University of Southern California Dissertations and Theses

Gonadal steroid hormones promote neuroplasticity in models of health and disease

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Gonadal steroid hormones promote neuroplasticity in models of health and disease

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Content GONADAL STEROID HORMONES PROMOTE NEUROPLASTICITY IN
MODELS OF HEALTH AND DISEASE

by
Eleni Antzoulatos

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)

December 2009

Copyright 2009 Eleni Antzoulatos
ii
Acknowledgements
In the five years that I spent in the Neuroscience Graduate Program, the
person to whom I owe the most is my advisor, Ruth Wood. I entered the graduate
program with a lot of experience in science but no experience in being a scientist.
Over the years, she has taught me to become a scientist. She taught me to plan
and execute experiments and to write and present my findings. She encouraged
me through disappointing results, celebrated milestones with me, and provided the
support and guidance that have helped me to become a competent scientist. She
has also become my friend.
I am also grateful to the members of my dissertation committee: Judy
Garner, Michael Jakowec, Joel Schechter, Larry Swanson, and Alan Watts. I
conducted my first experiments as a graduate student in collaboration with Dr.
Garner. Dr. Jakowec was a tremendous help to me as I switched gears in my
project, and always provided me with his optimistic and positive outlook on life
and science. Dr. Schechter was an excellent neighbor and confidant, not to
mention a steady supplier of red licorice. Finally, Drs. Swanson and Watts were
always excited to hear about my science as well as my plans in the neuroscience
graduate program and in my life. I could not have picked a better committee.
Finally, I wouldn’t be writing these acknowledgements without the
support of my family and friends. My friends have shared happiness, defeat,
frustration, and celebration with me over the years, some from far away, others
from right next door. My family has done the same. I would like to thank my
iii
parents for their support and love, my brother and sister-in-law for keeping me
grounded and politically aware, and my dog Tucker who had the most amazing
ability to relieve stress just by coming in to a room. Last and far from least, I
would like to thank my husband Christopher Aultman. We have stood by each
other for over a decade and his unwavering support means the world to me. He
has always known when to be critical, when to boost my confidence, and how to
stop me from procrastinating. He might disagree with the latter, but I would still
be in the planning stages my thesis if it wasn’t for him. He is my best friend, my
confidant, and the love of my life. I am excited by the thought of sharing many
more decades with him.

iv
Table of Contents

Acknowledgements

ii
List of Figures

v
Abstract

vi
Chapter 1 – Introduction

1
Chapter 2 – Cell Proliferation and Survival in the Mating Circuit of the
Male Syrian Hamster: Effects of Testosterone and Sexual Behavior

25
Chapter 3 – Sex Differences in the MPTP Mouse Model of Parkinson’s
Disease

48
Chapter 4 – Testosterone and Neuroplasticity in the Male MPTP-Lesioned
Mouse

70
Conclusion

98
References

106

v
List of Figures

Figure 2.1. Photographs of BrdU-labeled cells in MeP (A) and MPOA
(B).

36
Figure 2.2. The effects of testosterone on cell proliferation and survival in
the mating circuitry.

38
Figure 2.3. The effects of reproductive activity on cell proliferation and
survival in MeP and MPOA.

40
Figure 3.1. Rotarod performance in male (black) and female (white) mice.

59
Figure 3.2. Pole test performance in male (black bars) and female (white
bars) mice.

60
Figure 3.3. Stride length in male (black bars) and female (white bars)
mice.

61
Figure 3.4. Photomicrographs of TH-positive neurons in SNC of saline-
(A) and MPTP-treated (B) mice.

64
Figure 4.1. Behavioral Measures. 83

Figure 4.2. TH-staining in the dorsolateral striatum. 85

Figure 4.3. Golgi stained MSN’s.

88

Figure 4.4. Neuronal morphology totals.

90
Figure 4.5 Branch order totals. 91

vi
Abstract
Gonadal steroid hormones exert numerous effects on the brain and
behavior. Hormones are responsible for sexual differentiation during
development. In adulthood, hormones mediate sex-specific effects on behavior
and corresponding neural circuits. Hormones have been traditionally investigated
with respect to reproduction and the neural circuits that drive mating behavior.
However, hormone action is not restricted to the control of reproduction. In the
current dissertation, I investigated the role of gonadal hormones in three chapters
using two models. First, I investigated the role of testosterone on cell
proliferation and survival in the mating circuitry of the adult male hamsters
(Chapter 2). Testosterone exerted region-specific effects in the mating circuitry to
enhance cell proliferation in some regions, but not others. In addition, neither
testosterone nor mating experience increased cell survival in the mating circuit.
For Chapters 3-4, I focused on the nigrostriatal pathway. The nigrostriatal
pathway is involved in the circuits that coordinate voluntary movement, and is not
directly related to reproduction. Nonetheless, this pathway is sensitive to gonadal
hormones. In Chapter 3, I examined sex differences in nigrostriatal anatomy and
behavior in rodents under normal physiologic conditions and in the 1-methyl-4-
phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal degeneration
that mimics Parkinson’s disease. In Parkinson’s disease, both the incidence and
susceptibility are higher in men than in women. Gonadal steroids are thought to
partially underlie this difference. After MPTP, male mice had increased motor
vii
deficits compared with females. This supports previous findings showing the
increased susceptibility of males to MPTP. However, MPTP did not deplete
neurons in the nigrostriatal pathway. In Chapter 4, I used the MPTP mouse model
to determine whether testosterone acts as neuroprotectant in the nigrostriatal
pathway. The ability of gonadal hormones to guard the brain against insult is a
form of neuroplasticity with tremendous clinical implications. This process has
been well-studied with respect to estrogen. I found that testosterone does not
attenuate MPTP-induced neurotoxicity. These results suggest that testosterone is
not neuroprotective in the male nigrostriatal system. Taken together, the three
experiments in this thesis address the diverse roles of gonadal hormones and
establish novel, nontraditional roles for gonadal steroids in the brain.
1
Chapter 1
INTRODUCTION

The focus of my dissertation research has been to explore the actions of
gonadal steroid hormones on the brain. Hormones are traditionally recognized for
their role in reproduction. They establish sex differences during development and
act throughout puberty and adulthood to drive mating behavior. However, many
areas of the brain that are not related to reproduction are sexually dimorphic.
Hormones exert more subtle, modulatory effects on these nonreproductive brain
regions and behaviors. In addition, many hormone-sensitive brain regions are
substrates for neurological disease. Not surprisingly, hormones alter disease
susceptibility and progression.
Collectively, the experiments in this thesis address the diversity of
hormone action in the brain. My experiments evaluate traditional and more
recently-established roles for gonadal steroids. Chapter 2 looks at the effects of
testosterone on cell proliferation and survival in the mating circuitry of the adult
male hamster. Chapters 3 and 4 evaluate sex differences and the role of
testosterone in the basal ganglia circuits for movement under normal conditions
and in a model for neurodegenerative disease.

2
Steroid Hormones
Hormones are a class of chemical substances that are produced by cells in
one part of the body and that act on target tissues that are sometimes very far from
the source. Unlike protein hormones which are synthesized and stored, steroid
hormones are lipophilic and easily cross cell membranes (Nissey and Whitehead,
2001). Thus, steroids are synthesized when needed and immediately released into
the bloodstream.
The two major endocrine glands for steroid hormone production are the
adrenal glands and the gonads (reviewed in Nelson, 1995). The major hormone
product of the adrenal glands is cortisol, which is important for the inflammatory
and immune systems, and the stress response. Gonadal steroids in mammals
include testosterone, estrogen, and progesterone. They have an essential role in
the production of gametes and in the regulation of reproduction (Baum, 2002;
McCarthy and Becker, 2002). Gonadal hormone production is under the direct
control of the pituitary gland which synthesizes and releases gonadotropins,
protein hormones that travel to the gonad and stimulate the production of
testosterone in males (Baum, 2002), and estrogen and progesterone in females
(McCarthy and Becker, 2002).
Gonadal steroids are synthesized from the precursor cholesterol. In males,
testosterone is synthesized by the Leydig cells of the testes (Steinberger, et al.,
1970). Estrogen is produced in females by the granulosa and theca cells of the
ovary (McNatty, et al., 1979). Although testosterone is most often associated
3
with males and estrogen with females, there is considerable overlap. Testosterone
is a necessary precursor for estrogen synthesis (Naftolin and MacLusky, 1984;
Naftolin, et al., 1975). Aromatase, the enzyme which converts testosterone to
estrogen, is present in both males and females (Roselli, et al., 1998; Roselli, et al.,
1985; Wagner and Morrell, 1996). Thus, estrogen and testosterone are found in
both sexes. However, males have much higher levels of testosterone while
estrogen levels are higher in females.

The body and brain as targets of gonadal steroids
Gonadal steroid hormones have numerous effects across the lifespan.
Hormones exert organizational effects during development and activational
effects throughout adulthood (reviewed in Arnold and Breedlove, 1985). The
organizational effects of hormones are permanent and do not depend on continued
exposure to gonadal steroids. By contrast, activational effects are transient, and
occur in the presence of hormones. The experiments in this thesis focus on
hormone action in adult rodents. However, the ability of hormones to act
appropriately in adulthood assumes proper exposure to hormones during
development. In order to explain the rationale for the experiments in this thesis, it
is important to begin with a discussion of the role of hormones in establishing sex
differences and the modulatory role that hormones play during adulthood.
Hormones exert their first effects on the developing reproductive system.
In particular, the organizing effects of gonadal steroids are directly responsible for
4
sexual differentiation (McCarthy, 2008; Wilson and Davies, 2007; Wilson, 1978).
In mammals this is largely due to the presence of testosterone, which induces
differentiation along a masculine phenotype and suppresses the feminine
phenotype (Phoenix, et al., 1959). The classic systemic outcome of organization
is the induction of masculine genitalia (i.e. penis and testes) in the male (Jost, et
al., 1973). By contrast, the ovary is quiescent during development and the female
reproductive organs develop in the absence of gonadal steroids.
As it relates to my thesis, the brain is an important target for gonadal
steroids. Moreover, the same mechanisms for organization that occur in the body
also occur in the brain. In the developing brain, the most pronounced sex
differences occur in ventral forebrain regions that regulate reproduction. These
include the medial preoptic area (MPOA; Gorski, et al., 1978; MPOA; Raisman
and Field, 1973) (MPOA) and the anteroventral periventricular nucleus (Simerly,
1998; Simerly, et al., 1997; Sumida, et al., 1993). However, sex differences are
not restricted to brain regions that control reproduction. For example, in Chapter
3 I reported sex differences in the basal ganglia circuits for voluntary movement.
Organizational effects can only occur during a specific developmental
phase where the brain is sensitive to the masculinizing effects of testosterone
(Diaz, et al., 1995; MacLusky and Naftolin, 1981; McCarthy, 2008). The
biochemical events which underlie this critical period of sensitivity are unknown.
However, if testosterone is not available during the critical period, hormone-
sensitive areas of the brain will not masculinize (Breedlove and Arnold, 1983;
5
Pfaff, 1966) and males will not express male-typical behaviors in adulthood, even
when treated with testosterone (Hendricks, 1969). In fact, males will express
female sex behavior if treated with estrogen and progesterone (Gerall, et al., 1967;
Whalen and Edwards, 1966).
After organization is completed, gonadal steroid levels remain low
throughout adolescence. At puberty, hormone production increases and
stimulates the development of secondary sexual characteristics (Ojeda and
Skinner, 2006; Sisk and Zehr, 2005). In addition, gonadal steroids refine
hormone-sensitive circuits in the brain and establish the adult expression of
hormone-dependent social behaviors including mating (Schulz and Sisk, 2006).
Throughout adult life, hormones exert activational effects on sexually-
differentiated brain regions to mediate sex-specific effects on behavior (Arnold
and Breedlove, 1985). The traditional role for activational hormones involves
binding to steroid receptors to elicit changes in neuronal structure, physiology,
and behavior (Garcia-Segura, et al., 1994). Not surprisingly, steroid hormone
receptors are abundant in brain regions that are organized during development
and/or crucial for reproduction (Simerly, et al., 1990; Wood, et al., 1992). Thus,
the majority of work has been done in these circuits.
Both males and females exhibit hormone-dependent modulation of
neuronal structure (Cooke and Woolley, 2005; McEwen, et al., 1991; Parducz, et
al., 2005). In females, natural fluctuations in hormones over the estrous cycle
produce a dramatic structural remodeling of dendrites and synapses in the
6
ventromedial hypothalamus (VMH), a region that drives female sexual behavior
(Calizo and Flanagan-Cato, 2000). In males, testosterone enhances dendritic
morphology and somal size in key nuclei for mating behavior that include the
MPOA (Commins and Yahr, 1984) and the posterior medial amygdala (MeP;
Gomez and Newman, 1991).
More recently, activational effects of hormones have been observed
outside of the mating circuitry. Some regions, like the CA1 region of the
hippocampus, are similar to the mating circuitry in that they contain steroid
hormone receptors. In CA1 pyramidal cells, gonadal hormones increase spine
density and the density of spine synapses (Leranth, et al., 2004; Li, et al., 2004;
Yankova, et al., 2001). Spine synapses are also sensitive to hormones throughout
the cerebral cortex, which contains a more modest expression of steroid hormone
receptors (Hajszan, et al., 2007; Medosch and Diamond, 1982; Munoz-Cueto, et
al., 1990). In Chapter 4, I report that testosterone increases distal dendritic length
in the dorsolateral striatum, which does not contain many hormone receptors and
does not play a role in reproduction.
The experiments of this thesis address both organizational and activational
effects of gonadal hormones. In Chapter 2, I focus on the activational effects of
testosterone in MeP and MPOA. Although MeP and MPOA have been well-
studied for hormone effects on behavior and neuronal morphology, Chapter 2
addresses the relatively novel concept that hormones promote the birth and
survival of new neurons in these regions. Chapters 3 and 4 take a different
7
approach. Sex differences are well-investigated with respect to hormone-
dependent behaviors such as mating. In Chapter 3, I apply these traditional
concepts to the basal ganglia, a set of brain regions that are not typically
considered to be hormone-sensitive. In Chapter 4, I explore the traditional roles
of activational hormones on behavior and neuronal morphology, but focus on the
dorsolateral striatum, a major component of the basal ganglia.

Mechanisms for steroid hormone action
The observation that gonadal steroid hormones can have activational
effects in regions that are relatively devoid of steroid hormone receptors raises the
question, how are hormones acting in brain regions without hormone receptors?
The simplest answer reflects the complexity of hormone action in the brain.
Hormones act through multiple receptor subtypes and through multiple
intracellular pathways to exert activational effects.
In the classical mechanism for steroid action, hormones modulate gene
expression through their interaction with hormone receptors that act as ligand-
dependent transcription factors (reviewed in Losel and Wehling, 2003). The
hydrophobic steroid hormones easily diffuse through the cell membrane and bind
to hormone receptors in the cytoplasm or in the nucleus. After undergoing a
conformational change that exposes a DNA binding site, the hormone-receptor
complex binds DNA and stimulates or inhibits transcription. As a result, target
tissues produce a different subset of proteins, altering their physiology in response
8
to the steroid hormone. Not unexpectedly, a relative long time course is required
to observe the effects of hormones acting through classical pathways.
The involvement of protein translation in the classical pathway is
consistent with long-lasting changes in steroid-responsive brain regions. This is
well-demonstrated in the mating circuitry of adult male rodents, where circulating
gonadal hormones facilitate reproduction. After castration, males do not
immediately show impairments in mating behavior, even though endogenous
gonadal steroids are eliminated (Wood and Newman, 1995a). Instead, mating
behavior declines slowly over a period of weeks (Park, et al., 2004; Siegel,
1985b). The opposite is also true. Hormone replacement to castrated rodents will
restore reproductive behavior, but animals will not begin to express mating
behaviors until hormones are present over a period of weeks (Noble and Alsum,
1975; Sachs and Meisel, 1988; Siegel, 1985b). These long-lasting changes to
behavior are paralleled by long-term structural rearrangements to neurons in the
mating circuitry that include changes in dendritic branching, length, and soma
size (Commins and Yahr, 1984; Gomez and Newman, 1991).
Some actions of steroid hormones cannot be explained by the classical
model, including effects that are very rapid and/or not affected by inhibitors of
transcription or translation (Moore and Evans, 1999). These nonclassical effects
are often observed in brain areas where the distribution of classical hormone
receptors is minimal. Chapters 3-4 examine sex differences and testosterone-
driven plasticity in the dorsolateral striatum. Hormone-driven changes in the
9
striatum are not likely to arise from classical effects on AR and ER because both
receptor subtypes are practically absent in this region.
Unlike classical effects, in which hormone action is mediated by a single
genomic mechanism, nonclassical effects are likely mediated by a variety of
intracellular pathways (Falkenstein, et al., 2000). Gonadal steroids exert
nonclassical effects by binding to receptors that activate second messengers and
subsequently initiate rapid signaling cascades (Losel, et al., 2003). Nonclassical
receptors for steroid hormones may include effects at the plasma membrane,
either through putative membrane-bound receptors (Pappas, et al., 1995; Ramirez,
et al., 1996) or through modulatory effects on other neurotransmitter receptors
such as GABA receptors (Henderson, 2007). Finally, it may also be that AR and
ER, which are traditionally associated with the classical pathway, initiate
intracellular signaling cascades that are separate from their effects on transcription
(Kousteni, et al., 2001).
Although it is useful to classify hormone mechanisms as classical and
nonclassical, there is considerable overlap between the two categories.
Nonclassical effects are also referred to as non-genomic effects, because steroid
hormones do not act directly on DNA through the classical, genomic pathway.
However, the downstream consequences of nonclassical steroid hormone action
may include changes in transcription activity mediated by second messenger
cascades (Simoncini and Genazzani, 2003; Vasudevan and Pfaff, 2008). For
example, rapid estrogen signaling increases the formation of cyclic AMP (Gu, et
10
al., 1999; Gu and Moss, 1996). In turn, cyclic AMP recruits DNA binding
proteins and increases transcription independently of the estrogen receptor. Not
surprisingly, while the nonclassical actions of gonadal steroids initially described
the rapid effects of steroid hormones on cellular physiology (i.e. seconds to
minutes), it is now recognized that nonclassical effects can occur over a much
longer timeframe.
The experiments in this thesis address both classical and nonclassical
mechanisms for steroid hormone action. In Chapter 2, the effects of testosterone
on the birth and survival of new cells are described in MeP and MPOA, key
nuclei in the circuits for reproduction. As the mating circuitry contains an
abundance of steroid hormone receptors, testosterone can act via classical or
nonclassical pathways. By contrast, Chapters 3 and 4 focus on the dorsolateral
striatum, which is almost devoid of classical hormone receptors. Thus, hormones
must be acting through nonclassical mechanisms or on upstream, hormone
receptor-containing neurons to exert effects.

