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Estrogen and progesterone interaction on synaptic transmission and LTP in rodent hippocampus

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Estrogen and progesterone interaction on synaptic transmission and LTP in rodent hippocampus

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ESTROGEN AND PROGESTERONE INTERACTION
ON SYNAPTIC TRANSMISSION AND LTP IN RODENT HIPPOCAMPUS

by

Youngkyoung Kim

______________________________________________________________

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)

May 2010

Copyright 2010 Youngkyoung Kim
ii
Epigraph
"Blessed are the poor in spirit,
for theirs is the kingdom of heaven.

Blessed are they who mourn,
for they shall be comforted.

Blessed are the meek,
for they shall inherit the earth.

Blessed are they who hunger and thirst for righteousness,
for they shall be satisfied.

Blessed are the merciful,
for they shall obtain mercy.

Blessed are the pure of heart,
for they shall see God.

Blessed are the peacemakers,
for they shall be called children of God.

Blessed are they who are persecuted for the sake of righteousness,
for theirs is the kingdom of heaven."

Gospel of St. Matthew 5:3-10

iii
Dedication
This dissertation is dedicated to my beloved father, mother, and my
husband. My father, who had big dreams for me, always inspired and
encouraged me. He taught me that I could do accomplish anything in life if I tried
hard enough. The only reason I could start career in neuroscience after having
studied electrical engineering until five years ago was that I have a little piece of
his positive and challenging mind. If I did not feel him rooting for me in heaven, I
would not be standing here today. I vividly remember the passion with which he
taught me mathematics, physics, and chemistry. I am sure that he is smiling at
me right now, full of pride.
In the absence of my father, my mother’s sacrifice, support and sincere
prayer for me were incredible. She gave me the strength to overcome every
obstacle I faced during the eight years of study in the U.S. Whenever I felt lonely
and stressed out, talking with her over the phone for a couple of hours relaxed
me. She always calmed me and helped me to focus on what I was doing. During
the last eight years, my mother’s hair turned white, and I went from a young
international student to a married woman. I deeply appreciate my mother for
taking such good care of me. Lastly, I want to give my deepest gratitude to my
wonderful husband. Thanks to his love and support, I was able to finish this.
Again, I send my deepest appreciation and endless love to all of you.

iv
Acknowledgements
I am heartily thankful to my advisor, Dr. Thompson. Dr. Thompson
accepted me as his student when I did not know anything about animal
experiment or electrophysiology. I still vividly remember the day when I first met
him. Never will I forget the kindness and warmth he showed me as he let me
work in his lab with a handshake. During the past four years, he has always
encouraged me to pursue new ideas. Whether in collaboration or working
independently, I have learned how to solve scientific questions with his guidance.
He always had a great advice for me when I was stuck with a research-related
problem. I respect him as a greatest scholar, exemplary leader, and a wonderful
family man. Indeed, it is my lifetime honor to have him as my advisor. I also want
to thank Mrs. Judith Thompson. Her presence in the lab made us comfortable
and relaxed. I also want to thank my lab members; Narawut, Kahun, Carla,
Chanpreet, Michael, and especially Soyun. I appreciate my committee members;
Dr. Baudry, Dr. Swanson, Dr. Walsh, and Dr. Golomb. I also give my sincere
thanks to Dr. Foy and Dr. Akopian. It was my great privilege to have many
wonderful friends in Korean Christian bible study group at USC, USC Good
Shepherds. I will remember their prayer and encouragement during my difficult
times. I really appreciate my brothers and other relatives in Korea. I cannot
imagine how I could have survived without their help. Finally, I’d like to thank
everybody who supported me in many ways to accomplish the Ph.D. degree at
USC.
v
Table of Contents

Acknowledgements ..............................................................................................iv
List of Figures ...................................................................................................... vii
List of Abbreviations .............................................................................................ix
Abstract ................................................................................................................xi
Chapter 1: General Introduction ........................................................................... 1
Introduction .................................................................................................... 1
Hormone Therapy ......................................................................................... 2
Neurobiology of Estrogen in Hippocampus ................................................... 4
Estrogen Receptor (ER) ................................................................................ 5
Estrogen Effects on Hippocampal Morphology .............................................. 9
Estrogen Effects on Hippocampal Electrophysiology .................................. 13
Estrogen Induced Increase in NMDAR Expression and Transmission ........ 15
Estrogen Induced Decrease in GABAergic Inhibition .................................. 16
CREB, and MAP/ ERK ................................................................................ 17
P13K/Akt ..................................................................................................... 18
Neurotrophins .............................................................................................. 20
Progesterone Receptor (PR) ....................................................................... 22
Expression of PRs ....................................................................................... 23
Progesterone Effects on Brain Morphology ................................................. 24
Progesterone Effects on Cellular Excitability ............................................... 25
Progesterone Effects on Electrophysiology in the Brain .............................. 26

Chapter 2: Effective Dose of Progesterone Decreases Basal Transmission
and Long-term Potentiation in Female Rat Hippocampus ........................... 28
Chapter 2 Abstract ...................................................................................... 28
Introduction .................................................................................................. 28
Materials and Methods ................................................................................ 32
Results ........................................................................................................ 35
Discussion ................................................................................................... 42

Chapter 3: Effects of Estrogen and Progesterone Interaction on Synaptic
Transmission and Synaptic Plasticity in CA1 Region of Rat Hippocampus . 45
Chapter 3 Abstract ...................................................................................... 45
Introduction .................................................................................................. 46
Materials and Methods ................................................................................ 50
Results ........................................................................................................ 53
Discussion ................................................................................................... 62
vi
Chapter 4: Effect of Cyclic Hormonal Treatment on LTP in CA1 Region of
OVX Rat Hippocampus ............................................................................... 65
Chapter 4 Abstract ...................................................................................... 65
Introduction .................................................................................................. 66
Materials and Methods ................................................................................ 69
Results ........................................................................................................ 74
Discussion ................................................................................................... 77

Chapter 5: Estrogen and Progesterone Effects on Hippocampal Synaptic
Transmission and LTP in 3xtg-AD Mouse Model ........................................ 82
Chapter 5 Abstract ...................................................................................... 82
Introduction .................................................................................................. 82
Materials and Methods ................................................................................ 86
Results ........................................................................................................ 88
Discussion ................................................................................................... 95

Chapter 6: General Discussion and Summary.................................................... 97

References ....................................................................................................... 102

vii
List of Figures
Figure 2. 1 P4 regulates synaptic basal transmission on CA1 region ................ 37

Figure 2. 2 P4 effect on hippocampal synaptic plasticity; LTP ........................... 38

Figure 2. 3 P4 had no significant effect on LTD in CA1 of hippocampus ........... 40

Figure 2. 4 P4 effect on basal transmission and synaptic plasticity;
LTP and LTD and IPSC recordings ................................................. 41

Figure 3. 1 Basal synaptic transmission; vehicle, E2, E2+P4 and P4 ................. 55

Figure 3. 2 P4 suppressed E2-induced increase in basal transmission ............. 56

Figure 3. 3 Basal transmission changes by serial E2 and P4 administration ..... 58

Figure 3. 4 Comparison of basal transmission; vehicle, E2, E2 plus
P4 and P4 ........................................................................................ 58

Figure 3. 5 LTP recordings with vehicle, E2, E2plus P4 and P4
administration .................................................................................. 59

Figure 3. 6 LTD recordings with vehicle, E2, E2plus P4 and P4
administration .................................................................................. 60

Figure 3. 7 Comparison of LTP and LTD with different in vitro hormone
applications ...................................................................................... 61

Figure 4. 1 Cyclic HT protocol used in the present study .................................. 71

Figure 4. 2 LTP from different HT groups; OVX, E2, E2 plus P4, and P4 .......... 75

Figure 4. 3 E2 effect on LTP in hormone pretreated groups .............................. 76

Figure 4. 4 Comparison of LTP of each group before and after in vitro E2
application ....................................................................................... 77

Figure 5. 1 LTP of 3month 3xtg-AD male mice with presence of
vehicle, E2 and E2 plus P4 .............................................................. 90

Figure 5. 2 LTP of 6month 3xtg-AD male mice with presence of
vehicle, E2 and E2 plus P4 .............................................................. 91

viii
Figure 5. 3 LTP of 12month 3xtg-AD male mice with presence of
vehicle, E2 and E2 plus P4 .............................................................. 92

Figure 5. 4 LTP comparison between different age groups
(3,6,and12 month old 3xtg-AD male mice ) with application
of vehicle, E2 and E2 plus P4. ......................................................... 94

ix
List of Abbreviations

Aβ peptide: β-amyloid peptide
AD: Alzheimer’s Disease
Akt: RAC-alpha serine/threonine-protein kinase
AMPA: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate
AMPAR: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor
ANOVA: Analysis of variance
αCaMKII: α-Ca2+/calmodulin-dependent kinase II
APα: Allopregnanolone, 3-α-hydroxy-5α-pregnan-20-one
BDNF: Brain-Derived Neurotrophic Factor
CA1: Cornu Ammonis area 1
CA3: Cornu Ammonis area 1
Ca
2+
: Calcium
CEE: Conjugated Equine Estrogens
CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione
CNS: Central Nervous System
CREB: cAMP response element-binding
DG: Dentate Gyrus
E2: 17β-Estradiol
ER-α: Estrogen Receptor α
ER-β: Estrogen Receptor β
ERK: Extracellular-Signal Regulated Kinase
x
fEPSP: Field Excitatory Postsynaptic Potential
GABA: gamma-Aminobutyric acid
GABAA R; gamma-Aminobutyric acid A receptor
GAD: Glutamic Acid Decarboxylase
HFS: High Frequency Stimulation
HT: Hormone therapy
IPSCs: Inhibitory post-synaptic currents
LFS: Low Frequency Stimulation
LTD: Long-term Depotentiation
LTP: Long-term Potentiation
MAPK: Mitogen-Activated Protein Kinase
MPA: Medroxyprogesterone Acetate
mRNA: Messenger RNA
NMDAR: N-methyl-D-aspartate receptor
OVX: Ovariectomized
P4: Progesterone
PI3k: phosphoinositide 3-kinase
PKA: Protein Kinase A
PR: Progesterone receptor
TrkB: tyrosine receptor kinase B
WHI: Women’s Health Initiative Study
WHIMS: Women’s Health Initiative Memory Study
3xtg-AD: triple transgenic Alzheimer’s Disease
xi
Abstract
Since the early 1990’s, cognitive influences of estrogen (E2) have been
widely studied, especially in the hippocampus, one of the brain regions
responsible for learning and memory. Despite substantial evidence of estrogen’s
neuroprotective role in animals, the issue of hormone therapy (HT) was
complicated and much debate was generated when E2 was combined with
progesterone (P4) for clinical use in humans. It has been hypothesized that P4
counteracts the neuroprotective effects of E2. Therefore, elucidating the
interactions between these two hormones in the brain is vital. The present study
examines the neural physiological effects of estrogen and progesterone on the
synaptic transmission and plasticity in CA1 region of different animal models
including ovariectomized rat, hormone pre-treated female rat and 3xTG
Alzheimer model mice. We found that E2-induced enhancement in basal
transmission was decreased by P4, but E2-induced enhancement in long-term
potentiation (LTP) was unaffected by P4. In summary, these results support the
hypothesis that progesterone may attenuate the estrogen-induced
hyperexcitability in pyramidal cells. This study shows estrogen and progesterone
at certain concentrations work against each other and may explain the conflicting
data on HT. With better understanding of the interactions of E2 and P4 in the
future, it may become possible to design the most efficient and successful
hormonal therapies.

1
Chapter 1: General Introduction

Introduction

―How does the brain work?‖ Scientists in the field of neuroscience, biology,
chemistry, experimental psychology and other related fields have spent the last
several decades trying to answer this question. However, the brain is still the
most complicated and mysterious organ in our body. The brain is not stationary;
rather it changes its structure and progressively loses its normal function of
sensation, motor control and cognition as the results of aging. Currently, age-
related brain diseases and functional decline are the major problems along with
the increasing elderly population. Although there is still a lot to be elucidated
about how age changes the brain, advanced technologies have diagnosed
negative consequences of structural and cognitive changes in the aged brain.
During the past few decades, basic sciences and clinical studies have found that
in women, termination of menstrual cycle, which triggers dramatic loss of
neurosteroids such as estrogen and progesterone, is strongly related to
unfavorable neural consequences including declines of learning and memory.
Many studies with animals and humans have attempted to reveal how these
hormones modulate cognitive functions to utilize their utmost neuroprotective
benefits in clinical hormonal treatment. Despite the strong evidence of beneficial
effects of hormonal therapy (HT) on learning and memory on animals, its
effectiveness on human has been questioned. Initiation time of HT after
menopause, women’s age, different synthetic hormone compounds and
2
administration regimens were thought to be some of the factors in lack of efficacy
in human trials. Considering the importance of clinical reliability in HT, further
research is essential on the possible cellular, molecular, behavioral and cognitive
influences of E2 and P4. In this chapter, previous key studies relevant to the
neuronal effects of estrogen and progesterone on hippocampus will be reviewed.

Hormone Therapy

In spite of numerous studies showing the benefits of HT, the controversial
views on taking care of postmenopausal symptoms still prevail. The history of HT
using estrogen and progestin dates back almost 100 years. Since estrogen was
first used for treatment of irregular menstruation cycle in early 1900’s, HT has
undergone dramatic changes with the evolution of basic biological science and
clinical trials. HT grew increasingly popular for relief of menopausal symptoms. In
the mid 1970’s, estrogen alone treatment was found to increase the risk of
endometrial uterus cancer, therefore, combination with progesterone was
strongly recommended for women with an intact uterus. Many clinical studies
found that HT can slightly increase the risk of breast cancer, blood clot formation
and hypertension. However, other favorable effects were found including
improvement in bone density, coronary heart disease and postmenopausal
symptoms. Consequently, by the 1990s, HT was prescribed for long-term
administration with larger doses compared to currently used dosage for
postmenopausal women. This practice was questioned by the landmark study,
Women’s Health Initiative (WHI) study, which was conducted in 2002 and 2004.
3
The WHI study was a randomized, controlled trial with a large number of
postmenopausal women, and utilized conjugated equine estrogens (CEE)
combined with medroxyprogesterone acetate (MPA). This study reported the
potential danger of chronic use of HT by indicating that HT is associated with
increased risk of stroke and cardiovascular disease (Anderson, Limacher et al.
2004). In addition to these adverse physical results which HT may cause,
Women’s Health Initiative Memory Study (WHIMS) investigated how HT can
affect cognitive aspects of the postmenopausal women. Marked depletion of
endogenous hormonal level of postmenopausal women has been considered as
one of potential reasons for age-related cognitive decline such as verbal and
figural memory, and higher risk of developing Alzheimer’s disease than men. As
a result, there was a strong need for the precise evaluation of the impact of HT
on cognitive function and neurological disease. WHIMS included the largest size
of postmenopausal women (4532) aged from 65 years old to 72 years old without
dementia indications to evaluate HT effects on dementia, mild cognitive
impairment and cognitive malfunction (Shumaker, Legault et al. 2004).
Unfortunately, the results from WHIMS indicated that CEE combined with MPA
for 5 years of treatment significantly increased the risk of developing Alzheimer’s
disease, and it showed no benefit on cognitive performance. CEE alone
treatment showed no difference on the rate of probable dementia or mild
cognitive impairment between the hormone and placebo groups. However, there
was a nonsignificant increase in probable dementia, and long-term treatment
impaired cognitive performance. Considerable inconsistencies in the outcome of
4
WHIMS have produced a lot of debate especially in the field of basic science
where the focus had been on the putative neuroprotective actions and
mechanisms of estrogen and progesterone. However, this study had many
methodological imperfections such as age of patients at the initiation of HT,
combination of estrogen and progestins with quite different pharmacological
nature, route of delivery, duration of HT, and doses of HT. These are important
clinical questions which urgently need to be re-examined in both animal and
human studies. Currently, use of HT is recommended with lower doses for a
short period for women with an early onset of menopause. The future research
for HT with minimal risk factors is essential for physical and cognitive health
benefits which may lead to improved quality of life of fast growing population of
postmenopausal women.

Neurobiology of Estrogen in Hippocampus

The large body of previous researches regarding neurological actions of
estrogen has been focused on hippocampus as this brain structure is closely
related to cognition decline with aging. Located in medial temporal lobe,
hippocampus is considered as a critical component of memory system. The
dorsal hippocampus is responsible for working and spatial memory with the
information from the sensory cortex, whereas the ventral part which is connected
to amygdala, prefrontal cortex and hypothalamus manages emotional memory
and anxiety (Swanson and Cowan 1977; Moser and Moser 1998). The structural
changes in hippocampus and its strong correlation between cognition such as
5
learning and memory is greatly affected by estrogen. In rodents, for example,
learning impairments are attributed from various adverse conditions which
decrease the neurogenesis in the dentate gyrus (DG). Previous studies showed
that stress, aging, changes in endogenous level of hormones can affect the
generation of new neurons in DG (Drapeau, Mayo et al. 2003; Montaron,
Drapeau et al. 2006). On the other hand, factors that facilitate neurogenesis tend
to improve hippocampal dependent leaning tasks (Kempermann, Kuhn et al.
1995; van Praag, Shubert et al. 2005). Based on well-defined hippocampal
structure and significant progress in earlier studies on cellular plasticity, the
hippocampus has been identified as one of the most critical brain regions to
investigate the hormonal effects. Many studies have found the molecular, cellular
and systemic mechanism of how estrogen exerts its role in cognitive functions by
promoting long-term potentiation (LTP), which is a major synaptic plasticity model
related to learning and memory (Cordoba Montoya and Carrer 1997; Smith and
McMahon 2005). Estrogen is strongly involved in memory-related synaptic
plasticity in hippocampus (Brann, Dhandapani et al. 2007), therefore further
study on the neural modulation of this hormone is vital for potential clinical
advantages for postmenopausal women.

