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Estrogen, progesterone and BDNF interactions: roles in neuroprotection

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Estrogen, progesterone and BDNF interactions: roles in neuroprotection

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ESTROGEN, PROGESTERONE AND BDNF INTERACTIONS:
ROLES IN NEUROPROTECTION

by

Claudia C. Aguirre

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 Claudia C. Aguirre
ii
EPIGRAPH

Stopping by Woods on a Snowy Evening
Robert Frost
Whose woods these are I think I know.
His house is in the village though;
He will not see me stopping here
To watch his woods fill up with snow.

My little horse must think it queer
To stop without a farmhouse near
Between the woods and frozen lake
The darkest evening of the year.

He gives his harness bells a shake
To ask if there is some mistake.
The only other sound's the sweep
Of easy wind and downy flake.

The woods are lovely, dark and deep.
But I have promises to keep,
And miles to go before I sleep,
And miles to go before I sleep.
iii
DEDICATION

This dissertation is dedicated to my Mom and Dad, who have provided
exemplary virtue and guidance throughout my life. My father, who turned 60 this
year, has spent over twenty years reminding me that I can do anything. Had it not
been for his continuing encouragement and support, I would never have thought
I‟d reach such high accolades. When I wanted to work at Sea World, he bought
me books about whales and dolphins. When I wanted to be a writer, he
encouraged my reading, until about midnight, when he made me turn the lights
off. It was in his wise words of encouragement and personal experience that I
found confidence in myself to tackle any challenges that came my way. As a
perfect balance to those words that resonate within me to this day, I found my
mother‟s actions to be equally inspiring. More than anyone I have met, she
embodies an indomitable strength that is epitomized by her tenacity and
perseverance. Juggling a family, work, school and a foreign language is
something I highly admire, although I am certainly glad I did not have to do that
while earning this degree. I remember her long work days and late school nights,
all while providing a daily home-cooked meal and keeping a close eye on my
brother and I, something that is easily underappreciated in mothers. As a small
family away from our extended family, my parents managed to instill the values
set forth by my grandparents and the generations before them. It is the sum of
these collective values that have shaped my brother and I, and we can only hope
to do the same for our future children.
iv
I always knew that leaving the comforts of Peru for a distant country was
like jumping into a dark pool of immeasurable depth. Moving a young family must
have taken great courage and incredible willpower. For this, I give them my
utmost gratitude, since I would not be in the place I am today without that critical
decision. So, thank you for bringing me here and letting me do what I wanted.

v
ACKNOWLEDGEMENTS

This dissertation would not have been possible without the essential help from
countless people. My friends have provided both support and a necessary outlet
from research and academia. Someone once told me to say goodbye to my
social life when I start grad school - I knew from that moment that I would never
let that happen. As Mark Twain put it, I “never let my schooling interfere with my
education.” Aside from all the good times, I learned a lot from all my friends. Be it
in business, design, art, fashion, advertising or action sports, this knowledge will
prove to be as equally important as my doctorate in the workforce. My brother,
cousins and the rest of the family always kept things in perspective for me. Of
course, I could not have accomplished this without the technical assistance of my
co-workers in my lab as well as in other labs. Finally, the mentoring I received
from my P.I. and committee, as well as other faculty and university personnel
guided me tremendously throughout my four years at USC. Thank you to
everyone for making this happen. This study was supported by my NIA training
grant T32 AG000093-25 and NIH grant 1P01AG026572 to MB.

vi
TABLE OF CONTENTS

Epigraph ii
Dedication iii
Acknowledgements v
List of Figures viii
List of Abbreviations x
Abstract xii
Preface xiv
Chapter 1: Introduction 1
Women‟s Health Initiative 2
Medroxyprogesterone Acetate 4
The Hippocampal formation 6
Estrogen and Hippocampus 7
Estrogen receptors 10
Estrogen Receptor α (ERα) 13
Estrogen Receptor β (ERβ 15
Progesterone functions in brain 18
Estrogen and Progesterone Interactions 19
Female hormones and neuroprotection 21
Brain derived neurotrophic factor (BDNF) 22
BDNF and steroid hormones 24
Organotypic Hippocampal Slice Cultures 29
A model for cell damage/death: NMDA toxicity 30
Summary 31

Chapter 2: Progesterone reverses 17β-estradiol-mediated 33
neuroprotection and BDNF induction in cultured
hippocampal slices
Chapter 2 Abstract 33
Introduction 34
Materials and Methods 36
Results 41
Discussion 52
Chapter 2 References 58

vii
Chapter 3: Progesterone inhibits estrogen neuroprotection by 62
downregulating estrogen receptor- 
Chapter 3 Abstract 62
Introduction 64
Materials and Methods 67
Results 75
Discussion 92
Chapter 3 References 99

Chapter 4: General Discussion and Summary 102
Importance of the treatment protocol 102
Why didn‟t P4 treatment increase BDNF mRNA and 105
protein?
Antagonistic effects of Progesterone on Estrogen are 107
not mediated by its metabolites
Sex Differences 108
NMDA toxicity in organotypic hippocampal slices as a 109
model of cellular death
How do my results contribute to or extend the existing 111
knowledge of the neurobiology of estrogen and
progesterone interactions on the modulation of BDNF and
neuroprotection?

References 114

viii
LIST OF FIGURES

Figure 2.1: P4-mediated reversal of E2-induced 42
neuroprotection against NMDA toxicity in
cultured hippocampal slices

Figure 2.2 Effects of E2 and P4 on NMDA-induced 44
neuronal damage in cultured hippocampal
slices, assessed with PI uptake

Figure 2.3: Quantitative analysis of PI uptake in cultured 46
hippocampal slices

Figure 2.4 BDNF mediates E2 neuroprotective effects 48
against NMDA toxicity in cultured hippocampal
slices

Figure 2.5 Effects of E2 and P4 on TrkB 49
activation/phosphorylation in cultured
hippocampal slices

Figure 2.6 Effects of E2 and P4 on BDNF levels in 51
cultured hippocampal slices

Figure 3.1 PHTPP reverses E2-mediated neuroprotection 76
against NMDA toxicity.

Figure 3.2 ER  activation mediates estrogenic 78
neuroprotection against NMDA toxicity

Figure 3.3 E2 treatment increases ER  mRNA levels and 80
addition of P4 to E2-treated slices reverses this
effect

Figure 3.4 Quantification of ER  and ER  mRNA levels 81

Figure 3.5 Quantification of ER protein levels 82

Figure 3.6 P4 (4 h) treatment reverses DPN-mediated 84
decrease in PI uptake of cultured hippocampal
slices

ix
Figure 3.7 10nM E2 (24 h) treatment of cultured 86
hippocampal slices increases BDNF mRNA
levels and addition of P4 (4 h) to E2 treated
slices reverses this effect

Figure 3.8 Quantification of BDNF mRNA levels 87

Figure 3.9 DPN but not PPT treatment of cultured 88
hippocampal slices results in increased BDNF
protein levels.

Figure 3.10 E2 treatment of cultured hippocampal slices 89
from wild-type but not ER -KO mice results
in neuroprotection against NMDA toxicity

Figure 3.11 LDH release into medium of cultured 90
hippocampal slices from wild-type but not ER 
-/-
or
ER 
+/-
mice results in of E2 –mediated
neuroprotection against NMDA toxicity

Figure 3.12 ER  immunoreactivity in CA1 of cultured 91
hippocampal slices

Figure 3.13 Proposed mechanism of estrogen-progesterone 95
interactions

Figure 4.1 Timing Protocol 104

Figure 4.2 Progesterone timed treatments on E2-treated 105
hippocampal slices- effects on BDNF protein levels.

x
LIST OF ABBREVIATIONS

AD: Alzheimer‟s Disease
ANOVA: Analysis of variance
αCaMKII: α-Ca
2+
/calmodulin-dependent kinase II
AP : Allopregnanolone, 3- -hydroxy-5 -pregnan-20-one
BDNF: Brain-Derived Neurotrophic Factor
Ca
2+
: Calcium
CEE: Conjugated Equine Estrogens

CNS: Central Nervous System
DG: Dentate Gyrus
DMSO: Dimethylsulfoxide
DPN: diarylpropionitrile
E2: 17 -Estradiol
ELISA: Enzyme-Linked Immunosorbent Assay
ER- : Estrogen Receptor 
ER- : Estrogen Receptor 
ERE: Estrogen Response Element
HET: Heterozygous
HT: Hormone therapy
ICC: Immunocytochemistry
KO: Knock Out
LDH: Lactate Dehydrogenase
xi
MAPK: Mitogen-Activated Protein Kinase
MPA: Medroxyprogesterone Acetate
mRNA: Messenger RNA
NMDA: N-methyl-D-aspartate
NMDAR: NMDA receptors
OHSC: Organotypic hippocampal slice culture
P4: Progesterone
P75-NTR: p75 Neurotrophin Receptor
P9: Postnatal day 9
PI: Propidium iodide
PPT: Propyl Pyrazole Triol
PR: Progesterone receptor
RT- PCR: Reverse Transcriptase- Polymerase Chain Reaction
WHI: Women‟s Health Initiative Study
WHIMS: Women‟s Health Initiative Memory Study
WT: Wild type

xii
ABSTRACT

Human cognition encompasses all the components involved in information
processing. Thus, it is a multidimensional concept that involves several aspects
of learning, memory, abstract reasoning and other higher-order functions.
Interest in hormone effects on memory mechanisms has been spurred by
conflicting evidence implicating that hormone replacement therapy (HT) can
ameliorate or be detrimental to memory and cognitive ability in post-menopausal
women. The functions of the female gonadal steroids estrogen and progesterone
in the central nervous system (CNS) have been extensively studied. One of the
regions involving estrogenic action most studied is the hippocampal formation,
which governs the formation of spatial and episodic memories. Relatively less
attention has been devoted to progesterone (P4) and its effects in hippocampus.
Moreover, the possible regulation by P4 of E2-mediated neuroprotective effects
has not been extensively investigated. This dissertation is directed at studying
interactions between estrogen and progesterone in an in-vitro model of
excitotoxicity, and at evaluating the role of brain-derived neurotrophic factor
(BDNF) in mediating estradiol-induced neuroprotection, using NMDA treatment of
cultured hippocampal slices to elicit neuronal death. I hypothesized that the
neuroprotective effects of these hormones was critically dependent upon the
timing of hormone administration. Further, I proposed that estrogenic
neuroprotection against NMDA toxicity was dependent on the activation of BDNF
and its resulting signaling pathways, an effect mediated through the ER 
xiii
receptor. Using organotypic hippocampal slices, we showed that treatment with
estradiol paired with progesterone resulted in down-regulation of ER  mRNA
leading to diminished ER  -mediated estradiol responses, and in particular, in
decreased BDNF expression and protein levels, which accounted for elimination
of estrogen protection against NMDA toxicity. A better understanding of the role
of the temporal order of treatment with gonadal steroid hormones on
neurotrophin regulation may provide insight into the role of these hormones as
neuroprotective agents in neurological disorders such as Alzheimer‟s and
Parkinson‟s disease and as potential therapeutic treatments.

xiv
PREFACE

The purpose of the present dissertation is to elucidate the mechanisms
underlying hormonal neuroprotection by examining the interactions governing the
bidirectional effects of estrogen and progesterone. Numerous studies have
revealed that estrogenic effects in the hippocampal formation depend on the
activation of distinct estrogen receptors. However, the mechanisms of action
underlying varying estrogenic effects remain to be elucidated. One of the most
studied estrogenic effects in hippocampus involves estrogen-BDNF interactions.
Several hypotheses have been postulated, with estrogen inducing BDNF gene
transcription either directly or indirectly. Fewer studies have focused on
progesterone‟s interactions with BDNF. As such, this dissertation was aimed at
untangling some of the complexities involving estrogen, progesterone, and BDNF
in the realm of neuroprotection. The physiological relevance of these interactions
may shed light on the applicability of these hormones as potential therapeutic
agents in human health and disease. These studies were addressed within the
confines of the following specific aims:
1. Characterization of progesterone’s effects on 17 -estradiol (E2)-
mediated neuroprotection against NMDA toxicity. The temporal
pattern of hormone-dependent neuroprotection was assessed in
organotypic hippocampal slices. Cellular death was analyzed by
assessing the levels of lactate dehydrogenase released into the media as
well as the fluorescent intensity levels signifying uptake of propidium
xv
iodide into the slice tissue. The treated tissue was also analyzed via
Western blot technique for changes in BDNF and its receptor TrkB protein
levels to determine whether neurotrophin activation is necessary for
cytoprotection.
2. Determination of whether progesterone counteracts estrogenic
neuroprotection via degradation of ER , ER  and/or BDNF mRNA.
Organotypic hippocampal slices were assessed for levels of ER , ER 
and BDNF mRNA levels following E2 and P4 treatments alone and in
combination using RT-PCR techniques. The effects of P4 on mRNA
degradation were evaluated following transcription inhibition utilizing RT-
PCR techniques.
3. Evaluation of the roles of estrogenic receptors ER  and ER  in
mediating estradiol-dependent neuroprotection and the effect of
progesterone on these receptors. Selective ER  and ER  agonists,
alone or in combination with progesterone, were used in the same
treatment paradigm as in the E2 experiments. Cellular death was
assessed in the same manner as described in Aim 1. Further analysis of
tissues was performed with Western blot techniques.

1
CHAPTER 1
INTRODUCTION

The notion that aging brings about mental deterioration is no
contemporary issue. Dating back to 2000 B.C., the Egyptians were aware that
aging could bring about memory disorders and philosophers of the Greek and
Roman eras were also privy to the idea of irreversible mental disorders that
accompanied aging. Shakespeare put it simply in „As You Like It‟ when he
described the final stage of life as “second childishness and mere oblivion”
(Boller and Forbes, 1998). Over the years, accumulating data have established
the concept that the brain is not simply a predetermined organ, but a structure
that undergoes extensive modifications during development and throughout
adulthood. In particular, gonadal steroids have been repeatedly shown to
influence neuroanatomical and neurophysiological aspects of central nervous
system (CNS) functions, with important implications for age-related memory
decline and neurodegenerative diseases. Not surprisingly, the majority of the
studies in the field have focused on estrogenic actions. However, data generated
over the past decade have illustrated the effects of other sex hormones, such as
testosterone and progesterone, in the CNS.
Over the past few decades, considerable knowledge about the actions of
hormones in the central nervous system has accrued following technological
advances and a focused research effort to elucidate the underlying mechanisms
of these actions. This effort has been, in part, fueled by recent findings
2
supporting the potential clinical applications of hormone therapy in the elderly.
The aged population in industrialized countries has significantly increased over
the past century, leading to an increasingly larger subset of the population
vulnerable to age-related diseases. It is estimated that by the year 2030, the
population of those aged 65 and over will reach 1 billion, or 1 in 8 of the world‟s
population (NIA, 2007). In addition, the Alzheimer‟s Association estimates that
the prevalence of Alzheimer‟s disease (AD) may reach up to 16 million by 2040
in the United States alone (2009). AD and other dementias cost Medicare,
Medicaid and other businesses $148 billion per year, a figure that will
undoubtedly be mag6nified as the elderly population grows (2009). The
prevalence of many age-related degenerative diseases has proven to be a
financial burden for the public; thus, an effective and targeted therapy will be
beneficial not only to the individual, but to society as a whole.

Women‟s Health Initiative
Menopause, the transition period between a woman‟s reproductive years
and the cessation of ovarian function, is characterized by a precipitous decline in
ovarian hormones estrogen and progesterone. Female life expectancy has
dramatically increased over the years, while the age of menopause onset has
remained stable at around 50 years of age, resulting in a large population of
women living in a hormone-deprived state (Cyr et al., 2002; Sherwin, 2003).
With the number of post-menopausal women increasing every year, it has
become imperative to find new methods to alleviate the symptoms of
3
menopause. Clinicians widely prescribe hormone therapy (HT) for a variety of
menopause-related symptoms and disease prevention, resulting in over one third
of post-menopausal women using HT in 1999 (Keating et al., 1999; Utian et al.,
2008). Since menopausal women are up to three times as likely to develop
Alzheimer‟s disease as men, it is postulated that the loss of circulating ovarian
hormones places females at a significant risk for the development the disease
(Henderson, 1997; Sherwin, 2003). Alzheimer‟s disease is the most common
cause of dementia, a progressive decline in cognitive function that is not
attributed to normal aging (Utian et al., 2008). However, one trial within the WHI-
the Women‟s Health Initiative Memory Study (WHIMS)- revealed a two-fold
increase in dementia among women aged 65 to 79 who were taking 0.625mg
conjugated equine estrogen (CEE) with 2.5mg of the synthetic progestin,
medroxyprogesterone acetate (MPA) compared with placebo controls (Shumaker
et al., 2003; Shumaker et al., 2004). In contrast, observational studies with a
smaller sample size have reported the association of HT with a reduced risk of
developing AD, possibly due to the younger age of women in the study (Utian et
al., 2008). In addition, a recently published subset of the WHIMS trials concluded
that randomization to CEE with or without MPA was associated with greater brain
atrophy in frontal and hippocampal areas among women 65 yr and older
(Resnick et al., 2009). These results are in direct contrast to previous reports
indicating beneficial effects of HT on brain volumes (Eberling et al., 2003; Lord et
al., 2008), a result that could be attributed to the differences in cohort age.
Together, these findings suggest that there may be a critical window during
4
which HT can help reduce the risk of developing AD (Utian et al., 2008). As of
yet, there is no conclusive data on the use of HT for the treatment of dementia or
AD. Currently, clinicians are advised not to use HT for the treatment of AD or to
enhance cognitive function, a finding that is reflected in the 46% and 28% decline
in the initiation of estrogen therapy (ET) and hormone therapy, respectively (Buist
et al., 2004). An important question raised by the WHI trials is whether the
deleterious outcomes of combining medroxyprogesterone acetate (MPA) with
conjugated equine estrogen (CEE) can be attributed to effects unique to MPA or
whether they extend to other progestins. We therefore examined whether
progesterone (P4) would counteract estrogenic effects in the hippocampus in an
in vitro model of neurotoxicity.

