PUBERTAL CYTOGENESIS IN NEURAL CIRCUITS MEDIATING SEX-SPECIFIC
SOCIAL BEHAVIORS
By
Jenny Lily Kim

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Psychology – Doctor of Philosophy
2017

ABSTRACT
PUBERTAL CYTOGENESIS IN NEURAL CIRCUITS MEDIATING SEX-SPECIFIC
SOCIAL BEHAVIORS
By
Jenny Lily Kim
During puberty and adolescence, the brain experiences significant structural
changes that mediate the maturation of sex-specific sociosexual behaviors, such as
sexual behavior and intermale aggression. These sex-specific sociosexual behaviors
are regulated by sexually dimorphic brain regions such as the posterodorsal medial
amygdala (MePD) and ventromedial hypothalamus (VMH). The MePD in laboratory
rodents is larger in males than females and is responsible for processing chemosensory
signals to initiate context-appropriate social behaviors. The VMH is also larger in males
than females in rodent species, which is primarily driven by sex differences in the
ventrolateral subdivision (VMHvl). The VMHvl is involved in gating male sexual behavior
and intermale aggression. Many of these sociosexual behaviors do not emerge until the
onset of puberty, suggesting that morphological changes in the MePD and VMHvl
during puberty may facilitate their development. Recent studies have discovered that
new cells are added to the hypothalamus and amygdala during puberty and a subset of
pubertally born cells in the MePD is activated in response to a social encounter. The
current dissertation sets out to expand on these findings and examine pubertal
cytogenesis in the MePD and VMH using the mouse model. In Chapter 1A, a sex
difference in the number of pubertally born cells was revealed in the adult MePD, with
males having more pubertally born cells than females. This increase in pubertally born
cells in males was dependent on the presence of functional androgen receptors (ARs)

and pubertal and adult testosterone. In Chapter 1B, while I initially discovered a sex
difference in pubertally born cells in the VMHvl, this finding was not replicated in
subsequent studies. In addition, no sex differences were observed in the number of
pubertally born cells in sexually monomorphic brain regions (i.e. dorsomedial and
central subdivision of the VMH (VMHdm and VMHc, respectively) and dentate gyrus
(DG)). These results suggest that the number of pubertally born cells in the adult VMH
and DG may not be affected by gonadal hormones. In Chapter 2, I discovered that
pubertal, but not adult, ARs promote the increase in pubertally born neurons, but not
astrocytes, in the adult male MePD. However, pubertal and adult ARs did not influence
the number of pubertally born neurons or astrocytes in the VMH or DG. In Chapter 3, I
found that social interaction activated pubertally born cells in the adult male MePD,
while social interaction with a male conspecific activated pubertally born cells in the
adult male VMHvl and VMHdm. Social interaction did not activate pubertally born cells
in the VMHc and DG. These studies demonstrate that gonadal hormones play an
important role in regulating the proliferation and/or survival of pubertally born cells in the
adult MePD, but not VMH or DG. Furthermore, I provide evidence that pubertally born
cells in the MePD, VMHvl, and VMHdm are functionally incorporated into adult neural
circuits. Thus, the addition and functional integration of pubertally born cells into neural
circuits known to regulate social behavior may be a mechanism by which adult
sociosexual behaviors emerge during puberty.

Dedicated to In Suk Kim.

iv

ACKNOWLEDGEMENTS

First and foremost, I’d like to thank my advisor, Dr. Cheryl Sisk, for her unending
support during my graduate career. She has taught me the skills necessary to become
an independent researcher, a better writer, and an effective mentor. I would also like to
thank my other committee members, Drs. Cynthia Jordan, Marc Breedlove, and Tony
Nunez, for their important insights and guidance on my dissertation work. They
challenged me the whole way during my graduate studies, ultimately making me a
better researcher. I would also like to extend a heartfelt thank you to everyone in the
Sisk lab, especially Jane Venier, Dr. Margaret Mohr, Dr. Elaine Sinclair, Dr. Nancy
Staffend, as they have each taught me important laboratory skills needed to
successfully carry out my experiments. I would also like to thank the Breedlove-Jordan
lab, in particular, Dr. Kathy Halievski, Dr. Jen Brummet, Dr. Nick Hobbs, and Diane
Redenius, who have also provided technical assistance with my experiments. I am most
grateful for everyone’s social support, which has kept me going when times were tough.
Kathy, Jane, Elaine, Maggie, and Ashlyn, thank you so much for all the laughs and kind
words, I could not have made it through graduate school without your friendship. Also,
thank you to Dr. Casey Henley, who provided so much emotional support throughout
my graduate career. She has also taught me how to become a better teacher in the
classroom, and for that I am so grateful. Last, but certainly not least, I am so thankful for
Dr. Keenan Walker, who inspires to be a better scientist every day. I would not be the
person I am today without his unwaivering support, patience, and love.

v

TABLE OF CONTENTS

LIST OF FIGURES .......................................................................................................... ix
KEY TO ABBREVIATIONS…………………………………………………………………...xi
INTRODUCTION .............................................................................................................. 1
Postnatal Neuro- and Gliogenesis ........................................................................................ 1
The Social Neural Network .................................................................................................... 5
Hormonal Regulation of Pubertal Neurogenesis and Gliogenesis .................................. 11
Dissertation Overview .......................................................................................................... 14

REFERENCES ............................................................................................................... 18
CHAPTER 1A................................................................................................................. 26
ABSTRACT ............................................................................................................................ 26
INTRODUCTION .................................................................................................................... 27
MATERIALS AND METHODS ............................................................................................... 30
Animal Housing .................................................................................................................. 30
Experiment 1: Are there sex differences in the number of pubertally born cells in the adult
MePD? ................................................................................................................................ 30
Experiment 2: Are functional androgen receptors necessary to establish sex differences in
the number of pubertally born cells in adulthood? .............................................................. 31
Experiment 3: Is pubertal testosterone required to establish sex differences in the number
of pubertally born MePD cells in adulthood? ...................................................................... 32
Experiment 4: Do pubertal and adult testosterone have differential effects on the number of
pubertally born cells in the adult MePD? ............................................................................ 33
Tissue Collection and Processing ...................................................................................... 33
Immunohistochemistry ........................................................................................................ 34
Microscopic Analysis .......................................................................................................... 35
Single-label BrdU Analysis ................................................................................................. 35
RESULTS ............................................................................................................................... 36
Experiment 1: Adult male mice have more pubertally born MePD cells than adult female
mice. ................................................................................................................................... 36
Experiment 2: Dysfunctional androgen receptors are sufficient to eliminate sex differences
in the number of pubertally born cells in the adult MePD. .................................................. 37
Experiment 3: Pubertal testosterone and functional androgen receptors enhance the
number of pubertally born MePD cells. .............................................................................. 37
Experiment 4: The presence of both pubertal and adult testosterone increases the number
of pubertally born MePD cells. ............................................................................................ 39
DISCUSSION ......................................................................................................................... 40
Sex differences in pubertally born cells in the adult MePD ................................................ 40
Sex differences in the adult MePD: The role of androgen receptors and pubertal
testosterone ........................................................................................................................ 41
Pubertal and adult testosterone enhance the number of pubertally born cells in the adult
male MePD ......................................................................................................................... 44
Significance of pubertally born cells in the MePD .............................................................. 46
Conclusions ........................................................................................................................ 47
REFERENCES ....................................................................................................................... 54

vi

CHAPTER 1B................................................................................................................. 58
ABSTRACT ............................................................................................................................ 58
INTRODUCTION .................................................................................................................... 59
MATERIALS AND METHODS ............................................................................................... 62
Animal Housing .................................................................................................................. 62
Experiment 1: Are there sex differences in pubertally born cells in the adult VMH and DG?
............................................................................................................................................ 62
Experiment 2: Does the masculinization of pubertal born cells in the VMHvl require
functional androgen receptors? .......................................................................................... 63
Experiment 3: Is pubertal testosterone required to generate sex differences in the density
of pubertally born cells in the adult VMHvl? ....................................................................... 63
Tissue Collection and Processing ...................................................................................... 64
Immunohistochemistry ........................................................................................................ 65
Microscopic Analysis .......................................................................................................... 65
Single-label BrdU Analysis ................................................................................................. 66
RESULTS ............................................................................................................................... 67
Experiment 1: Male mice have more pubertally born cells than females in the VMHvl, but
not the VMHdm, VMHc, or DG. .......................................................................................... 67
Experiment 2: Androgen receptors are not necessary to increase the number of pubertally
born cells in the VMH and DG. ........................................................................................... 68
Experiment 3: Pubertal testosterone does not increase the number of pubertally born cells
in the VMH or DG. .............................................................................................................. 68
DISCUSSION ......................................................................................................................... 68
REFERENCES ....................................................................................................................... 79

CHAPTER 2 ................................................................................................................... 83
ABSTRACT ............................................................................................................................ 83
INTRODUCTION .................................................................................................................... 84
MATERIALS AND METHODS ............................................................................................... 86
Animal Subjects and Experimental Design ......................................................................... 86
BrdU administration ............................................................................................................ 87
Tissue Collection and Processing ...................................................................................... 87
Single-Label BrdU Immunohistochemistry ......................................................................... 88
Triple-Label BrdU/GFAP/NeuN Immunofluorescence ........................................................ 89
Microscopic Analysis .......................................................................................................... 89
Single-label BrdU Analysis ................................................................................................. 90
Triple-Label BrdU/GFAP/NeuN Analysis ............................................................................ 91
Statistical Analysis .............................................................................................................. 92
RESULTS ............................................................................................................................... 92
Functional androgen receptors during puberty, but not adulthood, promote the number of
pubertally born cells in the adult male MePD. .................................................................... 92
Functional androgen receptors, either during puberty or in adulthood, do not promote the
number of pubertally born cells in the VMH and DG. ......................................................... 93
Androgen receptors during puberty, but not adulthood, enhances the differentiation of
pubertally born cells into mature neurons in the MePD. ..................................................... 94
Androgen receptors during puberty or adulthood are not required to promote the
differentiation of pubertally born cells into mature neurons or astrocytes in the VMH and
DG. ..................................................................................................................................... 94
DISCUSSION ......................................................................................................................... 95

vii

The differential effects of pubertal and adult androgen receptors on pubertally born cells in
the MePD ............................................................................................................................ 95
Contributions of pubertal and adult androgens on the VMH and DG ................................. 99
Conclusions ...................................................................................................................... 104
REFERENCES ..................................................................................................................... 112

CHAPTER 3 ................................................................................................................. 118
ABSTRACT .......................................................................................................................... 118
INTRODUCTION .................................................................................................................. 119
MATERIALS AND METHODS ............................................................................................. 123
Animal Subjects and Experimental Design ....................................................................... 123
BrdU administration .......................................................................................................... 123
Social Interaction Tasks ................................................................................................... 124
Tissue Collection and Processing .................................................................................... 125
Double-Label BrdU/Fos Immunofluorescence .................................................................. 125
Triple-Label BrdU/GFAP/NeuN Immunofluorescence ...................................................... 126
Microscopic Analysis ........................................................................................................ 127
Double-label BrdU/Fos Analysis ....................................................................................... 128
Triple-Label BrdU/GFAP/NeuN Analysis .......................................................................... 128
Statistical Analysis ............................................................................................................ 129
RESULTS ............................................................................................................................. 129
Social interaction activates pubertally born cells in the adult male MePD. ....................... 129
Male-male social interactions activate pubertally born cells in the adult male VMHvl and
VMHdm. ............................................................................................................................ 130
Pubertally born cells in the adult male VMHc and DG are not activated by social
interactions. ...................................................................................................................... 132
Pubertally born cells in the MePD, VMH, and DG differentiate into mature neurons and
astrocytes. ........................................................................................................................ 133
DISCUSSION ....................................................................................................................... 133
Social interactions activate pubertally born cells in the adult male MePD ........................ 134
Male-male social interactions activate pubertally born cells in the VMHvl and VMHdm .. 137
Pubertally born cells in the VMHc and DG are not activated by social interactions ......... 139
A subset of pubertally born cells in the MePD, VMH, and DG differentiate into mature
neurons or astrocytes ....................................................................................................... 140
Conclusions ...................................................................................................................... 141
REFERENCES ..................................................................................................................... 148

CONCLUSIONS ........................................................................................................... 155
REFERENCES ..................................................................................................................... 167

viii

LIST OF FIGURES

Figure 1.1.

Posterodorsal medial amygdala (MePD) in adult male and
female mice.

51

Figure 1.2.

Sex differences in the total number and cell density of BrdU-ir
cells in the posterodorsal medial amygdala (MePD) in adult mice.

52

Figure 1.3.

Sex differences in the total number and density of BrdU-ir cells
in the MePD are dependent on functional androgen receptors and
independent of adult testosterone.

53

Figure 1.4.

Sex differences in the total number and density of BrdU-ir cells in
the MePD are dependent on pubertal testosterone and functional
androgen receptors.

54

Figure 1.5.

Pubertal and adult testosterone are necessary to increase the
number of pubertally born cells in the adult male MePD.

55

Figure 1.6.

Total number and cell density of BrdU-immunoreactive cells in
the ventrolateral subdivision of the ventromedial hypothalamus
(VMHvl) in adult male and female mice.

77

Figure 1.7.

Total number and cell density of BrdU-ir cells in the dorsomedial
and central subdivisions of the ventromedial hypothalamus in
adult male and female mice.

78

Figure 1.8.

BrdU-ir cell density in the dentate gyrus of the hippocampus in
adult male and female mice.

79

Figure 1.9.

Androgen receptors are not required to enhance the density
of pubertally born cells in the VMH and DG.

80

Figure 1.10. Pubertal testosterone is not necessary to increase the number
of pubertally born cells in the male, female, and iTfm male mice
in the VMH and DG.

81

Figure 2.1.

Blocking androgen receptors during puberty in male mice
reduces the number of pubertally born cells in the adult
posterodorsal medial amygdala (MePD).

110

Figure 2.2.

Androgen receptors are not required to increase the number
of pubertally born cells in the adult ventromedial hypothalamus
(VMH).

111

ix

Figure 2.3.

Androgen receptors are not required to increase the number
of pubertally born cells in the adult dentate gyrus of male mice.

112

Figure 2.4.

Blocking androgen receptors during puberty, but not adulthood,
reduces the number of pubertally born cells that become
neurons in the adult MePD.

113

Figure 2.5.

Androgen receptors are not required to enhance the number
of pubertally born cells that colocalize with mature neurons or
astrocytes in the adult VMH.

114

Figure 2.6.

Androgen receptors are not required for pubertally born cells
to differentiate into mature neurons or astrocytes in the adult
dentate gyrus.

115

Figure 3.1.

Social interaction activates pubertally born cells in the adult
male MePD.

148

Figure 3.2.

Male-male social interactions activate pubertally born cells in
the adult VMHvl and VMHdm.

149

Figure 3.3.

Pubertally born cells in the VMHc are not activated by social
interactions.

150

Figure 3.4.

Pubertally born cells in the dentate gyrus are not activated
by social interactions.

151

Figure 3.5.

Pubertally born cells in the adult MePD, VMH, and DG
differentiate into mature neurons and astrocytes.

152

x

KEY TO ABBREVIATIONS

Avidin-biotin complex

ABC

analysis of variance

ANOVA

androgen receptor

AR

anteroventral periventricular nucleus

AVPV

5-bromo-2’-deoxyuridine

BrdU

3,3’-Diaminobenzidine tetrahydrochloride

DAB

dentate gyrus

DG

doublecortin

DCX

estrogen receptor alpha

ERα

glial fibrillary acidic protein

GFAP

immediate early gene

IEG

immunoreactive

ir

intraperitoneal

i.p.

induced testicular feminization mutation

iTfm

luteinizing hormone

LH

posterodorsal medial amygdala

MePD

neural/glial antigen 2

NG2

neuronal nuclei

NeuN

postnatal day

P

principal nucleus of the bed nucleus of the stria terminalis

pBNST

progesterone receptor

PR

xi

proliferating nuclear cell antigen

PCNA

sexually dimorphic nucleus of the preoptic area

SDN-POA

standard error of the mean

SEM

subgranular zone

SGZ

subventricular zone

SVZ

subcutaneous

s.c.

posterodorsal medial amygdala

MePD

Tris-buffered saline

TBS

ventromedial hypothalamus

VMH

ventromedial hypothalamus (ventrolateral subdivision)

VMHvl

ventromedial hypothalamus (dorsomedial subdivision)

VMHdm

ventromedial hypothalamus (central subdivision)

VMHc

wildtype

WT

xii

INTRODUCTION
During puberty, the brain experiences substantial structural reorganization that,
in turn, facilitates behavioral changes necessary to navigate the adult world. We’ve
known for about 20-25 years that frontal and temporal cortical regions undergo
significant remodeling during adolescence through synaptogenesis, synaptic pruning,
apoptosis, and increased myelination (Barnea-Goraly et al., 2005; Blakemore et al.,
2010; Giedd, 2004, 2008; Sisk et al., 2005). However, recent studies from our lab have
revealed a new agent of change in the adolescent brain, namely the addition of new
neurons and glial cells to subcortical regions such as the amygdala and hypothalamus,
both of which are involved in regulating sex-specific social behaviors (Ahmed et al.,
2008; Mohr et al., 2016; Mohr et al., 2013).
Postnatal Neuro- and Gliogenesis
Research on the generation of new neurons in the brain, or neurogenesis,
outside of the early formative stages of brain development was a highly controversial
topic for decades (Kaplan, 2001). In the 1960s, the popular opinion within the field of
neuroscience was that the brain remained a static structure producing very little, if any,
new neurons beyond the time of birth (Gross, 2000). However, in the 1980s and 90s,
researchers began to reexamine the possibility of adult neurogenesis and the
groundbreaking discovery was made that new neurons are, in fact, generated in the
adult hippocampus and olfactory bulbs across several species (e.g. canaries, mice, rats,
tree shrews, and primates) (Cameron et al., 1993; Goldman et al., 1983; Gould et al.,
1997; Gould et al., 1999; Kempermann et al., 1997; Paton et al., 1984). These findings
began to shift the view of neuroscience and reignited the field’s interest in postnatal

1

neurogenesis. It is now widely recognized that new cells are continuously added to the
hippocampus and olfactory bulbs throughout the lifespan.
Throughout the years, most of the efforts to investigate postnatal neurogenesis
have been directed towards the hippocampus and olfactory bulbs. The hippocampus
and olfactory bulbs gain a significant number of new neurons throughout the lifespan,
continuously supplied with newborn neurons by the subgranular zone (SGZ) of the
hippocampus and subventricular zone (SVZ) of the lateral ventricles, respectively
(Alvarez-Buylla et al., 2004). The SGZ and SVZ have been characterized as neurogenic
niches, giving rise to new neurons in the postnatal brain (Alvarez-Buylla et al., 2004).
However, in recent years, it was discovered that new neurons are also added outside of
these canonical neurogenic regions and investigators have come to realize that other
limbic structures, such as the amygdala and hypothalamus, gain new cells during
adolescence and adulthood (Ahmed et al., 2008; Fowler et al., 2003; Fowler et al.,
2005; Mohr et al., 2016; Mohr et al., 2013; Saul et al., 2015).
Although research regarding adult neurogenesis continues to grow, research on
pubertal neurogenesis remains relatively sparse. This lack of attention towards the
pubertal brain is surprising, given the brain’s increased plasticity during adolescence.
Studies have determined that the adolescent brain gains significantly more new neurons
than the adult brain (He et al., 2007; Ho et al., 2012; Saul et al., 2014). Moreover, it
appears that pubertally born neurons are functionally distinct from adult born neurons.
For instance, when using a transgenic mouse model to block neurogenesis in the SGZ
and SVZ at different time points, researchers discovered that blocking pubertal
neurogenesis in female mice reduced social exploration and increased social aversion

2

during female-female social interactions. In contrast, blocking adult neurogenesis did
not have any significant effect on these social behaviors. However, pubertal and adult
neurogenesis appear to have a similar function in other behaviors (i.e. anxiety and
object-recognition memory), as blocking neurogenesis during both time points reduced
anxiety-like behavior and impaired preference for novel objects (Wei et al., 2011).
Interestingly, it was also discovered that reducing pubertal neurogenesis globally in
mice using temozolomide, a chemotherapy drug, caused impaired spatial learning on
the Morris water maze task. However, reducing adult or middle-aged neurogenesis in
the same manner did not have a deleterious effect on spatial learning (MartinezCanabal et al., 2013). Thus, neurons born at different developmental periods appear to
perform specialized behavioral functions, with pubertally born neurons facilitating the
acquisition of adaptive behaviors (e.g. appropriate social interactions and spatial
navigation for mating and food foraging) required for adulthood.
Contrary to postnatal neurogenesis, the concept of postnatal gliogenesis, or the
generation of new glial cells, has been readily accepted by neuroscientists for several
decades (Kaplan, 2001). It was long believed that glial cells were merely supportive
cells in the brain, or the glue that held the brain together, as astrocytes provided
nutrients to neurons, microglia cleaned up debris from neuronal injury, and
oligodendrocytes myelinated axons (Allen et al., 2009). In short, the major contribution
of glial cells to neural functioning was largely undervalued and ignored and it was not
until the late 1990s that scientists began looking past these passive roles originally
assigned to glial cells to investigate the more active presence of glia. Today, we know
that glial cells play an important role in maintaining a healthy neural environment.

3

Astrocytes regulate the formation, maintenance, and elimination of synapses in the
brain (Clarke et al., 2013). Furthermore, astrocytes possess transporters that participate
in the reuptake of neurotransmitters (e.g. glutamate) and can also release glutamate
stores into the extracellular space to replenish the glutamate supplies of neighboring
neurons (Clarke et al., 2013). In addition, not only do microglia clean up debris in the
central nervous system, but they also mediate synaptic development by way of synaptic
pruning (Harry et al., 2012) and oligodendrocytes have the capacity to release
neurotrophic factors to aid in axonal repair (Alizadeh et al., 2015). Even though great
strides have been taken to better understand the functional role of glial cells in the
nervous system, little is known regarding their developmental trajectory in the postnatal
brain. As with postnatal neurogenesis, the study of gliogenesis in the adult brain has
dominated the field and research on pubertal gliogenesis remains scarce. The brain
experiences synaptic reorganization and an increase in white matter volume during
puberty and adolescence (Giedd et al., 1999), which are most likely mediated by glial
cells. Therefore, it is essential that we gain a better understanding of pubertal
gliogenesis to determine whether glial cells generated during puberty, rather than
preexisting glial cells, contribute to structural and functional changes in the adolescent
brain.
Because adaptive social functioning is critical in maintaining psychological
health, it is important to understand the mechanisms by which neural circuits regulating
social behaviors are remodeled in the pubertal brain. Currently, there is a paucity of
research examining pubertal neurogenesis and gliogenesis, especially in regions
outside of the traditional neurogenic regions such as the amygdala and hypothalamus,

4

which are known to regulate sociosexual behaviors (i.e. mating and aggression) that
emerge during puberty (Cooke et al., 2003; Cooke et al., 2000; Kollack-Walker et al.,
1992, 1995; Lee et al., 2014; Lin et al., 2011). Because the prevalence of mental health
disorders that affect social behaviors increases during puberty(Paus et al., 2008), an
improved understanding of the determinants and downstream effects of pubertal
neurogenesis is clearly warranted. Given that some pubertally born neurons are
functionally distinct from adult born neurons (Wei et al., 2011), not all findings regarding
adult neurogenesis and gliogenesis can be extended to explain the regulation of these
processes during puberty. It is essential that more attention be shifted onto pubertal
neurogenesis and gliogenesis research in order to 1) further our understanding of the
functional role of pubertally born cells in adulthood and 2) determine their contribution to
adolescent structural brain changes.
The Social Neural Network
Rodents have complicated social networks and interact with one another based
largely on chemosensory cues to determine appropriate social action that, in the wild,
would increase their chances of survival and/or reproduction. For instance, adult male
mice are strongly attracted to female mice, as they find their scent (e.g. urine or vaginal
secretions) to be rewarding(Bell et al., 2013a; Brown, 1979). However, an adult male
mouse can find the scent of another male mouse to be aversive, causing avoidance or
aggressive behavior depending on the context of the social situation(Brown, 1979;
Jones et al., 1989). These behaviors emerge and are refined throughout the adolescent
period providing mice with the necessary skills to thrive as adults. The development of
adult sociosexual behaviors (i.e. sexual behavior and aggression) has been attributed to

5

the maturation of the adolescent brain, which includes significant structural changes to
the amygdala and hypothalamus(Sisk et al., 2005).
The posterodorsal medial amygdala (MePD) is a subregion in the amygdala that
receives strong input from the accessory olfactory bulb and, in turn, sends afferent
projections to hypothalamic regions known to govern reproductive and defensive
behaviors(Pardo-Bellver et al., 2012). Thus, the MePD evaluates and categorizes
chemosensory signals in order to initiate contextually-appropriate adult social behaviors
(Samuelsen et al., 2009). In male mice, when the scent of a female (e.g. urine) is
detected, the MePD becomes activated to initiate male sexual behavior (Samuelsen et
al., 2009). Further, ablation of the MePD impairs opposite sex odor preference in male
hamsters (Maras et al., 2006). The MePD of male rodents is also activated by social
encounters with female and male conspecifics (Hong et al., 2014; Kollack-Walker et al.,
1995; Taziaux et al., 2011). Sociosexual behaviors and opposite sex odor preference
emerge around the time of puberty, coinciding with the sudden rise in pubertal gonadal
hormones, primarily testosterone in males and estrogen in females (Cooke et al., 2003;
Cooke et al., 2007; Cooke et al., 2005). The MePD is densely populated with androgen
receptors (ARs) and estrogen receptors (ERs)(Simerly et al., 1990), and therefore,
gonadal hormones may act via ARs and/or ERs in the MePD during puberty to mediate
the maturation of sex-specific sociosexual behaviors in adulthood.
The adult MePD is a male-biased sexually dimorphic brain region. Males have a
larger volume, more neurons and glial cells, larger soma size, and greater synapse
number in the MePD than adult females (Cooke et al., 2003; Cooke et al., 2005; Morris
et al., 2008a; Morris et al., 2008b). Manipulating adult hormones can eliminate some of