Estrogen Receptor (ER)

Identified in the early 1960 (EV 1962; Jacobson 1962), in animal models,
ER has been extensively demonstrated as a regulator of structural and functional
changes in the brain. Existence of the ER has explained the specificity of
6
estrogen effects in the different neuronal systems and provided possible
foundations regarding the cellular, molecular actions of estrogen. Nuclear ER,
which exists inside of the cell nuclei, functions as a ligand-dependent
transcription factor to mediate gene expression, cellular development,
differentiation and maintenance in various brain areas including hippocampus,
cerebral cortex, midbrain and brainstem (McEwen 2002). In contrast, extra-
cellular ER, which is plasma membrane ER, manages rapid physiological
changes dependent on the activities of a various signal transduction and
pathways. Widely distributed two types of major ERs, ER alpha (ERα) and beta
(ERβ), were known to control the various neural changes in structure, cellular
physiology and biochemistry which may affect cognitive functions. ERα was
considered to mediate the genomic and non-genomic effects of estrogen, and
ERβ appeared to have similar estrogen-binding ability with ERα. The size of ERβ
is smaller than ERα, and has differences in the N-terminal transactivation and C-
terminal ligand binding domains (Enmark, Pelto-Huikko et al. 1997; Kuiper,
Shughrue et al. 1998). These two nuclear ERs were demonstrated to respond in
opposite ways in gene transcription in the response to ligand and estrogen-
responsive genes at an AP1 binding site (Paech, Webb et al. 1997). In addition
to ERα and ERβ, which have long been recognized and studied, ERx, the G
protein-coupled receptor, was found recently (Toran-Allerand, Guan et al. 2002).
Many complex experimental techniques were utilized for solid localization of ERs
and regulation of ER genes in the brain. These efforts have resulted in revealing
the underlying mechanisms of how estrogen affects brain function and behavior.
7
Study of localization of ER was first started from the uterus and mammary gland,
organs related to reproduction, and extended towards hypothalamus, pituitary
gland, up to the cerebral cortex, hippocampus, brain stem and midbrain neuronal
cells. The pattern of expression of these different ERs has been shown to be
quite different depend on different body structure, brain regions (McEwen 2002).
Previous studies with rat tissue have demonstrated that ERα is heavily
distributed in uterus, testis, ovary, pituitary and kidney, and ERβ is strongly
expressed in prostate, ovary, lung, brain, bone, uterus (Kuiper, Shughrue et al.
1998). Distribution of ERα in the brain is quite precisely established, yet
localization of ERβ needs more investigation. In rat brain, neurons with the ERs
have been found in the hippocampus, cerebral cortex, midbrain, and brainstem
(McEwen and Alves 1999). There is a wide distribution of ERα within axospinous
synapses and spines of pyramidal cells in stratum radiatum of female rat
hippocampal CA1 region, where many studies regarding synaptic transmission
and plasticity have been performed (Adams, Fink et al. 2002). In rodent
hippocampus, in situ hybridization showed that ERβ covered the whole
hippocampal area, much more abundant than ERα. Its distribution pattern
increases from dorsal to ventral and rostral to caudal of the hippocampus
(Shughrue, Lane et al. 1997) especially in the CA1 stratum radiatum, CA3
stratum lucidum, strata oriens (Shughrue and Merchenthaler 2000) and dentate
gyrus granule cell layer (Mitra, Hoskin et al. 2003; Herrick, Waters et al. 2006).
Higher concentration of ERβ immunoreactivity in pyramidal cells of CA1 and CA3
in the ventral hippocampus implies that ERβ is strongly engaged with cognitive
8
function (Shughrue and Merchenthaler 2000). ERs which exist in the spines of
apical and basal dendrites of pyramidal cells in CA1 may exert a critical role in
the change of spine density and the shape of synaptic boutons (Woolley, Gould
et al. 1990; Woolley and McEwen 1994). ERα and ERβ mRNA expressions
within the axon terminals and spines were proved not stationary but dynamically
variant depending on aging, neurodegenerative disease development, and cyclic
17β-estradiol (E2) levels in rat brain (Lauber, Romano et al. 1990; Shughrue,
Bushnell et al. 1992; Ö sterlund, GJM Kuiper et al. 1998). Number of synapses
with ERs and sensitivity to estrogen are decreased in aged female rats (Adams,
Shah et al. 2001). In the ERα gene knockout mouse, ERβ was expressed in the
preoptic area, cerebral cortex, hippocampus, paraventricular nucleus and
supraoptic nucleus (Couse, Lindzey et al. 1997; Shughrue, Scrimo et al. 1997;
Singh, Setalo Jr et al. 2000). Light microscopy found that nuclear ERα was
distributed in the inhibitory gamma-aminobutyric acid (GABA) interneurons in
female rats. This study also showed that extranuclear ERα engaged in pyramidal
and granule cells specifically in the region of axon and axon terminals, makes
synaptic contact with dendritic shafts and spines (Milner, McEwen et al. 2001).
Immunohistochemical staining of ERα proteins found that ERα was present in
both extranuclear and nuclear region of principal neurons in the CA1, CA3 and
dentate gyrus regions (Ishii, Ogiue-Ikeda et al. 2007). In human, from early
prenatal stages to adult, ERα and ERβ were found to be expressed continuously
in hippocampus, concentrated on pyramidal cells and dentate gyrus with distinct
developmental patterns (wrightGonzález, Cabrera-Socorro et al. 2007). Human
9
hippocampal ERβ immunoreactivity and number of neurons labeled with ERβ in
aged and Alzheimer’s disease(AD) was significantly increased (Savaskan,
Olivieri et al. 2001) indicating the ERβ’s beneficial effects on neuronal regulation
of estrogen. In addition, co-localizations of ERs and nerve growth factor may
facilitate the neuroprotective responses in the cerebral cortex, hypothalamus and
hippocampus (Toran-Allerand, Singh et al. 1999). ER’s neuroprotective action on
permanent cerebral ischemic brain injury was studied using ERα knockout and
ERβ knockout stroke model mice (Dubal, Zhu et al. 2001). This study showed
that ERα alone, but not ERβ, is completely responsible for the recovery effects in
the cerebral cortex, striatum and hippocampus in the physiological level of E2.
Further study showed that E2 exposure to the dendrite of hippocampal cultured
cell significantly increased the expression of ERα and ERβ as well as facilitated
the formation of NMDARs synapses along dendrites (Jelks, Wylie et al. 2007).
Recently, ERβ activation was found to play particularly critical role than ERα in
expression of GluR1 and phosphorylation, increases dendritic spine and spine
number, hippocampal LTP and spatial memory (Liu, Day et al. 2008).

Estrogen Effects on Hippocampal Morphology

The search for the cellular, molecular mechanisms underlying
hippocampal structural plasticity and circuit reorganization has been continuously
emphasized in field of neuroscience. Morphological changes in the brain
correlate with repeated experience, learning and memory, specifically LTP.
Depending on the estrogen level, dynamical morphological changes in
10
hippocampal neurons have been reported including an increased number of
dendritic spines and increased density of excitatory synapses (Gould, Woolley et
al. 1990; Woolley, Gould et al. 1990; Woolley and McEwen 1992). In spite of
extensive efforts, dendritic spines’ specific functions have not been clearly
identified. However, a survey of previous researches has revealed that dendritic
spines possess most excitatory synapses to control cellular excitability, and
deliver the distinct biochemical responses to activation of individual synapses by
selectively sorting out calcium influx (Nimchinsky, Sabatini et al. 2002). Dendritic
spines are believed to manage and integrate synaptic information which can
affect cognitive functions like learning and memory (Li, Brake et al. 2004).
Therefore, structural changes in dendritic spines induced by E2 treatment imply
important neuronal, functional and systematical influences in the hippocampus
(Wong and Moss 1992). Increase in spine density ties directly with increase in
NMDAR transmission and LTP, and this was proven by ER modulator, tamoxifen,
which inhibited the E2 induced increase in spine density, blocked the increase in
NMDAR transmission and heightened LTP(Cordoba Montoya and Carrer 1997;
Smith and McMahon 2005). Additionally, increased dendritic spine density of
CA1 pyramidal cell with acute administration of estrogen showed a solid temporal
relationship with memory retention in rats, indicating the importance of structural
remodeling in cognitive functions (Sandstrom and Williams 2001). Structural
abnormalities in dendritic spine have been demonstrated to result in many forms
of mental disease and cognitive impairments
11
(Irwin, Galvez et al. 2000; Kaufmann and Moser 2000). In 1990, E2 replacement
in adult rat was demonstrated to reverse significantly decreased dendritic spine
density induced by overiectomy especially in the CA1 pyramidal cells (Gould,
Woolley et al. 1990). Follow up study confirmed that hippocampal synapse
density and number of excitatory synaptic inputs to pyramidal cells varies during
natural estrous cycle in the adult rat (Woolley and McEwen 1992). The dendrites
of the apical and the basal branches of CA1 pyramidal cells produced increased
spine density with E2 treatment (Woolley and McEwen 1994). Increased number
of spines forms new axospinous synapses, and increases the number of multiple
synapse bouton (Woolley, Wenzel et al. 1996). High density of dendritic spine
and synapses on CA1 pyramidal cell has significantly increased cellular
excitability with elevated E2 level. This is similar to the electrophysiological study
with hippocampal slice, which showed that high frequency stimulation for LTP
induction facilitated the rapid and local formation of new dendritic spines (Smith
1999; Lendvai, Stern et al. 2000; McAllister 2000). At the site of stimulation,
stimuli that induce various forms of LPT trigger an increase in number of matured
filopodia and formation of new spines, suggesting NMDA receptor activation or
calcium application is necessary for restructuring of the actin cytoskeleton
(Engert and Bonhoeffer 1991; Maletic-Savatic, Malinow et al. 1999). E2 induced
spinogenesis as well as enlargement of head and synaptic contact area of
existing dendritic spines (Yuste and Bonhoeffer 2001). For example, unlike rats,
exogenous E2 application in OVX mice does not increase dendritic spine density
in CA1 stratum radiatum like, however it changes spines into mushroom shaped
12
(Li, Brake et al. 2004). Cytoskeleton of spine neck was shortened and widened,
verifying these geometric changes produced the reduced resistance and
increased efficacy of incoming current toward spine (Carlisle and Kennedy 2005).
The functional significance of rapid E2 regulation of spine shape and size has
become apparent from the findings of many lines of research. E2 regulation of
spine shape and size is crucial for LTP and information processes at synapses.
Constant alterations in spine shape may increase the synaptic strength by taking
a proportionally bigger number of postsynaptic receptors and vesicles in the
presynaptic axon with enlarged spine head volume (Harris and Stevens 1989;
Takumi, Ramí rez-León et al. 1999). At Schaffer collateral commissural synapses
of adult male rat, expression pattern of AMPA and NMDA receptors are
significantly dependent on postsynaptic density diameter and synaptic size
(Takumi, Ramí rez-León et al. 1999). In this study, AMPA receptor expression as
well as the ratio of AMPA receptors to NMDA receptors was shown to be linearly
increased along with the synapse diameter under 180 nm. Similarly, in cultured
hippocampal CA1 pyramidal neurons, mushroom-shaped larger spines contain
more AMPA receptors than thin, small spines at the single synapse (Matsuzaki,
Ellis-Davies et al. 2001). Increased AMPA receptor expression lends support to
the proposed mechanisms of LTP induction and expression, which emphasized
the importance of insertion of new AMPA receptors (Lisman and Harris 1993;
Isaac, Nicoll et al. 1995). Recently, fluorescene resonance energy transfer
(FRET) based imaging techniques found that actin polymerization dynamically
changes in response to tetanic electric stimulation (Okamoto and Hayashi 2006).
13
High frequency stimulation, which induces LTP, enhanced the formation of actin
filaments in spines, whereas low frequency stimulation for long-term depression
(LTD) induction increased actin depolymerization. Together, morphological
changes induced by E2 or the synaptic stimulations are directly engaged with the
LPT. To establish the successful future clinical strategies to prevent or reverse
the age-related decline in learning and memory during menopause, the
relationship between the estrogen-induced morphological and functional changes
in hippocampus must be defined as well as the impact of these changes on
cognition.

Estrogen Effects on Hippocampal Electrophysiology

Estrogen has unique nongenomic effects, which can occur rapidly in
seconds but also last hours, upon cellular activities. These acute effects have
been attributed to the membrane ERs with the input of estrogen derived from
hippocampus rather than circulating gonadal hormones (Falkenstein, Tillmann et
al. 2000; Kelly and Levin 2001). Electrophysiological investigations in rats and
mice found that E2, a selective estrogen agonist, is an effective modulator of
hippocampal synaptic plasticity. The long-lasting increase in LTP is a hallmark of
E2-induced hippocampal synaptic plasticity. Acute hippocampal slices have
become an efficient method to investigate the direct effect of E2 on glutamatergic
neurons. It is because in vivo studies combine both direct and indirect effects of
E2 through cholinergic or serotonergic projection to the hippocampus (Leranth,
Shanabrough et al. 2000; Leranth, Petnehazy et al. 2003). However, molecular
14
mechanisms of rapid action of E2 via membrane receptors have not been well
explained compared to those of genomic process. In an effort to elucidate the
physiological effects of estrogen on hippocampus through molecular and
biochemical mechanisms, Teyler et al. first conducted the experiment with
hippocampal slice, and found that direct application of 10pM of E2 with high
frequency stimulation of Schaffer collaterals increased intrinsic excitability of CA
pyramidal cells in adult male rats (Teyler, Vardaris et al. 1980). There are four
major potential mechanisms which explain the E2 induced facilitation in LTP.
First, E2 induces the increase in dendritic spine density and synapses on CA1
pyramidal cells. Second, E2 induces the increase in N-methyl-D-aspartic acid
receptor (NMDAR) expression and function which directly increase LTP (Woolley,
Weiland et al. 1997; Bi, Foy et al. 2001). Third, E2 induces the increase in
presynpatic glutamate release (Adams, Shah et al. 2001). Lastly, E2 induces the
decrease in inhibitory gamma-aminobutyric

acid (GABA)ergic inhibition, which in
turn, leads to active NMDAR participation required for LTP induction (Harris,
Ganong et al. 1984; Murphy, Cole et al. 1998; Rudick and Woolley 2001). E2
significantly enhances basal transmission and LTP in rat (Foy, Xu et al. 1999).
Hippocampal slices from wild type female mice incubated with ERβ selective
agonists showed significantly increased LTP than the control from the same
animals and ERβ knockout mice (Liu, Day et al. 2008). E2 also enhanced LTP in
vivo in awake rats (Cordoba Montoya and Carrer 1997) as well as in cultured
hippocampal slices (McEwen 2002). E2 induced LTP enhancement is closely
associated with improved performances in hippocampus-dependent memory
15
tasks (Packard and Teather 1997; Luine, Richards et al. 1998). LTD is also very
sensitive to E2 treatment in hippocampal slices from adult rats (Vouimba, Foy et
al. 2000). LTD is significantly enhanced by perfusion of 10nM of E2 in CA1, CA3
and DG (Ogiue-Ikeda, Tanabe et al. 2008).

Estrogen Induced Increase in NMDAR Expression and Transmission

A large number of studies examined the function of NMDAR-mediated
synaptic transmission in triggering LTP compared to the impact of GABAergic
inhibition or AMPA transmission (Bliss and Collingridge 1993; Foy, Xu et al. 1999;
Smith and McMahon 2005). Estrogen’s ability to increase the synaptic excitability
and strength of neural synapses comes from enhancement of NMDAR binding
and subunit expressions (Foy, Xu et al. 1999) as well as the increased dendritic
spine density. Pyramidal cells in CA1 of E2 and E2 plus progesterone treated
rats showed increased number of NMDAR binding sites, amplified NMDAR1
mRNA and protein level in their somata and dendrites eliciting extended duration
of EPSPs (Gazzaley, Weiland et al. 1996). Increase in NMDAR allows the
greater sensitivity to NMDAR-mediated synaptic transmission. However, AMPAR
in hippocampus remains unchanged after ovariectomy (Cyr, Ghribi et al. 2000;
Cyr, Ghribi et al. 2001). When the activation of synaptic NMDAR was inhibited
pharmacologically, it led to complete blockage of the LTP in the rat hippocampus,
(Collingridge, Kehl et al. 1983). Similarly, rats infused with NMDAR antagonist
showed selective impairment in spatial learning without damaging already
established information (Morris 1989). Physiological regulation via NMDAR is
16
dependent on the changes in spine density and hippocampus. Direct
intraperitoneal injections of NMDAR antagonist to OVX rats prevented effect of
E2 on spine density; however, AMPAR antagonist had no significant effect
(Woolley and McEwen 1994). In vitro study with cultured hippocampal neurons
also showed that increased spine density by E2 was blocked by the NMDA
antagonist, but not by AMPA antagonist (Murphy and Segal 1996). At synapses
between CA3–CA1 pyramidal cells from adult OVX rats with subcutaneous E2
injection, NMDAR transmission and the magnitude of LTP after 24 and 48 hours
are increased (Smith, Vedder et al. 2009). For non-NMDA mediated excitation,
E2 activates cAMP-PKA pathway, which results in augmented excitatory
glutamatergic transmission in rat CA1 hippocapal neurons (Gu and Moss 1996;
Gu and Moss 1998). In conclusion, E2 induced changes in NMDAR, the major
mediator of LPT, are crucial to understanding enhanced LTP in hippocampus.