Medroxyprogesterone Acetate
Medroxyprogesterone acetate (MPA) is a synthetic progestin commonly
used to help reduce the uterotrophic effects of unopposed estrogen therapy,
such as the increased risk of developing cancer (Hirvonen, 1996). Although it has
been shown that MPA is as effective as P4 in this regard, accumulating evidence
reveals that progestins vary widely in their functions on the central nervous
system. Following the Women‟s Health Initiative trials, in which more adverse
consequences were observed in the group receiving CEE + MPA than with CEE
alone, including an increased risk for breast cancer, it became imperative to
further explore the potential for different progestins to differentially impact
neuroendocrine function. One aspect that has been investigated has been the
5
question of whether progestins differ in their ability to be neuroprotective. The
effects of progesterone alone on neuroprotection will be discussed in more detail
in a subsequent section. However, a number of studies have directly compared
the effects of MPA and P4 following the advent of micronized progesterone as an
alternative to synthetic progestins. Recent research shows that MPA, unlike P4,
is not neuroprotective against glutamate excitotoxicity and inhibits E2-mediated
sprouting in hippocampus (Nilsen and Brinton, 2002; Brinton et al., 2008).
Moreover, P4 but not MPA, stimulated nuclear activation of ERK, suggesting that
the nuclear translocation is necessary for progesterone-mediated
neuroprotection, a feature that MPA does not possess (Nilsen and Brinton,
2003). In addition, the protective effects of progestins may also depend on
activation of the neurotrophin system. For example, neuroprotective efficacy
afforded by progesterone was correlated with its ability to increase BDNF mRNA
and protein, whereas MPA did not elicit BDNF increases and was therefore not
protective (Kaur et al., 2007; Jodhka et al., 2009).
Functional differences between these progestins are also evident in
humans, as progesterone administration to postmenopausal women enhanced
estrogenic protection against exercise-induced myocardial ischemia, whereas
MPA treatment did not (Rosano et al., 2000). To add complexity to these
interactions, MPA may reduce the availability of a potent metabolite of
progesterone, allopregnanolone (AP ), which has been implicated in mediating
many of the anxiolytic effects seen with progesterone (Lee et al., 1999). This
suggests that in affecting the bioavailability of AP , and thus progesterone-
6
mediated protective mechanisms, MPA may impact anxiety. Researchers
studying the subunit composition of GABA
A
receptor, which is implicated in the
regulation of anxiety, found that MPA and P4 differentially affect the 4 subunit of
GABA
A
in the CA1 region. More specifically, P4 but not MPA down-regulated 4
mRNA after 12 h, although this effect was lost at 24 h (Pazol et al., 2009). All of
these studies suggest that the choice of progestins may yield widely varying
effects and thus requires careful considerations. In understanding the cellular
and molecular differences between progestins in brain, we might be able to
provide critical insight into the development of an efficacious and safe therapy for
the treatment of postmenopausal conditions. Consequently, I examined whether
MPA had a similar antagonistic effect on estradiol-mediated neuroprotection
against NMDA toxicity as we have shown with progesterone.

The Hippocampal formation
Coined in 1587 by a student of Vesalius, Giulio Cesare Aranzi (Lewis,
1922), the hippocampus retained the mysterious nature of its namesake for
centuries to come. Only in the past century has the hippocampal formation been
implicated in processes of learning and memory. Memory is the vast store of
information gained from learning, which includes knowledge about language,
motor skills, lifetime episodes, and all the facts gathered throughout a lifetime. In
fact, without memory the experience of consciousness and self-awareness
vanishes (Thompson and Madigan, 2005). Clinical studies of patients with
hippocampal lesions led to the discovery that long-term memory and short-term
7
memory involve different brain systems (Sherwin, 2003; Thompson and Madigan,
2005). It is important to distinguish between the two in order to understand the
anatomical regions of the brain involved in each and how they are affected by
hormone actions. Long-term memory includes all the knowledge retained and the
ability to become aware only during recall. In contrast, working memory not only
includes retrieval from long-term memory stores, but also recalling newly learned
information (Thompson and Madigan, 2005). Due to its role in memory function,
the hippocampus has been at the forefront of studies exploring hormonal effects
on neuronal excitability and synaptic plasticity. Understanding the effects of
hormones on brain structures and functions may help elucidate the underlying
mechanisms involved in their actions.

Estrogen and Hippocampus
Numerous studies have focused on estrogen‟s effects on hippocampus,
which governs the formation of spatial and episodic memories. In a study by Foy
et al., the direct effects of estrogen on synaptic plasticity in hippocampus were
explored (1999). Hippocampal CA1 pyramidal cell EPSPs were intracellularly
recorded to determine the effects of estrogen on NMDA and AMPA receptors. To
isolate NMDAR-mediated EPSPs, slices were perfused with the AMPA receptor
antagonist DNQX in presence of low Mg2+ concentration. 17  -estradiol
treatment resulted in prolonged NMDAR-mediated responses, as illustrated by
increased EPSP amplitude. NMDA receptor specificity was further confirmed by
abolishing the response with application of the NMDA receptor antagonist, D-
8
APV. Similarly, acute perfusion of E2 increased responses of isolated synaptic
AMPA receptors (Foy et al., 1999). This study clearly demonstrated that acute
estradiol treatment modifies both NMDA and AMPA receptor functions, thus
increasing synaptic transmission in the hippocampus. That same year, Good et
al. demonstrated modulation of LTP and LTD induction by endogenous levels of
estrogen in the hippocampus (Good et al., 1999). Cyclical changes in circulating
hormones have been shown to exert major morphological and physiological
changes in hippocampus. Compared with met/diestrous and estrous stages,
animals in the proestrous stage, the period of highest estrogen and progesterone
levels, demonstrated elevated LTP induction in CA1 neurons. Conversely,
animals in the proestrous stage showed impaired LTD when compared with
animals in other stages. These results show that both endogenous and applied
estradiol affect synaptic plasticity, although the details of the underlying
mechanisms by which these effects are exerted remain unclear. Several
mechanisms of actions have been proposed, including calcium signaling,
activation of a second messenger system, and inhibition of GABAergic
neurotransmission (Good et al., 1999; Foster, 2005; Parducz et al., 2006).
Impaired GABAergic neurotransmission may have contributed to the impaired
LTD seen in the proestrous stage ((Good et al., 1999). Previous research
illustrating the presence of a large number of estrogen receptors on inhibitory
interneurons supports this hypothesis (Good et al., 1999; Blurton-Jones et al.,
2004). However, recent work suggests that estradiol may in fact have opposing
effects on different interneuron populations, such as the NPY-expressing subset.
9
In this subset, estradiol increases NPY expression, thus inhibiting glutamate
release presynaptically in rat hippocampus (Nakamura et al., 2007). On the other
hand, estradiol was shown to decrease the number of synaptic vesicles adjacent
to the presynaptic membrane of CA1 inhibitory synapses, suggesting a role for
estradiol in mediating disinhibition of hippocampal of CA1 pyramidal cells
(Ledoux and Woolley, 2005). Therefore, estrogen effects on hippocampal
plasticity may vary depending on cell type. Estradiol-mediated increases in the
formation of new spines and synapses may also be related to providing an
environment that supports synaptic plasticity. Estradiol treatment was shown to
increase astrocytic volume in rat CA1, suggesting that glia might also be an
important target for the neuroprotective effects of estrogen (Klintsova et al., 1995;
Spencer et al., 2008). Additionally, primate studies indicate that estrogen
enhancement of hippocampal function may transcend species. Estradiol
replacement was shown to increase the number of CA1 spines in both young and
aged OVX rhesus monkeys (Hao et al., 2003). Together, these data provide
evidence that estradiol can be a powerful modulator of hippocampal functions
through various mechanisms not clearly defined as of yet. The complexities
underscoring the mechanisms of estrogen effects on hippocampal function are
amplified by recent work related to specific functions of different estrogen
receptor subtypes.

10
Estrogen receptors
The effects of estrogen are mediated by two distinct intracellular receptors
that form a steroid-receptor complex necessary for activation of gene
transcription. The two receptors, estrogen receptor alpha (ER ) and beta (ER ),
are not true isoforms although they share similar DNA- and ligand-binding
domains (Bodo and Rissman, 2006; Weiser et al., 2008). Much of the earlier
work on steroid hormone-induced neural events has focused on the effects of
estrogen. In vivo autoradiography studies using [
3
H] estradiol provided evidence
that ERs were sparsely localized in rodent hippocampus with greater labeling in
the ventral hippocampus (Pfaff and Keiner, 1973). Subsequent studies using
immunocytochemistry (DonCarlos et al., 1991) and in situ hybridization (Simerly
et al., 1990) found that cells in Ammon‟s horn and dentate gyrus expressed light
to moderate levels of estrogen receptors. The labeling was primarily located in
the pyramidal cell layer of CA1-CA3 as well as in the polymorphic layer of the
dentate gyrus (DG) (Simerly et al., 1990). Although the labeling was not as
strong as that seen in hypothalamus, these experiments showed a greater
density of labeled neurons than was previously reported using binding or
autoradiographical techniques. In 1996, a novel estrogen receptor was cloned
(Kuiper et al., 1996) changing the environment under which hormones were
studied in the brain. This suggested that studies on the expression of estrogenic
receptors previous to this discovery were likely attributing expression levels to
two different subtypes of the receptor. Initial histochemistry studies revealed that
ER  mRNA and ER  mRNA were differentially expressed in reproductive tissues
11
(Kuiper et al., 1996). This differential pattern was naturally followed up in brain
regions expressing the original ER. In concert with previous studies, Shughrue et
al. (1997) found ER  mRNA to be lightly expressed throughout the extent of
Ammon‟s horn (CA1-CA3) and dentate gyrus. The expression level of ER 
mRNA was stronger than ER- , but with a similar anatomical location,
demonstrating increasing intensity from dorsal to ventral aspects of the
hippocampus. Thus, the authors proposed that the localization of ER-  in the
hippocampal formation, an area where ER  is sparse, might shed light on how
estrogen mediates its effects on learning and memory (Shughrue et al., 1997).
Subsequent studies on the differential expression of ER  and ER  supported this
evidence and expanded onto various mammalian systems. ER  mRNA levels
were examined in monkey hippocampus using RT-PCR, confirming higher levels
of ER  than ER  mRNA (Register et al., 1998). The expanding view of hormonal
effects on different cell types led to the re-evaluation of estrogen receptor
expression on non-neuronal populations. ER  -immunoreactive glia were found
in all layers of CA1-CA3 in adult male and female rats, as confirmed by double
staining with the astroglial marker, GFAP (Azcoitia et al., 1999). A later study
examining ER  immunoreactivity found that ER -IR was primarily localized to
the nucleus of some GABAergic interneurons in the CA1 stratum radiatum and
infragranular hilus of the dentate gyrus (Milner et al., 2001). Moreover, small
amounts of ER  IR were localized to the nuclear membrane in some pyramidal
12
and granule cell perikarya, as well as in some axons, axon terminals, astrocytic
processes and dendritic spines (Milner et al., 2001).
In addition, the discrepancies in expression levels of ERs could be due to
the complex nature of hormone receptor expression in a temporal and spatial
manner. Immunocytochemistry in cultured hippocampal neurons revealed that
ERs were expressed in neurons and astrocytes, with no obvious difference
between ER  and ER  expression in neonatal hippocampus (Su et al., 2001).
This finding could explain the relatively low expression levels of ER  when
compared to ER  in earlier studies that looked at expression levels in the adult
brain (Shughrue & Merchenthaler, 2000; Simerly et al., 1990). Su et al. found that
ER  immunoreactivity in hippocampus decreased from the neonatal stage to
adulthood, while ER  expression remained relatively stable (2001). In addition,
ER expression in human hippocampus showed that ER  was first detected at 15
gestational weeks (GW) in the nuclei of pyramidal cells in Ammon‟s horn and ER-
 was first detected at 17 GW in pyramidal cell nuclei and granule cells of the
dentate gyrus. Although ER  levels remained high through adulthood, ER  levels
were initially high in both pyramidal cell layers and granular cell layers prenatally
and subsequently dropped into adulthood (Gonzalez et al., 2005). As evidenced
by the wealth of reports identifying a strong pattern of ER  expression in the
hippocampus, there is clear evidence that the  isoform is a key target for
estrogenic effects on hippocampal function.
13
Notably, there is a functional divergence between these receptors. As
illustrated in mouse knockout studies, fertility is impaired in mice lacking the ER ,
but not the ER  gene (Lubahn et al., 1993; Krege et al., 1998). More recently,
these receptors have been implicated in modulating non-reproductive
neurobiological functions. Accumulating evidence has emphasized the role of
ER  in cognition and behavior (Bodo and Rissman, 2006; Liu et al., 2008; Walf et
al., 2008; Walf et al., 2009). Further characterization of the cellular actions of
these receptors may provide insight into behavior, learning, memory and even
sexual differentiation.

Estrogen Receptor α (ERα)
Several studies indicate the importance of ERα in memory function,
suggesting critical implications for age-related cognitive decline or
neurodegenerative diseases, such as AD. A recent study implicates the
importance of ERα in spatial learning and memory. Using a technique that
delivers ERα to the hippocampi of ERα KO mice via lentiviral transduction,
researchers were able to rescue the spatial learning deficits observed in ERα KO
mice. The study was limited to female mice because they exhibit learning and
memory deficits compared to wild type (WT), unlike male ERα-KO mice. These
results suggest that the learning deficits found in female ERα-KO mice are not
only due to the lack of ERα during development, an organizational effect, but also
due to the disruption in ERα function in adults. However, it is still unclear whether
the beneficial effects of adding ERα to these KO animals is occurring through the
14
natural ERα pathways or through another compensatory pathway. Hence,
lentivirus-mediated expression of ERα in the hippocampus reversed the learning
deficits seen in ERα KO female mice, demonstrating a role of ERα in spatial
learning in adults (Foster et al., 2008). Other studies have examined the roles of
estrogen receptors on cognition using specific agonists for ERα and ERβ, Propyl
Pyrazole Triol (PPT) and diarylpropionitrile (DPN), respectively. One study found
an effect of ERα but not ERβ agonist on the modulation of NMDA receptors
(NMDAR) in hippocampus (Morissette et al., 2008). NMDA receptors are
subtypes of glutamate receptors that have been implicated in E2-mediated
increases in spine density and cognition. In this study, ovariectomy resulted in
decreased expression of the NMDAR subunit, NR2B. Estradiol and PPT, but not
DPN treatment reversed this effect in hippocampus. In cortex, E2 treatment, but
neither DPN nor PPT treatment, reversed the decrease in NR2B levels
(Morissette et al., 2008). Another study focused on these ER subtypes and α-
Ca
2+
/calmodulin-dependent kinase II (αCaMKII) signaling to understand the
mechanisms underlying estrogenic effects on cognitive function (O‟Neill et al.,
2008). αCaMKII plays an important role in neuronal differentiation and cognitive
processes and is highly responsive to Ca
2+
levels. Under normal conditions, this
kinase is inhibited until Ca
2+
/CaM binds to its autophosphorylation domain and
permits kinase activity. In this study, researchers showed that E2 rapidly induced
αCaMKII autophosphorylation in an ER  and Ca
2+
influx-dependent manner.
Thus, PPT induced autophosphorylation in a dose-dependent manner, while
DPN treatment did not. Together, these data propose a central role for ERα in
15
E2-mediated effects on learning and cognitive processes.