6

these sex differences. For example, castrating adult male rats can reduce MePD
volume, neuronal soma size, and glial cell number, while treating adult females with
testosterone for at least 4 weeks can increase MePD volume and neuronal soma size
(Morris et al., 2008a). Although sex differences in the MePD exist prior to puberty, they
become even more prominent during puberty (Johnson et al., 2013). Sex differences in
pubertal hormone exposure (i.e. testosterone in males and estrogen in females) likely
facilitate sex differences in the morphological changes observed in the MePD during
puberty, which may mediate the emergence of sex-specific social behaviors (Juraska et
al., 2013). In male rats, sex differences in MePD volume before puberty are
independent of AR activity; however, this sex difference in MePD volume during puberty
requires the presence of functional ARs (Johnson et al., 2013). Thus, the MePD is
highly sensitive to circulating gonadal hormones, with males requiring pubertal ARs
establish sex differences in the adult MePD. However, it still remains unclear how
pubertal hormones, such as testosterone and estrogen, act via ARs and ERs to shape
sex differences in the MePD during puberty to facilitate the acquisition of sex-specific
sociosexual behaviors in adulthood.
The VMH is known regulate a wide variety of behaviors, such as feeding, female
sexual behavior(King, 2006; McClellan et al., 2006; Pfaff et al., 1979), and male
sociosexual behaviors (Choi et al., 2005; Lee et al., 2014; Lin et al., 2011). The
ventrolateral subdivision of the VMH (VMHvl) and dorsomedial subdivision of the VMH
(VMHdm) are the most well characterized subdivisions. Both the VMHvl and VMHdm
are strongly innervated by the MePD and pheromonal cues are processed by the MePD
(Pardo-Bellver et al., 2012) to then elicit reproductive or defensive behaviors,

7

respectively (Kunwar et al., 2015; Lin et al., 2011; Mongeau et al., 2003). In male mice,
cell recordings revealed that VMHvl neurons are excited in response to aggressive
interactions, while sexual encounters inhibited the same VMHvl neurons (Lin et al.,
2011). In addition, optogenetic activation of VMHvl neurons evokes aggressive
behaviors, whereas silencing these neurons inhibited aggressive behaviors(Lin et al.,
2011). Further investigation revealed that estrogen receptor-a (ERa) neurons are
responsible for controlling aggressive behavior in adult male mice, as activing ERa
neurons in the VMHvl was able to initiate attack behaviors towards both male and
female mice(Lee et al., 2014). Weak stimulation of these ERa neurons also promotes
male sexual behavior(Lee et al., 2014). Another study showed that ERa and
progesterone receptor ablation in the VMHvl reduces male sexual behavior and
intermale aggression in adult male mice (Sano et al., 2013; Yang et al., 2013).
Interestingly, when exposed to a predator, the VMHdm, but not VMHvl, shows a robust
Fos response, a marker of neural activation, in adult male mice (Silva et al., 2013).
Moreover, a predator scent alone (i.e cat collar) can also increase Fos expression in the
VMHdm of adult male rats (Dielenberg et al., 2001). The VMHc has also been
implicated in the control of defensive behaviors as stimulation of neurons in the VMHdm
and VMHc induced avoidance behaviors, such as fleeing, jumping, and immobility, in
adult male mice (Wang et al., 2015). Therefore, it is clear that, in adult male mice, the
VMHvl is involved in gating male sexual behavior and intermale aggression, while the
VMHdm and VMHc mediate defensive behaviors. As mentioned previously, male sexual
behavior and intermale aggression begin to emerge at the onset of puberty; however,
defensive behaviors are present prior to puberty. Interestingly, the VMHvl has a higher
8

density of ARs and ERs than the VMHdm and VMHc, suggesting that pubertal gonadal
hormones may act on sex steroid receptors in the VMHvl to facilitate the emergence of
these adult sociosexual behaviors.
Structurally, the VMH is a male-biased brain region, with males having a larger
volume than females (Dugger et al., 2007; Matsumoto et al., 1983). Initial studies
examining sex differences in the VMH established that males have a larger VMH than
females early in development during the perinatal period. Castrating male rats on P1
eliminates sex differences in VMH volume in adulthood, while castrating male rats on
P7 does not(Matsumoto et al., 1983). While early studies did not distinguish between
VMH subdivisions, later studies discovered that the sex difference in VMH volume is
largely accounted for by the VMHvl, while the VMHdm and VMHc are known to be
monomorphic. Furthermore, the larger VMHvl volume in males can be accounted for by
males having a larger neuronal soma size and a greater amount of neuropil than
females, and not neuron number (Dugger et al., 2007; Flanagan-Cato et al., 2001;
Madeira et al., 2001). While most of the structural VMHvl sex differences are
established prior to puberty, sex differences in the number of spine synapses and shaft
synapses do not emerge until puberty when males have significantly higher numbers
compared to females (Pozzo Miller et al., 1991). In addition, treating females with
testosterone eliminates this sex difference(Pozzo Miller et al., 1991). Therefore,
synaptic reorganization in the VMHvl during puberty could contribute to the maturation
of sex-specific sociosexual behaviors in adulthood.
The current literature indicates that the MePD and VMH play an integral role in
the regulation of sociosexual behaviors in adulthood and that structural changes in

9

these brain regions during puberty may mediate the development of these behaviors to
allow individuals to survive and thrive in the adult world. Recent reports indicate that
new neurons and glial cells are added to the amygdala and hypothalamus during
puberty(Ahmed et al., 2008; Mohr et al., 2016; Mohr et al., 2013), but it remains
unknown whether or how these pubertally born cells may contribute to the gain of
function observed during adolescent development. As previously mentioned,
sociosexual behaviors such as opposite-sex odor preference and intermale aggression
are not established until the pubertal period(Barkley et al., 1977; Bell et al., 2013a; Bell
et al., 2013b; Brown, 1979; Jones et al., 1974, 1989; Maras et al., 2006; Tenk et al.,
2009). Since these sociosexual behaviors are regulated by the MePD and VMH, it is
possible that new neurons and glial cells born during puberty may alter existing neural
circuits that underlie the development of these behaviors.
Defensive behaviors are present in early development, as rodents can display
freezing behaviors at a young age(Wiedenmayer, 2009). The implementation of more
adaptive defensive skills throughout development, such as avoidance behavior
(Wiedenmayer, 2009), may result from the maturation of the VMHdm and VMHc. Given
that many of these more advanced behaviors do not emerge until the pubertal period, it
is of great interest to investigate how pubertal hormones shape the adolescent brain.
Furthermore, a study has shown that pubertally born cells in limbic and hypothalamic
regions of the male hamster are activated during a sexual encounter(Mohr et al., 2013).
Thus, not only are new cells generated during puberty, they also mature into more
permanent fixtures in the brain and likely participate in the expression of adult social
behaviors. Since it has been established that the amygdala and hypothalamus are

10

capable of gaining new cells during puberty, it is of great interest to investigate how the
addition of new cells during puberty contribute to sex differences in adulthood and
whether these newly generated cells contribute to the maturation of these sociosexual
behaviors.
Hormonal Regulation of Pubertal Neurogenesis and Gliogenesis
Neurogenesis and gliogenesis have not been well defined in the adolescent
brain, and furthermore, there are very few studies that have examined this phenomenon
outside of the canonical neurogenic zones. However, due to the strong influence of
pubertal hormones on MePD and VMH, it is of great interest to examine how
manipulation of gonadal hormones affects proliferation and/or survival of pubertally born
neurons and glia into the adult brain and also, how these pubertally born cells contribute
to the gain of function (e.g. sociosexual behaviors) during adolescent development.
In one of the initial studies examining pubertal cytogenesis, sex differences in the
number of pubertally born cells added to sexually dimorphic brain regions in rats were
examined(Ahmed et al., 2008). The study focused on three sexually dimorphic brain
regions: the medial amygdala, the sexually dimorphic nucleus in the preoptic area
(SDN-POA), and the anteroventral paraventricular nucleus (AVPV). The medial
amygdala and SDN-POA are both male-biased brain regions, as these regions are
volumetrically larger in males than females and are known to mediate male sex
behavior(Morris et al., 2004). In contrast, the AVPV is a female-biased brain region
known to be involved in controlling the luteinizing hormone (LH)-surge in adult
females(Mohr et al., 2016; Morris et al., 2004). The study found that, during puberty, the
medial amygdala and SDN-POA had more pubertally born cells surviving to adulthood

11

in males than in females; however, in the AVPV females had more pubertally born cells
than males in adulthood(Ahmed et al., 2008). Furthermore, it appeared that these sex
differences were driven by gonadal hormones, as pre-pubertal gonadectomy reduced
the number of pubertally born cells in medial amygdala and SDN-POA in males, but not
females, and the number of pubertally born cells in AVPV in females, but not
males(Ahmed et al., 2008). These results suggest that one role pubertal sex hormones
play in achieving fully mature adult neural circuits is to promote the addition and/or
survival of new cells in specific sexually dimorphic regions of the brain.
Despite these compelling results, very little work has been done to follow-up
these findings and examine the effects of gonadal hormones on the proliferation or
survival of pubertally born cells. However, there is evidence that demonstrates the
importance of gonadal hormones in modulating the addition of new cells in the adult
brain. In adult male rats, androgens increased cell survival in the DG (Hamson et al.,
2013; Spritzer et al., 2007). These results were supported by a follow-up study that
demonstrated functional ARs were necessary to promote the survival of adult born
neurons (Hamson et al., 2013). Although, testosterone did not influence cell proliferation
in the DG or VMH of adult male meadow voles (Fowler et al., 2003), estrogen enhanced
the cell survival in the DG (Ormerod et al., 2004). Additionally, estrogen enhanced cell
proliferation in the DG of adult female rats (Tanapat et al., 1999), but not adult female
meadow voles (Fowler et al., 2005). In adult male meadow voles and hamsters,
testosterone did increase cell proliferation (Antzoulatos et al., 2008; Fowler et al., 2003)
in the medial amygdala; in male meadow voles, this increase in proliferation was largely
due to the effects of estrogen rather than androgens. Furthermore, testosterone did not

12

enhance cell survival in the medial amygdala of adult male hamster (Antzoulatos et al.,
2008). However, testosterone appears to have a different effect on the pubertal brain,
as testosterone promoted the number of pubertally born cells in the adult male rat
medial amygdala, suggesting that testosterone enhance the proliferation and/or survival
of these pubertally born cells. Interestingly, a study also demonstrated that, in male rats,
the addition of pubertally born astrocytes was dependent on the presence of functional
ARs. Based on this information, it appears that gonadal hormones have a species-,
brain region-, age-, and sex-specific effect on the proliferation and survival of newly
born cells in the postnatal brain.
Studies of pubertal cytogenesis have also confirmed that a subset of pubertally
born cells in the medial amygdala, SDN-POA, and AVPV of adult male and female rats
differentiate into mature neurons or astrocytes (Ahmed et al., 2008; Mohr et al., 2016).
In adult male hamsters pubertally born cells in the medial amygdala, medial preoptic
area, and arcuate nucleus also differentiate into mature neurons and astrocytes, with
the arcuate nucleus and MePD showing the highest percentage of pubertally born cells
expressing a mature neuronal marker (30% and 23% respectively) compared to the
medial preoptic area (9%) (Mohr et al., 2013). Interestingly, a study in male rats
discovered that 56-day old pubertally born cells did not differentiate into mature neurons
in the adult amygdala, but discovered that 10-day old pubertally born cells did express
doublecortin (~30%), an immature neuron marker, and NG2 (54%), an oligodendrocyte
precursor cell marker (Saul et al., 2015). The latter study only administered BrdU during
P30-31(Saul et al., 2015), while a previous study examining the medial amygdala
administered BrdU for 3 weeks during puberty (P28-49)(Mohr et al., 2013). Therefore,

13

differences in the number of pubertally born neurons in the amygdala may be due to
differences in the age of these cells. It is possible that pubertally born cells born outside
of P30-31 are more likely to differentiate into mature neurons. Overall, these studies
demonstrate that a subset of pubertally born cells in the amygdala and hypothalamus
survive into adulthood and differentiate into mature neuronal and glial phenotypes.
However, the proportion of cell phenotypes (i.e. neurons vs. glia) may be site-specific
and age-specific.
While it is likely that gonadal hormones play a key role in regulating pubertal
cytogenesis, further investigation is required to determine how hormones, such as
testosterone and estrogen, mediate the addition of new neurons and glial cells in the
different regions of the pubertal brain. It is unknown whether ARs or ERs modulate the
survival of pubertally born cells into adulthood. In order to ask more in-depth questions
regarding the hormone regulation of pubertal cytogenesis, it will be in the best interest
for researchers to utilize the mouse model because of the wide array of genetic tools
currently available for dissecting and understanding the relevant neurobiological
mechanisms. At the moment, it is unknown whether, in mice, there are sex differences
in pubertally born cells in sexually dimorphic brain regions, such as the MePD and
VMH. Thus, further investigation is required to determine if and how gonadal hormones
influence the survival of pubertally born cells in the amygdala and hypothalamus and
how these pubertally born cells contribute to adult sociosexual behaviors in mice.
Dissertation Overview
Previous studies in rats and hamsters have shown that new cells are added to
the amygdala and hypothalamus during puberty, and some of these cells differentiate

14

into mature neurons or glial cells (Mohr et al., 2013). There is also evidence that a
subset of these pubertally born cells become functionally integrated into adult neural
circuits. However, it remains unclear how pubertal gonadal hormones may contribute to
the proliferation and survival of these pubertally born cells or how these cells contribute
to the gain of function that characterizes adolescent development. To investigate
postnatal neurogenesis, many researchers utilize 5-bromo-2’-deoxyuridine (BrdU), a
thymidine analog that is incorporated into DNA during the S-phase of the cell cycle.
Today, it remains a popular tool and is commonly referred to as a cell birth date marker.
Some skeptics in the field argue that BrdU labels postmitotic cells undergoing DNA
repair, rather than new neurons (Taupin, 2007); however, studies have shown that
BrdU-immunoreactive (ir) cells co-express proliferative markers such as PCNA or Ki67,
indicating that these BrdU-ir cells are actually new proliferating cells and not older,
preexisting neurons undergoing DNA repair(Lagace et al., 2010; Mohr et al., 2016).
More specifically, our lab has demonstrated that about 95% of pubertally born cells in
the anteroventral paraventricular nucleus of the hypothalamus (AVPV) co-express
PCNA (Mohr et al., 2016). Therefore, we will use BrdU to label pubertally born cells in
the current dissertation.
Since pubertal cytogenesis has not yet been examined in the mouse brain, the
experiments in the current dissertation focus its attention on pubertal cytogenesis in
mice. In chapter 1 of this dissertation, I first examine whether pubertal cytogenesis
occurs in the mouse brain and whether there is a sex difference in the survival of
pubertally born cells in sexually dimorphic brain regions in adulthood. For this
dissertation, I examine the following brain regions of interest (ROIs): MePD, VMHvl,

15

VMHdm, VMHc, and DG. Again, the MePD and VMHvl are male-biased brain regions,
having a larger volume in males than females(Dugger et al., 2007; Morris et al., 2008a;
Morris et al., 2008b; Pfau et al., 2016), while the VMHdm, VMHc, and DG are known to
be monomorphic brain regions, showing no sex differences in volume in
adulthood(Dugger et al., 2007; Tabibnia et al., 1999). The results of chapter 1 show that
new cells are added to the pubertal brain in mice, and there is a sex difference in the
survival of these pubertally born cells with males having a greater number of pubertally
born cells than females in the MePD and VMHvl (both male-biased brain regions). No
sex differences in pubertally born cells were detected in the VMHdm, VMHc, and DG.
The results of chapter 1 also demonstrate that functional ARs are necessary to enhance
the survival of pubertally born cells in the adult MePD, but not VMHvl, VMHdm, VMHc,
or DG, using iTfm male mice, a transgenic mouse model where AR is rendered nonfunctional throughout the lifespan. Furthermore, the presence of both pubertal and adult
testosterone increases the number of pubertally born cells in the adult MePD of male
mice.
Following the results of Chapter 1, Chapter 2 of this dissertation investigates
whether AR specifically during puberty or adulthood are necessary to enhance the
survival of pubertally born cells. We discover that AR during puberty, but not adulthood,
is required to increase the number of pubertally born cells in the adult MePD. Disabling
AR during puberty or adulthood does not affect the density of pubertally born cells in the
VMHvl, VMHdm, VMHc, or DG. We also find that blocking AR during puberty reduces
the survival of pubertally born neurons, but not pubertally born astrocytes, in the adult
MePD.

16

In Chapter 3 of this dissertation, I examine whether pubertally born cells are
functionally incorporated in response to male-male or male-female social interactions.
The results of chapter 3 show that pubertally born cells in the MePD and VMH are
functionally incorporated into existing neural circuits and are activated (i.e., express fos)
in response to social interactions in adulthood. More specifically, we find that male-male
and male-female social interactions activate pubertally born cells in the MePD to
equivalent levels, whereas male-male social interactions, in particular, activate
pubertally born cells in the VMH.
This dissertation is a first step towards understanding the role of pubertal
cytogenesis in the amygdala and hypothalamus of the mouse brain, a phenomenon that
is virtually unexplored. While we establish that pubertal testosterone act via ARs in the
male mouse brain to increase the number of pubertally born cells in the adult MePD and
that a subset of pubertally born cells are functionally integrated into the adult brain,
there is still much to learn about the significance of pubertally born cells in adult brain
functions. In the conclusions chapter of this dissertation, I further discuss the
implications of the dissertation findings and consider future studies to expand on these
results to better understand the mechanisms underlying pubertal cytogenesis and its
contribution to the function of the adult brain.

17

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18

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CHAPTER 1A
Sex differences in pubertally born cells in the posterodorsal medial amygdala
in adult mice
ABSTRACT
The posterodorsal medial amygdala (MePD) regulates many sex-specific social
behaviors, such as sexual behavior and aggression, and is sexually dimorphic, with
males having a larger volume than females in adult rats and mice. While structural sex
differences in the rodent MePD exist prior to puberty, they become even more
exaggerated during pubertal development and are maintained into adulthood. Recently,
sex differences in the addition of new cells during puberty have been reported in the
MePD of rats, with males displaying more pubertally born cells in the adult MePD than
females. This result suggests neuro- and gliogenesis as a possible mechanism by
which sex differences in MePD structure are shaped during puberty. However, it is
currently unknown whether sex differences in pubertal cytogenesis also exist in the
mouse MePD or whether gonadal hormones influence the proliferation and/or survival of
pubertally born cells in the mouse brain. To investigate this, we conducted four
experiments in which all mice were administered a daily i.p. injection of 5-bromo-2’deoxyuridine (BrdU) during puberty (postnatal day (P)28-49). First, we examined sex
differences in the survival of pubertally born cells in the adult MePD. Then, we used an
androgen receptor (AR)-null transgenic mouse model (iTfm) along with male and female
wildtype littermates to investigate the role of AR on generating sex differences in
pubertally born cells in the adult MePD. Next, we manipulated pubertal testosterone in
male, female, and iTfm males to determine whether pubertal testosterone contributed to

26

sex differences in pubertally born MePD cell numbers in adulthood. Lastly, we explored
the differential effects of pubertal and adult testosterone on the addition of pubertally
born cells in the adult MePD by treating male mice with testosterone during puberty
and/or adulthood. Overall, we discovered a sex difference in pubertally born cell number
and density in the adult MePD with male mice having more pubertally born cells than
females. In addition, we found that functional ARs and pubertal testosterone are
necessary to establish sex differences in pubertally born cell number in the adult MePD.
Finally, we concluded that both pubertal and adult testosterone enhance the number of
pubertally born cells in adult male mice. These results indicate that pubertal
testosterone and ARs play an important role in establishing sex differences in the
number of pubertally born cells in the adult MePD of mice. Since many of the adult
social behaviors controlled by the MePD emerge at the onset of puberty and require the
presence of gonadal hormones, these findings suggest that cells added during puberty
may potentially contribute to the emergence of sex-specific adult behaviors in mice.
INTRODUCTION
Puberty and adolescence represent a developmental period during which the
brain undergoes significant changes in both structure and function. Well documented
structural changes in the adolescent brain include synaptic pruning and growth, cell
death, and myelination, which likely promote the acquisition of mature behaviors (e.g.
social and reproductive behaviors) necessary to navigate the adult world (BarneaGoraly et al., 2005; Blakemore et al., 2010; Blakemore et al., 2006; Giedd, 2004; Giedd
et al., 1999; Zehr et al., 2006). However, recently, researchers have identified a new

27

and surprising agent of change in the adolescent brain: neurogenesis and gliogenesis in
the amygdala (Ahmed et al., 2008; Mohr et al., 2013; Saul et al., 2015).
Research on adolescent neurogenesis and gliogenesis traditionally focus on two
brain regions, the hippocampus and the olfactory bulb – both long recognized to gain
new cells during adolescence and even adulthood. The subventricular zone and the
subgranular zone of the hippocampus are characterized as neurogenic niches, giving
rise to new neurons throughout the lifespan (Alvarez-Buylla et al., 2004). However, in
recent years, the focus on neuro- and gliogenesis has turned to regions outside of these
canonical neurogenic zones, as studies have discovered other limbic regions, like the
medial amygdala, also gain new cells in adolescence and adulthood (Ahmed et al.,
2008; Fowler et al., 2003; Fowler et al., 2002; Mohr et al., 2013; Saul et al., 2015).
The posterodorsal medial amygdala (MePD) is a sexually dimorphic brain region
known to regulate sociosexual behaviors, such as sexual behavior (Cooke et al., 2003;
Cooke et al., 2000). The adult male rodent MePD has a larger volume, more neurons
and glial cells, and larger neuronal soma size than the adult female MePD (Johnson et
al., 2008; Morris et al., 2008a; Morris et al., 2008b; Pfau et al., 2016). Many structural
sex differences in the adult MePD can be altered by manipulating circulating gonadal
hormones (Cooke et al., 2003; Johnson et al., 2013; Morris et al., 2008a; Morris et al.,
2008b), which is not surprising given that the MePD is densely populated with sex
steroid receptors (Simerly et al., 1990). For example, castration of adult males leads to
a reduction of MePD volume, smaller soma size, and fewer glial cells (Morris et al.,
2008b), and conversely, treating adult females with testosterone increases MePD
volume and soma size (Morris et al., 2008a). However, the manipulation of gonadal

28

hormones in adulthood does not have a significant effect on the number of MePD
neurons (Morris et al., 2008a), suggesting the sex difference in MePD neuron number is
organized before adulthood.
Studies examining pubertal cytogenesis in the amygdala of rats and hamsters
reveal a sex difference in the addition of new cells in the medial amygdala, including the
MePD, with adult males having more pubertally born cells in the medial amygdala than
adult female rats (Ahmed et al., 2008). Furthermore, pre-pubertal castration of male rats
eliminates this sex difference, with the number of pubertally born cells decreasing to
levels comparable to that of gonadally intact adult females (Ahmed et al., 2008). The
evidence, thus far, suggests that the addition of new cells during puberty may be a key
cellular event in the development of sex differences in the MePD. However, while
gonadal hormones are known to affect the number of pubertally born cells in the adult
MePD, the mechanisms through which gonadal hormones act to influence pubertal
cytogenesis requires further investigation.
Sex differences in the MePD exist prior to puberty, but these sex differences are
amplified in adulthood. This process appears to depend on the presence of functional
androgen receptors (AR) in rats (Johnson et al., 2013). Using male rats insensitive to
circulating androgens due to testicular feminization mutation (Tfm), researchers showed
wildtype male rats have a significantly larger volume and more astrocytes in the adult
MePD than either wildtype female or Tfm male rats (Johnson et al., 2013). However,
prior to puberty, Tfm male rats have a MePD volume and astrocyte numbers more
comparable to wildtype males than females (Johnson et al., 2013). These findings
indicate that the pubertal increase in MePD volume and astrocyte number in males

29

requires functional ARs, and prompted investigation of whether ARs also influence
pubertal cytogenesis in mice.
Given that mouse models enable the use of a wide array of genetic tools to
dissect and understand pubertal cytogenesis, the current study investigates pubertal
cytogenesis in the MePD of mice. First, we examine sex differences in the number of
pubertally born cells in the adult MePD. Next, we look at how AR and pubertal
testosterone influence sex differences in pubertally born cells in the adult MePD using
Cre-Lox technology to disable AR in male mice. Lastly, we examine the differential
effects of pubertal and adult testosterone on the number of pubertally born cells in the
adult MePD.
MATERIALS AND METHODS
Animal Housing
All mice were group housed (2-3 same-sex mice/cage) in clear plexiglass cages
(29x18x13cm) in a 12:12 light:dark cycle (lights on: 7:00 AM; lights off: 7:00 PM). All
subjects were provided ad libitum rodent chow (Harlan Teklad 8640 Rodent Diet,
Madison, WI) and water. All housing and experimental procedures were in compliance
with the National Institutes of Health Guide for the Care and Use of Laboratory Animals
and approved by Michigan State University’s Institutional Animal Care and Use
Committee.
Experiment 1: Are there sex differences in the number of pubertally born cells in the
adult MePD?
Experiment 1 examined whether cells are added to the mouse MePD during
puberty and whether there are sex differences in the number of pubertally born MePD

30

cells that survive into adulthood. Male (n=16) and female (n=16) wild-type (WT)
C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, Maine) on postnatal
day 21 (P21). During puberty (P28-49), all mice received a daily i.p. injection of the cell
birth date marker 5-bromo-2’-deoxyuridine (BrdU; 200 mg/kg body weight; Prod. No.
B5002; Sigma-Aldrich) during the light period between 10:00 AM-12:00 PM. In
adulthood, half of the males and females were perfused on P60; the remaining half
were perfused on P90. To analyze group differences in total number of BrdUimmunoreactive (ir) cells and BrdU-ir cell density, a two-way ANOVA was used with sex
and age at sacrifice as independent factors. For all analyses, p<0.05 was considered
statistically significant, and Cohen’s d was calculated to determine effect sizes.
Experiment 2: Are functional androgen receptors necessary to establish sex differences
in the number of pubertally born cells in adulthood?
Experiment 2 investigated the role of ARs on the number and density of
pubertally born MePD cells, using male mice with induced testicular feminization
mutation (iTfm). iTfm males are an AR-null model that is induced by cre/lox technology
as previously described (Chen et al., 2014). WT male (n=10), WT female (n=10), and
iTfm males (n=10) were obtained from an in-house iTfm colony. All animals were
weaned and group housed on P24. During puberty (P28-49), all mice received a daily
i.p. injection of BrdU (200 mg/kg body weight) during the light period between 8:00 AM10:00 AM. To standardize the levels of circulating hormones in the three genotypes in
adulthood, all mice were gonadectomized on P60 and received a subcutaneous Silastic
capsule implant filled with testosterone (1.6 mm – inner diameter and 3.2 mm – outer
diameter; 6 mm – effective release length, 1.6 cm – total capsule length). All animals

31

were perfused on P90. A one-way ANOVA was used to determine group differences in
the total number of BrdU-ir cells, BrdU-ir cell density, and volume by genotype. For all
analyses, p<0.05 was considered statistically significant, and Cohen’s d was calculated
to determine effect sizes.
Experiment 3: Is pubertal testosterone required to establish sex differences in the
number of pubertally born MePD cells in adulthood?
Experiment 3 examined the effects of pubertal testosterone and the role of ARs
on the survival of pubertally born MePD cells in adult mice. WT male (n=20), WT female
(n=20), and iTfm males (n=20) were obtained from the same iTfm colony as in
Experiment 2. All animals were weaned and group housed on P24. On P26, all animals
were gonadectomized and, for each genotype, half the animals were subcutaneously
implanted with a blank Silastic capsule (No T@P) and the other half received a
testosterone-filled Silastic capsule (T@P). During puberty (P28-49), all animals received
a daily i.p. injection of BrdU (200 mg/kg body weight) during the light period between
8:00 AM-10:00 AM. On P60, subcutaneously implanted capsules were removed from all
animals and replaced with a testosterone-filled Silastic capsule to produce equivalent
levels of circulating testosterone in all groups during adulthood. All animals were
perfused on P90. A two-way ANOVA was used to analyze group differences in the total
number of BrdU-ir cells, BrdU-ir cell density, and volume, using genotype and pubertal
testosterone treatment as independent factors. For all analyses, p<0.05 was considered
statistically significant, and Cohen’s d was calculated to determine effect sizes.