Estrogen Induced Decrease in GABAergic Inhibition

Along with the regulation of glutamate receptors, E2 directly decreases the
synthesis of GABA in interneurons and the GABAergic inhibition in the
hippocampus, thereby facilitating the formation of new dendritic spines and
cellular excitability (Murphy, Cole et al. 1998). E2 affects inhibitory inputs to
pyramidal cells by modulating expression of glutamic acid decarboxylase (GAD),
the

rate-limiting enzyme for GABA synthesis. In E2 treated ovariectomized (OVX)
rat, GAD mRNA levels increased in GABAergic neurons only in the CA1
pyramidal cell layer (Weiland 1992). In cultured rat hipocampal neurons, E2
17
decreases GABAergic synapic transmission, which results in increased spine
density (Murphy, Cole et al. 1998). Advanced studies recently investigated the
relationship between ERα and inhibitory signal transmission. In this study,
researchers found that ERα is localized in one-third of perisomatic inhibitory
boutons in CA1 region, and specifically exists on neropeptide Y(NPY) producing
cells. NPY is the inhibitor of glutamate release at the synapse(Hart, Snyder et al.
2007). In CA1 interneurons which express NPY, E2 mobilizes presynpatic
vesicles toward inhibitory synapses and also increases NPY expression
(Nakamura and McEwen 2005) . As a result, E2 may enhance inhibitory inputs
onto CA1 pyramidal cells from a group of NYP-expressing GABAergic
interneurons. However, mechanisms of GABAergic neurons in E2 effects remain
uncertain.

CREB, and MAP/ ERK

In addition to its acute effect on cellular excitability, estrogen can activate
various signaling pathways connected to structural and functional plasticity in the
hippocampus. E2 rapidly activates the transcription factor, the cyclic AMP
response element binding protein (CREB) in vivo and in vitro in the CA1 and CA3
region of rat hippocampus (Murphy and Segal 1997; Wade and Dorsa 2003;
Zhou, Zhang et al. 2005). Phosphorylation of CREB implicates E2-induced spine
formation, and up-regulation of synaptic protein expression (Murphy, Segal et al.
1997). CREB phosphorylation depends on E2 activation of mitogen-activated
protein kinases (MAPKs) or extracellular-signal regulated kinases (ERKs) in
18
hippocampal neurons triggered by rapid calcium influx (Zhao, Chen et al. 2005;
Mannella and Brinton 2006; Szego?, Barabas et al. 2006). CREB activation by
E2 depends not only on MAPK, but also on calcium/calmodulin-dependent
protein kinase II (CamKII), whose role in synaptic plasticity, learning and memory
has been well-investigated (Lee et al. 2004). CamKII is rapidly activated by E2 in
vivo and in vitro (Sawai, Bernier et al. 2002). CamKII is also thought to be
involved in the expression of dendritic spine protein, spinophilin, thereby
increasing the spine density. E2 has been reported to increase the expression of
CamK and CamKIV, which also phosphorylate CREB in the CA1 region of OVX
rats (Zhou, Zhang et al. 2005). E2-induced activation of MAPK and CREB is
considered essential for improved LTP in hippocampus.

P13K/Akt

E2 rapidly activates P13K/Akt which is related to cell growth, proliferation,
differentiation and growth factor mediated survival to induce actin remodeling and
filopodia enlargement (Akama and McEwen 2003). This was confirmed by E2-
increased subcellular distribution of Akt immunoreactivity in dendrites throughout
the shafts and in spines of CA1 stratum radiatum of female rats (Znamensky,
Akama et al. 2003). The density of Akt in CA1 in proestrous and E2 pre-treated
female rats was significantly higher than OVX and diestrus ones (Znamensky,
Akama et al. 2003). Increased Akt activation may facilitate the formation of new
spines as well as cell survival.
αCamKII
19
αCaMKII has been considered to play an prominent role in stabilization
and maintenance of LTP (Wu, Malinow et al. 1996). Accumulating data have
found that calmodulin has an essential role in ERα transcriptional activity. E2 was
found to activate CaMKII via very rapid nongenomic mechanism in a dose and
time-dependent manner in rodent hippocampus (Sawai, Bernier et al. 2002).
Calmodulin, a ubiquitous Ca
2+
sensor protein, appears to involve in ERα
transcriptional activity. Cell permeable calmodulin antagonists prevented E
2
from
stimulating ERα transcription, but not ERβ (Pedrero, Juana et al. 2002; Li, Li et al.
2003). Sensitivity to CaM antagonists

of estrogen-responsive tissues positively
correlates with a high ERα/ERβ

ratio. Moreover, interaction with calmodulin is
required for ERα to bind the ERE (Bouhoute and Leclercq 1995), and activate an
ERα-responsive promoter (Biswas, Reddy et al. 1998). Mutant calmodulin that is
unable to bind Ca
2+
, functioned as a major negative factor, reducing both basal
and E2 -stimulated transcriptional activity of ERα. Moreover, the CaMKII was
demonstrated as a major protein of the postsynaptic density (Benson, Gall et al.
1992; Kennedy 1998; Shen and Meyer 1999) that critically important for LTP and
synaptic differentiation. Radioimmunocytochemistry found that E2 treatment
increases expression of spinophilin in the CA1 region (Brake, Alves et al. 2001),
and in this process, CamKII exert its role both in the formation of synapses and
localization of AMPA receptors in synapses (Rongo and Kaplan 1999; Hayashi,
Shi et al. 2000). After induction of LTP by tetanic stimulation, phosphorylation of
CamKII was observed in synapses, dendrites and somas of hippocampal
neurons (Wu, Malinow et al. 1996). E2 was shown to induce phosphrylation of
20
CREB in hippocampal neurons however, CaMKII inhibitor prevented E2-induced
CREB activation, and reduced basal activity of CREB(Lee, Campomanes et al.
2004). CaMKII is thought to facilitate synaptic transmission by phosphorylation
of glutamate receptors, and increase the levels of these receptors in synaptic
membrane (Lisman and McIntyre 2001). This suggests the close relationships
between E2 and CamKII in modulating synaptic transmission, because E2 also
was demonstrated to regulate NMDA receptor expression and activation.

Neurotrophins

E2 also acts on neurotrophins such as nerve growth factor (NGF) through
tyrosine receptor kinase A (TrkA) (Gibbs, Wu et al. 1994). However, several
experiments regarding TrkA mRNA level and hippocampal NGF protein yielded
conflicting results (McCarthy, Barker-Gibb et al. 2002). The role of brain-derived
neurotrophic factor (BDNF) in the effects of estrogen on hippocampal physiology
has been extensively studied. Levels of BDNF mRNA and protein fluctuate
across the estrous cycle in female rats, with the highest level of mRNA and
protein occurring during proestrus (Scharfman, Mercurio et al. 2003). E2
increases BDNF signaling through tyrosine receptor kinase B (TrkB) in dentate
gyrus and CA3 stratum radiatum in rat hippocampal formation (Znamensky,
Akama et al. 2003). Progesterone (P4) is the most physiologically active
progestin of

ovarian origin, which modulates multiple cellular functions and
physiological changes in the

central nervous system (CNS) as well as
reproduction. P4 effects on neuroprotection, axonal regeneration or remyelination
21
and cognitive functions in the nervous system are of great interest due to their
potential clinical implications. P4’s primary role as the post-injury treatment of
both male and female with acute traumatic brain injury (TBI) has been
extensively investigated. Progesterone weakens the immune inflammation,
promotes axonal remyelinization or synaptogenesis, recovery of neural functions
and maintains neurotrophic factor (Roof, Hoffman et al. 1997; Wright, Bauer et al.
2001; Djebaili, Guo et al. 2005). P4 decreases mitochondrial dysfunction and
restores the hippocampal cell loss caused by TBI in female rats (Robertson,
Puskar et al. 2006). However, these studies have been focused on the higher-
than physiological levels of P4 to reverse pathological consequences after TBI,
and this resulted in limited insights regarding neuroprotective effects of P4.
Therefore, studies with normal endogenous levels of P4 are essential to
discovering the sophisticated mechanisms of P4. Moreover, clinical trials
reported that interpretation of P4 effects is dependent on variables such as age
of the patients, level of brain injury, timing of treatment and accurate
measurement method (Stein 2001; Wright, Kellermann et al. 2007). In an attempt
to uncover the neuroprotective effect of P4, clinical trials have demonstrated that
short-and long-term P4 treatment induces a significant progress in patients with
brain injury and motor neurons. P4 treatment was able to restore proliferation,
differentiation and growth of oligodendrocyte progenitor cells in the
experimentally simulated spinal cord injury as well as repairing the damaged
expression of BDNF mRNA, choline acetyltransferase (ChAT), Na,K-APTase
mRNA (De Nicola, Labombarda et al. 2009). Multiple neural effects of P4 have
22
been actively studied in basic science and clinical research. However, the
mechanism of P4 and its receptors’ regulation on the biochemical changes in
neurons, genetic transcription and cognitions is far from being completed.

Progesterone Receptor (PR)

The widely distributed progesterone receptors (PR) throughout the brain
especially in hypothalamic margin, hippocampus and cerebral cortex can be
turned on by the endogenous P4 circulation. Two forms of PR have been
identified; PRA with N terminal truncated, and PRB with a larger size. (Schrader,
Birnbaumer et al. 1981; Brinton, Thompson et al. 2008). Two distinct estrogen-
regulated promoters induce the gene expression of these two isoforms (Kastner,
Krust et al. 1990). PRB is first expressed at the time of birth, followed by PRA
after several days (MacLusky and McEwen 1980; Kato, Hirata et al. 1994).
Intracellular PRs existing in P4 sensitive neurons are assumed to manage neural
effects by activation of gene transcription and reorganization of genomic
networks. Increasing in vivo and in vitro studies found that the membrane PR
regulates diverse cellular alterations and protein kinase cascades independent of
the intracellular PR. In situ hybridization of female rat showed that neurons with
PR mRNA was distributed in the mediobasal hypothalamus, amygdale (Romano,
Krust et al. 1989), arcuate nucleus, medial preoptic nucleus and ventromedial
nucleus as well as the pyramidal layer of CA1 and CA3 of the hippocampus
(Kato, Hirata et al. 1994). A recent ultrastructural analysis found that PRs mainly
exist in extranuclear sites throughout the CA1, CA3 and dentate gyrus of the
23
hippocampus (Waters, Torres-Reveron et al. 2008). Derived from the soma of
pyramidal cells, dendrites and dendritic spines of CA1 stratum radiatum contain
PRs. Distinct biological responses are mediated by two PRs; PRB activates
transcriptional processes whereas PRA inhibits or reduces transcription of all
steroid hormone receptors including ERs within the cell. This indicates that PR
regulates physiological, biochemical responses induced by sex steroid (Wen, Xu
et al. 1994).

Expression of PRs

High levels of E2 increase PR labeling, implying possible interactions
between these two hormones (Waters, Torres-Reveron et al. 2008). E2
increases expression of PRA and PRB in the region of hypothalamus in
ovariectomized rat, while progesterone diminished PRs (Guerra-Araiza, Villamar-
Cruz et al. 2003). Expression of PRA is considered to be more sensitive than the
PRB in response to endogenous E2 level in the region of hippocampus. In the
hippocampus, PRA was significantly increased by E2; however, P4 did not
change the expression of any PR isoform (Camacho-Arroyo, Guerra-Araiza et al.
1998). Estrous cycle can alter PRB expression in rat brain region of the
hypothalamus, preoptic and frontal cerebral cortex except hippocampus (Guerra-
Araiza, Cerbón et al. 2000). Regulation of PR expression induced by E2 and P4
is different in OVX female and gonadectomized male rat (Guerra-Araiza, Coyoy-
Salgado et al. 2002). In OVX rat hippocampus, PRA expression was increased
by E2 however, P4 had no significant effect. In male hippocampus, PRs
24
expression was not affected by E2 nor P4. Accumulating results suggest that
expression of PR isoform is regulated by E2, but dependent on sex and brain
region.

Progesterone Effects on Brain Morphology

P4 influences the structural changes in the adult rat brain. The effective
period of P4 exposure and concentration for different animal models, and cell
type have not yet elucidated. However, multiple studies have found that P4 is
responsible for the proliferation of neural cells in peripheral and central nervous
system. P4 alone, increases expression of key transcription factors of Schwann
cell and influences the myelination program as well as it enhances cell
proliferation in cerebellum and hippocampus (Ghoumari, Baulieu et al. 2005;
Tanapat, Hastings et al. 2005; Magnaghi, Ballabio et al. 2007). P4, not its
metabolites, was shown to increase proliferation of NPC (Neural Progenitor Cell)
in female adult rat dentate gyrus in a dose-dependent manner through the
ERK/MAPK signaling pathway by membrane PR (Liu, Wang et al. 2009). Gould
and Woolley found that short-term application (5hr) of P4 showed the synergistic
effect to E2-induced enlargement in OVX adult rat CA1 pyramidal cell (Gould,
Woolley et al. 1990). However, long term P4 application (18h) significantly
decreased spine density in CA1 region (Woolley and McEwen 1993). P4-induced
structural changes in CA1 pyramidal cells is involved in voltage gated Ca
2+

channel conductance. Transient Ca
2+
current is significantly increased by P4
followed by E2, compared to non-treated OVX, and OVX rats without E2 pre-
25
treatment (Joels and Karst 1995). In dissociate cultured rat hippocampal
pyramidal neurons, P4 blocks the E2-induced increase in dendritic spines, and
enhances GABAergic inhibition (Segal and Murphy 2001). Exogenous P4
implantation in OVX female rats increased the spine densities in cortical layer III
and V (Chen, Yan et al. 2009). P4 also affects neurons in cerebellum, an
essential site for learning and memory, by promoting dendritic growth, dendritc
spine formation in purkinje cell, which synthesizes P4 during neonatal life
(Furukawa, Miyatake et al. 1998; Ukena, Usui et al. 1998; Ukena, Kohchi et al.
1999). P4 facilitates spinogenesis and synaptogenesis to mature the cerebellar
neurons of rats (Tsutsui 2006; Tsutsui 2008).

Progesterone Effects on Cellular Excitability

The mechanisms underlying P4 regulation on neuronal excitability, seizure
threshold or anxiety levels are unknown. However, an increasing body of
evidence has shown that P4 possibly can control these changes. According to
level of gonadal hormones, phasic changes in composition of GABAA in
hippocampus of female rat over the estrous cycle were shown. The GABA
A

receptor is the target for many clinically crucial therapeutic drugs, including
general anesthetics and anticonvulsants (Whiting 2003; Beleboni, Carolino et al.
2004). To date, 19 subunits have been identified and classified into subfamilies
based on their amino acid sequence homology; α
1-6
, β
1-3
, γ
1-3
, δ, ε, θ, π, ρ
1-3

(Sieghart, Fuchs et al. 1999; Sieghart and Sperk 2002). Different subfamilies
enable the expression of 20 to 30 distinct GABA
A
receptor isoforms in the CNS,
26
with unique expression patterns. Expression of inhibitory GABAA receptor
subunits in CA1, CA2 and CA3 regions of hippocampus was found to be
dependent on P4 level (Weiland and Orchinik 1995). During the diestrus phase,
when P4 level is high and E2 level is low, δGABAA expression is increased while
γ2GABAA expression is declined, accompanied by lowered neuronal excitability
(Mody 2005). In addtion, removal of 3α,5α-allopregnanolone, a metabolite of P4,
after chronic treatment of P4, increased anxiety and α4 subunit of the GABA
A

receptor in both male and female rat hippocampus (Gulinello, Gong et al. 2002).
Reduced GABA inhibition results in a decreased total GABA-gated current in
cultured pyramidal cells from CA1 hippocampus (Hsu and Smith 2003). Similar
phenomena were observed in cultured rat cerebellar granule cells and cortical
neurons when 3α,5α-allopregnanolone were depleted for long period of time
(Follesa, Concas et al. 2001). This result indicates that the P4 metabolite
mediates its physiological effects via alteration of GABAR expression in different
brain regions. Collectively, progesterone was demonstrated to regulate the
number of GABARs and GABAergic transmission in the nervous system (Smith
1991; Concas, Follesa et al. 1999).

Progesterone Effects on Electrophysiology in the Brain

Potential electrophysiological effects of P4 in hippocampus have received
less attention compared to E2. However, more evidence regarding P4-induced
neurophysiological changes is needed for advanced HT. In previous studies,
100nM of P4 application attenuated the baseline excitability and facilitated the
27
inhibitory effect on dendritic field of CA1 in OVX and intact female rat
hippocampal slices (Majewska, Harrison et al. 1986; Gee, Bolger et al. 1988;
Smith, Jones et al. 2002). Previous studies found that P4 is metabolized to
allopregnanolone, which potentiates the effects of GABA on the GABAR in the
brain. The inhibitory effects of P4 and its metabolites on the cellular excitability in
vitro is attributed to the enhancement of GABA action (Wang, Bäckström et al.
2000). In vivo, P4 decreases calcium uptake in synpatosomes in CA1 field of the
rat brain (Nikezic, Horvat et al. 1988), and modifies NMDA mediated
glutamatergic neurotransmission as well as GABA release from nerve terminals
in the CA1 to suppress excitatory responses (Taubøll, Ottersen et al. 1993).
Slices from gonadectomized male and female rats indicated a significantly
decreased dendrite EPSP (Excitatory Postsynaptic Potential) compared to those
of intact male and female rats. In vitro application of testosterone reversed this
decreased fEPSP by induction of greater spontaneous responses in CA1 (Smith,
Jones et al. 2002). When allopregnanolone is perfused to hippocampal slices
from OVX rats, fEPSP amplitude was significantly decreased suggesting that
cellular excitability in the basal transmission in CA1 region is decreased. In vitro,
application of 10
-8
M of P4 significantly reduced the amplitude of the fEPSPs in
CA1 in tetanized slices from both OVX and E2 treated OVX rats (Edwards, Epps
et al. 2000). However, fEPSPs of E2 treated OVX rats appeared to diminish P4
effect by the excitatory effects on hippocampal neurons. This implies that P4 may
interplay with E2 in hippocampal LTP.