Estrogen Receptor β (ERβ)
Given that ERβ expression is more predominant in cerebral cortex,
hippocampus, dorsal raphe, substantia nigra, midbrain, ventral tegmental area
and cerebellum as previously described, there has been a growing compendium
of research investigating the functional effects associated with actions of ERβ in
cortical and hippocampal processes. More specifically, the rapid actions of ERβ
in hippocampus have been recently shown to mediate some of the functional
effects of estrogen. It is generally accepted that the glutamate receptors,
NMDARs and AMPARs, are key components of the machinery underlying
cellular correlates of learning and memory. As previously stated, one group found
that PPT but not DPN reversed ovariectomy-mediated decreases in the NMDAR
subunit NR2B in hippocampus (Morissette et al., 2008). In contrast, another
group investigating the effects of another ERβ agonist, WAY-200070, on synaptic
plasticity and memory, found that activation of either ERβ or ERα did not affect
the levels of NMDAR subunits NR2B or NR1 in both WT and OVX animals (Liu et
al., 2008). Despite the lack of alterations in NMDAR subunits, WAY-200070
significantly increased GluR1 expression, an effect abolished in ERKO mice
and PPT-treated mice, suggesting that this effect is ER -dependent. Further
characterization of this selective ER  agonist in hippocampus revealed that ER 
activation mediates the effects of estradiol on memory in a hippocampus-
dependent task and mimics estradiol effects on a spatial memory task.
16
Furthermore, activation of ER  through this agonist led to increases in spine
density, dendritic branching, synaptic proteins and LTP (Liu et al., 2008).
Although compelling, this evidence leaves room for interpretation as to the
efficacy of this agonist as it compares to other ER  agonists. Seeing as these
studies were performed in OVX animals, it is important to understand these
effects on cycling intact animals. Proestrous rodents have higher E2 and
progesterone levels in hippocampus as compared to diestrous animals. In a
recent study, WT animals in the proestrous phase had improved performance in
object recognition and T-maze tasks, as well as reduced anxiety-like behavior in
the plus maze and mirror chamber tasks when compared to ERKO mice.
Genotype did not seem to play a role in circulating hormone levels across the
estrous cycle (Walf et al., 2009). These behavioral effects implicate an important
role of ER  in mediating estradiol influences on cognition and affective
behaviors.
Studies on ERs in rodents have also focused on emotional behaviors.
Contextual fear conditioning occurs when an aversive stimulus, such as an
electrical shock, is delivered in a novel context, and the fear memory results in
defensive behavior when animals are re-exposed to the particular context in the
absence of any aversive stimulus. When the context is repeatedly presented
without this stimulus, a new form of learning called extinction occurs. A recent
study found that intrahippocampal infusions of DPN, but not PPT, in OVX
animals enhanced contextual fear extinction, signifying that ER  activation
mediates estrogenic facilitation of fear extinction (Chang et al., 2009). Despite
17
the accumulating evidence for the necessary activation of ER  in mediating
estradiol effects on hippocampal plasticity, learning and memory, there are still
discrepancies in the literature. For example, it has been reported that both ER 
and ERα have similar rapid effects as E2 in regulating intracellular Ca
2+
signaling
in hippocampus as well as increasing ERK phosphorylation (Zhao and Brinton,
2007). Although both agonists had a similar role in mediating estrogenic
neuroprotection via the MAPK pathway, ER  activation had more dependence on
L-type channels, thus making it more relevant for estrogen promotion of memory
mechanisms. Finally, selective agonists for ERs have been recently investigated
in animal models of disease. In a rodent model of Alzheimer‟s disease, PPT but
not DPN treatment in OVX 3xTg-AD mice reduced accumulation of β-amyloid
protein and improved hippocampal-dependent performance (Carroll and Pike,
2008). However, the distribution and expression pattern of ERs in 3xTg-AD mice
has not yet been elucidated, making it difficult to interpret these results. A
shortcoming prevalent in all these studies is that they fail to investigate the
effects of progesterone. Both E2 and progesterone are depleted as a
consequence of menopause in women and OVX in experimental animal
paradigms. Furthermore ERKO mice have deficits in ovulation (Krege et al.,
1998), signifying low levels of P4 in these animals.
Collectively, these studies provide insight into the various molecular
mechanisms and behaviors sensitive to normal fluctuations in gonadal
hormones. However, further investigation on progestins might yield a more
cohesive understanding on hormonal effects in hippocampal processes.
18
Progesterone functions in brain
Progesterone (P4) is the most common naturally occurring progestogen in
humans. Initially isolated from rabbit corpus luteum over 80 years ago,
progesterone‟s functions have now been characterized to extend well beyond the
periphery. As previously stated, hormone therapy (HT) incorporates the
administration of progestogens in conjunction with estrogen in order to counter
some of the proliferative effects of estrogen on the uterine epithelium (Brinton et.
al., 2008). Thus, as with estrogen, this hormone has significant clinical
implications. The effects of progesterone are mainly elicited via the classic
progesterone receptor (cPR), a nuclear transcription factor, in a similar fashion to
estrogen and its classic receptor ER. The progesterone receptor dissociates from
the complex of chaperone molecules upon binding of P4, allowing for the
interaction with specific response elements (PREs) in target gene promoter
sequences to regulate transcription. The two known isoforms of this receptor are
PRB and the N-terminal truncated A isoform (PRA). In addition, P4 can act
through the newly identified membrane progesterone receptor 7TMPR ,  and ,
among other putative membrane-bound receptors recently described (Brinton et
al., 2008, Singh, 2006; Zhu et al., 2003). These receptors are broadly distributed
throughout the brain and within different cell types (Brinton et al., 2008).
However, they are functionally distinct in that PRA is a trans-dominant repressor
of PRB in a promoter- and cell type-dependent manner, as well as a target of
estrogenic action. For example, E2, but not P4, induces PRA expression in rat
hippocampus and olfactory bulbs (Guerra-Araiza et al., 2002). Additionally,
19
progesterone can elicit its effects via the actions of its reduced metabolites.
Accumulating evidence has characterized the neuroprotective effects of the 5 -
reduced metabolite of progesterone, allopregnanolone. Metabolites such as this
one can bind to discrete sites within the GABA
A
receptor complex, resulting in
increased chloride conductance, which may serve to reduce neural injury elicited
by seizure activity (Brinton et al., 2008). Thus, as with estrogen, protective effects
of progesterone may be mediated via multiple mechanisms. Given that
circulating gonadal hormone levels include both estrogen and progesterone, a
decline in both may be responsible for the detrimental effects seen in
neurodegenerative diseases like Alzheimer‟s disease. Additional studies that
expand our view on hormonal interactions and how they influence brain will be
instrumental for the development of a plausible therapy for those experiencing
hormonal loss.

Estrogen and Progesterone Interactions
The complexity of hormonal interactions is magnified by work
demonstrating that estrogen can stimulate progesterone receptor synthesis. As
previously noted, progesterone receptor can be subdivided into one population
that is inducible by estradiol, and one that is not (MacLusky and McEwen, 1978).
This has recently been shown in several cell types throughout the brain, including
hypothalamus (Micevych et al., 2007), raphe nucleus (Bethea, 1994; Alves et al.,
2000), pituitary cells (Garrido-Gracia et al., 2007), VMH (Moffatt et al., 1998) and
midbrain central gray and adjacent tegmental area (Turcotte and Blaustein,
20
1993), suggesting that some effects of estrogen on gene expression are in fact
mediated by progesterone receptors (Alves et al., 2000; Chung et al., 2006).
Furthermore, the two isoforms arising from distinct estrogen receptor genes, ER 
and ER , may differentially regulate PR synthesis. To explore this possibility, the
ERKO mouse was used as a model of ER  action. Gonadectomized (GDX)
animals receiving estradiol exhibited a larger number of PR-IR cells in the
median and dorsal raphe nucleus than vehicle-treated animals. Moreover, more
E2-induced PR-IR cells were observed than ER -IR cells, suggesting that there
may be an additional mediator of estrogenic action in serotonergic neurons
(Alves et al., 2000). Estrogen-induced PR-IR enhancement in an ER -deficient
animal points to the possible involvement of ER , or perhaps some other gene
product, in mediating these effects. Nevertheless, the specific co-localization of
these receptors within a select population of neurons implies direct estrogen-
progesterone modulation of serotonergic function. In corroboration with evidence
that the receptors ER  and ER  may differentially regulate PR synthesis (Moffatt
et al., 1998), recent evidence suggests that the two estrogen receptors may act
in a „ying-yang‟ fashion when modulating luteinizing hormone secretion. (Garrido-
Gracia et al., 2007; Moffatt et al.,1998). Taken together, the presence of
estrogen-inducible progesterone receptors represents an additional channel
through which estrogen can exert its effects indirectly via progesterone and its
receptors.

21
Female hormones and neuroprotection
It is now clearly established that menopause, as well as ovariectomy,
results in a depletion of both estrogen and progesterone. Thus, the decline in
progesterone as well as in estrogen may play a significant role in the various
deficits, including cognitive deficits, which follow menopause and should be
closely taken into account when trying to relate age-related hormonal and
cognitive changes. Steroid hormones synthesized in the brain can participate in
the regulation of a variety of functions, as previously described. There is a wealth
of data highlighting the neuroprotective effects of 17 -estradiol in various animal
models of neurodegeneration. Recent data also suggest that progesterone (P4)
has potent neuroprotective effects. For example, several studies have
demonstrated that progesterone is neuroprotective against glutamate-induced
cell death (Nilsen and Brinton, 2002, , 2003; Kaur et al., 2007) and traumatic
brain injury (Roof et al., 1997; Robertson et al., 2006). Like 17 -estradiol (E2),
P4 likely stimulates several different mechanisms of neuroprotection, including
activation of neurotrophins. In addition, reduced metabolites of progesterone,
such as 3- -hydroxy-5 -pregnan-20-one (AP , allopregnanolone) have been
shown to mediate, at least in part, the neuroprotective effects of progesterone.
Accumulating evidence points out that estrogen and progesterone
differentially affect several neurobiological processes and that it is their
interactions on these processes that influence brain systems involved in learning
and memory. For example, while progesterone has no effect on spine density on
its own, it prevents estradiol-induced increases in spine density when co-
22
administered (Murphy and Segal, 2000). Similarly, although E2 treatment results
in neuroprotection against glutamate toxicity, the combination of E2 and
progestin either enhances or attenuates neuroprotection, depending on the type
of progestin (Nilsen and Brinton, 2002). This effect was also shown in vivo,
where P4 combined with E2 treatment attenuates the E2 neuroprotective effect
against A  accumulation in a mouse model of AD (Carroll et al., 2007). These,
among other studies, make it increasingly clear that understanding the
interactions between E2 and P4 is paramount to understanding the complexities
underlying age-related cognitive deficits and diseases like Alzheimer‟s disease
(AD). As of yet, there are still conflicting data regarding the complex interactions
between various gonadal hormones and their differential effects on various brain
systems. Moreover, the cellular and molecular mechanisms underlying these
protective effects remain unclear.

Brain-Derived Neurotrophic Factor (BDNF)
First isolated from pig brain, brain-derived neurotrophic factor, BDNF, was
initially characterized as a key effector in mediating long-term survival and
promoting neuronal differentiation in developing neurons as well as neuronal
viability in adult brain (Barde et al., 1982; Lu and Chow, 1999). The past twenty
years have been witness to a growing compendium of functions assigned to
BDNF. In mammalian brain, the neurotrophin family is composed of nerve growth
factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3)
and neurotrophin 4 (NT4). Their functions are dependent upon activation of two
23
distinct classes of transmembrane receptors, the p75 neurotrophin receptor and
the Trk family of receptor tyrosine kinases (Lu et al., 2005; Scharfman and
MacLusky, 2006). While the p75 neurotrophin receptor is non-selective, each Trk
receptor selectively binds to a different neurotrophin, with BDNF selectively
binding to the TrkB receptor. Aging has been associated with decreasing BDNF
and/or TrkB receptor levels in several brain regions. Clearly, the loss of receptor
expression can have profound consequences for neurotrophin-mediated synaptic
plasticity. Given that downstream effects of neurotrophin activation influence
learning, memory and cognition, numerous studies have explored the role of
BDNF in hippocampal functions. Although aged mice show decreased spine
numbers in the basal dendrites of the CA1 region, aged mice heterozygous for
TrkB (+/-) exhibited enhanced reduction in spine number compared with age-
matched controls (von Bohlen Und Halbach et al., 2007). The critical roles of
BDNF and its receptor in hippocampal spine maintenance are evident here.
Perhaps the most important consequence of aging on BDNF regulation lies in the
clinical implications for age-related neurodegenerative diseases, such as
impaired TrkB receptors in Parkinson‟s disease (von Bohlen und Halbach et al.,
2005). Post-mortem analyses of patients with Parkinson‟s disease have also
revealed a correlation between reductions in BDNF levels and striatal
neurodegeneration (Mogi et al., 1999). Similarly, these deficits in expression
levels are seen in patients with Alzheimer‟s disease (Soontornniyomkij et al.,
1999; Hock et al., 2000). The implications for disease pathology may also signify
that BDNF plays more than a passive role in the disease process. Recently, the
24
idea that neurotrophins may also produce diametrically opposing effects on cell
survival has garnered support from studies linking neurotrophin precursors to
apoptosis (Lu et al., 2005). Given that these proneurotrophins are secreted and
can therefore be considered signaling molecules, it is important to distinguish
between the pro- and the mature forms of the neurotrophins being examined.
Furthermore, recent data show that pro-NGF is upregulated in pathological
conditions such as Alzheimer‟s disease, although pro-BDNF levels are
decreased in AD (Lu et al., 2005; Peng et al., 2005). These findings underscore
the complexities underlying neurotrophin action in brain under normal and
pathological conditions.

BDNF and steroid hormones
The long-standing relationship between structure and function is evident
when examining the similarities between neurotrophins and steroid hormones.
Many studies throughout the years have revealed a similar pattern of expression
between estrogen and BDNF. As described, BDNF and its receptor TrkB are
localized in cortex, hippocampus, midbrain, spinal cord, and hypothalamus and
are expressed in principal neurons as well as in glia and subcellular
compartments(Scharfman and MacLusky, 2006; Sohrabji and Lewis, 2006).
Furthermore, BDNF-synthesizing neurons have been co-localized with forebrain
estrogen receptors and the BDNF gene contains an estrogen response element
(ERE), suggesting a possible mechanism for regulation of this gene by estradiol
(Miranda et al., 1993). Finally, both mRNA and protein expression of ERα and
25
ERβ are found in numerous cell types (Simerly et al., 1990; Scharfman and
MacLusky, 2006), with some co-localization with BDNF-expressing cells (Blurton-
Jones et al., 2004). This comparable pattern of expression throughout the CNS is
paralleled with similar functions in brain. Estrogen, progesterone and BDNF have
striking effects that afford them the ability to be neuroprotective against certain
toxic stimuli. Some of these include, but are not limited to, neuroprotection
against ischemic insults by estrogen (Wang et al., 1999; Miller et al., 2005),
progesterone (Ardeshiri et al., 2006) and BDNF (Beck et al., 1994). As with
estrogen, protection against kainate-induced cell death is also afforded by BDNF
(Duan et al., 2001). Several reports have demonstrated estrogen-induced effects
on BDNF (Gibbs, 1999; Solum and Handa, 2002; Sohrabji and Lewis, 2006).
One group examined the effects of ovariectomy on BDNF expression. Using in
situ hybridization methods, reduced levels of neurotrophins were found in cortical
regions as well as in hippocampus of aged animals 7 months following
ovariectomy. This decrease was attenuated by estrogen replacement in
hippocampus, but not cortex, of OVX animals. This incomplete restoration of
BDNF levels may signify that the aged brain may not be able to respond to
estrogen replacement as effectively as young animals(Singh et al., 1995). The
discrepancies highlighted in estrogen-neurotrophin interactions are not fully
understood, but increasing evidence points to influences by other hormones.
Several studies have demonstrated that progesterone is neuroprotective against
glutamate-induced cell death (Nilsen and Brinton, 2002; Nilsen et al., 2002; Kaur
et al., 2007). Pretreatment of organotypic cortical explants with 100nM, but not
26
10nM, P4 for 24 h significantly reduced glutamate-induced cell death as
measured by LDH release. Furthermore, progesterone treatment significantly
increased BDNF mRNA and protein levels by about 75% of control levels (Kaur
et al., 2007). The effects of P4 on BDNF are not limited to cortex. In an in vivo
study, spinal cord injury resulted in depletion of BDNF mRNA levels compared to
controls. Interestingly, injured rats treated for 3 days with progesterone exhibited
significantly higher levels of BDNF mRNA and protein (Gonzalez et al., 2005).
Progesterone treatment also decreased the number of cells undergoing
chromatolysis, a feature of motoneuron degeneration, thus illustrating the clinical
relevance for P4. Therefore, hormonal treatment of spinal cord injuries may
someday be a plausible therapy.
Evidently, both estrogen and progesterone may exert their neurotrophic
effects by stimulating BDNF. The interactions between estrogen and
progesterone may provide further insight as to the mechanisms underlying the
hormonal-neurotrophin relationship. Cyclic changes in hormone levels are
responsible for various effects; likewise, fluctuating levels of BDNF mRNA have
been shown to be dependent on the estrous cycle (Gibbs, 1998) with BDNF
protein increases at proestrous and estrous stages. Moreover, increased
excitability observed in the CA1 and CA3 regions of the hippocampus coincided
with changes in BDNF expression, suggesting that the evoked responses may be
regulated by estrogen-mediated BDNF increase (Scharfman et al., 2003) . These
results disagree with previous data showing that BDNF protein levels declined
during estrous and proestrous even though the mRNA levels increased (Gibbs,
27
1998). The discrepancy in these studies may be due to methodological
differences in the studies, including detection procedures and antibody
selectivity. Recently, another group examined the effects of estrogen and
progesterone on BDNF expression. Aged OVX rats were treated with vehicle,
estrogen, or estrogen plus progesterone. While E2 increased BDNF, NT3 and
NGF levels in entorhinal cortex, concomitant treatment with P4 attenuated this
increase. Interestingly, this increase was not seen in the hippocampus (Bimonte-
Nelson et al., 2004). Although not discussed, this discrepancy seems to have
important implications for learning and memory. Perhaps hormone replacement
in aged OVX rats did not have an effect due to age. Further examination should
be done on hormone replacement effects on young OVX rats to see whether
there is a fundamental difference between region-specific responses to
hormones and neurotrophins. Recent data examining the roles of ERs in eliciting
BDNF production have emerged. In particular, the role of ER  in auditory system
was recently studied by utilizing ER agonists to investigate effects on protection
from acoustic trauma and BDNF production. Activating ER  via DPN treatment
protected wild type (WT) mice and increased BDNF mRNA and protein. Further,
DPN treatment in mice deficient in ERα (ERα KO) and aromatase (ARKO) led to
a relatively larger increase in the production of BDNF protein as compared to WT
mice. Thus, these researchers concluded that ER  protects the auditory system
against acoustic trauma, presenting the first evidence that directly correlates ER 
expression to the protection of auditory function. Although this research was
done in the auditory system, this evidence provides support for the role of ER  in
28
plasticity and BDNF activation (Meltser et al., 2008).
Despite the wealth of data indicating a direct interaction between
estrogen, progesterone and BDNF, the methodological differences and treatment
paradigms illustrated here may reflect effects that are dependent on age, sex,
length of ovariectomy and anatomical differences. On the other hand, the overlap
observed in the stimulation of second messenger systems may reflect an indirect
mechanism for hormonal regulation of neurotrophins. There is evidence that
hormones and BDNF can independently activate different signal transduction
pathways. For example estrogen, progesterone and BDNF have all been shown
to execute actions via second messengers systems and signal transduction
pathways. One of the primary pathways involved in synaptic plasticity, learning
and memory is through the MAPK pathway. Along with its many other functions,
BDNF is also implicated in the ERK/MAPK signaling cascade (Zheng & Quirion,
2004). Furthermore, cyclic changes in estrogen have been demonstrated as
having powerful effects on the ERK/MAPK signaling cascade. Ovariectomy
results in decreased levels of activated ERK2 and estrogen replacement
reverses this loss. Moreover, these cyclic changes are closely associated with
changes in the phosphorylated state of the NR2 subunit of NMDA receptors (Bi
et al., 2001). Several other groups have also examined estrogenic effects on this
pathway (Bi et al., 2001; Kim et al., 2002; Nilsen et al., 2002). However, cyclic
changes in hormonal state also involve progesterone, so it is not surprising that
this hormone may also able to affect the MAPK pathway (Nilsen et al., 2002;
Singh, 2001). Therefore, a possible mechanism of hormone action may involve
29
interactions between estrogen and progesterone to modulate NMDAR function
by regulating the ERK/MAPK signaling cascade. Another important pathway
critical for various cell processes is the PI3K/AKT pathway, which is also
activated by estrogen (Zhang et al., 2001; Znamensky et al., 2003) progesterone
(Kaur et al., 2007), and BDNF (Zheng and Quirion, 2004) . Similarly, the
Ca
2+
/CREB second messenger system is involved in estrogenic functions
(Panickar et al., 1997; Segal and Murphy, 1998; Zhou et al., 2005) as well as
interactions with BDNF (Alonso et al., 2002; Ying et al., 2002; Blanquet et al.,
2003) and P4 (Nilsen & Brinton, 2002). The relationships between progesterone
and BDNF appear to be more complex, as progesterone can stimulate BDNF
expression in motoneurons, while BDNF has been shown to inhibit progesterone
synthesis in the periphery (Jensen and Johnson, 2001; Gonzalez et al., 2005).
The inconsistencies in these interactions remain to be elucidated. Whether E2
and BDNF act synergistically or in parallel to exert neuroprotective effects in the
CNS has not been clarified.