An Introduction to the studies in the current dissertation
The experiments in the current dissertation explore the diverse functions
of gonadal hormones on the brain and behavior. Although the study of hormones
is long-established, there is still a lot that is unknown regarding hormone action in
the brain. Historically, studies have focused on a classical subset of mechanisms,
targets, and behaviors that have been best-studied through the examination of
11
hormone effects on the mating circuitry and reproductive behavior. It is now
accepted that hormones have much more pervasive influences on the brain. They
act in regions where hormone receptors cannot be visualized. They mediate
behaviors other than reproduction. They affect not only the normally functioning
brain, but have a profound impact in response to injury and disease.
In Chapter 2, the mating circuitry is used to examine a new role for
activational testosterone in the brain. The ability of hormones to stimulate
neurogenesis is a recently observed phenomenon and has been traditionally
studied in regions such as the hippocampus, which is highly proliferative but
contains a modest distribution of hormone receptors. Instead, I determined
whether testosterone increases cell proliferation and survival in MeP and MPOA,
both of which contain an abundance of steroid receptors. In addition, I
determined whether mating experience enhances cell survival in both of these
regions.
Chapters 3-4 use the nigrostriatal pathway to ask classical questions about
sex differences and hormonal modulation of neuroanatomy and behavior. The
nigrostriatal pathway is a part of the basal ganglia circuitry for voluntary
movement. While this pathway does not contain many hormone receptors, it is
nonetheless hormone-responsive. Chapter 3 examines sex differences in
nigrostriatal anatomy and behavior in rodents under normal physiologic
conditions and in a model of nigrostriatal degeneration. Chapter 4 focuses on
activational hormones in this same circuit. Specifically, I determined whether
12
testosterone enhanced neuronal morphology in the striatum and improved motor
performance in both normal and disease states.

Adult Neurogenesis: Changing the organization of the brain
The activational effects of gonadal steroid hormones are not limited to the
modulation of structural synaptic plasticity. Recently, it was observed that
estrogen and testosterone enhance the birth and/or survival of new neurons,
establishing a new role for hormones in the adult brain. Chapter 2 determined the
role of testosterone and the importance of mating behavior on cell proliferation
and survival in the mating circuitry of the adult brain.

Neurogenesis in the adult brain
Many tissues replenish themselves throughout life. For example, a
constant turnover of new cells occurs in both the epidermis and in the lining of the
gastrointestinal tract. Other tissues, including the brain, were originally thought
to be terminally differentiated with no capacity for hyperplasia after development
(Gross, 2000; Rakic, 1985a; Rakic, 1985b). This dogma was first challenged with
the discovery of adult mammalian neurogenesis (Altman and Das, 1965). In a
series of experiments, Altman described new cell birth in the adult hippocampus
after labeling proliferating cells in the dentate gyrus with [H
3
] thymidine (Altman,
1962; Altman and Das, 1965; Altman and Das, 1966; Altman and Das, 1967).
However, adult neurogenesis remained relatively underinvestigated until the
13
discovery of adult neurogenesis in the songbird (Goldman and Nottebohm, 1983)
and its subsequent confirmation in the rat hippocampus (Cameron, et al., 1993).
The identification of adult neurogenesis in the hippocampus was not by
chance. Although a modest amount of neurogenesis occurs in many brain areas,
the dentate gyrus and the olfactory bulb are the most highly neurogenic regions of
the brain. In the hippocampus, as many as 9000 new cells are born each day
(Cameron and McKay, 2001). Not surprisingly, most of what is known regarding
newly-proliferated neurons comes from studies of these two regions.
Adult neurogenesis encompasses two distinct processes, both of which are
essential for neurons to be functional. Cell proliferation must establish a
population of newly-born cells. In turn, newly-proliferated cells must survive,
mature into neurons, and integrate into existing neural circuits.
In both the hippocampus and olfactory bulb, the functionality of newly-
born neurons has been established. In the hippocampus, new granule cells
become incorporated into existing hippocampal circuitry by extending dendrites,
growing spines, and forming synapses (Kaplan and Bell, 1983; Kaplan and Hinds,
1977; Raineteau, et al., 2004; Stanfield and Trice, 1988; Wang, et al., 2005; Zhao,
et al., 2006). In addition, their electrophysiological properties change over time to
resemble mature granule cells (Overstreet-Wadiche and Westbrook, 2006; van
Praag, et al., 2002). A similar maturation of newly born neurons occurs in the
olfactory bulb (Belluzzi, et al., 2003; Carleton, et al., 2003; Petreanu and Alvarez-
Buylla, 2002).
14
The rates of neurogenesis in the hippocampus and olfactory bulb are
altered by a variety of factors. Positive modulators of neurogenesis in the
hippocampus include environmental enrichment and physical activity (Brown, et
al., 2003; Leal-Galicia, et al., 2007; Sonsalla, et al., 1992). Similarly, olfactory
neurogenesis is enhanced by olfactory enrichment (Rochefort, et al., 2002;
Shapiro, et al., 2007). By contrast, odor deprivation and stress decrease
neurogenesis (Corotto, et al., 1994; Gould and Tanapat, 1999; Petreanu and
Alvarez-Buylla, 2002).
Gonadal steroids have emerged recently as modulators of adult
neurogenesis in the hippocampus (reviewed in Galea, 2007). First, the capacity
for neurogenesis may be sexually dimorphic. In naturally-cycling female rats,
cell proliferation is significantly greater at proestrous, where estrogen levels are
highest, than in females at other phases of the estrous cycle or in males (Tanapat,
et al., 1999). Moreover, gonadal steroids exert sex-specific effects to enhance
neuronal proliferation and/or survival. Estrogen enhances short term cell
proliferation in the hippocampus of female rats (Ormerod, et al., 2003) but not
males (Fowler, et al., 2003). In males, testosterone has no effect on cell
proliferation, but enhances cell survival in the dentate gyrus (Fowler et al., 2003;
Spritzer and Galea, 2007). This effect is reproduced by the nonaromatizable
androgen DHT, but not by estrogen (Spritzer and Galea, 2007), suggesting that
cell survival in the dentate gyrus is mediated via an androgenic mechanism.
15
While the high rate of neurogenesis in the hippocampus makes it an
excellent model to study neurogenesis, the role of activational hormones in the
dentate gyrus is unclear. Hormones do not alter granule cell morphology and do
not play an essential role in facilitating hippocampal-dependent behaviors.
Moreover the expression of classical hormone receptors in the dentate gyrus is
modest, at best (Kerr, et al., 1995; Simerly et al., 1990; Weiland, et al., 1997). By
contrast, nuclei within the mating circuitry contain a modest amount of
neurogenesis (Fowler et al., 2003; Huang and Bittman, 2002; Peretto, et al., 2001)
but contain an abundance of classical hormone receptors (Wood et al., 1992).
Moreover, hormones are essential to drive mating behavior (Wood and Newman,
1995a). Chapter 2 evaluated the sensitivity of neurogenesis to gonadal steroids
and mating behavior in a classical brain circuit.

Chapter 2: Testosterone, Cell Proliferation and Survival
Chapter 2 includes two experiments. Initially, I determined if testosterone
enhances cell proliferation and survival in the mating circuitry of the male
hamster (Experiment 1). A limited number of studies suggest that cell
proliferation in MeP is increased by testosterone (Fowler et al., 2003). It is
unclear whether the effects of testosterone on cell proliferation are unique to MeP;
other hormone receptor-containing areas of the mating circuitry are almost
completely uninvestigated. Likewise, it is unknown whether hormones enhance
cell survival. Chapter 2, experiment 1 determined whether testosterone-driven
16
cell proliferation in MeP extends to MPOA, the principal efferent target of MeP
and whether testosterone enhances long-term cell survival in both regions.
I found that, although new cells are born in MeP and MPOA, cell survival
is extremely limited in both regions. One intriguing hypothesis to explain the loss
of newly-proliferated cells is reflected by the use it or lose it philosophy.
Specifically, it is thought that the survival of new neurons is enhanced in brain
regions that are regularly activated by experience or environment.
In neurogenic regions, neuronal activation is associated with an increase in
neurogenesis. In the dentate gyrus, hippocampal-dependent learning tasks
enhance cell survival (Epp, et al., 2007; Leuner, et al., 2004). In the olfactory
bulb, exposure to novel odors also enhances neurogenesis (Rochefort et al., 2002).
Thus, it is not unreasonable to expect that adult neurogenesis in the mating
circuitry is also enhanced by neuronal activation. In Chapter 2, experiment 2, I
tested the use it or lose it hypothesis in MeP and MPOA by comparing cell
proliferation and survival in male hamsters that were allowed to mate on a weekly
basis with males that did not mate.

The Nigrostriatal System: Sex differences and activational testosterone
Chapters 3-4 focus on the nigrostriatal system, an important target for
gonadal hormones that lies outside of the circuits for reproduction. The
nigrostriatal pathway consists of efferent connections from the substantia nigra
pars compacta (SNC) to the dorsolateral striatum, and is involved in coordinating
17
voluntary movements. Although the distribution of classical hormone receptors is
minimal, gonadal steroids modulate nigrostriatal function and sex differences
exist (reviewed in Becker, 1999; Becker, 2002). In addition, gonadal hormones
are also neuroprotective against the susceptibility and progression of nigrostriatal
diseases such as Parkinson’s disease (PD; Bourque, et al., 2009; PD; Dluzen,
2000). Chapters 3 and 4 evaluate sex differences (Chapter 3) and activational
effects of testosterone (Chapter 4) on nigrostriatal anatomy and motor behavior in
an animal model for PD.

Sex differences and gonadal hormones in the nigrostriatal system
The study of hormones and sex differences has traditionally focused on
social behaviors (i.e. mating) because they are highly sexually dimorphic. The
circuits for mating are extremely sensitive to hormones, which change neuronal
structure and drive reproductive behavior. By contrast, sex differences in the
nigrostriatal system are more difficult to see. This is not surprising, given that
hormones have a more subtle effect on nigrostriatal circuits, than they do in the
mating circuitry.
Despite a relative absence of classical hormone receptors, baseline sex
differences exist in the expression of striatal dopamine receptors and uptake sites
(Bazzett and Becker, 1994; Levesque, et al., 1989; Morissette and Di Paolo, 1993).
Behavioral sex differences are observed after amphetamine (AMPH)
administration, which increases dopamine release in the striatum and causes
18
stereotyped behaviors that include repetitive head and limb movements, sniffing
and grooming (Beatty, 1979). AMPH-induced stereotypy is greater in females
than in males (Becker, et al., 1982; Robinson, et al., 1980).
Sex differences in the striatum are driven largely by the effects of estrogen
in females. Estrogen increases dopamine release (McDermott, et al., 1994) and
extracellular dopamine (Xiao and Becker, 1994) in the striatum. In addition,
AMPH-stimulated dopamine release is enhanced by estrogen (Becker, 1990), as
are AMPH-induced stereotyped behaviors (Camp, et al., 1986; Castner, et al.,
1993). By contrast, estrogen does not change striatal dopamine release or
stereotypy after AMPH in males (Castner et al., 1993). The effects of testosterone
on the striatum in male rodents are less clear. While some studies report that
testosterone suppresses dopamine release and stereotyped behaviors (Beatty, 1979;
Dluzen and Ramirez, 1989), others report no effect of testosterone (Camp et al.,
1986; Xiao and Becker, 1994).
As it relates to my thesis, sex differences and the sexually dimorphic
effects of gonadal hormones in the striatum are thought to contribute to the
incidence and severity of neurodegenerative diseases such as PD. First, sex
differences exist in PD and in animal models of the disorder. Second, gonadal
hormones, specifically estrogen, exert neuroprotective effects against PD and
correspondingly modulate nigrostriatal degeneration in animal models of the
disease. Chapter 3 of my thesis examines sex differences in the MPTP mouse
19
model of PD. Chapter 4 uses the same model to determine whether testosterone is
neuroprotective in the nigrostriatal system of males.

Chapter 3: Sex differences in the MPTP mouse model for PD
PD is a progressive neurodegenerative disorder characterized by motor
impairments that include resting tremor, rigidity, bradykinesia, and postural
instability (Jankovic, 2008). PD results from the selective destruction of
nigrostriatal dopaminergic neurons in the substantia nigra pars compacta (SNC)
and a depletion of their corresponding efferents to the striatum. In PD, medium
spiny neurons (MSN’s) of dorsolateral striatum, which receive a major input from
SNC, show dramatic morphologic evidence of deafferentation, including
decreased spine density and dendritic length (McNeill, et al., 1988; Zaja-
Milatovic, et al., 2005). The ensuing reduction in striatal output is the primary
cause of motor dysfunction.
Sex differences exist in both incidence and phenotype of PD (Benedetti, et
al., 2001; Haaxma, et al., 2007; Shulman and Bhat, 2006; Tsang, et al., 2000;
Tsang, et al., 2001). The incidence of PD is one- to two-fold lower in women
(Baldereschi, et al., 2000; de Lau, et al., 2004; Elbaz, et al., 2002; Schrag, et al.,
2000; Shulman and Bhat, 2006; Taylor, et al., 2007; Van Den Eeden, et al., 2003;
Wooten, et al., 2004). In addition, motor impairments in early PD are less severe
in women (Lyons, et al., 1998). Women are more likely to have dyskinesias at
20
the onset of PD symptoms, while rigidity is more common in men (Baba, et al.,
2005; Haaxma et al., 2007).
Animal models of Parkinson’s disease include the 6-hydroxydopamine
(OHDA)-lesioned rat and the MPTP-lesioned mouse. Both models selectively
destroy dopaminergic neurons in SNC and subsequently, deplete striatal
dopamine (Baba et al., 2005)(Schober REF). The severity of this response is
sexually dimorphic. After 6-OHDA or MPTP, dopamine depletion is more severe
in male rodents (Freyaldenhoven, et al., 1996; Gillies, et al., 2004; Miller, et al.,
1998). In the case of 6-OHDA, male rats also have more cell loss in SNC
compared to females (Murray, et al., 2003).
It is unknown whether sex differences exist in motor impairments after
MPTP. Studies using rodent models have focused almost exclusively on striatal
dopamine levels to quantify sex differences after nigrostriatal lesions. Chapter 3
determined whether male mice were more susceptible than females to MPTP-
induced motor impairments and SNC cell loss. The experiment was designed
with two observations in mind. First, motor impairments are difficult to quantify
after MPTP or 6-OHDA because rodents do not mimic the spectrum of
Parkinsonian motor impairments seen in humans and in MPTP-lesioned primates
(Beal, 2001; Meredith and Kang, 2006; Sedelis, et al., 2001). Second, sex
differences in PD phenotype emerge at early stages of the disease but are not
present at later stages (Lyons et al., 1998). Thus, Chapter 3 used a battery of
21
behavioral tests to measure separate aspects of motor performance and used a
smaller lesion to induce a less severe form of PD-like pathology.