28
Chapter 2: Effective Dose of Progesterone Decreases Basal Transmission and
Long-term Potentiation in Female Rat Hippocampus

Chapter 2 Abstract

Many studies have demonstrated estrogen (E2)’s acute effects on
synaptic excitability and plasticity in rodent hippocampal slices and delineated
the underlying molecular mechanisms involved with glutamate receptors (Foy, Xu
et al. 1999; Smith and McMahon 2005) and multiple protein kinase pathways (Bi,
Broutman et al. 2000; Bi, Foy et al. 2003; Sweatt 2004). However, acute
physiological effects of progesterone (P4) on synaptic transmission and plasticity
in hippocampus have not been established. Although dose-dependent acute
neurophysiological effects of P4 in the hippocampus may be clinically essential,
no research has been conducted. In the present study, P4 was hypothesized to
affect synaptic transmission and plasticity in the hippocampus at certain
physiological levels. Administration of 1µM P4 to the adults ovariectomized (OVX)
female rat hippocampal slices significantly attenuated the basal transmission and
LTP. These results support the previous studies regarding GABA inhibition
effects of P4 (Callachan, Cottrell et al. 1987; Hsu and Smith 2003), suggesting
that P4 opposes the effects of E2 in the hippocampus.

Introduction

P4 has been utilized in postmenopausal hormone therapy (HT); however,
recently the role of HT has been questioned because of the poor outcome of
29
large clinical trial of WHIMS. HT for postmenopausal symptoms and prevention
of age-related chronic diseases has produced ongoing controversies and
confusion. One of major reasons is the inconsistent evidence for the role of
progestins in HT. P4 is an endogenous hormone secreted from ovary and
placenta, and progestins are synthetic steroids, which mimic the actions of P4
(Whitehead, Hillard et al. 1990). In spite of considerable differences in chemical
profiles and pharmacological consequences among different progestins and P4,
they are generally considered the same (Sitruk-Ware 2004). Many progestins are
not converted to

the GABA
A
receptor-active metabolite allopregnanolone, which
is important to mediate P4-induced biological effects (Schumacher, Guennoun et
al. 2007). Moreover, progestins’ relative binding affinity with PR is quite different,
and it is still uncertain if these progestins can actually bind with PRA and PRB in
the target cell (Sitruk-Ware 2004). The undesirable results of WHI may be
attributed to the use of older progestin, medroxyprogesterone acetate (MPA).
WHI revealed that conjugated equine estrogens (CEE) plus MPA increased the
risk of breast cancer, coronary heart disease and dementia compared to CEEs
alone (Rossouw, Anderson et al. 2002; Shumaker, Legault et al. 2003). However,
this study did not take into account the fact that actions of progestins including
MPA depend on the interactions with naturally secreted E2 and synthetic CEE.
Considering the importance of the combined effects of E2 and P4 in therapeutic
purpose of HT, the investigation of interaction of these two hormones at the
cellular level is critical. Within the nervous system, P4 shows the neuroprotective
and neurotrophic capabilities which might be applied to prevention and recovery
30
of age-related brain disease and cognitive dysfunctions. With loss of endogenous
P4, PR may not function normally in aging nervous system. However, an
increasing body of studies provides the evidences that, even in an aged brain, P4
can exert its advantageous role to some extent (Melcangi, Magnaghi et al. 1998;
Melcangi, Magnaghi et al. 1998; Azcoitia, Leonelli et al. 2003). In these studies,
P4 treatment reversed age-related myelin protein decline in nerve fiber of aged
rats, and increased the number of small myelinated fibers. Moreover, it
decreased the abnormalities in neuronal structure. Indeed, P4 implicates the
therapeutic benefits which can be very efficient and selective in neural system.
In the present study, the electrophysiologically effective dose of P4 for in
vitro hipppocampal slice in rat is administered. P4 has strongly been suggested
to modulate GABAergic inhibition through gene expression and non-genomic
mechanisms to change the cellular activity of CA1, and many in vitro studies
support this. Many in vitro studies have provided the neuroprotective effects
using allopregnanolone, the metabolite of P4. Field potentials evoked in CA1 rat
hippocampal slices were significantly suppressed by perfusion of
allopregnenolone after induction of irreversible depression produced by hypoxia
(Frank and Sagratella 2000). In hippocampal cultured cells, NMDA-induced
elevation of CA
2+
current was rapidly decreased by allopregnanolone. This shows
that allopregnenolone has GABAergic properties in managing cellular excitability.
Similarly, another study showed that GABAA-induced synaptic inhibition by
allopregnanolone can protect neuronal excitotoxicity by reduction of Ca
2+
influx
via the NMDAR (Scott, Tanguay et al. 2002; Lapchak 2004). There is more
31
evidence which supports GABAergic modulation by P4. In cultured rat cerebellar
purkinje cells, allopregnanolone’s neuroprotective effect can be Inhibited by
GABA
A
receptor antagonist, picotoxin (Ardeshiri, Kelley et al. 2006). In cultured
mouse neurons, apoptosis induced by NMDA was inhibited by allopregnanolone,
and this result explains how P4 may be related to cell death (Xilouri and
Papazafiri 2006). It is clear that elucidating how endogenous P4 interacts with
GABAA receptor is essential for understanding physiological changes in inhibitory
neurotransmission mediated by GABAA receptors in the brain. Previously,
allopregnanolone was suggested to modulate activity of GABAA receptors by
binding to membrane accessory protein in rat brain (Darbandi-Tonkabon,
Hastings et al. 2003). Recently, two discrete transmembrane binding sites in
GABAA receptors for allopregnanolone were identified (Hosie, Wilkins et al. 2006).
Two binding sites which exist on most of GABAA receptor family directly activate
and increase the GABA responses when both of them are occupied by
allopregnanolone. This result can lead to a breakthrough in understanding the
P4’s neuronal inhibition which can be different according to different brain region,
cell type and GABAA receptor subunit composition. A previous study found the
natural plasma membrane P4 concentrations in rats during pregnancy and
lactation (GROTA and EIK-NES 1967). The physiological P4 level was found to
be in the range of 66 µM to 50 nM ; 20ng/ml-260ng/ml. Based on this, it is highly
appropriate to examine the several different P4 concentrations which may bring
changes in synaptic transmission. 10
-9
, 10
-8
, 10
-7
and 10
-6
M of P4 were used in
the present study to find out in vitro electrophysiological effect of P4 on basal
32
transmission in CA1 region of rat hippocampus. In addition, how synaptic
plasticity, LTP and LTD can be regulated by efficient concentration of P4, will be
examined.

Materials and Methods

Animals
A total of 39 3-month old female ovariectomized Sprague-Dawley rats
(Harlan) were used in this study. All animal treatment and procedures were
performed in accordance with protocols approved by the University of Southern
California Institutional Animal Care and Use Committee. The rats were kept in
plastic cages under a 12-h light/dark cycle and provided with food and water, at a
temperature of 25°C. The animals were transferred from the vivarium on the day
of experiment. All efforts were made to minimize the number of animals used and
their suffering.

In vitro Hippocampal Slices Preparation and Solutions
The rats were anesthetized with isoflurane and, as soon as their eyes
were closed, they were instantly decapitated using a guillotine. After the skull
was opened, the brain was rapidly removed and kept for 2-3 minutes in icy and
slightly oxygenated mixed solution of half artificial cerebrospinal fluid (aCSF) and
half sucrose solution. This procedure allows the brain to become hard and
minimizes cellular activities. The cerebellum and the forebrain were removed.
Lastly, one third of the dorsal cortex was cut parallel to the longitudinal axis. The
33
remaining block of brain was glued on the vibratome stage, with the dorsal
surface down, and the caudal end toward the razor, which was fixed at 10
º
.
400μm-thick coronal hippocampal slices with surrounding cortical tissue were cut
using a vibratome (Series 1000). The slices were then transferred to a holding
chamber, which was submerged in oxygenated aCSF at room temperature.
ACSF solution consisted of 124 mM NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.3
mM MgSO4, 26 mM NaHCO3, 2.4 mM CaCl2, and 10 mM glucose. After one
hour of equilibrium period, activity of hippocampal cell was expected to be
restored and stabilized. The slices were transferred to a Haas-type interface
recording chamber and continuously perfused with oxygenated (95% O2 and 5%
of CO2) aCSF at a rate of 1.5 to 2 mL/min at 31-32°C. The surface of the slice
was also oxygenated with combined gas of 95% O2 and 5% CO2.

Hormone Preparation
P4 (Steraloids, Q2600-000) was kept in the refrigerator dissolved in 100%
ethanol and diluted with aCSF for each experiment to make the final
concentration of 10
-6
M. It was determined that the concentration of ethanol
(0.01%) had no effect on the electrophysiological activity in the slices. All
solutions were oxygenated prior to perfusion to the hippocampal slice.
Extracellular Recording
Field excitatory post-synaptic potentials (fEPSPs) were recorded from the
dendrite of pyramidal cells in stratum radiatum of CA1, using a glass pipette filled
with aCSF (2–3 M resistance) in response to current stimulation from a
34
stimulating electrode (twisted nichrome wires, 50 μm), which was positioned 1-2
mm from recording electrode in stratum radiatum. Axoclamp 2A DC amplifier and
Cyber-amp 320 (Axon Instruments) were used for signal magnification, filtered at
6KH, and then sampled at 20 kHz. Input/output curves were generated by
increasing stimulus intensities from minimum of 20 to 150 μA in increments of 10
μA. For a 15-min baseline, at 30-second intervals, pulses of 0.1 millisecond
duration were delivered through the stimulating electrode to generate fEPSPs. To
determine the P4 concentration which effectively changes the hippocampal basal
transmission, four different concentrations of P4 were perfused to hippocampal
slice. After a stable 15-min baseline period, P4 containing aCSF was perfused to
the chamber by switching from control aCSF to P4-containing aCSF (10
-9
, 10
-8
,
10
-7
, and 10
-6
M) for 30 minutes before delivering high-frequency stimulation (HFS)
or low-frequency stimulation (LFS). Control slices continued being perfused with
vehicle aCSF, containing 0.01% ethanol. LTP was induced at the same
stimulation intensity for baseline with two trains of 1 sec of 100-Hz pulses
separated by 20-sec intervals. Recording continued for 30 min after HFS. The
LFS stimulation train for LTD induction consisted of 900 pulses delivered at 1 Hz
for 15 min. HFS or LFS was delivered directly from baseline conditions in which
slices were being perfused with either aCSF or P4. Only one titanic stimulus or
LTS was given to each slice.
Data Analysis
To estimate LTP and LTD values, fEPSP slopes were expressed as the
percent of averaged fEPSP slope values recorded during the 15min baseline
35
before HFS or LFS and compared with data following HFS or LFS induction
protocols. Statistical significance between the groups was evaluated by unpaired
two-tailed t-tests. It was conducted on data recorded during the last 5 min of the
baseline period and the last 5 min of the infusion period (continued aCSF or P4)
to assess the effect of the P4 on basal synaptic transmission. To estimate the
magnitude of LTP and LTD, the data collected during the last 5 min of the
infusion period were compared with data collected during the last 5 min of the
HFS or LFS periods. Statistical significance between the groups was evaluated
by unpaired two-tailed t-tests. Results were expressed as means±S.E.M. All tests
were performed with a critical significance level of P<0.05, using SPSS software
running on a Dell computer.

Results

P4 Effect on Basal Synaptic Transmission in CA1 Region of Hippocampus
P4-induced synaptic change in basal transmission after baseline is
investigated with different P4 concentration within physiological level (Figure 2.1).
Three lowest concentrations of P4 (10
-9,
10
-8
, and 10
-7
M) did not significantly
change fEPSPs amplitude evoked by Schaffer collateral-commissural stimulation
compared to baseline. Average of fEPSPs from the slices perfused with 10
-9
M of
P4 were found to be 102% ± 2.7% above baseline values (P = 0.61, not
significant [ns]); 10
-8
M of P4 showed 96% ± 1.7% below baseline values (P =
0.06, ns); and 10
-7
M of P4, 96% ± 1.7% below baseline values (P = 0.06, ns).
However, at the highest concentration of P4 examined (10
-6
M), baseline fEPSPs
36
were found to be significantly attenuated compared to aCSF controls: 95% ± 1.7%
(P < 0.05).

P4 Effect on LTP in CA1 Region of the Hippocampus
P4 dose-dependent changes in LTP were examined with same
concentrations as above. LTP induced by 100H high-frequency stimulation.
When long-term potentiation was induced by high-frequency stimulation (HFS)
following perfusion of P4, fEPSP slope values were significantly decreased in the
P4-treated slices at the highest concentrations that were tested (10
-7
and 10
-6
M).
At 10
-7
M P4 following HFS, fEPSP slope values were significantly decreased by
111% ± 3.4% compared to vehicle, 130% ± 3.6%. ( P < 0.007); at 10
-6
M P4
following HFS, fEPSP slope values were 119% ± 3.4% (P < 0.05) compared to
vehicle (130%). Hippocampal slices treated with P4 at the highest concentrations
examined exhibited a pronounced, persisting, and significant decrease in LTP as
measured by population fEPSP slope recordings (Figure 2.2).

37
Figure 2. 1 P4 regulates synaptic basal transmission on CA1 region

Among different P4 concentration examined, only 10
-6
M of P4 significantly
reduced synaptic basal transmission by 94.8±1.67% (P<0.05) compared to aCSF
perfused control (100%±1%).

P4 and Long-Term Depression
To assess long-term depression in hippocampal slices perfused
with P4 at the four different concentrations used in the above experiments, there
were no significant changes in LTD magnitude between any of the P4 groups
studied compared to vehicle baseline control values. In contrast to its effect on
LTP, the same concentrations of P4 did not affect LTD (Figure 2.3).

38
Figure 2. 2 P4 effect on hippocampal synaptic plasticity; LTP

LTP recording with vehicle and P4; 100Hz of HFS induced LTP in CA1 region of
hippocampus. fEPSP’s amplitude after HFS in the presence of 10
-7
M and 10
-6
M
of P4 were significantly decreased, and showed prolonged decay.

Intracellular Experiments: P4 and GABAAR Mediated Current
10
-6
M of P4 decreased baseline synaptic transmission and magnitude of
LTP in CA1 rat hippocampus. This experiment was exclusively done by Dr.
Garnik Akopian to test the electophysiological effect of P4 on GABAA receptor-
mediated current of individual CA1 hippocampal pyramidal cells. Intracellular
sharp electrodes in dSEVC mode were used in the presence of NMDA and non-
NMDA glutamate receptor antagonists, 50 μM APV and 10 μM CNQX,
39
respectively. Under these experimental conditions, the isolated currents were
proven to be GABAA receptor-mediated, since they were reversed at near 0 mV
of holding potential and were completely blocked by the GABAA receptor
antagonist picrotoxin (100 μM). In two of five cells that were recorded, P4 at 10
-6

M increased the GABAA receptor-mediated current. Representative current
recorded before and during P4 application are shown in Figure 2. In conclusion,
P4, at concentrations greater than 10
-6
M significantly decreases both basal
transmission and LTP, while having no effect on LTD. The result of this study will
be utilized for following experiments in determining P4 concentration.

40
Figure 2. 3 P4 had no significant effect on LTD in CA1 of hippocampus

LTD recordings with vehicle and P4 administration.
1Hz of LFS induced LTD. There was a tendency of increased LTD with
10
-9
M of P4, however, all P4 concentrations did not significantly affect on
LTD

41
Figure 2. 4 P4 effect on basal transmission and synaptic plasticity; LTP and LTD
and IPSC recordings

(Top) fEPSP slope average value during last 5 minutes of basal
transmission, LTP and LTD were compared in vehicle and P4 at different
concentrations (10
-9
, 10
-8
, 10
-7
, and 10
-6
M). 10
-6
M of P4 significantly affect
on basal transmissionand LTP, but not LTD.
(Bottom) 10
-6
M of P4 increased inhibitory post-synaptic currents (IPSCs)
mediated by GABAA in two among five recorded hippocampal cells.

42
Discussion

Demonstration of immediate neurophysiological changes on the timescale
of minutes in hippocampus by application of P4 may provide the insight to
interpret and connect previous studies with E2 and P4. Although profound effects
of excitatory glutamatergic receptors were established on synaptic plasticity,
especially LTP, there are much less direct evidences for the action of GABAergic
receptors on activity dependent synaptic plasticity. This study suggests that
short-term exposure to neuroactive steroid, P4, may be a useful model in
elucidating its role in synaptic transmission, plasticity as well as the underlying
mechanism for role of GABAA receptors in altered synaptic transmission. Several
fundamental actions of P4 on synaptic transmission were found in this study. P4
with an effective concentration, rapidly attenuated both basal synaptic
transmission and significantly decrease LTP in CA1 field of rat hippocampus. P4
did not completely inhibit the LTP but reduced the amount of LTP. On the other
hand, P4 induced no change in LTD followed by low-frequency stimulation
suggesting inhibitory effect of P4 on synaptic transmission in hippocampus may
be increased by high-frequency stimulation. Although it is unclear how P4 or its
metabolite regulate the expression of GABA
A
R subunits and action, for a long
time, a reduction of GABA-mediated inhibition has been considered as the
primary factor for increased EPSPs and synaptic LTP(Abraham, Gustafsson et al.
1987). A number of recent studies have provided ample evidence to explain a
role for local steroid metabolism in regulating the effect of neurosteroids on
synaptic GABA
A
receptors(Belelli and Herd 2003; Puia, Mienville et al. 2003). P4
43
or its metabolites were shown to facilitate the interaction between inhibitory
neurotransmitter GABA, and the GABAA receptor in nongenomic way (Lambert,
Belelli et al. 2003). Moreover, GABAergic inhibitory interneurons have been
demonstrated to regulate synaptic transmission efficiently at hippcampal
pyramidal cells (McBain and Fisahn 2001). In the CA3 region, GABAergic
disinhibition increased synaptic efficacy, similar to NMDAR dependent effects
(Schneiderman, Sterling et al. 1994). Multiple studies reported that changes to
extrasynaptic GABA
A
receptors are mediated by P4 metabolites, but not via a
direct interaction with PR in rodents (Wei, Faria et al. 2004; Chadda and Devaud
2005; Maguire and Mody 2007). These results may imply the extensive and
efficient P4 mediated synaptic changes, as neurosteroids are synthesized

in
hippocampal principal output neurons, not interneurons (Agí s-Balboa, Pinna et al.
2006). A PR antagonist (RU486), or the neurosteroid synthesis inhibitor
(finasteride), were used to determine the changes in GABAA expressions and
functions associated with the estrous cycle. Finasteride administration prevented
GABA
A
R subunit expression in the neuronal membrane, however RA486 had

no
significant effects on GABA
A
R subunit regulation. This suggests that GABAA-
mediated synaptic regulation

is dependent on in vivo or in vitro P4 levels in
hippocampus, not affected by the activation

of PR. However, other neurosteroids,
such as corticosteroid also induces changes in GABA
A
receptor in rat
hippocampus (Orchinik, Weiland et al. 1995). Taken together, our intracellular
experimental result is consistent with other studies in the aspect of facilitating the
inhibitory or reducing cellular excitability through GABAR-mediated or the other
44
factors. Electrophysiological P4 regulation on synaptic transmission and LTP
strongly suggests the involvement of P4 in the processes underlying learning and
memory in both humans and animals.

