasticity, the mechanisms behind hormone-induced alterations in neuroanatomy are
still not clear. Are glia involved in the ontogeny of sex differences? What role due glia
play in hormone-induced plasticity in adulthood? This line of questioning is beginning to

The evidence for gonadal hormone influence on neurons is well established
(Hamson et al. 2010; Cooke et al. 1999; Woolley, 2007; Forger 2006). However, glia
also respond to gonadal hormones signals. ER are up-regulated in reactive astrocytes
after injury and estrogen influences astrocyte morphology in the hypothalamus,
hippocampus and in primary culture (Blurton-Jones and Tuszynski, 2001; Milner et al.
2000; Garcia-Segura et al. 1989, 1994). Estrogen appears to influence glia in the
amygdala as well. The scaffold protein, MNAR/PELP1, is important coactivator in
es
glia may respond directly to estrogen through nongenomic means (Khana et al., 2006;
Raza et al. 2008).
Gonadal hormones and amygdala glia during developmental
Evidence suggests that gonadal hormones may influence glia in the amygdala
early in development. For example, a single dose of estrogen during neonatal
development increased the number of proliferating glia in the basolateral nucleus 3 days
later in male rats but not in females (Dmitar et al. 1995). A slightly more complex
pattern of results was found in the medial, cortical, and central nuclei. In estrogen-

19

treated male rat pups, the percentage of BrdU-labeled glia was increased in all three
nuclei, based upon light microscopy identification, whereas in females, estrogen
increased the number of labeled glia only in the medial nucleus, while decreasing their
numbers in the cortical nucleus and having no effect in the central nucleus (Drekic et
al.1995).
Glia also appear responsive to hormones during puberty, another critical period
in development. Using BrdU labeling, Ahmed et al. (2008) demonstrated that new cells,
including GFAP-positive cells, are added to the medial amygdala during puberty in rats.
This pubertal addition was sexually dimorphic with males having more BrdU-positive
cells in the medial amygdala than females. Interestingly, the addition of BrdU-positive
cells in males was eliminated by prepubertal castration, strongly suggesting that the
addition of new cells in the amygdala during puberty, including GFAP-positive cells, is
modulated by gonadal hormones. Thus, while gliogenesis in the amygdala appears to
be regulated by gonadal hormones during development, the level of responsiveness is
both sex- and subregion-specific. This specificity is also found in the adult amygdala in
response to gonadal hormones.
Gonadal hormones and amygdala glia during adulthood
Several reports have documented glial sensitivity to fluctuations in hormone
levels within the amygdala of adult animals. For example, Blutstein et al. (2006)
reported that estradiol treatment of mice produces an increase in glutamine synthase
gene expression in the medial amygdala of adults, an interesting effect given that
neurons are incapable of synthesizing glutamate and are dependent upon glia to
replenish their glutamine supply. Furthermore, treatment of meadow voles with either

20

testosterone propionate or estradiol benzoate, but not DHT, resulted in a significant
increase in BrdU-labeled cells, 35% of which were glia, in the amygdala but not in the
dentate gyrus or ventromedial hypothalamus (VMH; Fowler et al. 2003). Interestingly,
estradiol benzoate seems to have this effect in meadow voles but not in prairie voles
(Fowler et al. 2005). The presence of an effect on glia in the amygdala, a region closely
linked to sexual behavior, in a polygamous rodent species (meadow voles) but not in
the monogamous rodent species (prairie voles) suggests hormone-dependent changes
in the number of amygdala glia may be involved in reproductive behaviors.
The medial amygdala is involved in reproductive behavior in several species and
glia within this region seem particularly responsive to changes in adult hormone levels.
For example, multiparous females given postpartum contact with pups exhibit reduced
numbers of GFAP-positive cells in the medial amygdala compared to pup-exposed
primiparous females (Featherstone et al. 2000), a difference which may be due to
greater estrogen exposure in postpartum dams. In addition, the density of GFAPimmunoreactivity is higher in the medial subregion during the proestrus phase
compared to the other phases of the estrous cycle in rats (Martinez et al., 2006), and in
ovariectomized females, injection of estradiol, alone or with progesterone, increased
GFAP-immunoreactive density in portions of the medial amygdala (Martinez et al.,
2006).
Similarly, testosterone increases glial cell numbers (based on their appearance in
Nissl staining) in the MePD of ovariectomized adult females compared to
ovariectomized controls (Morris et al., 2008), a pattern of results strongly suggesting
estrogenic metabolites of testosterone may lead to glial proliferation in some parts of the

21

adult medial amygdala. In the anteroventral medial amygdala (MeAV), by contrast,
Carrillo et al. (2007) found no changes in glial number across the estrous cycle, despite
changes in volume and other elements. Thus, even within subregions of the medial
amygdala (MePD compared to MeAV) glia may vary in their responsiveness to changes
in steroid hormone levels.
In addition to hormone mediated plasticity in adulthood, dramatic sex differences
can be found in adult amygdala glia. As mentioned above, total glia numbers based on
Nissl staining are sexually dimorphic in the MePD of adult rats with males having more
glia than females (Morris et al. 2008). GFAP-ir density measures also suggest a sex
difference in astrocytes within the MePD and MePV regions with females having greater
GFAP-ir density than males (Rasia-Filho et al. 2002).
Of course, gonadal hormones can influence astrocytes through several possible
pathways. In the portions of the hypothalamus estrogens influence the complexity of
astrocyte arbors through indirect actions through neurons (Mong et al. 2002). Neurons
in the amygdala, especially the medial regions, are rich in steroid hormone receptors,
suggesting that a similar indirect pathway may occur in the MePD (Simerly et al. 1990;
Sholl and Kim 1990; Donahue et al. 2005). Alternatively, steroid hormones may act
directly on glial cells.
Astrocytes in several species are known to express ARs and ER although this is
highly region specific (guinea pig: Langub and Watson 1992; rat: Azcoitia et al. 1999;
DonCarlos et al. 2006; primates: Finley and Kritzer, 1999; Blurton-Jones and Tuszynski
2001). However, as far as I am aware, no report has confirmed the presence of either
ER or ARs in amygdala astrocytes in vivo, leaving open the possibility that hormone

22

effects on astrocytes in the amygdala are through indirect pathways

that steroids

directly affect neurons, which then affect astrocytes.
Amygdala Glia: Conclusions
The available evidence makes a single cohesive report of glia function in the
amygdala difficult to produce. Clearly glia have offered a wealth of intriguing and
informative findings within the past several decades, and several excellent reviews have
summarized glia-based findings in various brain regions and in the brain as a whole
(Barres, 2008; Eng, Ghirnikar and Lee, 2000; Freeman and Doherty, 2006; GarciaSegura and Melcangi, 2006). Glia throughout the brain, not just the amygdala, are

dimorphisms suggest glial sex differences in this region may be linked to sex
differences in vulnerability to disorders such as depression, schizophrenia, anxiety
disorders, and autism.
Unfortunately, little work has quantified the effects of gonadal hormones on
astrocytes in the amygdala. While measures of GFAP-ir and classification systems for
astrocyte complexity are important early steps, they provide no information about
astrocyte numbers and fews details about the arbors of these highly plastic cells. Are
the numbers of astrocytes in the MePD different in male and female animals? If so,
when does the sex difference arise? Furthermore, GFAP-ir evidence suggests that
medial amygdala astrocytes are plastic and hormonally responsive in adulthood
(Martinez et al. 2006). Which class of gonadal hormones are amygdala astrocytes
responding to in adulthood? Answering these questions will certainly provide a more

23

complete picture of amygdala neuroanatomy and may provide important clues to the
regions function.
Overview
Astrocytes are both important in brain functioning but poorly understood. Their
role in the formation, maintenance and function of synapses makes understanding
astrocytes particularly important in brain regions known to be plastic throughout the
lifespan, such as the amygdala. While examination of hypothalamic astrocytes has
produced many intriguing findings, I now present the first detailed analysis of astrocytes
in the amygdala.
In Chapter 1 I describe the number and arbor complexity of MePD astrocytes in
unmanipulated adult animals. These results represent the first actual quantification of
astrocyte arbor complexity to date and reveal complex sex and hemisphere differences.
I also include Tfm male rats in this analysis which establishes that the sex differences
found depend upon functional ARs, leading us to question whether these differences
are 1) influenced by circulating hormones in adulthood and/or 2) are present earlier in
development.
In Chapter 2 I examine the former question by gonadectomizing adult male and
female rats and giving some testosterone. I demonstrate that both astrocyte numbers
and arbor complexity are influenced by circulating adult hormone levels but in a
hemisphere specific manner, further suggesting that the two amygdalae respond
differently to circulating hormone levels. Furthermore, I provide evidence that
astrocytes in the MePD, like astrocytes in the hippocampus and hypothalamus, contain

24

AR, which suggests the possibility of a cell-autonomous response of astrocytes to
androgens.
In Chapter 3 I investigate the developmental timing of the sex differences in
astrocyte number and complexity by examining astrocyte in the MePD of juvenile wt and
Tfm male rats. The results demonstrate that while a sex difference in astrocyte number
exists before puberty, no sex differences in astrocyte complexity are found in juvenile
animals. The use of Tfms is again informative, revealing that Tfm males have a are
masculine number of astrocytes before the important influence gonadal hormones
during puberty.
Due to the striking laterality found in almost every aspect of MePD astrocytes
that I examined, I review lateralized sex differences in the amygdala in Chapter 4.
While baffling and lacking a clear explanation, the laterality in the amygdala is
potentially of great importance since effects previously identified may be eliminated, or
new ones revealed, if the hemispheres are investigated separately. Finally, I briefly
discuss my findings and provide possible avenues for future exploration to expand the
research, both microscopically in terms of synapse formation within the MePD, and
macroscopically in terms of intra-amygdala connectivity.

25

REFERENCES

26

REFERENCES
Ackermann, G. E., Marenholz, I., Wolfer, D. P., Chan, W. Y., Schäfer, B., Erne, P.,
Heizmann, C. W. (2006). S100A1-deficient male mice exhibit increased
exploratory activity and reduced anxiety-related responses. Biochim Biophys
Acta 1763:1307-19.
Adolphs. R., Gosselin. F., Buchanan. T. W., Tranel. D., Schyns. P., Damasio, A. R.
(2005). A mechanism for impaired fear recognition after amygdala damage.
Nature 433:68-72.
Ahmed, E. I., Shcultz, K. M., Zehr, J. L., Lorenz, B. H., DonCarlos, L. L., Sisk, C. L.
(2008). Pubertal hormones modulate the addition of new cells to sexually
dimorphic brain regions. Nature Neuroscience 11:995-997.
Alheid, G. F., Beltramino, C. A., De Olmos, J. S., Forbes, M. S., Swanson, D. J.,
Heimer, L. The neuronal organization of the supracapsular part of the stria
terminalis in the rat: the dorsal component of the extended amygdala.
Neuroscience 84:967-96.
American Psychological Association. (2000). Diagnostic and statistical manual of mental
disorders: DSM-IV-TR.
Arimatsu, Y., Seto, A., Amano, T. (1981). Sexual dimorphism in alpha-bungarotoxin
binding capacity in the mouse amygdala. Brain research 213:432-437.
Azcoitia, I., Sierra, A., García-Segura, L. M. (1999). Localization of estrogen receptor
beta-immunoreactivity in astrocytes of the adult rat brain. Glia 26:260-267.
Barres, B. A. (2008). The Mystery and Magic of Glia: A Perspective on Their Roles in
Health and Disease. Neuron 60:430-440.
Bechara, A., Tranel, D., Damasio, H., Adolphs, R., Rockland, C. and Damasio, A. R.
(1995). Double dissociation of conditioning and declarative knowledge relative to
the amygdala and hippocampus in humans. Science 25:1115-8.
Bishop, S. J., Duncan, J., and Lawrence, A. D. (2004). State anxiety modulation of the
amygdala response to unattended threat-related stimuli. J Neurosci 17:10364-8.
Blurton-Jones, M., Tuszynski, M. H. (2001). Reactive astrocytes express estrogen
receptors in the injured primate brain. J Comp Neurol 433:115-23.

27

Blutstein, T., Devidze, N., Choleris, E., Jasnow, A. M., Pfaff, D. W., and Mong, J. A.
(2006). Oestradiol up-regulates glutamine synthetase mRNA and protein
expression in the hypothalamus and hippocampus: implications for a role of
hormonally responsive glia in amino acid neurotransmission. J Neuroendocrinol.
18:692-702.
Bowley, M. P., Drevets, W. C., Ongur, D., and Price, J. L. (2002). Low glial numbers in
the amygdala in major depressive disorder. Biological Psychiatry 52:404-412.
Braak, H., Sastre, M., Del Tredici, K. (2007). Development of alpha-synuclein
immunoreactive astrocytes in the forebrain parallels stages of intraneuronal
pathology in sporadic Parkinson's disease. Acta Neuropathol 114:231-41.
Bubenik, G. A., Brown, G. M. (1973). Morphologic sex differences in primate brain areas
involved in regulation of reproductive activity. Experientia 29:619-621.
Canteras, N. S., Simerly, R. B., Swanson, L. W. (1995). Organization of projections from
the medial nucleus of the amygdala - a Phal study in the rat. J Comp Neurol 360:
213-245.
Cahill, L. (2000). Neurobiological mechanisms of emotionally influenced, long-term
memory. Prog Brain Res 126:29-37. Review.
Cahill, L., Uncapher, M., Kilpatrick, L., Alkire, M. T., Turner, J. (2004). Sex-related
hemispheric lateralization of amygdala function in emotionally influenced
memory: an FMRI investigation. Learn Mem 11:261-6.
Cajal, S. R. (1909).
Maloine, Paris.

.A

Cardinal, R. N., Parkinson, J. A., Hall, J., Everitt, B. J. (2002). Emotion and motivation:
the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci
Biobehav Rev 26:321-52. Review.
Carrillo, B., Pinos, H., Guillamón, A., Panzica, G. and Collado, P. (2007).
Morphometrical and neurochemical changes in the anteroventral subdivision of
the rat medial amygdala during estrous cycle. Brain Res 1150:83-93.
Cooke, B. M. (2006). Steroid-dependent plasticity in the medial amygdala.
Neuroscience 138:997-1005. Review.
Cooke, B. M., Breedlove, S. M., Jordan, C. L. (2003). Both estrogen receptors and
androgen receptors contribute to testosterone-induced changes in the
morphology of the medial amygdala and sexual arousal in male rats. Horm
Behav 43:336-46.

28

Cooke BM, Hegstrom CD, Breedlove SM (2002) Photoperiod-dependent response to
androgen in the medial amygdala of the Siberian hamster, Phodopus sungorus. J
Biol Rhythms 17:147 154.
Cooke, B. M., Tabibnia, G., Breedlove, S. M. (1999). A brain sexual dimorphism
controlled by adult circulating androgens. Proc Natl Acad Sci USA 22:7538-40.
in other
-50
Cotter, D. R., Pariante, C. M., Everall, I. P. (2001). Glial cell abnormalities in major
psychiatric disorders: the evidence and implications. Brain Res Bull 55:585-95.
Review.
De La Garza, R., Mahoney, J. J. (2004). A distinct neurochemical profile in WKY rats at
baseline and in response to acute stress: implications for animal models of
anxiety and depression. Brain Res 1021:209-218.
Study of
neurons and glial cells of basolateral amygdala in male and female rats
neonatally treated with estrogen. Int J Neurosci 83:145-51.
Donahue, J. (2000). Cells containing immunoreactive estrogen receptorbasal forebrain. Brain research 856: 142-151.
DonCarlos, L. L., Sarkey, S., Lorenz, B., Azcoitia, I., Garcia-Ovejero, D., Huppenbauer,
C., Garcia-Segura, L. M. (2006). Novel cellular phenotypes and subcellular sites
for androgen action in the forebrain. Neuroscience 138:801-7. Review.
cell groups in corticomedial amygdala react differently to neonatally administered
estrogen. J Neuroscience 66:475-481.
Dudal, S., Morissette, C., Lacombe, D., Tremblay, P., Gervais, F. (2004). Differences in
the amyloid-beta-induced inflammatory response in microglia from C57BL/6 and
A/J strains of mice. J Neuroimmunol 153:26-35.
Durazzo, A., Morris, J. A., Breedlove, S. M., Jordan, C. L. (2007). Effects of the
testicular feminization mutation (Tfm) of the androgen receptor gene on
BSTMPM volume and morphology in rats. Neuroscience Letters 419:168-171.
Dyck, R. H., Bogoch, I. I., Marks, A., Melvin, N. R., Teskey, G. C. (2002). Enhanced
epileptogenesis in S100B knockout mice. Brain Res Mol Brain Res 106:22-9.

29

Eddleston, M. and Mucke, L. (1993). Molecular profile of reactive astrocytes;
implications for their role in neurologic diseases. Neurosci 54:15-36.
Elmariah, S. B., Hughes, E. G., Oh, E. J., Balice-Gordon, R. J. (2005). Neurotrophin
signaling among neurons and glia during formation of tripartite synapses. Neuron
Glia Biol 1:1-11.
Eng, L., Ghirnikar, R. S. (1994). GFAP and astrogliosis. Brain Pathol 4:229-228.
Eng, L., Ghirnikar, R. S., Lee, Y. L. (2000). Glial Fibrillary Acidic Protein: GFAP- ThirtyOne Years (1969 2000). Neuochemical Res 25:1439-1451.
Faber-Zuschratter, H., Hüttmann, K., Steinhäuser, C., Becker, A., Schramm, J., Okafo,
U., Shanley, D., Yilmazer-Hanke, D. M. (2009). Ultrastructural and functional
characterization of satellitosis in the human lateral amygdala associated with
Ammon's horn sclerosis. Acta Neuropathol 117:545-55.
Freeman, M. R., Doherty, J. (2006). Glial cell biology in Drosophila and vertebrates.
Trends Neurosci 29:82-90. Review.
Featherstone, R. E., Fleming, A. S., Ivy, G. O. (2000). Plasticity in the maternal circuit:
effects of experience and partum condition on brain astrocyte number in female
rats. Behav Neurosci 114:158-72.
Finley, S. K., Kritzer, M. F. (1999). Immunoreactivity for intracellular androgen receptors
in identified subpopulations of neurons, astrocytes and oligodendrocytes in
primate prefrontal cortex. J Neurobiol 40:446-457.
Fowler, C. D., Freeman, M. E., Wang, Z. (2003). Newly proliferated cells in the adult
male amygdala are affected by gonadal steroid hormones. J Neurobiol 57:
257-69.
Fowler, C. D., Johnson, F., Wang, Z. (2005). Estrogen regulation of cell proliferation and
distribution of estrogen receptor-alpha in the brains of adult female prairie and
meadow voles. J Comp Neurol 489:166-79.
Forger, N. G. (2006). Cell death and sexual differentiation of the nervous system.
J Neuroscience 138:929-938.
Garcia-Segura, L. M., Chowen, J. A., Duenas, M., Torres-Aleman, I. & Naftolin, F.
(1994). Gonadal steroids as promoters of neuro-glial plasticity.
Psychoneuroendocrinology,19:445-453.
Garcia-Segura, L. M., Melcangi, R. C. (2006). Steroids and glial cell function. Glia
54:485-498.
30

Garcia-Segura, L. M., Torres-Aleman, I., Naftolin, F. (1989). Astrocytic shape and glial
fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat
hypothalamic cultures. Brain Res Dev Brain Res 47:298-302.
Giedd, J. N., Castellanos, F. X., Rajapakse, J. C., Vaituzis, A. C., Rapoport, J. L.
(1997). Sexual dimorphism of the developing human brain. Prog
Neuropsychopharmacol Biol Psychiatry 21:1185-201.
Golgi, C. 1885. Sulla fina anatomia degli organi centrali del sistema nervoso. Riv. Sper.
Fremiat. Med. Leg. Alienazioni Ment 11:72-123.
Gosselin, R. D., Gibney, S., O'Malley, D., Dinan, T. G., and Cryan, J. F. (2009). Region
specific decrease in glial fibrillary acidic protein immunoreactivity in the brain of a
rat model of depression. Neuroscience 159:915-25.
Grootendorst, J., Mulder, M., Haasdijk, E., de Kloet, E. R., Jaarsma, D. (2000).
Presence of apolipoprotein E immunoreactivity in degenerating neurones of mice
is dependent on the severity of kainic acid-induced lesion. Brain Res
868:165-75.
Grove, E. A. (1988a). Efferent connections of the substantia innominata in the rat. J
Comp Neurol 277:347-64.
Grove, E. A. (1988b). Neural associations of the substantia innominata in the rat:
afferent connections. J Comp Neurol 277:315-46.
Guthrie, P. B., Knappenberger, J., Segal, M., Bennett, M. V., Charles, A. C., Kater, S. B.
(1999). ATP released from astrocytes mediates glial calcium waves. J Neurosci
19:520-8.
Hachem, S., Aguirre, A., Vives, V., Marks, A., Gallo, V., Legraverend, C. (2005). Spatial
and temporal expression of S100B in cells of oligodendrocyte lineage. Glia
51:81-97.
Halassa, M. M., Fellin, T., Takano, H., Dong, J. H., Haydon, P. G. (2007). Synaptic
islands defined by the territory of a single astrocyte. J Neurosci 27:6473-7.
Hamann, S. (2005). Sex differences in the responses of the human amygdala.
Neuroscientist 11:288-93. Review.
Hamidi, M., Drevets, W. C., and Price, J. L. (2004). Glial reduction in amygdala in major
depressive disorder is due to oligodendrocytes. Biological Psychiatry 55:563-569.
Hamson, D. K., Jordan, C. L., Breedlove, S. M. (2010). The testosterone two-step is
really a minuet. Neuron 66:167-169.
31

Hashimoto, N., Takeuchi, T., Ishihara, R., Ukai, K., Kobayashi, H., Iwata, H., Iwai, K.,
Mizuno, Y., Yamaguchi, H., and Shibayama, H. (2003). Glial fibrillary tangles in
diffuse neurofibrillary tangles with calcification. Acta Neuropathol 106:150-6.
Hatton, G. I. (2002). Glial-neuronal interactions in the mammalian brain. Adv Physiol
Educ 26:225-37. Review.
Hercher, C., Turecki, G., Mechawar, N. (2009). Through the looking glass: examining
neuroanatomical evidence for cellular alterations in major depression. Journal of
psychiatric research 43:947-961.
Hines, M., Allen, L. S., Gorski, R. A. (1992). Sex differences in subregions of the medial
nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat.
Brain Res 8:321-6.
Ino, H., Chiba, T. (2001). Cyclin-dependent kinase 4 and cyclin D1 are required for
excitotoxin-induced neuronal cell death in vivo. J Neurosci 21:6086-94
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (2008). Sex differences and laterality in
astrocyte number and complexity in the adult rat medial amygdala. J Comp
Neurol 511:599-609.
Jung, K. H., Chu, K., Lee, S.T., Kim, J. H., Kang, K. M., Song, E. C., Kim, S. J., Park, H.
K., Kim, M., Lee, S. K., Roh, J. K. (2009). Region-specific plasticity in the
epileptic rat brain: a hippocampal and extrahippocampal analysis. Epilepsia
50:537-49.
Kaku, M., Tsutsui, K., Motokawa, M., Kawata, T., Fujita, T., Kohno, S., Tohma, Y.,
Ohtani, J., Tenjoh, K., Tanne, K. (2003). Amyloid beta protein deposition and
neuron loss in osteopetrotic (op/op) mice. Brain Res Brain Res Protoc 12:104-8.
Kavushansky, A., Richter-Levin, G. (2006). Effects of stress and corticosterone on
activity and plasticity in the amygdala. J Neuro Res 84: 1580-1587.
Kavushansky, A., Vouimba, R., Cohen, H., Richter-Levin, G. (2006). Activity and
plasticity in the CA1, the dentate gyrus, and the amygdala following controllable
vs. uncontrollable water stress. Hippocampus 16: 35-42.
Killgore, W. D. and Yurgelun-Todd, D. A. (2004). Sex-related developmental differences
in the lateralized activation of the prefrontal cortex and amygdala during
perception of facial affect, Percept Mot Skills 99:371 391.
Kilpatrick, L. A., Zald, D. H., Pardo, J. V., Cahill, L. F. (2006). Sex-related differences in
amygdala functional connectivity during resting conditions. Neuroimage 30:
452-61.

32

Kim, D., Kim, E. H., Kim, C., Sun, W., Kim, H. J., Uhm, C. S., Park, S. H., Kim, H.
(2003). Differential regulation of metallothionein-I, II, and III mRNA expression in
the rat brain following kainic acid treatment. Neuroreport 14:679-82.
Khana, M. M., Hadman, M., De Sevilla, L. M., Mahesh, V. B., Buccafusco, J., Hill, W. D.,
Brann D. W. (2006). Cloning, distribution, and colocalization of MNAR/PELP1
with glucocorticoid receptors in primate and nonprimate brain.
Neuroendocrinology 84:317-29.
Klüver, H., Bucy, P. C. (1939). Preliminary analysis of functions of the temporal lobes in
monkeys. J Neuropsychiatry Clin Neurosci 9:606-20.
Kohmana, S.G., Goss, J.R., Finch, C.E., and McNeill, T. H. (1995). Increases of glial
fibrillary acidic protein in the aging female mouse brain. Neurobiology of Aging
16:59 67
Kraszpulski, M., Dickerson, P. A., Salm, A. K. (2006). Prenatal stress affects the
developmental trajectory of the rat amygdala. Stress 9:85-95.
Krettek, J. E., Price J. L. (1978a). A description of the amygdaloid complex in the rat
and cat with observations on intra-amygdaloid axonal connections. J Comp
Neurol 178: 255-280.
Krettek, J. E., Price J. L. (1978b). Amygdaloid projections to subcortical structures
within the basal forebrain and brainstem in the rat and cat. J Comp Neurol 178:
225-254.
Langub, M. J., & Watson, R. J. (1992). Estrogen receptor-immunoreactive
glia,endothelia, and ependyma in guinea pig preoptic area and median
eminence: electron microscopy. Endocrinology 130:364-372.
LeDoux, J. (1998). Fear and the brain: where have we been, and where are we going?
Biol Psychiatry 44:1229-38. Review.
Lee, Y., Gaskins, D., and Shekhar, A. (2007). Glia mechanisms in mood regulation: a
novel model of mood disorders. Psychopharmacology 191:55-65.
Legrand, A., Alonso, G. (1998). Pregnenolone reverses the age-dependent
accumulation of glial fibrillary acidic protein within astrocytes of specific regions
of the rat brain. Brain Res 802:125-33.
Leventopoulos, M., Ruedi-Bettschen, D., Knuesel, I., Feldon, J., Pryce, C. R.,
Opacka-Juffry, J., (2007). Long-term effects of early life deprivation on brain glia
in Fischer rats. Brain Research 1142:119-126.

33

Li, H., Blaustein, J. D., De Vries, G. J., Wade, G. N. (1993). Estrogen-receptor
immunoreactivity in hamster brain: preoptic area, hypothalamus and amygdala.
Brain Research 631:304-312.
Ling, M., Dong-Dong, W., Zhang, T.,Yu, H., Wang Y., Huang, S., Lee, F. S., Chen, Z.
(2011). Region-Specific Involvement of BDNF Secretion and Synthesis in
Conditioned Taste Aversion Memory Formation. J Neuro 31:2079-2090.
Linnemann, D. and Skarsfelt, T. (1994). Regional changes in expression of NCAM,
GFAP, and S100 in aging rat brain. Neurobiology of Aging 15:651 65.
Malsbury, C. W., McKay, K. (1989). Sex difference in the substance P-immunoreactive
innervation of the medial nucleus of the amygdala. Brain research bulletin 23:
561-567.
Martinez, F. G., Hermel, E. E., Xavier, L. L., Viola, G. G., Riboldi, J., Rasia-Filho, A. A.,
Achaval M. (2006). Gonadal hormone regulation of glial fibrillary acidic protein
immunoreactivity in the medial amygdala subnuclei across the estrous cycle and
in castrated and treated female rats. Brain Res 1108:117-26.
Mena, M. M., Garcia De Yebenes, J. (2008). Glial Cells as Players in Parkinsonism: The
''Good,'' the ''Bad,'' and the ''Mysterious'' Glia. The Neuroscientist 14:544-560.
Milner, T. A., Shah, P., Pierce, J. P. (2000). Beta-adrenergic receptors primarily are
located on the dendrites of granule cells and interneurons but also are found on
astrocytes and a few presynaptic profiles in the rat dentate gyrus. Synapse
36:178-93.
Mizukami, S., Nishizuka, M., Arai, Y. (1983). Sexual difference in nuclear volume and its
ontogeny in the rat amygdala Experimental neurology 79:569-575.
Mong, J. A., Nuñez, J. L., McCarthy, M. M. (2002). GABA mediates steroid-induced
astrocyte differentiation in the neonatal rat hypothalamus. J Neuroendocrinol
14:45-55.
Mong, J. A., Roberts, R. C., Kelly, J. J., McCarthy, M. M. (2001). Gonadal steroids
reduce the density of axospinous synapses in the developing rat arcuate
nucleus: an electron microscopy analysis. J Comp Neurol 432:259-67.
Morris, J. A., Jordan, C. L., Breedlove, S. M. (2008). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in adult rats. J Comp Neurol 506:851-9.

34

Morris, J. A., Jordan, C. L., Dugger, B. N., Breedlove, S. M. (2005). Partial
demasculinization of several brain regions in adult male (XY) rats with a
dysfunctional androgen receptor gene. Journal of Comparative Neurology,
487:217-226.
Mukaetova-Ladinska, E. B. and McKeith, I. G. (2006). Pathophysiology of synuclein
aggregation in Lewy body disease. Mech Ageing Dev 127:188 202
Newton, S. S., Collier, E. F., Hunsberger, J., Adams, D., Terwilliger, R., Selvanayagam,
E., Duman, R. S. (2003). Gene profile of electroconvulsive seizures: induction of
neurotrophic and angiogenic factors. J Neurosci 23:10841-51.
Nicoletti, J. N., Shah, S. K., McCloskey, D. P., Goodman, J. H., Elkady, A., Atassi, H.,
Hylton, D., Rudge, J. S., Scharfman, H. E. and Croll, S. D. (2008). Vascular
endothelial growth factor is up-regulated after status epilepticus and protects
against seizure-induced neuronal loss in hippocampus. Neuroscience
151:232-41.
Nishida, H. and Okabe, S. (2007). Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci 27:331-40.
Nishizuka, M., Arai, Y. (1981). Sexual dimorphism in synaptic organization in the
amygdala and its dependence on neonatal hormone environment. Brain
research 212:31-38.
Nishizuka, M., Arai, Y. (1982). Synapse formation in response to estrogen in the medial
amygdala developing in the eye. Proceedings of the National Academy of
Sciences of the United States of America 79:7024-7026.
Olsen, K. L., Whalen, R. E. (1982). Estrogen binds to hypothalamic nuclei of
androgen-insensitive (Tfm) rats Experientia 38:139-140.
Orre, K., Wennström, M. and Tingström, A. (2009). Chronic lithium treatment decreases
NG2 cell proliferation in rat dentate hilus, amygdala and corpus callosum. Prog
Neuropsychopharmacol Biol Psychiatry 33:503-10.
Pfrieger, F. W. and Barres, B. A. (1997). Synaptic efficacy enhanced by glial cells in
vitro. Science 277:1684-7.
Poryazova, R., Schnepf, B., Werth, E., Khatami, R., Dydak, U., Meier, D., Boesiger, P.
and Bassetti, C. L. (2009). Evidence for metabolic hypothalamo-amygdala
dysfunction in narcolepsy. Sleep 32:607-13.

35

Power, J. H., Asad, S., Chataway, T. K., Chegini, F., Manavis, J., Temlett, J. A., Jensen,
P. H., Blumbergs, P. C. and Gai, W. P. (2008). Peroxiredoxin 6 in human brain:
molecular forms, cellular distribution and association with Alzheimer's disease
pathology. Acta Neuropathol 115:611-22.
Price, J. L. (1987). Subcortical projections from the amygdaloid complex. Adv Exp Med
Biol 203:19-33.
Rasia-Filho, A. A., Xavier, L. L., dos Santos, P., Gehlen, G. and Achaval, M. (2002).
Glial fibrillary acidic protein immunodetection and immunoreactivity in the anterior
and posterior medial amygdala of male and female rats. Brain Res Bull 58:67-75.
Raza, L., Khana M. M., Mahesha, V. B., Vadlamudib, R. K., Branna, D. W. (2008).
Rapid Estrogen Signaling in the Brain. Neurosignals 16:140-153.
Rogan, M. T., LeDoux, J. E. (1995). LTP is accompanied by commensurate
enhancement of auditory-evoked responses in a fear conditioning circuit. Neuron
15:127-36.
Roselli, C. E., Salisbury, R. L., & Resko, J. A. (1987). Genetic evidence for
androgen-dependent and independent control of aromatase activity in the rat
brain Endocrinology 121:2205-2210.
Rubinow, M. J., Juraska, J. M. (2009). Neuron and glia numbers in the basolateral
nucleus of the amygdala from preweaning through old age in male and female
rats: a stereological study. J Comp Neurol 512:717-25.
Sah, P., Westbrook, R. F. and Lüthi, A. (2008). Fear conditioning and long-term
potentiation in the amygdala: what really is the connection? Ann N Y Acad Sci.
1129:88-95. Review.
Samson, R. D., Duvarci, S., Pare, D. (2005). Synaptic plasticity in the central nucleus of
the amygdala. Rev Neurosci 16:287-302.
Schummers, J., Yu, H., Sur, M. (2008). Tuned Responses of Astrocytes and Their
Influence on Hemodynamic Signals in the Visual Cortex. Science 320:16281643.
Scott, S. A., Dekosky, S. T., Sparks, D. L., Knox, C. A. and Scheff, S. W. (1992).
Amygdala cell loss and atrophy in alzheimers-disease. Annals of Neurology
32:555-563.

36

Shammah-Lagnado, S. J., Beltramino, C. A., McDonald, A. J., Miselis, R. R., Yang, M.,
de Olmos, J., Heimer, L. and Alheid, G. F. (2000). Supracapsular bed nucleus of
the stria terminalis contains central and medial extended amygdala elements:
evidence from anterograde and retrograde tracing experiments in the rat. J Comp
Neurol 422:533-55.
Sholl, S. A., Kim, K. L. (1990). Aromatase, 5-alpha-reductase, and androgen receptor
levels in the fetal monkey brain during early development. Neuroendocrinology
52:94-98.
Sigurdsson, T., Doyère, V., Cain, C. K. and LeDoux, J. E. (2007). Long-term
potentiation in the amygdala: a cellular mechanism of fear learning and memory.
Neuropharmacology 52:215-27. Review.
Simerly, R. B., Chang, C., Muramatsu, M., Swanson, L. W. (1990). Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ
hybridization study. J Comp Neurol 294:76-95.
Stefanacci, L., Amaral, D. G. (2002). Some observations on cortical inputs to the
macaque monkey amygdala: An anterograde study. J Comp Neurol 451: 301323.
Stefanova, N. and Ovtscharoff, W. (2000). Sexual dimorphism of the bed nucleus of the
stria terminalis and the amygdala. Adv Anat Embryol Cell Biol 158:1-78.
Review.
Tan, H., Lauzon, N. M., Bishop, S. F., Bechard, M. A., Laviolette, S. R. (2010).
Integrated Cannabinoid CB1 Receptor Transmission within the Amygdala
Prefrontal Cortical Pathway Modulates Neuronal Plasticity and Emotional
Memory Encoding. Cereb Cortex 20:1486-1496.
Terada, S., Ishizu, H., Yokota, O., Tsuchiya, K., Nakashima, H., Ishihara, T., Fujita, D.,
Uéda, K., Ikeda, K. and Kuroda, S. (2003) Glial involvement in diffuse Lewy body
disease. Acta Neuropathol 105:163 169
Thippeswamy, T., Mckay, J. S., Morris, R., Quinn, J., Wong, L., Murphy, D. (2004).
Glial-mediated neuroprotection: Evidence for the protective role of the NO-cGMP
pathway via neuron glial communication in the peripheral nervous system. Glia
49:197-210.
Truitt, W. A., Sajdyk, T. J., Dietrich, A. D., Oberlin, B., McDougle, C. J., Shekhar, A.
(2007). From anxiety to autism: spectrum of abnormal social behaviors modeled
by progressive disruption of inhibitory neuronal function in the basolateral
amygdala in Wistar rats. Psychopharmacology 191:107-18.

37

Ullian, E. M., Sapperstein, S. K., Christopherson, K. S. and Barres, B. A. (2001). Control
of synapse number by glia. Science 291:657-61.
Umeoka, S., Miyamoto, O., Nakagawa, T., Janjua, N. A., Nagao, S. and Itano, T.
(2001). Expression of an embryonic intermediate filament protein in amygdaloid
kindled rats. Epilepsy Res 43:249-53.
Vessal, M., Dugani, C. B., Solomon, D. A., Burnham, W. M. and Ivy, G. O. (2004).
Astrocytic proliferation in the piriform cortex of amygdala-kindled subjects: a
quantitative study in partial versus fully kindled brains. Brain Res 1022:47-53.
Vessal, M., Dugani, C. B., Solomon, D. A., McIntyre Burnham, W. and Ivy, G. O. (2005).
Might astrocytes play a role in maintaining the seizure-prone state? Brain Res.
1044:190-6.
von Ziegler von, N. I., Lichtensteiger W. (1992). Asymmetry of brain aromatase activity:
region- and sex-specific developmental patterns. Neuroendocrinology 55:512-518.
Wang, T. F., Zhou, C., Tang, A. H., Wang, S. Q. and Chai, Z. (2006).
Cellular mechanism for spontaneous calcium oscillations in astrocytes.
Acta Pharmacol Sin 27:861-8.
Wennström, M., Hellsten, J. and Tingström, A. (2004). Electroconvulsive seizures
induce proliferation of NG2-expressing glial cells in adult rat amygdala. Biol
Psychiatry 55:464-71.
Woldbye, D. P., Bolwig, T. G., Kragh, J. and Jørgensen, O. S. (1996). Synaptic
degeneration and remodelling after fast kindling of the olfactory bulb. Neurochem
Res 21:585-93.
Woolley, C. S. (2007). Acute effects of estrogen on neuronal physiology. Annu Rev
Pharmacol Toxicol 47:657-80. Review.
Yarbrough, W. G., V. E. Quarmby, et al. (1990). "A single base mutation in the
androgen receptor gene causes androgen insensitivity in the testicular feminized
rat." J Biol Chem., 265(15):8893-900.
Yamamuro, A., Ago, Y., Takuma, K., Maeda, S., Sakai, Y., Baba, A., Matsudo, T.
(2003). Possible Involvement of Astrocytes in Neuroprotection by the Cognitive
Enhancer T-588. Neurochemical Res 28:1779-1783.
Yilmazer-Hanke, D. M., Faber-Zuschratter, H., Blümcke, I., Bickel, M., Becker, A.,
Mawrin, C., and Schramm, J. (2007). Axo-somatic inhibition of projection neurons
in the lateral nucleus of amygdala in human temporal lobe epilepsy: an
ultrastructural study. Exp Brain Res 177:384-99.
38

Yokota, O., Tsuchiya, K., Hayashi, M., Kakita, A., Ohwada, K., Ishizu, H., Takahashi, H.
and Akiyama H. (2008). Glial clusters and perineuronal glial satellitosis in the
basal ganglia of neurofibromatosis type 1. Acta Neuropathol 116:57-66.
Yokota, O., Terada, S., Ishizu, H., Tsuchiya, K., Kitamura, Y., Ikeda, K., Uéda, K. and
Kuroda, S. (2002). NACP/alpha-synuclein immunoreactivity in diffuse
neurofibrillary tangles with calcification (DNTC). Acta Neuropathol 104:333-41.
Yu, S. Y., Wu, D. C., Liu, L., Ge, Y., Wang, Y. T. (2008). Role of AMPA receptor
trafficking in NMDA receptor-dependent synaptic plasticity in the rat lateral
amygdala. J Neurochem 106:889-99.
Zhang, L., Corona-Morales, A. A., Vega-González, A., García-Estrada, J. and Escobar,
A. (2009). Dietary tryptophan restriction in rats triggers astrocyte cytoskeletal
hypertrophy in hippocampus and amygdala. Neurosci Lett 450:242-5.
Zuloaga, D., Puts, D., Jordan, C., Breedlove, S. (2008). The role of androgen receptors
in the masculinization of brain and behavior: What we've learned from the
testicular feminization mutation. Hormones and behavior 53:613-626.

39

CHAPTER 1: SEX DIFFERENCES AND LATERALITY IN ASTROCYTE NUMBERS
AND COMPLEXITY IN THE ADULT RAT MEDIAL AMYGDALA
Abstract
The posterodorsal portion of the medial amygdala (MePD) is sexually dimorphic
in several rodent species. In several other brain nuclei, astrocytes change morphology
in response to steroid hormones. I visualized MePD astrocytes using glial-fibrillary
acidic protein (GFAP) immunocytochemistry. I compared the number and process
complexity of MePD astrocytes in adult wildtype male and female rats, and testicular
feminized mutant (Tfm) male rats that lack functional androgen receptors (ARs), to
determine whether MePD astrocytes are sexually differentiated and ARs have a role.
Unbiased stereological methods revealed laterality and sex differences in MePD
astrocyte number and complexity. The right MePD contained more astrocytes than the
left in all three genotypes, and the number of astrocytes was also sexually differentiated
in the right MePD, with males having more astrocytes than females. In contrast, the left
MePD contained more complex astrocytes than did the right MePD in all three
genotypes, and males had more complex astrocytes than females in this hemisphere.
Tfm males were comparable to wildtype females, having fewer astrocytes on the right
and simpler astrocytes on the left than do wildtype males. Taken together, these results
demonstrate that astrocytes are sexually dimorphic in the adult MePD and that the
nature of the sex difference is hemisphere-dependent: a sex difference in astrocyte
number in the right MePD and a sex difference in astrocyte complexity in the left MePD.
Moreover, functional ARs appear to be critical in establishing these sex differences in
MePD astrocyte morphology.

40

Introduction
The amygdala is implicated in the control of many social behaviors, including
fear, anxiety, and social interactions (Adolphs et al. 2005; Bishop et al. 2004; LeDoux et
al. 1998; Mah et al. 2007; Spezio et al. 2007; Truit et al. 2007; Williams et al. 2005).
Interestingly, many behavioral disorders in which the amygdala has been implicated
have clear sex-biases in the human population. For example, schizophrenia, autism,
and drug-addiction are all more common in males, while anxiety disorders and
depression are more common in females (American Psychiatric Association, 2000).
Many aspects of the amygdala are sexually dimorphic in a variety of mammalian
species, lending support to the idea that structural differences in the brain may underlie
biases in behavior and disease susceptibility, and that the amygdala may be a key
player in the control of such behaviors.
In rats, the medial amygdala is composed of several subnuclei that are
extensively interconnected (Alheid et al. 1995; Grove 1988a, 1988b; ShammahLagnado et al. 2000). The posterodorsal medial amygdala (MePD) in particular
receives input from chemosensory and hypothalamic regions and projects to the
preoptic area and ventral medial hypothalamic nucleus, both of which play important
roles in reproductive behaviors. The MePD normally contains a high concentration of
both androgen receptors (ARs) and estrogen receptors (ER; Simerly et al. 1990), and is
highly sexually dimorphic in adults. The MePD in male rats has a larger overall volume
(Hines et al. 1992), contains larger neuronal somata, more neurons (Morris et al. 2008),
and greater dendritic spine density than the MePD in female rats (Rasia-Filho et al.
2004). The adult MePD is also highly plastic. Castration of adult male rats eliminates

41

the sex difference in regional volume and neuronal soma size (Cooke et al. 1999), and
causes deficits in noncontact erections and ultrasonic vocalizations, while treatment
with androgens and/or estrogens prevents these changes following castration (Cooke et
al. 2003).
Typically, estrogen acting on ER is regarded as the pathway responsible for
hormone-induced masculinization in the rodent brain. However, recent results indicate
that AR also contributes to sexual differentiation of many brain regions, including the
MePD. Testicular feminized mutant (Tfm) male rats lack functional AR and are
insensitive to androgens (Yarbrough et al. 1990) and thus represent an important tool
for assessing the role of androgens in the origin of neural sex differences. I have found
that several aspects of MePD morphology are only partially masculinized in Tfm males,
indicating that ARs are necessary for complete masculinization of this nucleus (Morris et
al. 2005), but where androgens act to masculinize the MePD has not been established.
Gonadal hormones not only influence neuronal morphology, but also modulate
the morphology of non-neuronal cells such as astrocytes, which are thought to
contribute critically to some forms of neural plasticity. For example, estrogens increase
the complexity of astrocyte arbors in the arcuate nucleus and preoptic area of the
hypothalamus (reviewed in Garcia-Segura et al. 1994 and in Garcia-Segura and
McCarthy 2006). Moreover, androgens increase the number of astrocytes in
hippocampal cultures (Gatson and Singh 2007). Astrocytes are also known to express
steroid hormone receptors, including both ARs and ERs (Doncarlos et al. 2005; Lorenz
et al. 2005). Thus, steroid hormones may act directly on astrocytes to regulate their
morphology and function. Given the implicated role of astrocytes in adult neural

42

plasticity, their steroid responsiveness in some brain regions, and the highly plastic
nature of the adult MePD, I speculated that astrocytes might exhibit sexually dimorphic
morphologies in the MePD. Indeed, reports of sex differences in the MePD in both the
density of glial fibrillary acidic protein (GFAP) immunoreactivity and in the number of
Nissl-stained glial cells (Martinez et al. 2006; Morris et al. 2008) suggest that MePD
astrocytes may be sexually dimorphic in both number and process complexity. I now
report sex differences in both measures, and that Tfm males are like females in having
fewer and less complex MePD astrocytes than wildtype males, indicating that both the
number and complexity of MePD astrocytes in rats are normally masculinized through
the activation of ARs.
Methods
Ninety to 120 day-old wildtype male, wildtype female and Tfm male rats were
obtained from our Tfm colony at Michigan State University. Female carriers of this
spontaneous mutation of the AR gene have been bred with Long Evans male rats from
Charles Rivers for over 10 generations. Estrous cycle was not monitored for the
females used in this study, thus differences in ovarian hormones were not accounted
for. Animals were housed three per cage in a single colony room in standard rat cages
with food and water available ad libitum. Lights were on at 0700 and off at 1900 hours.
Animals were cared for in accordance with the guidelines set forth by the National
Institute of Health and all procedures were approved by the Institutional Animal Care
and Use Committee at Michigan State University.
Animals were given an overdose of sodium pentobarbital (120mg/kg, ip). Once
animals were deeply anesthetized (showing no reflex response to either tail or foot

43

pinch) they were intracardially perfused with 0.9% saline, followed by 4%
paraformaldehyde in 0.1M phosphate buffer (pH 7.4, ~250 mL/animal). The brains were
then removed and postfixed for five hours at 40 C in the same fixative solution and
stored in 30% phosphate-buffered sucrose at 40 C for 48 hrs. The left cortex of each
brain was scored with a shallow cut to mark that hemisphere. Brains were sectioned
coronally on a freezing microtome at 40µm through the region of interest. Sections
were collected into cryoprotectant (de Olmos et al. 1978) and stored at -200 C. Every
third section through the rostrocaudal extent of the MePD was processed for
immunocytochemistry.
Histology
For immunocytochemistry, 0.1 M phospate buffered saline with 0.3% Triton X100 (PBS-T; 140 mM NaCl, 10.7 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4)
served as a rinsing agent between all incubations and as dilutant. Upon removal from
cryoprotectant, tissue was placed into Netwell plates (Corning Life Sciences) and
thoroughly rinsed. Sections were then incubated for 20 min in 1% sodium borohydride
followed by a 25 min incubation in 1% H2O2, one hr incubation in 10% normal horse
serum (Chemicon, Temecula, CA), and a 24 hr incubation at 40 C in mouse anti-GFAP
monoclonal antiserum (1:50,000, Chemicon, MAB360). The antiserum was prepared
against purified glial fibrillary acidic protein from porcine spinal cord. In western blots,

technical information). Staining of sections through the amygdala and hypothalamus
produced a pattern of GFAP immunoreactivity that was identical to other descriptions
(Mong et al. 1999; Rasia-Filho et al. 1999; Martinez et al. 2006).

44

Following incubation in primary antiserum, tissue was incubated in biotinylated
rat-absorbed horse anti-mouse secondary antiserum (1:500, Vector Laboratories) for
one hour at room temperature. This was followed by one hr incubation in peroxidase
avidin-bitotin complex solution at half recommended strength (Elite ABC kit, Vector).
HRP was visualized using NiCl-enhanced diaminobenzidine (DAB, Sigma) in a 0.05 M
in Tris Buffer, pH 7.2. After rinsing to quench the peroxidase reaction, tissue was
mounted on gel-subbed slides, dried, dehydrated in alcohol rinses, and counter-stained
using Harris hematoxylin solution (Sigma), with 10% lithium carbonate used as the
bluing agent. Slides were then coverslipped with Permount.
Stereological Analysis
A Zeiss Axioplan II microscope with the visual field captured by an Optronics
MicroFire digital video camera and displayed on a monitor was used to quantify the
number and complexity of astrocytes within the region of interest. Using
StereoInvestigator software (v. 7.0, MBF Bioscience; Williston, VT), the perimeter of the
MePD was traced in serial sections at low magnification. MePD boundaries were
identified using the standard rat atlas (Paxinos and Watson, 2005), in conjunction with
previously established standards within our laboratory (Morris et al. 2005, 2007) and
others (Canteras et al. 1995; Hines et al. 1992). Several consistent morphological
features were used to accurately identify and trace the MePD, including the angle and
length of the optic tracts, the overlap of the optic tract with the cerebral peduncle, the
descent of the stria terminalis into adjacent regions, and the dense collection of neurons
forming the posteroventral medial amygdala. After tracing the boundaries of the MePD
at low magnification, astrocytes were counted and traced using a 100X Plan-NeoFluar,

45

1.3 N.A oil-immersion objective. Slides were coded to ensure that all measures were

Images of astrocytes were captured using the image capture option in
StereoInvesitgator. Images were resized and adjusted using Adobe Photoshop (v 7.0
San Jose, CA). All images received a +5 increase in brightness through a
brightness/contrast adjustment layer available in the software.
Astrocyte number: An optical fractionator probe (West et al. 1991) was used to
generate an unbiased estimate of astrocyte numbers within the boundaries of the
MePD. The optical fractionator estimates the total number of objects within a space
based on a sample representing a known fraction of that space. The method uses a 3
dimensional probe with a fixed, specified volume inserted into the tissue. Initial probe
insertion is random within the region of interest but then proceeds at fixed intervals
allowing for an unbiased and accurate estimate of the number of objects within the
entire region of interest. Probe dimensions were 45 by 45 µm with a height of 6-9 µm.
The coefficient of error (CE; Gunderson m=1) for each hemisphere was at or below
0.10.
Criteria for identifying an astrocyte included a distinct and recognizable nucleus
in a plane of focus withi

-value, and from which at least 2 GFAP

labeled fibers extended. Such nuclei were often, although not always, smaller than
surrounding (presumably neuronal) nuclei, often elliptical in shape, and lacking a clear
nucleolus. At times, especially around vascular pathways and at cortical boundaries,
GFAP immunoreactivity was dense enough to obscure the nucleus of origin and in
these cases, no cells were marked or traced. These methods produced estimates of

46

overall MePD volume, number of astrocytes, and average area of astrocyte nuclei per
hemisphere for each subject. The number of cells counted per hemisphere ranged from
approximately 250-400 depending on the sex and hemisphere of the subject.
Astrocyte process complexity: Neurolucida software (v. 7.0, MBF Bioscience;
Williston, VT) was used to trace and measure the entire visible arbor of 24 randomly
selected astrocytes per subject. The MePD was traced via the criteria and methodology
described above and a fractionator probe was used to define random tracing sites
throughout the MePD, with 12 sites per hemisphere. At each of these sites, the
astrocyte nearest the randomly placed marker, but at least 30 µm from the MePD
boundary, was traced in its entirety. The average number of primary processes,
average number of branch points, average number of branch endings, and average
branch length were calculated for each animal.
Additionally, each astrocyte was classified into one of four groups based on
overall morphological complexity following a classification scheme developed by Mong
and McCarthy (1999). In brief, Group I astrocytes exhibit few and short primary
processes (Fig 1A). Group II astrocytes possess more and longer primary processes
but few secondary processes (Fig 1B). Group III possess more secondary processes
and finally Group IV astrocytes have large and complex arbors with numerous
secondary processes (Mong et al. 1996; Mong et al. 1999, Figs 1C and 1D). The
percentage of astrocytes within the MePD that fall into each of the categories was
calculated for each of the 3 experimental groups (male, female and Tfm male).
Statistical Analysis

47

Separate two-way mixed-design analyses of variance (ANOVA) were conducted
for each dependent variable (MePD volume, astrocyte number, number of primary
processes, number of branch points, number of branch endings, and branch length).
The left and right hemispheres served as a repeated measure and genotype (male,
female, Tfm) as a between group measure. This was followed by one-way ANOVAs for
each individual hemisphere and LSD post hoc analysis to determine differences
between genotypes on each side. For all analyses, results are expressed as mean ±
SEM (standard error of the mean), and a p value of 0.05 was considered statistically
significant, with number of animals per group reported in figures.
Results
Regional Volume
Mixed design 2-way ANOVA (left and right hemisphere as repeated measures)
revealed a main effect of sex on MePD volume (p < 0.01) and a main effect of
hemisphere (p < 0.01) and a sex by hemisphere interaction (p < 0.001). In all three
groups, the right hemisphere had greater regional volume than the left (males: p < 0.01;
TFMs: p < 0.05; females: p < 0.05, Fig 2A). Posthoc analysis revealed that volume of
the MePD varied significantly across genotypes only in the right hemisphere (p < 0.05).
Thus, the overall main effect of genotype is due to differences in the right but not the left
hemisphere. Additional post-hoc analyses revealed that the right MePD of males has a
greater volume than that of either Tfm males or females (ps <0.04, Fig 2A) and that
volume of the right MePD is not different in Tfm males and females (p = 0.323). These
data agree well with estimates of MePD volume based on Nissl stained material (Morris

48

et al. 2005, 2008), suggesting that the MePD boundaries revealed by GFAP and
hematoxylin is comparable to that shown by Nissl stain.
Astrocyte Numbers
There was a main effect of hemisphere (p < 0.001) on the number of MePD
astrocytes indicating that astrocytes are more numerous on the right than on the left
side in all three groups (males: p < 0.001; TFMs: p < 0.01; females: p < 0.001, Fig 2B).
There was no significant main effect of genotype on the number of astrocytes but there
was a significant hemisphere by sex interaction (p = 0.01, Fig 2B), which led to separate
one-way ANOVAs for each hemisphere. Although astrocyte number does not differ
significantly by genotype in the left hemisphere (p = 0.49), it does in the right
hemisphere (p < 0.05). Additional post-hoc analyses reveal that wildtype males have
more astrocytes on the right than do Tfm males (p < 0.01) and marginally significantly
more do females (p = 0.058). Thus, in the left hemisphere, astrocyte numbers are not
sexually dimorphic whereas in the right hemisphere, astrocyte numbers are sexually
dimorphic, with wildtype males having more than wildtype females and Tfm males.
Since Tfm male rats lack functional ARs but have slightly higher than normal male
levels of androgen (Roselli et al. 1987), the difference between wildtype and Tfm males
in astrocyte number suggests that functional ARs are necessary to reach normal
masculine levels.
Astrocyte Complexity
Astrocyte complexity was assessed using several measures: the number of
primary branches, the number of branch points, the number of branch endings, and the
average length of branches. In all complexity measures, I found a significant main

49

effect of laterality (ps < 0.005) with the left hemisphere consistently exhibiting more
complex astrocytes than the right (Fig 3). I also found significant main effects of
genotype on each measure of astrocyte complexity (ps< 0.05) except for number of
primary branches where there was no main effect of genotype. Despite finding no
significant interactions of genotype with hemisphere on any measure of astrocyte
complexity, posthoc analyses revealed that astrocyte complexity varied significantly by
genotype on three of the four measures (number of branch points, number of branch
endings, and average branch length), but only in the left hemisphere (ps< 0.01).
Further analyses of astrocyte measures indicate that left MePD astrocytes of wildtype
males are more complex than those of females on all four measures, having on average
more primary branches as well as total number of branches, more branch points and
longer branches than do astrocytes in the left MePD of females (ps < 0.5, Fig 3).
Except for average number of primary branches, males also showed more complex
astrocytes than did Tfm males on the left MePD (ps < 0.01, Fig 3). Thus, although the
number of astrocytes in the left hemisphere was not sexually differentiated, astrocyte
complexity was. This is in contrast to the right hemisphere, which exhibits an ARdependent sexual dimorphism in total astrocyte numbers but exhibits no sex differences
in astrocyte complexity. Furthermore, the sex difference in astrocyte complexity in the
left MePD also appears to be AR-dependent, as Tfm males did not significantly differ
from females in any measure of astrocyte complexity.
A principle components analysis for each hemisphere revealed that all four
measures of astrocytes arbor complexity loaded very strongly onto a single factor,
suggesting that the four measures used are closely related and could be viewed as a

50

single measure of overall arbor complexity in that hemisphere. One-way ANOVAs
examining this single factor in each hemisphere confirmed a significant effect of
genotype on overall arbor complexity in the left MePD (p < 0.01) but no effect in the
right MePD. Post hoc analysis of the left MePD revealed that males have greater
overall arbor complexity than either Tfm males or females (ps < 0.01).
Morphology Classification
Astrocyte arbor complexity was measured and analyzed using methodology
established by Mong and McCarthy (1999). Chi-square analysis indicates that
distribution of the four classes of astrocytes is different in males compared to females or
Tfm males (ps < 0.01), in the left MePD, but not the right, with males having more class
IV astrocytes in the left hemisphere than do females or TFMs. Furthermore, Tfm males
did not differ from females (p = 0.611). Thus, this nominal method of assessing
astrocytes arbor complexity agrees well with the quantitative measures of complexity.
Both indicate that males exhibit more complex astrocytes than females or TFMs, but
only in the left hemisphere, and that Tfm males are feminine in their astrocyte
complexity. GFAP labeling may not reveal ultra fine astrocyte processes, so these data
should not be taken as absolute measures of astrocytes complexity, but as relative
measures across the three groups.
Astrocyte Nuclear Size
Analysis of the average area of individual astrocyte nuclei revealed no main
effects of sex or hemisphere. However, there were several correlations; in the left
MePD, average astrocytic nuclear size across all genotypes correlated positively with
the number of primary branches and the average branch length (Pearson correlations:

51

0.72 and 0.53 respectively). No measure of astrocyte complexity correlated with
astrocyte nuclear size in the right MePD. These correlations suggest that nuclear size
may become critical when astrocytic branching becomes extensive as in the left MePD.
Discussion
Prior investigations demonstrated that the adult MePD is highly plastic and
responds to steroid hormones (Cooke et al. 1999, 2003; Morris 2005, 2008). Because
astrocytes are implicated in regulating neuronal plasticity and are known to be steroidresponsive in some brain regions (Mong et al. 1999; Mong and McCarthy 2002; GarciaSegura et al. 1994), I asked whether astrocytes in the adult MePD are sexually
dimorphic in number and/or complexity and if so, whether such differences are sensitive
to hormone action via ARs. I found sex differences in, and dramatic laterality of,
astrocyte number and complexity in this nucleus, and by utilizing Tfm males that lack
functioning AR, I also determined that masculinization of astrocyte number and
complexity in the MePD requires functional AR.
MePD Laterality
Laterality in adult MePD anatomy is not unexpected and has been reported
previously in the literature. Briefly, the adult right MePD is larger in volume than the left
based on Nissl stained material (Morris et al. 2005). Surprisingly, although the left
MePD is smaller in volume, it contains more neurons and is sometimes reported to
contain neurons that have larger cell bodies than those in the right MePD (Morris et al.
2008). Although reports have detailed MePD connectivity in the rat (Canteras et al.
1992; Coolen et al. 1998), hemisphere differences in these parameters are either
absent or unreported.

52

The current results report a new and dramatic lateralization in the anatomy of the
adult rat MePD, with the right having more astrocytes than the left (Fig 2), while the left
has more complex astrocytes than the right (Fig 3). The increased number of
astrocytes in the right MePD may not be particularly surprising, given its larger volume
compared to the left. However, data on neuronal number makes it clear that larger
regional volume need not predict greater cell number (Morris et al, 2008). The
increased complexity of astrocytes in the left MePD, as revealed in several
morphological measurements, was unexpected. The use of a previously established
astrocyte complexity classification scheme (Mong and McCarthy 1999) also suggests
that the left hemisphere has more complex astrocytes than the right (Table 1).
Thus, it appears that astrocytes follow different strategies to populate each side
of the MePD: fewer but more complex astrocytes on the left versus more but simpler
astrocytes on the right. Understanding the significance of this dramatic laterality is a
challenge. The left MePD contains more neurons than the right (Morris et al. 2008) and
prior to puberty contains more dendritic endings and more excitatory synapses than the
right MePD (Cooke et al. 2007; Cooke and Wooley 2005). Given that astrocytes
ensheath neurons and synapses, the increased number of neurons and synapses on
the left may explain why astrocytic processes are more extensive on this side. On the
other hand, astrocytes in the right MePD are more numerous but significantly less
complex. The simpler morphology of right MePD astrocytes is reminiscent of less
mature glia (Sancho-Tello et al. 1995), suggesting that there may be a higher turnover
of astrocytes in the right MePD than the left. This would lead to a greater proportion of
young astrocytes with less complex arbors. Because adult treatment with testosterone

53

can increase the number of apparent glia (based on counts in Nissl) in the right but not
the left MePD of adult females (Morris et al., 2008), androgens may influence ongoing
turnover of astrocytes in the right MePD by regulating their genesis and/or survival.
Indeed, finding more newly generated glia in the medial amygdala of male than female
rats supports this view (Ahmed et al., 2008). In contrast, the left MePD may contain a
more stable, and hence older and more complex, population of astrocytes. Combining
cell birth and/or cell death markers with astrocytic markers such as GFAP could help to
address this possibility in future experiments.
How this laterality in astrocyte morphology relates to behavior will be more
challenging to understand. Unfortunately, reports describing the functional
consequences of unilateral medial amygdala lesions in rodent models are rare. The few
reports available suggest that the right medial amygdala may exert a stronger influence
on the hypothalamic-pituitary-gonadal axis than the left in both sexes (Sanchez and
Dominguez 1995; Gerendai et al. 1995). It is also possible that hemispheric and sex
differences exist in the communication between the MePD and other brain regions.
Reports of functional laterality in the amygdala of humans are more common and
offer a consistent portrait (Canli et al. 2002; Hamann et al. 2004; Cahill et al. 2004;
LaBar et al. 1998). For example, Cahill et al. (2001) found that in males, activity of the
right amygdala, but not the left, was related to recall of emotional memory. In females,
activity of the left amygdala, but not the right, was related to such processing.
Furthermore, by examining patients in a resting state using fMRI, Kilpatrick et al. (2006)
found that the right amygdala exhibited a more widespread distribution of functional
connectivity at rest in men than in women, and that the left amygdala exhibited a more

54

widespread distribution of functional connectivity at rest in women than in men.
Interestingly, the connectivity pattern was also different for males and females.
Kirkpatrick and colleagues (2006) concluded that during resting states there are
dramatic differences in the functional networks formed by the amygdala in men and
women, and that there is evidence for a female-left, male-right pattern of lateralization.
Perhaps, as is suggested in the human literature, sex differences and
hemisphere differences in the rodent MePD reflect differences in functional connectivity
with other regions. It is clear that several behaviors are influenced by the medial
amygdala and it is likely that astrocytes in this region are involved in restructuring of
neural connections in response to gonadal hormones. Recent evidence suggests that
astrocytes play a critical role in regulating neuronal responses and cerebral blood flow
(Schummers, Yu and Sur, 2008), raising the possibility that sex differences in
asymmetrical activity of the human amygdala may be related to sex differences in the
asymmetry of astrocyte number or morphology. However, the importance of greater
astrocyte numbers in the right hemisphere and greater astrocyte complexity in the left
hemisphere for brain function and behavior requires further study.
Astrocytes and MePD Anatomy
Dissociations between neuron number and sexually dimorphic regional volume
are not common in the literature. Because the MePD is one such rare example (Cooke
et al. 2003; Morris et al. 2008), I expected that astrocytes might be a large determinant
of regional volume. Although significant correlations existed between regional volume
and astrocyte number in the right hemisphere, astrocyte density (regional
volume/astrocyte number) was not equivalent across sexes or hemispheres (data not

55

shown), which one would expect if astrocyte number and regional volume were closely
linked. In fact, significant differences were found in astrocyte density, with females
having greater density compared to males, a result is consistent with a previous reports
of se differences in GFAP immunoreactivity in the MePD based on optical density
(Rasia-Filho et al. 2002). Accordingly, in our multiple regression analysis examining
which measures significantly contribute to right regional volume, total astrocyte number
emerged as a significant positive predictor (p < 0.05).
In the left hemisphere, astrocytic primary branches, branch endings, and branch
length all demonstrated significant positive correlations with left hemisphere volume.
Interestingly, left hemisphere volume also significantly correlated with average astrocyte
nuclear size. Thus, right MePD volume appears loosely linked to astrocyte numbers
and left MePD volume may be linked to overall astrocyte complexity and average
astrocyte nuclear size. Future experiments, which manipulate androgens and measure
astrocyte morphology, may clarify which components change with androgen-induced
changes in regional volume and neuronal soma size.
Since astrocytes appear to regulate the formation and stabilization of dendritic
spines (Ullian et al. 2001; Nishida and Okabe 2007), one might expect a relationship
between astrocytes and dendritic complexity in the MePD. In prepubescent rats, the
volume occupied by glia and the number of dendritic branch endings are greater in
males than in females in the left hemisphere (Cooke et al. 2007). In adults, males have
greater dendritic spine density than females in the MePD, but hemispheric information is
not available (Rasia-Filho et al. 2004). Depending on which hemisphere is considered,
adult sex differences in spine density would depict two very different pictures of

56

astrocyte-neuron interactions. Increased branch complexity in astrocytes has been
associated with an increase in functional synapses in other brain regions (Pfrieger and
Barres 1997; Elmariah et al. 2005) but has also been associated with decreases in
dendritic spines (Mong et al. 2001). Currently, it is not clear how various aspects of
astrocyte morphology (number versus complexity) relate to neurons, or whether this
relationship varies in a region-specific manner.
However, it is also possible that the male bias in astrocyte number in the right
MePD is related to synaptic connectivity. MePD volume occupied by glia has been
reported to be sexually monomorphic in the right hemisphere prior to puberty in mice
(Cooke et al. 2007), suggesting that sex differences in glial number emerge during
puberty. Given that neuron-astrocyte communication is important in normal pubertal
development in the hypothalamus (Dziedzic et al. 2003; Prevot et al. 2003), perhaps
part of the rewiring of the MePD during puberty is accomplished through gliogenesis,
with glia leading the way in establishing sex differences in connectivity, similar to the
role glia may play in establishing sex differences in neuron number in the bird song
system (Nordeen and Nordeen 1996). These new astrocytes may aid in the formation
and maintenance of crucial neuronal connections that ultimately contribute to the
expression of sex-specific behaviors. Future studies examining the interaction between
MePD astrocytes and local dendrites in each hemisphere are needed to understand the
role of astrocytes in modulating MePD structure and function.
Influence of AR
I found striking sex differences in astrocyte morphology in the MePD, with adult
wildtype males having more astrocytes than females on the right and more complex

57

astrocytes than females on the left. Moreover, MePD astrocytes in Tfm males were like
those in females and not like those in wildtype males. The number of astrocytes in the
right MePD of Tfm males was equivalent to that of females and significantly less than
that of males. All measures of astrocyte complexity except the number of primary
branches were also reduced in Tfm males compared to wildtype males. Also based on
the classification method, Tfm males exhibited a distribution of astrocytes complexities
that matched that of females, but differed from males (Table 1). Finally, Tfm males did
not significantly differ from females for any astrocyte measure, in either hemisphere.
These results clearly indicate that the sex differences in the number of astrocytes in the
right MePD and astrocyte complexity in the left MePD of adult rats are dependent upon
functional AR. ARs may act to promote the extension and elaboration of branches of
already established astrocytes on the left side, whereas ARs may promote gliogenesis
on the right. Either of these actions may be due to organizational effects of androgens
during development or activational effects during adulthood. How ARs exert these
different actions on the two sides is not clear but perhaps lateralization of expression of
ARs or critical cofactors are involved.
AR may influence astrocyte morphology through direct or indirect pathways. If
AR is found in MePD astrocytes, then androgens could act directly on these cells to
produce changes. Testosterone treatment increases the number of astrocytes in the
female rodent hippocampus (Conejo et al. 2003), and cultured astrocytes taken from the
optic nerve, which contain AR, respond to androgens with increases in GFAP and/or
proliferation (Aqapova et al. 2006). Furthermore, in cultured cortical astrocytes,
activation of membrane bound AR leads to proliferation and an increase in GFAP

58

(Gatson and Singh 2007) further suggesting that direct androgen-induced modulation of
astrocytes might occur in the MePD. However, the majority of AR containing cells in the
adult MePD are neurons (Greco et al. 1998; Lorenz et al. 2005), despite reports of
astrocytic AR in other brain regions (DonCarlos et al. 2005).
Alternatively, androgens may be acting on neuronal AR in the MePD or
elsewhere to affect MePD astrocytes indirectly. Androgens may stimulate neurons to
communicate with astrocytes via a chemical messenger or may induce morphological
changes in neurons, which astrocytes respond to. In the arcuate nucleus, evidence
suggests that estrogens induce GABA synthesis and release in local neurons, which
then act on GABA receptors in surrounding astrocytes to induce growth of astrocyte
processes (Mong et al. 2002). A similar, but AR-dependent, mechanism may occur in
the MePD. Because AR protein is defective throughout life in Tfm males, our data do
not address the stage at which AR promotes masculinization of MePD astrocytes. Nor
do they address whether adult androgen manipulations affect astrocyte morphology in
the MePD of either hemisphere. Such information will add greatly to our understanding
of how gonadal hormones influence the MePD to regulate behavior.