Chapter 4: Testosterone and neuroprotection in the nigrostriatal system
Neuroprotection has emerged recently as an important activational role for
gonadal hormones in the brain (Bialek, et al., 2004; Garcia-Segura, et al., 2001).
Due to the large volume of literature showing estrogen-dependent modulation of
nigrostriatal function, studies addressing gonadal hormones and neuroprotection
in this system have focused on females (Dluzen, 2000; Dluzen and McDermott,
2000). By contrast, the effects of testosterone on nigrostriatal function have
received little attention. Even less is known as to whether testosterone is
neuroprotective in the male nigrostriatal system, similar to estrogen in the female.
Chapter 4 used the MPTP mouse model to determine whether testosterone exerts
neuroprotective effects on nigrostriatal anatomy and behavior.
It is well-established that estrogen is neuroprotective against PD in
females (Saunders-Pullman, 2003). Estrogen levels are inversely correlated with
symptom severity (Quinn and Marsden, 1986) and estrogen replacement therapy
has been shown to attenuate PD symptoms (Saunders-Pullman, et al., 1999; Tsang
et al., 2000; Tsang et al., 2001). The stimulatory effects of estrogen in the female
striatum are thought to underlie this neuroprotective effect (Ragonese, et al.,
2006).
22
In rodent models of PD, estrogen attenuates the loss of striatal dopamine
and minimizes cell death in SNC (Disshon and Dluzen, 1997; Dluzen, 1997;
Dluzen, et al., 1996b; Ferraz, et al., 2008; Miller et al., 1998; Murray et al., 2003;
Shughrue, 2004). Compared with estrogen in females, testosterone remains almost
uninvestigated in males. Of the few studies that have addressed testosterone, its
role is unclear with respect to neuroprotection. While two independent groups
have shown no effect of testosterone on MPTP (Dluzen, 1996; Ekue, et al., 2002),
a different group has shown that testosterone exacerbates 6-OHDA neurotoxicity
(Gillies et al., 2004). Moreover, each of these studies have focused exclusively
on afferent inputs to the striatum, measuring dopamine content and release,
innervation, and transporter binding. Outside of striatal dopamine, the effects of
testosterone on the nigrostriatal pathway are largely unexplored.
Chapter 4 determined whether testosterone attenuates MPTP-induced
neurotoxicity in the nigrostriatal system. The actions of MPTP are not confined
to dopamine-producing neurons and their efferents. SNC projections form
synapses on MSN spine necks and MSN output subsequently drives behavior.
Chapter 4 measured dopaminergic innervation to the striatum but also measured
MSN neuronal morphology as well as motor performance to evaluate the
neuroprotective capacity of testosterone across this circuit.

23
Summary
This thesis explores sex differences and the role of testosterone on
neuroanatomical and behavioral plasticity. Much of what is known with respect
to sex differences and gonadal hormones relates to mating behavior and the
corresponding neural circuits for reproduction. The experiments in Chapter 2
address hormone action in this context. However, Chapters 3-4 leave the mating
circuitry and focus on sex differences and gonadal hormones in nonclassical brain
regions.
Chapter 2 examines the role of testosterone on adult neurogenesis in the
mating circuitry. While the effects of gonadal hormones on neurogenesis have
been investigated in the hippocampus, comparatively little is known regarding the
effect of hormones on neurogenesis in classical brain regions. Chapter 3
examines sex differences in the nigrostriatal system. Sex differences are
commonly studied in hormone receptor-rich brain areas. However, much less is
known about sex differences in areas that lack large numbers of classical hormone
receptors. Moreover, Chapter 3 uses an experimental model for
neurodegenerative disease, allowing for both the quantification of baseline sex
differences and of sex differences in the neurodegenerative process. Chapter 4
addresses the role of activational testosterone in the nigrostriatal system. The
nigrostriatal system is well-studied with respect to estrogen in females.
Testosterone has remained comparatively uninvestigated. Because Chapter 4 also
24
uses a neurodegenerative model, it allows us to determine whether testosterone is
neuroprotective in the nigrostriatal system.
Together the experiments in this thesis step outside of the traditional box
that defines gonadal hormone function. My studies address new roles for gonadal
hormones and illustrate the diverse effects of gonadal hormones on brain and
behavior.

25
Chapter 2
CELL PROLIFERATION AND SURVIVAL IN THE MATING CIRCUIT
OF ADULT MALE HAMSTERS: EFFECTS OF TESTOSTERONE AND
SEXUAL BEHAVIOR

Abstract
The transient actions of gonadal steroids on the adult brain facilitate social
behaviors, including reproduction. In male rodents, testosterone acts in the
posterior medial amygdala (MeP) and medial preoptic area (MPOA) to promote
mating. Adult neurogenesis occurs in both regions. The current study determined
if testosterone and/or sexual behavior promote cell proliferation and survival in
MeP and MPOA. Two experiments were conducted using the thymidine analog
BrdU. First, gonad-intact and castrated male hamsters (n=6/group) were
compared 24 hours or 7 weeks after BrdU. In MeP, testosterone stimulated cell
proliferation 24 hours after BrdU (intact: 22.8±3.9 cells/mm
2
, castrate:
13.2±1.4cells/mm
2
). Testosterone did not promote cell proliferation in MPOA.
Seven weeks after BrdU, cell survival was sparse in both regions (MeP: 2.5±0.6
and MPOA: 1.7±0.2 cells/mm
2
), and was not enhanced by testosterone. In
Experiment 2, gonad-intact sexually-experienced animals were mated weekly to
determine if regular neural activation enhances cell survival 7 weeks after BrdU
in MeP and MPOA. Weekly mating failed to increase cell survival in MeP
(8.1±1.6 vs. 9.9±3.2 cells/mm
2
) or MPOA (3.9±0.7 vs. 3.4±0.3 cells/mm
2
).
26
Furthermore, mating at the time of BrdU injection did not stimulate cell
proliferation in MeP (8.9±1.7 vs. 8.1±1.6 cells/mm
2
) or MPOA (3.6±0.5 vs.
3.9±0.7 cells/mm
2
). Taken together, our results demonstrate a limited capacity
for neurogenesis in the mating circuitry. Specifically, cell proliferation in MeP
and MPOA are differentially influenced by testosterone, and the birth and survival
of new cells in either region are not enhanced by reproductive activity.

Introduction
Gonadal steroid hormones exert activational effects on the adult brain that
sculpt neural circuits for expression of adult behavior. Hormones act in steroid-
responsive brain regions, where they exert neurotrophic effects to enhance
neuronal morphology and synaptic connectivity (Cooke and Woolley, 2005). In
addition, gonadal hormones stimulate neurogenesis in adult mammals (Fowler, et
al., 2007; Galea, et al., 2006). These structural changes are thought to be a
principal mechanism through which hormones promote social behaviors,
including mating.
Adult mammalian neurogenesis includes both cell proliferation and cell
survival. It is notably demonstrated in the hippocampus (Gould, 2007). In male
rodents, testosterone promotes the survival of new neurons in the dentate gyrus
(Spritzer and Galea, 2007). Cell proliferation has also been demonstrated
elsewhere in the male brain, including the olfactory bulb (Peretto et al., 2001),
posterior medial nucleus of the amygdala (MeP; Fowler et al., 2003), medial
27
preoptic area (MPOA) and bed nucleus of the stria terminalis (BST; Huang and
Bittman, 2002). MeP, BST, and MPOA are essential for male rodent sexual
behavior. Although this mating circuit exhibits a lower capacity for neurogenesis,
it serves as an excellent model to study hormone-driven cell proliferation and
survival. The actions of testosterone throughout the mating circuit are essential
for the expression of reproductive behavior in the presence of an appropriate
sexual partner (reviewed in Wood and Swann, 2000).
Both MeP and medial MPOA transduce testosterone via androgen and
estrogen receptors to stimulate mating (reviewed in Wood and Newman, 1995a).
Neurogenesis occurs in both regions (Fowler et al., 2003; Huang and Bittman,
2002). In MeP, testosterone increases cell proliferation (Fowler et al., 2003). It is
unknown whether testosterone influences new cell birth in MPOA. Therefore, it
is important to determine whether testosterone-enhanced cell proliferation extends
to MPOA. Additionally, we examined whether the survival of newly-born cells
parallels testosterone’s long term effects on behavior. Gonadal hormones exert
lasting effects throughout the male brain. For example, mating behavior in male
rodents is not immediately abolished after castration (reviewed in Wood and
Newman, 1995a). Conversely, the full recovery of sexual behavior in long-term
castrates requires weeks of testosterone exposure. Thus, newly-born cells are
more likely to be functionally important if they persist through the time course
known to influence behavior. Using the cell proliferation marker BrdU, the first
experiment determined if testosterone-driven cell proliferation is similar in MeP
28
and MPOA. Additionally, we determined if testosterone promotes cell survival in
MeP and MPOA.
In a second experiment, we determined if the regular activation of
reproductive circuits enhances the survival of cells in MeP and MPOA by
comparing male hamsters that were allowed to mate on a weekly basis with males
that did not mate. Several investigators have put forth the hypothesis that the
birth and/or survival of new cells in the adult brain are enhanced in regions that
are regularly activated by experience or environment (Gould, et al., 2000; Lledo,
et al., 2006; Prickaerts, et al., 2004). Animals engaged in hippocampal-dependent
learning tasks exhibit higher levels of cell proliferation and survival in the dentate
gyrus compared to animals that have not engaged in these tasks (Dalla, et al.,
2007; Shors, 2004). Additionally, cell proliferation is enhanced in the olfactory
bulb after rodents are repeatedly exposed to novel olfactory stimuli (Rochefort et
al., 2002). If the activity-dependent hypothesis applies to the hamster mating
behavior circuit, then regular sexual activity should enhance cell proliferation and
survival in MeP and MPOA.

Materials and Methods
Subjects
Forty-four adult male Syrian hamsters (Mesocricetus auratus, 130-150g)
were obtained from Charles River Laboratories (Wilmington, MA). Hamsters
were singly-housed on a long day photoperiod (14:10 LD) with access to food and
29
water ad libitum. Experimental procedures were approved by USC’s Institutional
Animal Care and Use Committee and conducted in accordance with the NIH
Guide for the Care and Use of Laboratory Animals (DHEW Publication 80-23,
revised 1985, Office of Science and Health reports, DRR/NIH, Bethesda, MD
20205).

Experiment 1
Gonad-intact and castrated male hamsters were compared at 24 hours and
7 weeks post-BrdU to determine if testosterone promotes cell proliferation and
survival in MeP and MPOA. Twenty-eight animals were used. Three weeks
prior to BrdU injections, half of the animals were castrated. Castration causes a
profound loss in circulating androgens, and is accompanied by the elimination of
sexual behavior (reviewed in Siegel, 1985b; Wood and Newman, 1995a). The
other half remained gonad intact. Proliferating cell populations were labeled 3
weeks following castration, when mating behavior is severely diminished
(reviewed in Wood and Newman, 1995a).
All animals received a single injection of BrdU [300mg/kg BW ip (Sigma,
St. Louis, MO) in 0.9% saline with 0.007N NaOH] to maximally incorporate
BrdU into dividing cells (Cameron and McKay, 2001; Fowler et al., 2003). Half
of the hamsters in each group (n=7 each) were sacrificed 24h later to determine if
testosterone promotes cell proliferation in MPOA and MeP. The remaining
animals were sacrificed 7 wk post-BrdU to determine if testosterone enhances the
30
survival of newly-born cells. Seven weeks is sufficient for testosterone to restore
mating behavior in long-term castrates and for newly-born olfactory neurons to
express Fos in response to estrous females (Huang and Bittman, 2002; Morin and
Zucker, 1978). By examining cell survival after seven weeks, the current study
seeks to bridge short-term (Fowler et al., 2003) and long-term (Huang and
Bittman, 2002) studies. The hypothesis is that testosterone enhances long-term
cell survival at seven weeks similar to its effects on short-term cell proliferation,
as measured at 24 hours.

Experiment 2
Gonad-intact, sexually-experienced male hamsters (n=18) were mated
weekly or left unstimulated to determine if regular sexual activity enhances cell
proliferation and survival in MeP and MPOA. Sexual interactions acutely
stimulate testosterone in male hamsters (Pfeiffer and Johnston, 1992).
Accordingly, we hypothesize that the consistent activation of mating circuits will
increase cell survival in MeP and MPOA. Males were included in the study only
if they mated to ejaculation in two of three preliminary tests for sexual experience.
Mating behavior tests were conducted during the first hours of the dark
phase under dim light. Eight female hamsters were used as stimulus animals for
mating behavior. All females were ovariectomized via bilateral dorsal flank
incision, and received a 4-mm Silastic estradiol implant sc (id: 1.98 mm, od: 3.18
mm; Dow Corning, MI) to maintain chronic physiologic levels of estrogen. To
31
stimulate lordosis, females received 250 ug progesterone in cottonseed oil sc 4
hours prior to testing (see Carter, 1985). For testing, an estrous female was
introduced into the male’s home cage for 10 minutes. Behavior of the test male
was recorded including mounts, intromissions, and ejaculations. For weekly
mating experience after BrdU injection in Experiment 2, males were required to
ejaculate at least once during each test. If necessary, males were given extra time
and/or placed with a different stimulus female to facilitate ejaculation. The week-
long period between mating exposures ensured that males would successfully
copulate at each mating test without becoming sexually satiated (Arteaga, et al.,
2000; Beach and Rabedeau, 1959).
As in Experiment 1, all males received a single injection of BrdU
(300mg/kg BW ip). The hamsters were separated into three groups (n=6/group).
Males in the Control group did not mate after BrdU administration. The
remaining hamsters were allowed to mate weekly for 7 weeks. However, to
determine whether reproduction stimulates cell proliferation, hamsters in the
Immediate Mating (IM) group were allowed to mate 10 minutes after BrdU
administration. Animals in the Delayed Mating (DM) group mated 24 hours later.
Thus, males in the IM and DM groups received the same amount of sexual
activity. However, only males in the IM group mated during BrdU incorporation,
which occurs over 2 hours following BrdU administration (Takahashi, et al.,
1992). Therefore, BrdU-labeling in IM hamsters should reflect mating-induced
cell proliferation and survival, while BrdU-labeling in DM hamsters should
32
indicate activity-dependent cell survival only. After the final sexual behavior test,
all hamsters were sacrificed.

Perfusion
Hamsters were deeply anesthetized with sodium pentobarbital (150mg/kg
BW) and perfused intracardially with 150mL of 0.1M sodium phosphate buffer
(PB, pH=7.4) containing 0.9% NaCl and 0.1% NaNO
3
, followed by 250mL 4%
paraformaldehyde in PB. The brains were removed, post-fixed in the perfusion
fixative for 1h and cryoprotected for 5 days at 4°C with 20% sucrose in PB. The
brains were rapidly frozen and sectioned coronally at 40um. Sections were stored
in PB with 0.01% sodium azide at 4°C until processed for BrdU
immunocytochemistry.

Immunocytochemistry for BrdU
Every fourth section was stained for BrdU according to the methods of
Fowler, et al. (2003). Sections from males in different groups of the same
experiment were stained at the same time. To denature the DNA, free-floating
brain sections were pre-treated with 2N HCl for 30 min at 37°C and then washed
to neutralize the acid with 0.1M borate buffer (pH=8.5) at room temperature (RT)
for 20 min. After additional washing with PB, sections were incubated overnight
at RT in monoclonal rat anti-BrdU antibody (1:500; AbD Serotec, Raleigh, NC)
with 4% normal donkey serum and 0.3% Triton X-100 in PB. The following day,
33
sections were incubated in biotinylated donkey-anti-rat secondary antibody (1:200,
Jackson Immunoresearch, West Grove, PA) and the avidin-biotin-horseradish
peroxidase complex, each for 1 hour at RT with extensive washes in between.
BrdU-labeled cells were visualized using NiCl-enhanced 3’3-diaminobenzidine
tetrahydrochloride (DAB) with 0.25% hydrogen peroxide. Sections were
mounted onto gelatin-coated slides, air-dried, cleared in xylenes, and coverslipped.
Adjacent sections from each brain were Nissl-stained to verify the boundaries of
MeP and MPOA.