45
Chapter 3: Effects of Estrogen and Progesterone Interaction on Synaptic
Transmission and Synaptic Plasticity in CA1 Region of Rat Hippocampus

Chapter 3 Abstract
17 β-estradiol (E2) and progesterone (P4) mediate their biological effects on
cellular processes through direct gene regulation as well as non-genomic actions
and rapid stimulatory effects by activating variety of signal transduction pathways.
Hormonal studies have well characterized the molecular and cellular effects of
E2 or P4 alone in the brain using animal models, while the effects of interactions
between E2 and P4 were not well investigated. In the central nervous system
(CNS), E2 has been shown to facilitate cellular excitability, which is associated
with anxiety, cognition, sensory, or motor response. P4 generally attenuates
these E2 actions (Belelli, Bolger et al. 1989; Frye and Scalise 2000). These
effects can change depending on the type and dose of steroid compound used
and the regions of the brain. This implies that controversial outcomes from HT
can be attributed to the interactions of E2 and synthetic P4, which differ from the
ones in premenopausal women. Therefore, progestin should not be considered
merely as a necessary addition to E2 for the protection against uterine cancer,
rather it can potentially be utilized as a beneficial component for postmenopausal
HT. These two hormones’ interaction can be very rapid (second to minutes) or
gradual through structural and synaptic changes in the CNS. In the present study,
acute physiological effects of combining E2 and P4 on synaptic transmission and
plasticity were investigated in CA1 region of rat hippocampus. 10
-6
M of P4
46
significantly reduced E2-induced cellular excitability in basal transmission and
also attenuated LTP level. However, the effect in LTP was not significant. This
implies that estrogen overrides P4 inhibition in LTP, yet P4 reduces efficacy of
estrogen in basal transmission. Therefore, P4 may act as a fine regulator of
cellular excitability in the hippocampus.
Introduction

P4 is thought to stabilize the neuronal hyperexcitability through inhibition
of glutamate receptors and potentiation of GABARs (Smith, Waterhouse et al.
1987; Smith 1994; ROOF and HALL 2000). Previous research has shown that P4
counteracts the effects of estrogen in many different organ systems. However, it
is possible that estrogen and progesterone may work synergistically in certain
situations. In the present study, we hypothesize that progesterone can attenuate
or strengthen the effects of estrogen on synaptic transmission and plasticity. In
the hippocampus, during the transition of proestrus cycle (when E2 levels are
high) to estrus cycle (when P4 levels are high) P4 facilitates the decaying of
increased dendritic spines, which was caused by E2 in CA1 region (Murphy,
Segal et al. 2000). Also P4 was shown to inhibit estrogen-mediated mossy fiber
development into the molecular layer of the dentate gyrus (Woolley and McEwen
1993). In cultured neurons, estrogen can induce an increase in BDNF protein
both by genomic and non-genomic mechanisms (Solum and Handa 2002) while
progesterone decreases estrogen-induced up-regulation of BDNF levels
(Bimonte-Nelson, Nelson et al. 2004). Behaviorally, administration of
47
progesterone and its metabolite, allopregpregnanolone (APα), dramatically
impairs spatial learning and memory in rats by activation of GABAA receptors in
the hippocampus (Johansson, Birzniece et al. 2002). In pathology, a recent study
found that estrogen attenuated β-amyloid accumulation in an AD transgenic
mouse model, and this effect was abolished by progesterone co-administration
(Carroll, Rosario et al. 2007). Although studies with animal models have
supported the neuroprotective effects of E2 and P4 separately, we have
inconsistent results regarding the effects of HT on cognitive functions in humans.
This could have resulted from insufficient understanding of the interactions
between E2 and P4. Limited experimental paradigm and difficulty to mimic the
natural hormonal fluctuations within physiological level are the main barriers to
understand these interactions.
Normal learning and memory in the hippocampus are largely the results of
the intricate and precise balance between excitatory glutamatergic and inhibitory
GABAergic neurons. The electrophysiogical effects of E2 and P4 are partially
attributed to the changes in cellular excitability, which are potentiation and
depression. E2 has excitatory effects by enhancing cellular responses to the
glutamatergic neurotransmitter (Smith, Waterhouse et al. 1987; Wong and Moss
1994) and induces rapid synaptic transmission at excitatory synapses in the CNS.
Massive Ca
2+
influx through NMDAR is considered as a critical contributor to
synaptic plasticity. E2 has been found to increase the number of NMDAR (Smith
1989; Gu and Moss 1996; Rudick and Woolley 2003) and non-NMDAR in distinct
brain regions (El-BakriWong, Thompson et al. 1996). Therefore, E2 induces both
48
structural and functional changes at excitatory synapses. E2 increases NMDAR
mediated synaptic input and neuronal sensitivity to excitatory synaptic input in
the hippocampus, which was blocked by an NMDA antagonist (Murphy and
Segal 1996). Glutamate receptor-mediated neurotoxicity or disturbance of
glutamate metabolism were proposed to cause neuronal death in many
neurodegenerative diseases such as Alzheimer’s disease (Gasic and Hollmann
1992). In OVX rat brain, E2 treatment significantly restored and increased NMDA
binding density in stratum radiatum and stratum oriens of CA1 and dentate gyrus,
whereas P4 had no effect (El-Bakri, Islam et al. 2004). Another study
investigated that E2 had no effect on AMPA binding density in the hippocampus
(Cyr, Ghribi et al. 2000). They also found that P4 and combination of P4 and E2
decreased NMDA but not AMPA binding in the frontal cortex; however, they
found no significant changes in the hippocampus (Cyr, Ghribi et al. 2000).
In addition to excitatory glutamatergic regulation of E2 and P4, excitability of
hippocampal neurons can be affected by inhibiting GABAA receptor. As
previously introduced, P4 facilitates GABA release, GABAA receptor’s expression
and inhibitory function in the hippocampus. However, P4 action on GABAA
receptor was suggested to be dependent on the circulating levels of E2. E2
treated rats showed increased GAD mRNA levels in CA1 pyramidal layer, but E2
plus P4 treatment reversed

the E2-induced increase in GAD mRNA in CA1
(Weiland 1992). An in situ hybridization study found that, P4 and
allopregnanolone, a metabolite of P4, suppressed mRNA levels of GABAA
receptor subunit α1 in the CA2, CA3, and the DG subfields of the hippocampus in
49
E2 pretreated OVX rats (Weiland and Orchinik 1995). On the other hand, P4
increased mRNA levels for the subunit γ2 in the CA1, CA2, and CA3 regions of
the hippocampus, but only in animals which did not receive the E2 treatment. E2
alone had no significant effect on the expression of GABAAR; however, it affected
P4-induced changes in GABAAR expression. These results suggest that effects
of E2 and P4 on cellular excitability in the hippocampus can be dynamically and
mutually variable. An in vitro study found that 10
-8
M P4 perfusion, in non-
tetanized OVX rat hippocampal slices, had no significant effect in basal
transmission in CA1 region of OXV rat (Edwards, Epps et al. 2000). However,
allopregnanolone significantly reduced the fEPSP amplitude. After repeated
100Hz of HFS for the induction of epileptiform activity, allopregnanolone had little
effect, while P4 significantly depressed both population spike and fEPSP
amplitudes in slices from OVX rats. In the OVX rats pre-treated with E2 in vivo,
P4 did not have the same inhibitory effects. This suggests that excitatory E2
effect on hippocampal neurons diminishes the responsiveness to P4. In the
present study, E2 was not used for in vivo priming before the in vitro P4
treatment. In vivo E2 treatment induces involvement of nuclear E2 receptor for
genomic actions, such as regulation of transcription of gene, which ultimately
modify neurotransmission (McEwen and Alves 1999; Maggi, Ciana et al. 2004).
These effects are relatively slow, needing hours to several days to occur. In
addition, E2 was suggested to regulate P4 synthesis in the brain (Micevych and
Sinchak 2008) and significantly increase the number of PRs in CA1 region of
OVX rats (Parsons, Rainbow et al. 1982). E2 also was found to increase the P4
50
and membrane P4 receptor binding in female rats (Tischkau and Ramirez 1993).
Given all these changes that the interaction between E2 and P4 can cause,
simultaneous application of combination of E2 plus P4 upon acute hippocampal
slices can contribute to elucidating the optimal HT.

Materials and Methods

Animals
Female ovariectomized Sprague-Dawley rats (3-4 month old, Harlan) were
used in this study. All animal treatment and procedures were performed in
accordance with protocols approved by the University of Southern California
Institutional Animal Care and Use Committee. After ovaryectomy surgery, 10-14
days of recovery period was allowed. There was one rat per cage. The rats were
kept in plastic cages under a 12-h light/dark cycle and provided with food and
water, at a temperature of 25°C. The animals were transferred from the vivarium
on the day of experiment. All efforts were made to minimize the number of
animals used and their suffering.

In vitro Hippocampal Slices Preparation and Solutions
The rats were anesthetized with isoflurane and, as soon as their eyes
were closed, they were instantly decapitated using a guillotine. After the skull
was opened, the brain was rapidly removed and kept for 2-3 minutes in icy and
slightly oxygenated mixed solution of half aCSF and half sucrose solution. The
cerebellum and the forebrain were removed. Lastly, one third of the dorsal cortex
51
was cut parallel to the longitudinal axis. The remaining block of brain was glued
on the vibratome stage, with the dorsal surface down, and the caudal end toward
the razor, which was fixed at 10
º
. 400μm-thick coronal hippocampal slices with
surrounding cortical tissue were cut using a vibratome (Series 1000). The slices
were then transferred to a holding chamber, which was submerged in
oxygenated aCSF at room temperature. ACSF solution consisted of 124 mM
NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2.4 mM
CaCl2, and 10 mM glucose. After one hour of equilibrium period, activity of
hippocampal cell was expected to be restored and stabilized. The slices were
transferred to a Haas-type interface recording chamber and continuously
perfused with oxygenated (95% O2 and 5% of CO2) aCSF at a rate of 1.5 to 2
mL/min at 31-32°C. The surface of the slice was also oxygenated with combined
gas of 95% O2 and 5% CO2.

Hormone Preparation
E2 and P4 were kept in the refrigerator dissolved in 100% ethanol and
diluted with aCSF for each experiment to make the final concentration of 100pM
and 10
-6
M, respectively. All solutions were oxygenated prior to perfusion to the
hippocampal slice.

Extracellular Recordings for Basal Transmission
Field excitatory post-synaptic potentials (fEPSPs) were recorded from the
dendrite of pyramidal cells in stratum radiatum of CA1, using a glass pipette filled
52
with aCSF (2–3 M resistance) in response to current stimulation from a
stimulating electrode (twisted nichrome wires, 50 μm), which was positioned 1-2
mm from recording electrode in stratum radiatum. Axoclamp 2A DC amplifier and
Cyber-amp 320 (Axon Instruments) were used for signal magnification, filtered at
6KH, and then sampled at 20 kHz. Input/output curves were generated by
increasing stimulus intensities from minimum of 20 to 150 μA in increments of 10
μA. To examine how P4 affects E2-induced cellular excitability, 10
-6
M of P4 was
perfused for 30 minutes after 30 minutes of 100pM of E2 application. E2
perfusion was continued with P4. In addition to serial application, combination of
100pM of E2 and 10
-6
M of P4 was perfused at the same time for 30 minutes after
15 minutes of baseline. ACSF, E2 and P4 were administered separately for 30
minutes after 15 minutes of baseline to be compared with each other.

Extracellular Recordings for LTP and LTD
LTP was induced at the same stimulation intensity for baseline, with two
trains of 1 sec of 100-Hz pulses separated by 20-sec intervals. Recording
continued for 30 min after HFS. The LFS stimulation train for LTD induction
consisted of 900 pulses delivered at 1 Hz for 15 min. HFS or LFS was delivered
after 30 minutes of hormone application; aCSF(control), 100pM of E2, 10
-6
M of
P4, or combination of E2 and P4 followed by 15 minutes of baseline period with
aCSF. The same hormone application was perfused after induction of LTP and
LTD. Only one titanic stimulus or LFS was given to each slice.

53
Data Analysis
fEPSP slopes during the entire recording period were evaluated as the
percent of averaged fEPSP slope values recorded during the 15min baseline.
Basal transmission for serial hormone application was compared with the
average of the last five minutes of fEPSP slope values induced by E2 application,
as well as the average of the last 5 minutes of fEPSP slope values induced by
the addition of P4. E2, P4, and E2 plus P4 effects on baseline transmission were
compared with last five minutes of baseline fEPSP slope values, and last five
minutes of fEPSP slope values induced by each hormone application. To assess
HFS and LFS, the last 5minutes fEPSP slope values of the infusion period (aCSF,
E2, P4, and E2 plus P4) were compared with the last 5minutes of the HFS or
LFS period. Statistical significance between groups was evaluated by unpaired
two-tailed t-tests. Results are expressed as means ±S.E.M. All tests were
performed with a critical significance level of P<0.05, using SPSS software
running on a Dell computer.

Results

Acute Electrophysiological Effects of E2 and P4 on CA1 Basal Transmission
Consistent with the previous studies (Wong and Moss 1992; Woolley
1998), 100pM of E2 in vitro application significantly increased fEPSP’s slope in
baseline transmission (112%±3%, P<0.01) compared to the vehicle (99%±4%) in
CA1 region of hippocampus. 10
-6
M of P4 administration significantly decreased
basal transmission (93% ± 4%, P<0.05) (Figure 3.1).
54
Acute E2 effect on synaptic plasticity; LTP
In addition to its effects on basal synaptic transmission, E2 rapidly
increased the capacity for synaptic plasticity in CA1. LTP was significantly
increased (152%±10.52%, P<0.01) compared to the vehicle (129%±.7.23%)
(Figure 3.5). HFS enhances synaptic strength so that the presynaptic cell can
excite the postsynaptic cell with more reliability and efficacy. HFS delivered in the
presence of E2 produced greater LTP in hippocampal slices (Foy, Xu et al. 1999).
Thus, E2 is believed to be related to learning and memory formation. The
increase in synaptic strength, which lasts for many hours, is vital in the rise of
postsynaptic Ca
2+
during the HFS (Malenka, Kauer et al. 1988). Recent studies
suggest that the underlying mechanism for E2-induced LTP enhancement is due
to the increase in NMDAR-mediated synaptic transmission (Bi, Broutman et al.
2000; Fugger, Kumar et al. 2001). Taken together, E2 is involved in this
nongenomic mechanism and plays a role in the modulation of excitatory synaptic
transmission in the hippocampus.

55
Figure 3. 1Basal synaptic transmission; vehicle, E2, E2+P4 and P4

Vehicle, 100pM of E2, 10
-6
M of P4 and E2 plus P4 solution was separately
perfused during 30 minutes after 15 minutes of baseline.
E2 significantly increased basal transmission (112%± 3%, P<0.05) compared to
baseline with aCSF (99%± 4%). E2 plus P4 administration generated smaller
fEPSPs slope during last five minutes (103%± 5%, P<0.05) than administration of
E2 alone. However, it was significantly greater than administration of P4 alone
(93% ± 4%, P<0.05).

\

56
Acute E2 Effect on Synaptic Plasticity; LTD
LTD has not been extensively investigated and analyzed compared to LTP.
However, when considering the role of E2 in memory processing, the effect of E2
on LTD is essential. In the present study, a train of low-frequency stimulation
produced a reduction in synaptic efficacy, and the perfusion of E2 markedly
suppressed LTD (95.47%±3.25%, P<0.05) compared to the vehicle (86%±2.19%)
in CA1 (Figure 3.6).

Figure 3. 2 P4 suppressed E2-induced increase in basal transmission

After 15 minutes of baseline with aCSF perfusion, 100 pM of E2 were
administered for 60 minutes. E2 significantly increased basal transmission
(113%± 3%, P<0.05) compared to baseline with aCSF (98%± 4%). P4, which
was administered 30 minutes later than E2, significantly attenuated basal
transmission (101%±2%, P<0.05).

57
Effect of in vitro E2 plus P4 Application on CA1 Basal Transmission

To investigate if P4 can modify E2-induced changes in basal transmission,
P4 was administered after 30 minutes of E2 application. Average of last five
minutes of EPSP slopes of E2 administration was compared with those of P4
administration. E2 significantly increased basal transmission (113%± 3%, P<0.05)
compared to baseline with aCSF (98%±4%). P4 gradually attenuated fEPSPs
slopes to cause a significant reduction during the last 5 minutes (101%±2%,
P<0.05). (Figure 3.2) E2-induced enhancement of fEPSP slope was significantly
decreased by P4 application, suggesting that P4 attenuates cellular excitability
and counteracts E2 effects on basal transmission. Finally, both E2 and P4 were
administered simultaneously for 30 minutes after the baseline. The fEPSPs
slopes were significantly smaller (103%± 5%, P<0.05) than administration of E2
alone and greater than administration of P4 alone (93% ± 4%, P<0.05). However,
it had no significant difference with fEPSPs from vehicle application (Figure 3.1).