Organotypic Hippocampal Slice Cultures
Due to its role in memory function, the hippocampus has been at the
forefront of studies exploring hormonal effects on neuronal excitability and
synaptic plasticity. The organotypic hippocampal slice culture method has been
shown to be a valuable tool for investigating numerous questions, due to its ease
of preparation and anatomical, physiological and biochemical integrity as
compared to the in vitro preparation. Remarkably, this method allows the
30
maintenance of nervous tissue under long-term survival conditions that yield
highly differentiated cells in both morphology and physiology (Gahwiler, 1999).
The Stoppini method used in our studies (Stoppini et al., 1991), allows for
recovery of sectioned tissue on a semi-porous membrane as opposed to rotating
the slice on a tube. In our studies, cultured hippocampal slices are maintained in
vitro for two weeks prior to treatments to allow recovery from sectioning damage
and completion of the trisynaptic circuitry (Nakagami et al., 1997). This
preparation allowed us to examine the aforementioned hypotheses in a model
reflecting the complexity of in the vivo brain, with functional cell-cell interactions,
along with the flexibility of isolating a particular brain region. However, this
particular preparation has its limitations, namely, the differentiation between cell
types with the assays proposed herein (Western blot, LDH release, PI). To
discern the effects seen in these experiments, we complemented neuroprotection
assays with immunocytochemistry in cultured hippocampal slices.

A model for cell damage/death: NMDA toxicity
N-methyl-D-aspartic acid (NMDA) is a selective agonist of the NMDA
glutamate receptors. Excessive stimulation of NMDA receptors leads to neuronal
lesion and death, resulting from a massive influx of calcium and sodium ions
(Fundytus et al., 2001). It has been demonstrated that the influx of Ca2+ leads to
a plethora of neurotoxic mechanisms, including generation of oxidative species
along with activation of proteases (Hou and MacManus, 2002). This is a well-
established model of excitotoxicity commonly used to study glutamatergic
31
transmission within the confines of synaptic plasticity, a cellular mechanism for
learning and memory. We have used this technique in our lab to elucidate the
mechanisms underlying estrogen-mediated neuroprotection in hippocampus (Bi
et al., 2000, 2001). The three subregions of the hippocampus, CA1, CA3 and DG
are differentially affected by NMDA toxicity, with CA1 being the most sensitive
(Rangaraan et al., 1999). In addition, this sensitivity parallels the NMDA receptor
density in hippocampus. This model of cell damage is versatile, as glutamate
toxicity is associated with acute insults leading to cellular damage, such as
ischemia and stroke, as well as chronic neurodegenerative diseases like
Alzheimer‟s disease leading to cellular death (Aarts and Tymianski, 2003, 2004).

Summary
This preceding section outlines our current understanding of estrogen,
progesterone and BDNF actions on hippocampal functions related to memory
and plasticity. Despite the wealth of data describing these actions in various
model systems, this section also delineates the discrepancies in the current
literature, as well as a gap in our current understanding of the underlying
mechanisms involved in progesterone‟s antagonistic effects on estrogen-
mediated neuroprotection. Therefore, this dissertation was aimed at examining
the interactions between estrogen and progesterone in an in-vitro model of
excitotoxicity, and at evaluating the role of BDNF in mediating estradiol-induced
neuroprotection. Using organotypic hippocampal cultured slices and NMDA
treatment to elicit neuronal death, I hypothesized that the neuroprotective effects
32
of these hormones was critically dependent upon the timing of hormone
administration. I sought to answer the question of whether estrogenic
neuroprotection against NMDA toxicity was dependent on the activation of BDNF
and its resulting signaling pathways, and whether this effect was mediated
through the ER  receptor. The results from the studies I completed have affirmed
a role for ER  in mediating estrogenic neuroprotection against NMDA toxicity,
and that progesterone‟s reversal of E2-mediated neuroprotection was due to its
ability to downregulate ER . Further, these interactions were critically dependent
on the activation of BDNF and its receptor TrkB, suggesting estrogen may
influence TrkB signaling through the ER  receptor to mediate neuroprotection
against NMDA. Such studies, along with those ongoing in the field, will be
instrumental in understanding hormonal relationships with respect to
neuroprotection and in facilitating new drug developments of hormone therapy
without detrimental effects.

33
CHAPTER 2
PROGESTERONE REVERSES 17 -ESTRADIOL-MEDIATED
NEUROPROTECTION AND BDNF INDUCTION IN CULTURED
HIPPOCAMPAL SLICES

CHAPTER 2 ABSTRACT

Due to the many similarities in mechanisms of action, targets and effects,
progesterone, estrogen and neurotrophins have been implicated in synaptic
plasticity as well as in neuroprotection and neurodegeneration. In this study, we
examined the interactions between 17- -estradiol (E2) and progesterone (P4)
and BDNF on both plasticity and excitotoxicity in rat cultured hippocampal slices.
First, we evaluated the neuroprotective effects of E2 and P4 against NMDA
toxicity in cultured rat hippocampal slices. As previously reported, pretreatment
with 10 nM E2 (24 h) was neuroprotective against NMDA toxicity. However,
progesterone (10 nM) added 20 h after E2 treatment for 4 h, reversed its
protective effect. In addition, the same E2 treatment resulted in an increase in
BDNF protein levels as well as in activation of its receptor, TrkB, while addition of
P4 attenuated E2-mediated increase in BDNF and TrkB levels. Furthermore, E2-
mediated neuroprotection was eliminated by a BDNF scavenger, TrkB-Fc. Our
results indicate that E2 neuroprotective effects are mediated through the BDNF
pathway, and that, under certain conditions, P4 antagonizes the protective effect
of estrogen.
34
INTRODUCTION

There is substantial evidence that the actions of steroid hormones extend
well beyond reproductive organs. Multiple extra-hypothalamic regions within the
central nervous system have been identified as targets for gonadal steroid
hormones such as progesterone and estrogen, including hippocampus and
cortex (Baulieu et al., 1996; Daniel et al., 1997; McEwen et al., 1999). Depletion
of these steroid hormones in post-menopausal women has been established as a
risk factor for the development of Alzheimer‟s disease (AD). There is a wealth of
data suggesting a neuroprotective role for 17 -estradiol, a main form of estrogen,
in various animal models of neurodegeneration. Emerging data also suggest that
progesterone (P4) has potent neuroprotective effects. For example, several
studies have demonstrated that progesterone is neuroprotective against
glutamate-induced cell death (Nilsen and Brinton, 2002, , 2003; Kaur et al., 2007)
and traumatic brain injury (Roof et al., 1997; Robertson et al., 2006). As with
17 -estradiol (E2), P4 likely stimulates several different mechanisms of
neuroprotection, including activation of neurotrophins. In addition, reduced
metabolites of progesterone, such as 3- -hydroxy-5 -pregnan-20-one (THP,
allopregnanolone) have been shown to mediate, at least in part, the
neuroprotective effects of progesterone (Ciriza et al., 2004). Interactions
between estrogen and progesterone have also been documented in brain, as
estrogen stimulates progesterone receptor expression in CA1 region of
hippocampus and in medial preoptic nucleus (Alves et al., 2000; Chung et al.,
35
2006). However, the possible regulation by P4 of E2-mediated neuroprotective
effects has not been extensively investigated.
Several studies have indicated a similar pattern of expression and
functional interactions for brain-derived neurotrophic factor (BDNF) and estrogen.
BDNF and its receptor TrkB are present in cortex, hippocampus, midbrain, spinal
cord, and hypothalamus and in principal neurons as well as in glial cells
(Scharfman and MacLusky, 2006; Sohrabji and Lewis, 2006). In forebrain,
BDNF-synthesizing neurons also express estrogen receptors, and the BDNF
gene contains an ERE element, suggesting possible interactions between
estradiol and BDNF regulation (Miranda et al., 1993). The relationships between
progesterone and BDNF appear to be more complex, as progesterone can
stimulate BDNF expression in motoneurons, while BDNF has been shown to
inhibit progesterone synthesis in the periphery (Jensen and Johnson, 2001;
Gonzalez et al., 2005). Moreover, progesterone has been shown to decrease
BDNF in hippocampus and increase it in cortex (Frye and Rhodes 2005; Kaur et
al. 2007). The inconsistencies in these interactions remain to be elucidated.
Whether E2 and BDNF act synergistically or in parallel to exert neuroprotective
effects in the CNS has not been investigated. The present study was therefore
directed at testing interactions between estrogen and progesterone in an in-vitro
model of excitotoxicity, and at evaluating the role of BDNF in mediating estradiol-
induced neuroprotection using NMDA treatment of cultured hippocampal slices to
elicit neuronal death.

36
MATERIALS AND METHODS

Animals
Animals were treated in accordance with the principles and procedures of
the National Institutes of Health Guide for the Care and Use of Laboratory
Animals; all protocols were approved by the Institutional Animal Care and Use
Committee of the University of Southern California. Timed pregnant Sprague-
Dawley rats were obtained from Charles River Laboratories (Wilmington, MA)
and kept in the vivarium in a temperature- and light-controlled environment with a
12 h light/dark cycle. On the day of experimentation, rats were removed from
their home cage and anesthetized using halothane.

Preparation of cultured hippocampal slices
Organotypic hippocampal slice cultures were prepared from postnatal day
8-10 Sprague-Dawley rat pups according to previously described methods
(Stoppini et al., 1991). Briefly, following decapitation, brains were removed from
the cranium and placed into chilled cutting medium (Earle‟s MEM, 25 mM
HEPES, 10 mM Tris-base, 10 mM D-glucose, 3 mM MgCl
2
, pH 7.2). After
isolation of hippocampi, 400 µm thick transversal slices were cut with a McIlwain
tissue chopper. Slices were placed into chilled cutting medium, inspected and
separated under a microscope. Six slices from the midsection of the
hippocampus were then placed onto a 0.4μm culture plate insert (Millipore,
Billerica, MA) in 6-well flat bottom tissue culture plates (BD Falcon, San Jose,
37
CA). Cultures were maintained in 1 mL of steroid-deficient maintenance medium
containing 20% heat inactivated charcoal stripped horse serum (Sigma, St.
Louis, MO), Earle's balanced salt solution, Basal Medium Eagle (Sigma), 20 mM
NaCl, 0.2 mM CaCl
2
, 1.7 mM MgSO
4
, 2.7 mM L-glutamine, 27 mM HEPES, 5
mM NaHCO
3
, 48 mM D-glucose, 0.5 mM ascorbic acid, 0.5%
penicillin/streptomycin and 0.01 % insulin (Sigma); pH 7.4. Slices were kept at 35
°C with 5% CO
2
and maintained for 14 days with complete medium exchange
every three days prior to experiments.

Treatment of cultured hippocampal slices
Cultured hippocampal slices were maintained in vitro for two weeks prior
to treatments to allow recovery from sectioning damage and completion of the
trisynaptic circuitry (see (Nakagami et al., 1997). At 14 DIV, cultures were
supplemented with the appropriate treatments (hormones, vehicle, NMDA) in
serum-free medium for the indicated periods of time. Controls received vehicle
(DMSO) treatments in similar hormone-deprived conditions to account for
procedural or vehicle-related effects.
17β-estradiol (E2) was diluted in dimethylsulfoxide (DMSO) to a stock solution of
10 M and was further diluted in culture medium to a final concentration of 10 nM
and applied for 24 h. We have previously shown that at this concentration, E2 is
neuroprotective against NMDA toxicity (Bi et al., 2000). We devised a treatment
protocol that could parallel the ratio of P4 to E2 treatment previously used in in-
vivo experiments (Bimonte-Nelson et al., 2004) and assumed to represent
38
physiological levels of hormones. The temporal pattern of P4 exposure was
selected to investigate the minimum exposure time required to antagonize
estrogen‟s effects in a 24 h time period. Ten nM P4 was diluted in DMSO and
applied to the culture medium for ½ h, 1 h, 2 h, 4 h, 12 h, 20 h and 24 h during
continuous 24 h E2 exposure. P4 was diluted in the same manner as E2 and
applied to the culture medium at a final concentration of 10 nM for 4 h alone or
following 20 h of E2 treatment. NMDA (50 µM) was applied for 3 h following
hormone washout. TrkB Fc (0.5 µg/mL) was co-administered with E2 for 24 h.
Vehicle controls were performed in parallel and received 0.1% DMSO in serum-
free medium.

Cell viability assessment
Slices used for protein analysis were collected at the end of treatments.
Slices used for neuroprotection assays were washed with serum-free medium
and underwent a 24 h recovery period in serum-free media containing propidium
iodide (PI; 4 µM, Calbiochem). After recovery, the medium was collected to
measure the amount of lactate dehydrogenase (LDH) released using a
spectrophotometric assay. Slices were imaged with an epifluorescent microscope
using an inverted Leica DMIRB with a 550LP filter and fitted with a 5x phase
contrast objective and a Spot RT slider color CCD camera (Roper Scientific,
Tucson, AZ). These images were captured using Photoshop CS2, converted to
monochrome and then pseudo-colored only to illustrate regional intensity.
Quantification of raw images was done on Photoshop CS2 by manually selecting
39
regions of interest (ROI) corresponding to CA1, CA3 and dentate gyrus (DG) of
hippocampus based on the rat atlas of hippocampus and morphological markers.
Pixel intensity values were then recorded on Excel (Microsoft). Absolute intensity
was calculated using total pixel intensity in the analyzed regions. Values were
recorded as percentage of the values found in slices treated with NMDA (50 µM)
for 24 h to induce maximal cell death (NMDAmax).

Western blot analysis
At the end of experimental treatments, slices were collected and
homogenized by sonication, and an aliquot of the homogenate was taken to
determine protein concentration by the Bradford method (BioRad, Hercules, CA).
Samples were prepared for immunoblotting by dilution 1:1 in Laemmli buffer with
5% β-mercaptoethanol (BioRad). Twenty to fifty µg of proteins were loaded onto
15% SDS-PAGE gels or 4-20% gradient gels (BioRad) along with Precision
unstained molecular weight markers to approximate protein molecular weights
(BioRad). Following electrophoresis, proteins were electroblotted onto NitroPure
nitrocellulose membranes (Osmonics, Minnetonka, MN). Western blot
membranes were washed with TBS-Tween (0.05%) and blocked with 5% non-fat
milk.

Antibodies
Immunodetection of proteins was performed using 1:200 anti-BDNF (Cat.
# sc-546, Santa Cruz Biotech, Santa Cruz, CA), 1:1,000 anti-phospho TrkA/TrkB
40
and 1:1,000 TrkB (Cell Signaling Technology, Danvers, MA), and 1:10,000 anti-
-Actin antibodies, along with peroxidase-labeled secondary antibodies, 1:2,000
anti-rabbit for BDNF, pTRK and TrkB and 1:10,000 anti-mouse for -Actin
(Jackson ImmunoResearch, Westgrove, PA). BDNF signal was optimized by
using 40 ug of protein, a 1:200 dilution of the antibody and blocking in 5% milk for
1 h. Initial studies using a BDNF standard showed a strong band around the
predicted 14kDa and a faint band around 25kDa. Likewise, pTrk and TrkB
antibody specificity was corroborated with activation of the TrkB receptor by
BDNF and reversal of activation by the addition of K252a (data not shown).
Unlabeled standards were tagged using peroxidase labeled StrepTactin
(BioRad). Immunoblots were visualized autoradiographically using enhanced
chemiluminescence (Pierce).

Densitometric analyses
To quantify protein levels, films were scanned (CanonScan N656U
scanner; Canon, Lake Success, NY) at 300 dpi and analyzed. Band intensities
were calculated using Image J software. Immunoblot ratios were normalized to
protein and were reported as a percentage of vehicle control.

Statistical analysis
One-way analyses of variance (ANOVA) followed by Tukey‟s Multiple
comparison post-hoc tests were used for pair-wise comparisons between
experimental treatments. Data were analyzed using GraphPad Prism 4 software
41
(San Diego, CA) and significance level was set at 0.05. Results were expressed
as means  S.E.M. for the indicated number of experiments.

RESULTS

P4 reverses E2-mediated neuroprotection against NMDA-induced neurotoxicity
To determine whether P4 interferes with the neuroprotective effects of E2,
cultured hippocampal slices were treated with E2 for 24 h and P4 was added to
the medium during the final 4 h of the 24 h treatment period. Hormones were
washed out by 2 successive medium changes prior to NMDA treatment (50 M
NMDA, 3 h). As previously shown (Bi et al., 2001), E2 (10 nM) significantly
reduced the amount of NMDA-induced LDH release (Fig. 2.1). Addition of P4 (10
nM) 20 h after initiating E2 pretreatment (E2 + P4 + NMDA) reversed the
reduction in LDH release to levels identical to those found in NMDA treated slices
(Fig. 2.1). Treatment with E2, P4 or E2+P4 alone did not increase levels of LDH
release as compared to that measured in control slices (One way ANOVA F
(7,193) = 43.45, p < 0.0001).