Data Analysis
Sections from each brain region were matched across animals to ensure
that MeP and MPOA were sampled consistently. BrdU-labeled cells were
counted bilaterally on coded slides by an observer blind to the treatment group
using an Olympus BH-2 microscope equipped with a drawing tube. Labeled cells
were counted in a 0.4 mm
2
triangular region of MeP lateral to the optic tract that
included both dorsal and ventral subdivisions (Plate 27 in Morin and Wood, 2001).
MPOA cell counts were completed in a 1.5 mm
2
rectangular area adjacent to the
third ventricle, extending dorsally to the border of BST and laterally to the edge
of the optic chiasm (Plate 22 in Morin and Wood, 2001). Figure 2.1 illustrates
BrdU labeling in MeP and MPOA.
BrdU-labeled cells were identified at 10x and verified at 40x. Because
there was no left-right asymmetry in MeP or MPOA, cell counts from both
34
hemispheres were combined. The total number of BrdU-positive cells per section
was averaged for each animal and the density of BrdU-labeled cells per mm
2
was
calculated. For each region, group differences were analyzed by a fully factorial
analysis of variance (ANOVA). Post-hoc comparisons using the Fisher’s LSD
test were conducted when statistically significant differences (p<0.05) were found.

Results
BrdU labeling was observed as dark punctate nuclear staining (Fig. 2.1a-c).
Labeled nuclei had an irregular shape and were distributed throughout MeP and
MPOA. The specific subnuclear distribution of BrdU-labeled cells has
implications for the origin of newly-born cells in the brain. Adult neurogenesis
may arise by migration of proliferating cells from the subventricular zone or by
division of neuronal precursors in situ (Lois and Alvarez-Buylla, 1994; Zhao, et
al., 2003). This latter mechanism is thought to give rise to pairs of labeled
daughter cells (Cameron and McKay, 2001). In MeP, labeled cells were observed
near the lateral ventricle. In MPOA, BrdU labeling was present near the
ependymal layer of the third ventricle. Labeled cells were also present at the
lateral edges of both cell counting regions. In addition, BrdU-labeled cells
commonly appeared in 2-cell clusters throughout MeP and MPOA (Fig. 2.1c).

35
Figure 2.1. Photographs of BrdU-labeled cells in MeP (A) and MPOA (B) at
24h post BrdU. (C) High magnification photograph of BrdU-labeling in MeP.
Labeled cells were observed as individual cells or in 2-cell clusters. Scale bar
= (A-B) 250um, (C) 25um. Dotted lines indicate atlas boundaries. Solid lines
indicate counting regions. Abbreviations: MPN, medial preoptic nucleus;
MPNmag, medial preoptic nucleus, magnocellular part; MePD, medial
amygdaloid nucleus, posterodorsal part; MePV, medial amygdaloid nucleus,
posteroventral part; oc, optic chiasm; ot, optic tract.

36

37
Experiment 1
Twenty four hours after injection, newly-born cells were observed in MeP,
as determined by the presence of BrdU-labeled cells (18.0±2.4 cells/mm
2
,
mean±SEM of gonad-intact and castrated males). Furthermore, cell proliferation
in MeP was enhanced by testosterone. Gonad-intact males had a significantly
higher density of BrdU-labeled cells (22.8±3.9 cells/mm
2
) than did castrated
males (13.2±1.4 cells/mm
2
, p<0.05, Fig. 2.2a). However, after 7 weeks, the
density of BrdU-labeled cells in MeP was drastically reduced in both gonad-intact
and castrated hamsters (overall mean 2.5±0.6 cells/mm
2
, p<0.05). In addition,
testosterone did not promote the survival of cells in MeP (gonad-intact 2.5±0.6 vs.
castrate 2.5±1.0 cells/mm
2
, n.s., Fig. 2.2a).
Cell proliferation in MPOA was substantially less than in MeP. Gonad-
intact and castrated males sacrificed 24h post-injection had an average of 7.9±1.0
BrdU-labeled cells/mm
2
(p<0.05, vs. MeP by paired t-test). Moreover,
testosterone did not promote cell proliferation in MPOA. The density of BrdU-
labeled cells in gonad-intact males (9.0±1.5 cells/mm
2
) was not different from
that in castrated males (6.8±0.9 cells/mm
2
, n.s., Fig. 2.2b). As with MeP, survival
of newly-born cells was poor in both gonad-intact and castrated males sacrificed 7
weeks post BrdU (1.7±0.2 cells/mm
2
, p<0.05). Similarly, testosterone had no
effect on cell survival in MPOA (gonad-intact: 2.2±0.1 cells/mm
2
vs. castrate:
1.3±0.1 cells/mm
2
, n.s., Fig. 2.2b). Despite the reduced proliferation of cells in
38
MPOA relative to MeP, the survival of BrdU-labeled cells at 7 weeks was
equivalent in both regions.

Figure 2.2. The effects of testosterone on cell proliferation and survival in
the mating circuitry. Mean±SEM number of labeled cells per mm
2
tissue at
24 hours and 7 weeks post-BrdU in gonad-intact (black bars) and castrated
(white bars) male hamsters (n=4-7/ group) in MeP (top) and MPOA (bottom).
Numbers in parentheses indicate n for each group. Bars with different letter
subscripts are significantly different.

39
Experiment 2
BrdU-labeled cells were observed in both MeP and MPOA of gonad-intact
sexually-experienced male hamsters. Nonetheless, the regular activation of
mating circuits did not enhance cell survival measured 7 weeks after BrdU.
Likewise, individual aspects of mating behavior (mounts, intromissions, or
ejaculations) did not correlate with cell survival in MeP or MPOA.
In MeP, DM hamsters that mated weekly had a similar density of BrdU-
labeled cells (8.1±1.6 cells/mm
2
) to Control males that did not mate (9.9±3.2
cells/mm
2
, Fig. 2.3a). Likewise, mating did not enhance cell survival in MPOA
(3.9±0.7 vs. 3.4±0.3 cells/mm
2
, n.s., Fig. 2.3b). Furthermore, mating at the time
of BrdU injection did not promote cell proliferation in male hamsters. In MeP,
BrdU-labeling was not different between IM (8.9±1.7 cells/mm
2
) and DM males
(8.1±1.6 cells/mm
2
, n.s., Fig. 2.3a). The same pattern was observed in MPOA
(3.6±0.5 vs. 3.9±0.7 cells/mm
2
, Fig. 2.3b).
Interestingly, unlike cell survival in Experiment 1, the survival of newly-
born cells was not equivalent in MeP and MPOA. Across the IM, DM, and
Control groups, male hamsters had a higher density of BrdU-labeled cells in MeP
(8.9±1.3 cells/mm
2
) than in MPOA (3.6±0.3 cells/mm
2
, p<0.05).

40

Figure 2.3. The effects of reproductive activity on cell proliferation and
survival in MeP and MPOA. Mean±SEM number of BrdU-labeled cells per
mm
2
tissue in reproductively inactive (Control, black bars) or reproductively
active hamsters (n=6/group) in MeP (top) and MPOA (bottom).
Reproductively active hamsters mated either during BrdU incorporation (IM,
dark gray) or 24h later (DM, light gray). Numbers in parentheses indicate n
for each group. There were no significant differences between groups.

Discussion
The current study used BrdU labeling to determine the effects of
testosterone and reproductive activity on cell proliferation and survival in MeP
and MPOA of the male Syrian hamster. Cell proliferation occurs throughout the
mating circuitry in a site-specific manner. Specifically, more cells arise in MeP
41
than in MPOA. We also found that cell proliferation in MeP, but not MPOA, is
sensitive to testosterone. Nonetheless, long-term (7 wk) cell survival was limited
in both regions and testosterone did not promote cell survival in MeP or MPOA.
When sexually-experienced animals were allowed to mate on a regular basis,
repeated activation of the sexual behavior circuitry did not enhance cell survival.
Likewise, acute mating experience did not further facilitate cell survival in MeP
or MPOA. Overall, our results demonstrate a limited capacity for cell
proliferation and survival in the mating circuitry that is differentially influenced
by testosterone.
The comparison of hamsters with (gonad-intact) and without (castrate)
testosterone is relevant to the normal physiologic environment of male hamsters.
Syrian hamsters are seasonal breeders. During long day photoperiods, male
hamsters are sexually active and fertile under the stimulatory actions of testicular
androgens (Miernicki, et al., 1990; Morin and Zucker, 1978). In contrast,
testosterone levels approach those of castrated males during short days. Therefore,
it is realistic to assume that hormone-dependent cell proliferation and/or survival
also varies with season and that MeP and MPOA are influenced by natural
fluctuations of testosterone that occur across the lifespan.
Because MeP and MPOA contain abundant steroid hormone receptors
(Wood et al., 1992) and play important roles in mating behavior, it would appear
that gonadal hormones might act similarly in both regions to stimulate cell
proliferation. The action of testosterone in either MeP or MPOA is sufficient to
42
stimulate mating (Wood and Newman, 1995b). Correspondingly, lesions to either
region impair sexual activity (Lehman and Winans, 1982; Powers, et al., 1987)
In addition, MeP and MPOA are heavily interconnected (reviewed in Wood and
Newman, 1995a). However, the results of the current study indicate that the
capacity for cell proliferation is greater in MeP than in MPOA. Furthermore,
testosterone exerted regionally-specific effects on the birth of new cells. In MeP,
cell proliferation was sensitive to testosterone. In MPOA, testosterone did not
stimulate new cell birth.
Although MPOA displays a low level of cell proliferation, it demonstrates
a large capacity for hormone-driven structural plasticity. Testosterone enhances
dendritic morphology in MPOA neurons (Cherry, et al., 1992). Interestingly, this
pattern is also observed in the female ventromedial hypothalamus (VMH), the
major organizing center for female rodent sexual behavior (Mathews and Edwards,
1977; Pfaff and Sakuma, 1979). In VMH, estrogen does not stimulate cell
proliferation (Fowler, et al., 2005) but drastically remodels dendritic morphology
in VMH neurons (Calizo and Flanagan-Cato, 2000; Frankfurt, et al., 1990). This
argues that the hypothalamic centers for sexual behavior in both sexes
demonstrate hormone-driven plasticity in cell remodeling rather than cell
proliferation.
However, in both MeP and MPOA, the long-term survival of newly-born
cells in the mating circuitry is limited. Few studies have examined cell survival
after 7 weeks. Instead, cell survival is commonly measured only up to 30 days
43
after BrdU injection. This time frame is relevant for the study of memory
formation in the hippocampus (Gould, et al., 1999). However, hormone-induced
changes in reproductive behavior occur over a much longer time course (reviewed
in Wood and Newman, 1995a). Castrated male hamsters do not show obvious
impairments in sexual performance immediately following gonadectomy. Rather,
mating behavior declines slowly after castration with the complete elimination of
sexual behavior occurring 7-10 weeks later (Park et al., 2004; Siegel, 1985b).
Conversely, testosterone administration to long-term castrates results in the full
recovery of mating after 8 weeks (Sachs and Meisel, 1988; Siegel, 1985b; Wood
and Newman, 1995a). In the olfactory bulb, the maturation and subsequent
activation of newly-proliferated cells occurs across a similar time frame. Longer
time periods (7 weeks) are sufficient to detect odor-induced Fos in substantial
populations of olfactory neurons that have migrated appropriately into the
olfactory bulb (Huang and Bittman, 2002). Thus, the analysis of cell survival at 7
weeks allowed us to follow hormone-influenced cell proliferation and survival
over a functionally-relevant time course.
However, few newly-born cells remained in the mating circuit after 7
weeks. Previous work in MeP and MPOA of gonad-intact males has shown
similar findings (Huang and Bittman, 2002). In the present study, we found that
the stimulatory effects of testosterone on cell proliferation in MeP were not
sustained over seven weeks. Additionally, we used a high dose of BrdU, designed
to label a much larger population of proliferating cells without inducing toxicity
44
(Cameron and McKay, 2001; Fowler et al., 2003). Nonetheless, the results
indicate that cell survival is extremely limited in the mating circuitry and is not
enhanced by testosterone. It seems that the small number of surviving cells in
MeP and MPOA would be unlikely to have substantial consequences on
reproductive behavior.
Our observation that testosterone stimulated cell proliferation selectively
in MeP but did not enhance long-term cell survival stands in contrast to the
hippocampus, where testosterone enhances cell survival but does not promote cell
proliferation in the dentate gyrus (Spritzer and Galea, 2007). This difference may
be mediated, in part, through the specific actions of testosterone’s metabolites. In
the male, testosterone can be aromatized to estrogen or reduced to the androgen
dihydrotestosterone (DHT). Behavioral and morphologic evidence suggests that
the actions of testosterone in MeP are mediated through its conversion to estradiol
(Fowler et al., 2003; Gomez and Newman, 1991; Wood, 1996). In contrast, DHT
mediates the effects of testosterone on hippocampal morphology and
neurogenesis (Leranth et al., 2004) and on cell survival (Spritzer and Galea, 2007).
Taken together, these observations suggest that different brain regions are tuned
to preferentially respond to androgenic or estrogenic metabolites of testosterone,
thereby producing different effects on cell proliferation and survival.
Because cell survival in MeP and MPOA in Experiment 1 was limited, it
was important in Experiment 2 to test the role of experience and neuronal activity
in maintaining newly-born cells. There is precedent for this in both hippocampus
45
and olfactory bulb, where neurogenesis is influenced by experience. In the
olfactory bulb, odor-enriching and odor-depriving environments increase
(Rochefort et al., 2002) and decrease (Petreanu and Alvarez-Buylla, 2002)
granule cell survival, respectively. In the hippocampus, the acquisition of a trace
conditioning response increases the survival of granule cells in the rodent dentate
gyrus (Gould et al., 1999). On the other hand, animals that experience
uncontrollable stressors have decreased cell proliferation in DG (Malberg and
Duman, 2003).
By contrast, sexual activity had no significant effect on cell survival or
proliferation in MeP and MPOA. First, when animals were mated on a weekly
basis, cell survival was not enhanced in either region. If activation enhances cell
survival, then regular mating experience should have increased BrdU labeling in
MeP and MPOA. Second, BrdU labeling was not different between animals that
mated during BrdU incorporation and animals that mated 24 hours post-BrdU.
Male hamsters experience a pulse of testosterone immediately following mating
(Pfeiffer and Johnston, 1992). If mating-induced testosterone stimulated cell
proliferation, more cells would have been present in IM hamsters than in DM
males.
Interestingly, cell survival in sexually-experienced hamsters was
selectively-enhanced, compared with sexually-naïve hamsters. In gonad-intact
but sexually-naïve hamsters from Experiment 1, the survival of new cells was
equivalent in MeP and in MPOA. In sexually-experienced Control hamsters from
46
Experiment 2 that did not mate following BrdU, cell survival was significantly
greater in MeP than in MPOA. Thus, experience before, but not after, BrdU may
have increased the capacity for cell survival in MeP.
Differences between sexually-naïve and experienced male hamsters have
been previously reported, and suggest that permanent physiological changes occur
after the acquisition of sexual behavior in male hamsters. First, sexually-
experienced male hamsters exposed to vaginal secretions from female hamsters
(FHVS) show increased neuronal activation compared to sexually-naïve hamsters
presented with FHVS (Westberry and Meredith, 2003). Second, sexually-
experienced male hamsters retain copulatory behaviors for longer periods of time
after castration than do sexually-naïve hamsters (Lisk and Heimann, 1980).
Nonetheless, given the relatively small number of surviving neurons, the selective
increase in BrdU labeling in sexually-experienced hamsters is unlikely to have a
substantial impact on mating behavior.
Overall, our results are in keeping with earlier studies demonstrating a
limited capacity for cell proliferation and survival in the mating circuitry.
However, our results extend the findings of earlier studies by demonstrating that
testosterone-stimulated cell proliferation does not occur evenly throughout the
mating circuitry. We also report that neither testosterone nor regular neural
activation improves cell survival in the mating circuit. This suggests that
although male hamsters exhibit dramatic behavioral changes across their lifespan,
the low survival of newly-born cells in MeP and MPOA occurs over a restricted
47
time course and is unlikely to account for seasonal changes in reproductive
response across the lifespan.

Acknowledgements
The authors thank Ruby E. Jong for her assistance with tissue analysis of BrdU-
labeling. This work is supported by NIH RO1-MH55034.