58
Figure 3. 3 Basal transmission changes by serial E2 and P4 administration

Last 5 minutes of fEPSPs slopes in the presence of vehicle, E2 and P4 were
averaged. E2 significantly increased basal transmission, whereas P4
attenuated E2-induced enhancement in basal transmission (n=6)

Figure 3. 4 Comparison of basal transmission; vehicle, E2, E2 plus P4 and P4

E2 significantly increased basal transmission (112%± 3%, P<0.05) compared to
baseline with aCSF (99%± 4%). E2 plus P4 administration generated smaller
fEPSPs slope during last five minutes (103%± 5%, P<0.05) than administration of
E2 alone. However, it was significantly greater than administration of P4 alone
(93% ± 4%, P<0.05).

*
*
*
*
59
Figure 3. 5 LTP recordings with vehicle, E2, E2plus P4 and P4 administration

For each recording, after 15 minutes of baseline with vehicle (aCSF), aCSF,
100pM of E2, 10
-6
M of P4, and E2 plus P4 were separately administered for 60
minutes.100hz of HFS induced LTP.E2 markedly enhanced LTP (152% ± 7%,
P<0.05) while P4 significantly decreased LTP (121%±4%, P<0.05) compared to
vehicle (135%±7%) E2 plus P4 also increase LTP (149%±6%, P<0.05) compared
to vehicle, however, it was not significantly different from LTP with E2 alone.

Effect of Combination of E2 and P4 on Synaptic Plasticity; LTP
When E2 and P4 were administered together 30 minutes before the
induction of LTP, average LTP amplitude of last 5 minutes after HFS was still
significantly higher (149%±6%, P<0.05), than vehicle (135%±7%). E2 plus P4
administration slightly reduced LTP amplitude compared to LTP with E2 alone
(152%±7%); however, it was not significant. P4- alone treatment produced
significantly reduced LTP (121%±4%, P<0.05) compared to control (Figure 3.5).
60
Effect of Combination of E2 and P4 on Synaptic Plasticity; LTD
E2 plus P4 administration had no significant effect on LTD (85% ±6%)
compared to vehicle (86%±2%), however it significantly suppressed LTD than P4
only administration (79% ±6%, P<0.05) (Figure 3.6).

Figure 3. 6 LTD recordings with vehicle, E2, E2plus P4 and P4 administration

1Hz of LFS during 15 minutes induced changes in synaptic plasticity inCA1
region of hippocampus.Vehicle, E2, E2 plus P4 and P4 was separately perfused
after 15 minutes of baseline.E2 plus P4 administration did not significantly
change LTD (85%±6%) compared to vehicle (86%±2%). However, it significantly
reduced LTD compared to P4-only administration (79% ±6%, P<0.05).

61
Figure 3. 7 Comparison of LTP and LTD with different in vitro hormone
applications

Enhancement in LTP with both E2-only and E2 plus P4 administration was
statistically significant compared to vehicle as well as P4. P4 did
not obviously attenuate or improve E2’s potentiation effect on LTP. Similarly,
E2 and E2 plus P4 significantly suppress the LTD.

*
*
*
*
62
Discussion

E2 Facilitates Basal Transmission and LTP in CA1
Our results are consistent with other studies which have reported the
predominant electrophysiological effect of E2 on CA1 pyramidal neurons;
depolarization, triggering action potential, and excitatory effect (Wong and Moss
1991). E2 has been shown to increase the dendritic fEPSP amplitude of the CA1
pyramidal neurons, recorded extracellularly in hippocampal slices from male and
female animals (Teyler, Vardaris et al. 1980; Van Haaren, Van Hest et al. 1990).
The E2-induced potentiation is stereospecific in multiple studies as it rapidly
induces the increase in basal transmission, which is reversible after the removal
of E2. Previous studied showed that both non-NMDA and NMDA receptors can
positively modulate the potentiation of glutamate receptor mediated synaptic
response (Malenka and Siegelbaum 2001). Although storage capacity, which
comes from the changes in intrinsic excitability, is considerably smaller than that
for synaptic plasticity, involvement of intrinsic excitability in learning and memory

is essential. In fact, changes in E2-induced intrinsic

cellular excitability may
suggest specific functional purpose. Increased excitability in dendrites largely
affects synaptic integration and plasticity by priming

a group of neighboring
synapses so that they undergo

LTP more readily (Johnston, Christie et al. 2003).
This may influence subsequent

learning (Zhang and Linden 2003). Similarly,
changes in cellular excitability of the neuron was suggested as an alternative
form of memory storage (Kim and Linden 2007).

63
P4 Attenuates E2-enhanced Basal Transmission in CA1
In the present study, the effect of E2 plus P4 was investigated in CA1 subfield of
hippocampus, since these two neurosteroids have been shown to produce
distinct electrophysiological effects in this brain region. Our results showed that in
vitro P4 application attenuates E2-induced increase in basal transmission in CA1
OVX rat hippocampal slices. This suggests that P4 may decrease neural
recruitment and excitability in the brain. P4 showed a rapid responsiveness after
E2 priming and significantly depressed excitability in baseline transmission in
CA1 subfield. This is consistent with the previous in vivo study, which examined
the changes in depressive effect of P4 metabolite on fEPSP in CA1 region of rat
hippocampal slices (Landgren and Selstam 1995). Landgren and Selstam found
that the inhibitory effect of P4 metabolite on the population spike was rapidly
amplified when it was administered after perfusion of E2 on rat hippocampal
slices. Therefore, the depressive effect of P4 on cellular response in
hippocampus can be dependent on estrous cycle, thus endogenous levels of E2.
In non-tetanized hippocampal slices, P4 counteracts E2 action by several
possible mechanisms. First, P4 was demonstrated to inhibit presynaptic
glutamate release from nerve terminals in the CA1 field (Taubøll, Ottersen et al.
1993). Second, P4 reduces Ca
2+
uptake in synaptosomes profoundly in the
hippocampus of OVX rats (Nikezic, Horvat et al. 1988). This effect may be
important because excessive Ca
2+
uptake during electrical stimulation was found
to be related to CA1 pyramidal cell damage in rats (Andine, Jacobson et al.
1992). Neuronal Ca
2+
uptake is reduced by both NMDA antagonist and non-
64
NMDA antagonist, indicating the activation of glutamate receptor is necessary for
Ca
2+
regulation. Third, P4 decreased the magnitude of fEPSP by potentiation of
GABAA response, which was demonstrated by the reduced cellular excitability by
GABAA modulators in tetanized hippocampal slices (Rafiq, Zhang et al. 1995).
These mechanisms could be responsible for the P4-induced reduction in
extracellular responses.

E2 plus P4 Induced No Significant Change in LTP Compared to E2-alone
P4 does not significantly affect LTP when it is administered with E2.
In contrast to basal transmission, when the same concentration of P4 was
perfused with E2, it had only limited effect on LTP compared to LTP induced in
the presence of E2 alone. This result may suggest that E2 effect is more potent
than P4 in regulating synaptic plasticity. Other cellular, biochemical mechanisms
related to LTP may be involved. E2 facilitates synaptic plasticity; however,
simultaneously it may decrease cellular responsiveness to P4.

65
Chapter 4: Effect of Cyclic Hormonal Treatment on LTP in CA1 Region of OVX
Rat Hippocampus

Chapter 4 Abstract

An important aspect in hormonal study is its cyclic changes, which are
associated with many variations in neurophysiology and cellular signal pathways,
leading to important effects on cognition. Potential clinical advantages of cyclic
hormone treatment compared to continuous treatment for postmenopausal
women should be examined with regard to neuronal impact. In the present study,
hormone levels including E2 and P4 in OVX rats were manipulated with
implantation of E2 and P4 pellets, which release these hormones in a controlled
manner. This study attempted to provide a sophisticated model to investigate
hippocampal physiological changes across the estrous cycle. Previous studies
have emphasized the importance of cyclic changes on E2-regulated synaptic
plasticity in hippocampus (Warren, Humphreys et al. 1995; Bi, Foy et al. 2001;
Thomas and Huganir 2004). With this background, the present study examined
the effects of cyclic HT on LTP in CA1 region of OVX rats. We had four groups of
different HT; OVX without HT, OVX plus prolonged E2 administration, OVX plus
cyclic P4 administration, OVX plus both prolonged E2 and cyclic P4
administration. Our results showed that rats which received cyclic HT with
prolonged E2 and cyclic P4 administration had greater LTP amplitude than
others. This suggests that cyclic treatment is effective in improvement of
cognitive function compared to prolonged HT without cyclic changes. P4-alone
66
treated OVX rats showed the least LTP; however, in vitro E2 application
significantly raised the LTP. This result implies that the effect of E2 on LTP may
be dependent on the levels of both P4 and E2.

Introduction

Although a number of risks and benefits of HT have been identified, the
precise effects of HT on cognitive function have proved difficult to define.
Women’s Health Initiative Memory Study (WHIMS) aimed to provide definitive
answers about cognitive benefits and risk of post menopausal HT, yet it has
baffled clinicians, researchers and the public with results contradictory to
previous studies demonstrating benefits of HT in cognition and memory. Current
HT is not able to fully imitate the complex molecular and physiological changes
which occur during the natural estrous cycle. However, many studies have found
that age, different estrogen and progestin compounds, dose, pattern (cyclic vs
continuous), timing (interval from menopause to HT initiation) and different routes
of administration can be extremely important in HT. Another critical factor which
may influence the outcome of HT is the inclusion of a progestin. In fact, oral
estrogen-alone had been the main HT, especially in the United States (Belchetz
1994). Conjugated equine estrogen is the most widely used estrogen. However,
E2-alone administration was demonstrated to increase the risk of endometrial
hyperplasia and

cancer by long-term clinical data. Currently most women who
have not undergone hysterectomy are treated with a synthetic progestin in
addition to estrogen.

The reason for using synthetic progestins in spite of many
67
debates about their safety and detrimental effects, is that absorption of P4, a
natural progesterone, is very poor. Oral administration of P4 is considered to
have clinical problems because it is rapidly metabolized and removed by liver,
causing sleepiness (ARAFAT, HARGROVE et al. 1989).
In the present study, cyclic treatment with E2 and P4 was administered to
mimic the pattern of hormonal secretion in premenopausal woman. Cyclic
hormonal treatment is considered beneficial because continuous exposure of
brain to E2 for an extended period of time was demonstrated to decrease ER
levels, which results in reduced neuronal sensitivity to E2 (Blaustein 1993;
DonCarlos, Malik et al. 1995; Brown, Scherz et al. 1996; Toran-Allerand, Singh et
al. 1999). In CA 1 region of OVX rat hippocampus, P4 treatment for a short
period (2 hours) significantly enhanced dendritic spine density in E2-treated
animals (Gould, Woolley et al. 1990). In contrast, when animals were treated with
P4 for a prolonged time (18 hours), dendritic spine density was decreased to the
level of OVX animals (Woolley, Gould et al. 1990). This implies that the duration
of P4 exposure is crucial in regulation of neuronal changes. A large body of
studies have demonstrated superior efficacy of cyclic hormone treatment
compared to tonic treatment by showing enhanced cognitive performances in
rats (Markowska and Savonenko 2002) . Cognitive improvement is the most
accurate indicator of efficacy of HT on brain function. Effect of cyclic E2
replacement without a progestin on cognitive function was tested with aged OVX
monkeys (Rapp, Morrison et al. 2003). They found that this cyclic treatment
68
reversed the age-related cognitive impairment in spatial working memory and
recognition memory, indicating that E2 is largely involved in cognitive function in
primates. In contrast with beneficial effect of E2-alone treatment, multiple clinical
studies reported that combination HT with estrogen-progestin therapy using MPA
blunted the beneficial association between E2 and cognition (Bimonte-Nelson,
Singleton et al. 2004). Further, CEE+MPA treatment did not improve cognition
and produced worse performance in verbal fluency (Grady et al., 2002),
implicating this combination can cause damage in cognition (Rice, Graves et al.
2000).
Recently, P4-only treatment, not MPA, was also shown to decrease
spatial working memory in aged OVX rats (Bimonte-Nelson, Singleton et al.
2004). They also reported that OVX in aged rats improved spatial working
memory than intact aged rat. This contradicts the previous studies, which
demonstrated that the hormone deprivation generated hippocampal- dependent
cognitive decline in young female rats (Daniel, Roberts et al. 1999; Bimonte,
Hyde et al. 2000). Many studies found that aged intact rats had significantly
higher levels of P4 than intact young female ones, whereas E2 level remained
stable (Huang, Steger et al. 1978; Wise and Ratner 1980). High levels of P4
were shown to have negative influence on cognition by many clinical studies
(Freeman, Weinstock et al. 1992; Brett and Baxendale 2001). Therefore, in aged
animal, persistently elevated P4 levels, compared to E2, may have a negative
impact on cognition. In middle-aged OVX rats, both the chronic treatment of E2
69
(0.25mg/60 days) and cyclic E2 (10-µg injection) improved spatial reference
memory. However, the addition of P4 readily reversed E2-induced beneficial
effect (Bimonte-Nelson, Francis et al. 2006).
In summary, E2 has shown to cause a reliable enhancement in spatial
learning in young OVX rats as well as aged female mice by regulating memory
and neural plasticity in the hippocampus (El-Bakri, Islam et al. 2004; Fernandez
and Frick 2004). On the other hand, P4 or its metabolites may impair
hippocampal-dependent learning in male rats and worsen the age-related
impairment of learning and memory in advanced mouse model (Flood, Farr et al.
1995; Johansson, Birzniece et al. 2002). With E2 plus P4 treatment, different
results were produced. Weekly injections of E2 plus P4 enhanced spatial
performance of aged OVX rats (Gibbs 2000). Compared to behavioral effects of
HT, little is known about electrophysiological effects of cyclic HT in the
hippocampus. This unresolved important issue, which merits further research,
provides the rationale for the present study. This study attempts to elucidate the
underlying cellular and neurobiological mechanisms of E2 plus P4 by mimicking
cyclic HT in young adult OVX rats.
Materials and Methods

Animals
16 3-month old female ovariectomized Sprague-Dawley rats (Harlan)
received HT according to the protocol. They were double-blinded, and there were
four animals per each group. OVX only group (OVX), E2-only (OVX+E2), E2 plus
70
cyclic P4(OVX+E2&P4) and P4 only group (OVX+P4) were prepared through 60
days of HT. Animals in OVX group did not receive any hormonal therapy for 60
days. 7 days after OVX surgery, animals in E2-only group underwent pellet
insertion surgery, and E2 was subcutaneously released for a total amount of 0.48
mg over 60 days. 7 days after OVX surgery, animals in P4-only group were also
implanted with the P4 pellet. On the 20th day after the implantation, the pellet
released 50 mg of P4 for 10 days. Then it stopped the P4 release for 20 days
and released another 50 mg of P4 for 10 days. E2 plus cyclic P4 group received
the same P4 treatment with P4-only group in addition to the continuous E2
administration of 0.48mg for 60 days (Figure4.1). All animal treatments and
procedures were performed in accordance with protocols approved by the
University of Southern California Institutional Animal Care and Use Committee.
Rats were kept in plastic cages under a 12-h light/dark cycle and provided with
food and water, at a temperature of 25°C. The animals were transferred from the
vivarium on the day of experiment. All efforts were made to minimize the number
of animals used and their suffering.

71
Figure 4. 1 Cyclic HT protocol used in the present study

OVX
7 d
20 d
10 d
E2
P4
20 d
10 d
P4
4 groups: Ovx; E2; P4; E2+P4
4 animals/group

OVX group; received no HT during whole period of time. E2-group; 7 days after
OVX surgery, E2 was released subcutaneously (0.48mg /60 days). P4 group;
10 days of P4 treatment (50mg/10days) was repeated twice during indicated time
period as shown in the diagram. E2 plus P4 group; continuous E2 treatment
during 60 days combined with two times of cyclic P4- treatment indicated above.

In vitro hippocampal slices preparation and solutions
The rats were anesthetized with isoflurane and, as soon as their eyes
were closed, they were instantly decapitated using a guillotine. After the skull
was opened, the brain was rapidly removed and kept for 2-3 minutes in icy and
slightly oxygenated mixed solution of half aCSF and half sucrose solution. The
cerebellum and the forebrain were removed. Lastly, one third of the dorsal cortex
was cut parallel to the longitudinal axis. The remaining block of brain was glued
on the vibratome stage, with the dorsal surface down, and the caudal end toward
the razor, which was fixed at 10
º
. 400μm-thick coronal hippocampal slices with
surrounding cortical tissue were cut using a vibratome (Series 1000). The slices
were then transferred to a holding chamber, which was submerged in
oxygenated aCSF at room temperature. ACSF solution consisted of 124 mM
NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2.4 mM
72
CaCl2, and 10 mM glucose. After one hour of equilibrium period, activity of
hippocampal cell was expected to be restored and stabilized. The slices were
transferred to a Haas-type interface recording chamber and continuously
perfused with oxygenated (95% O2 and 5% of CO2) aCSF at a rate of 1.5 to 2
mL/min at 31-32°C. The surface of the slice was also oxygenated with combined
gas of 95% O2 and 5% CO2.
Hormone Preparation
E2 was kept in the refrigerator dissolved in 100% ethanol and diluted with
aCSF for each experiment to make the final concentration of 100pM. All solutions
were oxygenated prior to perfusion to the hippocampal slice.