42
Figure 2.1. P4-mediated reversal of E2-induced neuroprotection against
NMDA toxicity in cultured hippocampal slices.

A. Treatment protocol timeline for hormones and NMDA. Treatment with P4 (10
nM), E2 (10 nM), or NMDA (50 µM) was performed as indicated in the diagram.
Vehicle was administered for the length of the experiment (not shown).
B. LDH release in the medium measured 24 h after NDMA treatment. Medium
was collected 24 h after initiation of NMDA treatment, and LDH activity was
determined as described under Material and Methods. Results are means ±
S.E.M of 7 experiments and are expressed as percent of release in NMDA-
treated slices. Statistical significance was analyzed by ANOVA followed by
Tukey's test for individual comparisons. *p < 0.001 vs. NMDA; † p < 0.001 vs. E2
+ NMDA (EN); NS: not significant.

Similarly, analysis of propidium iodide uptake in slices treated under
various conditions illustrated a marked difference between NMDA-treated slices
43
in the absence or presence of hormones. Fluorescent images of slices treated
were pseudo-colored only to illustrate regional intensity of cellular damage.
Visual analysis of cultured hippocampal slices treated with vehicle, E2, P4 or E2
in combination with P4 indicated that none of these treatments produced a
significant increase in PI uptake (Fig. 2.2, top). In contrast, cultured slices treated
with NMDA (50 M) for 3 h or 24 h exhibited increased PI uptake throughout the
hippocampus, with the highest increase in CA1 (Fig. 2.2, bottom).

44
Figure 2.2. Effects of E2 and P4 on NMDA-induced neuronal damage in
cultured hippocampal slices, assessed with PI uptake.

Top Row: Representative images of PI uptake in slices treated with vehicle
(VEH), E2 (10 nM), P4 (10 nM) or E2 (10 nM) + P4 (10 nM) (EP).
Bottom row: Representative images of PI uptake in slices treated with E2 (10 nM)
+NMDA (50 µM) (EN), P4 (10 nM) +NMDA (PN), E2 + P4 + NMDA (EPN) and
NMDA alone. NMDA treatment resulted in increased levels of fluorescence
intensity, and estrogen attenuated this increase. P4 reversed the protective
effects of E2. Color bar represents fluorescence intensity scale, with red
illustrating highest intensity.

Quantification of unmodified images indicated that maximal cell death was
obtained in slices treated with NMDA for 24 h (Fig. 2.3 b-d). In agreement with
our previous results, pretreatment for 24 h with E2 (10 nM) prior to 3 h of NMDA
exposure significantly reduced fluorescence intensity in CA1, CA3 and DG
regions of hippocampus (p < 0.001; Fig. 2.3 b-d). This reduction was reversed in
all three regions by the addition of P4 to E2 and NMDA-treated slices (EPN) to
45
levels that were not significantly different from those found in slices treated with
NMDA alone. Interestingly, addition of P4 for 4 h alone resulted in significant
protection from NMDA-mediated neurotoxicity in CA1 and DG, but not in CA3
(one way ANOVA for CA1: F (8, 267) = 349.4, p < 0.0001; ANOVA for CA3: F (8,
298) = 87.02, p < 0.0001; ANOVA for DG: F (8, 303) = 303.5, p < 0.0001; Fig. 2.3
b-d).

46
Figure 2.3. Quantitative analysis of PI uptake in cultured hippocampal
slices.

Images of slices treated as in Figure 2 were analyzed as described under
Materials and Methods. Regions analyzed (CA1, CA3 and DG) are indicated in
A. Results are expressed in percent of fluorescent intensity measured in slices
treated with NMDA (50 µM) for 24 h, and are means ± S.E.M. of 12-14 images
obtained in 6-8 slices. Statistical significance was analyzed by ANOVA followed
by Tukey's test for individual comparisons. ** p < 0.001 as compared to NMDA
treatment alone; * p < 0.05 as compared to NMDA treatment alone; † p < 0.05 as
compared to treatment with E2 + NMDA (EN).

47
Antagonistic effects of Progesterone on Estrogen are not mediated by its
metabolites
Allopregnanolone (ALLO), a metabolite of progesterone, has been shown
be a potent GABA
A
receptor modulator. To investigate whether the effects
observed after applying P4 for 4h were due to its conversion to ALLO, we co-
treated slices with both P4 and finasteride for 4h after 20 h of E2 treatment.
Finasteride inhibits the synthesis of 5α-reduced neurosteroids and has been
shown to significantly attenuate the formation of ALLO both in-vitro and in-vivo
(Rhodes and Frye, 2005; Izumi et al., 2007). Furthermore the concentration of
finasteride used in the present study has been used previously in hippocampal
slices and neurons (Mostallino et al., 2006; Izumi et al., 2007). Co-treatment with
P4 and finasteride did not significantly modify the results obtained using P4
alone. PI uptake was similar in slices treated with E2, P4 and NMDA and those
treated with E2, P4, Finasteride and NMDA in CA1, CA3 and DG regions of
hippocampus (data not shown).

E2-mediated neuroprotection is mediated by BDNF
To further investigate the mechanisms underlying E2 neuroprotection, we
utilized the BDNF scavenger, TrkB-Fc, to determine whether BDNF was involved
in mediating E2 neuroprotective effects. Addition of 0.5 µg/mL TrkB Fc
throughout the course of E2 pretreatment resulted in a significant attenuation of
E2-mediated neuroprotection against NMDA (50 M) toxicity (p < 0.001; Fig.
2.4).
48
Figure 2.4. BDNF mediates E2 neuroprotective effects against NMDA
toxicity in cultured hippocampal slices.

Cultured hippocampal slices were treated as shown in Figure 1 and TrkB-Fc (0.5
µg/mL) was present during the duration of E2 treatment. Medium was collected
24 h after NMDA treatment and LDH assayed as indicated under Materials and
Methods. Results are expressed as percent of LDH release measured in medium
of slices treated with NMDA for 3 h and are means ± S.E.M. of 5-6 experiments.
Statistical significance was analyzed by ANOVA followed by Tukey's test for
individual comparisons. * p < 0.001 as compared to NMDA treated slices.

Addition of the BDNF scavenger alone had no significant effect on LDH
release. We further tested BDNF involvement in E2 neuroprotection by assessing
the state of phosphorylation/activation of the BDNF receptor, TrkB (one way
ANOVA F (7, 14) = 16.16, p < 0.0001; Fig. 2.5). E2 treatment resulted in a
significant increase in TrkB phosphorylation, suggesting that E2-mediated
neuroprotection involves increased BDNF release followed by activation of the
TrkB receptor.

49
Figure 2.5. Effects of E2 and P4 on TrkB activation/phosphorylation in
cultured hippocampal slices.

Cultured hippocampal slices were treated with E2 (10 nM), P4 (10 nM) or E2 +
P4 as described in Figure 2.1. At the end of treatment, slices were collected and
processed for western blot analysis of p-TrkB (bottom). Levels of p-TrkB were
corrected with those of β-actin and results were expressed as percent of values
found in vehicle-treated slices; they are means ± S.E.M. of 4-5 experiments.
Statistical significance was analyzed by ANOVA followed by Tukey's test for
individual comparisons. ** p < 0.001 as compared to vehicle-treated slices; † p <
0.001 as compared to E2 treated slices.

P4 reverses E2-induced increase in BDNF levels and TrkB activation
E2 has previously been shown to increase BDNF expression in various
brain regions, including hippocampus. For instance, ovariectomy results in a
50
significant decrease in hippocampal BDNF mRNA, and E2 replacement partially
restores BDNF levels (Singh et al., 1995). We therefore measured BDNF levels
in cultured hippocampal slices following 24 h treatment with E2 (10 nM). Western
blot analysis indicated that this treatment resulted in a 75% increase in BDNF
levels (Fig. 2.6). Since this rise in BDNF expression was temporally associated
with the neuroprotective effects seen at the end of E2 treatment, we examined
whether the addition of P4 (10 nM) had any effects on E2-induced BDNF
increase. Interestingly, addition of P4 during the final 4 h of E2 treatment
significantly reversed E2-induced increase in BDNF levels. Furthermore, P4
treatment alone did not significantly increase BDNF protein expression after 4 h
of treatment (one way ANOVA F (3, 75) = 5.644, p = 0.0015; Fig. 2.6). In
addition, P4 treatment also eliminated E2-mediated TrkB
phosphorylation/activation and did not induce receptor activation on its own (one
way ANOVA F (3, 9) = 15.09, p = 0.0007; Fig. 2.5).

51
Figure 2.6. Effects of E2 and P4 on BDNF levels in cultured hippocampal
slices.

Cultured hippocampal slices were treated with E2 (10 nM), P4 (10 nM) or their
combination according to the protocol shown in Fig. 2.1A. Control slices were
treated with vehicle. Slices were collected at the end of treatment, and processed
for determination of BDNF levels with western blots as described under Material
and Methods. Results were normalized to levels of β-actin and were expressed
as percent of control; they are means ± S.E.M. of 7 experiments. Statistical
significance was analyzed by ANOVA followed by Tukey's test for individual
comparisons. * p < 0.05 as compared to control to E2 + P4-treated samples.

52
DISCUSSION

A substantial body of evidence indicates that estrogen, the biologically
most potent and prevalent female hormone, is neuroprotective under a variety of
conditions. However, the paucity of studies exploring the effects of progesterone
may account for some of the inconsistencies reported in the literature. It is now
clearly established that menopause, as well as ovariectomy, results in a
depletion of both estrogen and progesterone. Thus, the decline in progesterone
as well as in estrogen may play a significant role in the various deficits, including
cognitive deficits, which follow menopause and should be closely taken into
account when trying to relate age-related hormonal and cognitive changes. In
addition, accumulating evidence suggests that estrogen and progesterone
differentially affect several neurobiological processes and that it is their
interactions on these processes that influence brain systems involved in learning
and memory. For example, while progesterone has no effect on spine density on
its own, it prevents estradiol-induced increases in spine density when co-
administered (Murphy and Segal, 2000). Similarly, although E2 treatment results
in neuroprotection against glutamate toxicity, the combination of E2 and
progestin either enhances or attenuates neuroprotection, depending on the type
of progestin (Nilsen and Brinton, 2002). Furthermore, in vivo results also show a
detrimental effect of co-treating aged OVX rats with E2 and P4. While E2
increased neurotrophin levels in entorhinal cortex, concomitant treatment with P4
attenuated this increase (Bimonte-Nelson et al., 2004). As with many other
53
studies, these results do not take into account the temporal relationships
between hormone administrations, which may explain some of the discrepancies
in the current literature. As of yet, there are still conflicting data regarding the
complex interactions between various gonadal hormones and their differential
effects on various brain systems.
In our study, we utilized organotypic hippocampal slices prepared from P9
pups of both genders and cultured for two weeks with media supplemented with
charcoal-stripped horse serum. This method has been shown to reduce estrogen
levels to less than 10
-12
M in culture media containing 10% heat-inactivated FCS
(Sigma Product information sheet), and dextran-coated charcoal has been
utilized to separate free from antibody-bound progesterone (Jensen and
Johnson, 2001). Preliminary results did not show a sex effect, thus results from
hippocampal slices of both sexes were combined for all experiments.
Consequently, we chose Western Blot as a protein detection method instead of
ELISA. BDNF ELISA kit (Promega, WI) does not distinguish between the mature
and pro-BDNF forms; therefore, we utilized the BDNF antibody (Santa Cruz
Biotech, Santa Cruz, CA) in Western Blotting techniques to detect the mature
form at 14 kDa. However, this antibody reveals a distinct pattern of expression
compared with other antibodies, which may be due to differential binding to
specific conformations of BDNF (Scharfman and MacLusky, 2006). Similarly, the
pTrkB antibody used does not readily distinguish pTrkB from pTrkA, since the
binding sequences in the two receptors are identical. Nonetheless, we believe
our data suggests a strong relationship between E2-mediated upregulation of
54
BDNF protein and neuroprotection.
Our results indicate that progesterone, under certain conditions, produces
effects that counteract estradiol‟s neuroprotective effects in hippocampus. In
agreement with our previous findings, 17 -estradiol was neuroprotective against
NMDA-induced neurotoxicity in organotypic hippocampal slices (Bi et al., 2000).
In the present study, 10 nM P4 applied for 4 h following a 20 h E2 pretreatment
prevented E2-mediated neuroprotection against NMDA-elicited increases in both
LDH release and PI uptake. Addition of the 5  - reductase inhibitor finasteride to
E2 + P4-treated samples did not modify P4-induced reversal of E2-mediated
protection. This was not unexpected, as most effects of allopregnanolone, the
major metabolite of P4, require micromolar concentrations (Lockhart et al., 2002;
Xilouri and Papazafiri, 2006). Since we used 10 nM P4 in our experiments, it is
likely that the concentration of allopregnanolone generated would be less than 10
nM.
E2 treatment increased BDNF levels and phosphorylation of its receptor,
TrkB, suggesting that E2 can trigger both an increased expression of BDNF as
well as its release, followed by activation of the TrkB receptor. Addition of the
BNDF scavenger, TrkB-Fc, to the culture medium attenuated E2-mediated
neuroprotection, further strengthening the idea that BDNF release plays a critical
role in E2-mediated neuroprotection. On the other hand, P4 treatment did not
result in increased BDNF levels or phosphorylation of TrkB, although it exhibited
neuroprotective effects, at least in CA1 and DG. Our results are therefore in
agreement with previously reported data indicating that P4 can be
55
neuroprotective against glutamate neurotoxicity (Kaur et al. 2007; Nilsen and
Brinton, 2002).
A recent review provides evidence for TrkB signaling as a mediator of
estrogen effects in hippocampus (Spencer et al., 2008) TrkB immunoreactivity
was found to fluctuate during the estrous cycle, with the highest intensity in CA3
and dentate gyrus during proestrus, when both estrogen and BDNF protein levels
are also highest. Although it is not yet clear whether estrogen directly or indirectly
activates the TrkB receptor, our results indicate that activation of TrkB plays a
critical role in E2-induced neuroprotection from NMDA toxicity under these
conditions. Our results also suggest that P4 neuroprotective effects are probably
not mediated through BDNF signaling. In fact, P4 treatment prevented E2-
mediated increased BDNF expression and TrkB phosphorylation. This effect is
likely to account for the prevention by P4 of E2 neuroprotective effects. However,
it should be noted that there are discrepancies in the literature regarding the
relationship between progesterone and BDNF. Progesterone was shown to
reduce BDNF levels in the hippocampus, while increasing mRNA and protein
levels in cortical explants (Frye and Rhodes, 2005; Kaur et al., 2007). A recent
study also reported prevention by P4 of E2-mediated neuroprotection against
kainate excitotoxicity, although P4 alone did not exhibit neuroprotective effects
(Rosario et al., 2006). The mechanisms underlying the antagonist effect of P4 on
E2-mediated increased BDNF expression and signaling are not yet known, but
the short time-course of P4 effects suggests that the mechanism does not
involve traditional genomic pathways. An attractive hypothesis that could
56
account for our observed results has been suggested by our colleagues, who
have discovered that P4 treatment results in the rapid down-regulation of E2
receptors (Jayaraman and Pike, in press).
In the hippocampus of the adult rat, BDNF mRNA is present in dentate
gyrus granule cells and hippocampal pyramidal cells (Scharfman and MacLusky,
2006). Both of the classical progesterone receptors isoforms (PRA and PRB) are
also expressed in all regions of the hippocampus (Brinton et al., 2008). Finally
both mRNA and protein expression of ERα and ERβ are found in numerous cell
types (Simerly et al., 1990; Scharfman and MacLusky, 2006), with some co-
localization with BDNF-expressing cells (Blurton-Jones et al., 2004). Although the
relative distribution of these receptors is similar in hippocampus, the functional
role of each receptor remains to be elucidated.
It is becoming increasingly clear that understanding the interactions
between E2 and P4 is paramount to understanding the complexities underlying
age-related cognitive deficits and diseases like Alzheimer‟s disease (AD). In a
mouse model of AD, P4 treatment did not affect A  accumulation or behavioral
performance in a Y-maze, although it did reduce tau hyperphosphorylation.
When P4 was combined with E2 treatment, the E2 neuroprotective effect against
A  accumulation was attenuated (Carroll et al., 2007). Together with our results,
recent studies present strong evidence that although E2 and P4 may exert
similar actions, their combination and the potential effects of the temporal order
of treatment on neurotrophin regulation represent factors that need to be taken
57
into account to understand the role of these hormones as neuroprotective agents
and potential treatments for neurodegenerative disorders.

58
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62
CHAPTER 3
PROGESTERONE INHIBITS ESTROGEN NEUROPROTECTION BY
DOWNREGULATING ESTROGEN RECEPTOR- 

CHAPTER 3 ABSTRACT

While both 17  -estradiol (E2) and progesterone (P4) have been found to be
neuroprotective in a number of experimental paradigms, P4 has also been shown
to inhibit the neuroprotective effects of E2. We recently reported that a relatively
short treatment of cultured hippocampal slices with P4 for 4 h following a
prolonged (20 h) treatment with E2 eliminated estrogenic neuroprotection against
NMDA toxicity and induction of BDNF expression. In the present study, we
evaluated the effects of treatment on cultured hippocampal slices with P4
following E2 treatment on the expression and levels of the estrogen receptors,
ER  and ERβ, as well as of BDNF. Treatment with P4 reversed the E2-elicited
increases in levels of ERβ mRNA and protein. In contrast, E2 treatment did not
modify ER  mRNA, but increased ER  protein levels, which were also reversed
by P4 treatment. Furthermore, E2 treatment produced an increase in BDNF
mRNA, and P4 treatment following E2 treatment eliminated this increase.
Experiments with an ERβ-specific antagonist, PHTPP, and specific agonists for
ERα and ERβ, Propyl Pyrazole Triol (PPT) and diarylpropionitrile (DPN),
respectively, indicated that E2-mediated neuroprotection against NMDA toxicity
was mediated by the activation of ERβ receptors. This conclusion was also
63
verified by results indicating that E2 had no neuroprotective effects against
NMDA toxicity in cultured hippocampal slices prepared from ERβ-/- mice.
Our results indicate that E2-mediated neuroprotection against NMDA toxicity is
due to the activation of ERβ receptors and that P4 treatment following E2
treatment results in the down-regulation of ERβ mRNA and protein; this
mechanism thus provides a potential explanation for P4-mediated inhibition of E2
neuroprotective effects. Furthermore, the existence of such a mechanism has
important consequences for our understanding of the interactions between these
two important sex hormones.