48
Chapter 3
SEX DIFFERENCES IN MOTOR BEHAVIOR IN THE MPTP MOUSE
MODEL OF PARKINSON’S DISEASE

Abstract
Sex differences in Parkinson’s disease (PD) have been reported in humans
and rodent models, with a higher incidence in men and increased severity in male
rodents. The current study examined anatomical and behavioral sex differences in
the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model
of PD. Male and female mice (n=30 per group) were gonadectomized and
received physiologic replacement with testosterone or estrogen to maintain
constant hormone levels. Mice were injected with MPTP (10 mg/kg BW ip) or
saline daily for 5 days. Higher daily doses produced significant mortality in
females. One week later, motor function was measured using rotarod, gait, and
pole tests. Immediately afterwards, animals were sacrificed. Coronal sections
through the substantia nigra pars compacta (SNC) were immunostained for
tyrosine hydroxylase (TH), and counterstained for Nissl to quantify dopaminergic
neurons. In unlesioned mice, males significantly outperformed females in all
three motor tests (p<0.05). Compared with females, males had a greater overall
rotarod performance (ORP: 1317.1±98.3 vs. 988.1±95.6), descended a pole faster
(7.1±0.6 vs. 9.6±0.7 sec), and had longer stride lengths (hindlimb 7.3±0.1 vs.
6.8±0.1 cm). However, the number of TH-positive SNC neurons was similar in
49
unlesioned male (11 174.1±620.0) and female (11 246.1±1064.9) mice. MPTP
did not reduce TH-positive neurons in SNC of male (11 192.6±1295.4) or female
(9 927.6±978.9) mice. Nonetheless, MPTP significantly impaired ORP in males
(703.7±65.5, p<0.05 vs. unlesioned males) and in females (432.8±88.6, p<0.05 vs.
unlesioned females). In addition, MPTP-lesioned male mice had decreased stride
lengths (hindlimb 6.6±0.1 cm) after MPTP (p<0.05). MPTP had no effect on gait
in females. Finally, MPTP-lesioned males and females were unimpaired on the
pole test. Taken together, our results show that small, chronic doses of MPTP
produce sexually-dimorphic impairments in motor performance in mice without
depleting dopaminergic SNC neurons. This supports previous findings showing
the increased susceptibility of males to MPTP.

Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder
characterized by motor impairments that include resting tremor, rigidity,
bradykinesia, and postural instability (reviewed in Jankovic, 2008). Sex
differences have been reported for PD, and include a one- to two-fold higher
incidence in men (Baldereschi et al., 2000; de Lau et al., 2004; Elbaz et al., 2002;
Schrag et al., 2000; Shulman and Bhat, 2006; Taylor et al., 2007; Van Den Eeden
et al., 2003; Wooten et al., 2004). Epidemiological studies suggest that estrogen
contributes to sex differences in PD (Benedetti et al., 2001; Haaxma et al., 2007;
Shulman and Bhat, 2006; Tsang et al., 2001). In addition, experimental studies
50
have shown that estrogen exerts neuroprotective effects throughout the
nigrostriatal dopamine system (for review, see Dluzen, 2000).
Rodent models of PD include the 6-hydroxydopamine (6-OHDA)-lesioned
rat and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse
(Bove, et al., 2005; Jakowec and Petzinger, 2004; Jeon, et al., 1995; Schober,
2004). Both models replicate key features of PD by lesioning dopaminergic
neurons in the substantia nigra pars compacta (SNC) which project rostrally to the
striatum. Thus, after either 6-OHDA or MPTP, dopamine is depleted in the
striatum (Alvarez-Fischer, et al., 2008; Jakowec, et al., 2004). In both models,
sex differences in striatal neurochemistry are observed following dopamine-
depleting lesions. Specifically, striatal dopamine depletion is more severe in male
rodents following 6-OHDA (Murray et al., 2003; Tamas, et al., 2006) or MPTP
(Miller et al., 1998; Xu, et al., 2006). With 6-OHDA, male rats also have an
increased loss of dopaminergic neurons in SNC compared to females (Murray et
al., 2003; Tamas et al., 2006) and have more severe behavioral deficits post-lesion,
including both decreased spontaneous activity (Cass, et al., 2005) and ambulation
(Tamas, et al., 2005).
The current study determined whether MPTP in C57BL/6 mice produces
sex differences in motor impairments and whether the loss of dopaminergic
neurons in SNC after MPTP is sexually dimorphic. 6-OHDA is commonly
delivered by intracerebral injection to produce an acute, unilateral depletion of
dopaminergic neurons in SNC of rats. In mice, MPTP can be delivered
51
systemically to produce less severe, bilateral lesions to the nigrostriatal dopamine
system. We used a subacute dose of MPTP to reveal the potentially subtle sex
differences in motor impairment between male and female mice. The behavioral
tests measured distinct aspects of motor performance that parallel the motor
deficits seen in human Parkinson’s disease. In addition, tyrosine hydroxylase
(TH)-positive neurons in SNC were counted to determine whether MPTP
produces a greater loss of dopamine-producing neurons in male mice.

Methods
Animals
Adult male (n=45) and female (n=60) C57BL/6 mice (8-12 weeks) were
obtained from Charles River Laboratories (Wilmington, MA). Mice were group-
housed on a 12:12 LD photoperiod with access to food and water ad libitum.
Experimental procedures were approved by USC’s Institutional Animal Care and
Use Committee and conducted in accordance with the NIH Guide for the Care
and Use of Laboratory Animals (DHEW Publication 80-23, revised 1985, Office
of Science and Health reports, DRR/NIH, Bethesda, MD 20205).

Gonadectomy
All mice were gonadectomized and received systemic hormone
replacement with testosterone or estrogen to maintain physiologic levels in male
(Barkley and Goldman, 1977) and female (Jacob, et al., 2001) mice. Males were
52
castrated via a mid-line scrotal incision, and received a 5 mm Silastic implant (o.d.
2.16 mm, i.d. 1.02 mm, Dow Corning, Midland, MI) filled with crystalline
testosterone (Steraloids, Newport, RI) sc. Females were ovariectomized via
bilateral dorsal flank incisions, and received a similar implant of estradiol sc (1:1
17β-estradiol:cholesterol). Mice were allowed to recover from surgery for 7 days
before MPTP administration.

MPTP Administration
Male and female mice differ in their systemic tolerance for MPTP, with
females being more severely affected (Przedborski, et al., 2001). Thus, pilot
studies were conducted to establish the appropriate dose and inter-injection
interval for MPTP. MPTP (free-base, Sigma, St. Louis, MO) was dissolved in
0.9% saline and injected ip. Male and female mice received either (1) four
injections of 20 mg/kg MPTP at 2h intervals (n=15 males, n=25 females), (2) one
injection of 30 mg/kg MPTP (n=5 females), or (3) one injection of 10 mg/kg
MPTP daily for 5 days (n=15 males, n=15 females). Male mice tolerated all
doses and inter-injection intervals well. However, 80-90% of females died after
MPTP delivered via 4x20 mg/kg or 1x30mg/kg (see Table 3.1). With 4x20
mg/kg MPTP, deaths occurred after the second injection.
Male and female mice receiving 10 mg/kg MPTP per day for 5 days were
used to investigate sex differences in motor and neuromorphologic responses to
53
MPTP. Control male and female mice (n=15 each) received daily injections of
0.1 mL saline ip, also for 5 days.

Table 3.1. Mortality rates in male and female mice after MPTP.

MPTP Dose
Survival Rate
(Male Mice)
Survival Rate
(Female Mice)
4 x 20mg/kg MPTP at 2h
intervals
12 of 15 (80%) 3 of 25 (12%)
1 x 30mg/kg MPTP daily for 5
days
1 of 5 (20%)
1 x 10mg/kg MPTP daily for 5
days
15 of 15 (100%) 13 of 15 (87%)

Behavioral Tests
Behavioral testing was conducted under red light conditions during the
subjective dark phase. The rotarod, pole, and gait tests were used to measure
locomotor ability, bradykinesia, and stride length, respectively. Mice were
acclimatized to the behavioral testing apparatus 1-2 days before MPTP
administration and testing occurred 7-8 days following MPTP. One week is
sufficient for MPTP-induced cell death to occur (Jackson-Lewis, et al., 1995).
Rotarod. The latency to fall off a rotating spindle (rat size, diameter 7.3
cm, Columbus Instruments, Columbus, OH) was measured according to the
protocol of Rozas et al. (1998). Initially, mice were acclimatized to the rotarod
for 10 minutes at 5 rpm. During acclimatization, mice that fell were replaced on
the spindle. For behavioral testing, mice were tested at increasing speed (12, 14,
16, 18, 20, 22, 24, and 26 rpm) and the latency to fall was recorded for each
54
animal. The maximum duration at each speed was 150 seconds, with 150 seconds
rest between trials. The overall rotarod performance (ORP) for each group was
calculated by plotting the average latency to fall at each speed, and using the
trapezoidal method of Rozas et al. (1997) to estimate the area under the curve.
Pole Test. The time to turn head down (T-Turn) and completely descend
(T-Total) a pole measures bradykinesia according to Matsuura et al. (1997) Mice
were acclimatized to the cloth tape-wrapped metal pole (1 cm diameter, 50 cm
height) over 5 trials, each separated by 60 seconds. The first trial was limited to
300 seconds and subsequent trials were limited to 120 seconds. Behavioral
testing was identical to acclimatization, with the exception that all five trials were
capped at 120 seconds. Mice that failed to turn or fell off the pole were assigned
a time of 120 seconds. The three best scores were averaged for each mouse.
Gait Test. Stride length was measured according to the methods of
Fernagut et al. (2002). Briefly, mice were placed on an illuminated runway (4.5
cm wide, 42 cm long, with walls 12 cm high) and were allowed to run toward a
dark goal box (20x17x10 cm). Mice were acclimatized over two trials. Gait was
measured in a single trial. To differentiate between forelimb and hindlimb strides,
front and back paws were painted with red and black ink, respectively (Winsor
and Newton, London, England) as mice ran down the paper-lined runway. Stride
length was measured as the distance between successive paw prints. The average
of three strides was taken for each animal.

55
Perfusion and Immunocytochemistry
Mice were deeply anesthetized with sodium pentobarbital (150 mg/kg BW)
and perfused intracardially with 150 mL of 0.1 M sodium phosphate buffer (PB,
pH=7.4) containing 0.9% NaCl and 0.1% NaNO
3
. The brains were removed,
post-fixed in the perfusion fixative overnight and cryoprotected for 5 days at 4°C
with 20% sucrose in PB. The brains were rapidly frozen and sectioned coronally
at 30 um through the rostro-caudal extent of SNC according to the stereotaxic
atlas of the mouse brain (Paxinos and Franklin, 2001). Sections were stored in PB
with 0.01% sodium azide at 4°C until processed for TH immunocytochemistry.
Every sixth section was stained for TH. TH is the rate-limiting enzyme in
dopamine biosynthesis and is used as a marker for dopamine-producing neurons.
Sections from mice in different groups of the same experiment were stained at the
same time. Sections were incubated overnight at room temperature (RT) in
polyclonal rabbit anti-TH antibody (1:5000; Chemicon, Temecula, CA) with 4%
normal donkey serum and 0.3% Triton X-100 in PB. The following day, sections
were incubated in biotinylated donkey anti-rabbit secondary antibody (1:200;
Jackson Immunoresearch, West Grove, PA) and the avidin-biotin-horseradish
peroxidase complex (Vector Elite Kit; Vector Laboratories, Burlingame, CA),
each for 1 hour at RT with extensive washes in between. TH-labeled cells were
visualized using NiCl-enhanced 3’3-diaminobenzidine tetrahydrochloride with
0.25% hydrogen peroxide. Sections were mounted onto gelatin-coated slides, air-
dried, and counterstained for Nissl to identify neuronal nuclei. Afterwards, the
56
slides were dehydrated in alcohols, cleared in xylenes and coverslipped with
permount.
Unbiased stereological counts of dopaminergic neurons in SNC were
performed on 7-9 animals randomly selected from each experimental group. TH-
positive, Nissl-stained cells were counted bilaterally on coded slides by an
observer blind to the treatment group using an Olympus BX-50 microscope
(Olympus Optical, Tokyo, Japan) with motorized stage and Retiga-cooled CCD
camera (Q-imaging, Burnaby, British Columbia, Canada) according to the
methods of Petzinger et al. (2007). SNC was outlined in each section at low
magnification (10x) using the third cranial nerve and cerebral peduncle as
landmarks. A grid was placed over the outlined SNC and counting frames were
established at grid intersections. Counting was performed at 80x using the optical
fractionator technique assisted by the computer program BioQuant NovaPrime
(BioQuant Image Analysis, Nashville, TN). TH-positive cells were counted if
they had a visible nucleolus and were located within the counting frame. The
total number of TH-positive neurons in SNC was calculated based on the method
of Gundersen and Jensen (1987).

Statistics
Group differences were analyzed by a factorial analysis of variance
(ANOVA). Post-hoc comparisons using the Fisher’s LSD test were conducted
when statistically significant differences (p < 0.05) were found.
57
Results
In MPTP-treated mice, 100% of males survived the daily 10 mg/kg
injection schedule compared with 87% of females (p<0.05). At the time of
sacrifice, saline-injected male mice weighed 26.7±0.3 g while control females
weighed 24.2±0.2 g (p<0.05). MPTP caused a nonsignificant reduction in body
weight. However, MPTP-treated males also outweighed females (26.0±0.3 vs.
23.6±0.7 g).

Motor Behavior
There was a baseline sex difference in motor performance measured by
the rotarod, pole and gait tests. For all tests, males outperformed females. ORP
was greater in male mice (1317.0±98.3) than in females (988.1±95.6, p<0.05,
Figure 3.1a). There were no sex differences at low speeds (12 rpm), with male
and female mice having similar latencies to fall (p>0.05). Instead, sex differences
in rotarod performance emerged at higher speeds. At 26 rpm, males stayed on the
rotarod longer than females (49.6±13.1 vs. 14.9±2.9 sec, p<0.05).
Baseline sex differences were also observed on the pole test (Figure 3.2).
Although male and female mice had a similar latency to turn (T-Turn: 2.1±0.3 vs.
2.7±0.5 sec, n.s., Figure 3.2a), males descended the pole faster than females (T-
Total: 7.1±0.5 vs. 9.6±0.7 sec, p<0.05, Figure 3.2b). Finally, small but
significant differences were observed in stride length between male and female
mice (Figure 3.3). Males had a longer average forelimb stride length (7.4±0.1 cm)
58
than females (7.0±0.1 cm, p<0.05, Figure 3.3a). Similarly, mean hindlimb stride
length was 7.3±0.1 cm in males versus 6.8±0.1 cm in females (p<0.05, Figure
3.3b).

59

Figure 3.1. Rotarod performance in male (black) and female (white) mice
(n=13-15 per group). (A) Overall (Mean±SEM) rotarod performance (ORP)
for saline- (clear) and MPTP-treated (shaded) mice. Bars with common
letter subscripts are not significantly different. (B) Latency to fall off the
rotarod at 12-26 rpm (Mean±SEM) for saline- (squares) and MPTP-treated
(circles) mice. Asterisks represent sex differences (p<0.05) within the same
treatment group (MPTP or Saline).

60

Figure 3.2. Pole test performance in male (black bars) and female (white
bars) mice (n=13-15 per group). (A) Time to turn (T-Turn) and (B)
completely descend (T-Total) the pole (Mean±SEM) for saline- (clear) and
MPTP-treated (shaded) mice. Bars with common letter subscripts are not
significantly different.

61

Figure 3.3. Stride length in male (black bars) and female (white bars) mice
(n=13-15 per group). (A) Forelimb and (B) hindlimb stride lengths
(Mean±SEM) for saline- (clear) and MPTP-treated (shaded) mice. Bars with
common letter subscripts are not significantly different.

MPTP lesioning produced a similar deficit in rotarod performance in
males and females. MPTP caused a 47% reduction in ORP to 703.7±65.5 in male
62
mice (p<0.05 vs. unlesioned males, Figure 3.1a). MPTP-lesioned female mice
also performed more poorly on the rotarod. ORP was reduced 56% to 432.8±88.6
in MPTP-lesioned female mice (p<0.05 versus unlesioned females, Figure 3.1a).
MPTP treatment altered the pattern of performance on the rotarod in males and
females. Unlike vehicle-injected animals, MPTP-lesioned males remained on the
rotarod longer than females at low speeds (99.5±14.8 versus 59.8±8.8 sec,
p<0.05). At 26 rpm, the latencies to fall for males (14.6±4.4 sec) and females
(4.5±1.8 sec) were not significantly different.
MPTP did not impair performance on the pole test in male or female mice
(Figure 3.2). MPTP-treated males had similar times for T-Turn (2.4±0.4 sec) and
T-Total (7.0±0.5 sec) compared to non-lesioned males. Likewise, T-Turn
(2.9±0.8 sec) and T-Total in MPTP-treated female mice (10.3±1.0 sec) were not
different from unlesioned females.
Stride length was slightly, but selectively reduced in male mice after
MPTP. Forelimb stride length was 6.9±0.1 cm in MPTP-lesioned males
compared to 7.4±0.1 cm in unlesioned males (p<0.05, Figure 3.3a). Hindlimb
stride length was also decreased in MPTP-lesioned males (6.9±0.2 cm versus
unlesioned males, p<0.05, Figure 3.3b). In contrast, MPTP-lesioned female mice
had comparable forelimb (6.9±0.1 cm) and hindlimb (6.8±0.2 cm) stride lengths,
relative to unlesioned females (Figure 3.3).