Extracellular Recording
Field excitatory post-synaptic potentials (fEPSPs) were recorded from the
dendrite of pyramidal cells in stratum radiatum of CA1, using a glass pipette filled
with aCSF (2–3 M resistance) in response to current stimulation from a
stimulating electrode (twisted nichrome wires, 50 μm), which was positioned 1-2
mm from recording electrode in stratum radiatum. Axoclamp 2A DC amplifier and
Cyber-amp 320 (Axon Instruments) were used for signal magnification, filtered at
6KH, and then sampled at 20 kHz. Input/output curves were generated by
increasing stimulus intensities from minimum of 20 to 150 μA in increments of 10
μA. For a 15-min baseline, at 30-second intervals, pulses of 0.1 millisecond
duration were delivered through the stimulating electrode to generate fEPSPs. To
73
examine the LTP differences between the groups, HFS was given after 30
minutes of baseline. LTP was induced at the same stimulation intensity for
baseline, with two trains of 1 sec of 100-Hz pulses separated by 20-sec intervals.
Recording continued for 30 min after HFS. In addition, E2 effect on LTP for each
group was investigated by administration of 100pM of E2 after 15 minutes of
baseline followed by HFS.

Data Analysis
Each fEPSP slopes were expressed as the percent of averaged fEPSP
slope values recorded during the 15min baseline before HFS. To estimate LTP
values, averages of fEPSP slopes collected during the last 5 minutes after HFS
were compared with the average of fEPSP slopes of last five minutes before the
HFS. Statistical significance between groups was evaluated by unpaired two-
tailed t-tests, and results are expressed as means ±S.E.M. All tests were
performed with a critical significance level of P<0.05, using SPSS software
running on a Dell computer.

74
Results

LTP Comparison Between Different Hormone Treated Animal Groups
Results showed that the OVX animals treated only with P4 produced the
lowest LTP amplitude (110 % ± 5%) which was significantly different with OVX
(124%± 3%,P<0.05) and E2 plus cyclic P4 group (135%± 5%, P<0.05). OVX and
E2 treated animals showed similar modest LTP.; There was no significant
difference between these two groups. E2 and cyclic P4 treatment animals
showed significantly larger amount of LTP (138%±5%, P<0.05) than OVX and E2
treated groups (P<0.01) (Figure 4.2).

E2 Effect on LTP in Hormone Pretreated Groups
100pM of E2 perfusion significantly enhanced LTP in CA1 region of
hippocampus prepared from all groups. OVX groups (127% ± 5%, P<0.05) and
E2 treated groups (131% ±7%, P<0.05) showed relatively smaller increase in
LTP. The slices from E2 plus P4 groups also enhanced LTP; (152% ± 4%,
P<0.05). With E2, only P4-treated animals showed the largest enhancement in
LTP (142% ± 4%, P <0.05) (Figure 4.3 and 4.4).

75
Figure 4. 2 LTP from different HT groups; OVX, E2, E2 plus P4, and P4

100Hz of HFS induced LTP for each group; OVX animals treated with E2 plus
cyclic P4 generated the largest fEPSPs slope during last 5 minutes after HFS
(138 %±5%), which was significantly different with other three groups. P4-only in
vivo pretreatment produced the lowest LTP amplitude (110 % ± 5%) which was
significantly different with OVX (124%± 3%) and E2 plus P4 group (P<0.05). OVX
and E2 (118%± 4%) treated animals showed similar modest LTP without
significant difference.

76
Figure 4. 3 E2 effect on LTP in hormone pretreated groups

100pM of E2 was administered to hippocampal slices from each groups; OVX
groups and E2 treated groups showed relatively smaller increase in LTP; (127%
± 5%, p<0.05) and (131% ±7%, p<0.05). Slices from E2 plus P4 groups also
enhanced LTP; (152% ± 4%, p<0.05). Only P4-treated animal showed significant
enhancement in LTP amount indicating (142% ± 4%, p<0.05).

77
Figure 4. 4 Comparison of LTP of each group before and after in vitro E2
application

E2 significantly enhanced LTP in each group; P4 only treated group
showed the largest enhancement in LTP.

Discussion

Tonic and Long-period of E2 Treatment Had No Effect on LTP
The present study revealed several important factors which may modulate
the effects of hormones including the dose of E2 for tonic treatment and the
presence of P4 for cyclic hormone regimens. E2-treated animals showed no
difference in the amount of LTP compared to OVX animals. This is not the
consistent effect we have observed in vitro E2 administration on hippocampal
slices. A possible reason for this may be the E2 dosage. E2 dose (0.48mg/60
days) used in this study corresponded to the endogenous E2 level of
approximately 60pg/ml indicated as serum tests. This is much higher circulating
E2 levels than the night of proestrus in female rats (Woolley 2007). A previous
study found that neuroprotective effects of E2 in hippocampus is dependent on
*
*
*
*
78
the dose and the timing of its administration (Chen, Nilsen et al. 2006). They
found that low E2 exposure resulted in neuroprotection in rat hippocampal
neurons; however, high doses of E2 induced neurodegeneration by generating
neuronal toxicity. Similarly, high-dose E2 with or without P4 treatment had no
effect on spatial reference memory task, whereas low-dose E2 improved the
performance (Bimonte-Nelson, Francis et al. 2006). Despite the fact that many
studies have demonstrated E2-induced positive effect on the hippocampus-
dependent learning, some research showed no benefits or even impairment due
to E2 replacement therapy. A previous study found that long-term E2 treatment
(more than 5 weeks), which resulted in endogenous E2 level of (35±5 pg/ml), had
no significant in spatial learning in OVX young rats (Singh, Meyer et al. 1994).
Another study also demonstrated that the high plasma E2 levels are associated
with poorer spatial learning, while medium levels of E2 are associated with better
performance in young rodents (Galea, Kavaliers et al. 1995). In addition to the
dose of E2, the exposure duration may exert an important role. High doses of E2
enhanced spatial memory and significantly increased both the dendritic spine
density and the number of spine synaptic contacts on CA1 pyramidal neurons in
OVX rats, which corresponded to behavioral enhancement after only 4 hours of
exposure (MacLusky, Luine et al. 2005). Lack of E2 benefits in this study may
have been due to the prolonged exposure of high doses of E2. These results hint
at the complexity of E2 effects on cognition and suggest that different E2 dosage
and exposure period can be important factors in HT for postmenopausal women.
79
Highest LTP in Animals Treated With E2 and Cyclic P4

We also found that in vivo treatment of E2 and cyclic P4 treatment showed
the greatest LTP among different groups. This suggests that P4 may change the
effects E2 has on LTP, although the extent or direction of P4 action on E2 effects
is not clear. The current study attempted to assess how cyclic hormone therapy
affects LTP; however, it is difficult to make a clear interpretation about how P4
and E2 physiologically and functionally interact. Multiple studies found that P4
counteracts the E2 effects. The hippocampal neuronal connectivity, represented
by dendritic spine density (Gould, Woolley et al. 1990; Woolley and McEwen
1992), as well as biochemical and behavioral experiments have suggested that
P4 attenuates the effects of E2 (Bimonte-Nelson, Nelson et al. 2004; El-Bakri,
Islam et al. 2004). Other work has shown that E2’s neuroprotective effect was
facilitated when P4 was co-administered in the hippocampal neuron culture by
regulating MAPK activation, while MPA did not have the same effect (Nilsen and
Brinton 2002). In addition, when the rats received in vivo E2 injection at day 3
and 4 after OVX and received P4 treatment 4 hours before the in vitro
experiment, it showed significantly enhanced Ca
2+
current in CA1 pyramidal
neurons compared to OVX rats without HT and OVX plus only E2 treatment
(Joels and Karst 1995). E2 by itself only slightly increased Ca
2+
current in CA1
pyramidal neurons. This previous study may explain our results because
increased Ca
2+
influx is critical in heightened LTP amplitude. Collectively these
findings suggest that P4 may have different neural effects than E2 and that P4
can modulate E2's effects in some circumstances. Few in vitro studies so far
80
have directly compared hormone replacement therapies with and without P4.
Results from our study may add to the previous findings identifying P4 as a
therapeutically crucial factor that can affect the neural and cognitive outcome of
HT.
E2 Administration Significantly Enhanced LTP in P4- only Treated Rats
CA1 hippocampal slices prepared from the animal group which received
only P4 treatment showed the least amount of LTP. However, acute E2
administration reversed this phenomenon. Rats which received cyclic P4
treatment were examined to have approximately 500ng/dL with complete
depletion of E2. E2 plus cyclic P4 treated rats had endogenous hormone levels
of 70ng/ml of E2 and 400ng/dL approximately, and had the greatest LTP.
Considering this, in vito E2 application may play a major role in causing similar
effect as shown in E2 plus cyclic P4 group. In the present study, baseline
hormonal status can have a great impact in E2’s action on LTP. A previous study
also reported that the effects of P4 on behavioral performance varied according
to the estrous cycle in young rats (Dí az-Véliz, Urresta et al. 1994). In addition,
many studies demonstrated that endogenous and exogenous E2 administration
can produce different P4 actions (Edwards, Whalen et al. 1968; Rodier 1971).
Little is known about the mechanism of how E2 recovered the impaired LTP by
the P4 treatment; however, research into the neural and specific cognitive effects
of P4 alone and with E2 will provide valuable information to understand the
combined effects of E2 and P4. Outcomes of HT research may depend on
numerous factors not yet taken into account including baseline hormone levels,
81
different dosages and types of HT, different behavioral tasks, onset of HT, and
treatment duration. Moreover, molecular mechanisms behind genomic and
nongenomic actions of ERs and PRs, are highly related to the divergence effect
of HT. Collectively, identifying the impact of various elements of HT, individually
and in combination of E2 and P4, would be an efficient and optimal approach to
understand the findings which seem inconsistent and contradictory.

82
Chapter 5: Estrogen and Progesterone Effects on Hippocampal Synaptic
Transmission and LTP in 3xtg-AD Mouse Model

Chapter 5 Abstract

An abrupt loss of E2 and P4 after menopause has been linked to the
cognitive decline and neurodegenerative disorders like AD. The previous studies
of hormonal effects on AD have suggested the beneficial clinical implication of
HT for the prevention and treatment of this particular neuropathology (Davies and
Maloney 1976; Greene 2000; Carroll, Rosario et al. 2007; Henderson, Espeland
et al. 2007). In this study, a novel triple transgenic model of AD mouse (3xtg-AD
mouse) was used to examine the effects of E2 and P4 on the cellular activity in
CA1 hippocampus. This advanced animal model was developed to mimic the
human AD pathologies in the brain including progressive generation of Amyloid-β
(Aβ) containing plaques and tau neurofibrillary tangles (Oddo, Caccamo et al.
2003). Acute neuronal changes in the hippocampus in this animal model induced
by the application of E2 and P4 may provide an insight to correctly interpret the
previous studies regarding the role of HT in cognition.

Introduction

Neurodegenerative diseases related to aging have been recognized as a
critical problem to both individuals and the society with an increasing elderly
population. The most common of these, AD, causes problems with memory and
cognitive functions, and even changes personality (Price and Sisodia 1998). AD
83
slowly destroys the brain function through a series of changes, which begin with
a minor memory loss and progress to forgetfulness of recent events, impaired
judgment, confusion, inability to speak, restlessness and eventually death (Nicoll,
Wilkinson et al. 2003). The incidence of AD is considerably higher for women
than men, suggesting the depletion of E2 and P4 after menopause may be a
cause for the greater risk. However, therapeutic solutions remain scarce. A few
short-term observational studies showed that for women with AD, patients who
received HT had milder signs of dementia compared to those who did not receive
HT (Henderson, Watt et al. 1996; Asthana, Baker et al. 2001). However, clinical
studies by Women’s Health Initiative Memory Study (WHIMS) (Shumaker,
Legault et al. 2003) reported contradictory results and questioned the efficacy
and necessity of HT. WHIMS was a clinical trial with long-term follow-up and a
large sample size (4894 of women aged 65 or older), which reported that HT
significantly increased the risk of AD and had no effect on the prevention of mild
cognitive impairment.
The major pathological features of this disease are cerebral β-amyloid
peptide (Aβ) plaque, dystrophic neurites in neocortical terminal and prominent
neurofibrillary tangles in medial temporal-lobe structures, along with loss of
neurons and white matter and inflammation. In addition, AD reduces neuronal
synaptic efficacy (Selkoe 2001) by reducing cortical and hippocampal synaptic
density, which is strongly related to memory impairment in the early stage of the
disease (Terry, Masliah et al. 1991). The synaptic loss in AD was examined with
AD-model mice which develop plaques (Larson, Lynch et al. 1999; Walsh,
84
Townsend et al. 2005). Basal transmission LTP is impaired in plaque-developing
AD mice with perfusion of Aβ peptide to hippocampal slices (Larson, Lynch et al.
1999). This is attributed to the failure to release the presynaptic
neurotransmitters and the decrease in postsynaptic glutamatereceptor-mediated
current. Intraneuronal Aβ deposition decreases surface expression of NMDA
receptor and causes endocytosis of NMDAR, leading to synaptic dysfunction in
neuronal cultures from mice bearing amyloid precursor protein (Snyder, Nong et
al. 2005).
There have been many studies on the potential neuronal effects of E2 and
P4 using different animal models which are genetically programmed to express
neurodegenerative pathologies including AD (Shi, Panickar et al. 1998; Lee and
Trojanowski 1999; Gotz, Chen et al. 2001; Lewis, Dickson et al. 2001; Molikova,
Bezdickova et al. 2007). A sophisticated triple-transgenic model (3xTg-AD),
which develops AD pathology, was created (Oddo, Caccamo et al. 2003). In the
present study, these 3xTG-AD mice were used to more closely resemble human
models. The 3xTg-AD mice were originally created to develop age-dependent
neuropathology of both β-amyloid plaques and neurofibrillary tangles composed
of tau protein (Oddo, 2003). These mice carry three human familial AD genes of
PS1M146V, APPSwe, and tauP301L. In 6-month-old mice, extracellular Aβ
deposits appear in the cortical regions and the hippocampus, and neurofibrillary
tangles are seen from hippocampus to neocortex from the age of 12 months.
LTP is significantly impaired as they get older, and there are learning and
memory deficits in spatial and contextual tasks (Billings, 2005). Billings and
85
colleagues found that 4-month-old 3xTg-AD mice showed mild cognitive
impairment (MCI) in memory retention, not learning deficit. Recent studies with
OVX 3xtg-AD mice found that subcutaneous E2 treatment prevented the
development of Aβ accumulation in the entire hippocampus and significantly
improved the impaired hippocampus-dependent cognitive performance (Carroll,
Rosario et al. 2007). Yet E2 had no effect on tau hyperphosphorylation, whereas
P4 significantly reduced tau hyperphosphorylation in CA1 and subiculum. They
also found that P4 treatment by itself had no effect on Aβ accumulation; however,
when P4 was combined with E2, it reduced the beneficial effects of E2 on Aβ
accumulation. To better understand the conflicting effects of HT, the present
study examined the acute electrophysiological effects of E2 alone and in
combination with E2 and P4 on CA1 region of 3xTG mouse. We speculated that
consistent with previous studies, E2 may facilitate excitability in basal
transmission and enhance LTP while P4 is expected to work against E2. In
contrast, E2 or P4 may not significantly affect on synaptic transmission or LTP.
Also, E2 was found to act more strongly and efficiently on healthy cell (BRINTON
2005), implying the importance of sensitivity of ER or initiation timing of HT. If
these findings are applied to 3xTg-AD mice, degenerated hippocampal cells from
Aβ accumulation and tangles may not properly respond to E2 or P4. In addition,
these animals with AD genes may have reduced number of ERs and PRs, as
well as impaired sensitivity to E2 and P4. Electrophysiological studies with the
3xTg-AD model may provide a more direct examination of the role of E2 and P4
in AD pathology.
86
Materials and Methods

Animals
3, 6 and 12-month old male 3xtg mice were used in this study. All animal
treatments and procedures were performed in accordance with protocols
approved by the University of Southern California Institutional Animal Care and
Use Committee. There were 3 to 4 mice per cage. The mice were kept in plastic
cages under a 12-h light/dark cycle and provided with food and water, at a
temperature of 25°C. The animals were transferred from the vivarium on the day
of experiment. All efforts were made to minimize the number of animals used and
their suffering.

In vitro Hippocampal Slices Preparation and Solutions
3xtg-AD mice were anesthetized with isoflurane and, as soon as their
eyes were closed, they were instantly decapitated using a guillotine. After the
skull was opened, the brain was rapidly removed and kept for 2-3 minutes in icy
and slightly oxygenated mixed solution of half aCSF and half sucrose solution.
The cerebellum and the forebrain were removed. Lastly, one third of the dorsal
cortex was cut parallel to the longitudinal axis. The remaining block of brain was
glued on the vibratome stage, with the dorsal surface down, and the caudal end
toward the razor, which was fixed at 10
º
. 400μm-thick coronal hippocampal slices
with surrounding cortical tissue were cut using a vibratome (Series 1000). The
slices were then transferred to a holding chamber, which was submerged in
oxygenated aCSF at room temperature. ACSF solution consisted of 124 mM
87
NaCl, 3 mM KCl, 1.25 mM NaH2PO4, 1.3 mM MgSO4, 26 mM NaHCO3, 2.4 mM
CaCl2, and 10 mM glucose. After one hour of equilibrium period, activity of
hippocampal cell was expected to be restored and stabilized. The slices were
transferred to a Haas-type interface recording chamber and continuously
perfused with oxygenated (95% O2 and 5% of CO2) aCSF at a rate of 1.5 to 2
mL/min at 31-32°C. The surface of the slice was also oxygenated with combined
gas of 95% O2 and 5% CO2.

Hormone Preparation
E2 and P4 were kept in the refrigerator dissolved in 100% ethanol and
diluted with aCSF to make the final concentration of 100pM and 10
-6
M,
respectively. All solutions were oxygenated prior to perfusion to the hippocampal
slice.

Extracellular Recording
Field excitatory post-synaptic potentials (fEPSPs) were recorded from the
dendrite of pyramidal cells in stratum radiatum of CA1, using a glass pipette filled
with aCSF (2–3 M resistance) in response to current stimulation from a
stimulating electrode (twisted nichrome wires, 50 μm), which was positioned 1-2
mm from recording electrode in stratum radiatum. Axoclamp 2A DC amplifier and
Cyber-amp 320 (Axon Instruments) were used for signal magnification, filtered at
6KH, and then sampled at 20 kHz. Input/output curves were generated by
increasing stimulus intensities from minimum of 20 to 150 μA in increments of 10
88
μA. For a 15-min baseline, at 30-second intervals, pulses of 0.1 millisecond
duration were delivered through the stimulating electrode to generate fEPSPs. To
examine how E2 and E2 plus P4 regulate LTP, 100p

M E2 or combination of E2
and P4 was perfused for 15 minutes after baseline, and HFS was given for LTP
induction. LTP was induced at the same stimulation intensity for baseline, with
two trains of 1 sec of 100-Hz pulses separated by 10-sec intervals. Recording
were continued for 30 min after HFS.

Data Analysis
fEPSP slopes during entire recording period, were evaluated as the
percent of averaged fEPSP slope values recorded during the 15min baseline. To
assess HFS and LFS, the last 5minutes fEPSP slope values of the infusion
period (aCSF, E2, P4 and E2 plus P4) were compared with the last 5minutes of
the HFS or LFS period. Statistical significance between groups was evaluated
by unpaired two-tailed t-tests. Results are expressed as means ± S.E.M. All tests
were performed with a critical significance level of P<0.05, using SPSS software
running on a Dell computer.

Results

Normal LTP in 3 and 6month-old 3xtg mice
There was no significant difference in LTP between 3 and 6month-old
mice, they all showed normal LTP( 127%±5% and 138%±6%) compared to wild-
89
type mice. However, 12 month-old mice showed significantly smaller LTP (111%
±5%, P<0.05).
E2 significantly Enhanced LTP in All Age Group (3, 6, 12 month) of 3xtg-AD Mice
In vitro E2 administration significantly increased LTP (164%±7%, P<0.01)
in 3-month old 3xtg-AD male mice compared to the vehicle (127%±5%).
E2 plus P4 administration also enhanced LTP (167%±8%, P<0.05) compared to
the vehicle. However, there was so significant difference between LTP from E2-
treated and E2 plus P4-treated hippocampal slices consistent with the outcomes
from the 3 month OVX rats. (Figure 5.1)

90
Figure 5. 1 LTP of 3month 3xtg-AD male mice with presence of vehicle, E2 and
E2 plus P4

100pM of E2 were administered to the hippocampal slices from 3 month- old
3xtg-AD male mice. After 15 minutes of baseline, E2 was perfused for 15
minutes before the HFS, and continuously for 30 minutes. Average of the last 5
minutes of fEPSPs slopes was calculated for LTP comparison. E2 significantly
increased LTP (164%±7%, P<0.01) in compared to vehicle (127%±5%). E2 plus
P4 administration also enhanced LTP (167%±8%, P<0.05) compared to vehicle.
There was no significant difference in LTP between these two groups.

91
Figure 5. 2 LTP of 6month 3xtg-AD male mice with presence of vehicle, E2 and
E2 plus P4

LTP recordings of 6-month old 3xtg-AD-male mice; E2 significantly increased
LTP (153%±5%, P<0.05) compared to the vehicle (138%±6%). However, E2 plus
P4 did not show a statistically significant increase in LTP (149%±7%) compared
to the vehicle.

There was no evident decrease in LTP from 6-month old 3xtg-AD mice. Rather, it
showed slightly increased LTP compared to that of 3-month old 3xtg-AD mice.
E2 also significantly increased LTP (153%±5%, P<0.05) compared to the vehicle
(138%±6%) in 6-month old 3xtg-AD mice. Unlike 3 month old, E2 plus P4 did not
show a significant difference in LPT (149%±7%) with vehicle and E2 only
administration, meaning P4 attenuated E2 effect on LTP. Although the effect was
92
less potent than in 3-month old 3xtg-AD mice, E2 appeared to enhance synaptic
plasticity in 6-month old 3xtg-AD mice, (Figure 5.2).

Figure 5. 3 LTP of 12month 3xtg-AD male mice with presence of vehicle, E2 and
E2 plus P4

LTP in CA1 region of 12-month old 3xtg-AD mice was significantly reduced (111%
±5%, P<0.05) compared to 3 and 6-month old 3xtg-AD mice. In vitro E2
administration significantly enhanced LTP (129%±6%, P<0.05), whereas E2 plus
P4 did not have any effect (114%±4%).

12-month old 3xtg-AD mice showed significantly impaired LTP (111% ±5%,
P<0.05) compared to 3 and 6-month old 3xtg-AD mice, indicating that the
synaptic strength and efficacy may be reduced with the development of AD
pathology in the hippocampus. E2 significantly enhanced LTP (129%±6%,
93
P<0.05) compared to vehicle and E2 plus P4 (114%±4%). E2 plus P4 did not
show significant effect compared to vehicle (Figure 5.3).

94
Figure 5. 4 LTP comparison between different age groups (3,6,and12 month old
3xtg-AD male mice ) with application of vehicle, E2 and E2 plus P4.

(A) LTP in each age group without any hormone application was compared.;
3 and 6month-old 3xtg-AD mice showed normal LTP, on the other hand,
12month-old 3xtg-AD mice showed significantly impaired LTP.
(B) In 3month-old 3xtg-AD mice, both E2 and E2 plus P4 significantly
potentiate LTP compared to vehicle.
(C) In 6 month-old 3xtg-AD mice, E2 application induced significant
enhancement in LTP compared to vehicle. However, there was no
significant difference in LTP with E2 application and E2 plus P4 application.
(D) In 12 month-old 3xtg-AD mice, LTP was noticeably impaired compared.
Only E2 application significantly recovered LTP compared to vehicle and
E2 plus P4. P4 significantly suppressed E2 effect on LTP.

95
Discussion
Our data confirmed the E2 effect in the improvement of LTP in the animal
model with AD pathology and with different ages; 3, 6 and 12 months old. 3xtg-
AD mouse model has offered a great opportunity to investigate the relationship
between HT and neuronal degeneration caused by both plaques and tangles.
These AD pathologies affect learning, memory, and synaptic plasticity in an age-
dependent manner. In the present study, 3-month old- 3xtg-AD mice showed no
deficit in basal synaptic transmission and LTP. This is consistent with the
previous behavioral study, which reported normal performance on the spatial
learning and contextual fear conditioning in 2-month-old 3xTg-AD mice (Billings,
Oddo et al. 2005). They also demonstrated that the earliest cognitive deficit in
memory retention starts to appear with the accumulation of intrareuronal Aβ at
the age of 4 months. The intraneuronal Aβ immunoreactivity and extracellular Aβ
deposits were found in the frontal cortex of 6-month old 3xTg-AD mice, but no
extracellular Aβ was detected at this age (Oddo, Caccamo et al. 2003).
Our electrophysiological results showed no impairment in LTP in CA1
region in 6-month-old 3xtg-AD mice, suggesting hippocampus remained
unaffected, consistent with previous findings. In addition, pyramidal neurons in
CA1 field rapidly responded to acute E2 and E2 plus P4 application and
produced significantly enhanced LTP. This implies that membrane ER receptors
may be activated by E2 administration, and cellular synaptic strength is enough
to maintain the transmission normally. Our in vitro experiment with 9-month-old
96
3xtg-AD mice showed no significant difference in LTP from that of 6-month-old
3xtg-AD mice (Data is not shown). Therefore, 12-month-old 3xtg-AD mice were
used for further studies. By 12 months of age, these mice have extracellular Aβ
deposits in the hippocampus, as well as intraneuronal Aβ staining in the
pyramidal neurons in the CA1 region (Oddo, Caccamo et al. 2003). We found
that 12 month-old 3xtg-AD mice showed significantly smaller LTP despite using
considerably large current stimulation amplitude than the one used for 3 and 6-
month old 3xtg-AD mice. This shows that AD pathology in hippocampus
significantly interferes with the synaptic transmission and plasticity, which can
cause impairment in cognitive functions. Of note, E2 significantly increased LTP
in 12-month-old 3xtg mice, suggesting E2 still had an impact on synaptic
excitability and plasticity in hippocampal neurons. When P4 was administered
with E2, it reduced the effects of E2 on LTP and produced significantly lower LTP
than E2-alone treatment. Therefore, the effects of E2 and P4 seem to be
dependent on the age and neuropathological process. The results of the present
study provide novel findings about electrophysiological effects of E2 and P4 in
the hippocampus with AD pathology. Moreover, they support the previous
assessment of the role of E2 in cognitive decline attributed to neurodegenerative
diseases or dementias in postmenopausal women.
97
Chapter 6: General Discussion and Summary

The outcomes presented in this dissertation demonstrated the effects of
E2 and P4 on synaptic transmission and plasticity in hippocampus. Along with
the well-established favorable neuronal actions of E2, P4 was also considered to
perform a neuroprotective action through diverse mechanisms. Many studies
reported the synergistic or attenuating effects of P4 on E2 actions in the brain in
various aspects; biochemical, cellular, and behavioral changes. These studies
have focused on discovering the mutual regulations of E2 and P4, especially in
terms of their neuroprotective effects and cognitive changes through HT in
animals. However, only a few studies have attempted to demonstrate the mutual
modulations between E2 and P4 on physiological neuronal activity in the
hippocampus, and they have yielded contradictory results.
In the present study, we attempted to elucidate the rapid and nongenomic
electrophysiological effects of E2 and P4 using different animal models. It needs
to reminded that E2 plus P4 effects may appear differently according to
experimental protocols as well as many possible variations, which may exist
without our knowledge. First, we found that 10
-6
M of P4 significantly reduced
cellular excitability, and LTP in CA1 region of OVX rat hippocampal slices. This is
consistent with previous studies which demonstrated that the inhibitory response
of GABA
A
receptor, the primary mediator of fast inhibitory input in the CNS, were
potentiated by P4 metabolites (Kaura, Ingram et al. 2007; Tolmacheva and van
Luijtelaar 2007; Eser, Baghai et al. 2008; Ming-De Wang, Strijmberg et al. 2008).
98
P4-induced decrease in basal transmission in the present study may be
attributed to the direct action of P4 or its metabolite, or both. In the brain,
conversion of P4 to allopregnanolone rapidly takes place (several minutes)
producing the divergent physiological actions of these two neurosteroids (Ciriza,
Carrero et al.). Recent studies found that both P4 and allopregnanolone are
strong neuroprotective agents. They both reduced cellular death in male rats with
frontal cortex injury, shown by retaining better spatial learning ability (He, Evans
et al. 2004; Djebaili, Guo et al. 2005). However, they possess distinct
characteristics in the way they act on PRs as well as GABAARs.
Allopregnanolone does not directly function as a ligand on intranuclear PRs
(Rupprecht, Reul et al. 1993) or membrane PRs (Meffre, Delespierre et al. 2005).
Unlike allopregnanolone, P4 does not directly increase GABAergic transmission
(Ciriza, Carrero et al.).
The present study found that P4 readily attenuated E2-induced cellular
excitability in CA1 region of OVX rat hippocampal slices, but E2-induced
enhancement in LTP was not significantly affected. The effects of E2 appeared
unchangeable in the synaptic plasticity model; LTP and LTD. If GABAA receptor-
mediated synaptic transmission is significantly affected by P4, LTP should be
decreased despite the presence of E2. To explain this, functional importance of
GABAAR in LTP needs to be discussed. Many studies found that GABAAR
synaptic inhibition is critically involved in LTP-induced with high-frequency
stimulation (Davies, Starkey et al. 1991; Collinge, Whittington et al. 1994).
Previously, electrophysiological study demonstrated that in mice with weakened
99
GABAA receptor-mediated fast inhibition, LTP in CA1 region of the hippocampus
was impaired (Collinge, Whittington et al. 1994). This suggests that reduction of
the synaptic inhibition is essential for enhancement of excitability and
maintenance of LTP. Similarly, strengthened GABAA-receptor inhibition was
found more evidently at somata and apical dendrites of pyramidal cells in CA1,
where the GABAergic synapses were heavily located (Stelzer, Simon et al. 1994).
Inhibitory postsynaptic potentials (IPSPs),GABAA receptor conductance, and
GABAA sensitivity were all reduced after titanic stimulation (Otis, De Koninck et al.
1994; Stelzer, Simon et al. 1994). Collectively, HFS appears to enhance the
disinhibitoin of GABAA Rs, and this increases the efficacy of LTP maintenance.
The effects of P4 on GABAA R-mediated synaptic transmission may be masked
by the potentiated disinhibition of GABAARs during the LTP.
In the cyclic HT studies we designed, the genomic and systematic
changes may take place in the brain before the in vitro experiment. Considering
the characteristics of in vitro experiment, which is focused on rapid and instant
cellular changes, administration of E2 and P4 is does not lead to significant
structural changes or genomic processes. Importantly, we used natural
progesterone (P4), not progestins. As mentioned earlier, synthetic progestins
lack the intrinsic physiological benefits of P4, thus they cannot properly work in
the major signal transduction pathways, causing unfavorable side effects.

In the present study, we found that prolonged E2 plus cyclic P4 treatment
generated notably larger LTP in CA1 of hippocampus than other groups. E2 plus
100
cyclic P4 group and E2-only group received the same amount of E2. This
suggests that P4 does not always attenuate the effects of E2 but can dynamically
change E2 action depending on HT treatment variables. Previous in vitro study
found that LTP of female rats was dependent on estrous cycle (Bi, Foy et al.
2001). They measured natural E2 levels across the estrous cycle. E2 level during
proestrus was 53pg/ml and 32pg/ml during diestrus. LTP was induced with HFS
for both groups, and the proestrus group showed significantly higher LTP than
the diestrus group. Our cyclic HT was designed to give chronic E2 treatment (40-
50pg/ml) for 60 days plus two cycles of P4 treatment, so there was no variation in
the E2 level. Therefore, this treatment protocol may not be sufficient to satisfy
natural hormone fluctuations. However, we found that acute E2 application to
hippocampal slices from the group with E2 depletion plus cyclic P4 treatment
significantly enhanced LTP, suggesting ERs remained sensitive to the effects of
E2 on LTP.
To investigate the effects of E2 and P4 in the AD, we used 3xtg-AD
mouse model. Clear deficit in LTP was observed in 12 month-old 3xtg mice, and
E2 treatment led to a substantial increase in LTP, suggesting that neural
plasticity was still present at this age. Collectively, the present data from rodents
consistently indicate that loss of ovarian hormones can cause deleterious effects
upon cognitive functions dependent on hippocampus. Whether E2 regulates
memory and other cognitive functions in postmenopausal women is still under
debate; however, the basic science analyses have demonstrated that E2
regulates cognitive functions through multiple mechanisms. E2 enhanced
101
synaptic efficiency and improved neural circuits through the regulation of
structural changes in several regions of the brain. In addition to E2, P4 also
needs to be considered as an important regulator of neural function. Findings
from an increasing body of research have shown that P4 treatment antagonizes
the neuroprotective effects of E2 (Bimonte-Nelson, Nelson et al. 2004; Rosario,
Ramsden et al. 2006; Carroll, Rosario et al. 2008; Lewis, Orr et al. 2008).
However, this was observed more in old, middle-aged or unhealthy brain with
neurological pathologies. Neural benefits of E2 and P4-based HT are affected by
different treatment methodologies. Therefore, understanding the interactions
between E2 and P4 should is vital to maximize the beneficial effects of HT. I
In summary, this dissertation suggests that 1) P4 can regulate cellular
excitability as well as plasticity in hippocampus 2) P4 rapidly counteracts the
effects of E2 on basal synaptic transmission in CA1 pyramidal cells 3) cyclic
hormone delivery can be more effective than continuous treatment to increase
the benefits of HT 4) E2 and P4 can be therapeutically used for prevention and
relief of neurodegenerative diseases.
102
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Abstract (if available)

Abstract Since the early 1990’s, cognitive influences of estrogen (E2) have been widely studied, especially in the hippocampus, one of the brain regions responsible for learning and memory. Despite substantial evidence of estrogen’s neuroprotective role in animals, the issue of hormone therapy (HT) was complicated and much debate was generated when E2 was combined with progesterone (P4) for clinical use in humans. It has been hypothesized that P4 counteracts the neuroprotective effects of E2. Therefore, elucidating the interactions between these two hormones in the brain is vital. The present study examines the neural physiological effects of estrogen and progesterone on the synaptic transmission and plasticity in CA1 region of different animal models including ovariectomized rat, hormone pre-treated female rat and 3xTG Alzheimer model mice. We found that E2-induced enhancement in basal transmission was decreased by P4, but E2-induced enhancement in long-term potentiation (LTP) was unaffected by P4. In summary, these results support the hypothesis that progesterone may attenuate the estrogen-induced hyperexcitability in pyramidal cells. This study shows estrogen and progesterone at certain concentrations work against each other and may explain the conflicting data on HT. With better understanding of the interactions of E2 and P4 in the future, it may become possible to design the most efficient and successful hormonal therapies.

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Asset Metadata

Creator Kim, Youngkyoung (author)

Core Title Estrogen and progesterone interaction on synaptic transmission and LTP in rodent hippocampus

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Neuroscience

Publication Date 05/05/2010

Defense Date 03/23/2010

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

Tag Estrogen,hippocampus,long-term potentiation,LTP,OAI-PMH Harvest,progesterone

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Thompson, Richard F. (committee chair), Baudry, Michel (committee member), Golomb, Solomon W. (committee member), Swanson, Larry W. (committee member), Walsh, John P. (committee member)

Creator Email youngkki@usc.edu,youngkyoung.kim@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3019

Unique identifier UC1458228

Identifier etd-Kim-3622 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-340078 (legacy record id),usctheses-m3019 (legacy record id)

Legacy Identifier etd-Kim-3622.pdf

Dmrecord 340078

Document Type Dissertation

Rights Kim, Youngkyoung

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