64
INTRODUCTION

Menopausal women are up to three times as likely to develop Alzheimer‟s
disease as men, indicating that the loss of circulating ovarian hormones places
females at a significant risk for the development the disease (Henderson, 1997;
Sherwin, 2003). With the number of post-menopausal women increasing every
year, it has become imperative to find new methods to alleviate the symptoms of
menopause. Clinicians widely prescribe hormone therapy (HT) for a variety of
menopause-related symptoms and disease prevention (Keating et al., 1999;
Utian et al., 2008). However, evidence from the Women‟s Health Initiative (WHI)
studies in 2002 revealed increased deleterious effects in cognitive function and
breast cancer risk following combined estrogen and progestin therapy when
compared to placebo controls (Rossouw et al., 2002). These results propelled
the dramatic decline in menopausal hormone therapy in the United States and
galvanized the re-evaluation of hormone interactions in central nervous system
(Utian et al., 2008; Chlebowski et al., 2009).
Since both menopause and ovariectomy result in depleted estrogen and
progesterone levels, the decline in progesterone may also play a significant role
in the various deficits, including cognitive deficits, which follow menopause. A
growing compendium of research has recently focused on the role of
progesterone in regulating neural actions of estrogen involved in learning and
memory. For example, progesterone has been shown to counteract estradiol-
induced increases in spine density (Murphy and Segal, 2000) and neurotrophin
65
levels (Gibbs, 1999; Bimonte-Nelson et al., 2004), as well as reverse estrogenic
neuroprotection against glutamate toxicity (Nilsen and Brinton, 2002), NMDA
toxicity (Aguirre and Baudry, 2009), kainate lesions (Rosario et al., 2006) and A 
accumulation (Carroll et al., 2007). Furthermore, the two isoforms arising from
distinct estrogen receptor genes, ER  and ER , may differentially regulate PR
synthesis (Alves et al., 2000).
Estrogen receptors (ERs) have been localized throughout the cortex,
limbic system and hippocampus, in neurons, astrocytes and dendritic processes
(Simerly et al., 1990; Su et al., 2001; Milner et al., 2005), and with different
expression patterns between the two types of estrogen receptors ER  and ER 
(Azcoitia et al., 1999; Orikasa et al., 2000). Furthermore, a functional divergence
between the two receptors has been observed. As illustrated with mouse
knockout studies, fertility is impaired in mice lacking the ER  gene, but not the
ER  gene, suggesting that ER  may be more relevant for reproductive functions
than ER  (Shughrue et al., 1997). In contrast, accumulating evidence has
underscored the role of ER  in cognition and behavior. A recent study found that
various factors of synaptic plasticity, including activation of synaptic proteins,
enhancement of LTP and improved memory were dependent on the activation of
ER  (Liu et al., 2008). It has also been shown that ER -KO (BERKO) animals
had impaired performance in object recognition and placement tasks, even when
given estradiol supplements (Walf et al., 2008). Recent data examining the roles
of ERs in eliciting BDNF production further exemplify the functional differences
66
between these receptors. Basal expression of BDNF protein in cochlea of
BERKO animals was significantly lower than in wild type (WT), while activating
ER  with diarylpropionitrile (DPN) in WT mice increased BDNF mRNA and
protected against acoustic trauma (Inna et al., 2008). Although this study was
done in the auditory system, the evidence provides support for the role of ER  in
plasticity and BDNF activation. It has been established that both estrogen and
progesterone may exert their neurotrophic effects by stimulating BDNF
(Scharfman and MacLusky, 2006; Sohrabji and Lewis, 2006; Kaur et al., 2007).
However, the mechanisms underlying the hormonal-neurotrophin relationships
are yet to be clearly elucidated and a deeper understanding of the interactions
between estrogen and progesterone may provide further insight.
We therefore investigated whether progesterone has an antagonistic
effect on E2-mediated neuroprotection in an ER - dependent manner. We have
previously shown that estradiol treatment results in neuroprotection against N-
methyl-D-aspartate (NMDA) toxicity, an effect partially due to BDNF induction
(Aguirre and Baudry, 2009). In this study, we used the organotypic hippocampal
slice model to show that the treatment protocol of estradiol paired with
progesterone results in downregulation of ER  mRNA, leading to diminished
BDNF mRNA and protein levels and thus protection from NMDA toxicity. In
addition, the activation of ER  but not ER  mimicked estradiol‟s effects on
neuroprotection and BDNF protein expression. In summary, we show that
estrogenic neuroprotection against NMDA in cultured hippocampal slices is due
67
to upregulation of BDNF through ER  activation and that progesterone
counteracts protection by downregulating ER .

MATERIALS AND METHODS

Animals

Rats
Animals were treated in accordance with the principles and procedures of
the National Institutes of Health Guide for the Care and Use of Laboratory
Animals; all protocols were approved by the Institutional Animal Care and Use
Committee of the University of Southern California. Timed pregnant Sprague-
Dawley rats were obtained from Charles River Laboratories (Wilmington, MA)
and kept in the vivarium in a temperature- and light-controlled environment with a
12 h light/dark cycle. On the day of experimentation, rat pups were removed from
their home cage and sacrificed.

Mice
The Institutional Animal Committee approved this study for animal
experiments at the University of Southern California (USC) and principles of
laboratory animal care were followed according to the NIH guidelines as
mentioned above. C57BL/6 ER -KO and ER -KO mice were obtained from
68
Jackson Laboratories and were kept in cages on sawdust bedding in the same
room with a 12-h light-dark cycle and allowed free access to food and water. ER-
KO (ER 
-/-
and ER 
+/-
) and WT (ER 
+/+

+/+
) littermates were bred from
heterozygous ER 
+/-
mice. Genotyping of tail/blood DNA was performed to
identify ER /ER  genes by using polymerase chain reaction (PCR) (data not
shown).

Preparation of cultured hippocampal slices
Organotypic hippocampal slice cultures were prepared from postnatal day
nine (P9) Sprague-Dawley rat pups according to previously described methods
(Stoppini et al., 1991). Postnatal 11~14-day old C57BL/6 ER -KO and WT pups
were utilized for organotypic hippocampal slices and were prepared in the same
manner as rat pups. Briefly, following decapitation, brains were removed from
the cranium and placed into chilled cutting medium (Earle‟s MEM, 25 mM
HEPES, 10 mM Tris-base, 10 mM D-glucose, 3 mM MgCl
2
, pH 7.2). After
isolation of hippocampi, 400 µm thick transversal slices were cut with a McIlwain
tissue chopper (Westbury, NY). Slices were placed into chilled cutting medium,
inspected and separated under a microscope. Six slices from the midsection of
the hippocampus were then plated onto a 0.4μm culture plate insert (Millipore,
Billerica, MA) in 6-well flat bottom tissue culture plates (BD Falcon, San Jose,
CA). Cultures were maintained in 1 mL of steroid-deficient maintenance medium
containing 20% heat-inactivated and charcoal-stripped horse serum (Sigma, St.
Louis, MO), Earle's balanced salt solution, Basal Medium Eagle (Sigma), 20 mM
69
NaCl, 0.2 mM CaCl
2
, 1.7 mM MgSO
4
, 2.7 mM L-glutamine, 27 mM HEPES, 5
mM NaHCO
3
, 48 mM D-glucose, 0.5 mM ascorbic acid, 0.5%
penicillin/streptomycin and 0.01 % insulin (Sigma); pH 7.4. Slices were kept at
35 °C with 5% CO
2
and maintained for 14 days with complete medium exchange
every three days prior to experiments.

Treatment of cultured hippocampal slices
Cultured hippocampal slices were maintained in vitro for two weeks prior
to treatments to allow recovery from sectioning damage and completion of the
trisynaptic circuitry (Nakagami et al., 1997). At 14 DIV, cultures were
supplemented with the appropriate treatments (hormones, agonists, vehicle,
NMDA, inhibitors) in serum-free medium for the indicated periods of time.
Controls received vehicle (DMSO) treatments in similar hormone-deprived
conditions to account for procedural or vehicle-related effects.
17 -estradiol (E2) was diluted in dimethylsulfoxide (DMSO) to a stock solution of
10 M and was further diluted in culture medium to a final concentration of 10 nM
and applied for 24 h. We have previously shown that at this concentration, E2 is
neuroprotective against NMDA toxicity (Bi et al., 2000; Aguirre and Baudry,
2009). We devised a treatment protocol that could parallel the ratio of P4 to E2
treatment previously used in in-vivo experiments (Bimonte-Nelson et al., 2004;
Gibbs, 1999) and assumed to represent physiological levels of hormones. P4
was diluted in the same manner as E2 and applied to the culture medium at a
final concentration of 10 nM for 4 h alone or following 20 h of E2 treatment. ER 
70
and ER  selective agonists propyl pyrazole triol (PPT) and diarylpropionitrile
(DPN), respectively, were applied to culture media in the same manner and for
the same duration as E2 (24 h). To explore the efficiency of these agonists in
mimicking estrogenic protection from NMDA toxicity, we utilized three different
concentrations. PPT was administered to culture media at 1 M, 100nM and
10nM. DPN was administered to culture media at 100nM, 10nM and 1nM. These
concentrations differ due to the different binding affinities for each agonist to their
respective receptor (Covaleda, 2008). NMDA (50 µM) was applied for 3 h
following hormone washout. ER  selective inhibitor PHTPP (Tocris, Ellisville,
MO) was administered prior to E2 treatment for 1 h. Vehicle controls were
performed in parallel and received 0.1% DMSO in serum-free medium.
The ER-KO and WT mice received a slightly different hormone regimen. After 12-
14 days in culture, hippocampal slices were incubated in serum-free medium with
or without 1 nM 17 -E2 for 24 h. Following hormone washout, they were then
exposed to 50 µM NMDA for 3 h. After one wash with serum-free medium to
remove NMDA, cultured slices were incubated in serum-free medium for another
24 h in the presence of 17 -E2. 17 -E2 and NMDA solutions were all freshly
prepared.

Cell viability assessment
Slices used for protein analysis were collected at the end of treatments.
Slices used for neuroprotection assays were washed with serum-free medium
and underwent a 24 h recovery period in serum-free media containing propidium
71
iodide (PI; 4 µM, Calbiochem). After recovery, the medium was collected to
measure the amount of lactate dehydrogenase (LDH) released using a
spectrophotometric assay. Slices were imaged with an epifluorescent microscope
using an inverted Leica DMIRB with a 550LP filter and fitted with a 5x phase
contrast objective and a Spot RT slider color CCD camera (Roper Scientific,
Tucson, AZ). Quantification of raw images was done on Photoshop CS2 by
manually selecting regions of interest (ROI) corresponding to CA1, CA3 and
dentate gyrus (DG) of hippocampus based on the rat atlas of hippocampus and
morphological markers. Pixel intensity values were then recorded on Excel
(Microsoft). Absolute intensity was calculated using total pixel intensity in the
analyzed regions. Values were recorded as percentage of the values found in
slices treated with NMDA (50 µM) for 24 h to induce maximal cell death
(NMDAmax). Neuronal damage of mouse hippocampal slices was assessed by
semi-quantitative analysis of propidium iodide (PI) uptake as previously
described (Liu et al., 2003), and by measurement of lactate dehydrogenase
(LDH) released into the medium (Koh and Choi, 1987). PI uptake was scored
semi-quantitatively with a scale of 1-5 (1 = 0%, 2 = 0-25%, 3 = 25-50%, 4 = 50-
75%, and 5 = 75-100% of total area labeled by PI). Images of some PI-labeled
cultures were also captured with a digital camera under a fluorescence
microscope. LDH activity was expressed as units per ml medium, where one unit
of activity is the amount of LDH which causes a decrease of 0.001 AU of NADH
per min in the presence of sodium pyruvate at 340 nM. Data are reported as fold
of controls.
72
Western blot analysis
At the end of experimental treatments, slices were collected and
homogenized by sonication, and an aliquot of the homogenate was taken to
determine protein concentration by the Bradford method (BioRad, Hercules, CA).
Samples were prepared for immunoblotting by dilution 1:1 in Laemmli buffer with
5% β-mercaptoethanol (BioRad). Twenty to fifty µg of proteins were loaded onto
12% SDS-PAGE gels along with Precision unstained molecular weight markers
to approximate protein molecular weights (BioRad). Following electrophoresis,
proteins were electroblotted onto NitroPure nitrocellulose membranes
(Osmonics, Minnetonka, MN). Western blot membranes were washed with TBS-
Tween (0.05%) and blocked with 3% non-fat milk.

Antibodies
Immunodetection of proteins was performed using 1 g/mL anti-ERβ (Cat.
# ab3576, Abcam Inc., Cambridge, MA), 1:500 anti-BDNF (Santa Cruz Biotech.,
Santa Cruz, CA) and 1:10,000 anti- -Actin antibodies, along with peroxidase-
labeled secondary antibodies, 1:5,000 anti-rabbit for ERβ and 1:10,000 anti-
mouse for -Actin (Jackson ImmunoResearch, Westgrove, PA). Unlabeled
standards were tagged using peroxidase labeled StrepTactin (BioRad).
Immunoblots were visualized autoradiographically using enhanced
chemiluminescence (Pierce).

73
Densitometric analyses
To quantify protein levels, films were scanned (CanonScan N656U
scanner; Canon, Lake Success, NY) at 300 dpi and analyzed. Band intensities
were calculated using Image J software. Immunoblot ratios were normalized to
protein and were reported as a percentage of vehicle control.

RNA extraction and RT-PCR/QPCR
Following protein determination, hippocampal tissues were processed for
RT-PCR quantification of ERα, ERβ and BDNF mRNA. For RNA extractions,
treated cells were lysed using TRIzol reagent (Invitrogen Corporation; Carlsbad,
CA, USA) and processed for total RNA extraction as per manufacturer‟s protocol.
1-2 g of RNA was used for reverse transcription using the Superscript
TM
First
strand synthesis system (Invitrogen) and the resulting cDNA was used for both
standard PCR and real-time quantitative PCR amplifications. The primer sets
used were: ER - F: 5‟-CATCGATAAGAACCGGAG-3‟ and R: 5‟-
AAGGTTGGCAGCTCTCAT-3‟; ER  - F: 5‟-AAAGTAGCCGGAAGCTGA-3‟ and
R: 5‟-CTCCAGCAGCAGGTCATA-3‟; -actin – F: 5‟-
AGCCATGTACGTAGCCATCC-3‟ and R: 5‟-CTCTCAGCTGTGGTGGTGAA-3‟.
Quantitative PCR was carried out using DNA Engine Opticon 2 continuous
fluorescence detector (MJ Research Inc; Waltham, MA, USA). SYBR Green I
(Molecular Probes, Invitrogen, USA) was added to the PCR mix along with cDNA
and appropriate primers. The cycle of threshold (Ct) was determined for each of
the primers used. A standard curve was generated for each primer set such that
74
the Ct values obtained for different samples were included within the
corresponding standard curve. Relative quantification of mRNA levels from
various treated samples was done by the comparative Ct method as follows. The
standard-curve fitted Ct values for ER  and ER  from different samples were
normalized using the corresponding -actin values to get ∆Ct values. The ∆Ct
values for different treatment conditions were then subtracted by the ∆Ct value of
the control to get ∆∆Ct values. The relative levels of mRNA in treatment samples
compared to that of the control were obtained using the formula 2
-∆∆Ct
and are
plotted in the graph as percent of control values. The experiments were repeated
with at least 3 independent culture preparations.

ER KO Studies

Statistical analysis
One-way analyses of variance (ANOVA) followed by Tukey‟s Multiple
comparison post-hoc tests were used for pair-wise comparisons between
experimental treatments. Data were analyzed using GraphPad Prism 4 software
(San Diego, CA) and significance level was set at 0.05. Results are expressed as
means  S.E.M. for the indicated number of experiments.

75
RESULTS

ER  activation mediates estrogenic neuroprotection against NMDA toxicity
We have previously shown that 17-  estradiol treatment of organotypic
hippocampal slices is neuroprotective against NMDA toxicity (Bi et al., 2000;
Aguirre and Baudry, 2009). We therefore examined the roles of ERs in this effect.
Following a one-hour pretreatment with the ER  selective antagonist PHTPP
(1μM), 10nM E2 was applied to the culture medium for 24 h. After a 3 h NMDA
treatment and an overnight recovery period, culture media were assessed for
lactate dehydrogenase (LDH) levels. As previously shown, E2 treatment reverses
the increase in LDH levels induced by NMDA. Interestingly, pretreatment with
PHTPP for 1 h reversed the effects of E2 on LDH release (Fig. 3.1; one way
ANOVA, F
5,8
= 17.94, P = 0.0004).

76
Figure 3.1. PHTPP reverses E2-mediated neuroprotection against NMDA
toxicity.

10nM E2 treatment (24 h) reverses NMDA-induced neurotoxicity in cultured
hippocampal slices as evidenced by a decrease in LDH released into the
medium. Pre-treatment with ER  inhibitor, PHTPP, reverses E2-mediated
neuroprotection.