63
Cell Counts
Cell counts of TH-positive, Nissl-stained neurons in SNC are shown in
Figure 3.4. Unlesioned male and female mice had comparable numbers of TH-
positive neurons in SNC (11,174.1±620.0 vs. 9,927.6±978.9, n.s.). MPTP
lesioning did not deplete dopaminergic SNC neurons in males (11,192.6±1,295.4)
or in females (11,246.1±1,064.9), and there were no sex differences.

64

Figure 3.4. Photomicrographs of TH-positive neurons in SNC of saline- (A)
and MPTP-treated (B) mice. (C) Mean±SEM number of TH-labeled neurons
in male (black bars) and female (white bars) mice (n=7-9/group) in SNC.
There were no significant differences between groups. Scale bar =200um.
Abbreviations: 3n, third cranial nerve; SNC, substantia nigra pars compacta;
TH, tyrosine hydroxylase; VTA, ventral tegmental area.

Discussion
The current study investigated sex differences in motor impairments and
anatomical pathology in the MPTP mouse model of Parkinson’s disease. To
accomplish this, we used a subacute MPTP treatment, whereby mice received
65
daily injections of 10 mg/kg MPTP for five days. Higher doses of MPTP killed
female mice. In control animals, male mice outperformed females on the rotarod,
pole, and gait tests. MPTP lesioning reduced performance on the rotarod in both
male and female mice. However, gait was selectively impaired in males after
MPTP. In SNC, the total number of dopaminergic neurons was similar in all
groups, despite baseline sex differences in motor performance and motor
impairments after MPTP. Taken together, our results show that small, chronic
doses of MPTP attenuate locomotor ability without depleting dopaminergic
neurons in SNC and are also sufficient to reveal sex differences in gait
disturbance.
Interestingly, the largest MPTP-induced sex difference was in mortality.
At 10 mg/kg for 5 days, more females than males died after MPTP. In fact, when
we conducted pilot studies using larger doses of MPTP, 80-90% of female mice
died. In contrast, male mice have received substantially higher doses of MPTP
without mention of mortality (Fredriksson and Archer, 1994; Schmidt and Ferger,
2001). Death following MPTP is likely due to the systemic actions of MPTP on
the cardiovascular system (Jackson-Lewis and Przedborski, 2007), but it is
unknown why females are particularly susceptible. Based on our pilot studies the
lethal threshold for female mice is approximately 30 mg/kg MPTP. A single
injection of 30 mg/kg MPTP killed 80% of female mice within 2 hours. However,
when females were given 20 mg/kg injections at two-hour intervals, the majority
of females died shortly after the second injection.
66
Although female mortality was the primary reason for selecting the low
MPTP dose and prolonged interinjection interval, subacute administration of
MPTP at 10mg/kg revealed modest impairments in motor behavior that were
sexually dimorphic. By contrast, motor impairments after large doses of MPTP
may be so severe as to mask underlying sex differences. Indeed, larger doses of
MPTP produce considerable motor deficits (reveiwed in Sedelis et al., 2001). For
example, repeated injections of 36 mg/kg MPTP with acetaldehyde over several
weeks produced an 85% reduction in rotarod performance (Rozas et al., 1998).
Not surprisingly, large doses of MPTP are used to replicate late-stage
Parkinsonism (Schober, 2004). In PD patients, the motor phenotype is sexually
dimorphic, with men and women experiencing different symptoms (Baba et al.,
2005; Haaxma et al., 2007; Lyons et al., 1998; Scott, et al., 2000). For example,
both Accolla et al. (2007) and Lyons (1998) have reported a higher prevalence of
dyskinesia in women. However, by end-stage PD, sex differences are no longer
mentioned. Presumably, sex differences in motor behavior that are present in
earlier stages of PD disappear as the disease progresses and as patients become
more profoundly disabled.
Unfortunately, due to the tremendous variability among published studies
in the dose and timing of MPTP administration and in the selection of behavioral
protocols, there is little consensus on motor deficits following MPTP treatment
(Sedelis et al., 2001). On one hand, a number of papers have shown no difference
in motor performance even with larger doses of MPTP. For example, neither
67
Petroske et al. (2001) nor Sedelis (2000)observed rotarod deficits after subacute
(25 mg/kg daily for 5 days) and acute (4 x 15 mg/kg at 2 h intervals) doses of
MPTP, respectively. Others have shown substantial motor deficits after MPTP
(Arai, et al., 1990; Rozas et al., 1998; Tomac, et al., 1995). Our data demonstrate
that with multiple behavioral tests, it is possible to show deficits in motor
performance and, more specifically, to reveal subtle sex differences in behavioral
impairment after MPTP.
It is well-established that nigrostriatal dopaminergic projections are
essential for motor behavior. Nonetheless, our data plus those of others
demonstrate a substantial deficit in motor performance without the destruction of
dopaminergic neurons in SNC. This is likely due to the loss of dopaminergic
terminals in the striatum and the subsequent reduction of striatal dopamine (Lau
and Meredith, 2003; Petroske et al., 2001). In the current study, 10mg/kg MPTP
did not significantly deplete TH-positive neurons within SNC. This is consistent
with Petroske et al. (2001) who delivered daily injections of 25 mg/kg MPTP
without inducing dopaminergic cell death in SNC. However, unlike Petroske
(2001), we observed significant motor deficits on the rotarod in both male and
female mice. While Petroske et al. (2001) used a small-diameter spindle to test
rotarod performance, we followed the protocol of Rozas et al. (1998), and used a
larger, rat-sized spindle (7.3 cm diameter). This spindle distinguishes locomotor
ability from balance and/or coordination, and is more effective than smaller
spindles at detecting motor deficits after nigrostriatal lesions (Rozas et al., 1998).
68
While we did see effects of 10 mg/kg MPTP on the rotarod, there was no
effect on pole test performance. The absence of bradykinesia in the pole test may
be related to the low sensitivity of the test. Bradykinesia in rodents has been
primarily revealed after substantial lesions and destruction of SNC cells and/or
dopaminergic terminals in the striatum (Diguet, et al., 2005; Ogawa, et al., 1985).
Given that our dose and timing of MPTP were relatively modest, it is not
surprising that mice did not show deficits on the pole test. In contrast, rotarod
performance declined on average of 50% in MPTP-treated mice. Studies using a
similar rotarod protocol have demonstrated larger deficits in ORP, albeit with
larger doses of MPTP (Duan and Mattson, 1999; Rozas et al., 1998). Nonetheless,
the rotarod was sufficient to detect motor impairments in both male and female
mice.
While we saw no effect of MPTP on pole test performance, and equivalent
deficits on the rotarod, measures of stride length proved to be the most sensitive
for detecting sex differences after MPTP. Male mice had a selective reduction in
stride length after MPTP. Other studies have produced comparable reductions in
gait using male mice, with larger doses of MPTP (Amende, et al., 2005;
Mohanasundari, et al., 2006).
Body weight may explain differences in stride length between vehicle-
injected male and female mice, but does not explain why only MPTP-treated male
mice had decreased stride lengths. Previous studies have found that larger rodents
have longer stride lengths than smaller rodents (Heglund, et al., 1974; Koopmans,
69
et al., 2007; Parker and Clarke, 1990). In the current study, vehicle-injected male
mice significantly outweighed females and had longer strides. However, MPTP-
injected male mice also outweighed MPTP-injected females by the same margin.
Despite similar differences in body weight, stride lengths were equivalent in male
and female mice after MPTP, because males had a specific reduction in gait.
Rather, the lack of gait impairment in female mice may relate to gonadal
steroid hormones. In mice, the susceptibility of the nigrostriatal system to MPTP
is sexually dimorphic. After MPTP, female mice maintain higher striatal
dopamine levels (Miller et al., 1998; Xu et al., 2006) than males. This is largely
attributed to estrogen, which has neuroprotective effects throughout the brain
(reviewed in Garcia-Segura et al., 2001) that include modulating the response of
the nigrostriatal system to MPTP neurotoxicity (reviewed in Dluzen, 2000). In
ovariectomized female mice, estrogen replacement attenuates the loss of striatal
dopamine after MPTP (Dluzen, et al., 1996a; Dluzen et al., 1996b). In contrast,
testosterone does not alter the levels of dopamine in the striatum in response to
MPTP in male mice (Dluzen, 1996). In our hands, the ability of MPTP to reduce
stride length specifically in male mice provides a behavioral parallel to these
previous studies. In addition, by showing sex-specific impairments after MPTP,
our data support the hypothesis that gonadal steroid hormones may account for
the susceptibility of male mice to MPTP. Future studies, using gonadectomized
mice may help to determine whether the actions of gonadal hormones in the adult
contribute to the sex differences in motor performance seen in the current study.
70
Chapter 4
EFFECTS OF MPTP-INDUCED NEUROTOXICITY AND
TESTOSTERONE ON NIGROSTRIATAL TERMINALS AND STRIATAL
MEDIUM SPINY NEURONS

Abstract
Sex differences in Parkinson’s disease (PD) have been reported in humans
and in rodent models, with a higher incidence in men and an increased severity in
male rodents. Gonadal steroid hormones are thought, in part, to underlie this sex
difference. While estrogen is neuroprotective in the nigrostriatal system of
females, less is known about the effects of testosterone on this system in males.
The current study determined whether testosterone is neuroprotective in the male
nigrostriatal system by comparing gonad-intact and castrated male mice after
nigrostriatal lesions using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).
Male mice were either gonadectomized (n=23) or sham-gonadectomized (n=22).
Two weeks later, mice received 4 injections of MPTP (20 mg/kg BW ip) or saline,
both at 2 hour intervals. One week after the last injection, motor performance was
measured by rotarod and stride length. After behavioral testing, animals were
sacrificed, and the brains were removed and hemisected. Coronal sections
through the striatum of the right hemisphere were immunostained for tyrosine
hydroxylase (TH) to quantify nigrostriatal dopaminergic inputs. The left
hemisphere was stained using the Golgi-Cox technique to quantify neuronal
71
morphology in medium spiny neurons (MSNs) of the dorsolateral striatum.
MPTP caused a significant decrease in striatal TH immunoreactivity (mean gray
level 59.4±3.7 vs. 98.5±3.1 for unlesioned males, p<0.05). Despite this lesion,
MPTP did not impair motor performance and exerted only modest effects on
MSN dendritic morphology. MPTP decreased the average highest branch order
and increased spine density on proximal dendrites. Testosterone had separate
effects to reduce dendritic length of distal dendrites. Castrated males had shorter
5
th
order dendrites (78.9±14.8 um) than gonad-intact males (138.3±13.2 um,
p<0.05). As with MPTP, testosterone did not alter motor performance. There
was no interaction between testosterone and MPTP (p>0.05). Taken together, our
results suggest that testosterone does not exert neuroprotective effects in the
nigrostriatal system of males. While testosterone enhances neuronal morphology
on MSNs, the stimulatory effects of testosterone on neuronal morphology do not
overcome the MPTP effect.

Introduction
Gonadal steroid hormones are potent modulators of neuronal survival and
neuronal morphology (reviewed in Garcia-Segura et al., 1994). During
development, steroids exert organizational effects which lead to permanent sex
differences in the brain. In the adult, steroid hormones exert transient,
activational effects to promote neuroplasticity in steroid-sensitive brain regions.
Although gonadal steroids also exert protective effects against neurodegeneration
72
(Bialek et al., 2004; Garcia-Segura et al., 2001), sex differences exist in many
neurological disorders. Sex differences arise from a combination of
organizational and activational effects of hormones. As it relates to
neuroprotection, the occurrence of sex differences suggests that male and female
brains are not equally responsive to the neuroprotective effects of gonadal steroids.
Parkinson’s disease (PD) is a common neurological disorder that results
from the progressive loss of dopaminergic neurons in the substantia nigra pars
compacta (SNC; Fahn, 2003). Dopaminergic efferents from SNC project rostrally
to the striatum as part of the nigrostriatal pathway and synapse onto medium
spiny neurons (MSN’s) in the dorsolateral striatum. In PD, striatal dopamine is
depleted and there is a reduction in dopaminergic afferents to MSN’s. Sex
differences have been reported for PD, and include a one- to two-fold higher
incidence in men (Baldereschi et al., 2000; de Lau et al., 2004; Elbaz et al., 2002;
Schrag et al., 2000; Shulman and Bhat, 2006; Taylor et al., 2007; Van Den Eeden
et al., 2003; Wooten et al., 2004).
Estrogen promotes the function of the nigrostriatal system in females. In
the striatum, estrogen enhances dopamine release (Becker, 1990; McDermott, et
al., 1997; McDermott et al., 1994; Xiao and Becker, 1998), increases dopamine
metabolism (Castner et al., 1993; Di Paolo, et al., 1985) and alters dopamine
receptors (Lammers, et al., 1999; Levesque et al., 1989; Tonnaer, et al., 1989) and
uptake sites (Disshon, et al., 1998; Karakaya, et al., 2007). Estrogen is also
neuroprotective in the nigrostriatal system (reviewed in Dluzen, 2000). This has
73
been well-demonstrated using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) and 6 -hydroxydopamine (6-OHDA), neurotoxins which selectively
deplete dopaminergic SNC neurons (Schober, 2004). In both models, estrogen
attenuates the loss of striatal dopamine and minimizes cell death in SNC (Disshon
and Dluzen, 1997; Dluzen, 1997; Dluzen et al., 1996b; Ferraz et al., 2008; Miller
et al., 1998; Murray et al., 2003; Shughrue, 2004).
In parallel to its effects in the female, estrogen also modulates nigrostriatal
function in males (Becker, 1999; Bourque et al., 2009). Estrogen in males is
derived by the local aromatization of testosterone. Testosterone also acts on its
own as an androgen. However, relatively little is known as to whether
testosterone has neuroprotective effects in the male nigrostriatal system, similar to
the effects of estrogen in the female. Of the few studies that have addressed this
question, the majority suggest no effect of testosterone. For example, striatal
dopamine depletion after MPTP in castrated mice is attenuated by estrogen but
not by testosterone (Dluzen, 1996; Ekue et al., 2002). However, others report that
both testosterone and estrogen exacerbate 6-OHDA-induced striatal dopamine
depletion in males (Gillies et al., 2004).
Previous studies investigating the effects of testosterone on nigrostriatal
function in males after 6-OHDA or MPTP have measured dopamine content and
release (Dluzen, et al., 1994; Dluzen, 1996; Ekue et al., 2002; Gillies et al., 2004),
dopaminergic innervation to the striatum (Gillies et al., 2004), and dopamine
transporter binding (Ekue et al., 2002; Gillies et al., 2004) after 6-OHDA or
74
MPTP. It is unknown whether testosterone exerts downstream neuroprotective
effects on behavior or neuronal morphology after dopamine-depleting lesions.
The current study used castrated and gonad-intact adult male mice to determine
whether testosterone reduces MPTP-induced deficits on motor function,
neurochemistry, and neuronal architecture. We measured rotarod performance and
stride length to determine whether motor impairments after MPTP are more
severe in male mice lacking testosterone. In addition, tyrosine hydroxylase (TH)
immunoreactivity was measured in the striatum to determine whether nigrostriatal
innervation is more depleted in male mice lacking testosterone. TH is the rate
limiting enzyme in dopamine biosynthesis and is used as a marker for dopamine-
producing neurons. Finally, neuronal morphology was quantified in Golgi-labeled
striatal MSN’s to determine whether testosterone modulates the efferent targets of
SNC.

Methods
Animals
Forty-five adult male mice (8-10 weeks old) were obtained from Charles
River Laboratories (Wilmington, MA). Mice were group-housed on a 12:12 LD
photoperiod with access to food and water ad libitum. Experimental procedures
were approved by USC’s Institutional Animal Care and Use Committee and
conducted in accordance with the NIH Guide for the Care and Use of Laboratory
75
Animals (DHEW Publication 80-23, revised 1985, Office of Science and Health
reports, DRR/NIH, Bethesda, MD 20205).
Initially, half of the mice (n=23) were castrated via a midline scrotal
incision. The other half (n=22) received sham gonadectomies. Two weeks later,
half of the mice in each group received MPTP and half were given saline. MPTP
(free-base, Sigma, St. Louis, MO) was dissolved in 0.9% saline, and was
administered in 4 injections of 20 mg/kg ip with an interinjection interval of 2
hours. Control mice received 4 injections of 0.1 mL saline ip, also at 2-hour
intervals.
One week following MPTP, animals were tested for motor performance
and then sacrificed. One week is sufficient for MPTP-induced cell death to occur
(Jackson-Lewis et al., 1995). Brain tissue was collected to measure TH
immunoreactivity in the striatum, and to quantify morphology of Golgi-stained
MSN’s.

Behavior
Behavioral testing was conducted on consecutive days under red light
conditions during the subjective dark phase.
Rotarod. The latency to fall off a rotating spindle (rat size, diameter 7.3
cm, Columbus Instruments, Columbus, OH) was measured according to the
protocol of Rozas et al. (1998) and reflects locomotor ability. Mice were
acclimatized to the rotarod for 10 minutes at 5 rpm 1 day before MPTP
76
administration. During acclimatization, mice that fell were replaced on the
spindle. For behavioral testing, mice were tested at increasing speeds (12, 14, 16,
18, 20, 22, 24, and 26 rpm) and the latency to fall was recorded for each animal.
The maximum duration at each speed was 150 seconds, with 150 seconds rest
between trials. The overall rotarod performance (ORP) for each group was
calculated by plotting the average latency to fall at each speed, and using the
trapezoidal method of Rozas et al. (1997) to estimate the area under the curve.
Stride Length was measured according to the methods of Fernagut et al.
(2002). Reductions in stride length reflect nigrostriatal dysfunction (Jankovic,
2008; Morris, et al., 1994). Briefly, mice were placed on an illuminated runway
(4.5 cm wide, 42 cm long, with walls 12 cm high) and were allowed to run toward
a dark goal box (20x17x10 cm). Gait was measured in a single trial. To
differentiate between forelimb and hindlimb strides, front and back paws were
painted with red and black ink, respectively (Winsor and Newton, London,
England) as mice ran down the paper-lined runway. Stride length was measured
as the distance between successive paw prints. The average of three strides was
taken for each animal.

Perfusion
One day following behavioral testing, mice were deeply anesthetized with
sodium pentobarbital (150 mg/kg BW) and perfused intracardially with 150 mL
of 0.1M sodium phosphate buffer (PB, pH=7.4) containing 0.9% NaCl and 0.1%
77
NaNO
3
. The brains were removed and hemisected. The right hemisphere was
processed for TH immunocytochemistry and the left hemisphere was processed
for Golgi-Cox staining.

Tyrosine Hydroxylase Immunocytochemistry
The right hemispheres from each brain were placed in 4%
paraformaldehyde in PB overnight at 4°C. Hemispheres were then cryoprotected
for 5 days at 4°C with 20% sucrose in PB. The hemispheres were rapidly frozen
and sectioned coronally at 25 um through the rostro-caudal extent of the striatum.
Sections were stored in PB with 0.01% sodium azide at 4°C until processed for
TH immunocytochemistry.
Sections through the striatum at or rostral to the anterior commissure
corresponding to Plates 18-28 of Paxinos and Franklin (2001) were stained for
TH. Tissue from mice in different groups was stained at the same time. Sections
were incubated overnight at room temperature (RT) in polyclonal rabbit anti-TH
antibody (1:5000; Chemicon, Temecula, CA) with 4% normal donkey serum and
0.3% Triton X-100 in PB. The following day, sections were incubated in
biotinylated donkey anti-rabbit secondary antibody (1:200; Jackson
Immunoresearch, West Grove, PA) and the avidin-biotin-horseradish peroxidase
complex (Vector Elite Kit; Vector Laboratories, Burlingame, CA), each for 1 hour
at RT with extensive washes in between. TH-labeled cells were visualized using
NiCl-enhanced 3’3-diaminobenzidine tetrahydrochloride with 0.25% hydrogen
78
peroxide. Sections were mounted onto gelatin-coated slides. The following day,
the slides were dehydrated in alcohols, cleared in xylenes and coverslipped with
Permount.
The relative expression of TH-immunoreactivity was measured in
dorsolateral striatum on coded slides by an observer blind to the treatment group.
Striatal sections were matched across animals to ensure consistent sampling.
Three striatal sections were sampled per animal (n=8-9 animals per group) using
methods previously described by our laboratory (Fisher, et al., 2004; Petzinger et
al., 2007). Briefly, striatal sections were digitally photographed at low
magnification. The dorsolateral quadrant of each striatal section was outlined and
the gray level was measured in a 1.6 mm
2
circular region of interest at the
dorsolateral boundary of this quadrant (Fig 4.2). TH staining intensity, expressed
as mean gray level, was obtained by subtracting the background gray level in the
corpus callosum from the measured gray level in the striatum.

Golgi Cox Staining
Golgi-Cox staining was performed on the left hemisphere of each brain
using the FD Rapid GolgiStain Kit (FD NeuroTechnologies, Ellicott City, MD).
The hemispheres were placed in Golgi-Cox solution containing mercuric chloride,
potassium dichromate and potassium chromate for 2 weeks and the solution was
replaced after the first 24 hours. The brains were moved to a cryoprotection
solution (GolgiStain Kit) for 48 hours and then sectioned coronally at 200 um on
79
a vibratome (Vibratome Series 1000). Sections through the rostral-caudal extent
of the striatum were mounted on gelatin-coated slides. Slides were stored in a
humidity chamber overnight and developed the following day according to the
Rapid GolgiStain Kit protocol. Briefly, the slides were rinsed in distilled water
and placed in a developing solution for 10 minutes. Immediately afterwards, the
slides were rinsed, dehydrated in alcohols, cleared in xylenes, and coverslipped
with Cytoseal-60 mounting medium (Richard-Allan Scientific). Slides were
stored in the dark at RT until morphological analysis.
MSN morphology was analyzed on coded slides by an observer blind to
the treatment groups using a Nikon Eclipse 80i microscope (Nikon Instruments,
Inc., Melville, NY) with motorized stage and MicroFire camera (Olympus
America, Inc., Center Valley, PA). Five cells from each animal were analyzed
using NeuroLucida (MicroBrightField,Inc., Williston, VT). MSN’s selected for
analysis (5 per animal) were located in the dorsolateral quadrant of the striatum at
or rostral to the level of the anterior commissure (Plates 18-28 of Paxinos and
Franklin, 2001). Selected MSN’s were fully impregnated with Golgi stain and
had clearly visible spines. Additionally, obstruction by neighboring Golgi-stained
cells or blood vessels was minimal or absent. Using a 100x oil immersion lens,
the soma and entire dendritic tree from one primary dendrite per cell were traced
and spines identified using the Neuron Tracing function in NeuroLucida.
Morphometric analysis was conducted using NeuroExplorer software
(MicroBrightField, Inc.). Briefly, each dendritic segment was assigned a branch
80
order with the dendritic segment proximal to the soma identified as the first
branch order. Dendritic lengths, number of spines, and spine density were
computed for each branch order. Due to the relative lack of spines on primary
dendrites, branch order analysis was not performed on first-order dendrites. In
addition, total spine density and total dendrite length were calculated for the entire
dendritic tree.

Statistics
For both behavioral tests and anatomical measures, group differences were
analyzed by two factor (gonadal status and lesion) analysis of variance (ANOVA).
Post-hoc comparisons using the Fisher’s LSD test were conducted when
statistically significant differences (p < 0.05) were found.

Results
Motor Performance
All groups of mice performed similarly on the rotarod and had similar
stride lengths (Fig. 4.1). On the rotarod (Fig. 4.1a-b), mice had comparable
latencies to fall across the range of speeds tested (12-26 rpm) and had similar
ORPs. MPTP treatment did not decrease ORP. MPTP-lesioned animals had an
average ORP of 969.3±75.9 compared with 929.2±105.1 in saline-injected mice
(p>0.05, Fig. 4.1a). Castration was also without effect and there was no
81
interaction between MPTP treatment and gonadectomy on rotarod performance
(p>0.05, Fig. 4.1a-b).
Likewise, there were no effects of MPTP or gonadectomy on stride length,
and no interaction (p>0.05, Fig. 4.1c). Forelimb and hindlimb stride length
averaged 7.4± 0.1 cm and 7.2±0.1 cm, respectively, across all groups of animals.

Striatal TH
As reported previously by our lab (Fisher et al., 2004; Jakowec et al., 2004;
Petzinger et al., 2007; Vuckovic, et al., 2008), MPTP decreased TH
immunoreactivity in the striatum (Fig 4.2a-b). Striatal TH was reduced by 60%
after MPTP (mean gray level 98.5±3.1 vs. 59.4±3.7 in MPTP-treated mice,
p<0.05, Fig. 4.2c). However, there was no effect of castration on TH
immunoreactivity and no interaction between MPTP and gonadectomy (p>0.05,
Fig. 4.2c).

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Figure 4.1. Behavioral Measures. Rotarod performance in gonad-intact
(black) and gonadectomized (white) mice (n=9-12 per group). (A) Overall
rotarod performance (ORP) for saline- (clear) and MPTP-treated (shaded)
mice (Mean±SEM). (B) Latency to fall off the rotarod at 12-26 rpm for
saline- (squares) and MPTP-treated (circles) mice(Mean±SEM). (C)
Forelimb (black) and hindlimb (white) stride length (Mean±SEM) in saline-
injected (clear) and MPTP-treated (shaded) gonad-intact and
gonadectomized mice (n=8-9 mice per group). For all behavioral measures,
there were no significant effects of MPTP or gonadectomy. Abbreviations:
GDX, gonadectomized; Sham, sham-gonadectomized.

83

84
Figure 4.2. TH-staining in the dorsolateral striatum. Photomicrographs of
TH staining in saline-injected (A) and MPTP-treated (B) mice. (C) The
density of TH staining (Mean±SEM) in the dorsolateral striatum of saline-
injected (clear) and MPTP-treated (shaded) gonad-intact (black bars) and
gonadectomized (white bars) male mice (n=8-10 mice per group). The bar
represents an effect of MPTP (p<0.05). Abbreviations: GDX,
gonadectomized; Sham, sham-gonadectomized.

85

86
MSN Morphology
Spine Density. MSN’s have elaborate dendritic arbors with a high density
of dendritic spines (Fig. 4.3). Spines were largely absent from primary dendrites,
but increased on more distal dendrites. MPTP produced a modest increase in total
spine density (spines per 10 um) on MSNs (6.9±0.1 vs. 6.3±0.1 in saline-injected
mice, p<0.05, Fig. 4.4a). When analyzed according to branch order, the increase
in spine density was restricted to proximal dendrites (Fig. 4.5a). Specifically,
MPTP-treated mice had a higher spine density on 2
nd
order (5.3±0.3) and 3
rd
order
(7.1±0.2) dendrites, compared with 4.2±0.2 and 6.5±0.2 in saline-injected mice,
respectively (p<0.05, Fig. 4.5a). However, there was no effect of castration on
spine density and no interaction (p>0.05, Figs. 4.4a and 4.5a).
Branch Order. MPTP significantly decreased the average highest branch
order in gonad-intact and castrated male mice (3.9±0.1) compared to saline-
injected males (4.3±0.1, p<0.05, Fig. 4.4b). As with other measures of overall
neuronal morphology, castration was without effect and there was no interaction
(Fig. 4.4b).
Dendritic Length. There was no effect of MPTP on total dendrite length
(402.0±17.4 um vs. 415.5±20.2 um in saline-injected mice, p>0.05, Fig. 4.4c).
However, in parallel to the increase in spine density, we observed a selective
increase in dendritic length after MPTP on 2
nd
order dendrites (80.2±6.8 um vs.
61.6±4.6 um in saline-injected mice, p<0.05, Fig. 4.5b). Castration had no effect
on total dendritic length. However, castrated mice had a prominent reduction in
87
dendritic length of distal dendrites. Specifically, 5
th
order dendritic length was
significantly reduced in castrated mice (78.9±14.8 um) compared with gonad-
intact males (138.3±13.2 um, p<0.05, Figure 4.5b). There was no interaction
between MPTP treatment and gonadectomy (Figs. 4.4c and 4.5b).

88

Figure 4.3. Golgi stained MSNs. Photomicrograph of Golgi-impregnated
medium spiny neuron A) and corresponding neurolucida tracing (B).

89
Figure 4.4. Neuronal morphology totals. Total spine density (A), average
highest branch order (B) and total dendrite length (C) in saline-injected
(clear) and MPTP-treated (shaded) gonad-intact (black bars) and
gonadectomized (white bars) male mice (n=5 mice per group). Values are
expressed as Mean±SEM. Bars represent an effect of MPTP (p<0.05).
Abbreviations: GDX, gonadectomized; Sham, sham-gonadectomized.

90

91

Figure 4.5. Branch order totals. Spine density (A) and dendritic length (B)
for first to fifth order branches in striatal medium spiny neurons (n=5 mice
per group). Values are expressed as Mean±SEM. Bars represent an effect of
MPTP and asterisks represent an effect of castration (p<0.05).

Discussion
The current study used the MPTP mouse model of PD to investigate the
effects of testosterone on dopamine-depleting lesions of the nigrostriatal system.
MPTP decreased striatal TH immunoreactivity, reduced the average highest
branch order on MSN’s, and increased proximal spine density. Separately,
testosterone reduced dendritic length of distal dendrites. We predicted that
92
testosterone would act as a neuroprotectant to attenuate the effects of MPTP.
However, there was no interaction between gonadal hormone status and MPTP,
suggesting that testosterone does not exert neuroprotective effects in the
nigrostriatal system of males.
At first, the lack of interaction between testosterone and MPTP suggests
that the nigrostriatal system is not sensitive to gonadal steroid hormones in males.
Our findings agree with those of Dluzen et al. (1996), who administered MPTP to
gonad-intact and castrated male mice and found no effect of testosterone on
striatal dopamine. Studies using methamphetamine, which also depletes striatal
dopamine, have shown that amphetamine-induced stereotyped behaviors are
similar in gonad-intact and castrated animals (Camp et al., 1986).
Although testosterone is without effect, estrogen is neuroprotective in the
male mouse striatum (reviewed in Dluzen, 2000). In particular, estrogen
attenuates striatal dopamine depletion (Dluzen et al., 1996a; Dluzen et al., 1996b;
Ramirez, et al., 2003) and partially prevents the loss of striatal TH
immunoreactivity (Ookubo, et al., 2008; Shughrue, 2004) after MPTP. This
ability of estrogen, but not testosterone, to exert neuroprotective effects after
MPTP may be explained by regional differences in the metabolism of testosterone.
The expression of aromatase, the enzyme that converts testosterone to estrogen in
the male brain, is minimal in the striatum of male rodents (Kuppers and Beyer,
1998; Roselli, et al., 1997; Wagner and Morrell, 1996; Wagner and Morrell,
1997). Therefore, it is possible that the male striatum is capable of responding to
93
the neuroprotective actions of estrogen, but that the absence of aromatase limits
the local availability of estrogen in vivo.
Alternatively, the major androgenic and estrogenic metabolites of
testosterone may antagonize each other, thus limiting the neuroprotective ability
of testosterone. In the two studies that have addressed this question, both have
shown no effect of the non-aromatizable androgen dihydrotestosterone (DHT)
after dopamine-depleting lesions (Ekue et al., 2002; Murray et al., 2003).
However, these two reports differ with respect to estrogen. While Murray et al.
(2003) reported that estrogen treatment to castrated male rats exacerbated 6-
OHDA neurotoxicity, Ekue et al. (2002) demonstrated neuroprotective effects of
estrogen after MPTP in gonad-intact male mice. Nonetheless, the absence of an
effect of DHT in either study casts doubt on the possibility of antagonistic effects
of DHT and estrogen in the nigrostriatal system.
Testosterone did exert selective effects on MSN morphology. Specifically,
castration decreased dendritic length on MSN distal dendrites. This parallels the
actions of testosterone in hormone-sensitive areas of the brain. For example,
castration decreases dendritic branching and somal size in the posterior medial
amygdala (Gomez and Newman, 1991) and medial preoptic area (Cherry et al.,
1992), two key nuclei in the reproductive circuitry of the brain. In addition,
castration reduces spine density on hippocampal CA1 neurons in adult mice
(Leranth, et al., 2003). Testosterone-driven effects on neuronal morphology are
thought to occur via classical, genomic receptors for androgen or estrogen.
94
Interestingly, while the medial amygdala, medial preoptic area, and hippocampus
each contain classical receptors for both androgens and estrogen, the striatum is
devoid of classical hormone receptors (Simerly et al., 1990). Thus, it is unlikely
that testosterone acts in the striatum via similar neuronal mechanisms.
The morphologic response of striatal MSN’s to gonadal hormones may
occur through non-classical receptors or via indirect effect of testosterone on
upstream targets. The major afferent inputs to MSN distal dendrites originate in
the cortex and SNC (Gerfen, 1988). While the cortex contains a weak distribution
of gonadal hormone receptors, SNC contains even less (Simerly et al., 1990).
Therefore, it is unlikely that the classical actions of testosterone in either cortex or
SNC are strong enough to produce dramatic downstream changes to MSN
dendrites. Rather, testosterone may be acting through non-classical mechanisms
within the striatum to alter MSN dendritic morphology.
Although we found no interaction between gonadal hormone status and
MPTP neurotoxicity, males were still susceptible to MPTP. The 4x20 mg/kg dose
schedule for MPTP has been used extensively in our laboratory (Fisher et al.,
2004; Jakowec et al., 2004; Jakowec and Petzinger, 2004; Petzinger et al., 2007;
Vuckovic et al., 2008) and by other investigators. This lesioning protocol
produces a substantial and significant decrease in TH immunoreactivity. It is
therefore somewhat surprising that MPTP produced little or no change in motor
behavior and MSN morphology.
95
The comparative lack of effect of MPTP on motor function in mice is
well-known, if still poorly understood. MPTP lesioning in monkeys mimics the
full spectrum of Parkinsonian dysfunction (REFS) and stereotaxic injection of 6-
OHDA into SNC of rats produces consistent unilateral motor deficits (REFS). By
contrast, even severe MPTP lesions in mice produce inconsistent motor responses.
It has been hypothsized that >80% dopamine depletion is essential to demonstrate
consistent behavioral responses (Di Monte, et al., 2000; Kirik, et al., 1998; Lee, et
al., 1996; Mori, et al., 2005). However, even after large MPTP lesions, other
studies have found no motor impairment (Leng, et al., 2004; Willis and Donnan,
1987) or improved motor performance (Chia, et al., 1996; Colotla, et al., 1990;
Rousselet, et al., 2003). The lack of effect on motor function in the present study
is consistent with the range of responses reported previously.
The relatively modest effect of MPTP on MSN morphology was
somewhat less predictable. Dopaminergic nigrostriatal afferents synapse on
MSNs, primarily at the neck of dendritic spines (Gerfen, 1988; Smith and Bolam,
1990). Due to the loss of dopaminergic input, we expected to see a decrease in
dendritic spines. Instead, MSN’s exhibited a modest increase in proximal
dendritic spines. Interestingly, MSN spine density also increases after
methamphetamine, but this effect is selective to distal dendrites (Jedynak, et al.,
2007; Li, et al., 2003; Robinson and Kolb, 2004). The topographically distinct
increases in MSN spine density are associated with different striatal inputs.
Proximal MSN dendrites receive synaptic inputs from within the striatum, while
96
distal dendrites receive extrinsic inputs from the cortex and SNC (Smith and
Bolam, 1990). Taken together, this suggests that the slight increase in proximal
dendritic spine density after MPTP is driven by intrinsic striatal neurons, rather
than dopaminergic neurons of SNC. This may reflect an indirect effect of MPTP
on striatal interneurons, perhaps to compensate for dopamine depletion.
Given the importance of dopaminergic projections to the striatum and
substantial loss of dopaminergic inputs using a well-established lesioning
paradigm, the absence of MPTP-induced structural changes to MSN distal
dendrites is surprising. In postmortem cases of PD and after 6-OHDA lesions of
the nigrostriatal system, dendritic spine density is decreased (McNeill et al., 1988;
Solis, et al., 2007; Zaja-Milatovic et al., 2005). The absence of MPTP-induced
structural changes to distal dendrites may relate to the heterologous distribution of
striatal MSN’s. MSN’s include both D1 receptor-containing MSN’s of the direct
pathway and D2 receptor-containing neurons of the indirect pathway (Smith and
Bolam, 1990). Recently, Day et al. (2006) demonstrated a selective effect of
dopamine-depleting lesions on D2 receptor containing MSNs using 6-OHDA.
This indirect pathway has also been implicated behaviorally, with D2 receptor
knockout mice exhibiting PD-like akinesia and bradykinesia (Baik, et al., 1995).
Behavioral deficits in D1 receptor knockout mice are minimal (Xu, et al., 2000) or
absent (Drago, et al., 1994). It is likely that MPTP-induced spine changes are also
confined to the D2 receptor-containing subpopulation of MSNs. However, as
direct and indirect pathway MSNs cannot be morphologically distinguished using
97
Golgi-Cox staining, we were unable to compare these two subpopulations in the
current study.
Clinically, the actions of gonadal steroid hormones in the nigrostriatal
system are important because estrogen is neuroprotective in many neurological
disorders, including Parkinson’s disease. In fact, women are more likely to
develop PD after hysterectomy or menopause, when endogenous estrogen is
eliminated (Popat, et al., 2005; Ragonese, et al., 2004). One of the potential
benefits of hormone replacement therapy on postmenopausal women is the
potential to delay the onset and/or decrease the severity of neurodegenerative
disease. Men also experience a loss of testosterone with age, albeit less severe
than the complete loss of gonadal steroids in the female. Although androgen
replacement therapy is available for men with hypoandrogenism, the results of the
current study suggest that androgen replacement will not attenuate nigrostriatal
neurodegeneration in the male.
98
Chapter 5
CONCLUSION

The experiments in this thesis establish novel roles for gonadal steroids in
the adult brain. The traditional roles for gonadal hormones include (1)
establishing sex differences during development in brain regions that are relevant
to reproduction and (2) acting in classical, sexually-differentiated brain regions
throughout adulthood to alter neuronal structure and modulate behaviors. Chapter
2 focused on the mating circuitry, but identified on a new mechanism through
which hormones can modulate neural function: enhancing cell proliferation.
Chapter 3 took the traditional concept of sex differences but applied it to the
nigrostriatal pathway, which is not thought of as being strongly hormone-sensitive.
Finally, Chapter 4 explored the traditional role for testosterone to drive structural
and behavioral changes, but focused on the nigrostriatal pathway. In using a
model of neurodegeneration, Chapters 3 and 4 also addressed sex differences in a
disease state and the neuroprotective capacity of testosterone, respectively.

Chapter 2: Nontypical activational effects in classical brain regions
The experiments of Chapter 2 address a new role for activational
hormones in the classical, steroid receptor-rich nuclei of the mating circuitry. It is
well-established that gonadal steroid hormones exert activational effects in both
classical and nonclassical brain regions that include structural remodeling of
99
neurons (Cooke and Woolley, 2005). Recently, a new role for activational
hormones was established in a nonclassical brain region when it was shown that
hormones increase the survival of newly born neurons in the hippocampus.
Chapter 2 determined that testosterone selectively increased cell proliferation in
MeP but not MPOA, but had no effect on cell survival in either region.
This experiment is the first study to examine the hormone effects on cell
proliferation in two closely-connected nuclei. Based on what we know of
neurogenesis in the olfactory bulb, a pattern has emerged with respect to cell
proliferation throughout the mating circuit. In particular, the capacity for cell
proliferation decreases progressively from the olfactory bulbs, through the medial
amygdala to mid-line hypothalamic targets, including MPOA. One possible
explanation for the increased capacity for cell proliferation in MeP, compared
with MPOA, relates to the plasticity of afferent inputs, and the diversity of
behavioral responses in these two regions. In this regard, the medial amygdala
receives a dense projection from the olfactory bulbs (Coolen and Wood, 1998), a
highly plastic, highly neurogenic brain region. By contrast, inputs to MPOA do
not demonstrate comparable neurogenic capacity. Furthermore, MeP contributes
to a wide variety of behavioral outputs. As chemosensory stimuli from
conspecifics reach MeP, the animal interprets these cues in concert with blood-
borne steroids to govern different behavioral responses (Johnston, 1985). This
can include mating mediated via MPOA (Siegel, 1985c; Wood, 1998; Wood and
Newman, 1995b), or aggression regulated through the anterior hypothalamus
100
(Siegel, 1985a). Thus, the capacity for neurogenesis in a given brain region may
reflect the need for behavioral flexibility.
After experiment 1, I became particularly interested in cell survival, which
as extremely limited in both MeP and MPOA. Why weren’t the cells surviving?
In an attempt to further understand cell proliferation and survival in MeP and
MPOA, I turned to a behavioral model. It is well-established that activational
effects of hormones on steroid-sensitive nuclei in the mating circuitry drive
reproductive behavior. Chapter 2 asked the question, would behaviors that
increase hormone concentrations (i.e. mating) alter the structure of the brain?
However, weekly mating experience did not increase cell survival in the mating
circuitry.
Given the low level of cell survival, the question still remains as to the
role of newly born cells in the mating circuit. One hypothesis is that cell
proliferation and survival provide an underlying plasticity to the system so that
the brain can respond to injury. In this regard, it would be interesting to see if cell
survival increases after injury to either MeP or MPOA.

Chapter 3: Sex differences in nonclassical brain regions
Chapter 3 reported sex differences in the nigrostriatal system. Sex
differences in the brain have been well-documented in reproductively-relevant
areas that are highly sexually dimorphic. By contrast, the nigrostriatal system
does not have obvious structural sex differences. However, striatal responses to
101
gonadal hormones are sex-specific and sex differences exist with respect to
neurodegenerative disease (Becker, 1999; Dluzen, 2000). Chapter 3 determined
the extent of sex differences in the MPTP mouse model of PD. The focus was on
motor impairments.
In rodent models of nigrostriatal disease, striatal dopamine levels have
been almost exclusively used to evaluate sex differences in neurotoxicity.
Interestingly, motor behavior does not always correlate with striatal dopamine
levels. For example, when AMPH is used to stimulate dopamine release in
normal rodents, dopamine release is equivalent in male and female rats (Becker
and Ramirez, 1981). However, AMPH-induced stereotyped behaviors are greater
in females (Becker et al., 1982; Robinson et al., 1980) presumably because
females have a higher density of dopamine transporter in the striatum (Morissette
and Di Paolo, 1993). Thus, restricting analysis to dopamine in animal models of
PD may provide an incomplete picture of sex differences.
Chapter 3 is the first study to show sexually dimorphic motor impairments
in the MPTP mouse model of PD. Interestingly, the motor performance of
MPTP-treated mice for any given behavioral test was not indicative of their
overall motor phenotype. On the pole test, mice were unimpaired. Male and
female mice were equally impaired on the rotarod. Finally, gait was selectively
impaired in male mice. Together, these data emphasize the importance of looking
across a range of behavioral tests to get a more accurate idea of motor
102
performance. Unfortunately, the vast of MPTP studies in rodents to date use a
single test for motor performance.
An interesting question that emerges from Chapter 3 is whether sex
differences in MPTP mice arise from organizational or activational effects of
hormones. If activational hormones are responsible for sex differences in the
MPTP-treated mouse, then adult gonadectomy should abolish sex differences
after MPTP. Alternatively, organizational hormones may play a role in
establishing sexually dimorphic responses to MPTP. In this case, gonadectomy
will not eliminate MPTP-induced sex differences. More than likely, a
combination of organization and activation contributes to the sexually dimorphic
responses to MPTP.
Preliminary data from our lab suggests a combination of organization and
activation. We gonadectomized adult male and female mice, and then
administered MPTP two weeks later. Baseline sex differences in motor
performance were reversed, with female mice outperforming males on all three
behavioral tests. This sex-reversal was due to decreased motor performance in
castrated male mice, rather than increases in ovariectomized female motor
performance. After MPTP, motor performance was worse in gonadectomized
females than in gonadectomized males, when compared with same-sex gonad-
intact mice.

103
Chapter 4: Typical activational effects in nonclassical brain regions
In Chapter 4, I found that testosterone increases dendritic lengths on distal
dendrites of striatal MSNs. This is one of few studies demonstrating testosterone-
driven structural alterations outside of classical brain areas. Compared with
estrogen, testosterone remains relatively uninvestigated in the brain. Estrogen has
received increased attention in part, because it exerts protective effects against
neurodegenerative disease (Garcia-Segura et al., 2001). By contrast, Chapter 4
did not demonstrate any protective capacity of testosterone against nigrostriatal
degeneration.
At this point, it is unclear whether testosterone-driven structural
remodeling in the dorsolateral striatum is achieved through androgenic or
estrogenic mechanisms. Based on what is known regarding testosterone-driven
changes to neuronal morphology, it could be either. In the hippocampus,
testosterone acts through androgenic mechanisms to increase the density of spine
synapses (Leranth et al., 2003). In MeP, testosterone increases dendritic length
and branching via its conversion to estrogen (Gomez and Newman, 1991).
However, in the striatum, we expect that hormone-driven changes to MSNs occur
through androgenic mechanisms because of the limited distribution of aromatase
in this region. Furthermore, because the striatum is almost devoid of classical
hormone receptors, we predict that testosterone-driven structural remodeling is
achieved via nonclassical mechanisms.
104
With respect to neuroprotection, Chapter 4 found that testosterone does
not alter nigrostriatal remodeling after MPTP. It is tempting to ask whether
estrogen would reverse the MPTP-induced increase to MSN dendritic spines in
male mice. On one hand, there is a large volume that has shown that estrogen
exerts stimulatory activational effects in the striatum to include neuroprotection
(Becker, 1999; Dluzen, 2000). In this regard, it seems likely that estrogen would
restore spine density. On the other hand, it may be equally appropriate to ask to
what extent this matters. In vivo, the amount of estrogen available in the male
striatum is likely to be limited by the low levels of aromatase present. Thus, the
ability of the male striatum to respond to the administration of estrogen under
experimental conditions is not likely to be replicated in unmanipulated mice.

Chapters 2-4: Complicating the big picture
Taken together, the experiments in this thesis raise new questions
regarding the ability of hormones to act in both classical and nonclassical brain
regions. Fundamentally, they complicate the big-picture understanding of what
hormones do. Historically, it was much simpler to think of hormone action
exclusively in the context of reproduction, just as it was easier to think of
genomic effects as the only pathways through which hormones could act.
However, the numerous actions of gonadal hormones in nonreproductive areas
and through nonclassical mechanisms are completing our understanding hormone
action on the brain and behavior. Collectively, the diversity of hormone action
105
relates to the full expression of sex differences in normally functioning animals as
well as to the susceptibility and progression of disease. Further investigations,
especially to elucidate the role of activational testosterone on the brain, will lead
to a greater understanding of hormone action across the lifespan.

106
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Abstract (if available)

Abstract Gonadal steroid hormones exert numerous effects on the brain and behavior. Hormones are responsible for sexual differentiation during development. In adulthood, hormones mediate sex-specific effects on behavior and corresponding neural circuits. Hormones have been traditionally investigated with respect to reproduction and the neural circuits that drive mating behavior. However, hormone action is not restricted to the control of reproduction. In the current dissertation, I investigated the role of gonadal hormones in three chapters using two models. First, I investigated the role of testosterone on cell proliferation and survival in the mating circuitry of the adult male hamsters (Chapter 2). Testosterone exerted region-specific effects in the mating circuitry to enhance cell proliferation in some regions, but not others. In addition, neither testosterone nor mating experience increased cell survival in the mating circuit. For Chapters 3-4, I focused on the nigrostriatal pathway. The nigrostriatal pathway is involved in the circuits that coordinate voluntary movement, and is not directly related to reproduction. Nonetheless, this pathway is sensitive to gonadal hormones. In Chapter 3, I examined sex differences in nigrostriatal anatomy and behavior in rodents under normal physiologic conditions and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal degeneration that mimics Parkinson’s disease. In Parkinson’s disease, both the incidence and susceptibility are higher in men than in women. Gonadal steroids are thought to partially underlie this difference. After MPTP, male mice had increased motor deficits compared with females. This supports previous findings showing the increased susceptibility of males to MPTP. However, MPTP did not deplete neurons in the nigrostriatal pathway. In Chapter 4, I used the MPTP mouse model to determine whether testosterone acts as neuroprotectant in the nigrostriatal pathway.

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Creator Antzoulatos, Eleni (author)

Core Title Gonadal steroid hormones promote neuroplasticity in models of health and disease

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 12/02/2010

Defense Date 07/10/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,Dopamine,Estrogen,medial amygdala,medial preoptic area,medium spiny neuron,motor behavior,neurogenesis,neuronal morphology,neuroplasticity,OAI-PMH Harvest,Parkinson’s disease,sex characteristics,sexual behavior, animal,striatum,substantia nigra,testosterone,tyrosine hydroxylase

Language English

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Advisor Wood, Ruth I. (committee chair), Garner, Judy A. (committee member), Jakowec, Michael W. (committee member), Schechter, Joel E. (committee member), Swanson, Larry W. (committee member), Watts, Alan G. (committee member)

Creator Email antzoula@usc.edu,eleni.aultman@gmail.com

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