59

APPENDIX

60

1

Table 1 - Percent of Astrocytes Within Each of the Four Categories of Complexity , by
Genotype and Hemisphere.
Left Hemisphere
Astrocyte 1
Group
Male
Tfm males
Female
1

2
0.0
5.0
1.6

3
3.3
13.3
11.7

Right
Hemisphere
1
2

4
23.3
31.6
36.7

73.3
50.0
50.0

23.3
8.3
15.0

3
20.0
21.6
18.3

4
25.0
36.6
28.3

31.6
33.3
38.3

Group 1 astrocytes are the least complex; group 4 are the most complex.

61

A

B

C

D

Figure 1 - For interpretation of the references to color in this and all other figures, the
reader is referred to the electronic version of this dissertation. Photomicrographs
depicting astrocytes of varying complexity. Panel A depicts a Group 1 astrocyte, which
has few and short processes. Panel B depicts a Group 2 astrocyte, which has several
processes but branches remain unelaborated. Panel C depicts a Group 3 astrocyte,
which has multiple elaborated processes and Panel D depicts a Group 4 astrocyte,
which is highly elaborated. Because of the single plane of focus depicted, only a portion
of the full extent of arbor complexity is visible here. Complex astrocytes frequently
demonstrated multiple branches that emerged as the plane of focus was moved through
the tissue. Scale bar = 50 microns

62

Figure 2 - Estimated regional volume (A) and total astrocyte number (B) in the
posterodorsal medial amygdala (MePD) of adult wildtype male and female rats and
testicular feminized mutant (Tfm) male rats, which lack functional AR. The MePD
volume is significantly larger on the right than the left in all three groups, but this
laterality was most dramatic in males. The right hemisphere is also significantly larger
in males than in females and Tfm males. The right MePD contained significantly more
astrocytes than the left in all three groups (B). This laterality was most prominent in
males, leading to a sex difference in astrocyte number only on the right side. Note that
Tfm males have fewer astrocytes than wildtype males but no more than do females in
the right MePD, indicating that the sex difference in astrocyte number depends on AR.
In contrast, the left hemisphere showed no effect of genotype. Values are means of N =
8 10 rats/group (+ standard error of the mean, SEM).
63

Figure 3

64

Figure 3 - Estimated number of primary branches (A), branch points (B), branch endings
(C), and average branch length (D) in the MePD of adult wildtype male, Tfm male and
female rats. In all three groups, astrocytes in the left MePD had more primary
branches, more branch points, more branch endings and a larger average branch
length than those on the right, indicating that astrocytes in the left MePD have more
complex morphologies than astrocytes in the right. Moreover, there was a significant
sex difference in astrocyte complexity in the left MePD, with all measures indicating that
males have more complex astrocytes than females on the left side. Tfm males are not
significantly different from females for any measure of astrocyte complexity. Data from
Tfm males demonstrates that masculinization of astrocyte complexity in the left MePD
depends on AR. Values are means of N = 5 rats/ group (+ SEM).

65

REFERENCES

66

REFERENCES
Adolphs, R., Gosselin, F., Buchanan, T. W., Tranel, D., Schyns, P., Damasio, A. R.
(2005). A Mechanism for impaired fear recognition after amygdala damage.
Nature, 6;433(7021):68-72.
Agapova, O. A.,Malone, P. E., Hernandez, M. R. (2006). A neuroactive steroid 5alphaandrostane-3alpha,17beta-diol regulates androgen receptor level in astrocytes. J
Neurochem. 98(2):355-63.
Ahmed, E. I., Shcultz, K. M., Zehr, J. L., Lorenz, B. H., DonCarlos, L. L., Sisk, C. L.
(2008). Pubertal hormones modulate the addition of new cells to sexually
dimorphic brain regions. Nature Neuroscience 11:995-997.
Alheid, G. F., J. S. De Olmos, C. A. Beltramino. (1995). Amygdala and extended
amygdala. In The Rat Nervous System. G. Paxinos Ed. :495-578. Academic
Press. Orlando, FL.
Amateau, S. K. & McCarthy, M. M. (2002a). A novel mechanism of dendritic spine
plasticity involving estradiol induction of prostaglandin-E2. Journal of
Neuroscience, 22:8586-8596.
Amateau, S. K. & McCarthy, M. M. (2002b). Sexual differentiation of astrocyte
morphology in the developing rat preoptic area. Journal of Neuroendocrinology,
14:904-910.
American Psychological Association. (2000). Diagnostic and statistical manual of mental
disorders: DSM-IV-TR.
Avola, R., Spina-Purrello, V., Gallo, F., Morale, M. C., Marletta, N., Costa, A., Tirolo, C.,
Testa, N., Reale, S., Marchetti, B. (2000). Immortalized hypothalamic luteinizing
hormone-releasing hormone (LHRH) neurons induce a functional switch in the
growth factor responsiveness of astroglia: involvement of basic fibroblast growth
factor. Int J Dev Neurosci.18(8):743-63.
Bishop, S.J., Duncan, J. and Lawrence, A.D. (2004). State anxiety modulation of the
amygdala response to unattended threat-related stimuli. J. Neurosci,. 24:10364
10368
Cahill, L., Haier, R. J., White, N. S., Fallon, J., Kilpatrick, L., Lawrence, C., Potkin, S. G.,
Alkire, M. T. (2001). Sex-related difference in amygdala activity during
emotionally influenced memory storage. Neurobiol Learn Mem. 75(1):1-9.
Cahill, L., Uncapher, M., Kilpatrick, L., Alkire, M. T. and Turner, J. (2004). Sex-related
hemispheric lateralization of amygdala function in emotionally influenced
memory:an fMRI investigation. Learn. Mem. 11, 261 266.
67

Canli, T., Desmond, J. E., Zhao, Z., Gabrieli, J. D. (2002). Sex differences in the neural
basis of emotional memories. Proc Natl Acad Sci U S A 99:10789-10794.
Canteras, N. S., Simerly, R. B., Swanson, L. W. (1992). Connections of the posterior
nucleus of the amygdala. J Comp Neurol. 8;324(2):143-79.
Canteras, N. S., Simerly, R. B., Swanson, L. W. (1995). Organization of projections from
the medial nucleus of the amygdala - a Phal study in the rat. J Comp Neurol 360:
213-245.
Conejo, N. M., González-Pardo, H., Pedraza, C., Navarro, F. F., Vallejo, G., Arias, J. L.
(2003).Evidence for sexual difference in astrocytes of adult rat hippocampus.
Neurosci Lett. 20;339(2):119-22.
Cooke, B. M., Woolley, C. S. (2005). Sexually dimorphic synaptic organization of the
medial amygdala. J Neurosci. 16;25(46):10759-67.
Cooke, B. M. (2006). "Steroid-dependent plasticity in the medial amygdala."
Neuroscience, 138(3):997-1005.
Cooke, B. M., Breedlove, S. M., Jordan, C. L. (2003). Both estrogen receptors and
androgen receptors contribute to testosterone-induced changes in the
morphology of the medial amygdala and sexual arousal in male rats. Horm
Behav., 43(2):336-46.
Cooke, B. M., G. Tabibnia, Breedlove, S.M. (1999). "A brain sexual dimorphism
controlled by adult circulating androgens." Proc Natl Acad Sci U S A,
96(13):7538-40.
Cooke, B. M., Stokas, M. R., Woolley, C. S. (2007). Morphological sex differences and
laterality in the prepubertal medial amygdala. J Comp Neurol. 20;501(6):904-15.
Coolen LM, Wood RI. (1998). Bidirectional connections of the medial amygdaloid
nucleus in the Syrian hamster brain: simultaneous anterograde and retrograde
tract tracing. J Comp Neurol. 399(2):189-209.
Cunningham RL, Claiborne BJ, McGinnis MY. (2007). Pubertal exposure to anabolic
androgenic steroids increases spine densities on neurons in the limbic system of
male rats. Neuroscience. 12;150(3):609-15.
Dani, J. W., Chernjavsky, A., Smith, S. J. (1992). Neuronal activity triggers calcium
waves in hippocampal astrocyte networks. Neuron. 8(3):429-40.
de Olmos, J., Haryd, H., Heimer, L. (1987). the aferent connections of the main and
accessory olfactory bulb formations in the rat: an experimental HRP-study. J

68

Comp Neurol. 181:213-244.
DeVoogd, T., Nottebohm, F. (1981). Gonadal hormones induce dendritic growth in the
adult avian brain. Science. 214(4517):202-4.
Dhandapani, K. M., Mahesh, V. B., Brann, D. W. (2003). Astrocytes and brain function:
implications for reproduction. Exp Biol Med. 228(3):253-60. Review.
DonCarlos, L. L., Sarkey, S., Lorenz, B., Azcoitia, I., Garcia-Ovejero, D., Huppenbauer,
C., & Garcia-Segura, L. M. (2005). Novel cellular phenotypes and subcellular
sites for androgen in the forebrain. Neuroscience, 138:801-807.
Dziedzic, B., Prevot, V., Lomniczi, A., Jung, H., Cornea, A., Ojeda, S. R. (2003).
Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates
ErbB receptor function in astroglial cells of the neuroendocrine brain. J Neurosci.
23(3):915-26.
Elmariah,S.B., Oh, E. J., Hughes, E. G. and Balice-Gordon, R. J. (2005). Astrocytes
regulate inhibitory synapse formation via Trk-mediated modulation of
postsynaptic GABAA receptors, J Neurosci 25: 3638 3650.
Fraser, D. D., Mudrick-Donnon, L. A., MacVicar, B. A. (1994). Astrocytic GABA
receptors.Glia. 11(2):83-93. Review.
Garcia-Segura, L. M., Chowen, J. A., Duenas, M., Torres-Aleman, I. & Naftolin, F.
(1994). Gonadal steroids as promoters of neuro-glial plasticity.
Psychoneuroendocrinology,19:445-453.
Garcia-Segura, L. M. & McCarthy, M. M. (2006). Minireview: Role of Glia in
Neuroendocrine Function. Endocrinology, 145:1082-1086.
Gatson, J. W., Singh, M. (2007). Activation of a membrane-associated androgen
receptor promotes cell death in primary cortical astrocytes. Endocrinology.
148(5):2458-64.
Gerendai, I., Csaba, Z., Vokó, Z., Csernus, V. (1995). Involvement of a direct neural
mechanism in the control of gonadal functions. J Steroid Biochem Mol Biol. 53(16):299-305.
Gréco, B., Edwards, D. A., Michael, R. P., Clancy, A. N.(1998). Androgen receptors and
estrogen receptors are colocalized in male rat hypothalamic and limbic neurons
that express Fos immunoreactivity induced by mating. Neuroendocrinology,
67(1):18-28.
Grove, E. A. (1988a). Neural associations of the substantia innominata in the rat:
afferent connections. J. Comp. Neurol. 277:315-346.

69

Grove, E. A. (1988b). Efferent connections of the substantia innominata in the rat. J.
Comp Neurol. 277:347-364.
Hamann S, Herman RA, Nolan CL, Wallen K.(2004). Men and women differ in
amygdala response to visual sexual stimuli. Nat Neurosci 7: 411-416.
Hatton, G. I. (2002). Glial-neuronal interactions in the mammalian brain. Adv Physiol
Educ. 26(1-4):225-37. Review.
Hines, M., Allen, L. S. et al. (1992). "Sex differences in subregions of the medial nucleus
of the amygdala and the bed nucleus of the stria terminalis of the rat." Brain Res,
579(2):321-6.
Kilpatrick, L. A., Zald, D. H., Pardo, J. V., Cahill, L. F. (2006). Sex-related differences in
amygdala functional connectivity during resting conditions.
Neuroimage.30(2):452-61.
Kondo, Y., Sachs, B. D., Sakuma, Y. (1998). Importance of the medial amygdala in rat
penile erection evoked by remote stimuli from estrous females. Behav Brain Res.
91(1-2):215-22.
Kondo, Y., Sachs, B. D. (2002). Disparate effects of small medial amygdala lesions on
noncontact erection, copulation, and partner preference. Physiol Behav. 76(45):443-7.
LaBar, K. S., Gatenby, J. C., Gore, J. C., LeDoux, J. E., Phelps, E. A. (1998). Human
amygdala activation during conditioned fear acquisition and extinction: a mixedtrial fMRI study. Neuron. 20(5):937-45.
LeDoux, J. E., Iwata, J., Cicchetti, P., Reis, D. J. (1998). Different projections of the
central amygdaloid nucleus mediate autonomic and behavioral correlates of
conditioned fear.J Neurosci., 8(7):2517-29.
Lorenz, B., Garcia-Segura, L. M., & DonCarlos, L. L. (2005). Cellular phenotype pf
androgen receptor-immunoreactive nuclei in the developing and adult rat brain.
Journal of Comparative Neurology, 492:456-468.
Mah, L., Zarate, C. A. Jr, Singh, J., Duan, Y. F., Luckenbaugh, D. A., Manji, H. K.,
Drevets, W. C. (2007). Regional cerebral glucose metabolic abnormalities in
bipolar II depression. Biol Psychiatry, 15;61(6):765-75.
Martinez, F. G., Hermel E. E. S., Xavier, L. L., Viola, G. G., Riboldi, J., Rasia-Filho, A.
A., and Achaval, M. (2006). Gonadal hormone regulation of glial fibrillary acidic
protein immunoreactivity in the medial amygdala subnuclei across the estrous
cycle and in castrated and treated female rats. Brain Research, 1108:117-126.

70

Meaney, M. J. (1989). The sexual differentiation of social play. Psychiatr Dev. 7(3):24761.
Melcangi, R. C., Ballabio, M., Magnaghi, V., Celotti, F. (1995). Metabolism of steroids in
pure cultures of neurons and glial cells: role of intracellular signalling. J Steroid
Biochem Mol Biol.53(1-6):331-6. Review.
Mong, J. A., Glaser, E., McCarthy, M. M. (1999). Gonadal steroids promote glial
differentiation and alter neuronal morphology in the developing hypothalamus in
a regionally specific manner. Journal of Neuroscience, 19:1464-1472.
Mong, J. A. & McCarthy, M. M. (1999). Steroid-induced developmental plasticity in
hypothalamic astrocytes: implications for synaptic patterning. Journal of
Neurobiology, 40: 602-619.
Mong, J. A., McCarthy, M. M. (2002). Ontogeny of sexually dimorphic astrocytes in the
neonatal rat arcuate. Brain Res Dev Brain Res., 15;139(2):151-8.
Mong, J. A., Roberts, R. C., Kelly, J. J., McCarthy, M. M. (2001). Gonadal steroids
reduce the density of axospinous synapses in the developing rat arcuate
nucleus: an electron microscopy analysis. J Comp Neurol 432:259-267.
Morris, J. A., Jordan, C. L., Dugger, B. N., Breedlove, S. M. (2005). Partial
demasculinization of several brain regions in adult male (XY) rats with a
dysfunctional androgen receptor gene. Journal of Comparative Neurology,
487:217-226.
Morris, J. A., Jordan, C. L., Breedlove, S. M. (2008). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in rats. Journal of Comparative Neurology, in
press.
Nishida, H., Okabe, S. (2007). Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci., 10;27(2):331-40.
Nordeen, E. J., Nordeen, K. W. (1996). Sex difference among nonneuronal cells
precedes sexually dimorphic neuron growth and survival in an avian song control
nucleus. J Neurobiol. 30(4):531-42.
Paxinos G, Watson W. (2005). The rat brain in stereotaxic coordinates. Amsterdam:
Elsevier.
Pfrieger, F. W., Barres, B. A. (1997). Synaptic efficacy enhanced by glial cells in vitro.
Science. 277(5332):1684-7.

71

Prevot, V., Rio, C., Cho, G. J., Lomniczi, A., Heger, S., Neville, C. M., Rosenthal, N. A.,
Ojeda, S. R., Corfas, G. (2003).Normal female sexual development requires
neuregulin-erbB receptor signaling in hypothalamic astrocytes. J Neurosci.
23(1):230-9.
Rao, S. P., Sikdar, S. K. (2007). Acute treatment with 17beta-estradiol attenuates
astrocyte-astrocyte and astrocyte-neuron communication. Glia.55(16):1680-9.
Rasia-Filho, A. A., Xavier, L. L., dos Santos, P., Gehlen, G., Achaval, M. (2002). Glial
fibrillary protein immunodetection and immunoreactivity in the anterior and
posterior medial amygdala of male and female rats. Brain Res Bull. 58(1): 6775.
Rasia-Filho, A. A., Fabian, C., Rigoti, K. M., Achaval, M. (2004). Influence of sex,
estrous cycle and motherhood on dendritic spine density in the rat medial
amygdala revealed by the Golgi method. Neuroscience.,126(4):839-47.
Romeo, R. D., Diedrich, S. L., Sisk, C. L. (2000). Effects of gonadal steroids during
pubertal development on androgen and estrogen receptor-alpha
immunoreactivity in the hypothalamus and amygdala. J Neurobiol. 44(3):361-8.
Roselli, C. E., Salisbury, R. L., Resko, J. A. (1987). Genetic evidence for
androgen-dependent and independent control of aromatase activity in the
rat brain. Endocrinology. 121(6):2205-10.
Sanchez, M. A., Dominguez, R. (1995). Differential effects of unilateral lesions in the
medial amygdala on spontaneous and induced ovulation. Brain Res Bull.
38(4):313-7.
Sancho-Tello, M., Valles, S., Montoliu, C., Renau-Piqueras, J., Guerri, C. (1995).
Developmental pattern of GFAP and vimentin expression in the rat brain and
radial glial cultures. Glia 15(2):157-166.
Schumann, C. M., Hamstra, J., Goodlin-Jones, B. L., Lotspeich, L. J., Kwon, H.,
Buonocore, M. H., Lammers, C. R., Reiss, A. L., Amaral, D. G. (2004).
The amygdala is enlarged in children but not adolescents with autism; the
hippocampus is enlarged at all ages. J Neurosci. 24(28):6392-401.
Shammah-Lagnado, S. J., Beltramino, C. A., McDonald, A. J., Miselis, R. R., Yang, M.,
de Olmos, J., Heimer, L., Alheid, G. F. (2000). Supracapsular bed nucleus of the
stria terminalis contains central and medial extended amygdala elements:
evidence from anterograde and retrograde tracing experiments in the rat. J Comp
Neurol. 422(4):533-55.
Simerly, R. B., Chang, C., Muramatsu, M., Swanson, L. W. (1990). Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ

72

hybridization study.J Comp Neurol., 1;294(1):76-95.
Sparks, B. F., Friedman, S. D., Shaw, D. W., Aylward, E. H., Echelard, D., Artru, A. A.,
Maravilla, K. R., Giedd, J. N., Munson, J., Dawson, G., Dager, S. R. (2002).
Brain structural abnormalities in young children with autism spectrum disorder.
Neurology. 59(2):184-92.
Spezio, M. L., Huang, P. Y., Castelli, F., Adolphs, R. (2007). Amygdala damage impairs
eye contact during conversations with real people. J Neurosci., 11;27(15):3994-7.
Schummers, J., Yu, H., Sur, M. (2008). Tuned Responses of Astrocytes and Their
Influence on Hemodynamic Signals in the Visual Cortex. Science 320:16281643.
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. (2001). Control of
synapse number by glia. Science. 291(5504):657-61.
West, M. J., Slomianka, L., Gundersen, H. J. (1991). Unbiased stereological estimation
of the total number of neurons in the subdivisions of the rat hippocampus using
the optical fractionator. Anat Rec 231: 482-497.
Williams, L. M., M. J. Barton, et al. (2005). "Distinct amygdala-autonomic arousal
profilesin response to fear signals in healthy males and females." Neuroimage,
28(3): 618-26.
Yarbrough, W. G., V. E. Quarmby, et al. (1990). "A single base mutation in the
androgen receptor gene causes androgen insensitivity in the testicular feminized
rat." J Biol Chem., 265(15):8893-900.
Zehr, J. L., Todd, B. J., Schulz, K. M., McCarthy, M. M., Sisk, C. L. (2006). Dendritic
pruning of the medial amygdala during pubertal development of the male Syrian
hamster. J Neurobiol. 66(6):578-90.
Zuloaga, D. G., Morris, J. A., Monks, D. A., Breedlove, S. M., Jordan, C. L. (2007).
Androgen-sensitivity of somata and dendrites of spinal nucleus of the
bulbocavernosus (SNB) motoneurons in male C57BL6J mice. Horm Behav.
51(2):207-12.

73

CHAPTER 2: ASTROCYTES IN THE RAT MEDIAL AMYGDALA ARE RESPONSIVE
TO ADULT ANDROGENS
Abstract
The posterodorsal medial amygdala (MePD) exhibits numerous sex differences,
including differences in volume, and in the number and morphology of neurons and
astroctyes. Gonadal hormones in adulthood have been shown to play a role in
determining some of these sex differences but whether adult circulating hormones affect
the morphology of MePD astrocytes is unknown. I examined astrocytes in the MePD of
male and female Long Evans rats that were gonadectomized and treated for 30 days
with either testosterone or control treatment. At the end of treatment, brains were
collected and immunostained for glial fibrillary acidic protein (GFAP). I also co-stained
for GFAP and the androgen receptor (AR) in normal adult, gonadally intact male rats to
determine whether MePD astrocytes express ARs. Stereological analysis revealed that
both the number and complexity of astrocytes in the MePD were influenced by adult
androgens although many of the previously reported sex and laterality differences
persisted, suggesting that androgens affect the morphology of MePD astrocytes both
before and during adulthood. Furthermore, using confocal microscopy, I found robust
AR expression in MePD astrocytes. This finding suggests testosterone may act directly
on MePD astrocytes to influence their structure and function and raises the possibility
that androgens work via astrocytes to influence MePD function.
Introduction
The amygdala, a highly conserved brain region, is primarily associated with two
realms of behaviors: fear/anxiety behaviors (Rogan et al. 1997; LaBar et al. 1998; Nagy

74

et al. 1979; Adamec 1990), and social/mating behaviors (Sanders and Shekhar 1995;
Adolphs et al. 2002; Adolphs and Tranel 2003; Faber-Zuschratter et al. 2009; Lehman
and Winans 1982; Baum and Everitt 1992; Kondo and Arai 1995). The amygdala as a
whole is also implicated in several diseases and disorders related to these behaviors,
including autism, depression and anxiety disorder. Interestingly, all of these diseases
and disorders exhibit sex biases in prevalence rates (American Psychological
Association 2000).
The posterodorsal division of the medial amygdala (MePD) shows marked adult
plasticity controlled by gonadal hormones (Morris et al. 2005; 2008a, b). For example,
the volume of the MePD and size of neuronal somata in this region is normally greater
in adult male than in adult female rats, but castration of adult males eliminates these
sex differences and causes deficits in social/sexual behaviors (Cooke et al. 1999;
2003). Treatment with androgens and/or estrogens prevents these morphological
responses to castration (Cooke et al. 2003), indicating that gonadal hormones play a
critical role in regulating adult MePD anatomy and function. While the influence of adult
androgens on MePD neurons has been demonstrated, there are few reports on the
influence of circulating androgens on other cellular components of the MePD such as
glia.
I previously reported sex differences in the number and complexity of astrocytes
in the adult rat MePD, which showed striking lateralization (Johnson et al. 2008).
Moreover, I found that the sex differences in astrocyte morphology depended on
androgen receptors (ARs), because astrocytes were entirely feminine in both number
and complexity in genetic (XY) male rats with a dysfunctional AR, due to the testicular

75

feminization mutation (Tfm) (Johnson et al. 2008). The highly plastic nature of
astrocytes in other systems (Garcia-Segura et al. 1989; Mong 1996; Emamian et al.
2010), together with the AR-dependent sexual dimorphism of MePD astrocytes
suggests that they may also exhibit androgen-dependent adult plasticity. To test this
hypothesis, I examined the number and morphology of MePD astrocytes in male and
female rats after adult castration and testosterone (T) replacement. I now report that
MePD astrocytes are indeed hormone-sensitive in adulthood, with both the number of
astrocytes and the complexity of the astrocytic arbor responding to adult hormone
manipulations. Intriguingly, these responses are highly lateralized, echoing
hemisphere-specific responses of MePD regional volume, and providing further
evidence that the two amygdalae respond differentially to circulating hormones.
Methods
Animals and Surgeries
Ninety-day-old male and female Long Evans rats were obtained from Charles
River (Charles River Laboratories, Wilmington, Massachusetts). Same-sex animals
were housed three per cage in a single colony room in standard rat cages with food and
water available ad libitum. Lights were on at 0700 and off at 1900 hrs. Animals were
cared for in accordance with the guidelines set forth by the National Institute of Health
and all procedures were approved by the Institutional Animal Care and Use Committee
at Michigan State University.
After 3 weeks of acclimation, animals were randomly assigned to treatment groups
with N = 9 animals/group. Under isoflurane anesthesia, male and female rats were
gonadectomized and implanted s.c. with two Silastic capsules (each 20 mm effective

76

release length and 30 mm in total length, inner diameter 0.062 inches, outer diameter
0.125 inches) containing either crystalline testosterone (T, Sigma) or nothing (blank).
All capsules were incubated in phosphate-buffered saline (pH 7.4) for 48 hrs at room
temperature (RT) prior to implantation. All surgeries were performed using aseptic
procedures, and animals were provided postoperative analgesia (0.51 g
Buprenorphine/g of body weight).
Thirty days later, animals (age 141-142 days) were given an overdose of sodium
pentobarbital (150mg/kg, ip). Once animals were deeply anesthetized, showing no
reflex response to either tail or foot pinch, they were intracardially perfused with 0.9%
saline, followed by 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4, ~250
mL/animal). Before perfusion, blood was collected via cardiac puncture to measure T
titers. After perfusion, hormone implants were confirmed and seminal vesicles in males
were removed, trimmed and weighed. Brains were removed, weighed and postfixed for
five hrs at 40 C in the same fixative solution before immersion in 30% phosphatebuffered sucrose at 40 C for at least 48 hrs. Brains were then sectioned coronally on a
freezing microtome at 40µm through the region of interest after the left cortex of each
brain was scored to mark that hemisphere. Sections were collected into cryoprotectant
(de Olmos et al., 1978) and stored at -200 C. Every third section through the
rostrocaudal extent of the MePD was processed for immunocytochemistry (ICC).
For colocalization of ARs within the MePD, a separate cohort of gonadally intact
100-110 day-old Long Evans male rats (N = 3) were obtained from our breeding colony
at MSU and housed as described above. Animals were sacrificed and tissue collected
and stored as described above.

77

Immunostaining for the Astrocytic Marker GFAP
Archived sections were transferred from cryoprotectant to Netwell plates (Corning
Life Sciences) and thoroughly rinsed in 0.1 M phospate buffered saline (PBS; 140 mM
NaCl, 10.7 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) containing 0.3% Triton X100 and 0.1% Knox gelatin (PBS-GT). PBS-GT was used throughout as the vehicle for
ICC reagents and for rinsing. Sections were first incubated for 15 min in 0.5% sodium
borohydride before blocking in 10% normal horse serum containing free avidin (Avidin
Biotin blocking kit, Vector Laboratories), rinsed and then incubated one hr at RT in
mouse anti-GFAP monoclonal antiserum (1:50,000, Chemicon, MAB360) containing
free biotin (Avidin/Biotin blocking kit, Vector Laboratories). Tissue was then incubated
overnight in GFAP primary antiserum at 4 0 C in PBS-GT containing. The GFAP primary
antiserum was prepared against purified glial fibrillary acidic protein from porcine spinal
cord. In western blots, the antiserum recognizes a single band at approximately 51 kDA

Following incubation in primary antiserum, tissue was incubated in biotinylated
rat-adsorbed horse anti-mouse secondary antiserum (1:500, Vector Laboratories),
followed by incubation in peroxidase avidin-biotin complex solution (Elite ABC kit,
Vector at half recommended concentration). HRP was visualized using
diaminobenzidine (DAB, Sigma) in a 0.05 M tris buffer (pH 7.2). After rinsing to quench
the peroxidase reaction, tissue was mounted on gel-subbed slides, dried, and counterstained using Harris hematoxylin solution (Sigma), using 10% lithium carbonate as the
bluing agent. Slides were then rinsed, dehydrated, cleared and coverslipped with
Permount.

78

Stereological Analysis
A Zeiss Axioplan II microscope with the visual field captured by an Optronics
MicroFire digital video camera was used to quantify astrocyte number and the complexity
of astrocyte arbors within the region of interest. Using StereoInvestigator software (v. 8.0,
MBF Bioscience; Williston, VT), the perimeter of the MePD was traced in serial sections
at low magnification. MePD boundaries were identified using a standard rat atlas
(Paxinos and Watson 2005), in conjunction with previously established standards within
our laboratory (Morris et al., 2005; 2008a) and others (Hines et al., 1992). After tracing
the boundaries of the MePD at low magnification, astrocytes were first counted and then
traced using a 100X Plan-NeoFluar, 1.3 N.A. oil-immersion objective as described below.

membership.
Astrocyte number: An optical fractionator probe was used to generate an unbiased
estimate of astrocyte numbers within the boundaries of the MePD. Initial probe insertion
is random within the region of interest but then proceeds at fixed intervals allowing for an
unbiased estimate of the number of objects within the entire region of interest. Probe
dimensions were 35 by 35 µm with a height of 11 µm. The coefficient of error (CE;
Gunderson m=1) for each hemisphere was at or below 0.10.
Criteria for identifying an astrocyte included a distinct and recognizable nucleus in a
plane of focus within the sampling probe and from which at least 2 GFAP-labeled fibers
extended. Such nuclei were often, although not always, smaller than other nearby
(presumably neuronal) nuclei, often elliptical in shape, and lacking a clear nucleolus.
These methods produced estimates of overall MePD volume and number of astrocytes

79

per hemisphere for each subject. The number of cells counted to produce these
estimates ranged from approximately 250-500 per hemisphere depending on the
hemisphere and the sex of the subject.
Astrocyte process complexity: Following previously described methods (Johnson
et al., 2008), I used Neurolucida software (v. 7.0, MBF Bioscience; Williston, VT) to
trace and measure the entire visible arbor of 20 randomly selected astrocytes per
subject (10 astrocytes per hemisphere) with 5 subjects per treatment group. A
fractionator probe was used to define random tracing sites throughout the MePD, with
10 sites per hemisphere. At each of these sites, the astrocyte nearest the randomly
placed marker was traced in its entirety using a Wacom Cintiq 12WX tracing display
(Wacom Co.; Saitama, Japan). The average number of primary processes, average
number of branch points, average number of branch endings, and average branch
length per astrocyte were calculated for each hemisphere of each animal.
Double ICC for AR and GFAP
Sections were stained first for AR followed by staining for GFAP using well
described methods (Xiao and Jordan, 2002; Johnson et al., 2008). Briefly,
cryoprotected sections were rinsed in PBS-GT, blocked in 10% normal goat serum
(Vector Laboratories) containing free streptavidin (Streptavidin/Biotin blocking kit, Vector
Laboratories), followed by 72 hrs of incubation at 40 C in rabbit anti-AR monoclonal
antiserum (1:2000; Epitomics #1852-1 directed against the C-terminus of the human
androgen receptor, Burlingame, CA) containing free biotin. Following incubation in AR
primary antiserum, tissue was incubated in biotinylated goat anti-rabbit secondary
antiserum (1:2000, Vector Laboratories) followed by Cy2-conjugated streptavidin

80

(1:48000, Jackson Immunoresearch, West Grove, PA). Following AR
immunofluorescence, GFAP was visualized incubating tissue overnight at 4 0 C in
primary antiserum (1:2000) using the same mouse anti-GFAP monoclonal as described
above, but blocking for nonspecific binding of streptavidin in this case
(Streptavidin/Biotin blocking kit; Vector Laboratories). Tissue was incubated for 2 hr in
biotinylated goat anti-mouse 2° antibody (1:500, Vector Laboratories) followed by
incubation in Cy5-conjugated streptavidin (1:32000). Tissue was thoroughly rinsed and
mounted in the dark on gel-subbed slides and allowed to dry in a dark environment.
Cell nuclei were counterstained with DAPI (1 µg DAPI / 1 ml distilled H 2O) for 5 mins,
before dehydrating, clearing in Citrisolv and cover slipping with DPX (Sigma-Aldrich, St.
Louis, MO), which we find preserves fluorescent signals for more than a year when
slides are stored at -20º.
AR Immunohistochemisty Controls
Multiple controls were run to test the specificity of the AR primary antibody.
First, adjacent sections from the same subjects were processed with the anti-AR
primary antibody omitted. Omission of the primary antibody eliminated immunostaining
(Fig 4). Furthermore, preadsorbing the AR primary antibody with the immunizing
peptide (Epitomics P-1852, diluted 1:80 or 1/25 less than the 1:2000 dilution used for
the AR primary) eliminated AR labeling in the MePD (Fig 5) and other AR-containing
brain regions of normal adult male rats.
Third, we have developed an AR-knock out (KO) mouse using the cre/lox
system. Along with confirming AR gene recombination based on PCR, we find that
such AR-KO male mice faithfully recapitulate the Tfm male phenotype, including the

81

expected feminized ano-genital distance and nipples (Breedlove, personal observation),
like spontaneous Tfm males (Dugger et al. 2007). This observation demonstrates the
efficacy of the genetic manipulation in disabling AR function using the cre/lox system.
Using the same AR staining protocol, I see no immunolabeling in the brains of such ARKO male mice (Fig 6).
Confocal Microscopy: AR/GFAP Colocalization
DAPI-labeled astrocyte nuclei within the MePD were examined for AR
immunoreactivity. Analysis was conducted on an Olympus Fluoview 1000 confocal
scanning laser microscope equipped with argon (458, 488 and 514 nm), HeNeG (543
nm), HeNeR (633 nm), and LD405 lasers. The MePD was identified and defined at low
magnification (10x objective) using well-established criteria (Paxinos and Watson 2005;
Morris et al. 2008a; Hines et al. 1992).
Twenty to thirty sites per animal were visited within the MePD using a 60X PlanApo, 1.42 N.A. oil-immersion objective. Sites were chosen semi-randomly throughout
the MePD. At each site a sequential scan with 2 µm spacing between scans and a
depth of at least 18 µm was run and saved as image stacks using Olympus Basic
software (v 5.0).
To estimate the number of AR expressing astrocytes in the MePD, all DAPIstained cell nuclei fulfilling the criteria for astrocytic nuclei (described above) in the
image stacks were examined and judged as immunopositive for AR if immunoreactivity
was found clustered in the nucleus a

-stack

orthogonal viewer was used to confirm colocalization of markers for a sub-sample of
nuclei. From this analysis, I obtained estimates of the proportion of astrocytes that are

82

AR+ in the MePD in each hemisphere. All confocal images were saved as 24-bit TIFF
files and opened in Adobe Photoshop 7.0 for production of figures. Images were

unaltered in Photoshop other than adding appropriate labels.
Hormone Assay
Plasma T concentrations were measured in duplicate from 50 µl samples using the
Coat-a-Count Total Testosterone Kit (Diagnostic Products Corp., Los Angeles, CA) by
the Diagnostic Center for Population and Animal Health at Michigan State University.
The lower limit for detection was 0.1 ng/ml, and the intra-assay coefficient of variation
was 9.0%.
Statistical Analysis
Treating left and right hemispheres as a repeated measure with sex (male, female)
and treatment (T, blank) as between group measures, separate three-way mixed-design
analyses of variance (ANOVA) were conducted for each dependent variable (MePD
volume, astrocyte number, number of primary processes, number of branch points,
number of branch endings, and branch length). When appropriate, these were followed
by two-way ANOVAs for each hemisphere and LSD post-hoc analysis to assess
differences between groups. For all analyses, results are expressed as mean SEM
(standard error of the mean), with N= the number of animals per group, and statistical
significance set at p

83

Results
Serum Testosterone Levels
As expected, serum T levels were significantly different between T-treated
(Means SEMS; 11.18 0.14 ng/ml) and blank-treated animals (0.00 0.00 ng/ml; p <
0.001). This result, in conjunction with a significant difference in seminal vesicle weight
between T-treated (1.62 0.14g) and blank-treated males (0.18 0.01g; p < 0.001),
confirms that the hormone manipulations were successful.
Regional Volume
Effects of Hemisphere and Sex: MePD volume overall is larger in males than in
females (males: 476.14 20.45 versus 353.03 21.34 mm3x10-3, p < 0.001) and is
greater in the right hemisphere than the left for both sexes (ps < 0.001, Fig 7A). I also
find main effects of sex within each hemisphere, with males having a greater MePD
volume than females for both (ps < 0.005, Fig 7C, D). These sex and hemisphere
differences in MePD regional volume replicate previous findings (Morris et al., 2005;
Johnson et al., 2008).
Effects of Testosterone Treatment: I also confirmed that T-treated rats have a
larger MePD volume than blank-treated animals (T-treated: 468.93 19.49, blanktreated: 360.24 24.33 in mm3x10-3, p < 0.001) and found a significant treatment by
hemisphere interaction (p < 0.05), leading us to analyze the effect of treatment on
MePD volume separately for each hemisphere. I found that T treatment significantly
affected MePD volume in both hemispheres (ps < 0.01; Fig 7B). Individual planned
comparisons revealed that T-treated females had greater volume compared to blanktreated females in the left hemisphere (p < 0.01, Fig 7C), but T did not affect left MePD
volume in males. However, in the right MePD, T treatment increased MePD volume in
84

both sexes (ps < 0.01, Fig 7D). The greater MePD volume in T-treated animals
conforms to prior results based on similar hormone manipulations (Morris et al., 2008a)
and confirms the robust adult plasticity of this sexually dimorphic region (Cooke et al.,
1999).
Astrocyte Numbers
Effects of Hemisphere and Sex: GFAP staining in the amygdala and
hypothalamus produced a pattern of GFAP immunoreactivity matching that reported
previously (Johnson et al., 2008; Mong et al., 1999; Martinez et al., 2006). I replicated
our previous finding that there are more MePD astrocytes overall in the right
hemisphere than the left (number of astrocytes in left hemisphere: 6379.06 174.60; in
the right hemisphere: 8799.17 288.10, p < 0.001). Post-hoc matched-pairs t-tests
confirmed that this asymmetry was present for both males and females (Fig 8A) and
indeed, was evident in all four treatment groups (all ps < 0.005). I again found more
astrocytes in the MePD of males than of females (main effect of sex p < 0.001, Fig 8A),
and replicated the significant interaction of hemisphere by sex (p < 0.001), reflecting a
larger sex difference in the right hemisphere (males: 9841.72 299.70, females:
7756.62 352.27) than the left (males: 6755.10 245.08, females: 6003.02 220.94).
However, the number of astrocytes was greater in males than in females in both
hemispheres (left: p < 0.05, right: p< 0.001, Fig 8C, D). These sex and hemisphere
differences in astrocyte numbers in hormone-manipulated animals essentially replicate
our previous report of sex differences in gonadally intact wild type rats (Johnson et al.,
2008).

85

Effects of Testosterone Treatment: I also found a main effect of adult T treatment
on astrocyte numbers, with T-treated animals having more MePD astrocytes than blanktreated animals (T-treated: 16038.35 530.67, blank-treated: 14318.12 636.86, p <
0.05). There was also a significant interaction between treatment and hemisphere (p <
0.05) leading us to examine the effects of hormone manipulation in the two
hemispheres separately. These analyses revealed that while hormone treatment did
not affect astrocyte numbers in the left MePD overall, there was a significant effect of T
on astrocyte numbers in the right MePD, with androgen-treated animals possessing
more astrocytes than blank-treated animals (p < 0.01, Fig 8B). Examining the sexes
separately revealed that T treatment did affect astrocyte numbers in the left MePD, but
only in females (p < 0.05, Fig 8C). Further post-hoc analysis revealed that the effect of
T treatment in the right MePD was significant in females (p < 0.05, Fig 8D) and
marginally significant in males (p = 0.056).
Astrocyte Complexity
Effects of Hemisphere and Sex: All measures of astrocyte complexity except
average branch length exhibited a main effect of sex, with males having more complex
astrocytes overall than females (all ps < 0.05). In addition, all measures of astrocyte
complexity except average number of primary branches were greater in the right
hemisphere than the left (main effect of hemisphere all ps < 0.05). There were no
significant interactions between hemisphere and sex, or hemisphere and treatment for
any measure of astrocyte complexity. That the MePD of males contains more complex
astrocytes than females replicates previous results (Johnson et al., 2008).

86

Effects of Treatment: Collapsing across the sexes, androgen treatment affected
all measures of astrocyte complexity (main effects of androgen treatment, all ps <
0.005) by increasing the complexity of astrocyte arbors compared to blank-treated
animals. However, all measures of astrocyte complexity except average branch length
showed significant sex by treatment interactions (all ps < 0.05) reflecting the fact that T
increased astrocyte complexity more in males than in females (Fig 9). Separate
analysis of the two sexes revealed that T-treated males had significantly greater primary
branches, branch endings, and branch points in both the right and left hemispheres,
than did blank-treated males (all ps < 0.05, Fig 9A-C). In contrast, T-treated females
differed from blank-treated females only for average branch length, and only in the right
hemisphere (p < 0.05), yet T had no effect on this measure in males (Fig 9D).
AR Colocalization
The medial amygdala is particularly rich in AR+ nuclei (Gréco et al., 1998;
McAbee and DonCarlos, 1998) but whether such nuclei belong to neurons or glia is
unknown. As expected, I observed numerous AR+ nuclei in the MePD and many were
large and round, with distinct nucleoli, presumably belonging to neurons. However, I
also found distinct AR immunoreactivity in the nuclei of astrocytes in the MePD (Fig
10A). An orthogonal view bisecting the Z stack along X and Y axes centered on an AR+
nucleus clearly reveals that the AR staining is within an astrocyte nucleus, since AR
immunoreactivity colocalizes with DAPI staining from which GFAP+ processes emanate
(Fig 10B). Our estimates suggest that approximately 50% of astrocytes in the MePD
are AR+ in both hemispheres (number of AR+ astrocytes in left hemisphere:
106.33 61.33 out of a total of 200 115.47 astrocytes counted; number of AR+ cells in

87

the right hemisphere: 183.66 106.04 out of a total of 356+205.54 astrocytes counted).
AR+ astrocytes were also seen in previously documented areas, such as the arcuate
nucleus (Lorenz et al., 2005, Fig 11A) and hippocampus (Hösli et al., 2001; Tabori et
al., 2005, Fig 11B).
Discussion
The MePD is sexually dimorphic and sensitive to adult gonadal hormones
(Cooke et al. 1999; 2003; Morris et al. 2005; 2008a). However, some sex differences in
MePD neuroanatomy (e.g., regional volume and neuronal soma size: Cooke et al. 1999;
2007) are maintained by circulating testicular hormones in adulthood while others (e.g.,
neuron number: Morris et al. 2008a) are not. I recently demonstrated that MePD
astrocytes are also sexually dimorphic in both number and complexity and that these
sex differences depend on functional AR (Johnson et al. 2008). However, when these
sex differences in astrocyte morphology in the MePD are determined by androgens is
not known. I now report that astrocytes are sensitive to adult hormone levels in the
MePD and change in both number and arbor complexity in response to hormones.
I replicated the sex and hemisphere differences in astrocyte numbers found
previously in gonadally intact rats (Johnson et al. 2008). Moreover I found that
androgens in adulthood affect the number of MePD astrocytes, although the effect is
most pronounced in the right MePD of females. This result concurs with our prior report
based on Nissl staining that adult hormone manipulations influence the number of total
glia in the MePD only in females and only in the right hemisphere (Morris et al. 2008a).
I also found that astrocyte complexity was regulated by adult androgens, although in
this case, the effects of androgens are seen mainly in males and are symmetrical,

88

affecting most measures of astrocyte complexity in both hemispheres, as opposed to
the asymmetric and rather subtle effect of androgens on astrocyte complexity in
females. Specifically, all but one measure (branch length) of astrocyte complexity was
increased by androgens in males in both hemispheres, while androgens increased only
branch length in females and only in the right hemisphere. This measure of astrocyte
complexity, which was the only one affected by T in females, was unaffected by T in
males. Thus, in females, astrocytes in the right MePD appear most sensitive to adult
androgens, changing in both number and arbor complexity while in males, MePD
astrocytes on both sides of the brain depend on androgens in adulthood to maintain
their complexity but not number. Finally, I also found that about half of MePD
astrocytes in gonadally intact adult males are immunopositive for ARs, suggesting that
androgens may act directly upon MePD astrocytes to influence their morphology and
number.
Astrocytes and Gonadal Hormone Influence
An expanding body of research indicates that astrocytes are not the passive cells
once imagined and has linked these cells to a wide range of phenomena including rapid
and coordinate responses in the visual system (Schummers et al. 2008) and social
exploratory behavior (Lee et al., 2007). In addition, astrocytes are known to play a vital
role in synapse formation (Ullian et al. 2001; Hatton 2002; Barker and Ullian 2008).
With large, highly mobile arbors, a single astrocyte may contact approximately 300-600
dendrites and 1,000,000 synapses in the cortex (Halassa et al. 2007; Nishida and
Okabe 2007). In both the hippocampus (Garcia-Segura et al. 1988; Day et al. 1990;
1993; Leranth et al. 2008) and hypothalamus (Chowen et al. 1995; Mong 1996;

89

Amateau and McCarthy 2002; McQueen et al. 1990) astrocytes are sexually dimorphic
and responsive to gonadal hormone manipulations. Changes in astrocytes in both
regions are associated with changes in the surrounding neurons and/or behavior
(Emamian et al. 2010; Mong and McCarthy 1999; Micevych et al. 2010). Our results
further illustrate the dynamic nature of these cells and add to a growing list of brain
regions where hormones can induce changes in astrocytes.
In the amygdala, investigation of astrocytes has been more limited, but hormones
have been found to regulate GFAP expression in the region (Legrand and Alonso 1998;
Martinez et al. 2006). Our previous work showed sex differences in both the number
and complexity of astrocytes in the MePD portion of the amygdala, which are ARdependent (Johnson et al. 2008). The current data also demonstrate these sex
differences in astrocyte morphology and suggest that circulating gonadal hormones in
adulthood have a role in maintaining these sex differences.
As far as I am aware, changes in astrocyte numbers in the adult amygdala that
may be precipitated by hormones have only been reported once previously.
Featherstone and colleagues (Featherstone et al. 2000) found reduced numbers of
GFAP+ cells in the medial amygdala of pup-exposed multiparous postpartum females
compared to pup-exposed primiparous females. The authors suggest this difference
may be due to greater estrogen exposure in the multiparous dams, a conclusion in line
with the greater numbers of astrocytes I see in T-treated females. More recent
evidence showing that GFAP-immunoreactivity in the MePD is greatest during the
proestrus phase and is increased by estradiol or estradiol plus progesterone in
ovariectomized females, is also consonant with our current findings (Martinez et

90

al.2006). However, optical density measures of GFAP-immunoreactivity cannot
distinguish whether greater astrocyte numbers, greater arbor complexity or both,
contribute to differences.
Our current data are the first to demonstrate that MePD astrocytes in rats
respond to adult androgens. The most robust responses to T treatment in males are in
the complexity of the astrocyte arbor. Given the proposed link between number of
astrocytic processes and synapse density (Pfrieger and Barres 1997; Elmariah et al.
2005; Mong et al. 2001), our results suggest that neuronal connectivity in the amygdala
may be changing in response to circulating hormones in adult males. Interestingly,
astrocytic complexity has been associated with both increases in synapse number
(Pfrieger and Barres 1997; Elmariah et al. 2005) and decreases in dendritic spines and
axonal-dendritic synapses (Mong et al. 2001) making it unclear whether the changes I
see in astrocytes would be associated with increases or decreases in synaptic
connectivity in the MePD. However, the increased size of neuronal soma induced by
adult androgens in the MePD (Cooke et al., 2003) suggests that such increases in
astrocyte number and complexity may be associated with increases in synaptic
connectivity.
Gonadal Hormone Receptors in Astrocytes
Astrocytes in several other brain regions have been found to express AR,
including the cortex, arcuate nucleus and hippocampus (Finley and Kritzer 1999; Lorenz
et al. 2005; Sarkey et al. 2008; Nunez et al. 2003; Tabori et al. 2005). While the MePD
is particularly rich in hormone receptors, including AR (Shughrue and Merchenthaler
2001; Roselli 1991), astrocyte expression of AR has not been previously reported in this

91

region. Several aspects of MePD neuroanatomy are dependent upon circulating
androgens and/or functional ARs for complete masculinization, including astrocytes
(Cooke et al. 2003; Morris et al. 2005; Johnson et al. 2008). I now find that a large
fraction of MePD astrocytes express ARs, opening the possibility that ARs in the
astrocytes themselves mediate the effects of androgens on these cells and possibly
those on the neurons too.
Direct effects of gonadal hormones on astrocytes have been demonstrated in
vitro (Melcangi et al. 1996; Stone et al. 1998). Although the hippocampus is also rich in
hormone receptors, hippocampal astrocytes respond to gonadal hormones apparently
independent of either (classical) AR or ER activity (MacLusky and Hajszan 2004;
MacLusky et al. 2006). In the hypothalamus estrogens appear to act first on ER+
neurons that influence the surrounding astrocytes through GABA signaling (Mong et al.
2002). A similar scenario in which androgens act via neuronal AR to influence
astrocytes may occur in the MePD.
Gliogenesis and Cell Death in the Amygdala
While androgens in adulthood can affect the number of MePD astrocytes, it is not
clear what cellular mechanism androgens regulate to control their number. Hormones
affect both the proliferation and survival of astrocytes in several systems (Agapova et al.
2006; Gatson and Singh 2007). Moreover, in the rodent amygdala in particular,
evidence suggests that gonadal hormones can influence glial proliferation, at least

shows that cells proliferate in the rat medial amygdala during puberty, with more cells
added in males than in females, of which many of these newly generated cells are glia.

92

Interestingly, gonadectomy prior to puberty reversed this sex difference, reducing the
number of newly generated cells in males to that of females (Ahmed et al. 2008). Thus,
androgens may increase the number of astrocytes in the right MePD of females by
increasing their proliferation. However, it is also possible that fewer astrocytes survive
without gonadal hormones. Both estrogens (Sales et al. 2010) and androgens (Nguyen
and Jayaraman 2010; Ahlbom et al. 2001) are capable of preventing cell death in the
brain and elsewhere (reviewed in Forger 2006).
Limitations and Implications
A chief limitation of this report is that the effects seen cannot be specifically
attributed to the actions of AR or estrogen receptors (ER) alone. Due to the ability of T
(an AR ligand) to be aromatized into estrogen (an ER ligand) or dihydrotestosterone (a
high affinity AR ligand), it is possible that the treatment effects are due to ER activity
alone or a combination or AR and ER activity. Both are implicated in regulating the
adult morphology of the MePD (Cooke et al. 2003). However, given that functional ARs
are necessary for masculinized MePD astrocytes (Johnson et al. 2008), AR likely
mediate some of the effects of adult androgens on MePD astrocytes. One intriguing
possibility is that masculinization of astrocyte number may depend on ARs in
development, given the stability of astrocyte number in adult, hormone-manipulated
males, while both ARs and ERs may regulate their morphology in adulthood.
Conclusions and Additional Questions
The present findings provide new information about the cellular anatomy of the
amygdala. The sexually dimorphic volume of the adult MePD is responsive to adult
hormone levels and it is now clear that astrocytes are another component of this

93

hormone-dependent adult plasticity. Future studies will be aimed toward understanding
the mechanisms through which gonadal hormones act on MePD astrocytes to control
their number and growth and/or regression of their processes and whether such effects
are mediated via estrogens or androgens. In addition, the lateralized influence of
gonadal hormones on astrocyte number and morphology seen here and in
unmanipulated animals (Johnson et al. 2008) is complex and intriguing. Lateralization
in the amygdala is common in humans (Kilpatrick et al. 2006) and rodents (Baker 2004).
Interestingly, much of the lateralization in amygdala anatomy is sexually dimorphic and
developmentally regulated, resulting in complex interactions between age, sex and
hemisphere (Ziegler and Lichtensteiger 1992).
Although many aspects of the amygdala are still poorly understood, it is
associated with numerous disorders and diseases and that many of these diseases,
such as depression, anxiety and autism, exhibit sex-biases in prevalence. Continued
examination of hormonal influence on amygdala anatomy and its neuronal and nonneuronal components may be vital to understand these disorders.

94

APPENDIX

95

Figure 4 - Representative photomicrographs of coronal sections through the adult
MePD of normal (A) and AR-knockout (B) male mice immunostained for androgen
receptor (AR; Epitomics #1852-1 rabbit monoclonal anti-AR antiserum). Note the
distinct nuclear labeling in the MePD of the normal adult male that is absent in the
MePD of an AR knock-out male. Scale bar = 100 microns.

96

Figure 5 - Confocal image showing that preadsorbing the AR primary antibody with the
immunizing peptide (Epitomics P-1852, diluted 1:80 or 1/25 less than the 1:2000 dilution
used for the AR primary) eliminated AR labeling in the MePD. Image depicts the result
of a sequential scan calibrated to detect DAPI and Cy2 fluorescence. Scale bar = 10
microns.

97

Figure 6 - Confocal image showing that omission of the AR primary antibody eliminated
AR labeling in the MePD and other AR-containing brain regions of normal adult male
rats. Panels A-C depict the results of individual scans calibrated to detect DAPI (A),
Cy2 (B) and Cy5 (C). Both DAPI and GFAP labeling remained intact while Ar labelling
was eliminated. Panel D depicts a sequential scan for all three fluorescent labels. Scale
bar = 20 microns.

98

Figure 7

99

Figure 7 - MePD volume is sexually dimorphic and maintained by androgens in
adulthood especially in the right hemisphere. (A) Volume of the adult MePD in rats is
both sexually differentiated and asymmetric, as males have a larger MePD than
females, and the right (R) MePD is larger than the left (L) in both sexes. (B) T treatment
increases MePD volume overall in both the left and right hemispheres of rats
gonadectomized in adulthood (collapsed across sex). However, analyzing the
hemispheres separately reveals that T treatment significantly increases the volume of
the left MePD only in females (C) but increases the volume of the right MePD in both
sexes (D). Values are means of N = 9 rats/group (± standard error of the mean, SEM)

100

Figure 8

101

Figure 8 - Astrocyte numbers are lateralized, sexually dimorphic and maintained by
adult androgens in female rats. (A) The number of astrocytes in the adult MePD is both
sexually differentiated and asymmetric, with males having more MePD astrocytes
overall than females, and with the right (R) MePD containing more astrocytes than the
left (L) in both sexes. (B) T treatment increased the number of MePD astrocytes only in
the right hemisphere (collapsed across sex). Examining the effect of androgens
separately in each hemisphere revealed that in the left hemisphere, males have more
MePD astrocytes than females, adult testosterone (T) treatment affects this measure
only in females (C). Males also have more astrocytes than females in the right MePD,
where T increases astrocyte number in both sexes (D; the difference in males is
marginally significant (p = 0.056). Values are means of N = 9 rats/group (±SEM). Note
that astrocyte numbers in this figure closely mirrors the differences in MePD volume in
figure 2.

102

Figure 9

103

Figure 9 - Astrocyte complexity is increased by androgens in adulthood, predominantly
in males. Number of primary branches (A), branch points (B), branch endings (C) and
average branch length (D) of astrocytes were measured in the MePD of adult
gonadectomized male and female rats treated with testosterone (T) or blank capsules
for 30 days. In males, astrocyte process complexity in both the left and right MePD is
increased by T treatment, suggesting that adult androgens may normally maintain the
elaborate processes of adult MePD astrocytes. The exception to this pattern is average
branch length of MePD astrocytes (D), which was unaffected by androgens in males
and was the only measure of MePD astrocyte complexity affected by T treatment in
females, and that only in the right hemisphere. Values are means of N = 5 rats/ group,
(±SEM).

104

Figure 10 - Confocal images showing androgen receptor (AR) immunoreactivity within
nuclei of MePD astrocytes. In panel (A), DAPI, AR and GFAP reactivity is shown for the
same field of view in addition to a combined sequential scan for these three labels and
indicates colocalization of DAPI and AR staining within the same nuclei (arrows) of
GFAP+ astrocytes. The intensity of AR labeling in astrocytes varied and was often less
intense than what was seen in presumptive neuronal nuclei. In panel (B), an astrocyte
nucleus with AR immunoreactivity is shown with orthogonal views (along the yellow and
pink lines) demonstrating that AR immunoreactivity is within the same Z-plane as the
astrocytic nucleus. Scale bars = 10 microns.

105

Figure 11 - Confocal image showing nuclear AR immunoreactivity within astrocytes of
the arcuate nucleus (A) and CA1 hippocampus (B). Arrows point to AR+
immunoreactivity (red) in nuclei of GFAP expressing cells (green). Scale bars = 10
microns.

106

REFERENCES

107

REFERENCES
Adamec, R. E. (1990). Amygdala kindling and anxiety in the rat. Neuroreport 1:255-258.
Adolphs, R., Baron-Cohen, S., Tranel, D. (2002). Impaired recognition of social
emotions following amygdala damage. J Cogn Neurosci 14:1264-1274.
Adolphs, R., Tranel, D. 2003. Amygdala damage impairs emotion recognition from
scenes only when they contain facial expressions. Neuropsychologia
41:1281-1289.
Agapova ,O. A., Malone P. E., Hernandez, M. R. (2006.) A neuroactive steroid
5alpha-androstane-3alpha,17beta-diol regulates androgen receptor level in
astrocytes. Journal of neurochemistry 98:355-363.
Ahlbom, E., Prins, G. S., Ceccatelli, S. (2001). Testosterone protects cerebellar granule
cells from oxidative stress-induced cell death through a receptor mediated
mechanism. Brain Res 892:255-262.
Ahmed, E. I., Zehr, J. L., Schulz, K. M., Lorenz, B. H., DonCarlos, L. L., Sisk, C. L.
(2008). Pubertal hormones modulate the addition of new cells to sexually
dimorphic brain regions. Nat Neurosci 11:995-997.
Amateau, S. K., McCarthy, M. M. (2002). A novel mechanism of dendritic spine
plasticity involving estradiol induction of prostaglandin-E2. Journal of Neuroscience
22:8586-8596.
Association, A. P. (2000). Diagnostic and statistical manual of mental disorders:
DSM-IV-TR.
Baker, K. (2004). Amygdalar Lateralization in Fear Conditioning: Evidence for Greater
Involvement of the Right Amygdala. Behav. Neurosci.
Barker, A. J., Ullian, E. M. (2008). New roles for astrocytes in developing synaptic
circuits. Commun Integr Biol 1:207-211.
Baum, M. J., Everitt, B. J. (1992). Increased expression of c-fos in the medial preoptic
area after mating in male rats: role of afferent inputs from the medial amygdala
and midbrain central tegmental field. Neuroscience 50:627-646.
Chowen, J. A., Busiguina, S., Garcia-Segura, L. M. (1995). Sexual dimorphism and sex
steroid modulation of glial fibrillary acidic protein messenger RNA and
immunoreactivity levels in the rat hypothalamus. Neuroscience 69:519-532.
Cooke, B. M., Tabibnia, G., Breedlove, S. M. (1999). A brain sexual dimorphism
controlled by adult circulating androgens. Proc Natl Acad Sci USA 96:7538-7540.

108

Cooke, B. M., Breedlove, S. M., Jordan, C. L. (2003). Both estrogen receptors and
androgen receptors contribute to testosterone-induced changes in the morphology
of the medial amygdala and sexual arousal in male rats. Horm Behav 43:336-346.
Cooke, B. M., Stokas, M. R., Woolley, C. S. (2007). Morphological sex differences and
laterality in the prepubertal medial amygdala. J. Comp. Neurol. 501:904-915.
Day, J. R., Laping, N. J., McNeill, T. H., Schreiber, S. S., Pasinetti, G., Finch, C. E.
(1990). Castration enhances expression of glial fibrillary acidic protein and sulfated
glycoprotein-2 in the intact and lesion-altered hippocampus of the adult male rat.
Mol. Endocrinol. 4:1995-2002.
Day, J. R., Laping, N. J., Lampert-Etchells, M., Brown, S. A., O'Callaghan, J. P.,
McNeill, T. H., Finch, C. E. (1993). Gonadal steroids regulate the expression of
glial fibrillary acidic protein in the adult male rat hippocampus. Neuroscience
55:435-443.
de Olmos, J., Hardy, H., Heimer, L. (1978). The afferent connections of the main and
the accessory olfactory bulb formations in the rat: an experimental HRP-study. J.
Comp. Neurol. 181:213-244.
Dmitar, D., Slobodan, M., Bojan, S
neurons and glial cells of basolateral amygdala in male and female rats
neonatally treated with estrogen. Int. J. Neurosci. 83:145-151.
neuronal and glial
cell groups in corticomedial amygdala react differently to neonatally administered
estrogen. Neuroscience 66:475-481.
Dugger, B. N., Morris, J. A., Jordan, C. L., Breedlove, S. M. (2007). Androgen receptors
are required for full masculinization of the ventromedial hypothalamus (VMH) in
rats. Horm Behav 51:195-201.
Elmariah, S. B., Hughes, E. G., Oh, E. J., Balice-Gordon, R. J. (2005). Neurotrophin
signaling among neurons and glia during formation of tripartite synapses. Neuron
Glia Biol. 1:1-11.
Emamian, S., Naghdi, N., Sepehri, H., Jahanshahi, M., Sadeghi, Y., Choopani, S.
(2010). Learning impairment caused by intra-CA1 microinjection of testosterone
increases the number of astrocytes. Behav. Brain Res. 208:30-37.
Faber-Zuschratter, H., Hüttmann, K., Steinhäuser, C., Becker, A., Schramm, J., Okafo,
U., Shanley, D., Yilmazer-Hanke, D. M. (2009). Ultrastructural and functional
characterization of satellitosis in the human lateral amygdala associated with
Ammon's horn sclerosis. Acta Neuropathol. 117:545-555.

109

Featherstone, R. E., Fleming, A. S., Ivy, G. O. (2000). Plasticity in the maternal circuit:
effects of experience and partum condition on brain astrocyte number in female
rats. Behav. Neurosci. 114:158-172.
Finley, S. K., Kritzer, M. F. (1999). Immunoreactivity for intracellular androgen receptors
in identified subpopulations of neurons, astrocytes and oligodendrocytes in primate
prefrontal cortex. Journal of neurobiology 40:446-457.
Forger, N. G. (2006). Cell death and sexual differentiation of the nervous system.
J Neuroscience 138:929-938.
Garcia-Segura, L. M., Suarez, I., Segovia, S., Tranque, P. A., Calés, J. M., Aguilera, P.,
Olmos, G., Guillamón, A. (1988). The distribution of glial fibrillary acidic protein in
the adult rat brain is influenced by the neonatal levels of sex steroids. Brain Res
456:357-363.
Garcia-Segura, L. M., Torres-Aleman, I., Naftolin, F. (1989). Astrocytic shape and glial
fibrillary acidic protein immunoreactivity are modified by estradiol in primary rat
hypothalamic cultures. Brain Res Dev Brain Res 47:298-302.
Gatson, J. W., Singh, M. (2007). Activation of a membrane-associated androgen
receptor promotes cell death in primary cortical astrocytes. Endocrinology
148:2458-2464.
Gréco, B., Edwards, D. A., Zumpe, D., Michael, R. P., Clancy, A. N. (1998). Fos
induced by mating or noncontact sociosexual interaction is colocalized with
androgen receptors in neurons within the forebrain, midbrain, and lumbosacral
spinal cord of male rats. Horm Behav 33:125-138.
Halassa, M. M., Fellin, T., Takano, H., Dong, J., Haydon, P. G. (2007). Synaptic islands
defined by the territory of a single astrocyte. J Neuroscience 27:6473-6477.
Hatton, G. I. (2002). Glial-neuronal interactions in the mammalian brain. Adv Physiol
Educ 26:225-237.
Hines, M., Allen, L. S., Gorski, R. A. (1992). Sex differences in subregions of the medial
nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat.
Brain Res 579:321-326.
Hösli, E., Jurasin, K., Rühl, W., Lüthy, R., Hösli, L. (2001). Colocalization of androgen,
estrogen and cholinergic receptors on cultured astrocytes of rat central nervous
system. Int J Dev Neurosci 19:11-19.
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (2008). Sex differences and laterality in
astrocyte number and complexity in the adult rat medial amygdala. J Comp
Neurol 511:599-609.

110

Nunez, J. L., Huppenbauer, C. B., McAbee, M. D., Juraska, J. M., DonCarlos, L. L.
(2003). Androgen receptor expression in the developing male and female rat
visual and prefrontal cortex. Journal of Neurobiology 56:293-302
Kilpatrick, L. A., Zald, D. H., Pardo, J. V., Cahill, L. F. (2006). Sex-related differences in
amygdala functional connectivity during resting conditions. Neuroimage
30:452-461.
Kondo, Y., Arai, Y. (1995). Functional association between the medial amygdala and
the medial preoptic area in regulation of mating behavior in the male rat. Physiol
Behav 57:69-73.
LaBar, K. S., Gatenby, J. C., Gore, J. C., LeDoux, J. E., Phelps, E. A. (1998). Human
amygdala activation during conditioned fear acquisition and extinction: a
mixed-trial fMRI study. Neuron 20:937-945.
Lee, Y., Gaskins, D., Anand, A. (2007). Glia mechanisms in mood regulation: a novel
model of mood disorders. Psychopharmacology 191:55-65.
Legrand, A., Alonso, G. (1998). Pregnenolone reverses the age-dependent
accumulation of glial fibrillary acidic protein within astrocytes of specific
regions of the rat brain. Brain Res 802:125-133.
Lehman, M. N., Winans, S. S. (1982). Vomeronasal and olfactory pathways to the
amygdala controlling male hamster sexual behavior: autoradiographic and
behavioral analyses. Brain Res 240:27-41.
Leranth, C., Szigeti-Buck, K., Maclusky, N. J., Hajszan, T. (2008). Bisphenol A prevents
the synaptogenic response to testosterone in the brain of adult male rats.
Endocrinology 149:988-994.
Lorenz, B., Garcia-Segura, L., DonCarlos, L. (2005). Cellular phenotype of androgen
receptor-immunoreactive nuclei in the developing and adult rat brain. J Comp
Neurol 492:456-468.
MacLusky, N., Hajszan, T., Johansen, J., Jordan, C. L., Leranth, C. (2006). Androgen
effects on hippocampal CA1 spine synapse numbers are retained in Tfm male rats
with defective androgen receptors. Endocrinology 147: 2392-2398.
MacLusky, N., Hajszan, T., Leranth, C. (2004). Effects of dehydroepiandrosterone and
flutamide on hippocampal CA1 spine synapse density in male and female rats:
implications for the role of androgens in maintenance of hippocampus structure.
Endocrinology 145:4154-4161

111

Martinez, F. G., Hermel, E. E. S., Xavier, L. L., Viola, G. G., Riboldi, J., Rasia-Filho, A.
A., Achaval, M. (2006). Gonadal hormone regulation of glial fibrillary acidic protein
immunoreactivity in the medial amygdala subnuclei across the estrous cycle and in
castrated and treated female rats. Brain Res 1108:117-126.
McAbee, M. D., DonCarlos, L. L. (1998). Ontogeny of region-specific sex differences in
androgen receptor messenger ribonucleic acid expression in the rat forebrain.
Endocrinology 139:1738-1745.
McQueen, J., Wright, A., Arbuthnott, G. (1990). Glial fibrillary acidic protein
(GFAP)-immunoreactive astrocytes are increased in the hypothalamus of
androgen- insensitive testicular feminized (Tfm) mice. Neurosci Lett 118:77-81.
Melcangi, R. C., Riva, M. A., Fumagalli, F., Magnaghi, V., Racagni, G., Martini, L.
(1996). Effect of progesterone, testosterone and their 5 alpha-reduced metabolites
on GFAP gene expression in type 1 astrocytes. Brain Res 711:10-15.
Micevych, P., Bondar, G., Kuo, J. (2010). Estrogen actions on neuroendocrine glia.
Neuroendocrinology 91:211-222.
Mong, J., Nunez, J., McCarthy, M. (2002). GABA mediates steroid induced astrocyte
differentiation in the neonatal rat hypothalamus. J. Neuroendo 14:45-55.
Mong, J., Kurzweil, R. L., Davis, A. M., Rocca, M. S., McCarthy, M. M. (1996). Evidence
for Sexual Differentiation of Glia in Rat Brain. Horm Behav 30:553-562.
Mong, J. A., Glaser, E., McCarthy, M. M. (1999). Gonadal steroids promote glial
differentiation and alter neuronal morphology in the developing hypothalamus in a
regionally specific manner. J Neurosci 19:1464-1472.
Mong, J. A., Roberts, R. C., Kelly, J. J., McCarthy, M. M. (2001). Gonadal steroids
reduce the density of axospinous synapses in the developing rat arcuate nucleus:
an electron microscopy analysis. J Comp Neurol 432:259-267.
Mong, J. A., McCarthy, M. M. (1999). Steroid-induced developmental plasticity in
hypothalamic astrocytes: implications for synaptic patterning. Journal of
neurobiology 40:602-619.
Morris, J., Jordan, C. L., Dugger, B., Breedlove, S. M. (2005). Partial demasculinization
of several brain regions in adult male (XY) rats with a dysfunctional androgen
receptor gene. Journal of Comp Neurol. 487:217.
Morris, J. A., Jordan, C. L., Breedlove, S. M. (2008a). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in adult rats. J Comp Neurol 506:851-859.

112

Morris, J. A., Jordan, C. L., King, Z. A., Northcutt, K. V., Breedlove, S. M. (2008b).
Sexual dimorphism and steroid responsiveness of the posterodorsal medial
amygdala in adult mice. Brain Res 1190:115-121.
Nagy, J., Zámbó, K., Decsi, L. (1979). Anti-anxiety action of diazepam after intraamygdaloid application in the rat. Neuropharmacology 18:573-576.
Nguyen, T., Jayaraman, A. (2010). Androgens selectively protect against apoptosis in
hippocampal neurones. Journal of Neuroendocrinology 22:1013-1022.
Nishida, H., Okabe, S. (2007). Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci 27:331-340.
Paxinos, G., Watson W. (2005). The rat brain in stereotaxic coordinates. Amsterdam:
Elsevier.
Pfrieger, F. W., Barres, B. A. (1997). Synaptic efficacy enhanced by glial cells in vitro.
Science 277:1684-1687.
Rogan, M. T., Stäubli, U. V., LeDoux, J. E. (1997). Fear conditioning induces
associative
long- term potentiation in the amygdala. Nature 390:604-607.
Roselli, C. E. (1991). Sex differences in androgen receptors and aromatase activity in
microdissected regions of the rat brain. Endocrinology 128:1310-1316.
Sales, S., Ureshino, R. P., Pereira, R. T. D. S., Luna, M. S. A., Pires de Oliveira, M.,
Yamanouye, N., Godinho, R. O., Smaili, S. S., Porto, C. S., Abdalla, F. M. F.
(2010). Effects of 17beta-estradiol replacement on the apoptotic effects caused by
ovariectomy in the rat hippocampus. Life Sci. 86:832-838.
Sanders, S. K., Shekhar, A. (1995). Anxiolytic effects of chlordiazepoxide blocked by
injection of GABAA and benzodiazepine receptor antagonists in the region of the
anterior basolateral amygdala of rats. Biological Psychiatry 37:473-476.
Sarkey, S., Azcoitia, I., Garcia-Segura, L. M., Garcia-Ovejero, D., DonCarlos, L. (2008).
Classical androgen receptors in non-classical sites in the brain. Hormones and
Behavior 53:753-764
Schummers, J., Yu, H., Sur, M. (2008). Tuned responses of astrocytes and their
influence on hemodynamic signals in the visual cortex. Science 320:1638-1643.
Shughrue, P.,
estradiol-mediated protection against brain injury. Proc Natl Acad Sci USA 98:
1952-1957.
Stone, D., Song, Y., Anderson, C., Krohn, K., Finch, C. E., Rozovsky, I. (1998).
Bidirectional transcription regulation of glial fibrillary acidic protein by estradiol in
vivo and in vitro. Endocrinology 139: 3202-3209.
113

Tabori, N. E., Stewart, L. S., Znamensky, V., Romeo, R. D., Alves, S. E., McEwen, B.
S., Milner, T. A. (2005). Ultrastructural evidence that androgen receptors are
located at extranuclear sites in the rat hippocampal formation. Neuroscience
130:151-163.
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. (2001). Control of
synapse number by glia. Science 291:657-661.
von Ziegler von, N. I., Lichtensteiger W. (1992). Asymmetry of brain aromatase activity:
region- and sex-specific developmental patterns. Neuroendocrinology 55:512-518.
Xiao, L., Jordan, C. L. (2002). Sex differences, laterality, and hormonal regulation of
androgen receptor immunoreactivity in rat hippocampus. Horm Behav 42:327-336.

114

CHAPTER 3: ANDROGEN DEPNDENT SEX DIFFERENCES IN ASTROCYTE
NUMBER BUT NOT ASTROCYTE ARBOR COMPLEXITY IN THE JUVENILE
MEDIAL POSTERODORSAL AMYGDALA
Abstract
The medial posterodorsal amygdala (MePD) is sexually dimorphic both in
juvenile and adult rats and requires androgen receptor (AR) stimulation for complete
masculinization. I previously reported that astrocytes in this region are more numerous
and more complex in adult males than in females, and that these sex differences are
androgen-dependent. I now report that MePD astrocyte number, but not arbor
complexity, is sexually dimorphic in juvenile 25-day old rats. Using stereological
analysis of tissue immunocytochemically labeled for glial fibrillary acidic protein (GFAP),
male juvenile rats displayed more MePD astrocytes than females, but no sex
differences were seen for any measure of arbor complexity. Both astrocyte number and
astrocyte complexity were reduced in juveniles compared to previous estimates in
adults. Furthermore, the sexual dimorphism in astrocyte number at 25 days is
independent of ARs since testicular feminized mutant (Tfm) males, which lack functional
ARs, nevertheless exhibited masculine numbers of MePD astrocytes. This is in contrast
to adults, where Tfm males display a feminine number of astrocytes, indicating that
some time after 25 days, AR activation is required for full masculinization of astrocyte
number. As seen in adult animals, the sexual dimorphism in astrocyte number was also
asymmetric, with the MePD having more astrocytes in the right hemisphere than the left
in all three genotypes. These findings demonstrate that several cellular constituents of
the MePD are sexually dimorphic and influenced by gonadal hormones, likely acting
through both estrogen receptors (ERs) and ARs, throughout the lifespan.

115

Introduction
The amygdala is a complex brain region involved in fear, anxiety and social
behaviors (Klüver and Bucy 1938; LaBar et al. 1998; Aggleton and Passingham 1981).
In many species, including humans, the amygdala undergoes marked structural and
functional changes prior to, and during, puberty (Giedd et al. 1996; Neufang et al. 2009;
Blakemore 2006; Ziegler and Lichtensteiger 1992). Moreover, evidence suggests that
in some species the developmental trajectory of amygdala neuroanatomy is at least
partially controlled by gonadal hormones such as estrogens and androgens acting both
early in life and during puberty (Ahmed et al. 2008; Merke et al. 2003; Rubinow and
Juraska 2009; Nishizuka and Arai 1981; 1982). Investigation of how and where gonadal
hormones act to alter cellular components within the amygdala will yield important
information about this dynamic brain region.
The mammalian amygdala consists of several subregions with extensive
interconnectivity. In rodents the medial posterodorsal subregion (MePD) is highly
sensitive to gonadal hormone influence throughout the lifespan and contains a
particularly dense supply of both ERs and ARs (Simerly et al. 1990; Wood and Newman
1995). Some aspects of MePD neuroanatomy are sexually dimorphic in juvenile
animals, including regional volume and synaptic organization (Mizukami et al. 1983;
Cooke and Woolley 2005). Several additional sex differences are found in adult
animals including total number of glia, astrocyte number and astrocyte arbor complexity
(Morris et al. 2008; Johnson et al. 2008). These results suggest that hormonal influence
may further alter MePD neuroanatomy during puberty, and indeed androgen-dependent

116

gliogenesis in the rodent medial amygdala during puberty has been demonstrated
(Ahmed et al. 2008).
The timing of sex differences in astrocytes (a subtype of glia) in the amygdala is
particularly intriguing due to the important role these cells play in neural network
modulation and synapse formation (Halassa et al. 2007; Ullian et al. 2001; Nishida and
Okabe 2007; Hatton 2002). In some brain regions, including the hypothalamus and
amygdala, astrocytes contain gonadal hormone receptors and respond to hormonal
manipulations with changes in morphology (Lorenz et al. 2005; Pfrieger and Barres
1997; Elmariah et al. 2005; Mong et al. 2001; Sarkey et al. 2008), Johnson et al. in
prep. b).
Interestingly, in the hypothalamus astrocyte sex differences are present very
early in development and persist into adulthood (Mong 1996) suggesting hormones
influence these cells during early development. In the MePD, astrocyte number and
arbor complexity are both sexually dimorphic in adults but little is known about when this

diseases and disorders that exhibit strong sex differences in population prevalence,
such as schizophrenia and autism, both of which are linked to critical periods of
hormonal influence, it is likely that understanding hormone-induced neuroanatomical
changes in the amygdala, including changes in astrocytes, will be an important step in
the development of treatments.
Methods
Animals
Twenty-eight-day-old wildtype (wt) male, female and testicular feminized mutant

117

(Tfm) male rats were obtained from our breeding colony at Michigan State University
and genotyped using PCR. The colony has been infused with Long Evans sires from
Charles River for over 20 generations. Animals were housed five to nine per cage with
siblings in standard rat cages with food and water available ad libitum. Lights were on
at 0700 and off at 1900 hours. Animals were cared for in accordance with the
guidelines set forth by the National Institute of Health and all procedures were approved
by the Institutional Animal Care and Use Committee at Michigan State University.
On the day of sacrifice, animals were given an overdose of sodium pentobarbital
(150mg/kg, ip). Once animals were deeply anesthetized (showing no reflex response to
either tail or foot pinch) they were intracardially perfused with 0.9% saline, followed by
4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4, ~100 mL/animal). Brains were
removed and weighed, postfixed for five hours at 4°C in the same fixative solution, then
stored in 20% phosphate-buffered sucrose at 4°C for at least 48 hrs. The left cortex of
each brain was scored to mark that hemisphere and then sectioned coronally on a
freezing microtome at 40µm through the region of interest. Sections were collected into
cryoprotectant (de Olmos et al. 1978) and stored at -20°C until stained. Every second
section through the rostrocaudal extent of the MePD was processed for
immunocytochemistry.
Histology
Immunocytochemistry for Counting Astrocyte Numbers
Archived sections were transferred from cryoprotectant to Netwell plates (Corning
Life Sciences) and thoroughly rinsed in 0.1 M phosphate buffered saline (PBS; 140 mM
NaCl, 10.7 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4) containing 0.3% Triton

118

X-100 and 0.1% Knox gelatin (PBS-GT). PBS-GT was used throughout as the vehicle
for ICC reagents and for rinsing. Sections were then incubated for 1 hr in 10% normal
horse serum (Vector Laboratories) with avidin (Avidin Biotin blocking kit, Vector
Laboratories) followed by 1 hr incubation at room temperature in mouse anti-GFAP
monoclonal antiserum (1:6000, Chemicon, MAB360) with biotin (Avidin biotin blocking
kit, Vector Laboratories). Tissue was then incubated overnight at 4°C in the same
1:6000 primary antiserum without biotin. The antiserum was prepared against purified
glial fibrillary acidic protein from porcine spinal cord, and in western blots recognizes a
sin
incubation in primary antiserum, tissue was incubated in biotinylated rat-absorbed horse
anti-mouse secondary antiserum (1:500, Vector Laboratories) for 1 hr at room
temperature. This was followed by 1 hr incubation in peroxidase avidin-biotin complex
solution at half recommended strength (Elite ABC kit, Vector). HRP was visualized
using an Immpact diaminobenzidine kit (DAB, Vector Laboratories) with a 4.5 min
reaction time. Tissue was rinsed to quench the peroxidase reaction, mounted on gelsubbed slides, and allowed to dry for at least 24 hrs. Sections were counter-stained
using Harris hematoxylin solution (Sigma) to visualize cell nuclei, using 10% lithium
carbonate as the bluing agent, before dehydrating in a graded series of ethanols,
clearing in Citrisolv and cover-slipping using Permount.
I found that the processes of juvenile astrocytes were not well stained using the
above protocol, and thus, modified the staining protocol to optimize visualization of their
arbors. Specifically, dilution of the primary antiserum was increased to 1:10,000 and
the DAB reaction time was reduced to 2.5 min. The remaining alternate set of tissue

119

sections was stained using this modified protocol and used for measuring arbor
complexity of juvenile astrocytes. The original set was used to assess astrocyte
number.
Stereological Analysis
A Zeiss Axioplan II microscope with the visual field captured by an Optronics
MicroFire digital video camera was used to quantify the number and complexity of
astrocytes within the region of interest. Using StereoInvestigator software (v. 8.0, MBF
Bioscience; Williston, VT), the perimeter of the MePD was traced in serial sections at
low magnification. MePD boundaries were identified using the standard rat atlas
(Paxinos and Watson 2005) in conjunction with previously established standards within
our laboratory (Morris et al. 2008; Johnson et al. 2008, in prep. b) and others (Hines et
al.1992). After tracing the boundaries of the MePD, astrocytes were counted and traced
using a 100X Plan-NeoFluar, 1.3 N.A oil-immersion objective. Slides were coded to

astrocytes were captured using the image capture option in StereoInvestigator.
Astrocyte Number
An optical fractionator probe (West 1993) was used to generate an unbiased

a ZThe coefficient of error (CE; Gunderson m=1) for each hemisphere was at or below
0.10.
Criteria for identifying an astrocyte included a distinct and recognizable nucleus in
a plane of focus within the sampling probe and from which at least 2 GFAP labeled

120

fibers extended (Fig 12). Such nuclei were often, although not always, smaller than
surrounding (presumably neuronal) nuclei, often elliptical in shape, and lacking a clear
nucleolus. These methods produced estimates of overall MePD volume and number of
astrocytes per hemisphere for each subject. The number of cells counted per
hemisphere ranged from approximately 150 - 300 depending on the sex and
hemisphere of the subject.
Astrocyte Process Complexity
Neurolucida software (v. 7.0, MBF Bioscience; Williston, VT) was used to trace
and measure the entire visible arbor of 24 randomly selected astrocytes per subject.
The MePD was traced via the criteria described above and 12 sites per hemisphere
were selected. At each of these sites, the astrocyte nearest the randomly placed
marker was traced in its entirety using a Wacom Cintiq 12WX tracing display (Wacom
Co.; Saitama, Japan, Fig 13). The average number of primary processes, average
number of branch points, average number of branch endings, and average branch
length per astrocyte were calculated for each hemisphere of each animal.
Statistical Analysis
Separate two-way mixed-design analyses of variance (ANOVA) were conducted
for each dependent variable (MePD volume, astrocyte number, number of primary
processes, number of branch points, number of branch endings, and branch length).
The left and right hemispheres served as a repeated measure and genotype (male,
Tfm, female) as a between group measure. This was followed by one-way ANOVAs for
each individual hemisphere and LSD post hoc analysis to determine differences
between genotypes on each side. For all analyses, results are expressed as mean ±

121

SEM (standard error of the mean), with N = number of animals, and a p value of 0.05
was considered statistically significant.
Results
Regional Volume
Mixed design 2-way ANOVA (left and right hemisphere as repeated measures)
revealed a main effect of genotype on MePD volume (p < 0.001) in addition to a main
effect of hemisphere (p < 0.001) and a genotype by hemisphere interaction (p < 0.05).
Post-hoc analysis revealed that Tfms have a greater MePD volume than wt males (p <
0.05) and females (p < 0.001) and males to have a greater MePD volume than females
(p < 0.05). Separate one-way analysis of variance in each hemisphere revealed a main
effect of genotypes in the left hemisphere (p < 0.05) with Tfms having a greater MePD
volume than females (p < 0.01, Fig 14A) but with wt males being no different than either
females or Tfm males. There was also a main effect of genotype in the right hemisphere
(p < 0.001) with all groups being significantly different from each other (all p
Fig14A). Thus Tfms have greater MePD volumes than females in both hemispheres
and a greater volume than males in the right hemisphere. These represent the first
measures of MePD volumes in Tfm male rats. Paired-samples t-tests revealed that the
right hemisphere had greater regional volume than the left in both wt and Tfm males (ps
< 0.005) with no significant laterality of MePD volume in females.

Astrocyte Numbers
Similar to previous reports in adults (Johnson et al. 2008) there was a main effect
of hemisphere (p < 0.001) with greater numbers of astrocytes in the right MePD

122

compared to the left in all groups (ps < 0.05). There was also a main effect of genotype
on the number of astrocytes as wt and Tfm males have more astrocytes than do
females (ps < 0.05). Although there was no genotype by hemisphere interaction, the
complex pattern of laterality previously seen in adult animals prompted separate
analysis of the two hemispheres. While there was no main effect of genotype in the left
MePD there was a significant effect in the right MePD (p < 0.05, Fig14B). Nevertheless,
post-hoc analysis of the left MePD did reveal that males had greater numbers of
astrocyte than females (p < 0.05, Fig 14B) and Tfms were intermediate. In contrast,
post-hoc analysis of the right MePD revealed males and Tfms had greater numbers of
astrocytes than females (ps < 0.05, Fig 14B). Thus, in the right hemisphere astrocyte
numbers are sexually dimorphic, and Tfm males are masculinized. Since Tfm male rats
lack functional AR but have circulating androgens (Roselli and Salibury 1987), these
results suggest that the sex difference is not dependent on AR at this age.
Astrocyte Complexity
Astrocyte complexity was assessed using previously established measures from
adults (Johnson et al. 2008, in prep. b) and analyzed using mixed design 2-way
ANOVAs (left and right hemisphere as repeated measures). No main effects or
interactions of hemisphere and genotype were found for any measure of astrocyte
complexity (Fig 15). Thus none of the sex differences or laterality of astrocyte arbors
seen in adult animals were present in these juveniles. Moreover, estimates of astrocytic
arbor complexity and length in the juvenile MePD are below those obtained for adult
MePD astrocytes, suggesting that puberty may represent a time when astrocytes not
only extend processes to achieve their full adult extent, but also critically depend on

123

androgens acting via ARs to develop processes in a sex- and hemisphere-specific
manner.
Discussion
Astrocytes During Neonatal Development
Early prenatal development is a critical period during which hormonal influence
serves to masculinize much of the central nervous system in male mammals (Mizukami
et al. 1983; Segovia and Guillamón 1993; Gorski et al. 1978; Merke et al. 2003; Gilmore
et al. 2007; Chura et al. 2010; Cooke et al. 1998; reviewed in Breedlove 2010; Hamson
et al. 2010). Although reliable patterns have emerged, the pathways mediating
masculinization are complex and still poorly understood. Reports from the past three
decades reveal that gonadal hormones influence astrocytes and that this influence may
occur early in development (Weiner 1983; Tranque et al. 1987; Day et al. 1993; Mong
1996). In the arcuate nucleus the masculinization of astrocyte arbors has been
attributed to estrogens and linked to changes in surrounding neurons (Naftolin 1978;
Leedom et al. 1994; Mong et al. 1999; 2001; 2002). Like the arcuate nucleus, the
MePD exhibits sexually dimorphic astrocytes in adulthood (Martinez et al. 2006;
Johnson et al. 2008). However, the timing of these sex differences and the role of AR
and ER in their early development has not been previously investigated. Furthermore,
and in contrast to the arcuate nucleus, the MePD requires functional ARs for complete
anatomical masculinization (Morris et al. 2005; Cooke et al. 2003). This information,
combined with the growing appreciation of astrocytes in brain plasticity led us to
investigate possible sex differences in juvenile astrocytes, using Tfm rats to probe for
any role of ARs in MePD sex differences.

124

The current findings provide further evidence that MePD astrocytes are sexually
dimorphic in number and reveal that this sex difference is present prior to puberty. The
sex differences in astrocyte number in the left MePD are concordant with previously
reported sex differences in volume occupied by glia in the left MePD (females < males,
Cooke et al. 2007). However, sex differences in glia in the juvenile right MePD have not
previously been reported.
In the right hemisphere, Tfm males are masculinized in terms of astrocyte number,
suggesting that this sex differences is not dependent upon functional AR prepubertally.
This result raises the question of how the sex differences originate. Do estrogens
during early development lead to increased astrocyte numbers in the MePD?
Interestingly, there are sex differences in astrocyte proliferation in the juvenile rat MeA
with females exhibiting greater numbers of BrdU+ cells, which co-label with GFAP in the
MeA, suggesting increased astrogenesis in females at these times (Krebs-Kraft et al.
2010). These findings at PND 4 and 14 in the MeA contrast with the greater number of
astrocytes I see in males in the MePD. Stereological counts of astrocyte numbers,
which also account for possible hemisphere effects are needed at these time points to
see how proliferation at PNDs 4 and 14 relate to the sex differences I see at PND 25.
Intriguingly, the sex difference in astrocyte proliferation in the medial amygdala at
PNDs 4 and 14 is dependent upon endogenous cannabinoid receptor activation, since
administration of a cannabinoid receptor agonist to females eliminated the sex
differences (Krebs-Kraft et al. 2010). This result suggests that sex differences in the
cannabinoid system may be responsible for the sex differences in MeA astrocyte
numbers; sex differences in metabolism of endogenous cannabinoids have also been

125

implicated (Krebs-Kraft et al. 2010). While these results provide clues to a possible
receptor system modulating astrocyte proliferation, they also raise additional questions
about how and when the sex differences in cannabinoid synthesis and/or receptor
activation originate.
Although few comparisons are available for the sex differences I see in astrocyte
numbers, there are previous reports of sex differences in juvenile astrocyte complexity.
For example, while astrocyte arbor complexity is sexually dimorphic in the
hypothalamus of neonates, and this sex differences is retained into adulthood, this
pattern is not present in the MePD. I found no sex differences in any measure of
astrocyte arbor complexity in the juvenile MePD, suggesting that the sex difference in
arbor complexity seen in adult animals arises during puberty. Are changes in astrocytes
in the amygdala during puberty part of a synaptic reorganization of this region? Further
research examining synapse formation in the MePD during puberty might answer this
question.
Implications and Additional Questions
While the current experiment does not include adult animals, previous reports
using similar methodologies from the same animal colony found androgen dependent,
and AR dependent, sex differences in both MePD astrocyte number and arbor
complexity in two separate studies from our lab (Johnson et al. 2008; Johnson et al. in
prep. b). I therefore feel comparison of the current results in juvenile animals to
previous reports in adults is valid, and suggests that while the sex difference in MePD
astrocyte number is present at PND 25, the absolute numbers of astrocytes increases
between that age and adulthood.

126

The current evidence demonstrates that prior to puberty, Tfm males are
masculinized, as both wt males and Tfms having more astrocytes than females. In
adult animals, Tfm males are feminine in astrocyte number, suggesting an ARdependent increase in MePD astrocyte numbers in wt males during puberty. Ahmed
and colleagues (Ahmed et al. 2008) found androgen-dependent proliferation in nonspecific glia in the rat medial amygdala during puberty. In contrast, I found no sexual
dimorphism in astrocyte arbor complexity prior to puberty. However, in adulthood the
sexual dimorphism in astrocyte arbor complexity is pronounced and Tfm males are
feminized. Are androgens acting on AR during puberty to promote arbor elaboration in
the MePD? Hormonal regulation of both GFAP-immunoreactive optical density and
astrocyte arbor complexity has been previously demonstrated in the MePD of adult
animals, so hormonal regulation during puberty seems plausible (Martinez et al. 2006,
Johnson et al. in prep. b).
Despite this new information, numerous questions remain. For example, are
estrogens acting directly on astrocytes to promote cell proliferation and arbor
elaboration during early development? Evidence from the hypothalamus suggests that
estrogens act indirectly through ER+ neurons to influence astrocytes morphology (Mong
et al. 2002). A similar pathway is possible in the MePD but cannot be confirmed or
eliminated based on the currently available data. While I found evidence of AR+
immunoreactivity in MePD astrocytes of adult animals, the current report suggests that
estrogens establish early sex differences in the number of these cells. Do MePD
astrocytes contain ERs as well? Astrocytes in both humans and rodents are known to
express ERs in some brain regions, and the MePD is known to contain ER

127

immunoreactivity (Donahue 2000; Langub and Watson 1992; Milner et al. 2001; Simerly
et al. 1990). It is possible that ER+ astrocytes in the MePD respond to hormonal signals
early in development to prevent cell death or promote cell proliferation in developing
males. What role does this early estrogen-induced sexual dimorphism play in MePD
function and animal behavior?
Additionally, how early does this sexual dimorphism arise? Increasing evidence
suggests that early prenatal hormones may shape the amygdala in important ways in
disorders such as autism (Sweeten et al. 2002; Baron-Cohen et al. 2005). Singlenucleotide polymorphisms (SNPs) in the human ER are correlated with autistic-like
traits, and the same report found SNPs in synapse formation genes linked to autisticlike traits as well (Chakrabarti et al. 2009). In rodents, sex differences in excitatory
post-synaptic potentials have been reported in the medial amygdala (Cooke and
Woolley 2005) suggesting hormones may somehow influence the function of synapses
in the region. Do hormone responsive astrocytes affect synapse formation in the
amygdala?
What is clear from the present findings is that gonadal hormones play a role in the
masculinization of astrocytes during critical periods in development. While currently the
functional consequences of this influence remain elusive, continued investigation may
reveal whether changes in astrocytes lead to alterations in the neuronal network in this
important brain region. Changes in astrocyte morphology are associated with several
diseases and disorders including depression (Gosselin et al. 2007; Hercher et al. 2009)

rodent models is needed to understand how astrocytes may alter amygdala function

128

and how gonadal hormone may alter astrocytes during critical periods in development.

129

APPENDIX

130

Figure 12 - Photomicrographs depicting astrocytes of varying complexity in the juvenile
MePD. Panel A depicts a simple astrocyte, which has few and short processes. Panel
B depicts a more complex astrocyte that has multiple, elaborated processes. Scale bar
= 10 microns.

131

Figure 13 - Photomicrograph depicting a complex astrocyte in the juvenile MePD (A)
and the resulting tracing (B). Because of the narrow plane of focus, only a portion of the
full extent of arbor complexity is visible at a time. Complex astrocytes frequently
demonstrated multiple branches that emerged as the plane of focus was moved through
the tissue. Scale bar = 10 microns.

132

Figure 14 - Regional volume and astrocyte number are sexually dimorphic in the
posterodorsal medial amygdala (MePD) of juvenile wildtype male, female and testicular
feminized mutant (Tfm) male rats. MePD volume is larger on the right than the left in
juvenile wildtype and Tfm males but not in females (A). Values are means of N = 9
rats/group (+ standard error of the mean, SEM).

133

Figure 15 - Estimated number of primary branches in astrocytes of the MePD in juvenile
wildtype male, Tfm male and female rats. No hemisphere or sex differences were seen
in any group. All other measures of astrocyte complexity exhibited a similar pattern.
Values are means of N = 5 rats/ group (+ SEM).
134

REFERENCES

135

REFERENCES
Aggleton, J. P., Passingham, R. E. (1981). Stereotaxic surgery under X-ray guidance
in the rhesus monkey, with special reference to the amygdala. Experimental brain
research 44:271-276.
Ahmed, E. I., Zehr, J. L., Schulz, K. M., Lorenz, B. H., DonCarlos, L. L., Sisk, C. L.
(2008). Pubertal hormones modulate the addition of new cells to sexually
dimorphic brain regions. Nature Neuroscience 11:995-997.
Baron-Cohen, S., Knickmeyer, R. C., Belmonte, M. (2005). Sex differences in the brain:
implications for explaining autism. Science 310:819.
Blakemore, S. (2006). Development of the adolescent brain: implications for executive
function and social cognition. J Child Psychology and Psychiatry 47:296-312.
Breedlove, S. M. (2010). Minireview: Organizational hypothesis: instances of the
fingerpost. Endocrinology 151:4116-4122.
Chakrabarti, B., Dudbridge, F., Kent, L., Wheelwright, S., Hill-Cawthorne, G., Allison, C.,
Banerjee-Basu, S., et al. (2009). Genes related to sex steroids, neural growth, and
social-emotional behavior are associated with autistic traits, empathy, and
Asperger syndrome. Autism research 2(3):157-177.
Chura, L. R., Lombardo, M. V., Ashwin, E., Auyeung, B., Chakrabarti, B., Bullmore, E.
T., Baron-Cohen, S. (2010). Organizational effects of fetal testosterone on human
corpus callosum size and asymmetry. Psychoneuroendocrinology 35:122-132.
Cooke, B. M., Woolley, C S. (2005). Sexually Dimorphic Synaptic Organization of the
Medial Amygdala. Journal of Neuroscience 25:10759-10767.
Cooke, B M, Breedlove, S. M., Jordan, C. L. (2003). Both estrogen receptors and
androgen receptors contribute to testosterone-induced changes in the morphology
of the medial amygdala and sexual arousal in male rats. Hormones and behavior,
43:336-346.
Cooke, Bradley M, Stokas, M. R., Woolley, Catherine S. (2007). Morphological sex
differences and laterality in the prepubertal medial amygdala. The Journal of
Comparative Neurology, 501:904-915.
Cooke, B, Hegstrom, C., Villeneuve, L. (1998). Sexual differentiation of the vertebrate
brain: principles and mechanisms. Frontiers in Neuroendocrinology 19:323-362.
Day, J. R., Laping, N. J., Lampert-Etchells, M., Brown, S. A., O'Callaghan, J. P.,
McNeill, T. H., Finch, C. E. (1993). Gonadal steroids regulate the expression of
glial fibrillary acidic protein in the adult male rat hippocampus. Neuroscience 55:

136

435-443.
de Olmos, J., Hardy, H., Heimer, L. (1978). The afferent connections of the main and
the accessory olfactory bulb formations in the rat: an experimental HRP-study. The
Journal of Comparative Neurology 181:213-244.
Donahue, J. (2000). Cells containing immunoreactive estrogen receptorbasal forebrain. Brain research 856: 142-151.
Elmariah, S. B., Hughes, E. G., Oh, E. J., Balice-Gordon, R. J. (2005). Neurotrophin
signaling among neurons and glia during formation of tripartite synapses. Neuron
glia biology 1:1-11.
Giedd, J. N., Vaituzis, A. C., Hamburger, S. D., Lange, N., Rajapakse, J. C., Kaysen, D.,
Vauss, Y. C., et al. (1996). Quantitative MRI of the temporal lobe, amygdala, and
hippocampus in normal human development: ages 4-18 years. The Journal of
Comparative Neurology 366:223-230.
Gilmore, J., Lin, W., Prastawa, M. (2007). Regional Gray Matter Growth, Sexual
Dimorphism, and Cerebral Asymmetry in the Neonatal Brain. Journal of
neuroscience 27:1255-1260.
Gorski, R. A., Gordon, J. H., Shryne, J. E., Southam, A. M. (1978). Evidence for a
morphological sex difference within the medial preoptic area of the rat brain. Brain
research 148:333-346.
Gosselin, N., Peretz, I., Johnsen, E., Adolphs, R. (2007). Amygdala damage impairs
emotion recognition from music Neuropsychologia 45: 236-244.
Halassa, M. M., Fellin, T., Takano, H., Dong, J., Haydon, P. G. (2007). Synaptic islands
defined by the territory of a single astrocyte. Journal of Neuroscience
27:6473-6477.
Hamson, D. K., Jordan, C. L., Breedlove, S. M. (2010). The testosterone two-step is
really a minuet. Neuron 66:167-169.
Hatton, G. I. (2002). Glial-neuronal interactions in the mammalian brain. Advances in
physiology education 26:225-237.
Hercher, C., Turecki, G., Mechawar, N. (2009). Through the looking glass: examining
neuroanatomical evidence for cellular alterations in major depression. Journal of
psychiatric research 43:947-961.
Hines, M., Allen, L. S., Gorski, R. A. (1992). Sex differences in subregions of the medial
nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat.
Brain research, 579:321-326.

137

Johnson, R. T., Breedlove, S. M., Jordan, C. L. (2008). Sex differences and laterality in
astrocyte number and complexity in the adult rat medial amygdala. The Journal of
Comparative Neurology 511:599-609.
Johnson, R. T., Schneider, A., DonCarlos, L. L., Breedlove, S. M., Jordan, C. L. (in
prep. b). Astrocytes in the Rat Medial Amygdala Are Responsive to Adult
Androgens.
Klüver, H., Bucy, P. C. (1939). Preliminary analysis of functions of the temporal lobes in
monkeys. J Neuropsychiatry Clinical Neuroscience 9:606-20.
LaBar, K. S., Gatenby, J. C., Gore, J. C., LeDoux, J. E., & Phelps, E. A. (1998). Human
amygdala activation during conditioned fear acquisition and extinction: a
mixed-trial fMRI study Neuron 20:937-945.
Langub, M. J., & Watson, R. J. (1992). Estrogen receptor-immunoreactive glia,
endothelia, and ependyma in guinea pig preoptic area and median eminence:
electron microscopy. Endocrinology 130:364-372.
Leedom, L., Lewis, C., Garcia-Segura, L M, Naftolin, F. (1994). Regulation of arcuate
nucleus synaptology by estrogen Annals of the New York Academy of Sciences
743:61-71.
Lorenz, B., Garcia-Segura, Luis Miguel, DonCarlos, L. L. (2005). Cellular phenotype of
androgen receptor-immunoreactive nuclei in the developing and adult rat brain The
Journal of Comparative Neurology 492:456-468.
Martinez, F. G., Hermel, E. E. S., Xavier, L. L., Viola, G. G., Riboldi, J., Rasia-Filho, A.
A., Achaval, M. (2006). Gonadal hormone regulation of glial fibrillary acidic protein
immunoreactivity in the medial amygdala subnuclei across the estrous cycle and in
castrated and treated female rats. Brain research 1108:117-126.
Merke, D. P., Fields, J. D., Keil, M. F., Vaituzis, A. C., Chrousos, G. P., & Giedd, J. N.
(2003). Children with classic congenital adrenal hyperplasia have decreased
amygdala volume: potential prenatal and postnatal hormonal effects The Journal of
clinical endocrinology and metabolism 88:1760-1765.
Milner, T., McEwen, B., Hayashi, S. (2001). Ultrastructural evidence that hippocampal
alpha estrogen receptors are located at extranuclear sites. The Journal of
Comparative Neurology 429:355-371
Mizukami, S., Nishizuka, M., Arai, Y. (1983). Sexual difference in nuclear volume and its
ontogeny in the rat amygdala Experimental neurology 79:569-575.

138

Mong, J., Nunez, J., McCarthy, M. (2002). GABA mediates steroid induced astrocyte
differentiation in the neonatal rat hypothalamus. J. Neuroendo 14:45-55.
Mong, J., Kurzweil, R. L., Davis, A. M., Rocca, M. S., McCarthy, M. M. (1996). Evidence
for Sexual Differentiation of Glia in Rat Brain. Horm Behav 30:553-562.
Mong, J. A., Glaser, E., McCarthy, M. M. (1999). Gonadal steroids promote glial
differentiation and alter neuronal morphology in the developing hypothalamus in a
regionally specific manner. J Neurosci 19:1464-1472.
Mong, J. A., Roberts, R. C., Kelly, J. J., McCarthy, M. M. (2001). Gonadal steroids
reduce the density of axospinous synapses in the developing rat arcuate nucleus:
an electron microscopy analysis. J Comp Neurol 432:259-267.
Mong, J. A., McCarthy, M. M. (1999). Steroid-induced developmental plasticity in
hypothalamic astrocytes: implications for synaptic patterning. Journal of
neurobiology 40:602-619.
Morris, J. A., Jordan, C. L., & Breedlove, S. M. (2008). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in adult rats The Journal of Comparative
Neurology, 506(5), 851-859. doi:10.1002/cne.21536
Morris, J., Jordan, C. L., Dugger, B., & Breedlove, S. M. (2005). Partial
demasculinization of several brain regions in adult male (XY) rats with a
dysfunctional androgen receptor gene. Journal of Comparative Neurology, 487(2),
217.
Naftolin, F., Brawler, J. R. (1978). The effect of estrogens on hypothalamic structure
and function American journal of obstetrics and gynecology 1:758-65.
Neufang, S., Specht, K., Hausmann, M., Güntürkün, O., Herpertz-Dahlmann, B., Fink,
G. R., Konrad, K. (2009). Sex differences and the impact of steroid hormones on
the developing human brain Cerebral cortex 19:464-473.
Nishida, H., Okabe, S. (2007). Direct astrocytic contacts regulate local maturation of
dendritic spines. The Journal of neuroscience 27:331-340.
Nishizuka, M., Arai, Y. (1981). Sexual dimorphism in synaptic organization in the
amygdala and its dependence on neonatal hormone environment. Brain research
212:31-38.
Nishizuka, M., Arai, Y. (1982). Synapse formation in response to estrogen in the medial
amygdala developing in the eye. Proceedings of the National Academy of
Sciences of the United States of America 79:7024-7026.

139

Paxinos, G., Watson W. (2005). The rat brain in stereotaxic coordinates. Amsterdam:
Elsevier.
Pfrieger, F. W., Barres, B. A. (1997). Synaptic efficacy enhanced by glial cells in vitro
Science 277:1684-1687.
Roselli, C., Salisbury, R. (1987). Genetic evidence for androgen-dependent and
independent control of aromatase activity in the rat brain. Endocrinology
121:2205-2210.
Rubinow, M. J., Juraska, J. M. (2009). Neuron and glia numbers in the basolateral
nucleus of the amygdala from preweaning through old age in male and female
rats: a stereological study The Journal of Comparative Neurology, 512:717-725.
Sarkey, S., Azcoitia, I., Garcia-Segura, L. M., Garcia-Ovejero, D., DonCarlos, L. (2008).
Classical androgen receptors in non-classical sites in the brain. Hormones and
Behavior 53:753-764
Scott, S. A., DeKosky, S. T., Sparks, D. L., Knox, C. A., Scheff, S. W. (1992). Amygdala
cell loss and atrophy in Alzheimer's disease Annals of neurology 32:555-563.
Segovia, S., Guillamón, A. (1993). Sexual dimorphism in the vomeronasal pathway and
sex differences in reproductive behaviors Brain research Brain research reviews
18:51-74.
Simerly, R. B., Chang, C., Muramatsu, M., Swanson, L. W. (1990). Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ
hybridization study. The Journal of Comparative Neurology 294:76-95.
Sweeten, T. L., Posey, D. J., Shekhar, A., McDougle, C. J. (2002). The amygdala and
related structures in the pathophysiology of autism. Pharmacology, biochemistry,
and behavior 71:449-455.
Tranque, P. A., Suarez, I., Olmos, G., Fernandez, B., Garcia-Segura, L M. (1987).
Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in
the rat brain Brain research 406:348-351.
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S., Barres, B. A. (2001). Control of
synapse number by glia. Science, 291:657-661.
von Ziegler, N. I., Lichtensteiger, W. (1992). Asymmetry of brain aromatase activity:
region- and sex-specific developmental patterns Neuroendocrinology, 55:512-518.
Weiner, L. (1983). Structural and cytochemical changes in astrocytes from the brain
periventricular zone of the copper deficient blotchy mouse. The Anatomical record
205:355-61

140

West, M. (1993). New stereological methods for counting neurons. Neurobiology of
Aging, 14:275-285.
Wood, R. I., Newman, S. W. (1995). Androgen and Estrogen Receptors Coexist within
Individual Neurons in the Brain of the Syrian Hamster. Neuroendocrinology,
62:487-497.

141

CHAPTER 4: DIFFERENTIAL HORMONE ACTION IN THE LEFT AND RIGHT
AMYGDALAE
Abstract
The amygdala is a highly connected and plastic region involved in numerous
behaviors and implicated in disorders including depression, schizophrenia and autism.
Research has repeatedly demonstrated that the anatomy and function of the amygdala
is sexually dimorphic in humans and laboratory species. Sexual dimorphisms in the
amygdala have been important in our understanding of how gonadal hormones
influence the brain. In this review, I summarize literature demonstrating that the
amygdala of the two hemispheres may also be distinct in their response to gonadal
hormones and argue that this distinction may be informative to future research
concerning the amygdala and gonadal hormone influence.
Introduction
Gonadal hormones alter many aspects of brain neuroanatomy and function in
complex ways; from modulation of GABAA receptor subunit expression (McIntyre,
Porter, & Henderson, 2002) and optic-nerve head astrocyte activity (Agapova et al.,
2006) to producing sex differences in corpus callosum structure (Chura et al., 2010) and
stimulus processing networks (Kilpatrick et al., 2006). While it is understood that
gonadal hormones are responsible for many sex differences throughout the brain
(Gorski et al., 1978; Hofman and Swaab, 1989; Allen et al., 1991; Allen and Gorski,
1991; Cooke and Woolley, 2005; Alonso-Nanclares et al., 2008; Morris et al., 2008a)
several complications prevent a complete understanding of gonadal hormone influence.
One often ignored issue is laterality of gonadal hormone effects. Hemisphere-specific
hormone effects exist in multiple brain regions (Chura et al., 2010; Neufang et al., 2009;

142

Xiao and Jordan, 2002), but the amygdalae in particular appear highly lateralized.
In a variety of species, the amygdalae are responsive to gonadal hormones
during development and in adulthood (humans: Giedd et al., 1997; cats: Rees and
Michael, 1984; mice: Morris et al., 2008b; hamsters: Zehr et al., 2006; rats: Cooke et al.,
2003). As is the case with many sexually dimorphic regions, it has often been assumed
that gonadal hormones act symmetrically upon the amygdalae in the two hemispheres.
However, both human and rodent models indicate that sex hormones may influence the
anatomy and function of the amygdala in a hemisphere-specific manner. In this review I
present an overview of examples where the amygdala exhibits sex differences in
conjunction with hemisphere differences and discuss findings in both humans and
rodents.
Hemispheric Sex Differences in the Human Amygdala
The primate amygdala contains both estrogen receptor (ER) and androgen
receptor (AR; Donahue, 2000; Abdelgadir et al., 1999) and several reports document
sex differences in the size of the amygdala relative to other brain regions (Giedd et al.,
1996; Goldstein et al., 2001; Gur et al., 2002; Peper et al., 2009). Furthermore, sex
differences in amygdala volume using fMRI are related to serum testosterone levels
(Neufang et al., 2009), and men with Klinefelter syndrome (XXY sex chromosome
complement) show reduced amygdala volumes with the strongest differences in the left
hemisphere (Shen et al., 2004; Patwardhan et al., 2002).
Unfortunately, results from imaging techniques and tissue analysis often differ.
Brabec and colleagues (Brabec et al., 2010) assessed male and female mice
amygdalae using two tracing parameters in post-mortem tissue and found no sex

143

differences in amygdala volume and argue for refinement of imaging measurements.

hormone fluctuations. There is evidence for menstrual-cycle influence on amygdala
function (Dreher et al., 2007; Goldstein et al., 2010) and portions of the rodent
amygdala are highly plastic and hormone-responsive (Cooke, 2007). Additional studies
with hormonal-controls may be necessary before conclusions can be reached. Despite
this difficulty in clear delineation of sex differences in human amygdala anatomy,
imaging work continues to demonstrate sex differences in amygdala function.
Moreover, many of these reports reveal lateralization of amygdala function, which is
sex-specific.
Humans - Faces and Emotional Memory
As evidence of its role in social behavior, the amygdala is active in most reports
of brain response to facial affect and emotional stimuli, and several well-documented
lateralized sex differences come from this area of research. For example, while
exposure to fearful faces activated the left amygdala in both sexes, exposure to happy
faces produced a laterality effect with the right amygdala being more activated than the
left, but only in males. Interestingly, females showed balanced amygdala activation
during exposure to happy faces (Killgore and Yurgelun-Todd, 2001). Subsequent
reports found similar sex differences and laterality when examining fearful faces and
fearful eyes (Williams et al., 2005; Hardee et al., 2007) and demonstrated that women
with Turner syndrome, who lack an X chromosome, show greater right amygdala and
less left amygdala activation in response to fearful faces compared to control subjects,
suggesting a possible role for sex chromosomes in this laterality (Skuse et al., 2005).

144

Results in adolescents show similar instances of laterality. Males had greater
right hemisphere activation when viewing faces, and this laterality effect was strongest
when viewing angry faces. Adolescent females equally activated both amygdalae
(Schneider et al., 2011). Additionally, there is an age-related increase in left cortical
activation relative to left amygdala activity when viewing fearful faces in adolescent girls,
but not in boys (Killgore and Yurgelun-Todd, 2004; Yurgelun-Todd and Killgore, 2006)
suggesting as females age the cortex becomes increasingly involved in facial
processing but that this does not occur in males. However, two additional reports
illustrate the complexity of laterality in facial processing.
Schneider and colleagues (Schneider et al., 1997) found that mood induction,
during which subjects were asked to mimic the mood shown in a stimulus face, resulted
in greater activation in the left amygdala in men and women. This may represent a shift
to left-processing for men when asked to imitate a mood or it could be due to a small
sample where results from both sexes were combined. Secondly, there is evidence that
sex of the stimulus face may influence hemisphere effects although the female-left and
male-right activation pattern persisted (Armony and Sergerie, 2007).
A sex difference in the pattern of lateralized amygdala response extends beyond
response to faces. Positron emission tomography reports examining the sexes
separately were the first to notice a lateralized response of the amygdala to emotionally
arousing pictures. Both Cahill (Cahill et al., 1996) and Hamann (Hamann et al., 1999)
found right-preferential amygdala activation in males while separate MRI reports found
left amygdala activation in response to similar stimuli in females (Canli et al., 2000;
Canli, 1999). These reports were followed by demonstrations that amygdala activation

145

in the left hemisphere for women and right hemisphere for men was related to memory
for emotional films (Cahill et al., 2001; 2004). Similar patterns of sex-specific
lateralization were seen in response to negative images (Canli et al., 2002; Mackiewicz
et al., 2006). Additionally, after exposure to a disturbing image or story, an immediate
early spike of fMRI activity was strongest in the right amygdala for men and left
amygdala for women (Gasbarri et al., 2007).
Interestingly, although emotional memory is lateralized in this manner, Canli and
colleagues (Canli et al., 2000) found that self-reports of emotional arousal correlated
with left amygdala activity in both men and women. This left shift for men may be
similar to the left shift seen when males are asked to mimic the mood of stimulus faces
(Schneider et al. 1997) and suggests that when awareness of self is required, males
shift to using the left amygdala. Finally, a male-right and female-left bias in amygdala
activation also influences long term memory for central story information in men and
peripheral story detail in women (Cahill, 2003).
Two more interesting and related findings include a study of psychosis that found
left amygdala enlargement in women with mood-incongruent delusions but not in similar
males (Gibbs et al., 2008). In concordance with this, the administration of procaine, an
anesthetic agent that reliably produces psycho-sensory and hormonal responses,
including delusions, resulted in greater left amygdala activity in women compared to
men (Adinoff and Devous, 2003). Thus, there is repeated evidence of a female-left,
male-right bias in the perception of various types of emotional/social material even
when such perceptions may be psychotic or pharmacologically induced. There is also
evidence to suggest that the left amygdala may be tied to self-awareness, since it is

146

activated in both sexes during mood induction and self-reports of arousal. Additional

other brain regions, which may be related to this effect.
Humans - Functional Connectivity
Kilpatrick and colleagues (Kilpatrick et al., 2006) examined which brain regions
exhibit correlated activity with the amygdala during rest; a measure understood to imply
connectivity between the regions. The right amygdala in males showed greater resting
state functional connectivity to other brain regions than in females, whereas the left
amygdala in females showed greater functional connectivity than in males. Intriguingly,
the male-right amygdala bias was strongly associated with regions linked to the
detection of external stimuli, such as the sensorimotor cortex and striatum. In contrast,
the female-left bias of the female amygdala involved regions linked to internal
monitoring such as the hypothalamus (Kilpatrick et al., 2006). As the authors note,
these results suggest not only a male-right and female-left division of amygdala utility,
but also that the role of the two hemispheres may be divided into monitoring external
versus internal information.
Importantly, these findings were later replicated in a homosexual sample (Savic
and Lindström, 2008). Specifically, homosexual men are more like heterosexual
women in exhibiting more widespread connections from the left amygdala whereas the
opposite is true for homosexual women, who are more like heterosexual men.
Interestingly, gonadal hormone levels during development are linked to sexual
orientation in adulthood (Finn et al., 2002; Manning et al., 2007) perhaps the same
hormones may alter patterns of amygdala connectivity in a hemisphere-specific manner

147

(Kilpatrick et al., 2006). A link between the amygdala, sexual behavior and hormones is
present in several species (Green et al., 1957; Lehman and Winans, 1980; Cooke et al.,
2003; Sartorius et al., 2008), but the human amygdala sexual response is complex and
possibly hemisphere specific.
Humans - Sexual Response

long association with sexual behavior (Klüver and Bucy, 1938), one might expect

evidence is sparse but intriguing. One group using fMRI found that males demonstrated
greater amygdala activation in response to visual sexual stimuli compared to females
with the sex difference being greatest in the left hemisphere (Hamann et al., 2004;
Hamann, 2005). However, sexual stimulation of the penis resulted in decreased
regional cerebral blood flow in the right amygdala and orgasm results in deactivation of
the left amygdala in males (Georgiadis and Holstege, 2005; Holstege et al., 2003).
These findings might suggest that in males appetitive sexual responses are related to
left amygdala activation and consummatory behavior may require some balance of right
and left amygdala deactivation. However, subsequent examination failed to find a
relationship between male orgasm and the amygdala (Georgiadis et al., 2007) and other
reports of appetitive response have been conflicting. For example, homosexual men
demonstrated less activity in the left amygdala compared to heterosexual men when
both groups where shown male homosexual erotic films (Hu et al., 2008). However,
Paul and colleagues (Paul et al., 2008) found the opposite result with right amygdala

148

activation in heterosexual men shown male homosexual erotic films, which they
attribute to an aversive response.

to assess and inconsistent (Karama et al., 2002; Kim et al., 2006; Miyagawa et al.,
2007; Rupp and Wallen, 2007). Amygdala activity during sexual stimuli may underlie a
general emotional arousal (Walter et al., 2008) and sociological effects can influence
the region during sexual stimuli presentation (Beauregard et al., 2001). Until further
reports address these issues, reaching conclusions about hemisphere-specific effects
remains difficult.
Conclusions from Humans
Although facial processing, emotional memory and functional connectivity exhibit
reliable lateralized sex differences in humans, there are several concerns. For
example, hemisphere effects in the human imaging literature do not appear to be
particularly robust. Recent meta-analyses of emotional processing are discrepant, with
hemispheric differences seen in some (Baas, 2004; Fusar-Poli et al., 2009) and not in
others (Wager et al., 2003; Sergerie et al., 2008). One explanation put forth is that
laterality in the human imaging literature may not indicate separate hemisphere
functions but may be due to different activation decay in the left (slow decay) verse right
(faster decay) amygdalae. Sergerie and colleagues (Sergerie et al., 2008) argue that
the commonly used block-design trial format could produce artificial laterality if
activation decays differently in the two hemispheres.
Some studies demonstrating hormonal influence on the amygdala in humans
have not supported hemisphere effects. For example, adolescent females with

149

congenital adrenal hyperplasia (CAH), a genetic disorder that results in elevated
androgen exposure prenatally, displayed amygdala responses to faces indistinguishable
from similar aged control males. Furthermore, no laterality was seen in control males or
CAH females (Ernst et al., 2007). Similarly, a positive correlation was found between

suggesting circulating hormones may influence amygdala functionality (Derntl et al.,
2009). However, no laterality was seen. While both these studies provide evidence for
hormonal influence on amygdala functionality, the lack of laterality during processing of
faces diverges from other reports (Schneider et al., 2011; Killgore and Yurgelun-Todd,
2001) and casts doubt on a hypothesis of lateralized hormone action in the amygdala.
Thus, the human literature currently provides an unclear picture of lateralized sex
differences in the amygdala. However, including only studies which examined males
and females, and which found both sex and hemisphere differences, does provide fairly
consistent evidence of a male-right, female-left bias (Table 2). Additional studies using
male and female subjects engaging in wide variety of amygdala-related tasks are
needed. Additionally, evidence of lateralized sex differences in human anatomy are
needed to bolster support for hemisphere specific gonadal hormone action, but as the
rodent literature demonstrates, these may require increased resolution in imaging
techniques or a focus on specific subregions in human tissue.
Hemispheric Sex Differences in the Rodent Amygdala
Rodents - Amygdala Function
Contrasting the human literature, lateralized sex differences in rodent amygdala
anatomy are prevalent, and a few interesting reports related to amygdala function also

150

reveal lateralized sex differences. Gerendai and colleagues (Gerendai et al., 1995)
used unilateral surgical deafferentation of the temporal lobes, including portions the
corticomedial amygdala, in combination with left or right hemicastration to investigate
neural control over gonadal functions. In females, right or left temporal lobe
deafferentation in conjunction with right or left ovariectomy reduced serum luteinizing
hormone levels. In males, temporal lobe deafferentation was effective at reducing
testosterone production only if the left testis was removed, but hemisphere
deafferentation effectiveness varied with age. Specifically, in prepubertal males
unilateral orchidectomy (left testis) reduced serum testosterone ( measured in vitro) only
with right temporal lobe deafferentation while in adult males unilateral orchidectomy (left
testis) reduced testosterone only with left temporal lobe deafferentation (Gerendai et al.,
1995). These results suggest that while both temporal lobes are involved in gonadal
hormone regulation in females, in prepubertal males the right hemisphere may be
important and this role switches to the left hemisphere in adulthood.
Furthermore, although reduction of luteinizing hormone levels was hemisphere
independent, compensatory ovarian hypertrophy after unilateral ovariectomy was
eliminated only with right temporal lobe lesions (Gerendai et al., 1995). Similar
evidence shows that lesions of the right medial amygdala disrupt ovulation more
effectively than lesions of the left amygdala in adult females (Sanchez and Dominguez,
1995). Together the findings demonstrate some right hemisphere dominance in control
of the ovaries. Given the important role of the hypothalamus in ovulation and the strong
connections between the amygdala and hypothalamus, gonadal hormones may alter

151

connectivity between the right amygdala and hypothalamus to produce this hemispheric
dominance.
Another aspect of amygdala function, the stress response, may also exhibit
lateralized sex differences. After hemisphere specific 6-hydroxydopamine lesions to the
basolateral amygdala, right-lesioned males had increased time in the open arms and
reduced time in the closed arms of an elevated plus maze compared to sham-lesioned
animals. Interestingly, right-lesioned females spent less time in the open arms and
more time in the closed arms compared to shams, suggesting that right amygdala
lesions are anxiolytic in males and anxiogenic in females (Sullivan et al., 2009).
Additionally, plasma ACTH levels in male rats with lesions to the right amygdala
were significantly lower than left-lesioned males. However, in females left and right
lesions reduced ACTH levels equally. Finally, after 30 minutes of restraint stress, leftlesioned males trended towards a higher stress response compared to right lesioned
males while right-lesioned females had higher stress responses than left-lesioned
animals, resulting in a significant sex by hemisphere effect (Sullivan et al., 2009).
These reports demonstrate that lesions of the amygdala are able to influence
both gonadal hormone and corticotropin production in complex hemisphere and sexspecific patterns, and that the stress response is also sensitive to opposing hemispherespecific amygdala lesions in male and female rats. Several other domains of amygdala
function such as fear have been thoroughly researched and there is evidence of
laterality in the rodent amygdala fear response (Baker, 2004; Scicli et al., 2004;
Coleman-Mesches and McGaugh, 1995; Adamec et al., 2005; Berlau and McGaugh
2006). Unfortunately, while several of these reports show a right-bias in their male

152

subjects, they lack female subjects, precluding detection of sex differences. Therefore,

fear response.
Rodents - Amygdala Anatomy
Modern imaging techniques have been used to examine amygdala volume in
non-humans and, as in humans, results are conflicting. Magnetic resonance
microscopy examining amygdala volume in male and female mice revealed a sex
difference in amygdala volume (females < males) with the greatest sex difference in the
right hemisphere after corrections for total brain volume (Koshibu, 2004). However, a
larger study found no sex or hemisphere differences in the amygdala in the mouse brain
(Spring et al., 2007). As in humans, it is possible that current imaging techniques lack
the resolution to reliably identify sex differences in amygdala anatomy, especially if sex
differences are prominent only in certain amygdala subregions.
The medial amygdala (MeA) may be such a region. The MeA contains a dense
concentration of both ER and AR and exhibits several lateralized sex differences
(Canteras et al., 1995; Simerly et al., 1990; Cooke et al., 2003). Moreover, several of
these lateralized sex differences are present before adulthood. Early in development,
the rat MeA displays sex differences in aromatase activity, an enzyme that converts
testosterone, an AR agonist, into estradiol, an ER agonist. At gestational day 22 males
have greater aromatase activity in the left MeA than the right, while females have
greater aromatase activity in the right MeA than the left. By postnatal day (PND) 6, the
hemisphere difference in males has reversed (right > left), but by PND 15 the two sides
seem equivalent. As in males, the female hemisphere difference reverses from right >

153

left to left > right by PND 6. However, this laterality in aromatase activity remains
present at PND 15 in females (von Ziegler and Lichtensteiger, 1992). While these
results suggest estrogen action in the medial amygdala is intricately arranged and may
occur at different times for each hemisphere, the anatomical and behavioral implications
of these findings are unclear.
Early gonadal hormones influence many aspects of MeA neuroanatomy. For
example, the right posterodorsal medial amygdala (MePD) is larger in 25-day-old male
rats than in females, but only in the right hemisphere (Johnson et al., in prep. a).
Furthermore, neither sex differences nor laterality in MePD volume appear dependent
upon functional ARs in juveniles (Johnson et al., in prep. a). While the right hemisphere
displays greater sex differences in MePD volume at this age, this is not true for other
measures of MePD anatomy.
Dendrite length and complexity are greater in the left amygdala of juvenile males
compared to females, and male rats have more excitatory synapses in the left MePD
than females (Cooke et al., 2007; Cooke and Woolley, 2005). There is also a sex
difference in miniature excitatory post-synaptic current frequency (females < males) and
synapse number found only in the left hemisphere but no difference in the number of
neurons in this hemisphere (Cooke et al., 2007; Cooke and Woolley, 2005). In contrast,
in the right MePD, juvenile males have more neurons than juvenile females but there
are no sex differences in dendritic morphology (Cooke and Woolley, 2005; Cooke et al.,
2007). Finally, there are hemisphere and sex differences in juvenile MePD astrocytes
as well. Juvenile male rats have more astrocytes in the left and right hemisphere than
do juvenile females. However, both sexes have more astrocytes in the right

154

hemisphere than the left (Johnson et al., in prep. a). Interestingly, functional ARs are
not necessary for the lateralization of astrocyte numbers in the juvenile MePD (Johnson
et al., in prep. a).
These complex sex by hemisphere interactions are also prominent in the adult
medial amygdala. MePD volume is sexually dimorphic in rats, with males having larger
MePD regional volume than females (Hines et al., 1992), which is also lateralized as the
right MePD is larger than the left (Morris et al., 2005; Johnson et al., 2008). The right
MePD also has a greater rostral-caudal extent than the left in male rats and evidence
from Tfm male rats shows that while functional ARs are necessary for masculinized
MePD volume, they are not necessary for the lateralization of MePD volume (Morris et
al., 2005). In mice there is also a sex difference and lateralization, but only in females is
the left MePD larger than the right (Morris et al., 2008b).
Although in male rats the right MePD is larger in volume than the left, the left
MePD contains more neurons than the right in both sexes (Morris et al., 2008a). There
are also sex differences in MePD neuronal soma size but reports of laterality are
conflicting. Cooke and colleagues (Cooke et al., 2003) found larger somata in the left
hemisphere compared to the right in male rats but this laterality was not seen in
subsequent experiments (Morris et al., 2008a; b). Unspecified glia numbers are also
lateralized in adults, with more in the right MePD than the left of both male and female
rats, contrasting the greater number of neurons in the left MePD (Morris et al., 2008a).
In the adult rat, MePD astrocytes display particularly intriguing patterns of
lateralization. As in juveniles, both male and female adults have more astrocytes in the
right MePD than the left, and within the right hemisphere males have more astrocytes

155

than do females. In contrast, there is no sex difference in astrocyte number in the left
hemisphere. However, the left MePD exhibits more complex astrocytes compared to
the right. Furthermore, the hemisphere difference in astrocyte complexity occurs in the
context of a sex difference in the left hemisphere, where males have more complex
astrocytes than do females (Johnson et al., 2008).
Thus, the laterality in astrocyte number (left < right) mirrors MePD regional
volume (left < right), while the laterality in complexity of astrocyte arbors (right <left) may
mirror neuron number (right < left); Morris et al., 2008a; Johnson et al., 2008).
Importantly, the sex differences in astrocyte number and arbor complexity are both AR
dependent, but the hemisphere differences appear to be AR independent (Johnson et
al., 2008). These findings in adults contrast with juveniles where ARs are not necessary
for masculinized astrocyte numbers, and astrocyte arbors are neither lateralized nor
sexually dimorphic (Johnson et al. in prep. a). These differences between juveniles and
adults suggest lateralized sex differences in astrogenesis and arbor proliferation during
puberty, which are AR dependent. Specifically, functional ARs appear to mediate
astrogenesis in the right MePD of males and females during puberty, but to a greater
extent in males, therefore maintaining the sex differences seen in this hemisphere in
juveniles. In the left MePD, ARs mediate an increase in astrocyte arbor complexity
during puberty, generating new sex differences in adulthood.
Perhaps the sex differences and laterality in MePD astrocytes indicate different
inter- and intra-amygdala connections in the left and right hemispheres brought on by
gonadal hormone action. Since astrocytes are closely linked to synapse formation
(Nishida and Okabe, 2007; Halassa et al., 2007) such an explanation fits with the

156

laterality in synapses and excitatory post-synaptic currents seen in juvenile animals
(Cooke and Woolley, 2005). However, multiple reports suggest that dendritic spines are
neither sexually dimorphic nor lateralized in juvenile or adult animals (Arpini et al., 2010;
Cooke et al., 2007), and that gonadal hormones may specifically promote sex
differences in MePD synapses on dendritic shafts (Nishizuka and Arai, 1981).
Conclusions from Rodents

luteinizing hormone release, as well as stress response, may be lateralized (Gerendai
et al., 1995; Sullivan et al., 2009). Furthermore, several aspects of amygdala
neuroanatomy exhibit lateralized sex differences, primarily in the MePD (Table 3).
Many of these effects are lateralized before puberty, demonstrating lateralized hormonal
influence during development in the two amygdalae (Johnson et al., in prep. a; Cooke
and Woolley, 2005; Cooke et al., 2007).
While many of the sexual dimorphisms in MePD measures are at least partially
dependent upon ARs, whenever Tfm males are examined, they exhibit similar
lateralization compared to males (Johnson et al., 2008, in prep. a; Morris et al., 2005),
which suggests that ERs mediate the masculinization of laterality in this nucleus. In the
case of astrocytes, it appears that ARs mediate an increase in astrocyte number in the
right MePD and astrocyte complexity in the left MePD sometime during puberty.
However, the hemisphere differences are present in both juvenile and adult Tfm males;
they are simply smaller in absolute terms in juveniles than in adults (Johnson et al.,
2008, in prep. a). Therefore it is unlikely that AR action alone is responsible for

157

differences between the left and right MePD. Similar conclusions may hold for MePD
regional volume (Morris et al., 2005).
Perhaps the most intriguing clue comes from the previously mentioned
lateralization of aromatase activity in the MeA during development (von Ziegler and
Lichtensteiger, 1992). If aromatase activity indicates early estrogen action, then it may
be the lateralization of aromatase that establishes the earliest hemisphere differences in
the region. Furthermore, hormonal influence during this initial lateralization may
orchestrate the progression of additional laterality as gonadal hormones act on the
amygdalae during puberty and adulthood. Identifying the mechanisms through which
this priming may occur will be challenging but potentially informative.
It should be noted that the lateralized sex differences in the rodent amygdala
literature are generally restricted to the medial subregion. Although laterality is present
in other subregions (Ji, 2009; Berlau and McGaugh, 2006; LaLumiere, 2005) the lack of
sex differences in these regions may be due to frequent use of only male animals. It is
possible that if both sexes were examined, lateralized sex differences would emerge for
the basolateral or central amygdala. While this outcome would greatly strengthen the
case for hemisphere-specific gonadal hormone action in the amygdala, given the
interconnectedness of the amygdala, the effects in the MeA alone may lead to
lateralized function in the region as a whole.
Combining the Human and Rodent Literature
Both the human and rodent literature suggests that gonadal hormones influence
the amygdala in a hemisphere-specific manner. The human literature shows lateralized
sex differences in amygdala function, with the most reliable and robust findings in the

158

consistent male-right and female-left pattern in these reports suggests that men and
women use different hemispheres for processing the same information, and results from
homosexual and clinical populations appear to corroborate this assumption (Savic and
Lindström, 2008; Skuse et al., 2005).
Evidence from rodents also indicates lateralized gonadal hormone influence in
the amygdala. Hormone manipulations and Tfm males repeatedly illustrate that
gonadal hormones are responsible for sex differences in several components of MeA
neuroanatomy and that these measures are frequently lateralized (Cooke et al., 2003;
Morris et al., 2005; Johnson et al., 2008). Therefore, it appears that in both humans
and rodents the same gonadal hormones that masculinize the brain during development
influence the neuroanatomy of the left and right amygdala differentially. Still, the most
difficult questions remain: how are hormones achieving this hemisphere-specific
influence and to what purpose?
Possible Mechanisms
One explanation of lateralized sex differences in the amygdala is that sex
chromosomes are primarily responsible for amygdala laterality with minimal influence of
traditional gonadal hormone action. Indeed, there is evidence for regulation of cerebral
laterality by sex chromosomes (van Rijn et al., 2006; Annett, 2008; Agate, 2003). For
example, women with Turner syndrome (X chromosome monosomy) exhibit increased

syndrome (XXY) trended toward reduced brain asymmetry in frontal regions (Rezaie et
al., 2009).

159

Another report specifically implicated human genes coding for Protocadherin11
on the X and Y chromosomes in establishing cerebral asymmetry (Crow, 2010).
However, the Protocadherin11 hypothesis is based on verbal and spatial ability
variances in sex chromosome aneuploidies, and related studies suggest these effects
may be restricted to the neocortex (Murphy et al., 1994). Perhaps the genes
responsible for cortical laterality (Protocadherin11X,Y) are not responsible for
lateralization of sub-cortical structures. In support of this, evidence from homosexual
populations suggests sex chromosome complement alone does not determine male or
female typical patterns of amygdala laterality (Kilpatrick et al., 2006; Savic and
Lindström, 2008) and differences in behavior between XXY and XY mice are due mostly
to androgen deficiency rather than genetic complement (Liu et al., 2010).
A second possibility is that gonadal hormone receptors control the development
of sex differences and laterality in the amygdala. In humans, AR polymorphisms and
handedness are related; reduced AR binding efficiency is associated with increased lefthandedness (Medland et al., 2005). However, evidence from Tfm male rats and mice
shows that while functional ARs are necessary for many of the sex differences seen in
portions of the amygdala, a loss of AR function does not eliminate laterality of these
measures (Morris et al., 2005; Johnson et al., 2008, in prep. a).
It is possible that estrogens and ER are mediating lateralized sex differences in
the amygdala. The hemisphere-specific sex differences in aromatase activity in the
MeA during development suggest estrogen influence (von Ziegler and Lichtensteiger,
1992). Estrogens have cell and region-specific effects on neuroanatomy (Raz et al.,
2008; Micevych et al., 2010) reviewed in (McCarthy, 2010), but exactly how estrogens

160

might, for example, increase astrocyte numbers in the MePD only in the right
hemisphere is still unclear.
An explanation of lateralized gonadal hormone receptor activity could involve
differential expression of gonadal hormone co-regulators in the left and right MePD.
These co-regulators are found in both neurons and astrocytes (Grenier et al. 2006), and
are capable of altering the actions of steroid receptors in complex ways (reviewed in
Tetel 2009). Members of the steroid receptor co-activator family (SRCs) are found in
the amygdala of rodents, including the MePD, and modify gene expression and
influence behavior (Meijer et al. 2000; Tetel et al. 2004; Molenda et al. 2002; Auger et
al. 2000). However, I am not aware of any reports demonstrating laterality in gonadal
hormone co-regulator expression or activity in the amygdala. It is possible that such
differences are present and awaiting identification.
Tentative Explanations and Conclusions
At this time, explaining the significance of lateralized sex differences in the
amygdala involves mostly speculation. Lateralization is proposed to enhance cognitive
capacity and brain efficiency (reviewed in Vallortigara and Rogers, 2005), and sex
differences in the brain and behavior often (although certainly not always, see De Vries

Perhaps, for reproductive purposes, gonadal hormones act differently in the left and
right amygdala.
Humans and rodent males primarily use stimulus information from the external
environment to locate and assess potential mates. In contrast, females often assess
internal information to determine if mating is a viable option. Indeed, human females

161

alter mating strategies and choices depending upon current ovulatory state (PentonVoak, 2000; Penton-Voak et al., 1999; Haselton et al., 2007; Gangestad et al., 2002).
In rodents, the influence of internal status on female mating behavior is clearly
demonstrated by the ability to make an unreceptive female rat receptive simply by
injecting steroid hormones. In contrast male rats with greatly reduced levels of
testosterone still retain reproductive function.
The lateralized sex differences in the amygdala may be the result of a fine tuning
allowing a specific amygdala hemisphere to emphasize important information relevant

colleagues (Kilpatrick et al., 2006), in which the male amygdala is strongly linked to
regions that monitor external stimuli and the female amygdala is linked to internal
monitoring regions, supports this idea.
Importantly, while these lateralized sex differences may have initially evolved to
allow efficient processing of sexually specific social/mating information, it is possible
other amygdala behaviors have become lateralized and sexually dimorphic as well. In
humans the functional connectivity data along with mood induction and self-report
results suggest that the left amygdala may be biased towards homeostatic activity and
that males utilize this hemisphere only when self awareness is necessary. Is this the
result of a relative silencing of the left amygdala sometime during development in
males? Furthermore, are the numerous sexual dimorphisms in cellular anatomy

rewiring of this hemisphere in males?

162

Anxiety disorders, depression, schizophrenia and autism are all linked to the
amygdala (Charney et al., 1993; Canli et al., 2005; Phillips et al., 1999; Amaral et al.,
2008) and show sex biases, with greater prevalence in males (schizophrenia, autism) or
in females (anxiety, depression; American Psychological Association, 2000).
Furthermore, depression, autism and schizophrenia have shown interesting laterality
effects in neuroanatomy (Versace et al., 2010; Nacewicz et al., 2006; Hulshof et al.
2001f). If sex hormones are reshaping the amygdalae in a hemisphere-dependent
manner, understanding laterality and sex differences in amygdala neuroanatomy may
be important for developing more effective treatments for amygdala-related disorders.
Paraphrasing Larry Cahill (Cahill, 2006), the available evidence indicates that studies of
amygdala sexual dimorphisms risk conclusions that are incomplete at best, and wrong
at worst, if they fail to address potential influence of both sex and hemisphere. It is our
hope that this collection of findings will compel researchers to record and report
hemisphere information in the future when investigating gonadal hormone influence in
the amygdala.

163

APPENDIX

164

Table 2 - Lateralized sexual dimorphisms in the human amygdala from experiments
examining both men and women

Differences in color indicate significant sex differences. In cases where the sex
difference favors women (males < females) text is colored pink. In cases where the sex
difference favors men (females < males) text is colored blue. 1 Schneider et al., 2011, 2
Cahill et al., 2001, 3 Canli et al., 2002, 4 Kilpatrick et al., 2006, 5 Savic and Lindström,
2008, 6 Gasbarri et al., 2007, 7 Gibbs et al., 2008, 8 Adinoff and Devous, 2003 *Sex
differences was in the volume of the left amygdala when females and males with mood
incongruent delusions were compared.

165

Table 3 - Lateralized sexual dimorphisms in the rodent medial posterodorsal amygdala
(MePD).

For each measure, differences in size indicate significant hemisphere differences within
sex. Differences in color indicate significant sex differences. In all cases the sex
difference favors male rodents. 1 Johnson et al. in prep. a, 2 Cooke et al. 2007, 3
Cooke et al. 2005, 4 Morris et al. 2005, 5 Johnson et al. 2008, 6 Morris et al. 2008.
*Johnson et al. in prep. a found laterality in both sexes but not sex difference in the left
MePD. Cooke et al. 2007 found no laterality in females but did find a sex difference in
the left MePD. **Dendrite measures were dendritic length, number of dendritic tips and
intersections in Scholl analysis. ***Morris et al. 2005 found no laterality in females.
Johnson et al. 2008 found laterality in females.
166

REFERENCES

167

REFERENCES
Abdelgadir, S. E., Roselli, C. E., Choate, J. V., Resko, J. A. (1999). Androgen receptor
messenger ribonucleic acid in brains and pituitaries of male rhesus monkeys:
studies on distribution, hormonal control, and relationship to luteinizing hormone
secretion. Biol. Reprod 60:1251-1256.
Adamec, R. E., Blundell, J., Burton, P. (2005). Neural circuit changes mediating lasting
brain and behavioral response to predator stress. Neurosci Biobehav Rev
29:1225-1241.
Adinoff, B., Devous, M. (2003). Gender differences in limbic responsiveness, by
SPECT, following a pharmacologic challenge in healthy subjects. Neuroimage
18:697-706.
Agapova, O. A., Malone, P. E., Hernandez, M. R. (2006). A neuroactive steroid
5alpha-androstane-3alpha,17beta-diol regulates androgen receptor level in
astrocytes. Journal of neurochemistry 98:355-363.
Agate, R. J. (2003). Neural, not gonadal, origin of brain sex differences in a
gynandromorphic finch. Proceedings of the National Academy of Sciences
100:4873-4878.
Allen, L. S., Richey, M. F., Chai, Y. M., Gorski, R. A. (1991). Sex differences in the
corpus callosum of the living human being. J Neurosci 11:933-942.
Allen, L. S., Gorski, R. A. (1991). Sexual dimorphism of the anterior commissure and
massa intermedia of the human brain. J. Comp. Neurol 312:97-104.
Alonso-Nanclares, L., Gonzalez-Soriano, J., Rodriguez, J. R., DeFelipe, J. (2008).
Gender differences in human cortical synaptic density. Proc Natl Acad Sci USA
105:14615-14619.
Amaral, D., Schumann, C., Nordhal, C. (2008). Neuroanatomy of autism. Trends
Neurosci 31:137-145.
Annett, M. (2008). Tests of the right shift genetic model for two new samples of family
handedness and for the data of McKeever (2000). Laterality 13:105-123.
Armony, J. L., Sergerie, K. (2007). Own-sex effects in emotional memory for faces.
Neurosci Lett 426:1-5.

168

Arpini, M., Menezes, I. C., Dall'Oglio, A., Rasia-Filho, A. A. (2010). The density of
Golgi-impregnated dendritic spines from adult rat posterodorsal medial
amygdala neurons displays no evidence of hemispheric or dorsal/ventral
differences. Neurosci Lett 469:209-213.
American Psychological Association. (2000). Diagnostic and statistical manual of mental
disorders: DSM-IV-TR.
Baas, D. (2004). Lateralization of amygdala activation: a systematic review of functional
neuroimaging studies. Brain Research Reviews 45:96-103.
Baker, K., Kim, J. (2004). Amygdalar Lateralization in Fear Conditioning: Evidence for
Greater Involvement of the Right Amygdala. Behav. Neurosci. 118:15-23.
Beauregard, M., Lévesque, J., Bourgouin, P. (2001). Neural correlates of conscious
self-regulation of emotion. Journal of Neuroscience 21:165.
Berlau, D., McGaugh, J. (2006). Enhancement of extinction memory consolidation: The
role of the noradrenergic and GABAergic systems within the basolateral
amygdala. Neurobiol Learn Mem 86:123-132.
Brabec, J., Rulseh, A., Hoyt, B., Vizek, M., Horinek, D., Hort, J., Petrovicky, P. (2010).
Volumetry of the human amygdala
An anatomical study. Psychiatry Research:
Neuroimaging 182:67-72.
Cahill, L., Haier, R., Fallon, J. (1996). Amygdala activity at encoding correlated with
long-term, free recall of emotional information. In: Proceedings of the National
Academy of Science USA 93:8016-8021.
Cahill, L., Haier, R. J., White, N. S., Fallon, J., Kilpatrick, L., Lawrence, C., Potkin, S. G.,
Alkire, M. T. (2001). Sex-related difference in amygdala activity during
emotionally influenced memory storage. Neurobiol Learn Mem 75:1-9.
Cahill, L., Uncapher, M., Kilpatrick, L., Alkire, M. T., Turner, J. (2004). Sex-related
hemispheric lateralization of amygdala function in emotionally influenced
memory: an FMRI investigation. Learn Mem 11:261-266.
Cahill L. (2003). Sexadrenergic blockade. Neurobiol Learn Mem 79:81-88.

-

Cahill L. (2006). Why sex matters for neuroscience. Nat Rev Neurosci 7:477-484.
Canli, T., Zhao, Z., Brewer, J., Gabrieli, J. D. E., Cahill, L. (2000). Event-Related
Activation in the Human Amygdala Associates with Later Memory for Individual
Emotional Experience. Journal of Neuroscience 20:1-5.

169

Canli, T., Desmond, J., Zhao Z., Gabrieli, J. D. E. (2002). Sex differences in the neural
basis of emotional memories. In: Proceedings of the national academy of science
99:10789-10794.
Canli, T., Cooney, R., Goldin, P., Shah, M. (2005). Amygdala reactivity to emotional
faces predicts improvement in major depression. Neuroreport 16:1267-1270.
Canli, T. (1999). Hemispheric asymmetry in the experience of emotion: A perspective
from functional imaging. The Neuroscientist 5:201-207.
Canteras, N. S., Simerly, R. B., Swanson, L. W. (1995). Organization of projections from
the medial nucleus of the amygdala: a PHAL study in the rat. J. Comp. Neurol.
360:213-245.
Charney, D. S., Deutch, A. Y., Krystal, J. H., Southwick, S. M., Davis, M. (1993).
Psychobiologic mechanisms of posttraumatic stress disorder. Arch Gen
Psychiatry 50:295-305.
Chura, L. R., Lombardo, M. V., Ashwin, E., Auyeung, B., Chakrabarti, B., Bullmore, E.
T., Baron-Cohen, S. (2010). Organizational effects of fetal testosterone on
human corpus callosum size and asymmetry. Psychoneuroendocrinology
35:122-132.
Coleman-Mesches, K., McGaugh, J. L. (1995). Differential effects of pretraining
inactivation of the right or left amygdala on retention of inhibitory avoidance
training. Behav. Neurosci. 109:642-647.
Cooke, B. M., Breedlove, S. M., Jordan, C. L. (2003). Both estrogen receptors and
androgen receptors contribute to testosterone-induced changes in the
morphology of the medial amygdala and sexual arousal in male rats. Horm
Behav 43:336-346.
Cooke, B. M., Stokas, M. R., Woolley, C. S. (2007). Morphological sex differences and
laterality in the prepubertal medial amygdala. J. Comp. Neurol. 501:904-915.
Cooke, B. M., Woolley, C. S. (2005). Sexually Dimorphic Synaptic Organization of the
Medial Amygdala. Journal of Neuroscience 25:10759-10767.
Crow, T. J. (2010). A theory of the origin of cerebral asymmetry: epigenetic variation
superimposed on a fixed right-shift. Laterality 15:289-303.
Derntl, B., Windischberger, C., Robinson, S., Kryspin-Exner, I., Gur, R. C., Moser, E.,
Habel, U. (2009). Amygdala activity to fear and anger in healthy young males is
associated with testosterone. Psychoneuroendocrinology 34:687-693.

170

Donahue, J. (2000). Cells containing immunoreactive estrogen receptorbasal forebrain. Brain Res 856:142-151.
Dreher, J., Schmidt, P. J., Kohn, P., Furman, D., Rubinow, D., Berman, K. F. (2007).
Menstrual cycle phase modulates reward-related neural function in women. Proc
Natl Acad Sci USA 104:2465-2470.
Ernst, M., Maheu, F. S., Schroth, E., Hardin, J., Golan, L. G., Cameron, J., Allen, R.,
Holzer, S., Nelson, E., Pine, D. S., et al. (2007). Amygdala function in
adolescents with congenital adrenal hyperplasia: a model for the study of early
steroid abnormalities. Neuropsychologia 45:2104-2113.
Finn, C., Cooke, B., Breedlove, S. (2002). Differences in finger length ratios between
selfArchives of Sexual Behavior
31:123-127.
Fusar-Poli, P., Placentino, A., Carletti, F., Allen, P., Landi, P., Abbamonte, M., Barale,
F., Perez, J., McGuire, P., Politi, P. L. (2009). Laterality effect on emotional faces
processing: ALE meta-analysis of evidence. Neurosci Lett 452:262-267.
-retention tactics across the menstrual cycle:
evidence for shifting conflicts of interest. Proc. Biol. Sci. 269:975-982.
Gasbarri, A., Arnone, B., Pompili, A., Pacitti, F., Pacitti, C., Cahill, L. (2007). Sex-related
hemispheric lateralization of electrical potentials evoked by arousing negative
stimuli. Brain Res 1138:178-186.
Gatson, J. W., Singh, M. (2007). Activation of a membrane-associated androgen
receptor promotes cell death in primary cortical astrocytes. Endocrinology
148:2458-2464.
Georgiadis, J. R., Reinders, A. A. T. S., Van der Graaf, F. H. C. E, Paans, A. M. J,
Kortekaas, R. (2007). Brain activation during human male ejaculation revisited.
Neuroreport 18:553-557.
Georgiadis, J. R., Holstege, G. (2005). Human brain activation during sexual stimulation
of the penis. J. Comp. Neurol. 493:33-38.
Gerendai, I., Csaba, Z., Vokó, Z., Csernus, V. (1995). Involvement of a direct neural
mechanism in the control of gonadal functions. The Journal of Steroid
Biochemistry and Molecular Biology 53:299-305.
Gibbs, A., Dazzan, P., Morgan, K. (2008). ScienceDirect - Journal of Psychiatric
Research : Sexually dimorphic changes in the amygdala in relation to delusional
beliefs in first episode psychosis. Journal of psychiatric research 42:913-919.
171

Giedd, J. N., Vaituzis, A. C., Hamburger, S. D., Lange, N., Rajapakse, J. C., Kaysen, D.,
Vauss, Y. C., Rapoport, J. L. (1996). Quantitative MRI of the temporal lobe,
amygdala, and hippocampus in normal human development: ages 4-18 years. J.
Comp. Neurol. 366:223-230.
Giedd, J. N., Castellanos, F. X., Rajapakse, J. C., Vaituzis, A. C., Rapoport, J. L.
(1997). Sexual dimorphism of the developing human brain. Prog.
Neuropsychopharmacol. Biol. Psychiatry 21:1185-1201.
Goldstein, J. M., Seidman, L. J., Horton, N. J., Makris, N., Kennedy, D. N., Caviness, V.
S., Faraone, S. V., Tsuang, M. T. (2001). Normal sexual dimorphism of the adult
human brain assessed by in vivo magnetic resonance imaging. Cereb. Cortex
11:490-497.
Goldstein, J. M., Jerram, M., Abbs, B., Whitfield-Gabrieli, S., Makris, N. (2010). Sex
differences in stress response circuitry activation dependent on female hormonal
cycle. Journal of Neuroscience 30:431-438.
Gorski, R. A., Gordon, J. H., Shryne, J. E., Southam, A. M. (1978). Evidence for a
morphological sex difference within the medial preoptic area of the rat brain.
Brain Res 148:333-346.
Green, J. D., Clemente, C. D., De Groot, J. (1957). Rhinencephalic lesions and
behavior in cats. An analysis of the Kluver-Bucy syndrome with particular
reference to normal and abnormal sexual behavior. J. Comp. Neurol.
108:505-545.
Gur, R. C., Gunning-Dixon, F., Bilker, W. B., Gur, R E. (2002). Sex differences in
temporo-limbic and frontal brain volumes of healthy adults. Cereb. Cortex
12:998-1003.
Halassa, M. M., Fellin, T., Takano, H., Dong, J., Haydon, P. G. (2007). Synaptic islands
defined by the territory of a single astrocyte. Journal of Neuroscience
27:6473-6477.
Hamann, S., Herman, R., Nolan, C. (2004). Men and women differ in amygdala
response to visual sexual stimuli. Nat Neurosci. 7:411-416.
Hamann, S. (2005). Sex differences in the responses of the human amygdala.
Neuroscientist 11:288-293.
Hamann, S. B., Ely, T. D., Grafton, S. T., Kilts, C. D. (1999). Amygdala activity related to
enhanced memory for pleasant and aversive stimuli. Nat Neurosci 2:289-293.

172

Hardee, J. E., Thompson, J. C., Puce, A. (2007). The left amygdala knows fear:
laterality in the amygdala response to fearful eyes. Social Cognitive and Affective
Neuroscience 3:47-54.
Haselton, M. G., Mortezaie, M., Pillsworth, E. G., Bleske-Rechek, A., Frederick, D. A.
(2007). Ovulatory shifts in human female ornamentation: near ovulation, women
dress to impress. Horm Behav 51:40-45.
Hines, M., Allen, L. S., Gorski, R. A. (1992). Sex differences in subregions of the medial
nucleus of the amygdala and the bed nucleus of the stria terminalis of the rat.
Brain Res 579:321-326.
Hofman, M. A., Swaab, D. F. (1989). The sexually dimorphic nucleus of the preoptic
area in the human brain: a comparative morphometric study. J. Anat. 164:55-72.
Holstege, G., Georgiadis, J. R., Paans, A. M. J., Meiners, L. C., Van der Graaf, F. H. C.
E., Reinders, A. A. T. S. (2003). Brain activation during human male ejaculation.
Journal of Neuroscience 23:9185-9193.
Hu, S. H., Wei, N., Wang, Q. D., Yan, L. Q., Wei, E. Q., Zhang, M. M., Hu, J. B., Huang,
M. L., Zhou, W. H., Xu, Y. (2008). Patterns of Brain Activation during Visually
Evoked Sexual Arousal Differ between Homosexual and Heterosexual Men.
American Journal of Neuroradiology 29:1890-1896.
Hulshoff Pol, H. E. H., Schnack, H. G., Mandl, R. C. W, van Haren, N. E. M. Koning, H.
Collins, D. L., Evans, A. C., Kahn, R. S. (2001). Focal gray matter density
changes in schizophrenia. Archives of General Psychiatry 58:1118 1125.
Ji, G. (2009). Hemispheric Lateralization of Pain Processing by Amygdala Neurons.
Journal of neurophysiology 102:2253-2264.
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (2008). Sex differences and laterality in
astrocyte number and complexity in the adult rat medial amygdala. J. Comp.
Neurol. 511:599-609.
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (in prep. a) Androgen Dependent Sex
Differences in Astrocyte Number but not Astrocyte Arbor Complexity in the
Juvenile Medial Posterodorsal Amygdala.
Johnson, R. T., Schneider, A., DonCarlos, L. L., Breedlove, S. M., Jordan, C. L. (in
prep. b). Astrocytes in the Rat Medial Amygdala Are Responsive to Adult
Androgens.
Karama, S., Lecours, A. R., Leroux, J., Bourgouin, P., Beaudoin, G., Joubert, S.,
Beauregard, M. (2002). Areas of brain activation in males and females during
viewing of erotic film excerpts. Hum Brain Mapp 16:1-13.
173

Killgore, W. D. S., Yurgelun-Todd, D. A. (2001). Sex differences in amygdala activation
during the perception of facial affect. Neuroreport 12:2543.
Killgore, W. D. S, Yurgelun-Todd, D. A. (2004). Sex-related developmental differences
in the lateralized activation of the prefrontal cortex and amygdala during
perception of facial affect. Percept Mot Skills 99:371-391.
Kilpatrick, L. A., Zald, D. H., Pardo, J. V., Cahill, L. F. (2006). Sex-related differences in
amygdala functional connectivity during resting conditions. Neuroimage
30:452-461.
Kim, S. W., Sohn, D. W., Cho, Y., Yang, W. S., Lee, K., Juh, R., Ahn, K., Chung, Y.,
Han, S., Lee, K. H., et al. (2006). Brain activation by visual erotic stimuli in
healthy middle aged males. International Journal of Impotence Research 18:452457.
Klüver, H., Bucy, P. C. (1938). An analysis of certain effects of bilateral temporal
lobectomy in the rhesus monkey, with sp
Journal of Psychology: Interdisciplinary and Applied 5:33-54.
Koshibu, K. (2004). Sex-specific, postpuberty changes in mouse brain structures
revealed by three-dimensional magnetic resonance microscopy. Neuroimage
22:1636-1645.
LaLumiere, R., McGaugh, J. L. (2005). Memory enhancement induced by post-training
-adrenergic or muscarinic agonists
requires activation of dopamine receptors: Involvement of right, but not left,
basolateral amygdala. Learning & Memory 12:527-532.
Lehman, M, Winans S. (1980). Medial nucleus of the amygdala mediates
chemosensory control of male hamster sexual behavior. Science 31: 557-560.
Liu, P., Erkkila, K., Lue, Y. (2010). Genetic, hormonal, and metabolomic influences on
social behavior and sex preference of XXY mice. American Journal of Physiology
299:446-455.
Mackiewicz, K. L., Sarinopoulos, I., Cleven, K. L., Nitschke, J. B. (2006). The effect of
anticipation and the specificity of sex differences for amygdala and hippocampus
function in emotional memory. Proc Natl Acad Sci USA 103:14200-14205.
Manning, J. T., Churchill, A. J. G., Peters, M. (2007). The Effects of Sex, Ethnicity, and
Sexual Orientation on Self-Measured Digit Ratio (2D:4D). Arch Sex Behav
36:223-233.

174

McCarthy, M. M. (2010). How it's made: organisational effects of hormones on the
developing brain. J Neuroendocrinol 22:736-742.
Medland, S. E., Duffy, D. L., Spurdle, A. B., Wright, M. J., Geffen, G. M., Montgomery,
G. W., Martin, N. G. (2005). Opposite effects of androgen receptor CAG repeat
length on increased risk of left-handedness in males and females. Behav. Genet.
35:735-744.
Micevych, P., Bondar, G., Kuo, J. (2010). Estrogen actions on neuroendocrine glia.
Neuroendocrinology 91:211-222.
Miyagawa, Y., Tsujimura, A., Fujita, K., Matsuoka, Y., Takahashi, T., Takao, T., Takada,
S., Matsumiya, K., Osaki, Y., Takasawa, M., et al. (2007). Differential brain
processing of audiovisual sexual stimuli in men: comparative positron emission
tomography study of the initiation and maintenance of penile erection during
sexual arousal. Neuroimage 36:830-842.
Morris, J., Jordan, C. L., Dugger, B., Breedlove, S. M. (2005). Partial demasculinization
of several brain regions in adult male (XY) rats with a dysfunctional androgen
receptor gene. Journal of Comparative Neurology 487:217.
Morris, J. A., Jordan, C. L., Breedlove, S. M. (2008a). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in adult rats. J. Comp. Neurol. 506:851-859.
Morris, J. A., Jordan, C. L., King, Z. A., Northcutt, K. V., Breedlove, S. M. (2008b).
Sexual dimorphism and steroid responsiveness of the posterodorsal medial
amygdala in adult mice. Brain Res 1190:115-121.
Murphy, D. G. M., Allen, G., Haxby, J. V., Largay, K. A., Daly, E., White, B. J., Powell,
C. M., Schapiro, M. B. (1994). The effects of sex steroids, and the X
chromosome, on female brain function: A study of the neuropsychology of adult
turner syndrome. Neuropsychologia 32:1309-1323.
Nacewicz, B. M., Dalton, K. M., Johnstone, T., Long, M. T., McAuliff, E. M., Oakes, T.
R., Alexander, A. L., Davidson, R. J. (2006). Amygdala volume and nonverbal
social impairment in adolescent and adult males with autism. Arch Gen
Psychiatry 63:1417-1428.
Neufang, S., Specht, K., Hausmann, M., Güntürkün, O., Herpertz-Dahlmann, B., Fink,
G. R., Konrad, K. (2009). Sex differences and the impact of steroid hormones on
the developing human brain. Cereb. Cortex 19:464-473.
Nishida, H., Okabe, S. (2007). Direct astrocytic contacts regulate local maturation of
dendritic spines. J Neurosci 27:331-340.

175

Nishizuka, M., Arai, Y. (1981). Sexual dimorphism in synaptic organization in the
amygdala and its dependence on neonatal hormone environment. Brain Res
212:31-38.
Patwardhan, A. J., Brown, W. E., Bender, B. G., Linden, M. G., Eliez, S., Reiss, A. L.
(2002). Reduced size of the amygdala in individuals with 47,XXY and 47,XXX
karyotypes. Am. J. Med. Genet. 114:93-98.
Paul, T., Schiffer, B., Zwarg, T., Krüger, T. H. C., Karama, S., Schedlowski, M.,
Forsting, M., Gizewski, E. R. (2008). Brain response to visual sexual stimuli in
heterosexual and homosexual males. Hum Brain Mapp 29:726-735.
Penton-Voak, I. (2000). Female preference for male faces changes cyclically:: Further
evidence. Evolution and Human Behavior 21:39-48.
Penton-Voak, I. S., Perrett, D. I., Castles, D. L., Kobayashi, T., Burt, D. M., Murray, L.
K., Minamisawa, R. (1999). Menstrual cycle alters face preference. Nature
399:741-742.
Peper, J., Schnack, H., Brouwer, R. (2009). Heritability of regional and global brain
structure at the onset of puberty: A magnetic resonance imaging study in
9 year old twin pairs. Human brain mapping 30:2184:2196.
Phillips, M. L., Williams, L., Senior, C., Bullmore, E. T., Brammer, M. J., Andrew, C.,
Williams, S. C., David, A. S. (1999). A differential neural response to threatening
and non-threatening negative facial expressions in paranoid and non-paranoid
schizophrenics. Psychiatry Research 92:11-31.
Raz, L., Khan, M., Mahesh, V., Vadlamudi, R. (2008). Rapid estrogen signaling in the
brain. Neurosignals 16:140-153.
Rees, H. D., Michael, R. P. (1984). Estrogen target neurons in the forebrain of the cat
during postnatal development. Brain Res 315:73-79.
Rezaie, R., Daly, E. M., Cutter, W. J., Murphy, D. G. M., Robertson, D. M. W, DeLisi, L.
E, Mackay, C. E., Barrick, T. R., Crow, T. J., Roberts, N. (2009). The influence of
sex chromosome aneuploidy on brain asymmetry. Am. J. Med. Genet.
150B:
74-85.
Rupp, H. A., Wallen, K. (2007). Sex Differences in Response to Visual Sexual Stimuli: A
Review. Arch Sex Behav 37:206-218.
Sanchez, M. A., Dominguez, R. (1995). Differential effects of unilateral lesions in the
medial amygdala on spontaneous and induced ovulation. Brain Res. Bull.
38:313-317.

176

Sartorius, A., Ruf, M., Kief, C., Demirakca, T. (2008). Abnormal amygdala activation
profile in pedophilia. European Archives of Psychiatry and Clinical Neuroscience
258:271-277.
Sato, K., Matsuki, N., Ohno, Y., Nakazawa, K. 2003. Estrogens inhibit l-glutamate
Journal of
neurochemistry 86:1498-1505.
Savic, I., Lindström, P. (2008). PET and MRI show differences in cerebral asymmetry
and functional connectivity between homo- and heterosexual subjects. Proc Natl
Acad Sci USA 105:9403-9408.
Schneider, F., Grodd, W., Weiss, U., Klose, U., Mayer, K. R., Nägele, T., Gur, R. C.
(1997). Functional MRI reveals left amygdala activation during emotion.
Psychiatry Research: Neuroimaging 76:75-82.
Schneider, S., Peters, J., Bromberg, U., Brassen, S., Menz, M. M., Miedl, S. F., Loth, E.,
Banaschewski, T., Barbot, A., Barker, G., et al. (2011). Boys do it the right way:
Sex-dependent amygdala lateralization during face processing in adolescents.
Neuroimage (in press).
Scicli, A., Petrovich, G., Swanson, L. (2004). Contextual Fear Conditioning Is
Associated With Lateralized Expression of the Immediate Early Gene c-fos in the
Central and Basolateral Amygdalar. Behavioral Neuroscience 118:5-14.
Sergerie, K., Chochol, C., Armony, J. (2008). The role of the amygdala in emotional
processing: A quantitative meta-analysis of functional neuroimaging studies.
Neurosci Biobehav Rev 32:811-830.
Shen, D., Liu, D., Liu, H., Clasen, L., Giedd, J., Davatzikos, C. (2004). Automated
morphometric study of brain variation in XXY males. Neuroimage 23:648-653.
Simerly, R. B., Chang, C., Muramatsu, M., Swanson, L. W. (1990). Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ
hybridization study. J. Comp. Neurol. 294:76-95.
Skuse, D. H., Morris, J.S., Dolan, R. J. (2005). Functional dissociation of amygdalamodulated arousal and cognitive appraisal, in Turner syndrome. Brain
128:2084-2096.
Spring, S., Lerch, J. P., Henkelman, R. M. (2007). Sexual dimorphism revealed in the
structure of the mouse brain using three-dimensional magnetic resonance
imaging. Neuroimage 35:1424-1433.

177

Sullivan, R. M., Duchesne, A., Hussain, D., Waldron, J., Laplante, F. (2009). Effects of
unilateral amygdala dopamine depletion on behaviour in the elevated plus maze:
role of sex, hemisphere and retesting. Behav. Brain Res. 205:115-122.
Vallortigara, G., Rogers, L. J. (2005). Survival with an asymmetrical brain: advantages
and disadvantages of cerebral lateralization. Behav Brain Sci 28:575-89
van Rijn, S., Swaab, H., Aleman, A., Kahn, R. S. (2006). X Chromosomal effects on
social cognitive processing and emotion regulation: A study with Klinefelter men
(47,XXY). Schizophrenia Research 84:194-203.
von Ziegler, N. I., Lichtensteiger, W. (1992). Asymmetry of brain aromatase activity:
region- and sex-specific developmental patterns. Neuroendocrinology
55:512-518.
Versace, A., Thompson, W. K., Zhou, D., Almeida, J. R. C., Hassel, S., Klein, C. R.,
Kupfer, D. J., Phillips, M. L. (2010). Abnormal left and right amygdalaorbitofrontal cortical functional connectivity to emotional faces: state versus trait
vulnerability markers of depression in bipolar disorder. Biological Psychiatry
67:422-431.
Wager, T., Phan, K., Liberzon, I. (2003). Valence, gender, and lateralization of
functional brain anatomy in emotion: a meta-analysis of findings from
neuroimaging. Neuroimage 20:713-728.
Walter, M., Bermpohl, F., Mouras, H., Schiltz, K., Tempelmann, C., Rotte, M., Heinze,
H., Bogerts, B., Northoff, G. (2008). Distinguishing specific sexual and general
emotional effects in fMRI Subcortical and cortical arousal during erotic picture
viewing. Neuroimage 40:1482-1494.
Williams, L. M., Barton, M. J., Kemp, A. H., Liddell, B. J., Peduto, A., Gordon, E.,
Bryant, R. A. (2005). Distinct amygdala autonomic arousal profiles in response
to fear signals in healthy males and females. Neuroimage 28:618-626.
Xiao, L., Jordan, C. L. (2002). Sex differences, laterality, and hormonal regulation of
androgen receptor immunoreactivity in rat hippocampus. Horm Behav
42:327-336.
Yurgelun-Todd, D. A., Killgore, W. D. S. (2006). Fear-related activity in the prefrontal
cortex increases with age during adolescence: a preliminary fMRI study.
Neurosci Lett 406:194-199.
Zehr, J. L., Todd, B. J., Schulz, K. M., McCarthy, M. M., Sisk, C. L. (2006). Dendritic
pruning of the medial amygdala during pubertal development of the male Syrian
hamster. Journal of neurobiology 66:578-590.

178

GENERAL DISCUSSION
Early efforts by Migel Garciaastrocyte marker glial fibrillary acidic protein (GFAP) in response to estrogen led the
way in modern investigations of gonadal hormone influence on glial cells (Tranque et
al., 1987, also see Zimmerman 1982). Subsequent reports focused on hypothalamic
astrocytes, which are hormone responsive and sexually dimorphic (Mong et al. 1996;
Amateau et al., 2000; Langub and Watson 1992). The collected findings detailed the
role of astrocytes in ovulation and emphasized that astrocytes are a vital component of
brain circuitry (reviewed in Garcia-Segura et al., 2008). However, continued exploration
has identified gonadal hormone responsive astrocytes in other brain regions, such as
the hippocampus (Day et al., 1990) and amygdala (Johnson et al., 2008; in prep. b)
Heavily connected with the hypothalamus, cortex, hippocampus and brain stem,
the amygdala is a hub of brain communication and is involved in a wide range of
functions including fear, social and reproductive behaviors. Like the hypothalamus, the
amygdala contains high concentrations of gonadal hormone receptors, and in laboratory
species and humans the amygdala is sexually dimorphic (Simerly et al., 1990;
Nishizuka and Arai, 1981; Kilpatrick et al., 2006). Several of these sexual dimorphisms
are present in juveniles while others emerge after puberty (Cooke et al., 2007; Zehr et
al., 2006). However, little information has been available regarding astrocytes in this
important and hormone sensitive brain region.
As their roles in synapse formation, brain injury and disease have become clear,
the need to understand how gonadal hormones influence astrocytes has grown (Ullian
et al., 2001; Blurton-Jones and Tuszynski, 2001; Garcia-Ovejero et al., 2005). In this

179

review I discuss several findings in the medial posterodorsal amygdala (MePD), a
region responsive to both androgen and estrogen influence (Dugger et al., 2007; Cooke
et al., 1999). I find that astrocytes are gonadal hormone sensitive in portions of the
amygdala and that, in contrast to the hypothalamus, androgen receptors (ARs) are
principally important in establishing astrocytic sex differences in this region. I discuss
the implications of these finding for amygdala function in both humans and rodents.
Astrocytes are Sexually Dimorphic in the Juvenile and Adult MePD
The sexual dimorphism in hypothalamic astrocyte complexity is present at
postnatal day (PND) 1 (Mong et al. 1996), suggesting that gonadal hormone action
during early development is responsible. The juvenile MePD exhibits sex differences in
synapse density, neuron number and regional volume (Cooke et al., 2007; Cooke and
Woolley, 2005) at PND 28, which also appear to be the result of early gonadal hormone
action, leading to the possibility that MePD astrocytes may also be sexually dimorphic in
juveniles.
In 25 day old rats, I used glial fibrillary acidic protein (GFAP)
immunocytochemistry with a Harris hematoxylin counterstain to stereologically count
MePD astrocyte numbers and to manually trace the processes of randomly selected
astrocytes, providing quantitative measures of astrocyte arbor complexity (Fig 13). I
found that in contrast to the hypothalamus, there are no sex differences in astrocyte
arbor complexity in juvenile animals (Fig 15). None of the measures I obtained from
manual tracing of astrocyte arbors exhibited any significant sex differences. However, I
did find sex differences in the number of astrocytes in the MePD, with males having
greater astrocytes than females in both the left and right hemispheres (Fig 14B). Thus,

180

it appears that gonadal hormones during early development either increase gliogenesis
or are protective against cell death in the male MePD, but do not result in increased
astrocyte arborization in males or females (Johnson et al., in prep. a).
Although astrocytes are tied to dendritic spine formation (Ullian et al., 2001), the
relationship between astrocyte complexity and dendritic spine density is highly region
specific (Pfrieger and Barres 1997; Elmariah et al., 2005; Mong et al., 2001). The
greater synapse density and miniature excitatory post-synaptic currents in the
prepubertal male MePD (Cooke and Woolley, 2005) indicate differences in neural
connectivity in the region. Our results suggest that these sex differences in the
surrounding neuronal network are either tied to the sex differences in astrocyte number
or may be independent of astrocytes.
I investigated astrocytes in the adult MePD as well. As in juvenile animals, adult
rats exhibit a sex difference in astrocyte numbers with male rats having more than
females but only in the right hemisphere (Fig 2B). Furthermore, the hemisphere
differences seen in both sexes for juvenile astrocyte numbers (left < right) are still
present in adult animals. However, unlike juveniles, adult MePD astrocyte arbors are
also sexually dimorphic, with male astrocytes having more primary branches, branch
points, branch endings, and greater branch length on average compared to females
(Johnson et al., 2008).
Interestingly, this sex difference also exists in the context of a hemisphere
difference. Specifically, sex differences in measures of astrocyte arbor complexity were
seen in the left MePD but not the right (Fig 3). Thus both astrocyte number and arbor

181

complexity are sexually dimorphic in the MePD of adult animals, but these sex
differences are present in opposite hemispheres.
Laterality is common in the MePD and the greater numbers of astrocytes in the

accompanied by greater neuron numbers compared to the right (Morris et al., 2008b).
However, this opposing laterality of astrocyte number and arbor complexity has not
been reported previously and is particularly intriguing given the numerous examples of
lateralized function seen in the human amygdala (Cahill et al. 2001; Canli et al. 2000;
Schneider et al. 2011)
Androgen Receptors Mediate Gliogenesis and Astrocyte Arbor Proliferation
Testicular feminized mutant (Tfm) males were examined along with wild-type
animals, allowing us to identify the contribution of ARs to the sex differences in the
MePD. Tfm male rats are genetic males (XY) that lack functional ARs. Tfm males have
internalized testes and produce testosterone but due to androgens inability to bind to
AR, they exhibit a feminized phenotype with nipples and reduced ano-genital distance
(Dugger et al., 2007). Furthermore, prior reports from our lab have demonstrated that in
multiple brain regions, including the MePD, Tfm male neuroanatomy is incompletely
masculinized (Dugger et al., 2007; Durazzo et al., 2007; Morris et al., 2005).
I found that in juvenile animals Tfm males were masculine for both MePD
regional volume and astrocyte number (Fig 13B) indicating that the sexual dimorphisms
seen in these measures prior to puberty are not dependent upon AR (Johnson et al., in
prep. a). However, in adult animals Tfms are feminized for astrocyte number

182

suggesting AR sensitive astrogenesis in the MePD during puberty (Johnson et al., 2008,
Fig 2B), which is concordant with prior evidence of androgen dependent gliogenesis in
the region during puberty (Ahmed et al., 2008).
Interestingly, Tfm males are also feminized for astrocyte arbor complexity once
the sex differences in these measures emerge in adulthood (Johnson et al., 2008, Fig
3). Furthermore, there appears to be little increase in Tfm male astrocyte complexity
during this period, while wt males exhibit increases in primary branch numbers, branch
points, branch endings, and branch length (Fig 17). Together with previous evidence,
these findings suggest that puberty is a critical period for the MePD as gonadal
hormones act to change the astrocytes in the region in a sexually dimorphic manner,
and that ARs play a vital role in masculinization of MePD astrocytes during that period.
MePD Astrocytes are Plastic in Adults and Contain Androgen Receptors
The MePD exhibits a mixture of plastic and static cellular anatomy in adult
animals (Morris et al., 2008). While the sexual dimorphisms seen in adult and juvenile
animals suggest gonadal hormone action during early development and during puberty,
they do not determine if astrocytes respond to circulating hormones in adulthood. In
vivo and in vitro evidence shows that astrocytes respond to gonadal hormones in
several brain regions, including the hippocampus, hypothalamus and cortex, where
astrocytes contain ER and/or AR, suggesting possible cell-autonomous responses (Lu,
2003; Kuo et al., 2010; Gatson and Singh, 2007).
To investigate the influence of circulating hormones on astrocytes in the MePD I
used male and female animals that were gonadectomized and implanted with capsules
containing either testosterone or nothing (blank) for 30 days. The animals were

183

analyzed using methodology similar to our previous investigations of MePD astrocytes,
and a separate cohort of males was examined for AR immunoreactivity. Using duallabel immunocytochemistry in conjunction with confocal microscopy I found evidence of
AR immunoreactivity within nuclei of many MePD astrocytes (Fig 10A, B). This adds
the amygdala to a list of regions where AR positive astrocytes have been identified, and
opens the possibility that androgens act directly on these cells to induce changes in
MePD neuroanatomy.
In hormone manipulated animals I found that removal of circulating hormones
resulted in reduced astrocyte numbers compared to animals exposed to testosterone.
This effect was primarily seen in females and only in the right hemisphere (Fig 8).
Furthermore, I found that circulating hormones maintain arbor complexity in the MePD
of males with T-treated males exhibiting more complex astrocytes than blank-treated
males in both hemispheres. There was a similar effect in females but only in the right
hemisphere and significant only for one measure of astrocyte complexity (Fig 9). Thus,
it appears that both astrocyte numbers and arbor complexity are influenced by
circulating hormone levels, although the effect is more powerful for arbor complexity.
Comparing juveniles and adults suggests that the development of masculinized
astrocyte arbor complexity is androgen dependent (Johnson et al., 2008). However,
because testosterone can be converted to estrogen, data from our hormone
manipulated animals does not indicate whether ERs or ARs are responsible for
maintaining this sex difference in adulthood. Peak estrogen levels during proestrus are
linked to higher levels of GFAP-immunoreactivity in the MePD (Martinez et al., 2006)
suggesting estrogen control of astrocytes in the region, at least in females. Estrogen

184

could be maintaining astrocyte complexity in males through aromatization of
testosterone. This outcome would indicate an interesting shift in which androgens are
responsible for establishing the sex difference, while estrogens maintain it in adulthood.
Conclusions
Interpreting the collected findings is difficult due to the region specific influence of
astrocytes and the complex laterality in MePD neuroanatomy. Nevertheless, several
findings point to a period of neuronal pruning in the MePD of males during puberty. Our
results suggest that this may be mediated by an increase in astrocyte arbor complexity.
During puberty there is a reduction in the number of primary dendrites and
terminal spine density in the MePD (Zehr et al. 2006). This reduction is accompanied
by reductions in spinophilin protein levels but an increase in synaptophysin levels
suggesting that while dendritic spines have been reduced, the amount of synapses has
not (Zehr et al., 2006). Furthermore, Nishizuka and Arai (1981) reported a
development-related and hormone responsive sex difference in synapses in the medial
amygdala using electron microscopy. While they found no sex differences in juveniles,
they did find sex differences in the number of dendritic shaft synapses in adults but not
in adult dendritic spine synapses. They also found that while treatment of neonatal
females with testosterone resulted in masculine numbers of dendritic shaft synapses in
adulthood, such treatment did not influence dendritic spine synapses. Finally, they also
found that neonatal orchidectomy reduced both dendritic shaft and spine synapses in

reducing dendritic spines and altering neural communication in adult animals (Cooke
and Woolley, 2009). Together these studies strongly suggest a pubertal reorganization

185

of MePD neuronal connectivity. Specifically, there is likely a reduction in dendritic
spines and an increase in dendritic shaft synapses in males during puberty which is
gonadal hormone dependent.
Our results demonstrate an increase in the complexity of astrocytes in the left
MePD during puberty (Fig 17), which is mediated by androgens. Given these findings
and the involvement of astrocyte processes in synapse formation (Elmariah et al.,
2005), I propose that androgens during puberty induce MePD astrocyte arbor
proliferation, leading to reductions in spine density and possibly new dendritic shaft
synapse formation in the MePD. This is concordant with the sex differences seen in
adult animals for dendritic shaft synapse number, astrocyte arbor complexity (Nishizuka
and Arai, 1981; Johnson et al., 2008) and provides a possible cellular mechanism for
the dendritic pruning seen during puberty (Zehr et al., 2006).
It is likely that this process is primed by gonadal hormones earlier in development
since neonatal administration of testosterone to females resulted in masculinized
numbers of dendritic shaft synapses in adults but not in juveniles, (Nishizuka and Arai,
1981). The sex differences seen in juvenile Tfms suggest possible early ER influence in
the region and the intricately timed expression of aromatase levels early in development
also indicate estrogen involvement (von Ziegler and Lichtensteiger, 1992). An increase
in adult astrocyte arbor complexity in females post puberty resulting from neonatal
testosterone treatment would confirm this early hormone priming and strengthen the
proposed association between adult synapse arrangement and astrocyte arbor
complexity.

186

Despite indirect evidence for early hormone priming by estrogen, the sex
differences in MePD astrocyte complexity that arise during puberty are AR dependent
and remain plastic in adulthood (Johnson et al., 2008; in prep. b). Thus, if astrocytes
are indeed responsible for the changes in synapse complement this process would be
directed by AR action. Our finding of ARs in MePD astrocytes opens the possibility that
androgens act directly on astrocytes to change their arbor complexity, which may then
cause changes in synapses in the region. A similar pathway has already been
described in the preoptic area with estrogens acting on ER positive astrocytes to
influence surrounding neurons (Amateau and McCarthy 2002; McCarthy et al., 2003).
An additional complication for our proposal is that the androgen dependent
increase in astrocyte complexity seen between prepuberty and adulthood occurs only in
the left hemisphere. Therefore, our current hypothesis is restricted to the left MePD.
However, this assumes that the combined hemisphere data demonstrating dendritic
pruning (Zehr et al., 2006) and synapse sexual dimorphisms (Nishizuka and Arai, 1981)
would not be found exclusively in the right hemisphere once analyzed independently
and presents the obvious question of what is happening in the right MePD?
Perhaps the increase in astrocyte numbers seen in the right hemisphere (Fig 16)
also serves to prune dendritic spines and/or change synapse complement.
Alternatively, the increased astrocyte numbers seen in the right MePD may not
significantly influence the neural communication network. Unfortunately, reports
examining the functional consequences of gliogenesis are scarce. Reduced glial cell
numbers are seen in depression, and electroconvulsive therapy, a treatment option in
severe depression, activates glia and gliogenesis in the amygdala (Bowley et al. 2002;

187

Jansson et al. 2009; Rajkowska and Miguel-Hidalgo 2007). It therefore seems likely
that there are functional consequences to the increased numbers of astrocytes in the
MePD, especially in males, after puberty.
Fortunately, results from human functional imagine studies may provide insight
into this laterality. In the human facial perception and emotional memory literature there
is support for a male-right and female-left amygdala activation model (Killgore and
Yurgelun-Todd, 2001; Hardee et al., 2007; Cahill, 2006, Table 2). This laterality is also
seen in resting state functional connectivity, presumed to reflect lateralized sex
differences in amygdala connectivity to other brain regions (Kilpatrick et al., 2006).
Interestingly, the human male right amygdala exhibits high resting state functional
connectivity with areas involved in sensory perception while the female-left amygdala
exhibits such connections with homeostatic centers. Results from homosexual and
clinical populations strengthen the hypothesized gonadal hormone influence on
lateralized amygdala function (Savic and Lindström, 2008; Skuse et al., 2005).
If amygdala laterality plays a similar role in humans and rodents, then perhaps
the consequence of an astrocyte mediated synaptic reorganization of the left amygdala

astrocyte numbers in the right MePD may be involved in biasing amygdala activity to
this hemisphere in males, producing the male-right bias seen in adults.
Not surprisingly, given its central role in brain communication, the amygdala has
been implicated in several diseases and disorders, including autism, which exhibits a
strong male bias in the population and may be related to androgen influence (American
Psychological Association, 2000; Baron-Cohen, 2002). Demonstrations that autistic

188

individuals exhibit deficits in empathizing while displaying a propensity for systematizing
(Lombardo et al., 2010; Auyeung et al., 2009a; b) seem in line with greater amygdala
functional connectivity with homeostatic regions in females and sensory regions in
males (Kilpatrick et al., 2006). If gonadal hormones during early development are
priming the amygdala for synaptic reorganization during puberty, leading to a maleright/sensory-monitoring amygdala network, then what are the consequences for early
disruption or acceleration of this process? Continued investigation of how gonadal
hormones influence amygdala cellular anatomy may provide answers. Given the
increasing importance of astrocytes, understanding how they influence the synaptic
organization of the amygdala should be a high priority.
Future Directions and Closing Remarks
The sexual dimorphisms I have reported in MePD astrocytes underscore the fact
that the cellular components of the amygdala are influenced by gonadal hormones
acting upon both ERs and ARs. What I have proposed is that androgens act on MePD
astrocytes during puberty to promote astrogenesis and/or arbor proliferation and this
leads to changes in MePD synapse complement, altering MePD connectivity and
amygdala functionality. Although these reports provide new evidence of complex
cellular sexual dimorphisms and hormone responsiveness in an important brain region,
additional research is necessary to test these hypotheses and detail the mechanisms
through which gonadal hormones activate MePD astrocytes. Therefore a wide range of
future research directions is available.
Questions regarding the ontogeny of astrocyte sex differences are particularly
intriguing and may have important implications for disease research. A series of

189

experiments centered on ideas put forth by Nishizuka and Arai (1981) would be
informative. Because a single PND 5 injection of T failed to masculinize synapses in
juvenile females but did masculinize synapse in adult females, it appears that neonatal
gonadal hormones provide a priming effect, leading to changes that occur later, during
puberty.
Is there a priming effect of neonatal gonadal hormones for astrocyte sex
differences in adulthood? If neonatal T treatment masculinizes astrocytes before
puberty or fails to result in masculinized astrocytes in adult females, this would suggest
that the changes I see in astrocytes during puberty are orthogonal to the previously
reported changes in MePD synapses. However, if T treatment leads to masculine
astrocytes in adult females then several more experiments should follow.
Orchidectomy of male rats on PND 1, which are then given recurring
testosterone treatment starting on day 28, could be examined. If these animals
exhibited masculinized astrocytes, one might deduce that gonadal hormone influence
between PNDs 1 and 28 is not necessary for the pubertal increase in astrocyte numbers
and arbor complexity. Including a group of males who were orchidectomized just prior
to puberty but who received no testosterone supplementation would produce a

gonadal hormones must be available to produce masculinized astrocytes in adult
animals.
Assuming sustained evidence of a link between astrocyte sex differences and
MePD neuropil, investigating the mechanisms and consequences of changes in MePD

190

astrocytes would be valuable. My results using Tfm males demonstrate that ARs
contribute to both astrocyte complexity and numbers in the MePD.
Although astrocytes in the region contain AR, it is not clear if the sex differences seen
are due to androgens acting directly on astrocytes. Use of the cre-lox system in which
ARs are selectively disabled in GFAP- expressing cells would explore this question.
Finding that removal of AR from astrocytes in males resulted in feminized astrocytes in
the MePD would immediately lead to questions about the consequences for surrounding
neurons, but would also open the door for further exploration of the molecular and
genetic mechanisms behind AR influence in astrocytes.
Another intriguing investigation might examine how ARs mediate cellular
proliferation in some astrocytes while promoting arbor elaboration in other populations.
Work in the hypothalamus has identified prostaglandins and endogenous cannabinoids
as intermediaries in pathways leading to astrocytic sex differences (Amateau and
McCarthy, 2002; Krebs-Kraft et al., 2010). This evidence, in combination with the
diverse actions of gonadal hormone co-regulators (Tetel, 2009; Auger et al. 2000;
Meijer et al. 2000) suggests a wide array of possible molecular signaling pathways.
Astrocytes express some of these co-regulators (Grenier et al. 2006) and their
activation has been linked to sexually dimorphic gene expression (Molenda et al. 2002).
Furthermore, the co-regulator SRC-1 has already been identified as responsive to
seasonal fluctuations that also affect mating behavior and cellular anatomy in the MePD
of male hamsters (Tetel et al., 2004; Gomez and Newman 1991).
Finally, besides cellular and molecular questions, my dissertation raises
questions about the function of the amygdala as a whole. At the broadest level,

191

questions should now be asked about astrocytes in other regions of the amygdala. Are
astrocytes in other subregions sexually dimorphic as well? If so, when do these sex
differences arise? Do astrocytes throughout the amygdala increase in arbor complexity
during puberty? Moreover, what are the consequences of astrocyte-induced changes in
medial amygdala connectivity? Are MePD astrocytes strengthening connections to the
hypothalamus and/or olfactory bulbs in males or are the changes in connectivity more
local, resulting in altered communication between the medial amygdala and other
amygdala subunits? How are these changes integrated into the functioning of the
amygdala as a whole?
My dissertation shows that astrocytes are an important and dynamic part of the
medial amygdala and likely play an important role in shaping this region as it responds
to gonadal hormones throughout life. The novel sexual dimorphisms and complex
laterality I found emphasize the need for continued exploration of brain neuroanatomy,
including examination of cell types beyond neurons and careful documentation of both
sex and hemisphere. The amygdala in particular appears to present challenging levels
of sex and hemisphere differences in numerous aspects of cellular anatomy, all of which
make the region more intriguing. Despite decades of research and a constantly
expanding role in important brain function, the amygdala remains poorly understood and
mysterious. Continued exploration of the region will likely provide information useful in
our understanding of disorders such as autism and depression and may lead to insight
regarding the function of the brain as whole.

192

APPENDIX

193

Figure 16 - MePD astrocytes increase in number during puberty in both the left and right
hemispheres. In the left hemisphere Tfm males do not differ from wt males before or
after puberty and astrogenesis does not depend on functional androgen receptors.
Astrogenesis in the right hemisphere is greater compared to the left and is at least
partially dependent upon functional androgen receptors. Tfm males are masculine
before puberty but feminine in adulthood. The right hemisphere displays a more
dramatic decline in astrocyte numbers when adult circulating hormone levels are
reduced and this effect is especially prominent in females. Values are means of N = 810 rats/ group (+ SEM).
194

Figure 17 - Astrocytes exhibit an androgen-dependent increase in the number of branch
endings during puberty in the left MePD. Other measures of astrocyte complexity such
as number of primary branches, branch points, and average branch length exhibit
similar patterns. Eliminating adult circulating hormone through gonadectomy reduces
astrocyte complexity in the left hemisphere. Values are means of N = 5 rats/ group (+
SEM).

195

REFERENCES

196

REFERENCES
Amateau, S. K., McCarthy, M. M. (2002). A novel mechanism of dendritic spine
plasticity involving estradiol induction of prostaglandin-E2. Journal of
Neuroscience 22:8586-8596.
Amateau, S. K., Mong, J. A., McCarthy, M. M. (2000). Steroid-mediated differentiation
of astrocytes in the rat perinatal POA. Soc Neurosci 26:224.33.
American Psychological Association. (2000). Diagnostic and statistical manual of mental
disorders: DSM-IV-TR.
Ahmed, E. I., Shcultz, K. M., Zehr, J. L., Lorenz, B. H., DonCarlos, L. L., Sisk, C. L.
(2008). Pubertal hormones modulate the addition of new cells to sexually
dimorphic brain regions. Nature Neuroscience 11:995-997.
Arpini, M., Menezes, I. C., Dall'Oglio, A., Rasia-Filho, A. A. (2010). The density of
Golgi- impregnated dendritic spines from adult rat posterodorsal medial
amygdala neurons displays no evidence of hemispheric or dorsal/ventral
differences. Neurosci Lett 469:209-213.
Auger, A.P., Tetel, M.J., McCarthy, M.M. (2000). Steroid receptor coactivator-1
mediates the development of sex specific brain morphology and behavior. Proc.
Natl. Acad. Sci. USA 97:7551 7555.
Auyeung, B., Baron-Cohen, S., Ashwin, E., Knickmeyer, R., Taylor, K., Hackett, G.
(2009a). Fetal testosterone and autistic traits. Br J Psychol 100:1-22.
Auyeung, B., Baron-Cohen, S., Ashwin, E., Knickmeyer, R., Taylor, K., Hackett, G.
(2009b). Fetal testosterone predicts sexually differentiated childhood behavior in
girls and in boys. Psychhol Sci 20:144-148.
Baron-Cohen S. (2002). The extreme male brain theory of autism. Trends Cogn Sci
6:248-254.
Blurton-Jones, M., Tuszynski, M. H. (2001). Reactive astrocytes express estrogen
receptors in the injured primate brain. J Comp Neurol 433:115-23.
Bowley, M. P., Drevets, W. C., Ongur, D., Price, J. L. (2002). Low glial numbers in
the amygdala in major depressive disorder. Biological Psychiatry 52:404-412.
Cahill L. (2006). Why sex matters for neuroscience. Nat Rev Neurosci 7:477-484.
Cahill, L., Haier, R. J., White, N. S., Fallon, J., Kilpatrick, L., Lawrence, C., Potkin, S. G.,
Alkire, M. T. (2001). Sex-related difference in amygdala activity during
emotionally influenced memory storage. Neurobiol Learn Mem 75:1-9.

197

Canli, T., Zhao, Z., Brewer, J., Gabrieli, J. D. E., Cahill, L. (2000). Event-Related
Activation in the Human Amygdala Associates with Later Memory for Individual
Emotional Experience. Journal of Neuroscience 20:1-5.
Cooke, B. M., Tabibnia, G., Breedlove, S. M. (1999). A brain sexual dimorphism
controlled by adult circulating androgens. Proc Natl Acad Sci USA 22:7538-40.
Cooke, B. M., Stokas, M. R., Woolley, C. S. (2007). Morphological sex differences and
laterality in the prepubertal medial amygdala. J Comp Neurol. 20;501(6):904-15.
Cooke, B. M., Woolley, C. S. (2005). Sexually dimorphic synaptic organization of the
medial amygdala. J Neurosci. 16;25(46):10759-67.
Cooke, B. M., Woolley, C. S. (2009). Effects of prepubertal gonadectomy on a
male-typical behavior and excitatory synaptic transmission in the amygdala. Dev
Neurobiol 69:141-152.
Day, J. R., Laping, N. J., McNeill, T. H., Schreiber, S. S., Pasinetti, G., Finch, C. E.
(1990). Castration enhances expression of glial fibrillary acidic protein and
sulfated glycoprotein-2 in the intact and lesion-altered hippocampus of the adult
male rat. Mol. Endocrinol. 4:1995-2002.
Dugger, B. N., Morris, J. A., Jordan, C. L., Breedlove, S. M. (2007). Androgen receptors
are required for full masculinization of the ventromedial hypothalamus (VMH) in
rats. Horm Behav 51:195-201.
Durazzo, A., Morris, J. A., Breedlove, S. M., Jordan, C. L. (2007). Effects of the
testicular feminization mutation (Tfm) of the androgen receptor gene on
BSTMPM volume and morphology in rats. Neuroscience Letters 419:168-171.
Elmariah, S. B., Hughes, E. G., Oh, E. J., Balice-Gordon, R. J. (2005). Neurotrophin
signaling among neurons and glia during formation of tripartite synapses. Neuron
Glia Biol 1:1-11.
Garcia-Ovejero, D., Azcoitia, I., DonCarlos, L. L., Melcangi, R. C., Garcia-Segura, L. M.
(2005). Glia-neuron crosstalk in the neuroprotective mechanisms of sex steroid
hormones. Brain Res Brain Res Rev 48:273-286.
Garcia-Segura, L. M., Lorenz, B., DonCarlos, L. L. (2008). The role of glia in the
hypothalamus: implications for gonadal steroid feedback and reproductive
neuroendocrine output. Reproduction 135;419.
Gatson, J. W., Singh, M. (2007). Activation of a membrane-associated androgen
receptor promotes cell death in primary cortical astrocytes. Endocrinology.
148(5):2458-64.

198

Grenier, J., Trousson, A., Chauchereau, A., Cartaud, J., Schumacher, M., Massaad, C.,
(2006). Differential recruitment of p160 coactivators by glucocorticoid receptor
between Schwann cells and astrocytes. Mol. Endocrinol. 20:254 267.
Gomez, D. M., Newman, S. W. (1991). Medial nucleus of the amygdala in the adult
Syrian hamster: a quantitative Golgi analysis of gonadal hormonal regulation of
neuronal morphology. The Anatomical record 231;498-509.
Hardee, J. E., Thompson, J. C., Puce, A. (2007). The left amygdala knows fear:
laterality in the amygdala response to fearful eyes. Social Cognitive and Affective
Neuroscience 3:47-54.
Jansson, L., Wennström, M., Johanson, A., Tingström, A. (2009). Glial cell activation
in response to electroconvulsive seizures. Progress in neuropsychopharmacology & biological psychiatry 33:1119-1128.
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (2008). Sex differences and laterality in
astrocyte number and complexity in the adult rat medial amygdala. J. Comp.
Neurol. 511:599-609.
Johnson, R. T., Breedlove, S. M., Jordan, C. L. (in prep. a) Androgen Dependent Sex
Differences in Astrocyte Number but not Astrocyte Arbor Complexity in the
Juvenile Medial Posterodorsal Amygdala.
Johnson, R. T., Schneider, A., DonCarlos, L. L., Breedlove, S. M., Jordan, C. L. (in
prep. b). Astrocytes in the Rat Medial Amygdala Are Responsive to Adult
Androgens.
Killgore, W. D. S., Yurgelun-Todd, D. A. (2001). Sex differences in amygdala activation
during the perception of facial affect. Neuroreport 12:2543.
Kilpatrick, L. A., Zald, D. H., Pardo, J. V., Cahill, L. F. (2006). Sex-related differences in
amygdala functional connectivity during resting conditions. Neuroimage
30:452-461.
Krebs-Kraft, D., Hill, M., Hillard, C., McCarthy, M. M. (2010). Sex difference in cell
proliferation in developing rat amygdala mediated by endocannabinoids has
implications for social behavior. Proceedings of the national academy of science
107:20535-20540.
Kuo, J., Hamid, N., Bondar, G., Prossnitz, E. R., Micevych, P. (2010). Membrane
estrogen receptors stimulate intracellular calcium release and progesterone
synthesis in hypothalamic astrocytes. Journal of Neuroscience 30:12950-12957.

199

Langub, M. J., & Watson, R. J. (1992). Estrogen receptor-immunoreactive glia,
endothelia, and ependyma in guinea pig preoptic area and median eminence:
electron microscopy. Endocrinology 130:364-372.
Lombardo, M. V., Chakrabarti, B., Bullmore, E. T., Sadek, S. A., Pasco, G.,
Wheelwright, S. J., Suckling, J., Baron-Cohen S. (2010). Atypical neural
self-representation in autism. Brain 133:611-624.
Lu, Y., Zeng, M., Hu, X., Xu, H., Swaab, D. F., Ravid, R., Zhou, J. (2003). Estrogen
-immunoreactive astrocytes are increased in the hippocampus in
Alzheimer's disease. Exp. Neurol. 183:482-488.
Martinez, F. G., Hermel, E. E., Xavier, L. L., Viola, G. G., Riboldi, J., Rasia-Filho, A. A.,
Achaval M. (2006). Gonadal hormone regulation of glial fibrillary acidic protein
immunoreactivity in the medial amygdala subnuclei across the estrous cycle and
in castrated and treated female rats. Brain Res 1108:117-26.
McCarthy, M. M., Todd, B. J., Amateau, S. K. (2003). Estradiol modulation of
astrocytes and the establishment of sex differences in the brain. Annals of the
New York Academy of Sciences1007:283-297.
Meijer, O.C., Steenbergen, P.J., de Kloet, E.R. (2000). Differential expression and
regional distribution of steroid receptor coactivators SRC-1 and SRC-2 in brain
and pituitary. Endocrinology 141:2192 2199.
Molenda, H.A., Griffin, A.L., Auger, A.P., McCarthy, M.M., Tetel, M.J. (2002). Nuclear
receptor coactivators modulate hormone-depen- dent gene expression in brain
and female reproductive behavior in rats. Endocrinology 143:436-444.
Mong, J., Kurzweil, R. L., Davis, A. M., Rocca, M. S., McCarthy, M. M. (1996). Evidence
for Sexual Differentiation of Glia in Rat Brain. Horm Behav 30:553-562.
Mong, J. A., Roberts, R. C., Kelly, J. J., McCarthy, M. M. (2001). Gonadal steroids
reduce the density of axospinous synapses in the developing rat arcuate
nucleus: an electron microscopy analysis. J Comp Neurol 432:259-67.
Morris, J. A., Jordan, C. L., Dugger, B. N., Breedlove, S. M. (2005). Partial
demasculinization of several brain regions in adult male (XY) rats with a
dysfunctional androgen receptor gene. Journal of Comparative Neurology,
487:217-226.
Morris, J. A., Jordan, C. L., Breedlove, S. M. (2008). Sexual dimorphism in neuronal
number of the posterodorsal medial amygdala is independent of circulating
androgens and regional volume in adult rats. J Comp Neurol 506:851-9.

200

Nishizuka, M., Arai, Y. (1981). Sexual dimorphism in synaptic organization in the
amygdala and its dependence on neonatal hormone environment. Brain research
212:31-38.
Pfrieger, F. W. and Barres, B. A. (1997). Synpatic efficacy enhanced by glial cells in
vitro. Science 277:1684-7.
Rajkowska, G., Miguel-Hidalgo, J. J. (2007). Gliogenesis and glial pathology in
depression. CNS & neurological disorders drug targets 6:219-233.
Savic, I., Lindström, P. (2008). PET and MRI show differences in cerebral asymmetry
and functional connectivity between homo- and heterosexual subjects. Proc Natl
Acad Sci USA 105:9403-9408.
Schneider, S., Peters, J., Bromberg, U., Brassen, S., Menz, M. M., Miedl, S. F., Loth, E.,
Banaschewski, T., Barbot, A., Barker, G., et al. (2011). Boys do it the right way:
Sex-dependent amygdala lateralization during face processing in adolescents.
Neuroimage (in press).
Simerly, R. B., Chang, C., Muramatsu, M., Swanson, L. W. (1990). Distribution of
androgen and estrogen receptor mRNA-containing cells in the rat brain: an in situ
hybridization study. J Comp Neurol 294:76-95.
Skuse, D. H., Morris, J.S., Dolan, R. J. (2005). Functional dissociation of amygdalamodulated arousal and cognitive appraisal, in Turner syndrome. Brain
128:2084-2096.
Tetel, M. J. (2009). Modulation of steroid action in the central and peripheral nervous
systems by nuclear receptor coactivators. Psychoneuroendocrinology 34S:
S9-S19.
Tetel, M.J., Ungar, T.C., Hassan, B., Bittman, E.L. (2004). Photoperiodic regulation of
androgen receptor and steroid receptor coactivator-1 in Siberian hamster brain.
Mol. Brain Res. 131:79 87.
Tranque, P. A., Suarez, I., Olmos, G., Fernandez, B., & Garcia-Segura, L. M. (1987).
Estradiol--induced redistribution of glial fibrillary acidic protein immunoreactivity in
the rat brain. Brain research 406:348-351.
Ullian, E. M., Sapperstein, S. K., Christopherson, K. S. and Barres, B. A. (2001). Control
of synapse number by glia. Science 291:657-61.
von Ziegler von, N. I., Lichtensteiger W. (1992). Asymmetry of brain aromatase activity:
region- and sex-specific developmental patterns. Neuroendocrinology
55:512-518.

201

Zehr, J. L., Todd, B. J., Schulz, K. M., McCarthy, M. M., Sisk, C. L. (2006). Dendritic
pruning of the medial amygdala during pubertal development of the male Syrian
hamster. J Neurobiol. 66(6):578-90.
Zimmermann, P. (1982). Estrogen-dependent changes in the functional
interrelationships among neurons, ependymal cells and glial cells of the arcuate
nucleus. Cytometric studies in the female albino mouse. Cell Tissue Res
227:113 128.

202