Since inhibition of ER  resulted in decreased neuroprotection afforded by
estradiol, we examined the effects of specific ER agonists on neuroprotection.
The ERα and ERβ agonists, Propyl Pyrazole Triol (PPT) and diarylpropionitrile
(DPN), respectively, were added to the slice culture medium in the same manner
as for the E2 experiments. To explore the efficiency of these agonists in
mimicking estrogenic protection from NMDA toxicity, we utilized three different
concentrations. PPT was added to culture medium at 1 M, 100 nM and 10 nM.
DPN was added to culture medium at 100 nM, 10 nM and 1 nM. These
77
concentrations differ due to the different binding affinities for each agonist to their
respective receptor. Our results examining cellular death by propidium uptake
fluorescence indicated that E2-mediated neuroprotection against NMDA toxicity
was mediated by the activation of ERβ and not ER . Quantification of fluorescent
images indicated that pretreatment for 24 h with DPN at all concentrations
(100nM, 10nM and 1nM) prior to 3 h of NMDA exposure significantly reduced
fluorescence intensity in CA1 regions of hippocampus (Fig. 3.2a). Results in
CA3 and DG were not significant, although there was a trend in DG (Fig. 3.2c).
(Fig. 3.2a, CA1; one way ANOVA, F
7,16
= 17.33, P < 0.0001; Fig. 3.2b,CA3; one
way ANOVA, F
7,16
= 0.5197, P = 0.8069; Fig. 3.2c, DG; one way ANOVA, F
7,16
=
3.321, P = 0.0222).

78
Figure 3.2. ER  activation mediates estrogenic neuroprotection against
NMDA toxicity.

Addition of DPN but not PPT significantly protects against NMDA-induced toxicity
in CA1 (3.2A). No significant results in CA3 or DG (3.2 B,C).

A

B

C
79
Progesterone reverses estrogen-mediated increases in ER  but not ER  mRNA
To further investigate the molecular mechanisms underlying the
antagonistic effect of P4 when added 20 h following 17-  estradiol treatment
(Aguirre and Baudry, 2009), we investigated whether the mRNA levels of
estrogen receptors were differentially affected. Cultured hippocampal slices were
treated with 10nM E2 for 24 h and concomitant treatment with 10nM P4 during
the final 4 h of the E2 treatment period. E2 (24 h) treatment resulted in a
significant increase in levels of ER  mRNA but not ER  mRNA as compared to
vehicle controls (Fig. 3.3). P4 alone (4 h) did not significantly alter ER  mRNA or
ER  mRNA levels. Interestingly, the addition of P4 to E2-treated slices reversed
the estradiol-mediated increase in ER  mRNA (Fig. 3.3).

80
Figure 3.3. E2 treatment increases ER  mRNA levels and addition of P4
to E2-treated slices reverses this effect.

Cultured hippocampal slices were treated with E2 (10 nM), P4 (10 nM) or E2 +
P4. At the end of treatment, slices were collected and processed for RT-PCR
analysis of ER  and ER .
Hormonal treatment did not have a significant effect on ER  mRNA levels.

81
Figure 3.4. Quantification of ER  and ER  mRNA levels.

Quantification of PCR data illustrates that the combination of P4 + E2 reversed
ER  mRNA levels to those observed with P4 or vehicle (Fig. 3.4b). On the other
hand, ER  levels were not significantly different between estradiol-treated and
progesterone plus estradiol treated (E+P) slices (Fig. 3.4a) (Fig. 3.4a; one way
ANOVA, F
3,26
= 5.267, P = 0.0057; Fig. 3.4b; one way ANOVA, F
3,18
= 9.176, P =
0.0007).

82
Progesterone reverses estradiol-induced increases in ER  and ER  protein
levels.
The effects of hormone administration on relative levels of ER  and ER 
proteins in hippocampus are illustrated in Fig. 3.5. Highest levels of both ER 
and ER  protein were detected following E2 treatment. Interestingly, addition of
10 nM P4 (4 h) to E2-treated slices reversed both E2-mediated increases in ER 
and ER  protein levels, despite only having a significant effect on ER  mRNA
(Fig. 3.5a, b) (Fig. 3.5a; one way ANOVA, F
3,8
= 15.79, P = 0.0010; Fig. 3.5b;
one way ANOVA, F
3,12
= 6.207, P = 0.0086).

Figure 3.5. Quantification of ER protein levels

A. E2 increases ER  protein levels and P4 reverses this effect.
B. E2 increases ER  protein levels and P4 reverses this effect.

A
83
Figure 3.5, Continued

Progesterone reverses ER  -mediated neuroprotection against NMDA toxicity
Since ER  seems to be important in mediating E2‟s effects on
neuroprotection in CA1, we examined whether progesterone could antagonize
ER -mediated neuroprotection in cultured hippocampal slices. From our previous
experiments, we deemed DPN, an ER  agonist, to be most efficacious at
protecting slices from NMDA damage at 10nM. Furthermore, PPT, an ER 
agonist, was used at 100nM hereinafter. DPN (10nM) decreased NMDA-induced
increase in PI uptake fluorescence in CA1, as previously shown. Progesterone
reversed this reduction in CA1 to levels that were not significantly different from
B
84
those found in slices treated with NMDA alone (Fig. 3.6A, CA1; one way
ANOVA, F
5,12
= 15.33, P < 0.0001; Fig. 3.6B,CA3; one way ANOVA).

Figure 3.6. P4 (4 h) treatment reverses DPN-mediated decrease in PI
uptake of cultured hippocampal slices

A. Quantification of PI uptake in CA1. DPN but not PPT is neuroprotective
against NMDA toxicity. 10nM DPN reverses NMDA-induced increase in PI
fluorescence. Addition of P4 to DPN-treated slices reverses DPN-induced
neuroprotection and has no effect on PPT-treated slices.
B. Quantification of PI uptake in CA3. P4 reverses DPN-mediated
neuroprotection but has no effect on PPT-treated slices.
C. Quantification of PI uptake in DG. P4 does not have a significant effect on
DPN- or PPT-treated slices.

A
85
Figure 3.6, Continued

C

B
86
Progesterone reverses estradiol-induced increase in BDNF mRNA
We also evaluated whether the effect we observed previously (Aguirre and
Baudry, 2009) on BDNF protein levels could be observed at the mRNA level.
Indeed, P4 treatment also reversed E2-induced increase in BDNF mRNA levels
in parallel with its effects on ER  mRNA levels (Fig. 3.7).

Figure 3.7. 10nM E2 (24 h) treatment of cultured hippocampal slices
increases BDNF mRNA levels and addition of P4 (4 h) to E2 treated slices
reverses this effect.

Quantification of PCR data revealed a significant reduction in BDNF mRNA from
E2-treated to E2+P4 – treated hippocampal slices (Fig. 3.8) (Fig. 3.8; one way
ANOVA, F
3,4
= 12.10, P = 0.0178).

87
Figure 3.8. Quantification of BDNF mRNA levels

ER  activation increases BDNF protein expression and progesterone reverses
this effect

We previously reported that 10 nM 17 -estradiol treatment for 24 h in
cultured hippocampal slices induced an increase in BDNF protein levels (Aguirre
and Baudry, 2009). This effect is mediated through ER , as DPN but not PPT
was also able to increase BDNF protein levels. Additionally, progesterone
reversed this increase in parallel with its antagonistic effects on neuroprotection.
(Fig. 3.9; one way ANOVA, F
4,23
= 3.890, P = 0.0148).

88
Figure 3.9. DPN but not PPT treatment of cultured hippocampal slices
results in increased BDNF protein levels.

Addition of 10 nM P4 to DPN-treated slices, but not PPT-treated slices reverses
DPN-mediated increase in BDNF protein levels.

Mouse KO Studies

Estradiol replacement to wild type but not ER -KO mice results in
neuroprotection against NMDA toxicity
To further demonstrate that E2-mediated neuroprotection may be
dependent on ER  activation, we investigated the effects of E2 treatment on
NMDA-induced neurotoxicity in cultured hippocampal slices prepared from ER -
89
KO animals. Treatment with NMDA caused a significant increase in PI uptake,
particularly in CA1 and CA3 regions of hippocampus. There was about 60% of
the slice area exhibiting intense PI fluorescence in NMDA-treated cultures (Fig.
3.10). Measurements of LDH release into the medium confirmed that NMDA
induced a moderate to severe cell damage in mouse OHSC. LDH activity in the
medium was markedly increased 24 h after NMDA treatment as compared to
control cultures (Fig. 3.11). Pretreatment with 1 nM E2 significantly reduced
NMDA-induced LDH release into the medium and PI uptake as compared to
control (white bars, Fig. 3.10, 3.11).

Figure 3.10. E2 treatment of cultured hippocampal slices from wild-type
but not ER -KO mice results in neuroprotection against NMDA toxicity.

P.I. uptake in cultured hippocampal slices. * = P<0.01. E2 treatment reverses
NMDA-induced toxicity in cultured slices from WT (white bars) but not from ER
KO or Het (ER 
-/-
or ER 
+/-
) slices.

90
E2 treatment of cultured hippocampal slices from ER -KO

(ER 
-/-
and
ER 
+/-
) mice did not significantly decrease NMDA-induced hippocampal neuronal
damage (black and grey bars, Figs. 3.10, 3.11). Interestingly, when compared to
WT mice, we observed a statistically significant lower level of LDH activity in the
medium for NMDA-treated slices from both ER 
-/-
and ER 
+/-
mice (Fig. 3.11).
However, we did not observe this with PI uptake (Fig. 3.10). (Figure 3.10, WT;
F(3,112) = 935.9 EN vs NMDA: P <0.001; HET; F(3,28)= 281 VEH vs NMDA,
EN: P<0.001; KO; F(3,28) = 457.2, EN vs E2: P< 0.001) (Figure 3.11, WT;
F(3,49)= 52.70, EN vs NMDA: P < 0.001; HET; F( 3,12)= 4.2, EN vs E2: P< 0.05;
KO; F(3,12) = 10.35, EN vs E2: P< 0.01)

Figure 3.11. LDH release into medium of cultured hippocampal slices
from wild-type but not ER 
-/-
or ER 
+/-
mice results in of E2 –mediated
neuroprotection against NMDA toxicity

91
Immunocytochemistry of ERβ
To corroborate our previous results, we examined the effects of 24 h E2
followed by 4 h P4 treatment on ERβ protein levels in CA1 utilizing
immunocytochemistry techniques. In accordance with our previous data, E2
treatment produced an increase in ERβ immunoreactivity, an effect that was
reversed by concomitant treatment with P4 (Fig. 3.12).

Figure 3.12. ER  immunoreactivity in CA1 of cultured hippocampal
slices.

A. VEH; B. E2; C. P4; D. E+P

92
DISCUSSION

The results presented here demonstrate that progesterone treatment
reverses estrogenic increases in BDNF and ER  mRNA and protein, resulting in
a reversal of E2-mediated neuroprotection against NMDA toxicity in cultured
hippocampal slices. Progesterone has been shown to have an antagonistic effect
on estrogenic functions, as illustrated by studies on spine density (Murphy and
Segal, 2000), spatial memory (Bimonte-Nelson et al., 2006) and kainate
excitotoxicity (Rosario et al., 2006). This effect was also shown in vivo, where P4
combined with E2 treatment attenuated the E2 neuroprotective effect against A 
accumulation in a mouse model of AD (Carroll et al., 2007). These, among other
studies, accentuate the necessity of better understanding the interactions
between E2 and P4 in order to resolve the complexities underlying age-related
cognitive deficits and diseases like Alzheimer‟s disease (AD). As of yet, there are
still conflicting data regarding these complex interactions on various brain
systems. The complexity of hormonal interactions is also magnified by studies
demonstrating that there is a functional divergence between different estrogen
receptors. Accumulating evidence has emphasized the role of ER  in cognition
and behavior (Bodo and Rissman, 2006; Liu et al., 2008; Walf et al., 2008; Walf
et al., 2009). Therefore, in this study, we aimed to provide some insight into the
antagonistic effects of progesterone on estrogen-mediated neuroprotection.
Previously, we showed that P4 treatment reversed E2-mediated neuroprotection,
93
which was due, in part, to BDNF activation (Aguirre and Baudry, 2009). Here, we
expand upon those results with the observation that estrogenic effects on
neuroprotection and BDNF upregulation are in fact ER -mediated. We also
observed that E2 treatment leads to neuroprotection against NMDA toxicity in
wild type (WT) but not in ER 
-/-
or ER 
+/-
mice. Furthermore, the ER  selective
agonist diarylpropionitrile (DPN) mimicked E2‟ effects on both neuroprotection
against NMDA toxicity and BDNF protein expression. This suggests that
activation of ER  is necessary for E2-mediated upregulation of BDNF, and
neuroprotection against NMDA toxicity.
As evidenced by numerous studies, both estrogen and progesterone may
exert their neurotrophic effects by stimulating BDNF expression. Fluctuating
levels of BDNF mRNA have been shown to be dependent on the estrous cycle,
with increased BDNF protein during proestrous and estrous stages (Gibbs,
1998). In a study examining hormonal effects on neurotrophins, aged OVX rats
were treated with estrogen or estrogen plus progesterone. While E2 increased
BDNF, NT3 and NGF levels in entorhinal cortex, concomitant treatment with P4
attenuated this increase (Bimonte-Nelson et al., 2004). However, there were no
effects of estrogen or estrogen plus progesterone in hippocampus. Perhaps
hormone replacement is no longer responsive in aged brain, thus signifying the
existence of a critical window of time where these hormones exert their effects. In
our study, we utilized hippocampi from young animals, although it would be
interesting to see whether our effects are abolished in aged hippocampus. In
addition, the roles of different ERs in eliciting BDNF production have not been
94
previously elucidated in hippocampus. A recent study in the auditory system
showed that activating ER  protected WT mice from acoustic trauma and
increased BDNF mRNA and protein in the cochlea (Inna et al., 2008).
Menopausal women are up to three times as likely to develop Alzheimer‟s
disease as men, indicating that the loss of circulating ovarian hormones places
women at a significant risk for the development the disease (Henderson, 1997;
Sherwin, 2003). By clarifying the relationship between estrogen and
progesterone, we may be able to provide a therapy for those suffering hormonal
loss, and the resulting detrimental effects on cognition and memory. In our study,
a 4h progesterone treatment following 20h E2 exposure in cultured hippocampal
slices resulted in reversal of E2-mediated neuroprotection and BDNF induction.
Furthermore, this treatment also resulted in the down-regulation of ER  but not
ER  mRNA. Our current hypothesis to account for these effects is that
progesterone exerts its antagonistic effects at the level of mRNAs for ER  and
BDNF. Four h of P4 treatment may be too short a time for classic genomic
effects to take place. Nevertheless, this timing protocol results in the down-
regulation of both ER  and BDNF, and thus abolishes E2-mediated
neuroprotection against NMDA toxicity. A proposed mechanism of action is
illustrated in Figure 3.13.

95
Figure 3.13 Proposed mechanism of progesterone-estrogen interaction

Preliminary observations point to a possible mechanism to account for
these effects and support the hypothesis that progesterone stimulates mRNA
degradation or destabilization, suggesting a post-transcriptional control of P4 in
96
regulating neuroprotection. The length of the poly(A) tail of mRNAs has important
roles in mRNA stability, degradation and translational efficiency (Meijer et al.,
2007). Progesterone has been shown to shorten the poly(A) tail of ovine
luteinizing hormone subunits, resulting in mRNA down-regulation. In addition,
significant shortening was observed following treatment with 7-10nM P4 and after
3 – 10 h of treatment (Wu and Miller, 1991). Interestingly, we also observed
down-regulation of mRNA within these narrow dosage and timing ranges. We
hypothesize that it is the degradation or destabilization of ER  mRNA by
progesterone that results in the antagonism of E2-mediated BDNF release and
subsequent neuroprotection. Future studies exploring the length of poly(A) tails
of various mRNAs following progesterone treatment might further increase our
understanding of hormonal relationships with respect to neuroprotection and
enable new drug developments of hormone therapy without detrimental effects.
A better understanding of the role of ER  has important implications for
learning and memory. Accumulating evidence has characterized the significance
of this receptor in mediating many of estradiol‟s effects on hippocampal
functions. Recently, characterization of a selective ERβ agonist, WAY-200070
revealed that ER  activation mediates the effects of estradiol on memory in a
hippocampus-dependent task and mimics estradiol effects on a spatial memory
task. Furthermore, activation of ER  through this agonist led to increases in
spine density, dendritic branching, synaptic proteins and LTP (Liu et al., 2008).
Although compelling, this evidence leaves room for interpretation as to the
efficacy of this agonist as it compares to other ER  agonists. Given that a
97
number of studies exploring estrogenic effects are performed in OVX animals, it
is important to understand these effects on cycling intact animals. In a recent
study, WT animals in the proestrous phase had improved performance in object
recognition and T-maze tasks, as well as reduced anxiety-like behavior in the
plus maze and mirror chamber tasks when compared to ERKO mice. Genotype
did not seem to play a role in circulating hormone levels across the estrous cycle
(Walf et al., 2009). These behavioral effects implicate an important role of ER  in
mediating estradiol influences on cognition and affective behaviors. Finally,
selective agonists for ERs have been recently investigated in animal models of
disease. In a rodent model of Alzheimer‟s disease, PPT but not DPN treatment in
OVX 3xTg-AD mice reduced accumulation of β-amyloid protein and improved
hippocampal-dependent performance (Carroll and Pike, 2008). However, the
distribution and expression pattern of ERs in 3xTg-AD mice has not yet been
elucidated, making it difficult to interpret these results. A shortcoming prevalent
in all these studies is that they fail to investigate the effects of progesterone. Both
E2 and progesterone are depleted as a consequence of menopause in women
and OVX in experimental animal paradigms. Furthermore ERKO mice have
deficits in ovulation (Krege et al., 1998), signifying low levels of P4 in these
animals.
Collectively, these studies provide insight into the various molecular
mechanisms and behaviors sensitive to normal fluctuations in gonadal
hormones. However, further investigation on progestins might yield a more
cohesive understanding on hormonal effects in hippocampal processes.
98
Most studies utilize the ovariectomized (OVX) rat model when studying
hormone interactions. Inevitably, the hormonal injections may cause stress,
leading to possible increases in circulating stress hormones, which may be a
confounding factor. We tried to eliminate external factors that may influence the
effects of the hormones administered by using the controlled system of the
organotypic hippocampal slice. This preparation has been shown to retain the
functionality of the hippocampal circuit as well as serve as a good model for
studying cell viability. Therefore, we believe this preparation allowed us to
examine the aforementioned hypotheses in a model reflecting the complexity of
in the vivo brain, with functional cell-cell interactions, along with the flexibility of
isolating a particular brain region. However, this particular preparation has its
limitations, namely, difficulty to differentiate between cell types with the assays
proposed herein (Western blot, LDH release, PI). To discern the effects seen in
these experiments, we complemented the neuroprotection assays with
immunocytochemistry in cultured hippocampal slices. We obtained similar
results to western blot data; namely, E2 treatment (24h) increased ER 
immunoreactivity and P4 (4h) addition abolished this effect.
In summary, we found that estradiol neuroprotection against NMDA
toxicity in cultured hippocampal slices is mediated by ER , leading to increased
production of BDNF mRNA and protein. Furthermore, progesterone counteracts
these effects via down-regulation of ER , possibly through the destabilization or
degradation of mRNA.