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Experiment 4: Do pubertal and adult testosterone have differential effects on the
number of pubertally born cells in the adult MePD?
The purpose of Experiment 4 was to tease apart the effects of pubertal vs adult
testosterone on the number of pubertally born MePD cells in male mice. WT male mice
(n=32) were obtained from a WT C57BL/6 in-house colony. All animals were weaned
and group housed on P24. On P26, all mice were gonadectomized and half the mice
were subcutaneously implanted with a blank Silastic capsule and the remaining half
received a testosterone-filled Silastic capsule. During puberty (P28-49), all animals
received a daily i.p. injection of BrdU (200 mg/kg body weight) during the light period
between 12:00 PM-2:00 PM. On P60, subcutaneously implanted capsules were
removed from all subjects. Within each group, half the mice then received a blank
Silastic capsule, while the other half received a testosterone-filled Silastic capsule.
Thus, four experimental groups were created: 1) no testosterone after gonadectomy on
P26 (T-/-); 2) testosterone during puberty only (T+/-); 3) testosterone in adulthood only
(T-/+); and 4) testosterone during both puberty and adulthood (T+/+). All animals were
perfused on P90. Group differences in the total number of BrdU-ir cells, BrdU-ir cell
density, and volume were examined using a one-way ANOVA comparing four groups:
T-/-, T+/-, T-/+, and T+/+. For all analyses, p<0.05 was considered statistically
significant, and Cohen’s d was calculated to determine effect sizes.
Tissue Collection and Processing
All mice were given an overdose of sodium pentobarbital (150 mg/kg) by i.p.
injection and intracardially perfused with 0.9% saline followed by cold 4%
paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). Brains were removed

33

and postfixed overnight in 4% paraformaldehyde, then stored in 30% sucrose at 4 °C
until sectioning. Brains were cut on a freezing microtome into 30 μm coronal sections,
and collected as either 3 (Experiment 1) or 4 alternate series of sections (Experiments
2-4). For each experiment, the entire first series was Nissl stained with thionin,
dehydrated, and coverslipped.
Immunohistochemistry
In each experiment, the entire second series of brain sections was processed for
immunohistochemistry to label BrdU-ir cells. Free-floating sections were rinsed in Trisbuffered saline (TBS; 0.05 M; pH 7.6) for 30 minutes and then incubated in 0.6% H2O2
for 10 minutes. After a 15-minute rinse in TBS, the sections were incubated in 0.93N
HCl for 30 minutes at 37°C (except in experiment 4 – the sections were incubated in 2N
HCl for 1 hour at 37°C). Sections were next neutralized in 0.1M borate buffer (pH 8.5)
for 10 minutes. Following a 30-minute rinse in TBS, the sections were blocked in TBS
containing 0.01% Triton-X-100 and 3% donkey serum, and then incubated in
monoclonal rat anti-BrdU (catalog no. MCA2060; Serotec) at a working concentration of
1 μg/ml at 4°C for 48 hours. Sections were then rinsed in TBS for 30 minutes and
incubated in biotinylated donkey anti-rat secondary antibody (catalog no. 712-065-150;
Jackson Immunoresearch) at a working concentration of 2 μg/ml for 2 hours at room
temperature. Following a 30-minute rinse in TBS, the sections were incubated in
avidin/biotinylated enzyme complex (ABC Elite Kit, Vector Laboratories, Burlingame,
CA) for an hour and then reacted in 3,3’-Diaminobenzidine tetrahydrochloride (DAB;
Sigma; working concentration 0.25 mg/ml) for about 2 minutes. Following this reaction,
all sections were mounted onto gelatin-subbed slides and coverslipped.

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Microscopic Analysis
All microscopic analyses were performed by an observer blind to treatment group
using an Olympus BX51 microscope under brightfield illumination using Neurolucida
version 10 (MBF Bioscience). The regions of interest were traced using the Nisslstained series and contours were superimposed onto adjacent sections of the singlelabel BrdU series. The boundaries of the MePD on the Nissl series were determined
using a standard mouse brain atlas (Paxinos et al., 2004). To consistently sample
anatomically-matched sections across all animals, the following landmarks were used to
trace the MePD: the shape of the optic tract, the size and shape of the lateral ventricles,
the size and shape of the intercalated amygdalar nucleus, and the size and shape of the
stria terminalis. BrdU-ir cells were identified by the presence of a dark brown nuclear
stain.
Single-label BrdU Analysis
In experiment 1, only five anatomically-matched bilateral sections of the MePD
were available for analysis due to poor tissue quality of the more posterior sections.
Animals that did not have five anatomically-matched sections were excluded from
analysis. Final sample sizes were: P60 females (n=7), P60 males (n=5), P90 females
(n=5), and P90 males (n=6). In experiments 2-4, six sections throughout the full rostralcaudal extent of the MePD were available for analysis. Animals that did not have six
anatomically-matched sections were excluded from analysis. The final sample size for
each experiment were: Experiment 2 – males (n=9), females (n=9), and iTfm (n=9);
Experiment 3 – No T@P males (n=7), No T@P female (n=7), No T@P iTfm (n=9); T@P

35

males (n=7), T@P females (n=7), and T@P iTfm (n=9); Experiment 4 – T-/- males
(n=10), T-/+ males (n=10), T+/- males (n=8), and T+/+ males (n=9).
For all experiments, the total number of BrdU-ir cells was determined by
summing the number of BrdU-ir cells across all sections analyzed. The density of BrdUir cells in each animal was calculated by dividing the total number of BrdU-ir cells
(summed across sections) by the total area (summed across sections). For experiments
2-4, MePD volume was estimated by summing the area of all sections analyzed and
multiplying by the number of series (4) and section thickness (30 μm).
RESULTS
Experiment 1: Adult male mice have more pubertally born MePD cells than adult female
mice.
We found BrdU-ir cells in several regions of the adult mouse brain, including the
MePD (Fig 1.1), indicating that new cells are generated in the mouse brain during
puberty. Two-way ANOVA was performed to examine sex differences in the number of
pubertally born MePD cells in adulthood at P60 and P90. A main effect of sex on total
number of BrdU-ir cells in the MePD was observed (F (1,19)=20.84, p=0.000). Male
mice had ~1.8 times more pubertally born MePD cells than female mice (Fig 1.2A).
There was not a main effect of age at sacrifice, indicating that pubertally born cells
survive from P60 to P90 with very little attrition of cells. Thus, if new cells born during
puberty survive until P60, they will likely survive until P90. There was no significant
interaction between sex and age of sacrifice. Thus, at P60, males have more BrdU-ir
cells in the MePD than females, and this sex difference is stably maintained until at
least P90. A similar pattern of results was obtained when BrdU-ir cell density was

36

analyzed. Two-way ANOVA revealed a significant main effect of sex (F(1,19)=13.05,
p=0.002), with no main effect of age at sacrifice and no significant interaction. Male
mice had ~1.4 times the density of BrdU-ir cells in the MePD compared to female mice
(Fig 1.2B).
Experiment 2: Dysfunctional androgen receptors are sufficient to eliminate sex
differences in the number of pubertally born cells in the adult MePD.
One-way ANOVA revealed a significant difference among genotypes on the total
number of BrdU-ir cells in the adult MePD (F(2,24)=9.27, p=0.001). WT male mice had
1.5 times the number of BrdU-ir cells in the MePD compared to WT female (p=0.011,
d=1.62) and 1.7 times the number of BrdU-ir cells than iTfm male mice (p=0.001,
d=1.84) (Fig 1.3A). No significant difference was observed between WT female and
iTfm male groups. Quantitative analysis also found a significant difference between
genotypes on the density of BrdU-ir cells in the adult MePD (F(2,24)=10.25, p=0.001),
with WT males having 1.5 and 1.6 times the density of pubertally born MePD cells
compared to WT female (p=0.006, d=1.87) and iTfm mice (p=0.001, d=1.78),
respectively (Fig 1.3B). There was no significant difference between the WT female and
iTfm male mice in the density of pubertally born cells in the MePD. There was no
significant difference between genotypes on MePD volume (F(2,24)=0.90, p=0.42) (Fig
1.3C).
Experiment 3: Pubertal testosterone and functional androgen receptors enhance the
number of pubertally born MePD cells.
Two-way ANOVA found a significant main effect of genotype (F(2,40)=9.13,
p=0.001) and pubertal testosterone treatment (F(1,40)=23.22, p=0.000) on the total

37

number of pubertally born MePD cells in adulthood, as well as a significant interaction
(F(2,40)=4.61, p=0.016) (Fig 1.4A). Bonferroni post-hoc tests revealed no significant
differences in the number of BrdU-ir cells between genotype in the No T@P groups.
However, significant differences among genotypes were observed amongst the T@P
groups. Female mice treated with pubertal testosterone had the most pubertally born
MePD cells, with 1.3 times the number of BrdU-ir cells compared to WT male mice
(p=0.026, d=0.88), and 2 times the number of cells compared to iTfm mice (p=0.000,
d=2.03). In addition, male WT mice had 1.5 times the number of BrdU-ir cells compared
to iTfm mice (p=0.011, d=1.33).
A similar pattern of results was obtained when BrdU-ir cell density was analyzed,
i.e., significant main effects of genotype (F(2,40)=7.81, p=0.001) and pubertal
testosterone treatment (F(1,40)=15.63, p=0.000), as well as a significant interaction
(F(2,40)=4.87, p=0.013) (Fig 1.4B). Bonferroni post-hoc tests revealed no significant
differences between genotype within the no T@P groups on the density of BrdU-ir cells
in the MePD. However, there were significant differences amongst all genotypes within
the T@P groups on the density of BrdU-ir cells. Female mice treated with pubertal
testosterone had 1.3 times and 2.1 times higher density of pubertally born cells in the
MePD than males (p=0.034, d=0.89) and iTfm mice (p=0.000, d=2.08), respectively.
Male mice also had a 1.6 times higher density of BrdU-ir cells than iTfm mice (p=0.012,
d=1.19). Two-way ANOVA of MePD volume found no significant main effect of
genotype (F(2,40)=0.24, p=0.790); however, there was a significant main effect of
pubertal testosterone treatment on MePD volume (F(1,40)=4.95, p=0.032, d=0.71), with
mice that received pubertal testosterone having an MePD volume that is ~10% larger

38

than the No T@P groups (Fig 1.4C). No significant interaction was found
(F(2,40)=1.22, p=0.307).
Experiment 4: The presence of both pubertal and adult testosterone increases the
number of pubertally born MePD cells.
One-way ANOVA revealed significant group differences on the total number of
BrdU-ir cells in the MePD (F(3,33)=13.10, p=0.000) (Fig 1.5A). Bonferroni post hoc
analyses showed that T+/+ males had significantly more pubertally born MePD cells
than T-/- (p=0.000, d=2.39), T-/+ (p=0.004, d=1.76), and T+/- (p=0.038, d=1.36) males.
In addition, T+/- males had more BrdU-ir cells in the MePD than T-/- males (p=0.028,
d=1.43). Similarly, T-/+ males had a larger number of BrdU-ir cells compared with T-/males (non-significant trend, p=0.10, d=1.17). Again, an ANOVA showed a similar
pattern of results with significant group differences on the density of pubertally born
cells in the MePD (F(3,33)=10.69, p=0.000) (Fig 1.5B). Bonferroni post hoc analyses
showed that T+/+ males had significantly a higher density of BrdU-ir cells than T-/(p=0.000, d=2.42) and T-/+ (p=0.024, d=1.43) males, but not T+/- males (p=0.420). In
addition, T+/- males had a higher density of pubertally born cells in the MePD than T-/males (p=0.009, d=1.60). Similar to total BrdU-ir cell number, T-/+ males had a higher
density of BrdU-ir cells compared with NoT@P/NoT@A males (non-significant trend,
p=0.10, d=1.21).
Significant group differences in MePD volume were also observed (F(3,33)=3.18,
p=0.037) (Fig 1.5C). Bonferroni post hoc analysis found that T+/+ male mice had a
significantly larger MePD volume than T-/- male mice (p=0.031, d=1.40). No significant
differences in MePD volume were observed between all other groups.

39

DISCUSSION
Sex differences in pubertally born cells in the adult MePD
In the current study, we discovered a sex difference in pubertally born cells in the
adult MePD, with males having a greater number and higher density of pubertally born
cells than females, and this sex difference persists from P60 to P90. Because male
mice typically have a larger MePD volume than females (Morris et al., 2008b; Pfau et
al., 2016), this sex difference in the density of pubertally born cells shows that the gain
of BrdU-ir cells is greater per mm2 in males. This indicates that the greater number of
BrdU-ir cells in males cannot be accounted for by sex differences in MePD size. These
results corroborate previous findings on pubertal cytogenesis in rats, which also found
that males had a higher number of pubertally born cells than females in the adult medial
amygdala (Ahmed et al., 2008).
Interestingly, past studies have also shown that a subset of pubertally born cells
in the rat and hamster MePD differentiate into mature neurons or astrocytes (Ahmed et
al., 2008; Mohr et al., 2013); however, sex differences in the phenotype of pubertally
born cells have not yet been examined. It has been established that male rodents have
more neurons in the adult MePD than females (Morris et al., 2008a), while another
study discovered no sex differences in neuron number prior to puberty (Cooke et al.,
2005). These results suggest that sex differences in the number of neurons in the
MePD develop during puberty, thus, the addition of new cells during puberty may be a
potential mechanism by which adult sex differences in MePD neuron number are
shaped. Further investigation regarding the phenotype of pubertally born cells will

40

provide more insight regarding when sex differences in MePD neuron number are
established.
It should also be noted that neurons are not the only cell-type to increase in
number in brain regions such as the amygdala and hypothalamus from adolescence to
adulthood as several studies have shown that glial cells (i.e. astrocytes, microglia, and
oligodendrocytes) also increase in number during puberty (Chareyron et al., 2012;
Johnson et al., 2013; Mohr et al., 2016; Mohr et al., 2013; Saul et al., 2015). While sex
differences in the addition of microglia and oligodendrocytes in the MePD during
puberty have not been examined, it has been shown that male rats gain significantly
more astrocytes in the MePD than females during puberty (Johnson et al., 2013).
Therefore, the sex difference in the number of pubertally born cells in the adult MePD
could also be accounted for by the addition of new glial cells.
Sex differences in the adult MePD: The role of androgen receptors and pubertal
testosterone
The current findings also demonstrate that the presence of functional ARs and
pubertal testosterone are necessary to establish sex differences in pubertally born
MePD cells in adulthood as iTfm males exhibit a more feminine number and density of
pubertally born cells in the adult MePD. It should be noted that for this experiment,
male, female, and iTfm males remained gonadally intact during puberty, but received
testosterone treatment in adulthood to standardize circulating levels of testosterone.
Despite adult testosterone treatment, a sex difference in pubertally born cells was still
detected, indicating adult testosterone treatment in female mice did not enhance the
number of pubertally born cells to masculine levels. These results indicate that pubertal

41

testosterone plays an important role in establishing sex differences in the adult MePD.
To confirm this, we manipulated pubertal testosterone and, again, standardized
circulating levels of testosterone in adulthood. We found that male and female mice
given pubertal testosterone had more pubertally born cells that iTfm male mice with
pubertal testosterone as well as those not given pubertal testosterone across all
genotypes. These results demonstrate that both functional androgen receptors and
pubertal testosterone are required to generate sex differences in pubertally born cells in
the adult MePD. Similarly, a previous study in rats showed that pre-pubertal
gonadectomy eliminated sex differences in the number of pubertally born cells in the
adult medial amygdala (Ahmed et al., 2008). Furthermore, sex differences in the
addition of astrocytes during puberty in the adult MePD requires functional AR (Johnson
et al., 2013). These results suggest that pubertal testosterone may be activating ARs to
facilitate the proliferation, migration, and/or survival of pubertally born cells in the MePD.
Confirming previous studies, we found that remaining gonadally intact during
puberty and receiving testosterone in adulthood increased female MePD volume
comparable to the size of males; however, we also discovered that the adult MePD
volume was not significantly different between wildtype male and iTfm male mice with
adult testosterone. This novel finding suggests that adult testosterone acts via estrogen
receptors (ERs) to increase MePD volume. The adult MePD volume of Tfm males,
without hormone manipulation, is partially demasculinized, having an intermediate
volume between wildtype males and females (Morris et al., 2005). The volumetric
growth of MePD volume in iTfm males in response to aromatized testosterone is
feasible, as previous studies have demonstrated that both ERs and ARs contribute to

42

generating sex differences in MePD volume and neuronal soma size (Cooke et al.,
2003).
Furthermore, we discovered that pubertal testosterone masculinized adult MePD
volume, regardless of genotype. Subjects that received pubertal testosterone had a
larger MePD volume than those that did not receive pubertal testosterone. Since the
absence of pubertal testosterone reduced MePD volume in both male and female mice,
despite adult testosterone, we can conclude that pubertal testosterone organizes the
MePD to allow for adult testosterone to increase its volume in adulthood. Pubertal
testosterone could be acting via ARs or ERs to organize the MePD, but since iTfm
males also had larger MePD volume when treated with pubertal testosterone, it seems
that an ER-dependent mechanism is a likely candidate. iTfm males have non-functional
ARs, leading them to have small, undescended testes that secrete lower amounts of
testosterone compared to wildtype males (Chen et al., 2014); however since we
previously showed that adult testosterone can increase MePD volume in iTfm male
mice that are gonadally intact during puberty, it is possible that this low level of
testosterone in iTfm males may be aromatized to produce levels of estrogen that are
sufficient to activate ERs and organize the MePD during puberty to enable adult
testosterone to increase MePD volume. Furthermore, adult testosterone increases
female MePD volume (Morris et al., 2008a; Morris et al., 2008b) even though female
rodents do not typically experience high levels of androgens during puberty, indicating
that pubertal estrogen activity in the MePD is sufficient to organize the MePD to allow
for masculinizing effects of adult testosterone on MePD volume.

43

Pubertal and adult testosterone enhance the number of pubertally born cells in the adult
male MePD
Results from the current report establish that pubertal and adult testosterone
work together to increase the number of pubertally born cells in the adult male MePD.
As predicted, male mice had more pubertally born cells in the adult MePD when treated
with testosterone during puberty and adulthood than male mice that received no
hormone treatment. In addition, pubertal testosterone alone increased the number of
pubertally born cells compared to those that did not receive pubertal and adult
testosterone, but without adult testosterone, cell counts were lower than males that
received testosterone throughout the lifespan. Furthermore, adult testosterone alone
resulted in fewer pubertally born cells in the adult MePD than those that received
testosterone throughout the lifespan. These findings suggest that testosterone plays an
important role in enhancing either the proliferation, migration, and/or survival of
pubertally born cells during the pubertal period, but adult testosterone works to
maximize the number of pubertally born cells in the adult MePD.
Moreover, testosterone treatment during puberty and adulthood increased MePD
volume compared to males that received no hormone treatment during these time
points. However, male mice given only pubertal or adult testosterone maintained an
intermediary MePD volume. We previously established that the presence of pubertal
testosterone is necessary for adult male mice to maintain a larger MePD volume relative
to females. Furthermore, in previous findings in this study discussed above, this
activational effect of adult testosterone in males was only seen when males also
experienced pubertal testosterone which presumably organized the MePD in a

44

masculine fashion. Without the presence of pubertal testosterone, adult testosterone did
not increase MePD volume. Thus, the discovery of this intermediate volume in males
given testosterone only during puberty or adulthood was unexpected. A previous study
in male hamsters did find that pubertal testosterone increased MePD volume
independent of the presence of adult testosterone, suggesting that pubertal
testosterone, at least in male hamsters, is sufficient to induce a long-term increase in
MePD volume (De Lorme et al., 2012). However, since we detected a substantial
increase in MePD volume in males that received testosterone during both puberty and
adulthood compared to males that only received testosterone during puberty, we
believe this not to be the case in male mice. These differences between mice and
hamsters could be a result of differing testosterone treatments (i.e. hamster: 2 weeks of
testosterone in adulthood; mice: 4 weeks of testosterone in adulthood); however, it is
also possible that testosterone may organize the pubertal brain differently between
species.
To our knowledge, there are currently no other studies that have examined the
influence of gonadal hormones on the number of pubertally born cells into adulthood,
specifically in the MePD; however, a few studies regarding adult neurogenesis may help
elucidate the effects of gonadal hormones on proliferation and/or survival of newly born
cells in the brain. A study in adult male rats demonstrated that while, testosterone did
not affect cell proliferation in the DG, it does enhance cell survival (Spritzer et al., 2007).
Interestingly, this enhancement in cell survival is due to actions of androgens, and not
estrogen. Treatment with DHT, a potent androgen, but not estrogen, enhanced cell
survival in the DG (Spritzer et al., 2007) and moreover, adult male rats treated with DHT

45

and flutamide, an AR antagonist, as well as Tfm male rats, displayed reduced levels of
cell survival in the DG (Hamson et al., 2013). Surprisingly, testosterone appears to
differentially effect cell proliferation in the medial amygdala compared to the DG with
testosterone increasing cell proliferation in the medial amygdala of adult male meadow
voles (Fowler et al., 2003), but these effects were mediated by estrogens and not
androgens (Fowler et al., 2003). Furthermore, estrogen treatment has been shown in
increase cell proliferation in medial amygdala of adult female meadow voles (Fowler et
al., 2005) as well as the DG of adult female rat (Tanapat et al., 1999). Taken together,
these results indicate that, in adulthood androgens and estrogens have a brain regionspecific and sex-specific effect on adult neurogenesis. In males, androgens regulate cell
survival, at least in the hippocampus, while estrogens promote cell proliferation in the
medial amygdala. In females, estrogen promotes cell proliferation in both the DG and
medial amygdala. Further investigation is required to determine whether pubertal
estrogens and androgens can act in a similar manner to influence cell proliferation,
migration, and/or survival to contribute to sex differences in pubertally born cell number
in the adult MePD.
Significance of pubertally born cells in the MePD
The MePD is a key regulator of sex-specific sociosexual behaviors, such as male
sexual behavior (Kollack-Walker et al., 1992, 1995). Many of these social behaviors
emerge around the time of puberty, suggesting that structural changes that occur during
the pubertal period may facilitate the development of behaviors required to survive and
thrive in the adult world. Morphological changes in the pubertal brain, including the
addition of new cells, are largely influenced by gonadal hormones, which increase at the

46

onset of puberty. Interestingly, manipulation of gonadal hormones significantly alters the
development of social behaviors. For instance, testosterone treatment before and
during puberty, but not after, promotes the development of male sexual behavior in
hamsters(Schulz et al., 2009). In addition, opposite-sex odor preference, a behavior
integral to male sexual behavior, does not develop until puberty(Bell et al., 2013). A
study in male hamsters found that pubertally born cells in the MePD express Fos, a
marker of neural activation, in response to a sexual encounter(Mohr et al., 2013).
Therefore, the acquisition and incorporation of pubertally born cells into existing neural
circuits may critically mediate the emergence of sociosexual behaviors and it is through
the hormonal control of this cellular process that sex-specific sociosexual behaviors
emerge.
Conclusions
The overall aim of this study was to examine sex differences in pubertal
cytogenesis and determine the role of gonadal hormones on pubertally born cells in the
adult mouse MePD. We discovered that there is a sex difference in the number of
pubertally born cells in the adult MePD with males having more cells than females. We
also revealed that androgen receptors are required to establish sex differences in
pubertally born cell number in the adult MePD. Moreover, we found that pubertal and
adult testosterone work together to enhance the number of pubertally born cells in the
adult MePD. Our results establish a novel mechanism by which the pubertal brain is
shaped by pubertal gonadal hormones to potentially contribute to sex differences in
MePD morphology seen in adulthood. It is clear that pubertal androgens act via ARs to
stimulate proliferation and/or enhance survival of pubertally born cells during puberty,

47

while adult testosterone increases the number of pubertally born cells; however, at this
time, it is currently unknown whether adult testosterone acts through an AR or ER
dependent mechanism to promote this increase in cell number. Follow up experiments
will involve an analysis of the phenotype of pubertally born cells, which may differentiate
into neurons or glia, and exploration of the potential functions pubertally born cells may
serve in mediating sex-specific behaviors in adulthood.

48

Figure 1.1. Posterodorsal medial amygdala (MePD) in adult male and female mice.
Representative photomicrographs displaying traces of the MePD on Nissl-stained
coronal sections in the (A) male and (B) female adult mice and pubertally born cells in
the MePD of (C) male and (D) female adult mice. The small inlaid boxes show BrdUlabeled cells at a higher magnification. OT = optic tract; MePV = posteroventral medial
amygdala; BLA = basolateral amygdala. Low mag scale bar = 200 μm; high mag scale
bar = 10 μm.

49

Figure 1.2. Sex differences in the total number and cell density of BrdU-ir cells in
the posterodorsal medial amygdala (MePD) in adult mice. Quantitative analysis
revealed that, in the MePD, (A) male mice have more pubertally born cells than female
mice at P60 and P90 with no significant attrition of cells between the two time points. (B)
The density of BrdU-ir cells was also significantly higher in the male MePD when
compared to female mice. Data are represented as mean ± SEM.

50

Figure 1.3. Sex differences in the total number and density of BrdU-ir cells in the
MePD are dependent on functional androgen receptors and independent of adult
testosterone. Quantitative analysis revealed that (A) male mice have significantly more
pubertally born cells in the MePD when compared to female and iTfm mice treated with
testosterone in adulthood. (B) The density of BrdU-ir cells in the adult MePD is higher in
males than female and iTfm mice treated with adult testosterone. (C) No sex differences
were observed in MePD volume between male, female, and iTfm mice when treated
with testosterone. Data are represented as mean ± SEM.

51

Figure 1.4. Sex differences in the total number and density of BrdU-ir cells in the
MePD are dependent on pubertal testosterone and functional androgen receptors.
Quantitative analysis revealed that (A) T@P female mice have significantly more BrdUir cells than all other groups and T@P male mice had more pubertally born cells than no
T@P groups and T@P iTfm mice. (B) The density of BrdU-ir cells in the adult MePD is
significantly higher in T@P female mice when compared to all other groups. (C) No sex
difference is observed in MePD volume between male, female, and iTfm mice; however,
animals that received testosterone during puberty have a larger MePD volume than
groups that did not receive testosterone during puberty. Data are represented as mean
± SEM. *ps<0.04. #ps<0.02.

52

Figure 1.5. Pubertal and adult testosterone are necessary to increase the number
of pubertally born cells in the adult male MePD. Quantitative analysis revealed that
(A) T+/+ male mice had significantly more BrdU-ir cells in the MePD than all other
groups. In addition, T+/- male mice had significantly more pubertally born cells in the
MePD than the T-/- group. (B) T+/+ male mice had a higher density of pubertally born
cells in the MePD compared to the T-/- and T-/+ groups. T+/- male mice also had a
significantly higher density of BrdU-ir cells than the T-/- group. (C) T+/+ male mice had a
significantly larger MePD volume than the T-/- male mice. Data are represented as
mean ± SEM. T@P: testosterone during puberty; T@A: testosterone during adulthood.

53

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54

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CHAPTER 1B
Pubertal cytogenesis in the mouse ventromedial hypothalamus and dentate gyrus
ABSTRACT
Many sex-specific sociosexual behaviors, such as sexual behavior and
aggression, emerge during puberty and are regulated by sexually dimorphic structures
in the brain like the ventromedial hypothalamus (VMH). The VMH is a male-biased
neural structure and its volume is larger in males than females with the ventrolateral
subdivision of the VMH (VMHvl) mainly driving this sex difference. The dorsomedial and
central subdivisions of the VMH (VMHdm and VMHc, respectively) do not display a
volumetric sex difference. Recent studies in rats and hamsters have found that new
cells are added to the amygdala and hypothalamus in the pubertal brain, which may
facilitate the acquisition of adult sociosexual behaviors. However, it is currently unknown
whether mice experience pubertal cytogenesis in the VMH to potentially contribute to
sex differences observed in the adult structure. In the current study, we examined
whether there was a sex difference in the number of pubertally born cells in the VMHvl.
To investigate this, we administered 5-bromo-2’-deoxyuridine (BrdU), a cell birth date
marker, to male and female mice during puberty and examined the total number and
density of pubertally born cells in adulthood at two time points, postnatal day (P)60 and
P90. We also analyzed the VMHdm, VMHc. and dentate gyrus (DG), another
monomorphic structure, as a control regions. Furthermore, we examined the role of
androgen receptors (ARs) and pubertal testosterone on pubertal cytogenesis in the
VMH and DG using an AR-null mouse model (iTfm). The current study detected a sex
difference in the VMHvl, with males having significantly more pubertally born cells than

58

females and this sex difference was stably maintained from P60 to P90. No sex
differences were discovered in pubertally born cell density in the VMHdm, VMHc, and
DG. Furthermore, we discovered that ARs and pubertal testosterone do not promote the
increase of pubertally born cells in all subdivisions of the VMH and DG. These findings
demonstrate that sex differences in pubertal cytogenesis of the VMHvl are generated,
independent of ARs and pubertal testosterone, to potentially contribute to structural sex
differences observed in adulthood that may mediate the emergence of sex-specific adult
behaviors in mice.
INTRODUCTION
Puberty and adolescence represent a critical period during which the brain
undergoes significant changes in both its structure and function. Well documented
structural changes in the adolescent brain include synaptic pruning and growth, cell
death, and myelination, which likely promote the acquisition of mature behaviors
necessary to navigate in the adult world, including social and reproductive behaviors
(Barnea-Goraly et al., 2005; Blakemore et al., 2010; Giedd, 2004, 2008; Sisk et al.,
2004). However, more recent studies have identified yet another cellular mechanism by
which the adolescent brain undergoes change, namely, cytogenesis (Ahmed et al.,
2008; He et al., 2007; Mohr et al., 2016; Mohr et al., 2013; Saul et al., 2015; Staffend et
al., 2014).
Research on cytogenesis after the early formative stages of brain development
has focused on two brain regions, the hippocampus and the olfactory bulb, long
recognized to gain new cells throughout the lifespan. However, in recent years,
neurogenesis and gliogenesis outside of these canonical neurogenic regions have

59

gained attention as investigators come to realize that other brain regions, such as the
amygdala and hypothalamus, also gain new cells in adulthood (Fowler et al., 2003;
Kokoeva et al., 2005; Ruscio et al., 2015). While the literature regarding cytogenesis in
the adult brain continues to grow, studies investigating cytogenesis in the pubertal brain
remain sparse even though evidence indicates that cytogenesis may be a key cellular
event of brain development during puberty, given its wide spread occurrence in many
different brain regions of several different rodent species, including rats and hamsters
(Mohr et al., 2016; Mohr et al., 2013; Saul et al., 2015; Staffend et al., 2014). In Chapter
1A of this dissertation, we discovered that new cells are added to the posterodorsal
medial amygdala (MePD) during puberty in mice, and in the current study, we turn our
attention to the ventromedial hypothalamus (VMH).
The VMH is a male-biased sexually dimorphic brain region known to have a
larger volume in males than females, with the ventrolateral subdivision of the VMH
(VMHvl), in particular, driving this sex difference. The dorsomedial VMH (VMHdm) and
the central VMH (VMHc) are sexually monomorphic in structure, having no significant
volumetric differences detected in adulthood (Dugger et al., 2007). Recent evidence
indicates that the murine VMHvl receives input from the MePD to gate the expression of
sexual behavior or aggression as appropriate to the social context (Choi et al., 2005; Lin
et al., 2011). These adult sociosexual behaviors emerge during the pubertal period, a
time when rodents experience a significant increase in pubertal hormones, primarily
testosterone in males and estrogen in females. While it has been established that
gonadal hormones regulate synaptic organization in the VMH during puberty (Pozzo

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Miller et al., 1991), it remains unknown how gonadal hormones influence the addition of
new cells to the pubertal brain, particularly the VMH.
In the initial studies examining pubertal cytogenesis using 5-bromo-2’deoxyuridine (BrdU), a cell birth date marker, researchers observed the addition of new
cells in amygdala and hypothalamus, both regions involved in social behaviors in rats
and hamsters. More specifically, there was a sex difference in the number of pubertally
born cells in sexually dimorphic regions of the brain: the medial amygdala and the
sexually dimorphic nucleus of the preoptic area (SDN-POA). The medial amygdala and
SDN-POA of adult male rats had more BrdU-immunoreactive (ir) cells than female rats.
Likewise, a brain region that is larger in adult females, the anteroventral paraventricular
nucleus (AVPV), contained more BrdU-ir cells in adult females than in adult males
(Ahmed et al., 2008). Pubertally born cells have also been found to differentiate into
mature neurons or glia. Pubertally born cells in the adult hamster are even activated by
a social encounter (Mohr et al., 2013). Thus, not only are new cells generated during
puberty, they also appear to mature into more permanent fixtures in the brain and
become functionally integrated into existing neural circuits. Therefore, in an effort to
gain better understanding of the possible functions pubertally born cells may have, the
current study first aimed to determine whether new cells were added to the VMH during
puberty, and whether gonadal hormones led to an increase in their number in a sexdependent manner.
In the current study, we administered BrdU to mice during puberty (P28-49) to
label new born cells. First, we assessed sex differences in the number and density of
pubertally born cells in the VMHvl, VMHdm, and VMHc at two time points: P60 and P90.

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We also included the dentate gyrus (DG) in our analyses, as this brain region is also
known to be sexually monomorphic in structure, and therefore, may serve as a control
region. Next, we investigated the role of androgen receptors (ARs) and pubertal
testosterone on pubertal cytogenesis in all regions of interest using an AR-null mouse
model (iTfm) and comparing them to wildtype male and female mice.
MATERIALS AND METHODS
Animal Housing
In each experiment, all subjects were group housed (2-3 same-sex mice/cage) in
clear plexiglass cages (29x18x13cm) in a 12:12 light:dark cycle (lights on: 7:00 AM;
lights off: 7:00 PM) and provided ad libitum rodent chow (Harlan Teklad 8640 Rodent
Diet, Madison, WI) and water. All housing and experimental procedures were in
compliance with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals and approved by Michigan State University’s Institutional Animal
Care and Use Committee.
Experiment 1: Are there sex differences in pubertally born cells in the adult VMH and
DG?
In experiment 1, we assessed the number and density of pubertally born cells in
the adult mouse VMHvl, VMHdm, VMHc, and DG to determine whether there was a sex
difference in each region of interest. Male (n=16) and female (n=16) wild-type (WT)
C57BL/6 mice were obtained from Jackson Laboratory (Bar Harbor, Maine) on postnatal
day 21 (P21). 5-bromo-2’-deoxyuridine (BrdU; 200 mg/kg body weight; Prod. No.
B5002; Sigma-Aldrich) was administered daily (i.p.) during puberty (P28-49) during the
light period between 10:00 AM-12:00 PM. Then, in adulthood, half of each sex were

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perfused on P60 and the remaining half were perfused on P90. A two-way ANOVA was
used to examine group differences in the total number of BrdU-ir cells and BrdU-ir cell
density using sex and age at sacrifice as independent factors.
Experiment 2: Does the masculinization of pubertal born cells in the VMHvl require
functional androgen receptors?
In experiment 2, we investigated the role of androgen receptors (AR) on the
density of pubertally born cells in the VMHvl, VMHdm, VMHc, and DG, using male mice
with induced testicular feminization mutation (iTfm). As described in a recent study, iTfm
males are an AR-null mouse model induced by cre/lox technology and are
phenotypically female, but genetically male(Chen et al., 2014). WT male (n=10), WT
female (n=10), and iTfm males (n=10) were obtained from an in-house iTfm colony. All
animals were weaned and group housed on beginning on P24. All subjects received a
daily i.p. injection of BrdU (200 mg/kg body weight) during puberty (P28-49) at the lights
on period between 8:00 AM-10:00 AM. In order to standardize circulating levels of
testosterone in adulthood, on P60, all mice were gonadectomized and subcutaneously
implanted with a Silastic capsule filled with testosterone (1.6 mm – inner diameter and
3.2 mm – outer diameter; 6 mm – effective release length, 1.6 cm – total capsule length)
and then perfused on P90. A separate one-way ANOVA was used to analyze group
differences in the density of BrdU-ir cells by genotype for each region of interest.
Experiment 3: Is pubertal testosterone required to generate sex differences in the
density of pubertally born cells in the adult VMHvl?
In experiment 3, we examined the role of pubertal testosterone and androgen
receptors on the number of pubertally born cells in the VMHvl, VMHdm, VMHc, and DG

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of adult mice. WT male (n=20), WT female (n=20), and iTfm males (n=20) were
obtained from the same iTfm colony discussed in Experiment 2. All animals were
weaned and group housed on P24. On P26, all animals were gonadectomized and, for
each genotype, half the animals were subcutaneously implanted with a blank Silastic
capsule (No T@P) and the other half received a testosterone-filled Silastic capsule
(T@P). During puberty (P28-49), all animals received a daily i.p. injection of BrdU (200
mg/kg body weight) during the light period between 8:00 AM-10:00 AM. On P60,
subcutaneously implanted capsules were removed in all animals and replaced with a
testosterone-filled Silastic capsule to produce equivalent levels of circulating
testosterone in all groups in adulthood. All animals were perfused on P90. For each
region of interest, a separate two-way ANOVA was used to analyze group differences in
the density of BrdU-ir cells using genotype and pubertal testosterone treatment as
independent factors.
Tissue Collection and Processing
All mice were given an overdose of sodium pentobarbital (150 mg/kg) by i.p.
injection and intracardially perfused with 0.9% saline followed by cold 4%
paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). Brains were removed
and postfixed overnight in 4% paraformaldehyde, then stored in 30% sucrose at 4 °C
until sectioning. Brains were cut on a freezing microtome into 30 μm coronal sections,
and collected as either 3 (Experiment 1) or 4 alternate series of sections (Experiments
2-3). For each experiment, the first series was Nissl stained with thionin, dehydrated,
and coverslipped.

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Immunohistochemistry
In each experiment, the second series of brain sections was processed for
immunohistochemistry to label BrdU-immunoreactive (ir) cells. Free-floating sections
were rinsed in Tris-buffered saline (TBS; 0.05 M; pH 7.6) for 30 minutes and then
incubated in 0.6% H2O2 for 10 minutes. After a 15-minute rinse in TBS, the sections
were incubated in 0.93N HCl for 30 minutes at 37°C (except in experiment 4 – the
sections were incubated in 2N HCl for 1 hour at 37°C). Sections were next neutralized
in 0.1M borate buffer (pH 8.5) for 10 minutes. Following a 30-minute rinse in TBS, the
sections were blocked in TBS containing 0.01% Triton-X-100 and 3% donkey serum,
and then incubated in monoclonal rat anti-BrdU (catalog no. MCA2060; Serotec) at a
working concentration of 1 μg/ml at 4°C for 48 hours. Sections were then rinsed in TBS
for 30 minutes and incubated in biotinylated donkey anti-rat secondary antibody (catalog
no. 712-065-150; Jackson Immunoresearch) at a working concentration of 2 μg/ml for 2
hours at room temperature. Following a 30-minute rinse in TBS, the sections were
incubated in avidin/biotinylated enzyme complex (ABC Elite Kit, Vector Laboratories,
Burlingame, CA) for an hour and then reacted in 3,3’-Diaminobenzidine
tetrahydrochloride (DAB; Sigma; working concentration 0.25 mg/ml) for about 2
minutes. Following this reaction, all sections were mounted onto gelatin-subbed slides
and coverslipped.
Microscopic Analysis
All microscopic analyses were performed by an observer blind to treatment group
using an Olympus BX51 microscope under brightfield illumination using Neurolucida
version 10 (MBF Bioscience). The regions of interest were traced using the Nissl-

65

stained series (Fig 1.6A-B) and contours were superimposed onto adjacent sections of
the single-label BrdU series (Fig 1.6C-D). The boundaries of the VMH on the Nissl
series were determined using a standard mouse brain atlas (Paxinos et al., 2004). The
following landmarks were used to trace the VMH: the size, shape, and staining intensity
of the nucleus itself, as well as the size and shape of the third ventricle and the size and
shape of the optic tract. BrdU-ir cells were identified by the presence of a dark brown
nuclear stain.
Single-label BrdU Analysis
In experiment 1, six sections of the VMH were bilaterally analyzed, which
encompassed the rostral-caudal extent of the ventrolateral, dorsomedial, and central
subdivisions. Animals that did not have six anatomically-matched VMH sections due to
poor tissue quality or missing sections were excluded from final analysis. The final
sample sizes were as follows: P60 females (n=7), P60 males (n=5), P90 females (n=8),
and P90 males (n=8). In addition, the 2 most rostral bilateral sections of the DG were
analyzed (Fig 1.8A-D). Due to poor tissue quality, 2 males from the P60 cohort were
removed from the final analysis. For each region of interest, the total number of BrdU-ir
cells was determined by summing the number of BrdU-ir cells across all sections
analyzed. The density of BrdU-ir cells in each animal was calculated by dividing the total
number of BrdU-ir cells (summed across sections) by the total area (summed across
sections).
For experiments 2 and 3, three – four sections of the VMH were bilaterally
analyzed in the ventrolateral, dorsomedial, and central subdivisions. Animals that did
not have at least 3 anatomically-matched VMH sections were excluded from final

66

analysis. The final sample sizes were as follows: Experiment 2 – males (n=10), females
(n=10), and iTfm (n=10); Experiment 3 – No T@P males (n=9), No T@P female (n=7),
No T@P iTfm (n=9); T@P males (n=9), T@P females (n=5), and T@P iTfm (n=8). The
2 most rostral bilateral sections of the DG were analyzed. Animals that did not have at
least 2 anatomically-matched DG sections were excluded from final analysis. The final
sample sizes were as follows: Experiment 2 – males (n=10), females (n=10), and iTfm
(n=10); Experiment 3 – No T@P males (n=9), No T@P female (n=8), No T@P iTfm
(n=8); T@P males (n=9), T@P females (n=8), and T@P iTfm (n=10). For each region of
interest, the density of BrdU-ir cells in each animal was calculated by dividing the total
number of BrdU-ir cells (summed across sections) by the total area (summed across
sections).
RESULTS
Experiment 1: Male mice have more pubertally born cells than females in the VMHvl,
but not the VMHdm, VMHc, or DG.
In the VMHvl, there was a main effect of sex (F(1,24)=12.96, p=0.001), with male
mice having a higher number and density of pubertally born cells than female mice in
adulthood (Fig 1.5E-F). There was no main effect of age at sacrifice and no significant
interaction on total number of BrdU-ir cells in the VMHvl. Also, there were no main
effects of sex or age at sacrifice and no interaction on the density or number of BrdU-ir
cells in the VMHdm (Fig 1.7A-B), VMHc (Fig 1.7C-D), and DG (Fig 1.8E).

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Experiment 2: Androgen receptors are not necessary to increase the number of
pubertally born cells in the VMH and DG.
In the VMHvl, there was no significant different in the density of pubertally born
cells between genotype (Fig 1.9A). There was also no significant difference in the
density of pubertally born cells in the VMHdm (Fig 1.9B), VMHc (Fig 1.9C), and DG (Fig
1.9D) between genotype.
Experiment 3: Pubertal testosterone does not increase the number of pubertally born
cells in the VMH or DG.
In the VMHvl, there was no significant main effect of genotype or pubertal
testosterone treatment on the density of pubertally born cells (Fig 1.10A). There was
also no significant interaction of genotype and pubertal testosterone treatment on the
density of pubertally born cells in the VMHvl. There was also no significant main effect
of genotype on the VMHdm (Fig 1.10B), VMHc (Fig 1.10C), and DG (Fig 1.10D) or
pubertal testosterone treatment in the VMHdm, VMHc, and DG. There was also no
significant interaction of genotype and pubertal testosterone treatment on the density of
pubertally born cells in the VMHdm, VMHc, and DG.
DISCUSSION
The current study discovered that new cells are born in the VMH during puberty
with no significant attrition of cells seen from P60 to P90. In our initial study, we
observed a sex difference in the number and density of pubertally born cells in the
VMHvl with males having more pubertally born cells than females. However, in our
follow-up studies, we failed to replicate these findings, as no significant group
differences in pubertally born cell density were observed between male, female, and

68

iTfm male mice. iTfm male mice have non-functional ARs, thus it appears that functional
ARs are not required to promote the increase of pubertally born cells in the VMHvl.
Pubertal testosterone is also not necessary to increase pubertally born cell density in
the VMHvl. A study in adult male meadow voles found that hormone treatment (i.e.
testosterone, estrogen, and DHT, a potent androgen) did not affect cell proliferation in
the VMH (Fowler et al., 2003). It is unknown how gonadal hormones affect cell
proliferation in the VMHvl during puberty, but based on these results, it is possible that
gonadal hormones do not play a role in the proliferation or survival of pubertally born
cells in the VMHvl.
Methodical differences between experiments could account for the inconsistent
findings on sex differences in pubertally born cells in the VMHvl shown in the present
study. For instance, the initial sex differences study, we used gonadally intact males
and females, while the subjects in the latter studies underwent 1-2 surgeries. There is
evidence that shows stress can decrease cell proliferation and cell survival in the brain
(Mirescu et al., 2006) and exposure to anesthetic, such as isoflurane, can cause cell
death in the brain (Zhu et al., 2010). While no signs of distress were observed in the
animals used in this study, beyond the discomforts of surgery, it is possible that these
surgical procedures dampened sex differences initially detected between males and
females in the VMHvl. However, if this is the case, it would appear that the
posterodorsal medial amygdala (MePD) is somehow immune to the effects of surgery,
as the sex difference in the density of pubertally born MePD cells was maintained
following surgeries. Alternatively, the sex differences in pubertally born cells within the
VMHvl in the initial experiment may have resulted from type 1 error. However, the fact

69

that statistical significance for these findings survived a rather conservative Bonferroni
correction for multiple comparisons makes this possibility less likely. It is clear that
further investigation is required to understand the role of gonadal hormones on cell
proliferation and survival in the mouse VMHvl.
Sex differences in VMH volume are established early in development around the
perinatal period (Matsumoto et al., 1983). Castration of male rats at P1 feminized VMH
volume in adulthood, while castration at P7 did not reduce adult VMH volume
(Matsumoto et al., 1983). In addition, there is no sex difference in neuron number in the
adult VMH (Madeira et al., 2001); however, sex differences in the number of synapses
are established in the VMH during puberty with males having more synapses than
females. In addition, treating females with testosterone can masculinize the number of
synapse in the VMH (Pozzo Miller et al., 1991). Interestingly, recent studies have
discovered that glial cells, such as astrocytes, oligodendrocytes, and microglia, play a
critical role in the formation, maintenance, and elimination of synapses(Aguzzi et al.,
2013; Allen et al., 2009; Clarke et al., 2013). Therefore, while we do not always observe
a sex difference in pubertally born cell density, gonadal hormones may affect the
differentiation of pubertally born cells into specific cell phenotypes. For instance,
testosterone could promote the genesis and/or survival of glial cells in the VMH to
facilitate synaptic reorganization. Follow-up studies will be necessary to explore whether
pubertally born cells participate in the synaptogenesis in the VMHvl.
No sex differences in the number and density of pubertally born cells were
detected in the VMHdm, VMHc, and DG, brain regions that are sexually monomorphic
in volume(Dugger et al., 2007; Tabibnia et al., 1999). These results corroborate findings

70

from a previous study, which showed no sex differences in the DG (Ahmed et al., 2008).
We also discovered that ARs and pubertal testosterone did not increase the number of
pubertally born cells in the VMHdm, VMHc, and DG. Male, female, and iTfm male mice
had a similar density of pubertally born cells in the VMHdm, VMHc, and DG, regardless
of pubertal testosterone treatment. While there are currently no previous studies
examining the effects of gonadal hormones on pubertal cytogenesis in the VMH, a
recent study did find that there were no sex differences in the total number of pubertally
born cells in the adult DG and furthermore, pre-pubertal gonadectomy did not affect
pubertally born cells in the DG (Ahmed et al., 2008). Although no significant effects of
hormonal manipulations were detected in the number of pubertally born cells in the DG,
several studies have shown that androgens and estrogen play an important role in the
addition of new cells in the DG during adulthood. When adult male rats are castrated,
the level of cell proliferation in the dentate gyrus does not change significantly from of
gonadally intact male rats(Spritzer et al., 2007). Similarly, in adult male meadow voles,
the administration of testosterone, estrogen, or DHT did not alter levels of cell
proliferation in the DG(Fowler et al., 2003). However, it appears that androgens play an
important role in cell survival in the adult DG, as gonadally intact male rats had
significantly more BrdU-ir cells in the DG than castrated males one month after BrdU
treatment (Spritzer et al., 2007) and treating adult male rats with DHT and flutamide
reduced the survival of adult born cells in the DG compared to males treated with only
DHT (Hamson et al., 2013). Furthermore, Tfm male rats treated with testosterone had
fewer adult born cells survive in the adult DG compared to testosterone-treated males
(Hamson et al., 2013). On the other hand, in female rats, estrogen seems to have a

71

strong effect on cell proliferation, but not cell survival in the DG (Barker et al., 2008;
Ormerod et al., 2003; Tanapat et al., 1999). Interestingly, it has been reported that 2
days after BrdU treatment, adult female rats have more BrdU-ir cells in the DG than
males, but 2 weeks after BrdU administration, there is no longer a sex difference
(Tanapat et al., 1999). Taken together, these studies suggest that development of the
male and female brains may each follow unique trajectories, which are driven by sex
differences in hormone level with cell proliferation and cell death working together to
shape sexually dimorphic neural circuits. Therefore, even though no sex differences
were observed in the number of pubertally born cells in the adult DG, there is still a
possibility that gonadal hormones may have distinct effects on cell proliferation and
survival depending on sex. Thus, in order to better understand the role of pubertal
hormones on pubertal cytogenesis, more research must be done to examine the
differential effects of gonadal hormones on the proliferation and survival of pubertally
born cells in the mouse brain.
In the current study, we initially discovered a sex difference in the number and
density of pubertally born cells in the VMHvl, with males having more BrdU-ir cells than
females. However, we were unable to detect this sex difference in two follow-up
experiments. Moreover, we did not observe any sex differences in the number and
density of pubertally born cells in the VMHdm, VMHc, and DG. In addition, the current
findings provide evidence that ARs and pubertal testosterone are not necessary to
increase the number of pubertally born cells in the VMHvl, VMHdm, VMHc, and DG.
Based on these results, it is clear that the role of gonadal hormones on pubertal
cytogenesis the mouse VMHvl requires further investigation. Follow up experiments

72

should examine the role of estrogen on pubertal cytogenesis. Furthermore, future
studies should address whether gonadal hormones influence differentiation of pubertally
born cells into specific cell phenotypes, allowing us to explore the function of such
pubertally born cells in mediating sex-specific behaviors in adulthood.

73

Figure 1.6. Total number and cell density of BrdU-immunoreactive cells in the
ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) in adult male
and female mice. Representative photomicrographs displaying traces of the VMH on
Nissl-stained coronal sections in the (A) male and (B) female adult mice and pubertally
born cells in the VMH of (C) male and (D) female adult mice. The small inlaid boxes
show BrdU-labeled cells in the VMHvl at a higher magnification. (E) ANOVA revealed
that adult male mice have significantly more pubertally born cells than female mice
(p=0.001). (F) ANOVA revealed that male mice have a higher density of BrdU-ir cells in
the VMHvl than female mice in adulthood (p=0.01). VMHdm = dorsomedial subdivision
of the VMH; VMHc = central subdivision of the VMH. Low magnification scale bar
indicates 200 μm; high magnification scale bar indicates 10 μm.

74

Figure 1.7. Total number and cell density of BrdU-ir cells in the dorsomedial and
central subdivisions of the ventromedial hypothalamus in adult male and female
mice. (A) Quantitative analysis revealed no sex difference in (A) BrdU-ir cell density and
no sex difference in (B) total number of pubertally born cells in the VMHdm. (C) ANOVA
found no significant differences in BrdU-ir cell density in the VMHc between male and
female mice, and (D) comparable numbers of BrdU-ir cells in the VMHc.

75

Figure 1.8. BrdU-ir cell density in the dentate gyrus of the hippocampus in adult
male and female mice. Representative photomicrographs showing coronal sections of
the DG stained for BrdU-ir cells in (A) male and (B) female mice sacrificed on P60 and
(C) male and (D) female mice sacrificed on P90. (E) Two-way ANOVA (Sex x Age at
Sacrifice) revealed a comparable number of pubertally born cells between all groups.
Low magnification scale bar indicates 200 μm; high magnification scale bar indicates 10
μm.

76

Figure 1.9. Androgen receptors are not required to enhance the density of
pubertally born cells in the VMH and DG. A one-way ANOVA revealed no significant
differences in the density of pubertally born cells between male, female, and iTfm male
mice in the (A) VMHvl, (B) VMHdm, (C) VMHc, and (D) DG.

77

Figure 1.10. Pubertal testosterone is not necessary to increase the number of
pubertally born cells in the male, female, and iTfm male mice in the VMH and DG.
Quantitative analysis using a two-way ANOVA (Genotype x pubertal testosterone
treatment) revealed a comparable density of pubertally born cells between all groups in
the (A) VMHvl, (B) VMHdm, (C) VMHc, and (DG).

78

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Ruscio, M. G., Bradley King, S., & Haun, H. L. (2015). Social isolation increases cell
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Saul, M. L., Helmreich, D. L., Rehman, S., & Fudge, J. L. (2015). Proliferating cells in
the adolescent rat amygdala: Characterization and response to stress.
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Sisk, C. L., & Foster, D. L. (2004). The neural basis of puberty and adolescence. Nat
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Spritzer, M. D., & Galea, L. A. (2007). Testosterone and dihydrotestosterone, but not
estradiol, enhance survival of new hippocampal neurons in adult male rats. Dev
Neurobiol, 67(10), 1321-1333.
Staffend, N. A., Mohr, M. A., DonCarlos, L. L., & Sisk, C. L. (2014). A decrease in the
addition of new cells in the nucleus accumbens and prefrontal cortex between
puberty and adulthood in male rats. Dev Neurobiol, 74(6), 633-642.
Tabibnia, G., Cooke, B. M., & Breedlove, S. M. (1999). Sex difference and laterality in
the volume of mouse dentate gyrus granule cell layer. Brain Res, 827, 41-45.
Tanapat, P., Hastings, N. B., Reeves, A. J., & Gould, E. (1999). Estrogen Stimulates a
Transient Increase in the Number of New Neurons in the Dentate Gyrus of the
Adult Female Rat. J Neurosci, 19(14), 5792-5801.
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CHAPTER 2
Androgen receptors during puberty, but not adulthood, increase pubertally born neurons
in the posterodorsal medial amygdala in adult male mice
ABSTRACT
The pubertal brain undergoes significant structural changes that facilitate the
development of adult sociosexual behaviors. Recent studies in rats and hamsters have
demonstrated that new neurons and glial cells are added to the brain during puberty
and become functionally integrated into brain regions, like the medial amygdala, which
is known to regulate social behaviors in rodents. In Chapter 1 of this dissertation, we
discovered that new cells are also added to the mouse posterodorsal medial amygdala
(MePD) and the ventromedial hypothalamus (VMH) during puberty. Furthermore, there
was a sex difference favoring males in the number of pubertally born cells in the adult
MePD and ventrolateral subdivision of the VMH (VMHvl). In addition, using an AR-null
mouse model (iTfm), we discovered that ARs as well as pubertal testosterone promote
the number of pubertally born cells in the adult male MePD, but not VMHvl. However,
since iTfm male mice have non-functional ARs throughout the lifespan, it is currently
unknown when ARs are necessary to masculinize the number pubertally born cells in
the adult MePD. Furthermore, it is unclear whether androgens play a role in influencing
the genesis and/or differentiation of specific cell phenotypes of pubertally born cells in
the MePD and VMH. Therefore, in the current study, we examined the role of ARs
specifically during puberty and adulthood, by treating male mice with flutamide, an AR
antagonist. Male mice were castrated (P26), implanted with a testosterone capsule, and
then treated with a time-release flutamide pellet during puberty (P26-50) or adulthood

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(P50-74). Control mice were castrated at P26 but implanted with only a testosterone
capsule or received a sham surgery and remained gonadally intact. All mice were
administered BrdU during the first week of puberty (P28-35) and sacrificed in adulthood
at P74. We discovered that blocking pubertal, but not adult, ARs reduces the number of
pubertally born cells in the adult male MePD. In addition, blocking pubertal ARs also
reduces the number of pubertally born neurons, but not astrocytes, in the adult male
MePD. Neither pubertal nor adult ARs were to necessary increase the number of
pubertally born cells, including pubertally born neurons and astrocytes, in the VMH and
DG. Thus, ARs, specifically during puberty, are critical for the gain of pubertally
generated neurons in the adult male MePD.
INTRODUCTION
The pubertal brain endures extensive remodeling, which facilitates the acquisition
of sociosexual behaviors required for survival and successful reproduction in adulthood.
These adult sociosexual behaviors are regulated by sexually dimorphic brain regions,
such as the posterodorsal medial amygdala (MePD) and the ventromedial
hypothalamus (VMH) (Cooke et al., 2003; Cooke et al., 2000; Kollack-Walker et al.,
1995; Lin et al., 2011; Samuelsen et al., 2009; Yang et al., 2013). During puberty, males
and females experience a surge in gonadal hormones, primarily testosterone and
estrogen, respectively(Sisk et al., 2004), and since the MePD and VMH are densely
populated with sex steroid receptors, such as androgen receptors (ARs) and estrogen
receptors (ERs)(Simerly et al., 1990), it is not surprising that these pubertal gonadal
hormones induce significant structural changes to these brain regions. In the MePD and
VMH, pubertal increase in gonadal hormones can alter volume, neuronal soma size,

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number of glial cells, and synaptogenesis (Cooke et al., 2007; Cooke et al., 2009;
Johnson et al., 2013; Pozzo Miller et al., 1991). More recently, studies have discovered
that pubertal gonadal hormones also influence the addition of new cells in the
adolescent brain, including the MePD and VMH(Ahmed et al., 2008).
A previous pubertal cytogenesis study in rats detected a sex difference in the
number of pubertally born cells in the adult medial amygdala. Adult male rats have more
pubertally born cells than females in the medial amygdala and a subset of these cells
differentiate into mature neurons or astrocytes (Ahmed et al., 2008). The accumulation
of pubertally born cells in the adult medial amygdala depends on the presence of
gonadal hormones, as pre-pubertal castration reduces the total number of pubertally
born cells (Ahmed et al., 2008). In Chapter 1 of this dissertation, a sex difference in
pubertally born cells was also discovered in the MePD and VMHvl of mice. Male mice
have more pubertally born cells than females in the MePD and ventrolateral subdivision
of the VMH (VMHvl). No sex differences were detected in monomorphic brain regions,
such as the dorsomedial and central subdivisions of the VMH (VMHdm and VMHc,
respectively) and the dentate gyrus of the hippocampus (DG). Using androgeninsensitive iTfm male mice, it was established that sex differences in pubertally born cell
number in the adult mouse MePD, but not VMHvl, depend on the presence of ARs.
However, since iTfm males have non-functional ARs throughout the lifespan, it is
currently unknown when AR activity is critical to mediate the masculinization of the
number of pubertally born cells in the adult male MePD.
In the current study, we examined whether AR activity specifically during puberty
or adulthood was required for the masculine development of pubertally born cells in the

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adult MePD. Regions of interest also included the VMHvl, VMHdm, VMHc, and DG in
an effort to replicate previous findings to ensure ARs did not play a critical role in the
proliferation and/or survival of pubertally born cells. Furthermore, we analyzed the
percent colocalization of pubertal born cells with NeuN, a mature neuron marker, and
GFAP, an astrocytic marker, to determine whether ARs during puberty or adulthood
promote the differentiation and/or survival of pubertally born neurons or astrocytes in
the adult MePD, VMH, and DG.
MATERIALS AND METHODS
Animal Subjects and Experimental Design
48 male mice were obtained from a wildtype C57BL/6 in-house mouse colony
and were weaned at P24. All animals were group housed (4-5 mice/cage) in clear
plexiglass cages (29x18x13cm) in a 12:12 light:dark cycle (lights on: 7:00 AM, lights off:
7:00 PM). All subjects were provided ad libitum rodent chow (Harlan Teklad 8640
Rodent Diet, Madison, WI) and water. All housing and experimental procedures were
performed in compliance with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals and approved by the Michigan State University’s Institutional
Animal Care and Use Committee.
Mice were randomly assigned to one of the following four experimental groups
(n=12/group): Group 1 (Flut@Puberty): On P26, mice were castrated and implanted
subcutaneously (s.c.) with a testosterone capsule (1.6 mm – inner diameter and 3.2 mm
– outer diameter; 6 mm – effective release length, 1.6 cm – total capsule length) and
flutamide pellet (100 mg flutamide/pellet – 5 mg dose/day, 21-day release, Innovative
Research of America, Saratoga, FL). On P50, all implants were removed and replaced

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with only a testosterone capsule. Group 2 (Flut@Adulthood): On P26, male mice were
castrated and received a testosterone capsule implant (s.c.). On P50, the testosterone
capsule was removed and replaced with a new testosterone capsule and a flutamide
pellet. Group 3 (T Only): On P26, male mice were castrated and received a testosterone
capsule implant (s.c.). On P50, the testosterone capsule was removed and replaced
with a new testosterone capsule. Group 4 (Sham): On P26, male mice received a sham
surgery, remaining gonadally intact throughout the duration of the study, and received a
blank capsule implant (s.c.). On P50, the blank capsule was removed and replaced with
a new blank capsule.
BrdU administration
During the first week of puberty (P28-P35), all subjects were administered BrdU
(1.0 mg/ml, Sigma-Aldrich) dissolved in the drinking water (Snyder et al., 2011). BrdU
drinking water was prepared fresh every 3 days. During BrdU administration, all mice
were housed in Innocage mouse cages (37 x 23 x 14 cm) with Aquavive amber water
bottles (Innovive, San Diego, CA). The use of the Aquavive amber water bottles allowed
us to administer BrdU via drinking water in smaller volumes (about 100 ml of
water/bottle) to avoid excess BrdU waste. The Aquavive amber water bottles also had
minimal leakage and since BrdU is light sensitive, the amber bottles provided adequate
protection from light. In addition, the use of disposable Innocage mouse cages avoided
BrdU contamination of reusable animal cages.
Tissue Collection and Processing
On P74, all mice were given an overdose of sodium pentobarbital (150 mg/kg) by
i.p. injection and intracardially perfused with 0.9% saline followed by cold 4%

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paraformaldehyde in 0.1 M phosphate buffered saline (pH 7.4). Brains were removed
and postfixed overnight in 4% paraformaldehyde, then switched to 30% sucrose
solution at 4 °C until sectioning. All brains were sectioned within one week of perfusion.
Brains were cut on a freezing microtome into 30 μm coronal sections (from about
Bregma -0.82 to Bregma -2.74) and collected as 4 alternate series. For each
experiment, the first series was Nissl stained with thionin, dehydrated, and coverslipped.
Single-Label BrdU Immunohistochemistry
The entire second series of brain sections underwent immunohistochemistry to
label BrdU-immunoreactive (ir) cells. Free-floating sections were rinsed in Tris-buffered
saline (TBS; 0.05 M; pH 7.6) for 30 minutes and then incubated in 0.6% H2O2 for 10
minutes. After a 15-minute rinse in TBS, the sections were incubated in 2N HCl for 60
minutes at 37°C. Sections were then neutralized in 0.1M borate buffer (pH 8.5) for 10
minutes. Following a 30-minute rinse in TBS, the sections were blocked in TBS
containing 0.01% Triton-X-100 and 3% donkey serum, and then incubated in
monoclonal rat anti-BrdU (catalog no. MCA2060; Serotec) at a working concentration of
1 μg/ml at 4°C for 48 hours. Sections were then rinsed in TBS for 30 minutes and
incubated in biotinylated donkey anti-rat secondary antibody (catalog no. 712-065-150;
Jackson Immunoresearch) at a working concentration of 2 μg/ml for 2 hours at room
temperature. Following a 30-minute rinse in TBS, the sections were incubated in
avidin/biotinylated enzyme complex (ABC Elite Kit, Vector Laboratories, Burlingame,
CA) for an hour and then reacted in 3,3’-Diaminobenzidine tetrahydrochloride (DAB;
Sigma; working concentration 0.25 mg/ml) for about 3 minutes. Following this reaction,
all sections were mounted onto gelatin-subbed slides, dehydrated, and coverslipped.

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Triple-Label BrdU/GFAP/NeuN Immunofluorescence
The entire third series of brain sections underwent immunofluorescence to label
for BrdU, GFAP (a marker of astrocytes), and NeuN (a marker of mature neurons).
Free-floating sections were rinsed in Tris-buffered saline (TBS; 0.05 M; pH 7.6) for 30
minutes and then incubated in 0.1% sodium borohydride for 10 minutes. After a 15minute rinse in TBS, the sections were incubated in 2N HCl for 60 minutes at 37°C.
Afterward, the sections were neutralized in 0.1M borate buffer (pH 8.5) for 10 minutes.
Following a 30-minute rinse in TBS, the sections were blocked in TBS containing 0.01%
Triton-X-100 and 3% donkey serum for 1 hour, and then incubated in a primary antibody
cocktail at 4°C for 48 hours, which contained monoclonal rat anti-BrdU (catalog no.
MCA2060; Serotec) at a working concentration of 1 μg/ml, polyclonal rabbit anti-GFAP
(catalog no. Z0334; Dako) at a working concentration of 0.58 μg/ml, and monoclonal
mouse anti-NeuN (catalog no. MAB377, Chemicon) at a working concentration of 1
μg/ml. Sections were then rinsed in TBS for 30 minutes and incubated in a secondary
antibody cocktail for 2 hours at room temperature, which contained Cy2 AffiniPure
donkey anti-rat secondary antibody (catalog no. 712-225-153; Jackson
Immunoresearch), Cy3 AffiniPure donkey anti-rabbit (catalog no. 711-165-152, Jackson
Immunoresearch), and Cy5 AffiniPure donkey anti-mouse (catalog no. 715-175-151,
Jackson Immunoresearch). Following a 30-minute rinse in TBS, all sections were
mounted onto gelatin-subbed slides, dehydrated, and coverslipped.
Microscopic Analysis
All microscopic analyses were performed by a blind observer. The regions of
interest for all microscopic analyses included the posterodorsal medial amygdala

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(MePD), the ventrolateral, dorsomedial, and central subdivisions of the ventromedial
hypothalamus (VMHvl, VMHdm, and VMHc, respectively), and the dentate gyrus (DG).
In the single-label BrdU analysis, the regions of interest were traced using the Nisslstained series and contours were superimposed onto adjacent sections of the singlelabel BrdU series using. The boundaries of the MePD (Fig 2.1A-H) and VMH (Fig 2.2AH) were determined using a standard mouse brain atlas. In the triple-label
BrdU/GFAP/NeuN analysis, the regions of interest were traced using the NeuN stain to
determine MePD (Fig 2.4A), VMH (Fig 2.5A), and DG (Fig 2.6A) boundaries. To
consistently sample anatomically-matched sections across animals, the following
landmarks were used to trace the MePD: the shape of the optic tract, the size and
shape of the lateral ventricles, the size and shape of the intercalated amygdalar
nucleus, and the size and shape of the stria terminalis. For the VMH, the size, shape,
and staining intensity of the nucleus itself, as well as the size and shape of the third
ventricle were used to consistently sample anatomically-matched sections across
animals. For the DG, the two most rostral sections were analyzed (Fig 2.3A-D).
Single-label BrdU Analysis
The single-label BrdU series was analyzed using an Olympus BX51 microscope
under brightfield illumination using Neurolucida version 10 (MBF Bioscience). These
analyses examined new cells born during the first week of puberty (P28-35). The BrdUir cells analyzed were approximately 39-46 days old. The full rostral-caudal extent of the
MePD, VMHvl, VMHdm, and VMHc were analyzed, which included a total of six MePD
sections and five VMH sections. Animals that did not have all anatomically-matched
sections were excluded from analysis. The final sample sizes were as follows:

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Flut@Puberty (n=12), Flut@Adulthood (n=11), T Only (n=11), and Sham (n=12). The
total number of BrdU-ir cells was determined by summing the number of BrdU-ir cells
across all sections analyzed. The density of BrdU-ir cells in each animal was calculated
by dividing the total number of BrdU-ir cells (summed across sections) by the total area
(summed across sections). The volumetric estimates of the MePD, VMHvl, VMHdm,
and VMHc were calculated by summing the area of all sections analyzed in each region
of interest and multiplying by the number of series (4) and section thickness (30 μm).
Triple-Label BrdU/GFAP/NeuN Analysis
The triple-label BrdU/GFAP/NeuN series was analyzed using an Olympus
FluoView FV1000 confocal laser-scanning microscope. A z-stack orthogonal viewer was
utilized to verify colocalization of BrdU/GFAP and BrdU/NeuN. All regions of interest
were analyzed using the multi-line Argon gas laser (488 nm), green Helium Neon laser
(543 nm), and red Helium Neon laser (633 nm) to detect BrdU-, GFAP-, and NeuNpositive cells, respectively. Again, the full rostral-caudal extent of the MePD, VMHvl,
VMHdm, and VMHc were analyzed. For this confocal analysis, a total of 6 mice/group
were used to analyze the MePD and VMH and a total of 2 mice/group was used to
analyze the DG. The total number of BrdU-, GFAP-, and NeuN-positive cells was
determined by summing the number of BrdU-, GFAP-, and NeuN-positive cells across
all sections analyzed. The % colocalization of BrdU/NeuN cells were calculated by
dividing the total number of BrdU/NeuN-positive cells by the total number of BrdUpositive cells multiplied by 100. The % colocalization of BrdU/GFAP cells were
calculated by dividing the total number of BrdU/ GFAP-positive cells by the total number
of BrdU-positive cells multiplied by 100. The following represents the average number of

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BrdU-ir cells analyzed per animal in each brain region of interest: MePD – 585 BrdU-ir
cells, VMHvl – 182 BrdU-ir cells, VMHdm – 100 BrdU-ir cells, VMHc – 55 BrdU-ir cells,
and DG – 341 BrdU-ir cells.
Statistical Analysis
For the single-label BrdU analysis, group differences in total number of BrdU-ir
cells, BrdU-ir cell density, and volume were each analyzed using a one-way ANOVA to
compare all four treatment groups. For the triple-label BrdU/GFAP/NeuN analysis, a
one-way ANOVA was used to determine whether the % colocalization of BrdU/GFAP
and BrdU/NeuN differed between treatment groups. For all analyses, a significance
value of p < 0.05 was used and significant results were followed up with a Bonferroni
post-hoc test. Cohen’s d was used to calculate effect sizes.
RESULTS
Functional androgen receptors during puberty, but not adulthood, promote the number
of pubertally born cells in the adult male MePD.
ANOVA revealed a significant difference between treatment groups on the total
number of BrdU-ir cells in the adult MePD (F(3,42)=6.42, p=0.001)(Fig 2.1I). Males that
received testosterone throughout the lifespan (p=0.001, d=1.52) and males that
received flutamide during adulthood (p=0.015, d=1.90) had more BrdU-ir cells in the
adult MePD than male mice that received flutamide during puberty. Males in the T Only
group had approximately 43% more pubertally born cells in the MePD than males in the
Flut@Puberty group, while males in the Flut@Adulthood group had about 34% more
BrdU-ir cells than males in the Flut@Puberty group. Sham males were not significantly
different from all other groups and instead, had an intermediate number of MePD BrdU-

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ir cells, with 18% more BrdU-ir cells than the Flut@Puberty group (d=0.96), and 18%
and 12% fewer BrdU-ir cells than the T Only (d=0.79) and Flut@Adulthood (d=0.71)
groups, respectively. Furthermore, an ANOVA found that the density of BrdU-ir cells in
the adult MePD was significantly different between groups (F(3,42)=7.24, p=0.001)(Fig
2.1J). Similar to the total number of BrdU-ir cells, the T Only (p=0.000, d=1.69) and
Flut@Adulthood (p=0.014, d=1.60) groups had a higher density of BrdU-ir cells in the
adult MePD than the Flut@Puberty groups. Again, the sham males were not
significantly different from all other groups. There were no significant differences in
MePD volume among the groups (F(3,42)=0.264, p=0.851)(Fig 2.1K).
Functional androgen receptors, either during puberty or in adulthood, do not promote
the number of pubertally born cells in the VMH and DG.
ANOVA revealed no significant differences among groups in the total number of
BrdU-ir cells in the adult VMHvl (F(3,41)=1.264, p=0.299) (Fig 2.2I), VMHdm
(F(3,41)=1.952, p=0.136) (Fig 2.2L), or VMHc(F(3,41)=0.568, p=0.639) (Fig 2.2O).
There were also no significant differences between groups in the density of pubertally
born cells in the adult VMHvl (F(3,41)=1.586, p=0.208) (Fig 2.2J), or VMHc
(F(3,41)=0.974, p=0.414) (Fig 2.2P). There was a significant difference between groups
in the VMHdm (F(3,41)=2.905, p=0.046) (Fig 2.2M) with sham males having a higher
density of pubertally born cells than the Flut@Puberty group (p=0.033, d=1.19). There
were also no significant differences between groups in the volume of the adult VMHvl
(F(3,41)=1.347, p=0.272) (Fig 2.2K), VMHdm (F(3,41)=1.362, p=0.268) (Fig 2.2N), or
VMHc (F(3,41)=1.28, p=0.294) (Fig 2.2Q). In addition, no significant differences were

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observed between groups on the total number of BrdU-ir cells (F(3,42)=2.091, p=0.116)
in the dentate gyrus (Fig 2.3E).
Androgen receptors during puberty, but not adulthood, enhances the differentiation of
pubertally born cells into mature neurons in the MePD.
An ANOVA found a significant difference between groups in the percent
colocalization of BrdU/NeuN-positive cells in the adult MePD (F(3,20)=12.799, p=.000)
(Fig 2.4B, 2.4D). Bonferroni post-hoc analysis revealed that the Flut@Puberty male
mice had a significantly lower proportion of pubertally born cells that colocalized with the
mature neuron marker, NeuN, than Flut@Adulthood (p=0.002, d=2.92), T Only
(p=0.003, d=2.89), and sham male mice (p=0.000, d=2.88). However, no significant
group differences were observed in the percent colocalization of BrdU/GFAP-positive
cells in the adult MePD (F(3,20)=2.003, p=0.146) (Fig 2.4C, 2.4E), suggesting that
androgen receptors play an important role in the differentiation or survival of pubertally
born neurons, but not pubertally born astrocytes.
Androgen receptors during puberty or adulthood are not required to promote the
differentiation of pubertally born cells into mature neurons or astrocytes in the VMH and
DG.
An ANOVA revealed no significant differences between all groups in the percent
colocalization of BrdU/NeuN-positive cells in the VMHvl (F(3,20)=0.190, p=0.902) (Fig
2.5B), VMHdm (F(3,20)=2.304, p=0.108) (Fig 2.5D), and VMHc (F(3,20)=1.189,
p=0.339) (Fig 2.5F). An ANOVA revealed no significant differences between all groups
in the percent colocalization of BrdU/GFAP-expressing cells in the VMHvl
(F(3,20)=0.419, p=0.741) (Fig 2.5C), VMHdm (F(3,20)=1.078, p=0.381) (Fig 2.5E), and

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VMHc (F(3,20)=0.729, p=0.547) (Fig 2.5G). No significant differences group differences
were observed in the percent colocalization of BrdU/NeuN-positive cells in the DG
(F(3,8)=1.429, p=0.359) (Fig 2.6B) or in the percent colocalization of BrdU/GFAPpositive cells in the DG (F(3,8)=0.987, p=0.483) (Fig 2.6C).
DISCUSSION
The differential effects of pubertal and adult androgen receptors on pubertally born cells
in the MePD
In the current study, we discovered that androgen receptor (AR) activity during
puberty, but not adulthood, is necessary to increase the accumulation of BrdU-ir cells in
the adult male MePD. Treatment with flutamide, an AR antagonist, during puberty
significantly reduced the total number and density of pubertally born cells in the adult
MePD compared to males that received flutamide only during adulthood or males that
received only testosterone. Interestingly, gonadally intact sham males had an
intermediate number and density of pubertally born cells in the MePD. Gonadally intact
males normally display a pulsatile pattern of testosterone release (Nyby, 2008), while
exogenous testosterone treatment provides a steady level of circulating testosterone.
Therefore, it is possible that exogenous testosterone treatment may increase the
number of BrdU-ir cells compared to endogenous testosterone. From Chapter 1A of this
dissertation, we discovered that both pubertal and adult testosterone are necessary to
enhance the number of pubertally born cells in the MePD of adult mice. Together with
the results from the current experiment, we have identified that pubertal testosterone
acts via ARs to increase the number of pubertally born cells in the adult male MePD.
Since removing adult testosterone reduces the number of BrdU-ir cells (Chapter 1A),

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but treating with flutamide in adulthood does not (this study), it is likely that adult
testosterone is aromatized into estrogen to act on MePD estrogen receptors (ERs), and
not ARs, to maintain masculine numbers of pubertally born cells in the adulthood.
Few studies have examined the role of gonadal hormones on cell proliferation
and survival in the male medial amygdala. In male voles, testosterone increased cell
proliferation in the medial amygdala and this effect was largely due to estrogen, and not
androgens (Fowler et al., 2003). In adult hamsters, testosterone increased cell
proliferation, but not cell survival, in the posterior medial amygdala (Antzoulatos et al.,
2008). So far, all the studies that have examined the effects of gonadal hormones on
cytogenesis in the medial amygdala have been in adults and have not specifically
analyzed the MePD (Antzoulatos et al., 2008; Fowler et al., 2003; Fowler et al., 2005). It
is possible that testosterone may have a different effect on cell proliferation and survival
during puberty than adulthood. Further investigation will be required to examine the role
on testosterone on the proliferation and survival of new generated cells during puberty.
In the current study, pubertal ARs can increase the number of pubertally born
cells in the adult MePD by influencing cell proliferation, cell survival, or cell migration.
Activation of ARs during puberty could promote cell proliferation in the MePD to
increase pubertally born cells; although, it is also possible that pubertal ARs promote
the migration of new cells into the MePD from neurogenic niches. Alternatively, the
absence of ARs may promote the death of pubertally born cells, ultimately leading to
fewer BrdU-ir cells in the adult MePD with the flutamide treatment. Unfortunately, due to
the experimental design of the current study, we are unable to determine how pubertal
ARs stimulate an increase in pubertally born cells in the adult MePD. Future studies will

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need to investigate whether pubertal ARs play a role in cell proliferation, survival, or
migration in the MePD.
In the current report, blocking pubertal ARs also reduced the number of
pubertally born neurons in the adult male MePD. These results are the first to
demonstrate that pubertal androgens affect the survival of a specific cell phenotype in
the male MePD. In adulthood, males have more neurons in the MePD than females
(Morris et al., 2008b), and a study has shown that this sex difference in neuron number
does not exist pre-pubertally (Cooke et al., 2009), suggesting that more neurons are
added to the male MePD during puberty. The current findings suggest that androgens
may facilitate the addition of pubertal neurons to the male MePD. Although, the
mechanisms by which ARs act to enhance the number of pubertally born neurons is
currently unknown. It is possible that amygdalar progenitor cells or immature neurons
express ARs, by which androgens directly act on pubertally born cells to alter the
transcription of genes that regulate differentiation or androgens may act on ARs in other
neurons or glial cells, which then indirectly promote the differentiation of pubertally born
cells. At the moment, it is unknown whether pubertally born cells, themselves, express
ARs. It was discovered that immature neurons in the adult DG of rats did not express
ARs (Hamson et al., 2013); however, we’ve already established that androgen actions
can be different between species, ages, and brain regions, thus, even though immature
neurons in the adult DG do not express ARs, it is still possible that immature pubertally
born cells in the male MePD do express ARs. Several studies have shown that
astrocytes in the MePD of adult rats express ARs (Johnson et al., 2012). Thus,
androgens may act on astrocytes to influence pubertally born cell differentiation

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potentially through the release neurotrophic factors, as there is evidence that astrocytes
can release neurotrophic factors like brain-derived neurotrophic factor (BDNF)
(Dougherty et al., 2000; Fulmer et al., 2014).
Blocking ARs during puberty may also promote neuronal death in the MePD.
Studies have demonstrated that cell death is a mechanism by which sex differences in
the adult brain are established in regions such as the principle nucleus of the bed
nucleus of the stria terminalis (pBNST) and anteroventral periventricular nucleus
(AVPV) (Forger, 2006; Forger et al., 2004; Gotsiridze et al., 2007). Testosterone
decreases cell death in the pBNST and increases cell death in the AVPV (Chung et al.,
2000; Forger, 2006; Sumida et al., 1993). Therefore, in the MePD, it could be that ARs
do not affect the differentiation of pubertally born cells into neurons, but promote their
survival into adulthood. While we have confirmed that the number of pubertally born
neurons is greater when pubertal ARs are present, it still remains to be determined
whether the same number of neurons are born in all groups and AR enhances survival
of these neurons, whether AR works to enhances the differentiation of pubertally born
neurons or a combination of these mechanisms.
Although blocking ARs during puberty reduced the number of pubertally born
neurons, blocking ARs during puberty or adulthood did not have an effect on the
number of pubertally born astrocytes in the adult male MePD. These results were quite
surprising, as a previous study discovered that ARs regulated the increase in the
number of astrocytes during puberty in the MePD of male rats (Johnson et al., 2012). In
the current study, we only administered BrdU during the first week of puberty, therefore
it is possible that AR may affect the addition of pubertally born astrocytes during mid to

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late puberty. We can, however, conclude that androgens during early puberty do not
affect the number of pubertally born astrocytes in the adult male MePD.
We also discovered that blocking ARs during puberty or adulthood did not affect
male adult MePD volume as there were no significant differences in MePD volume at
P74 between males treated with flutamide only during puberty, males treated with
flutamide only during adulthood, males treated only with testosterone, and sham males.
Several studies have shown that testosterone has an activational effect on the volume
of the adult MePD (Cooke et al., 2003; Morris et al., 2008a; Morris et al., 2008b).
Testosterone treatment in gonadectomized adult male or female rodents for at least 4
weeks can increase the MePD volume to a masculine size, whereas castrating a male
rodent can decrease MePD volume to be comparable to the female MePD volume
(Morris et al., 2008b). Even though testosterone is traditionally viewed as the dominant
male gonadal hormone acting via ARs, testosterone can also be aromatized into
estrogen to act via ERs. In fact, a study demonstrated that testosterone acts via both
ARs and ERs to maintain the adult male MePD volume (Cooke et al., 2003). Thus, in
the current experiment it is possible that testosterone can increase the volume of the
MePD to a masculine size by acting via ERs.
Contributions of pubertal and adult androgens on the VMH and DG
The current findings show that ARs are not required during puberty or adulthood
to increase the total number or density of pubertally born cells in the adult VMHvl,
VMHdm, and VMHc. Similarly, in Chapter 1B of this dissertation, we discovered
comparable numbers of pubertally born cells in the VMHvl, VMHdm, and VMHc
between androgen-insensitive iTfm male and wildtype male mice. In adult male voles,

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testosterone treatment did not alter cell proliferation in the VMH, furthermore, treatment
with estradiol and DHT did not have any effect on cell proliferation as well(Fowler et al.,
2003). Taken together, these results indicate that gonadal hormones may not play a
role in pubertal cytogenesis in the male VMH. Although, we cannot rule out the
possibility that testosterone may be acting via ERs rather than ARs in the VMH to
promote the addition of new cells during puberty, as the VMHvl is densely populated
with both ARs and ERs(Simerly et al., 1990). A study in female mice found that
estrogen increased the number of adult born cells in the VMH 34 days post-BrdU
administration, indicating that estrogen treatment may influence cell survival in the VMH
(Bless et al., 2016). Thus, it is possible that gonadal hormones do not influence cell
proliferation in the VMH during puberty; however, adult testosterone, aromatized into
estrogen, act via ERs to influence the survival of pubertally born cells.
The current study also discovered that ARs during puberty or adulthood did not
influence cell differentiation in the VMH, as all groups had the same proportion of BrdUir colocalize with NeuN or GFAP in all VMH subdivisions. A study in female mice
discovered that estrogen treatment enhanced the survival of adult-neurons in the VMH
(Bless et al., 2016). Interestingly, in female meadow voles, estrogen treatment did not
influence number of BrdU cells colocalized with TuJ1, a neuronal marker, in the adult
VMH 24 hours post-BrdU treatment (Fowler et al., 2005). These results suggest that
estrogen does not affect the genesis of new neurons in the VMH, but long-term
estrogen treatment could influence the survival of newborn VMH neurons. However,
since these studies were conducted in adult female rodents, further investigation is

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needed to examine the role of estrogen on cell differentiation of pubertally born cells in
the VMH of male mice.
We also found that pubertal and adult androgens did not influence adult volumes
of the VMHvl, VMHc, and VMHdm. In a previous study, it was discovered that ARs were
required to fully masculinize the VMHvl volume in adult rats, as Tfm rats had a more
feminine VMHvl volume compared to wildtype male (Dugger et al., 2007). However, in
the current study, we do not observe any volumetric changes as a result of blocking
ARs during puberty or adulthood. This result suggests that androgens work to
masculinize VMH volume prior to puberty. A previous study in adult male rats found that
castrating males on P1 reduced VMH volume in adulthood; however, castrating male
rats at P7 did not have any effect on volume(Matsumoto et al., 1983). Therefore, it
seems that perinatal androgens have a long-term organizational effect on VMH volume
in males, increasing VMH volume in adulthood independent of androgen manipulation
during puberty or adulthood.
In this study, pubertal and adult androgens did not appear to influence the
number of pubertally born cells in the DG of adult male mice. A previous study in adult
male rats demonstrated testosterone enhanced cell survival in the DG, and it appears
this increase in cell survival was driven by the presence of androgens, as treatment with
DHT, but not estrogen, increased the number of surviving BrdU-ir cells (Spritzer et al.,
2007). Furthermore, adult Tfm male rats and adult wild-type male rats treated with
flutamide also showed a reduction in cell survival in the dentate gyrus(Hamson et al.,
2013). However, in male meadow voles, gonadal hormones (i.e. testosterone, estrogen,
and DHT) did not have any effect on cell proliferation in the DG (Fowler et al., 2003) and

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estrogen treatment in castrated adult male rats did not increase cell proliferation
compared to oil-treated male rats (Barker et al., 2008). Therefore, in adulthood,
androgens promote the survival of new cells, while estrogen enhances cell proliferation,
which is inconsistent with our current findings. There are several methodological factors
that could account for these differences. For instance, the studies in male rats
examining the effects of gonadal hormones on cell survival single-housed their subjects
after weaning. Several studies have shown that social isolation can have a negative
impact on hippocampal neurogenesis(Ibi et al., 2008; Leasure et al., 2009; Lieberwirth
et al., 2012; Spritzer et al., 2011) and more recently, a study showed that while
castration reduced the number of BrdU-ir cells in the DG of adult male rats 16 days
post-BrdU treatment, socially isolated male rats had even fewer BrdU-ir cells than pairhoused males (Spritzer et al., 2011). Therefore, in the current study, the absence of
androgens during puberty or adulthood could have led to a reduction in cell survival, but
social housing (4-5 mice/cage) may provide a compensatory mechanism to ultimately
enhance cell survival. In addition, the age difference of when BrdU was administered
could also explain these inconsistencies. Significantly more cells are born during
puberty than adulthood, and more pubertally born cells are likely to survive than adult
born cells (Curlik et al., 2014; Ho et al., 2012; Staffend et al., 2014). There is evidence
that cells born during puberty are functionally distinct from adult-born
neurons(Kirshenbaum et al., 2014; Martinez-Canabal et al., 2013; Wei et al., 2011),
therefore, the regulation of the survival and functional integration of new born cells by
gonadal hormones may be different during adolescence and adulthood. While the
differential effects of gonadal hormones on pubertal and adult cytogenesis have not

102

been examined, a study has established that fluoxetine, an antidepressant drug,
increases cell proliferation in male adults compared to controls, but does not affect cell
proliferation in male adolescents(Hodes et al., 2009). Therefore, although gonadal
hormones may influence cell proliferation and survival in the adult DG, it is possible that
they do not have this same effect in the pubertal DG.
Furthermore, we discovered that pubertal and adult androgens did not alter the
proportion of pubertally born cells co-expressing NeuN or GFAP in the adult DG. A
previous study in adult male rats discovered that androgens enhanced neurogenesis in
the DG as DHT increased the number of adult-born neurons in the DG than male rats
treated with DHT and flutamide 30 days after BrdU administration (Hamson et al.,
2013). Although, another study found no differences in the number of cells showing
BrdU-ir and NeuN-ir colocalization in the DG between castrated and gonadally intact
male rats 16 days after BrdU treatment (Spritzer et al., 2011), indicating that long-term,
but not short-term, androgen treatment enhances neurogenesis in the DG. There is also
evidence that estrogen enhances cell survival in the adult male meadow vole dentate
gyrus, but only when estrogen is administered 6-10 days post-BrdU administration
(Ormerod et al., 2004).. Based on these findings, androgens and estrogens could be
working together to enhance the number of pubertally born neurons in the DG,
therefore, estrogenic effects may compensate for non-functional ARs during puberty or
adulthood to enhance survival of pubertally born neurons in the adult DG. In regard to
astrogliogenesis, it has been shown that astrocytes can express ARs and ERs (Azcoitia
et al., 1999; Finley et al., 1999; Garcia-Ovejero et al., 2002; Johnson et al., 2012), but
very few studies have examined the role of gonadal hormones on the addition of new

103

astrocytes to the dentate gyrus under non-pathological conditions, as the recruitment of
reactive astrocytes and microglia to sites of neuronal injury have been a primary focus
in this field of study(Rajkowska et al., 2007; Rodriguez et al., 2009; Sofroniew et al.,
2010). While it remains unknown at what stage of astrogliogenesis that astrocytes begin
to express ARs and ERs, but it is possible that gonadal hormones may act directly on
newly born astrocytes, themselves, to enhance survival. In the MePD, the addition of
pubertal astrocytes in males was dependent on the presence of functional androgen
receptors. While this is the case in the MePD, the survival of pubertally born astrocytes
in the DG does not appear to exclusively depend on androgens, thus, androgens and
estrogens may work together at different stages of development to promote the survival
of pubertally born astrocytes.
Conclusions
In the current study, we discovered that androgens during puberty, but not
adulthood, facilitate an increase in the number of pubertally born cells in the adult male
MePD. Furthermore, pubertal androgens enhance the number of pubertally born
neurons, but not astrocytes, in the adult male MePD. However, we also determined that
neither pubertal nor adult androgens influence the number of pubertally born VMH and
DG cells, including pubertally born neurons and astrocytes. These findings demonstrate
that the actions of androgens have an age-specific and site-specific effect on the
survival of pubertally born cells with pubertal androgens enhancing the survival of
pubertally born neurons in the MePD, but not affecting other brain regions such as the
VMH or DG. This study is the first to determine that pubertal ARs influence a specific
cell phenotype in the MePD. In future studies, it will be important to examine the role of

104

ARs on generating certain cell phenotypes in cells born during mid to late puberty.
Furthermore, it will be critical to determine whether pubertally born cells become
functionally integrated into adult neural circuits and then determine whether neurons
and glia have different roles in mediating the emergence of male sociosexual behaviors.

105

Figure 2.1. Blocking androgen receptors during puberty in male mice reduces the
number of pubertally born cells in the adult posterodorsal medial amygdala (MePD).
Representative photomicrographs displaying the traces of the MePD on Nissl-stained coronal
sections and pubertally born cells in the adult MePD, respectively, in (A-B) Flut@Puberty male
mice, (C-D) Flut@Adulthood male mice, (E-F) T Only male mice, and (G-H) sham male mice.
Quantitative analysis revealed that (I) Flut@Puberty male mice have fewer pubertally born cells
and (J) a lower density of BrdU-ir cells than Flut@Adulthood and T Only male mice. (K) No
volumetric differences were detected in the adult MePD between all groups. Data are
represented as mean ± SEM. The small inlaid boxes show BrdU-labeled cells at a higher
magnification. Low mag scale bar = 200 μm; high mag scale bar = 10 μm.

106

Figure 2.2. Androgen receptors are not required to increase the number of pubertally
born cells in the adult ventromedial hypothalamus (VMH). Representative
photomicrographs displaying the traces of the VMHvl, VMHdm, and VMHc on Nissl-stained
coronal sections and pubertally born cells in the adult VMH, respectively, in (A-B) Flut@Puberty
male mice, (C-D) Flut@Adulthood male mice (E-F) T Only male mice, and (G-H) sham male
mice. An ANOVA revealed no significant differences in (I, L, O) total number of pubertally born
cells, (J, M, P) density of BrdU-ir cells, and (K, N, Q) volume of the adult VMHvl, VMHdm, and
VMHc between all groups. Data are represented as mean ± SEM. The small inlaid boxes show
BrdU-labeled cells at a higher magnification. Low mag scale bar = 200 μm; high mag scale bar
= 10 μm.

107

Figure 2.3. Androgen receptors are not required to increase the number of pubertally
born cells in the adult dentate gyrus of male mice. Representative photomicrographs
displaying pubertally born cells in coronal sections of the adult dentate gyrus in (A)
Flut@Puberty male mice, (B) Flut@Adulthood male mice, (C) T Only male mice, and (D) sham
male mice. Quantitative analysis revealed no significant differences between groups in (E) the
total number of pubertally born cells in the adult male dentate gyrus. The small inlaid boxes
show BrdU-labeled cells at a higher magnification. Low mag scale bar = 200 μm; high mag
scale bar = 10 μm. Data are represented as mean ± SEM. * p < .001.

108

Figure 2.4. Blocking androgen receptors during puberty, but not adulthood, reduces the
number of pubertally born cells that become neurons in the adult MePD. (A) A
representative photomicrograph of a coronal section of the adult MePD in a sham male stained
for BrdU (green), GFAP (red), and NeuN (blue). Quantitative analysis revealed that (B)
Flut@Puberty male mice had significantly less pubertally born cells that colocalized with a
mature neuron marker in the adult MePD than Flut@Adulthood, T Only, and Sham male mice.
However, (C) no significant differences were observed between groups in the proportion of
pubertally born cells that colocalized with an astrocytic marker in the adult MePD. Merged Zstack images of BrdU-ir cells colocalized with (D) NeuN-ir and (E) GFAP-ir cells are shown at
maximum intensity projections. Low mag scale bar = 200 μm; high mag scale bar = 10 μm. Data
are represented as mean ± SEM.

109

Figure 2.5. Androgen receptors are not required to enhance the number of pubertally
born cells that colocalize with mature neurons or astrocytes in the adult VMH. (A) A
representative photomicrograph of a coronal section of the adult VMH in a sham male stained
for BrdU (green), GFAP (red), and NeuN (blue). Quantitative analysis revealed no significant
differences between groups in the proportion of pubertally born cells that colocalized with (B, D,
F) a mature neuronal marker or (C, E, G) astrocytic marker in the adult VMHvl, VMHdm, and
VMHc. Scale bar = 200 μm. Data are represented as mean ± SEM.

110

Figure 2.6. Androgen receptors are not required for pubertally born cells to differentiate
into mature neurons or astrocytes in the adult dentate gyrus. (A) A representative
photomicrograph of a coronal section of the adult DG in a sham male stained for BrdU (green),
GFAP (red), and NeuN (blue). Quantitative analysis revealed no significant differences between
groups in the proportion of pubertally born cells that colocalized with (B) a mature neuronal
marker or (C) astrocytic marker in the adult DG. Scale bar = 200 μm. Data are represented as
mean ± SEM.

111

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effects of androgen in prenatal rats. Neuroscience Letters, 151(1), 41-44.
Wei, L., Meaney, M. J., Duman, R. S., & Kaffman, A. (2011). Affiliative behavior requires
juvenile, but not adult neurogenesis. J Neurosci, 31(40), 14335-14345.
Yang, C. F., Chiang, M. C., Gray, D. C., Prabhakaran, M., Alvarado, M., Juntti, S. A., . .
. Shah, N. M. (2013). Sexually dimorphic neurons in the ventromedial
hypothalamus govern mating in both sexes and aggression in males. Cell,
153(4), 896-909.

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CHAPTER 3
Sociosexual interactions activate pubertally born cells in the posterodorsal medial
amygdala and ventromedial hypothalamus of adult male mice
ABSTRACT
In adult mice, pubertally born cells are retained in three limbic regions: the
posterodorsal medial amygdala (MePD), ventromedial hypothalamus (VMH), and
dentate gyrus (DG). The MePD and VMH are both sexually dimorphic brain regions
linked to sex-specific social behaviors, such as male sexual behavior and intermale
aggression. A previous study in male hamsters showed that some pubertally born cells
in the adult hamster brain develop into mature neurons and glia that are functionally
incorporated into neural circuits involved in sex-specific social behaviors. In the current
study, we examined whether pubertally born cells are activated by socially relevant
stimuli in the MePD, VMH, and DG of adult male mice. We also determined the
phenotype of pubertally born cells in each region of interest using NeuN, a mature
neuron marker, and GFAP, an astrocytic marker. The subdivisions of the VMH were all
analyzed as separate regions of interest: ventrolateral subdivision (VMHvl), dorsomedial
subdivision (VMHdm), and central subdivision (VMHc). We administered 5-bromo-2’deoxyuridine (BrdU) to male mice during the first week of puberty (P28-35) to label cells
born during this period of time. On P63, each subject was exposed to one of three
social interactions one hour prior to sacrifice: resident-intruder, receptive female, or no
social interaction. Aggressive and sexual behaviors elicited about 0.43% and 0.56 of
pubertally born cell activation in the MePD, respectively, while aggressive behaviors
activated about 0.68% of pubertally born cells in the VMHvl and 0.66% of pubertally

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born cells in the VMHdm. Furthermore, social interaction did not activate pubertally born
cells in the VMHc and DG. In the MePD, about 6% and 12% of pubertally born cells
differentiated into mature neurons and astrocytes, respectively. In the VMH, 7% of
pubertally born cells were mature neurons and 7% were astrocytes in all subdivisions
analyzed. In the DG, about 75% of pubertally born cells were neurons and 10% were
astrocytes. These findings indicate that the pubertally born cells are incorporated into
existing adult neural circuits known to regulate sociosexual behaviors.
INTRODUCTION
The pubertal brain undergoes extensive structural remodeling(Blakemore et al.,
2010; Giedd et al., 1999; Sisk et al., 2004), which includes cytogenesis, or the addition
of new cells (Ahmed et al., 2008; Mohr et al., 2016; Mohr et al., 2013; Saul et al., 2015;
Staffend et al., 2014). Recent findings from our lab and the previous chapter in this
dissertation have shown that new neurons and glial cells are added to the posterodorsal
medial amygdala (MePD), ventromedial hypothalamus (VMH), and dentate gyrus (DG)
during puberty and that a subset of these cells survives well into adulthood(Ahmed et
al., 2008; Mohr et al., 2013). The MePD, VMH, and DG are involved in regulating
sociosexual behaviors, such as male sexual behavior and intermale aggression(Falkner
et al., 2014a; Kollack-Walker et al., 1992, 1995; Lee et al., 2014; Lin et al., 2011;
Samuelsen et al., 2009a; Sano et al., 2016; Sano et al., 2013), which emerge during
puberty to promote the survival and/or reproductive capabilities of adult rodents(Barkley
et al., 1977; De Lorme et al., 2013; Schulz et al., 2006). However, it is currently unclear
whether pubertally born cells are functionally integrated into brain regions known to
regulate sociosexual behaviors such as the MePD, VMH, and DG.

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The MePD is a subregion of the medial amygdala that receives strong afferent
projections from the accessory olfactory bulb (AOB) (Cadiz-Moretti et al., 2016). The
AOB sends chemosensory information to the MePD (Samuelsen et al., 2009b) and, in
turn, the MePD categorizes socially relevant cues from the environment to help elicit
context-appropriate behaviors (e.g. male sexual behavior in response to the presence of
a sexually receptive female)(Samuelsen et al., 2009a). The MePD of male rodents is
highly responsive to the scent of conspecifics. For instance, the adult male mouse
MePD shows an increase in Fos expression, a marker of neural activation, in response
to female urine and bedding soiled by a female (Samuelsen et al., 2009a; Taziaux et al.,
2011). In addition, the male MePD is also markedly activated by physical social
interactions as sexual interactions with a receptive female and aggressive encounters
with an intruder male increase Fos expression(Hong et al., 2014; Kollack-Walker et al.,
1995; Taziaux et al., 2011).
The VMH is part of the mediobasal hypothalamus and receives strong
innervations from the MePD (Pardo-Bellver et al., 2012). The VMH is commonly
associated with regulating female sexual behavior (Flanagan-Cato et al., 2001) and
energy balance (King, 2006b), but recent studies have implicated the VMH in male
sexual behavior, intermale aggression, and defensive behaviors as well (Choi et al.,
2005; Falkner et al., 2014a; Falkner et al., 2014b; Lin et al., 2011; Wang et al., 2015).
The ventrolateral subdivision of the VMH (VMHvl), in particular, is involved in gating
male sexual behavior and intermale aggression in adult male mice(Choi et al., 2005;
Lee et al., 2014; Lin et al., 2011). When neurons in the VMHvl are activated, males
become aggressive, but inhibition of these neurons promotes mating behavior (Lin et

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al., 2011). More specifically, the activation of neurons expressing estrogen receptor-a
(ERa) in the VMHvl can elicit aggressive behavior in males (Lee et al., 2014).
Furthermore, ablating ERa in the VMH or progesterone receptors in the VMHvl leads to
significant reductions in male sexual behavior and intermale aggression (Sano et al.,
2013; Yang et al., 2013). The dorsomedial and central subdivisions of the VMH
(VMHdm and VMHc, respectively) mediate defensive behaviors in adult male
mice(Kunwar et al., 2015; Mongeau et al., 2003; Wang et al., 2015). Stimulation of
neurons in the VMHdm and VMHc elicits avoidance behaviors, such as fleeing and
jumping, and immobility(Wang et al., 2015). In addition, a predator scent (i.e. cat collar)
and exposure to a predatory rat induces Fos expression in the VMHdm of adult male
rats (Dielenberg et al., 2001; Silva et al., 2013). Thus, subdivisions in the VMH serve
different functions, with the VMHvl regulating male sexual behavior and intermale
aggression, while the VMHdm and VMHc plays a role in controlling defensive behaviors.
The DG is a region in the hippocampus recognized for maintaining relatively high
levels of postnatal neurogenesis well into adulthood. Throughout the years, the DG has
been associated with controlling many different types of behaviors, such as spatial
learning, mood regulation, object recognition, and more recently, social behaviors (Ibi et
al., 2008; Jessberger et al., 2009; Shors et al., 2002; Wei et al., 2011). Social
experiences tend to have a strong influence on neurogenesis in the DG in rodents. In
adult male rats, sexual encounters with a receptive female enhances the number of new
neurons in the DG (Leuner et al., 2010). Conversely, social isolation during adolescence
leads to reduced levels of neurogenesis in the DG of adult male mice (Ibi et al., 2008).
Moreover, it appears some pubertally born neurons in the DG are functionally distinct

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from adult born neurons. Blocking pubertal neurogenesis in female mice reduces social
exploration and increases social aversion during female-female social interactions,
while blocking adult neurogenesis does not have an effect on these social behaviors
(Wei et al., 2011). Thus, it seems that pubertally born neurons in the DG may facilitate
the acquisition of adaptive behaviors (e.g. appropriate social behaviors for mating)
required for adulthood.
While it is clear that the MePD, VMH, and DG are intimately involved in the
regulation of adult sociosexual behaviors, the neurological changes that facilitate the
development of these behaviors remains unknown. A previous study found that that a
subset of pubertally born MePD cells in male hamsters is activated by a sexual
encounter(Mohr et al., 2013). This was the first study to show that pubertally born cells
had the capacity to mature into permanent fixtures in the brain to participate in the
expression of adult social behaviors, like sexual behavior. Therefore, it is possible that
pubertally born cells in the MePD, VMH, and DG also become functionally integrated
into existing neural circuits and are activated by social interactions.
The current chapter aimed to expand on this breakthrough discovery and
examine Fos expression in each region of interest (ROI) (i.e. MePD, VMHvl, VMHdm,
VMHc, and DG ) to detect region-specific activation in response to different social
behaviors in adult males and determine whether pubertally born cells in the of adult
male mice become functionally integrated into existing neural circuits that mediate adult
sociosexual behaviors (i.e. male sexual behavior and intermale aggression). In addition,
we determined what proportion of pubertally born cells in each ROI became mature
neurons or astrocytes.

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MATERIALS AND METHODS
Animal Subjects and Experimental Design
42 male mice from an in-house wildtype C57BL/6 mouse colony were used for
experimental procedures described below. In addition, 14 female and 14 male mice
from the same colony were used as stimulus animals for social interaction tests. All
mice were weaned at P24 and group housed (4-5 mice/cage) in clear plexiglass cages
(29 x 18 x 13 cm) in a 12:12 light:dark cycle (lights on: 7:00 AM, lights off: 7:00 PM). All
subjects were provided ad libitum rodent chow (Harlan Teklad 8640 Rodent Diet,
Madison, WI) and water. All female stimulus mice were ovariectomized on between
P50-P60 and subcutaneously (s.c.) implanted with a Silastic capsule filled with 17βestradiol (0.6 mm–inner diameter and 3.2 mm–outer diameter; 12 mm–effective release
length, 1.8 cm–total capsule length, filled with 25 µg 17β-estradiol /ml sesame oil). All
female stimulus mice participated in a social interaction test between P60-P70. Four
hours prior to the social interaction test, all females received an injection of
progesterone (0.5mg/0.1 ml in sesame oil, s.c.). All housing and experimental
procedures were performed in compliance with the National Institutes of Health Guide
for the Care and Use of Laboratory Animals and approved by the Michigan State
University’s Institutional Animal Care and Use Committee.
BrdU administration
During the first week of puberty (P28-P35), all subjects were administered BrdU
(1.0 mg/ml, Sigma-Aldrich) dissolved in the drinking water. BrdU drinking water was
prepared fresh every 3 days. During BrdU administration, all mice were housed in
Innocage mouse cages (37 x 23 x 14 cm) with Aquavive amber water bottles (Innovive,

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San Diego, CA). The use of the Aquavive amber water bottles allowed us to administer
BrdU via drinking water in smaller volumes (about 100 ml of water/bottle) to avoid
excess BrdU waste. The Aquavive amber water bottles also had minimal leakage and
since BrdU is light sensitive, the amber bottles provided adequate protection from light.
In addition, the use of disposable Innocage mouse cages avoided BrdU contamination
of reusable animal cages.
Social Interaction Tasks
On P56, one week prior to social interactions and subsequent sacrifice, all male
mice were housed in separate cages and pair-housed with a female mouse to provide a
home cage territory and sexual experience. On P63, the female was removed from the
home cage one hour prior to the social interaction tasks. The male mice were randomly
assigned to one of three types of social interactions: resident-intruder paradigm (n=14),
sexual behavior (n=14), or no social interaction (n=14). In the resident-intruder
condition, a smaller, unfamiliar male intruder was introduced to the home cage of the
resident at the start of the test. After fifteen minutes, the intruder was removed from the
cage and the resident male was sacrificed one hour later. In the sexual behavior
condition, an unfamiliar estrogen and progesterone primed female mouse was
introduced to the male’s home cage. After fifteen minutes, the female mouse was
removed from the cage and the male subject was sacrificed one hour later. In the no
social interaction condition, the male mice received no social stimulus and resided in
their home cage until time of sacrifice. All social interactions were monitored to confirm
that the appropriate social interaction (i.e. attack and chase behavior during residentintruder paradigm and mounting and intromission during sex behavior) occurred as

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intended. Henceforth, the following group names will be used: male-male interaction
group (male mice that underwent the resident-intruder paradigm), male-female
interaction group (male mice that experienced male sexual behavior), and no social
interaction group (male mice that experienced no social interaction).
Tissue Collection and Processing
On P63, all mice were given an overdose of sodium pentobarbital (150 mg/kg,
i.p.) and intracardially perfused with 0.9% saline followed by cold 4% paraformaldehyde
in 0.1 M phosphate buffered saline (pH 7.4). Brains were removed and postfixed
overnight in 4% paraformaldehyde, then switched to 30% sucrose solution at 4 °C until
sectioning. All brains were sectioned within one week of perfusion. Brains were cut on a
freezing microtome into 30 μm coronal sections (from about Bregma -0.82 to Bregma 2.74), and collected as 4 alternate series. The entire first series for each animal was
Nissl stained with thionin, dehydrated, and coverslipped.
Double-Label BrdU/Fos Immunofluorescence
The entire second series of brain sections underwent immunofluorescence to
label BrdU-immunoreactive (ir) and Fos-ir cells. Free-floating sections were rinsed in
Tris-buffered saline (TBS; 0.05 M; pH 7.6) for 30 minutes and then incubated in 0.1%
sodium borohydride for 10 minutes. After a 15-minute rinse in TBS, the sections were
incubated in 2N HCl for 60 minutes at 37°C. Sections were then neutralized in 0.1M
borate buffer (pH 8.5) for 10 minutes. Following a 30-minute rinse in TBS, the sections
were blocked in TBS containing 0.01% Triton-X-100 and 3% donkey serum for 1 hour,
and then incubated in a primary antibody cocktail at 4°C for 48 hours, which contained
monoclonal rat anti-BrdU (catalog no. MCA2060; Serotec) at a working concentration of

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1 μg/ml and polyclonal rabbit anti-c-Fos (catalog no. ABE457; Millipore) at a working
concentration of 0.1 μg/ml. Sections were then rinsed in TBS for 30 minutes and
incubated in a secondary antibody cocktail for 2 hours at room temperature, which
contained Cy2 Affinipure donkey anti-rat secondary antibody (catalog no. 712-225-153;
Jackson Immunoresearch) at a working concentration of 2.8 μg/ml and Cy3 Affinipure
donkey anti-rabbit (catalog no. 711-165-152, Jackson Immunoresearch) at a working
concentration of 2.8 μg/ml. Following a 30-minute rinse in TBS, all sections were
mounted onto gelatin-subbed slides, dehydrated, and coverslipped.
Triple-Label BrdU/GFAP/NeuN Immunofluorescence
The third series of brain sections of 6 animals (2 mice/group) underwent
immunofluorescence to label for BrdU, GFAP (a marker of astrocytes), and NeuN (a
marker of mature neurons). Since differences in social interaction prior to sacrifice could
not affect colocalization of pubertally born cells with NeuN or GFAP, a total of 6 mice
were analyzed for the MePD and VMH, and a total of 3 mice were analyzed for the DG
(1 mouse/group). Free-floating sections were rinsed in Tris-buffered saline (TBS; 0.05
M; pH 7.6) for 30 minutes and then incubated in 0.1% sodium borohydride for 10
minutes. After a 15-minute rinse in TBS, the sections were incubated in 2N HCl for 60
minutes at 37°C. Sections were then neutralized in 0.1M borate buffer (pH 8.5) for 10
minutes. Following a 30-minute rinse in TBS, the sections were blocked in TBS
containing 0.01% Triton-X-100 and 3% donkey serum for 1 hour, and then incubated in
a primary antibody cocktail at 4°C for 48 hours, which contained monoclonal rat antiBrdU (catalog no. MCA2060; Serotec) at a working concentration of 1 μg/ml, polyclonal
rabbit anti-GFAP (catalog no. Z0334; Dako) at a working concentration of 0.58 μg/ml,

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and monoclonal mouse anti-NeuN (catalog no. MAB377, Chemicon) at a working
concentration of 1 μg/ml. Sections were then rinsed in TBS for 30 minutes and
incubated in a secondary antibody cocktail for 2 hours at room temperature, which
contained Cy2 Affinipure donkey anti-rat secondary antibody (catalog no. 712-225-153;
Jackson Immunoresearch) at a working concentration of 5.6 μg/ml, Cy3 Affinipure
donkey anti-rabbit (catalog no. 711-165-152, Jackson Immunoresearch) at a working
concentration of 6.0 μg/ml, and Cy5 Affinipure donkey anti-mouse (catalog no. 715-175151, Jackson Immunoresearch) at a working concentration of 6.0 μg/ml. Following a 30minute rinse in TBS, all sections were mounted onto gelatin-subbed slides, dehydrated,
and coverslipped.
Microscopic Analysis
All microscopic analyses were performed by an observer blind to treatment
group. The regions of interest for all microscopic analyses included the posterodorsal
medial amygdala (MePD), the ventrolateral, dorsomedial, and central subdivisions of
the ventromedial hypothalamus (VMHvl, VMHdm, and VMHc, respectively), and the
dentate gyrus (DG). The boundaries of the MePD and VMH on the Nissl series were
determined using a standard mouse brain atlas(Paxinos et al., 2004). To consistently
sample anatomically-matched sections across animals, the following landmarks were
used to trace the MePD: the shape of the optic tract, the size and shape of the lateral
ventricles, the size and shape of the intercalated amygdalar nucleus, and the size and
shape of the stria terminalis. For the VMH, the size, shape, and staining intensity of the
nucleus itself, as well as the size and shape of the third ventricle were used to

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consistently sample anatomically-matched sections across animals. For the DG, the two
most rostral sections were analyzed.
Double-label BrdU/Fos Analysis
The double-label BrdU/Fos series was analyzed using an Olympus BX51
microscope under epi-illumination using Neurolucida version 10 (MBF Bioscience). The
regions of interest were traced using the Nissl-stained series and contours were
superimposed onto adjacent sections of the double-label BrdU series. The full rostralcaudal extent of the MePD, VMHvl, VMHdm, and VMHc was analyzed, which included a
total of six MePD sections and five VMH sections. Animals that did not have all
anatomically-matched sections were excluded from analysis. The final sample sizes
were as follows: male-male interaction (n=13), male-female interaction (n=11), and no
social interaction (n=14). The total number of Fos-ir cells was calculated by summing
the number of Fos-ir cells across all sections analyzed. The total number of BrdU-ir cells
was determined by summing the number of BrdU-ir cells across all sections analyzed.
The % BrdU-ir cells that expressed Fos was calculated by dividing the total number of
double-labeled BrdU-ir/Fos-ir cells by the total number of BrdU-ir cells multiplied by 100.
Triple-Label BrdU/GFAP/NeuN Analysis
The triple-label BrdU/GFAP/NeuN series was analyzed using an Olympus
FluoView FV1000 confocal laser-scanning microscope. A z-stack orthogonal viewer was
utilized to verify colocalization of BrdU/GFAP and BrdU/NeuN. All regions of interest
were analyzed using the multi-line Argon gas laser (488 nm), green Helium Neon laser
(543 nm), and red Helium Neon laser (633 nm) to detect BrdU-ir, GFAP-ir, and NeuN-ir
cells, respectively. Again, the full rostral-caudal extent of the MePD, VMHvl, VMHdm,

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and VMHc was analyzed. The % colocalization of BrdU with GFAP was calculated by
dividing the total number of BrdU-ir/GFAP-ir cells by the total number of BrdU-ir cells
multiplied by 100. The % colocalization of BrdU with NeuN was calculated by dividing
the total number of BrdU-ir/NeuN-ir cells by the total number of BrdU-ir cells multiplied
by 100.
Statistical Analysis
For the double-label BrdU/Fos analysis, group differences in total number of
BrdU-ir cells, total number of Fos-ir cells, and % colocalization of BrdU-ir/Fos-ir cells
were analyzed using a one-way ANOVA. For all analyses, a significance value of p ≤
0.05 was used and Cohen’s d was calculated to determine effect sizes. For the triplelabel BrdU/GFAP/NeuN analysis, no statistical analyses were performed because of
small sample sizes (n=2/group). Only % colocalization of BrdU/GFAP and BrdU/NeuN
were calculated, as described above, to determine the proportion of pubertally born
cells that differentiated into astrocytes or mature neurons for each region of interest.
RESULTS
Social interaction activates pubertally born cells in the adult male MePD.
Pubertally born cells in the MePD were activated by social interactions in adult
male mice as evidenced by Fos expression in BrdU-ir cells (Fig 3.1A). An ANOVA
revealed a significant group difference in the proportion of pubertally born MePD cells
that express Fos (F(2,35)=11.731, p=0.000) (Fig 3.1B). Post-hoc analyses showed that
male-male social interaction (p=0.006, d=1.35) and male-female social interaction
(p<0.000, d=2.10) significantly increased the proportion of BrdU-ir cells that colocalize
with Fos-ir in the adult MePD when compared to no social interaction. No significant

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differences were observed between the male-male and male-female social interaction
groups (p=0.516). In addition, there were significant differences in the total number of
Fos-ir cells in the adult MePD between social interaction groups (F(2,35)=33.409,
p=.000) (Fig 3.1C). The expression of Fos was larger in response to male-male social
interaction (p<0.000, d=2.48) (Fig 3.1E) and male-female social interaction (p<0.000,
d=4.11) (Fig 3.1F) than no social interaction (Fig 3.1G), recapitulating previous studies
showing increased activation of the adult MePD in response to social interactions with a
male or female conspecific(Kollack-Walker et al., 1995). No significant differences were
detected between the male-male social interaction and male-female social interaction
groups (p=1.00). Furthermore, no group differences were observed in the total number
of BrdU-ir cells (F(2,35)=1.029, p=0.368) (Fig 3.1D) showing that the number of
pubertally born cells in the MePD was comparable across all groups.
Male-male social interactions activate pubertally born cells in the adult male VMHvl and
VMHdm.
Double-labeled BrdU-ir and Fos-ir cells were identified in the adult male VMH
(Fig 3.2A) with male-male social interactions enhancing the activation of pubertally born
cells in the VMHvl and VMHdm. An ANOVA revealed a significant group difference in
the proportion of pubertally born cells that express Fos in the VMHvl (F(2,35)=4.649,
p=0.016) (Fig 3.2B) and VMHdm (F(2,35)=4.298, p=0.021) (Fig 3.2C). In the VMHvl,
male-male interaction significantly increased the proportion of BrdU-ir cells that
expressed Fos compared to no social interaction (p=0.013, d=1.23). The proportion of
BrdU-ir cells that express Fos-ir after male-female interaction was not different from that
of either the male-male (p=0.534) or no social interaction (p=0.417) groups, suggesting

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that pubertally born cells in the VMHvl are only moderately activated in following social
interaction with a female conspecific. Similar to the VMHvl, more pubertally born cells in
the VMHdm were activated in response to male-male interactions than no social
interaction (p=0.018, d=1.06). Moreover, the percent cell colocalization of BrdU-ir/Fos-ir
in the male-female interaction group was not significantly different compared to the
male-male interaction (p=0.306) or no social interaction (p=0.860) groups.
Quantitative analysis of Fos-ir cells revealed significant group differences in the
total number of Fos-ir cells in the VMHvl (F(2,35)=14.707, p<.000) (Fig 3.2D), with
male-male (p<0.000, d=1.80) (Fig 3.1H) and male-female social interactions (p=0.006,
d=2.80) (Fig 3.1I) significantly increasing VMHvl activation compared with no social
interaction (Fig 3.1J). Moreover, male-male and male-female social interactions
increased Fos-ir expression in the VMHvl to comparable levels (p=0.264). Group
differences in the number of Fos-ir cells were also detected in the VMHdm
(F(2,35)=6.902, p=0.003) (Fig 3.2E). Post-hoc analyses revealed that male-male social
interaction induced greater Fos-ir expression in the VMHdm than no social interaction
(p=0.003, d=1.39). The number of Fos-ir cells in the male-female social interaction
group did not significantly differ from the male-male interaction (p=0.808) or no social
interaction groups (p=0.076). These results suggest that social interactions with either a
male or female conspecific activates the adult male VMHvl, while only male-male social
interaction elicits high levels of activation in the VMHdm. Interestingly, male-female
social interaction also moderately increased VMHdm activation. Again, no group
differences were found in the total number of BrdU-ir cells in the VMHvl (F(2,35)=0.886,
p=0.421) (Fig 3.2F) and the VMHdm (F(2,35)=1.629, p=0.211) (Fig 3.2G).

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Pubertally born cells in the adult male VMHc and DG are not activated by social
interactions.
In the VMHc, double-labeled BrdU-ir and Fos-ir cells were observed in only 2
male mice, both of which were in the male-female social interaction group. ANOVA
detected no significant group differences in the proportion of pubertally born cells that
express Fos-ir (F(2,35)=3.096, p=0.058) (Fig 3.3A). However, ANOVA revealed group
differences in the number of Fos-ir cells in the VMHv (F(2,35)=6.902, p=0.003) (Fig
3.3B). Male-male (p<0.000, d=1.52) and male-female (p=0.041, d=1.56) social
interactions evoked greater VMHc activation compared to no social interactions. Malemale and male-female social interactions appeared to stimulate an equivalent Fos-ir
response in the VMHc (p=0.419). Once more, no group differences were found in the
total number of BrdU-ir cells in the VMHc (F(2,35)=1.459, p=0.246) (Fig 3.3C).
A small subset of pubertally born cells in the adult male DG expressed Fos-ir (Fig
3.4A). An ANOVA revealed no significant group differences in the percent colocalization
of BrdU-ir/Fos-ir in the DG (F(2,35)=1.154, p=0.327) (Fig 3.4B) or the total number of
BrdU-ir cells in the DG (F(2,35)=3.103, p=0.060) (Fig 3.4D). However, significant group
differences were detected in the total number of Fos-ir cells in the adult DG
(F(2,35)=10.104, p=0.000) (Fig 3.4C). Post-hoc analyses discovered male-male
(p=0.003, d=1.62) (Fig 3.4F) and male-female (p=0.001, d=1.38) (Fig 3.4E) interactions
significantly increased the number of Fos-ir cells in the adult male DG than no social
interaction (Fig 3.4G) with no significant differences between the male-male and malefemale interaction groups were detected in the total number of Fos-ir cells (p=1.00).

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Pubertally born cells in the MePD, VMH, and DG differentiate into mature neurons and
astrocytes.
Using triple-label immunofluorescence and confocal laser scanning
microscopy, we discovered that a subset of pubertally born cells in the adult MePD,
VMH, and DG differentiated into mature neurons (Fig 3.5A) and astrocytes (Fig 3.5B).
These pubertally born cells are estimated to be about 4-5 weeks old, as BrdU was
administered from P28-35 and tissue was collected at P63. The proportion of BrdU-ir
cells that colocalized either NeuN-ir or GFAP-ir in the MePD, VMH, and DG is depicted
in Fig 3.5C. Approximately 6% and 12% of pubertally born MePD cells colocalized with
NeuN-ir and GFAP-ir, respectively. The proportion of BrdU-ir cells that differentiated into
mature neurons or astrocytes was similar across the subdivisions of the VMH. About 67% of BrdU-ir cells colabeled with NeuN-ir in the VMHvl, VMHdm, and VMHc, while
approximately 7% of BrdU-ir cells colabeled with GFAP-ir in all subdivisions of the VMH.
In the adult male DG, most BrdU-ir cells colocalized with NeuN-ir (~75%) indicating that
most pubertally born cells in the DG differentiate into neurons. A small subset of
pubertally born cells in the DG also expressed GFAP (10%).
DISCUSSION
Here we discovered that a subset of pubertally born cells in the adult male MePD
was activated in response to social interactions with a male or female conspecific, while
a group of pubertally born cells in the VMHvl and VMHdm was activated in response to
male-male social interaction. Social interactions did not activate pubertally born cells in
the VMHc and DG. These findings demonstrate that cells generated during the first
week of puberty can be activated in adulthood as a consequence of social interaction in

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a brain region-specific manner. In addition, we found that overall neural activation, as
indicated by the presence of Fos-ir cells, was increased in all ROIs (i.e. MePD, VMHvl,
VMHdm, VMHc, and DG) in response to a male-male or male-female social interaction,
replicating findings from previous work (Kollack-Walker et al., 1995). Therefore, in the
MePD, the activation patterns of pubertally born cells mirrors that of overall neural
activation in response to male-male and male-female social interactions. However, this
is not the case for the subdivisions of the VMH or DG. Together these results indicate
pubertally born cells in the adult MePD participate in both male sexual behavior and
intermale aggression, while the activation of pubertally born cells in the VMHvl is only
critical for intermale aggression. We also examined the phenotype of pubertally born
cells in all ROIs, and that a portion of pubertally born cells in all regions differentiated
into neurons or astrocytes. Thus, some pubertally born cells are capable of maturing
into neurons or astrocytes in the adult male brain at ~4-5 weeks of age.
Social interactions activate pubertally born cells in the adult male MePD
Intermale aggression and sexual behavior activated about 0.4% and 0.5% of
BrdU-ir cells in the adult male MePD, respectively. To our knowledge, this is the first
study to demonstrate that pubertally born cells in the MePD respond to intermale
aggression in adulthood. Intermale aggression emerges around the time of puberty
(Barkley et al., 1977) when the pheromones of an unfamiliar conspecific male become
aversive(Brown, 1979). Since the MePD is involved in the processing of chemosensory
information, it is possible that new cells may be born during puberty to help mediate
intermale aggression in adulthood. In a recent study, investigators found that prepubertal, but not adult, knockdown of ERa-expressing neurons in the medial amygdala

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decreased aggressive behaviors in adult male mice (Sano et al., 2016; Sano et al.,
2013). Therefore, pubertal estrogen has an organizational effect on the medial
amygdala to facilitate the development of male aggressive behaviors. It is possible, in
male mice, that estrogen, aromatized from testosterone, acts to promote the
proliferation or survival of pubertally born cells that become incorporated into existing
neural circuits to regulate intermale aggression in adulthood. Very little is known
regarding the effects of estrogen on cytogenesis in the male MePD, but a study in adult
male meadow voles revealed that testosterone or estrogen, but not DHT, increased cell
proliferation in the medial amygdala (Fowler et al., 2003). However, we cannot rule out
the possibility that pubertally born cells express ER, which by estrogen acts on these
ER-expressing pubertally born cells to enhance survival. Furthermore, estrogen could
stimulate the migration of cells into the MePD, which by then these cells mature and
become incorporated into existing neural circuits.
In the current study, we identified about 0.5% active BrdU-ir cells in male mice in
response to sexual behavior. While the proportion activated pubertally born cells may
seem low, it should be noted that about 6% of pubertally born cells in the male mouse
MePD become mature neurons. Since Fos is traditionally used to examine activation of
neurons, it is not surprising to find a small percentage of BrdU-ir cells are activated in
the adult male MePD. However, a previous finding in our lab showed roughly 2% of
pubertally born cells in the adult MePD of male hamsters were activated by a sexual
encounter (Mohr et al., 2013). These two studies used different methods of BrdU
administration, which could also account for differences in pubertally born cells
activation: in the hamster study, BrdU was injected daily (i.p.) for three weeks (P28-49),

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while in the mouse study, BrdU was administered via drinking water for one week (P2849). It is possible that cells born during mid to late puberty have a more prominent role
in regulating male sexual behavior.
It is also important to consider the effects of environmental conditions on the
functional incorporation of pubertally born cells. Environmental enrichment (i.e. larger
cages, running wheel, cotton bedding, and cardboard igloo), after weaning, doubled the
percent colocalization of BrdU-ir/Fos-ir cells in the adult male hamster MePD in
response to sexual behavior (Mohr et al., 2013). It is now well established that
environmental conditions can influence neurogenesis as introducing adult rodents to
environmentally enriched conditions, exercise, and sexual experience enhances
neurogenesis in the hippocampus (Fan et al., 2014; Kempermann et al., 2002; Leuner
et al., 2010; Monteiro et al., 2014; Vivar et al., 2013), suggesting that these new
neurons may play an important functional role in the behaviors that promote their
survival. While the mice in the current study only received moderate environmental
enrichment (i.e. cardboard igloo and cotton bedding), they were socially enriched (i.e.
group housed). Social enrichment is also known to promote neurogenesis, particularly
in the hippocampus (Gheusi et al., 2009; Lu et al., 2003; Westenbroek et al., 2004).
Moreover, social isolation reduces hippocampal and amygdalar neurogenesis in female
prairie voles (Fowler et al., 2002; Lieberwirth et al., 2012). Therefore, since the subjects
in the current study received social enrichment and moderate environmental
enrichment, we believe it is unlikely that the lack of additional enrichment (i.e. running
wheel and larger cage) stunted the development of pubertally born cells in mice.

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The MePD is most commonly known for regulating social interactions with
conspecifics, however, it should be noted that the MePD has many additional functions
including regulating pubertal timing, feeding, and reward motivated behaviors (Hu et al.,
2016; King, 2006a; Li et al., 2015; Pardo-Bellver et al., 2012). Therefore, it is possible
that these behaviors or other types of social interactions may also activate a subset of
pubertally born cells in the MePD.
Male-male social interactions activate pubertally born cells in the VMHvl and VMHdm
A higher proportion of pubertally born cells in the VMHvl became activated in
response to intermale aggression than sexual behavior or no social interaction in adult
male mice. Past research has shown that Fos expression is increased in the male
VMHvl by an aggressive encounter with a conspecific male(Kollack-Walker et al., 1995).
In adult male mice, excitation of VMHvl neurons prompts males to attack all intruders,
including conspecific males and females and even inanimate objects (e.g. gloves)(Lin et
al., 2011). Due to these recent findings, the VMHvl is now recognized as a key regulator
of intermale aggression; however, the VMHvl is also involved in male sexual
behavior(Falkner et al., 2014b; Kollack-Walker et al., 1995; Yang et al., 2013), so it was
unexpected to not see a significant increase in pubertally born cell activation by sexual
behavior in adult male mice. These results could indicate the while the VMHvl are
known to regulate male sexual behavior, it is possible that pubertally born cells do not
respond to male sexual behavior, and they are more important for regulating intermale
aggression.
In the VMHdm, the increase in BrdU-ir/Fos-ir cell colocalization in response to
intermale aggression was quite surprising. Traditionally, the rodent VMHdm has been

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implicated in initiating defensive behaviors (i.e. freezing or avoidance) in response to
predators with minimal neural activation in response to social interactions with a
conspecific (Dielenberg et al., 2001; Perez-Gomez et al., 2015). Although, it is possible
that defensive behaviors initiated in response to interacting with an unfamiliar male
mouse increased neural activation in the VMHdm. Group housing (4-5 mice/cage) may
have inadvertently affected the development of aggressive and defensive behaviors in
the male subjects. Group housed male rodents rapidly form stable dominance
hierarchies usually comprised of one dominant male and several subordinate
males(Poole et al., 1973; So et al., 2015). In dominance hierarchies, the dominant male
mouse is typically the most aggressive, while the subordinate males display low levels
of aggression (Poole et al., 1973). Therefore, when introduced to the resident-intruder
paradigm, it is possible that the dominant male from each cage performed aggressively,
while subordinate males displayed a mixture of aggressive and a greater amount of
defensive behaviors in response to an unfamiliar intruder male. This was the case in
male hamsters; a previous study showed subordinate males displayed more defensive
behaviors than dominant males in response to an agonistic encounter, however,
interestingly, the number of Fos-ir cells in the VMH did not differ between groups (Pan
et al., 2010). The study did not examine the subdivisions of the VMH separately, thus,
group differences in the VMHdm could have been overshadowed by increased
activation in the VMHvl.
Similar to the MePD, the VMH, in general, is responsible for regulating many
behaviors including energy homeostasis and feeding(Gavini et al., 2016; King, 2006b).
Researchers have demonstrated that newly generated cells in the VMH of adult mice

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become leptin-responsive 4 weeks post-BrdU treatment, indicating that new born adult
cells in the VMH participate in energy balance regulation(Bless et al., 2016; Kokoeva et
al., 2005). Thus, it is possible that pubertally born cells in the adult VMH are more
sensitive to behaviors such as energy homeostasis.
Pubertally born cells in the VMHc and DG are not activated by social interactions
In the VMHc, there was very little, if any, activation of pubertally born cells in
adult male mice. Out of all the subjects, pubertally born cells in the VMHc of only two
male mice were activated by social interaction; therefore, statistically, there were no
group differences in pubertally born cell activation. Unfortunately, not much is known
regarding the function of the VMHc. In general, the VMHc is not included in
neuroanatomical studies due to its sparse neuronal population, but on occasion,
researchers will combine the VMHc with the VMHdm to create one entity (VMHdm/c).
Recently, a study discovered that activation the VMHdm/c increased defensive
behaviors in male mice (Wang et al., 2015), however, this behavioral effect could be
due to the activation of the VMHdm alone. Thus, it is not surprising that pubertally born
cells in the VMHc were not activated by social interactions.
Pubertally born cells in the DG were not activated in response to social
interactions. The DG has been shown to be involved in social behaviors(Ahern et al.,
2016; Ibi et al., 2008; Opendak et al., 2016), therefore, it was unexpected to discover
there was no increase in pubertally born cell activation in the DG in response to social
interactions. For instance, social isolation during adolescence significantly reduces
neurogenesis in the DG and increases aggression in male mice; however, treating
socially isolated mice with fluoxetine, an antidepressant, prevented these adverse

139

effects (Ibi et al., 2008). In addition, male rats that were group-housed after weaning
show enhanced Fos expression in response to a social encounter in the DG than males
raised in social isolation(Ahern et al., 2016). However, the DG of the hippocampus is a
complex brain regions and participates in a myriad of different behaviors(Curlik et al.,
2014; de Andrade et al., 2013; Duarte-Guterman et al., 2015), therefore, pubertally born
cells in the adult DG may serve a different purpose than participating in social
interactions with a male or female conspecific.
Although pubertally born cells in the VMHc and DG are not activated by social
interactions, the overall neural activation pattern, as assessed by Fos expression,
showed that both the VMHc and DG are activated by social interactions. This finding
suggests that neurons in the VMHc and DG are activated by social interactions;
however, they are born outside of the first week of puberty. It is possible that pubertally
born cells during mid to late puberty in the VMHc and DG are more responsive to social
interactions.
A subset of pubertally born cells in the MePD, VMH, and DG differentiate into mature
neurons or astrocytes
In the current study, we confirmed that a portion of pubertally born cells in the
adult mouse MePD, VMH, and DG differentiated into mature neurons or astrocytes,
replicating the results of the previous chapter. These results also corroborate the
previous findings that have shown some cells born in the postnatal born also
differentiate into mature neurons and glial cells (i.e. astrocytes, microglia, and
oligodendrocytes) (Bless et al., 2014; Fowler et al., 2003; Kokoeva et al., 2005; Mohr et
al., 2013; Saul et al., 2015). However, the fact remains that most of the pubertally born

140

cells in the MePD and VMH did not colocalize with NeuN or GFAP. A study in male
meadow voles found that approximately 35% of newly born cells in the amygdala of
adult male meadow voles colabeled with NG2 (Fowler et al., 2003). In rats, 50% of
pubertally born cells in the male rat amygdala colocalized with NG2 10 days after BrdU
administration (Saul et al., 2015). NG2 is traditionally known as a marker for
oligodendrocyte cell precursors and most NG2-expressing cells do mature into
oligodendrocytes, however studies have shown that a subset of NG2-expressing cells
can also go on to become astrocytes and neurons (Vigano et al., 2016). In male rats,
about 15% pubertally born cells expressed doublecortin (DCX), an immature neuron
marker, 4 and 8 weeks post-BrdU administration. Furthermore, a recent study in female
rats showed about 23% of pubertally born cells in the female rat anteroventral
periventricular nucleus expressed Iba1, a microglia marker (Mohr et al., 2016). Thus,
while a significant portion of pubertally born cells did not express NeuN or GFAP in the
mouse MePD and VMH, it is feasible that these pubertally born cells may be other cell
phenotypes such as oligodendrocytes, microglia or neurons in an immature state. It
should also be noted that not all astrocytes are known to express GFAP, so future
studies could utilize other astrocytic markers, such as Aldh1L1(Cahoy et al., 2008).
Conclusions
The current study demonstrates that pubertally born cells are functionally
integrated into three limbic structures in the adult mouse brain: the posterodorsal medial
amygdala, the ventromedial hypothalamus, and the dentate gyrus of the hippocampus.
Thus, a subset of cells born during the first week of puberty persist and survive into
adulthood to contribute to the maturation of social behaviors and some cells differentiate

141

into mature neurons or astrocytes. In addition, we established that the MePD, VMH, and
DG all have a role in sexual and aggressive behaviors. Documenting the functional
incorporation of pubertally born cells into the behavioral circuitry in mice sets the stage
to further explore and identify the neurochemical phenotype, connectivity, and function
of these pubertally born cells in future studies.

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Figure 3.1. Social interaction activates pubertally born cells in the adult male
MePD. (A) Representative photomicrograph displaying a colocalized BrdU-ir (green)
and Fos-ir (red) cell in a coronal section of an adult male MePD. (B) Quantitative
analysis revealed that more pubertally born cells are activated in response to a malemale and male-female interactions than no social interaction. (C) Male mice exposed to
a male or female mouse also have significantly more Fos-ir cells in the adult MePD than
male mice that received no social interaction. (D) No significant differences were
observed in the total number of BrdU-ir cells in the adult MePD between groups.
Representative photomicrographs of the MePD display the BrdU-ir and Fos-ir cells of
male mice in the (E) male-male, (F) male-female, and (G) no social interaction groups.
Data are represented as mean ± SEM. Low mag scale bar = 100 μm, high mag scale
bar = 10 μm.

143

Figure 3.2. Male-male social interactions activate pubertally born cells in the adult
VMHvl and VMHdm. (A) A representative image of a colocalized BrdU-ir and Fos-ir cell
in the adult male VMHvl. Quantitative analysis revealed that more pubertally born cells
were activated in the (B) VMHvl and (C)VMHdm in response to male-male interactions
than no social interaction. (D) In the VMHvl of male mice had more Fos-ir cells in
response to male-male and male-female interactions than no social interactions. (E) In
the VMHdm, male mice had more Fos-ir cells during the male-male social interaction
than no social interaction. No significant differences were found in the total number of
BrdU-ir cells between groups in the (F)VMHvl and (G)VMHdm. Representative
photomicrographs of the VMH show the BrdU-ir and Fos-ir cells of male mice in the (H)
male-male, (I) male-female, and (J) no social interaction groups. Data are represented
as mean ± SEM. Low mag scale bar = 100 μm, high mag scale bar = 10 μm.

144

Figure 3.3. Pubertally born cells in the VMHc are not activated by social
interactions. (A) An ANOVA revealed no significant group differences in the %
colocalization of BrdU- and Fos-ir cells in the VMHc. (B) The VMHc also showed greater
activation in response to a male-male and male-female social interaction compared to
no social interaction. (C) No significant differences were found between groups in the
total number of BrdU-ir cells. Data are represented as mean ± SEM.

145

Figure 3.4. Pubertally born cells in the dentate gyrus are not activated by social
interactions. (A) Representative image of a colocalized BrdU-ir and Fos-ir cells in the
adult male dentate gyrus. (B) No significant differences were detected between groups
in the % colocalization of BrdU-ir and Fos-ir. (C) Quantitative analysis revealed malemale and male-female social interactions increased the total number of Fos-ir cells in
the adult DG compared to no social interaction. (D) No significant differences were
found between groups in the total number of BrdU-ir cells. Representative
photomicrographs show BrdU-ir and Fos-ir cells in the adult DG of male mice in the (H)
male-male, (I) male-female, and (J) no social interaction groups. Data are represented
as mean ± SEM. Low mag scale bar = 100 μm, high mag scale bar = 10 μm.

146

Figure 3.5. Pubertally born cells in the adult MePD, VMH, and DG differentiate into
mature neurons and astrocytes. Representative photomicrographs of BrdU-ir cells in
the adult male MePD colocalized with (A) NeuN, a mature neuron marker, and (B)
GFAP, an astrocytic marker. Yellow arrows indicate cells that only express BrdU, white
arrows indicate BrdU-ir cells that express NeuN, and blue arrows indicate BrdU-ir cells
that express GFAP. Pie charts display the percent colocalization of all BrdU-ir cells with
either NeuN-ir cells or GFAP-ir cells in the (C) MePD, (D) VMHvl, (E), VMHdm, (F)
VMHc, and (G) DG. Data displayed show mean ± SEM.

147

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CONCLUSIONS
For several decades, the belief that the brain remained a static structure after the
perinatal period dominated the field of neuroscience (Kaplan, 2001). Even though
evidence of postnatal neurogenesis was first established in the 1960s (Altman et al.,
1965), the controversial topic was fiercely debated until the 1980s, when Fernando
Nottebohm made the groundbreaking discovery that new neurons were produced in the
adult brain of canaries (Goldman et al., 1983). However, even with this enormous
paradigm shift in our understanding of postnatal neurogenesis, skeptics touted this
finding was unique to the avian species and not transferable to the mammalian brain
(Rakic, 1985). This claim was, of course, debunked as several laboratories have
demonstrated that new neurons are added to the adult brain in mice, rats, tree shrews,
primates, and even humans (Cameron et al., 1993; Eriksson et al., 1998; Gould et al.,
1997; Gould et al., 1999; Kuhn et al., 1996). Today, it is widely accepted that new
neurons are added to the postnatal brain in the hippocampus and olfactory bulb;
meanwhile, the idea that new neurons are added outside of these canonical neurogenic
regions is still being disputed. The findings in this dissertation contribute to the body of
work investigating postnatal neurogenesis and gliogenesis by 1) highlighting the
potential mechanisms that regulate the addition of new neurons and glial cells in the
postnatal amygdala and hypothalamus and 2) examining the functionality of these new
cells.
Previous studies in adult rodents have found that several factors work together to
influence the proliferation and survival of adult born neurons, which include
environmental enrichment, stress and anxiety, physical exercise, and age(Kempermann

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et al., 2002; Kempermann et al., 1997; Mirescu et al., 2006; Vivar et al., 2013;
Westenbroek et al., 2004). It was discovered that gonadal hormones also participate in
adult neurogenesis with androgens and estrogens influencing cell proliferation and cell
survival in males and females(Fowler et al., 2003; Fowler et al., 2005; Hamson et al.,
2013; Ormerod et al., 2003, 2004; Spritzer et al., 2007; Tanapat et al., 1999). More
recently, a study demonstrated that gonadal hormones influence the addition of new
cells to the pubertal brain as well (Ahmed et al., 2008). During puberty, a time when the
brain is highly plastic (Ho et al., 2012; Saul et al., 2014; Staffend et al., 2014),
individuals experience gonadal maturation leading to a rise in circulating gonadal
hormones; this increase in gonadal hormone release (primarily testosterone in males
and estrogen in females) contributes to structural and functional changes that help
facilitate the emergence of adult sociosexual behaviors (Sisk et al., 2005). This is not
surprising, as many brain regions known to regulate sociosexual behaviors, such as the
posterodorsal medial amygdala (MePD) and ventromedial hypothalamus (VMH), are
populated with androgen receptors (ARs) and estrogen receptors (ERs)(Simerly et al.,
1990). A delicate balance between androgen and estrogen action in these brain regions
is believed to control sex-specific behaviors in adulthood. Given this information, the
overall aim of this dissertation was to examine the role of gonadal hormones on pubertal
cytogenesis in the MePD and VMH, and determine whether these new born cells
become functionally integrated into existing neural circuits that contribute to adult
sociosexual behaviors.
In the initial study examining pubertal cytogenesis outside of the classic
neurogenic zones, researchers discovered that there was a sex difference in the

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addition of pubertally born cells to sexually dimorphic brain regions, such as the medial
amygdala(Ahmed et al., 2008). The medial amygdala is a male-biased region (i.e.,
larger in males compared to females) which regulates social behaviors(Cooke et al.,
2003; Morris et al., 2008a; Morris et al., 2008b). It was recently discovered that adult
male rats had more pubertally born cells in the adult medial amygdala than females
(Ahmed et al., 2008). The fact that this sex difference was hormone-dependent
suggests that gonadal hormones may regulate the addition of new cells during puberty
to establish or maintain sex differences seen in the adult medial amygdala. Thus, our
first aim was to examine sex differences in pubertal cytogenesis in the MePD and
ventrolateral subdivision of the VMH (VMHvl), both male-biased sexually dimorphic
brain regions, using the mouse model. We found that adult male mice had significantly
more pubertally born cells than females in the MePD and VMHvl, while no sex
differences were observed in sexually monomorphic structures (i.e. dorsomedial and
central subdivision of the VMH (VMHdm and VMHc, respectively) and dentate gyrus
(DG)). Identifying sex differences in the MePD and VMHvl prompted us to further
investigate whether testosterone, the primary male gonadal hormone, increase the
number of pubertally born cells in these male-biased brain regions.
In the current dissertation, we concluded that testosterone acting via ARs
increased the number of pubertally born cells in the MePD of adult male mice. In the
adult MePD, adult testosterone alone did not eliminate sex differences in the number of
pubertally born cells, but providing both pubertal and adult testosterone significantly
increased the amount of pubertally born cells in males and females. In addition, we
discovered that the presence of functional ARs in male mice was required to increase

157

the number of pubertally born cells. More in-depth analysis revealed that it was actually
pubertal AR activity that promoted the increase in pubertally born cells in the adult male
MePD. While we have determined pubertal testosterone acts via an AR-dependent
mechanism to stimulate the proliferation and/or survival of pubertally born cells in the
adult MePD, we are unable to exclude the contributions of ERs to increasing the
number of pubertally born cells in the adult male MePD. Because blocking ARs in
adulthood did not lead to a decrease in pubertally born cells, it is possible that adult
testosterone is aromatized into estrogen and acts through ERs to promote the survival
of pubertally born cells. A previous study in adult male rats revealed that ARs and ERs
work together to masculinize the volume and neuronal soma size of the medial
amygdala. Thus, testosterone may act on both ARs and ERs to promote the
accumulation of pubertally born cells in the adult male MePD. A critical next step will be
to block ER activity during puberty and/or adulthood in male mice, using an ERantagonist, to examine the role of ERs on promoting the survival of pubertally born cells
in the adult male MePD. As previously mentioned, pubertal and adult testosterone
treatment also significantly increased the number of pubertally born cells in the adult
female MePD. Yet, it remains unclear whether each metabolite of testosterone (i.e.
estrogen and DHT, a potent androgen) individually contributes to the proliferation and/or
survival of pubertally born cells in the MePD. Thus, future studies are needed to
determine how gonadal hormones influence pubertal cytogenesis in the adult MePD of
females by blocking ARs or ERs during puberty and/or adulthood. These results will
further our understanding of how sex differences in the number of pubertally born cells
are produced and maintained in the adult MePD.

158

In the VMHvl, adult testosterone increased the number of pubertally born cells in
adult male and female mice, regardless of pubertal testosterone. Moreover, blocking
pubertal and adult ARs did not reduce pubertally born cell density in the adult male
VMHvl. Since adult testosterone treatment increased pubertally born cell density
independent of AR activity, it is possible that testosterone acts via an ER-dependent
mechanism to enhance the survival of pubertally born cells in the adult male VMHvl.
However, since gonadally intact females are already exposed to high levels of estrogen
at the onset of puberty, it seems unlikely that adult testosterone is acting through ERs to
increase pubertally born cell density in the adult female VMHvl. On the other hand, it is
possible that DHT, a metabolite of testosterone, acts via ARs in the adult VMHvl to
promote the survival of pubertally born cells in females. This pattern of findings would
suggest that gonadal hormones differentially effect the survival of pubertally born cells
in the adult VMHvl between male and female mice. A previous study in adult female rats
discovered that estrogen increased cell proliferation in the DG, however, in adult male
meadow voles, treatment with estrogen did not enhance cell proliferation in the DG.
Given these results, it is crucial to explore the differential effects of estrogen and DHT
on pubertal cytogenesis in male and female mice. It should be noted that gonadal
hormones prior to puberty may also organize the male and female brain leading to sex
differences in the sensitivity to pubertal and/or adult hormones. Therefore, it will also be
important to determine whether manipulating gonadal hormones during the perinatal
period (e.g. gonadectomy vs. gonadally intact) affects hormone sensitivity in the brains
of males and females during puberty and adulthood and leads to sex differences in
pubertally born cells in the adult MePD and VMHvl.

159

In the current line of experiments, we established that gonadal hormones
influence pubertal cytogenesis in the MePD and VMHvl. However, due to the
experimental design used in these studies, we can only examine how gonadal
hormones contribute to the survival of these pubertally born cells into adulthood. In this
case, cell survival is defined as the total number of pubertally born cells present in the
adult MePD and VMHvl at the time of sacrifice. Because all subjects were treated with
BrdU during puberty and then sacrificed in adulthood, we are unable comment on how
gonadal hormones affect other mechanisms that may contribute to generating sex
differences in pubertally born cell number in adulthood such as cell proliferation, cell
migration, cell death, and cell differentiation. Several studies have demonstrated that
gonadal hormones can affect cell proliferation in brain regions like the DG and
amygdala in adulthood. However, it is currently unknown how gonadal hormones
influence cell proliferation in the pubertal brain. In order to parse out the differential role
of gonadal hormones on cell proliferation and cell survival in the adult MePD, a timecourse study must be conducted by administering BrdU during puberty and then
examining the number of pubertally born cells after varying time points (e.g. 24 hours, 2
days, 7 days, 14 days, 21 days, and 56 days). An examination of pubertally born cell
number more proximally following BrdU administration (30 minutes, 1 hour, 2 hours, 6
hours, 10 hours) may provide insight into how gonadal hormones influence cell
migration. Although, the migratory pathway of pubertally born cells to brain regions such
as the MePD and VMH are not known, recent studies have identified the third ventricle
and circumventricular organs as possible neurogenic niches where progenitor cells may

160

proliferate and reside. Therefore, it is possible that pubertally born cells originate in
these neurogenic niches rather than the subventricular zone or subgranular zone.
Gonadal hormones are also known to shape sex differences in the adult brain by
mediating cell death. Within the anteroventral periventricular nucleus (AVPV), a femalebiased brain region in which adult female rodents have a larger volume and more
neurons than males, the sex difference is largely regulated by cell death through
apoptotic testosterone signaling among males. Interestingly, by deleting genes that
promote cell death (Bax gene), the sex difference in AVPV neuron number is eliminated
in adult mice. It is therefore possible that sex differences in pubertally born cell number
in the adult MePD and VMHvl are a result of increased cell death in females. Using
transgenic mice to disable the Bax gene, future studies can administer BrdU during
puberty and examine the number of pubertally born cells in the MePD and VMHvl to
determine whether Bax-mediated cell death is required to establish sex differences in
pubertally born cell number in adulthood. Understanding the mechanism by which sex
differences in the number of pubertally born cells are established will help to provide
more insight on how gonadal hormones work to shape the adolescent brain to promote
sex differences in structure and function in adulthood.
Cell differentiation may also act to promote sex differences within the brain. We
discovered that pubertal ARs enhance the number of pubertally born neurons in the
adult male MePD, but not the VMHvl, while pubertal and adult ARs did not influence the
number of pubertally born astrocytes in the MePD or VMHvl. Thus, in the adult male
MePD, testosterone may act via ARs to promote the differentiation of pubertally born
cells into a neuronal phenotype. However, it is also possible that blocking ARs during

161

puberty stimulates apoptosis of pubertally born neuron. A time-course study would be
necessary to determine whether fewer pubertally born cells differentiate into neurons or
more pubertally born neurons die as a result of blocking pubertal ARs. Currently, it is
unclear how the presence of androgens works to increase the number of pubertally born
neurons in the MePD. It is possible that androgens may act indirectly to enhance the
genesis and/or survival of pubertally born neurons. For example, astrocytes in the
MePD have been shown to express ARs (Johnson et al., 2012); therefore, androgen
could act on ARs expressed in astrocytes to stimulate the release of neurotrophic
factors that can contribute to the survival of pubertally born neurons in the MePD.
However, if ARs are present on pubertally born cells themselves, it is possible that
androgens may act directly on pubertally born cells to drive either their genesis and/or
survival of pubertally born neurons that may go on to influence adult social behaviors.
Thus, another critical next step will be to investigate if and when pubertally born cells in
the MePD begin to express ARs. Furthermore, it was rather surprising to find that
blocking ARs during puberty or adulthood did not affect the number of pubertally born
astrocytes in the adult MePD, as a previous study found the addition of new astrocytes
during puberty in male rats is dependent on the presence of functional ARs (Johnson et
al., 2013). However, since we only evaluated astrocytes born during the first week of
puberty, it is possible that the survival of astrocytes born during mid or later puberty
depend on the functional androgen receptors, contributing to sex differences in
astrocyte number in the adult MePD.
Lastly, in this dissertation, we discovered that a subset of pubertally born cells in
the mouse MePD, VMHvl, and VMHdm are functionally incorporated into existing neural

162

circuits and participate in adult sociosexual behaviors. This is the second study to
demonstrate that pubertally born cells outside of the canonical neurogenic zones
become functionally integrated into the adult brain. A previous study in male hamsters
found that a group of pubertally born cells in the amygdala and hypothalamus are
activated in response to a sexual encounter (Mohr et al., 2013). In male mice, we
discovered that social interaction with a male or female conspecific activated pubertally
born cells in the MePD, while social interactions with a male activated pubertally born
cells in the VMHvl and VMHdm. Although most pubertally born cells were not activated
by a male or female social encounter in adult male mice, it is possible that these
pubertally born cells may be activated by other behaviors other than male and female
social interactions. Thus, future studies could examine the response of pubertally born
cells to other stimuli, such as administering glucose or leptin to enhance the Fos in the
VMH, or perhaps examining the Fos response in the MePD to anxiety-inducing tasks or
a predator scent.
Another important next step will be to examine whether gonadal hormones
actually play an indirect role in regulating pubertal cytogenesis. It is possible that
pubertal testosterone elicits behaviors that, in turn, influence pubertal cytogenesis. For
instance, studies have shown that male mice that are socially isolated have reduced
neurogenesis in the hippocampus compared to males that are group housed despite the
males being gonadally intact (Lieberwirth et al., 2012; Spritzer et al., 2011). Thus, social
interaction may facilitate the preservation of these newly generated cells. A study also
found that testosterone treatment increased self-grooming in male mice (Achiraman et
al., 2014). In early development, high levels of maternal licking and grooming resulted in

163

increased neuronal survival in the hippocampus of offspring compared to those that
received low levels of maternal care (Bredy et al., 2003). Therefore, it is possible that an
increase in self-grooming during adulthood, as a result of testosterone treatment, may
have a similar effect which leads to an increase in cell number in the brain. Follow-up
investigations should examine how gonadal hormones control behaviors that may
facilitate the changes in proliferation and/or survival of pubertally born cells in the brain.
In many mental health disorders, such as depression, disruption in social
behaviors is a core symptom (Segrin, 2000). Interestingly, the prevalence of mental
health disorders increases during puberty with females being twice more likely to
develop these disorders compared to males (Paus et al., 2008). Therefore, another
important future consideration will be to examine how cytogenesis in brain regions
involved in regulating social behaviors differs between males and females in animal
models of mental illness. In adulthood, decreased hippocampal neurogenesis has been
observed in animal models of depression and anxiety (Hill et al., 2015; Snyder et al.,
2011). Antidepressant drugs, such as fluoxetine, increase neurogenesis in the
hippocampus, which may work to alleviate depressive symptoms (Klomp et al., 2014).
Interestingly, a study found that fluoxetine affected adult male and female rodents
differently, increasing cell proliferation in males, but not females (Hodes et al., 2009).
However, these effects were not observed during puberty, as males and females
treated with saline or fluoxetine both had high levels of cell proliferation (Hodes et al.,
2009). These results suggest that antidepressant drugs have an age-specific and sexspecific effect. It is clear that antidepressant medications, one of the most popular
treatment options utilized by clinicians today, may not be appropriate for all age groups

164

or sexes. Therefore, it is essential that we further our understanding of the aberrant
neurological changes that can occur in both sexes during adolescence as well as
adulthood in order to develop more effective therapeutic options to treat mental health
disorders.
In conclusion, the current dissertation has provided evidence that pubertal
testosterone acts via ARs to increase the number of pubertally born cells in the male
MePD, but not VMHvl. Moreover, pubertal ARs are required to increase the number of
pubertally born neurons, but not astrocytes in the adult male MePD. We have also
confirmed that a subset of pubertally born cells in the MePD and VMHvl are activated in
response to social interactions. While the current results contribute important insights on
the role of gonadal hormones on pubertally born cells in the MePD and VMHvl, there
are still many unanswered questions. It will be important for future projects to assess
the unique contributions of new neurons and glia depending on a distinct pubertal
period (i.e. early, mid, or late puberty). The development of the pubertal brain is a
gradual process, and it is possible that new cells born during discrete pubertal time
periods may have specific functions in the adult brain. In addition, recent studies have
shown that sexual differentiation of the central nervous system involves both peripheral
and central ARs (Swift-Gallant et al., 2012). Therefore, it will be important to distinguish
the contributions of peripheral ARs from central ARs on promoting pubertal cytogenesis;
and follow-up studies may aim to control the temporal and spatial specificity of AR
expression in male mice by reducing AR activity starting at puberty and only in the
nervous system.

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This dissertation contributes evidence that the adolescent brain gains new
neurons and astrocytes, and furthermore, cells born during puberty are activated by
sociosexual behaviors in adulthood. In male mice, pubertal androgens play an integral
role in enhancing the number of pubertally born neurons in the adult MePD. Based on
the understanding that pubertal neurogenesis and gliogenesis in the amygdala and
hypothalamus occur across at least three different species (i.e. rats, hamsters, and
mice)(Ahmed et al., 2008; Mohr et al., 2013; Saul et al., 2015; Staffend et al., 2014), it is
likely that this phenomenon exists in other species as well, including humans.
Therefore, it is necessary to work toward a better understanding of the mechanisms
regulating the proliferation and survival of pubertally born cells as we also begin to
grasp the functional role of these cells in adolescent development. During puberty and
adolescence, the prevalence of mental health disorders substantially increases (Paus et
al., 2008), and research has shown that stress, anxiety, and depression are associated
with decreased neurogenesis and gliogenesis (de Andrade et al., 2013; Hill et al., 2015;
Kreisel et al., 2014; Rajkowska et al., 2007; Saaltink et al., 2014). A full understanding
of atypical development is currently hampered by a dearth of knowledge about the
neurological changes in the adolescent brain associated with healthy or typical social
maturation. Thus, continued research on pubertal neurogenesis and gliogenesis will
have profound ramifications for understanding healthy social development as well as
identifying new targets for intervention when adolescent development goes awry.

166

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