99
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102
CHAPTER 4
GENERAL DISCUSSION AND SUMMARY

The data presented in this dissertation demonstrate that 17-  estradiol
protects organotypic hippocampal slice cultures from NMDA-induced
excitotoxicity through the activation of the ER  and not ER  receptors. In
addition, progesterone counteracts E2-mediated neuroprotection by down-
regulating E2-mediated increases in ER  and BDNF mRNA and protein.
Considering the timing used in the present protocol as well as some of the
preliminary observations we made, we propose that these effects of
progesterone on E2-mediated neuroprotection are due to an effect of at the level
of mRNA through degradation or destabilization, resulting in post-transcriptional
regulation of E2-mediated neuroprotection.

Importance of the treatment protocol
Prior to this study, there were no available data on a combined treatment
of estrogen and progesterone in organotypic hippocampal slice cultures. Several
previous studies from our laboratory had examined the effects of 17 -estradiol in
synaptic plasticity and neuroprotection using this model (Bi, Dominguez). I was
interested in extending these results by examining the effects of progesterone on
E2-mediated neuroprotection. A growing compendium of studies suggested that
progesterone antagonized the beneficial effects of estrogen in hippocampal spine
density (Murphy and Segal, 2000) and spatial memory (Bimonte-Nelson et al.,
103
2006), among other effects. Additionally, fluctuating levels of BDNF were shown
to be dependent on the estrous cycle, with elevated BDNF protein during
proestrous, the period of highest estrogen and progesterone levels (Gibbs,
1998). Moreover, researchers examining hormonal effects on neurotrophins in
aged OVX rats, found that E2 increased BDNF, NT3 and NGF levels in
entorhinal cortex, while concomitant treatment with P4 attenuated this increase
(Bimonte-Nelson et al., 2004). However, there were no effects of estrogen or
estrogen plus progesterone in hippocampus. Another seminal study to this
dissertation examined the effects of estrogen and progesterone treatment in
adult OVX rats (Gibbs, 1999). Treatment with estrogen for 48 h followed by 5 h
progesterone treatment decreased estradiol-induced increase in BDNF mRNA
expression level and decreased BDNF protein levels compared to controls in
hippocampus. Although not discussed, the antagonistic effect of progesterone on
E2-induced BDNF mRNA in hippocampus could have significant implications for
BDNF-mediated regulation of learning and memory processes. The
discrepancies in the literature led me to examine the role of progesterone in
estrogen-mediated neuroprotection, with emphasis on BDNF expression levels.
Moreover, since the bulk of studies utilized either dissociated neuronal cultures
or in-vivo preparations, I decided that the organotypic slice model shared
qualities of both in-vitro and in-vivo preparations, and thus chose it as my primary
model for the resulting experiments. Based on the information from Gibb‟s 1999
study, I devised a treatment protocol that would parallel their treatment time,
which would specifically apply for the organotypic slice model. Taking the
104
progesterone-to-estrogen time ratio used in the in-vivo study, I applied it to a 24 h
treatment time, resulting in an estradiol treatment for 24 h with a concomitant
progesterone treatment for the final 4 h of estradiol treatment (Figure 4.1).

Figure 4.1 Timing protocol

Furthermore, I observed the effects of progesterone on BDNF protein
expression by applying it at different times during a 24 h estradiol treatment.
Results showed that progesterone begins to counteract estrogenic induction of
BDNF protein after 4 h (EP4h), and continues to do so until termination of 24 h
E2 treatment (EP24h, Figure 4.2). Thus, I chose a 4 h progesterone treatment
and a 24 h 17 -estradiol treatment for the remaining experiments.

105
Figure 4.2 Progesterone timed treatments on E2-treated hippocampal
slices- effects on BDNF protein levels.

Why didn‟t P4 treatment increase BDNF mRNA and protein?
As with other neurotrophins, BDNF levels are positively correlated with E2
and P4 levels and negatively correlated with menopausal age (Begliuomini et al.,
2007). Furthermore, fluctuating levels of BDNF are dependent on the stage of the
menstrual cycle, while hormonal replacement in post-menopausal women
restored BDNF levels to those seen in fertile women (Begliuomini et al., 2007).
Thus, treatment with either estrogen or progesterone should result in increased
levels of BDNF. Studies from the Singh lab have examined the effects of
progesterone on BDNF. The initial observation that estradiol partially restored
BDNF levels after ovariectomy led them to conduct additional studies on
106
progesterone, since it too is diminished following ovariectomy. Similar to our
studies, they utilized organotypic cortical explants from P3 mice and treated
cultures with progesterone to examine its effects on neuroprotection and BDNF
expression. They concluded that a 24 h P4 treatment resulted in protection
against glutamate toxicity and increased BDNF mRNA and protein expression in
cortical explants (Kaur et al., 2007). As with our studies, they examined cellular
death with the lactate dehydrogenase (LDH) release assay. However, they
utilized ELISA assay instead of western blot to analyze changes in BDNF levels,
indicating that they examined total BDNF levels, while we distinguished between
pro and mature forms with western blot (Aguirre et al., 2009). Although the
results are seemingly contradictory, there are significant differences between the
studies that could explain the discrepancies. Minor differences lie in the method
of preparation and the model system. Kaur et al. examined the role of
progesterone in organotypic cortical explants while we focused on hippocampal
explants. Although both anatomical areas are implicated in neurodegenerative
disease and cognition, the hippocampus plays a more pivotal role in learning,
memory and the associated effects of neurodegenerative diseases such as
Alzheimer‟s disease. Furthermore, the distribution of hormonal receptors varies
between these two regions, as ER  is more abundant in hippocampus than ER .
The distribution pattern of specific progesterone receptors PRA and PRB in brain
has not been well studied to our knowledge, although both receptors are present
in cortex and hippocampus (Brinton et al., 2008). More importantly, the effects of
progesterone on BDNF expression seen in their study do not directly contradict
107
our results. Kaur et al. observed protection from glutamate toxicity following 24 h
treatment with 100 nM, but not 10 nM P4. Furthermore, they reported
progesterone-induced increases in BDNF mRNA and protein following 18 h
treatment with 100 nM P4 (Kaur et al., 2007). Since they did not observe a
protective effect at 10 nM, it is possible that they did not observe an increase in
BDNF expression at this concentration, although it was not reported. In a more
recent study, their group found that although 100 nM P4 increased BDNF protein
expression, 10 nM P4 did not (Jodhka et al., 2009). Even though both of these
studies examined P4 after 18 h, it is evident that there are concentration-
dependent differences in the ability of P4 to increase BDNF mRNA and protein.
Thus, our 4 h treatment with 10 nM progesterone in hippocampus did not result
in increased levels of BDNF mRNA or protein, in good agreement with their
results (Aguirre and Baudry, 2009, Kaur et al., 2007, Jodhka et al., 2009).

Antagonistic effects of Progesterone on Estrogen are not mediated by its
metabolites
3- -hydroxy-5 -pregnan-20-one (allopregnanolone, AP ), the reduced
metabolite of progesterone and a potent GABA
A
receptor modulator, has been
shown to mediate, at least in part, the neuroprotective effects of progesterone
(Ciriza et al., 2004). To investigate whether the effects observed after applying
P4 for 4 h were due to its conversion to AP , we co-treated slices with both 10
nM P4 and 1 µM finasteride for 4 h after 20 h of E2 treatment. Finasteride inhibits
the synthesis of 5α-reduced neurosteroids and has been shown to significantly
108
attenuate the formation of AP  both in-vitro and in-vivo (Rhodes and Frye, 2005;
Izumi et al., 2007). The concentration of finasteride used in this study has been
used previously in hippocampal slices and neurons (Mostallino et al., 2006; Izumi
et al., 2007). Co-treatment with P4 and finasteride did not significantly modify the
results obtained using P4 alone. PI uptake was similar in slices treated with E2,
P4 and NMDA and those treated with E2, P4, finasteride and NMDA in CA1, CA3
and DG regions of hippocampus (data not shown). Although the effects of
allopregnanolone on the GABAergic system have been clearly established, in
vitro studies generally use micromolar concentrations of allopregnanolone. The
effects of progesterone we observed were in the nanomolar range, which, even if
progesterone were completely metabolized to allopregnanolone, would not likely
generate micromolar concentrations. Under our experimental conditions,
finasteride did not modify the effects of progesterone, strongly suggesting that
the observed effects are not due to its conversion to allopregnanolone.

Sex Differences
In the field of hormonal research in brain, sex differences are prevalent.
For instance, neurons derived from female mice have been shown to be more
sensitive to etoposide and staurosporine-induced apoptosis than male-derived
neurons. In contrast, neurons derived from males were preferentially sensitive to
nitrosative stress and excitotoxicity when compared to female-derived neurons
(Du et al., 2004). Moreover, estrogen or progesterone did not alter PR isoforms
in female rats, although estrogen selectively induced PRA in male cerebellum
109
(Camacho-Arroyo et al., 2007). In our studies, preliminary results did not show
sex differences, thus results from hippocampal slices of both sexes were
combined for all experiments. At the early stage of development our slices are
prepared (PND9), there appears to be no sex difference in hippocampal function
to our knowledge. Studies on hormone neuroprotection against glutamate toxicity
also displayed no sex differences when studying young animals (Kaur et al.,
2007).

NMDA toxicity in organotypic hippocampal slices as a model of cellular death
Due to its role in memory function, the hippocampus has been at the
forefront of studies exploring hormonal effects on neuronal excitability and
synaptic plasticity. The organotypic hippocampal slice culture method has been
shown to be a valuable tool for investigating numerous questions, due to its ease
of preparation and anatomical, physiological and biochemical integrity as
compared to the in vivo preparation or primary neuronal culture model.
Remarkably, this method allows the maintenance of nervous tissue under long-
term survival conditions that yield highly differentiated cells in both morphology
and physiology (Gahwiler et al. 1999). In our study, we utilized organotypic
hippocampal slices prepared from P9 pups of both genders and cultured for two
weeks with medium supplemented with charcoal-stripped horse serum. Even
though the age of cultures at treatment time is parallel to an in-vivo age of P21-
22, it is likely that the results might not directly translate to other studies
110
examining older animals simply because our slices are incubated for 12-14 days
in vitro.
This maintenance in vitro for two weeks prior to treatments allows
recovery from sectioning damage and completion of the trisynaptic circuitry
(Nakagami et al., 1997). This preparation allowed us to examine the
aforementioned hypotheses in a model reflecting the complexity of in the vivo
brain, with functional cell-cell interactions, along with the flexibility of isolating a
particular brain region. However, this particular preparation has its limitations,
namely, the differentiation between cell types with the assays proposed herein
(Western blot, LDH release, PI). Therefore, we complemented western blot
analyses with immunocytochemistry in cultured hippocampal slices in order to
gain a deeper understanding of the cellular effects of progesterone treatment
(Fig. 3.12).
Prolonged stimulation of NMDA receptors leads to neuronal death. This is
a well-established model of excitotoxicity, in which the three subregions of the
hippocampus, CA1, CA3 and DG are differentially affected, with CA1 being the
most sensitive (Rangarajan et al., 1999). In addition, this sensitivity parallels
NMDA receptor density in hippocampus. We used this model in our studies to
elucidate the mechanisms underlying hormone-mediated neuroprotection in
hippocampus. However, it needs to be stressed that the LDH and PI assays we
used to assess cell death do not clearly distinguish between necrotic and
apoptotic cell death. Therefore, future studies distinguishing between these types
of cellular death may provide additional insight to the effects of hormonal
111
interactions in specific models of apoptosis.

How do my results contribute to or extend the existing knowledge of the
neurobiology of estrogen and progesterone interactions on the modulation of
BDNF and neuroprotection?
At the time this dissertation was conceived, little was known about the
underlying mechanisms of progesterone‟s effects on estrogen neuroprotection.
Moreover, the roles of estrogen receptors on hippocampal function had not been
clearly elucidated. Since then, accumulating evidence has emphasized the role
of ER  in cognition and behavior (Bodo and Rissman, 2006; Liu et al., 2008; Walf
et al., 2008; Walf et al., 2009). In our studies, we observed that estrogenic effects
on neuroprotection and BDNF upregulation are in fact ER -mediated. 17  -
estradiol treatments resulted in neuroprotection against NMDA toxicity in wild
type but not in ER KO

mice, while the ER  selective agonist diarylpropionitrile
(DPN) mimicked estradiol effects on both neuroprotection against NMDA toxicity
and BDNF protein expression. This suggests that the activation of ER  is
necessary for estradiol-mediated upregulation of BDNF, leading to
neuroprotection against NMDA toxicity. Together with current research being
performed in many other labs, we have taken steps towards a more cohesive
understanding on hormonal effects in hippocampal processes.
Menopausal women are more likely to develop Alzheimer‟s disease than
men, indicating that the loss of circulating ovarian hormones places women at a
significant risk for the development the disease (Henderson, 1997; Sherwin,
112
2003). Thus, examining the roles of estrogen and progesterone on
neuroprotection may lead to the development of a therapy for those suffering
hormonal loss and the resulting detrimental effects on cognition and memory.
Progesterone has been shown to have antagonistic effects on estrogenic
functions, as illustrated by studies on spine density (Murphy and Segal, 2000)
spatial memory (Bimonte-Nelson et al., 2006) kainate excitotoxicity (Rosario et
al., 2006) and A  accumulation (Carroll et al., 2007). These, among other
studies, accentuate the necessity of understanding the interactions between E2
and P4 in order to understand the complexities underlying age-related cognitive
deficits in diseases like Alzheimer‟s disease (AD). In our studies we were able to
show that P4 treatment reversed E2-mediated neuroprotection against NMDA
toxicity in cultured hippocampal slices (Aguirre and Baudry, 2009). In addition, a
4 h progesterone treatment following 20 h E2 exposure in cultured hippocampal
slices resulted in the down-regulation of ER  but not ER  mRNA as well as in
BDNF mRNA. Our current hypothesis is that progesterone exerts its antagonistic
effects at the level of RNAs for ER  and BDNF. Since 4 h P4 treatment in
cultured hippocampal slices may be too short a time for classic genomic effects
to take place, we hypothesize that a post-transcriptional effect of P4 regulates
neuroprotection through the stimulation of mRNA degradation or mRNA
destabilization. We believe that it is the degradation or destabilization of ER 
mRNA by progesterone that results in antagonism of E2-mediated BDNF release
and subsequent neuroprotection; an additional effect of P4 could also produce
degradation or destabilization of BDNF mRNA. An illustration of this hypothesis is
113
seen is Figure 3.13. Future studies exploring the length of poly(A) tails of various
mRNAs following progesterone treatment might further increase our
understanding of hormonal relationships with respect to neuroprotection and
enable new drug developments of hormone therapy without detrimental effects.
Collectively, this dissertation, in addition to previous studies, demonstrates
that ER  is a critical element in mediating E2 neuroprotection and that
progesterone can, through post-transcriptional mechanisms, modulate estrogenic
function in hippocampus.

114
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Abstract (if available)

Abstract Human cognition encompasses all the components involved in information processing. Thus, it is a multidimensional concept that involves several aspects of learning, memory, abstract reasoning and other higher-order functions. Interest in hormone effects on memory mechanisms has been spurred by conflicting evidence implicating that hormone replacement therapy (HT) can ameliorate or be detrimental to memory and cognitive ability in post-menopausal women. The functions of the female gonadal steroids estrogen and progesterone in the central nervous system (CNS) have been extensively studied. One of the regions involving estrogenic action most studied is the hippocampal formation, which governs the formation of spatial and episodic memories. Relatively less attention has been devoted to progesterone (P4) and its effects in hippocampus. Moreover, the possible regulation by P4 of E2-mediated neuroprotective effects has not been extensively investigated. This dissertation is directed at studying interactions between estrogen and progesterone in an in-vitro model of excitotoxicity, and at evaluating the role of brain-derived neurotrophic factor (BDNF) in mediating estradiol-induced neuroprotection, using NMDA treatment of cultured hippocampal slices to elicit neuronal death. I hypothesized that the neuroprotective effects of these hormones was critically dependent upon the timing of hormone administration. Further, I proposed that estrogenic neuroprotection against NMDA toxicity was dependent on the activation of BDNF and its resulting signaling pathways, an effect mediated through the ERbeta receptor. Using organotypic hippocampal slices, we showed that treatment with estradiol paired with progesterone resulted in down-regulation of ERbeta mRNA leading to diminished ERbeta-mediated estradiol responses, and in particular, in decreased BDNF expression and protein levels, which accounted for elimination of estrogen protection against NMDA toxicity.

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Asset Metadata

Creator Aguirre, Claudia C. (author)

Core Title Estrogen, progesterone and BDNF interactions: roles in neuroprotection

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 11/06/2009

Defense Date 08/27/2009

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

Tag Alzheimer's disease,BDNF,Estrogen,estrogen receptor beta,hippocampus,neuroprotection,NMDA,OAI-PMH Harvest,organotypic hippocampal slice,progesterone

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Baudry, Michel (committee chair), Madigan, Stephen A. (committee member), Thompson, Richard (committee member)

Creator Email bellestar1@gmail.com,ccaguirr@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2717

Unique identifier UC1365318

Identifier etd-Aguirre-3192 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-271440 (legacy record id),usctheses-m2717 (legacy record id)

Legacy Identifier etd-Aguirre-3192.pdf

Dmrecord 271440

Document Type Dissertation

Rights Aguirre, Claudia C.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu