Effects ofEstrogen in the Basolateral
Amygdala ofthe Rat Brain
by
James Arthur Andrews
Submitted in Partial Fulfillment ofthe Requirements
for the Degree of
Master ofScience
in the
Biological Sciences
Program
Youngstown State University
June, 2000
Effects ofEstrogen in the Basolateral Amygdala ofthe Rat Brain
by
James Arthur Andrews
I hereby release this thesis to the public. I understand this thesis will be
housed at the Circulation Desk ofthe University Library and will be
available for public access. I also authorize the University or other
individuals to make copies ofthis thesis as needed for scholarly research.
Signature:
Approvals: ~~, 1L~
Mark D. Womble, Thesis Advisor
alker, Committee Member
~/ -/ ~ it:.-) .c>t.-<-t~ " ~ ~u"-
Robert . LeiPhzr, Committee Member
Peter J. Nasvinsky, Dean of
Date
,61160
Date
~_ .. JI/OC
Date
~L!fttr?
Date
Date
Acknowledgements
Indeed, I am grateful and indebted to all ofthose who contributed to
my works published here. Including all ofthe beneficiaries ofmy gratitude
in one document is impossible, so please forgive me ifI do not list your
name. My friends and family have performed harder than I at times. I love
you all. The amount ofhelp they contributed is beyond quantification.
Additionally, thanks to those who lent me shelter when I had none.
Above all, thanks and praise to the Creator. Without faith, I doubt
that I would have finished this project. You gave the determination,
knowledge, and wit to deal with the relative enormosities encountered in this
project. When I needed motivation, you were there for me.
My Grandfather, Arthur E. Verner is the first non-omnipotent being to
which I must give thanks (not that he is too far from it). When I was unable
to continue, I thought ofall ofthe times he went out ofhis way to make sure
that I was well in difficult times. In times without food, without water, or
without money...he has always come through. Most importantly, his
statement: "Ifyou're gonna start something, finish it!" echoed in my head,
and drove me to research, read, and write to create the thesis contained
within the following pages.
111
Abstract
Clinical evidence has shown estrogen may delay the onset of
Alzheimer's disease and protect against neuronal damage associated with
stroke. Intracellular recordings (current-clamp) were made to characterize
the effects ofestrogen in the basolateral amygdala (BLA) ofthe rat brain.
Excitatory postsynaptic potentials (EPSPs) were elicited by stimulation of
afferents in the external capsule. Estrogen was found to decrease EPSP
amplitude in a rapid (20-30 min) fashion. Similarly, reduction of
spontaneous synaptic activity occurred upon estrogen treatment. EPSP
amplitudes returned to normal within 20 minutes ofestrogen washout. 4 
hydroxy tamoxifen (4-0HT), and estrogen receptor antagonist, prevented the
estrogen-induced decrease in EPSP amplitude, suggesting dependence on an
estrogen receptor. Estrogen treatment had no effect on neuronal input
resistance, accommodation response, resting membrane potential, or action
potential firing frequency. Preliminary data showed no change in inhibitory
postsynaptic potential (IPSP) amplitude, suggesting estrogen might act on
the presynaptic cell. These findings imply that estrogen may be protecting
neurons from excitotoxic injury associated with stroke through the
modulation ofglutamate release.
IV
Table ofContents
Title Page i
Signature and Release ..
Acknowledgements iii
Abstract iv
Table ofContents v
Table ofFigures ..
Introduction 1
Alzheimer's disease 1
Treatment ofAlzheimer's Disease 5
Heredity ofAlzheimer's Disease 7
Estrogen 8
The Genomic Pathway ofEstrogen Action 12
The Estrogen Receptor (ER) 13
Antagonism ofthe ER 15
Non-Genomic Mechanism ofEstrogen Action 17
The Basal Lateral Amygdala (BLA) 20
v
Materials and Methods 25
Results 29
Discussion 67
References 82
VI
Table ofFigures
Figure 1. The Pathway ofestrogen synthesis 11
Figure 2. Stimulating/Recording apparatus 24
Figure 3. Effect ofestrogen on EPSP amplitude 36
Figure 4. Effects ofestrogen on cellular recordings ofEPSPs from BLA
neurons superimposed 37
Figure 4a. EPSPs in control saline and estrogen 37
Figure 4b. BLA EPSP responses under perfusion ofestrogen and estrogen
washout superimposed 38
Figure 5. Indiyidual cellular recordings ofEPSPs from a BLA neuron
presynaptically stimulated via EC 39
Figure Sa. EPSP recordings made in the presence ofcontrol ACSF 39
Figure 5b. EPSP recordings made in the presence of estrogen 40
Figure.5c. EPSP recordings after switching the flow ofbath solution from
estrogen to normal ACSF 41
Figure. 6. Individual cellular recordings oftrain stimulations from BLA
neurons superimposed 42
Figure. 6a. Train stimulations in control ACSF and ACSF containing
estrogen 42
VB
Figure.6b. Superimposed train stimulations in ACSF containing estrogen
and estrogen washout. .. """""" """"" "" "." """ """ " " 43
Figure 7. Individual cellular recordings from a BLA neuron train stimulated
via EC. " """" """"" """""" " " ".. " ".""'"~ 44
Figure 7a. Train stimulation recordings ofmade from a BLA neuron under
train stimulation via EC in control ACSFII"'"''''''''''''''''''''''''''''''''''''''''44
Figure 7b. Train stimulation recordings ofmade from a BLA neuron under
train stimulation via EC in ACSF containing estrogen..""""""".""."" 45
Figure 7c. Train stimulation recordings ofmade from a BLA neuron under
train stimulation via EC after estrogen washout. "".""."""."""".".""." 46
Figure 8. Effect ofthe alcohol vehicle on EPSP amplitude. """""""".""" 47
Figure 9. Individual cellular recordings ofEPSPs from BLA neurons with
the effects ofcontrol, ethanol, and estrogen superimposed. """""""."" 48
Figure 9a. Single BLA neuron EPSP responses from a single cell perfused
with control ACSF and ACSF containing alcohol superimposed. """". 48
Figure 9b. Single BLA neuron EPSP responses from a single cell perfused
with estrogen and ethanol superimposed. "."""".""."".""".""""""""" 49
Figure 1Q. Effect ofestrogen on intracellular recordings ofIPSPs from BLA
neurons superimposed.."""""" """""""." "".""."" " "" 50
V111
Figure lOa. BLA IPSP responses from a single cell perfused with control
ACSF and wash superimposed. """"""",,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,"",,,,,,, 50
Figure 1Ob. BLA neuron IPSP responses from a single cell perfused with
estrogen and estrogen washout superimposed. """"""""""""""""""".51
Figure II. Individual IPSP recordings from a BLA neuron presynaptically
stimulated via EC.."""".. ".. "."""""""."""""...""."".""...".. """",""", 52
Figure 11 a. Recordings ofIPSPs made from a BLA cell under presynaptic
stimulation via EC in control ACSF. """"""""""""""".""""""""""". 52
Figure lIb. IPSPs recordings from a single cell perfused with estrogen. ".53
Figure llc. IPSP recordings made after switching the flow ofbath solution
from estrogen to normal ACSF.."".. "...""".. ".....".""...".....""""".""." 54
Figure 12. Effect ofestrogen on the early membrane resistance. """"""".55
Figure 13. Effect ofestrogen on the membrane resistance measured via late
current. """,""" 56
Figure 14. Effect ofestrogen on voltage response to hyperpolarizing current
.. . 57InJ ectlon .
Figure 15. Effect ofestrogen on resting membrance potential. """"."""".58
Figure 16. Effect ofestrogen on action potential frequency. """"""""""" 59
Figure 17. Effect ofestrogen on the voltage response ofa BLA pyramidal
neuron to an 800 pA depolarizing current. """""""""""""""""""""". 60
IX
Figure 17a. Voltage Trace showing the response ofa BLA pyramidal
neuron in control ACSF 60
Figure 17b. Voltage Trace showing the response ofa BLA pyramidal
neuron in ACSF containing estrogen 61
Figure 18. Effect of4-0RT and the blocking effect of4-0RT on EPSP
amplitude modulation 62
Figure 19. Effects of4-0RT and concurrent perfusion of4-0RT and
estrogen on the early membrane resistance 63
Figure 20. Effects of4-0RT and concurrent perfusion of4-0RT and
estrogen on the late membrane resistance 64
Figure 21. Effects of4-0RT and concurrent perfusion ofestrogen and 4-
ORT on resting membrane potentiaL 65
Figure 22. Effects of4-0RT and concurrent perfusion ofestrogen and 4-
ORT on action potential frequency 66
x
Introduction
Estrogen is a female hormone that appeared to protect neurons from
cell death (Behl et a11995; Goodman, 1996; Green, 1996; Reibel et al.,
2000; Singer, 1996, 1998) and may playa protective role against several
neurological diseases such as Alzheimer's disease (Birge et ai, 1997;
Henderson et ai, 1994, 1997; Kawas et al., 1997), Parkinson's disease
(Bedard et al., 1977), and cerebral ischemia (Hum and Macrae, 2000;
Sawada et al., 2000; Rusa et al., 1999; Fung et al., 1999). Thus,
postmenopausal women have the lowest levels ofcirculating estrogen and
the highest probability ofdeveloping AD. It is currently not known how
estrogen protects individuals from AD, nor is the actions ofestrogen in the
brain fully understood. The present study was undertaken to examine the
actions ofestrogen on neuronal function and synaptic transmission in the
mammalian brain.
Alzheimer's disease
Alzheimer's disease (AD) is a disorder ofthe central nervous system
that involves neuron and synapse loss, the presence ofsenile plaques,
neurofibrillary tangles, and neocortical atrophy (Reviewed by Bondi and
1
Lange, 1998). In 1907, Alois Alzheimer, a German physician and
neuropathologist, first described the morphological symptoms we now know
today as AD. Dr. Alzheimer studied the brain ofa patient who had died
from dementia and found several distinct morphological changes in the AD
brain (Reviewed by Bondi and Lange, 1998). These symptoms presently
affect 50% ofall people over the age of85 and are predicted to affect 14
million people by the year 2050 (Reviewed by Birge et al., 1997). Once
diagnosed, average life expectancy is 7 to 10 years, ofwhich most will be
spent in a long-term care facility. In their final stages, AD patients are
thoroughly debilitated by the neurological effects. AD patients experience a
slow and painful decay ofall senility and eventually die while in a
permanent vegetative state (Reviewed by Bondi and Lange, 1998).
Neurofibrillary tangles and senile plaque formation significantly
contribute to neuron loss, the subsequent decrease in synapses and
neurotransmitter content, and the neocortical atrophy seen in AD patients.
Unfortunately, the neuritic plaques and neurofibrillary tangles associated
with AD can only be found through post-mortem staining. Ifthese
morphological changes are later found in brain tissue taken from a dementia
patient, there is conclusive evidence that the patient suffered from AD
(Reviewed by Bondi and Lange, 1998).
2
Neurofibrillary tangles are associated with the cytoskeleton ofneurons
affected by AD. Neurofibrils, also known as neurofilaments, are long
strands oflinked protein subunits. Neurofibrillary tangles are formed from
neurofibril subunit deformation, and this directly impacts structure and
function ofthe neurofibrils (Kandel et al., , 1995). Transport proteins and
structural support proteins ofneurofibrils lose their delicate organization and
become a chaotic filamentous intertwining structure (Reviewed by Bondi
and Lange, 1998). Neurofibrillary tangles cause death in neurons by
inhibiting the efficient transport ofmaterials throughout the neuron.
Neurons with neurofibrillary tangles eventually die from the lack ofessential
transportation ofcellular components (Kandel et al., , 1995).
Neuritic plaques are found in the areas between neurons ofsuch areas
as the amygdala, hippocampus, and other cortical regions (Reviewed by
Bondi and Lange, 1998). The neuritic plaques are formed by deposits of
insoluble ~-amyloid protein. ~-amyloid is cleaved from amyloid precursor
protein (APP), a soluble protein normally found in the brain. The soluble
form ofB-amyloid is a protease released in response to neuronal injury
where it plays a neuroprotective role. It is believed that an alteration in the
normal metabolism ofthis protein can contribute to the development ofAD
by making the cleaved ~-amyloid protein insoluble and cytotoxic (Chao et
3
al., 1998). Soluble p-amy10id has been shown to be non-cytotoxic in some
cases, and insoluble p-amy10id that forms neuritic plaques has been found to
be neurotoxic to hippocampal neurons in culture (Cotman, et al., 1992; Pike,
et al., 1991).
The toxicity ofp-amy10id may result from its actions as a protease.
After cleavage oftheir substrates, proteases release free radicals as by 
products. Free radicals in solution can oxidize cellular components essential
for a variety ofcellular processes. Free radical production is believed to
cause the toxicity ofthe p-amy10id plaques (Maury, 1995). Under normal
conditions, the soluble form ofB-amy10id peptide is helpful to the central
nervous system, but due to a change in the position ofcleavage from its
precursor protein, APP, the insoluble form ofthe peptide ironically becomes
a detriment to neuronal functioning (Maury CPJ, 1995). The change in
cleavage site is believed to be inherited, and this may underlie the genetic
susceptibility that some families display for early onset ofAD (Chao et al.,
1994).
Psychological signs ofAD include unsteady development ofdementia
and a gradual impairment ofmemory, judgment, and concentration
(Reviewed by Bondi and Lange, 1998). Since AD is the most common
cause ofdementia (Henderson., 1997), all cases ofdementia especially in the
4
elderly have a very high chance ofbeing a symptom ofAD. Dementia in
AD is marked by one or more ofthese disorders: aphasia (problems with
language), apraxia (problems with carrying out motor activity despite having
the physical ability), agnosia (problems with object recognition despite
intact sensory function), and impaired planning, abstracting, and sequencing
(Reviewed by Bondi and Lange, 1998).
AD patients may suffer from depression at early stages ofthe disease
and they may exhibit abnormal fits ofrage as the disease progresses
(Reviewed by Bondi and Lange, 1998). These fits ofrage are closely
associated with other personality changes found in AD. Behavioral
symptoms ofAD probably result from the neurodegeneration occurring in
the limbic structures, such as the amygdala, as well as in higher cortical
areas (Reviewed by Bondi and Lange, 1998).
Treatment ofAlzheimer's Disease
Currently, a suggested means oftherapy and prophylaxis for AD
patients is the administration ofvitamins A, C, and E (Reviewed by Bondi
and Lange, 1998). These vitamins are good antioxidants, which act to
subdue the cytotoxicity of~-amyloid. Vitamin E (a-tocopherol) has shown
5
to playa protective role against the neurotoxic effects of ~-amy10id in
animal models (Jaffe et al., 1994), , and it also shows the potential of
delaying AD due to its strong antioxidant activities (Birge et al., 1997). The
antioxidants absorb the free radicals generated by ~-amy10id, and other
sources offree radicals such as cyclo-oxygenase, a chemical released upon
an inflammatory response. Some functions ofcyclo-oxygenase include
involvement in the production ofprostaglandins and mediation of
excitotoxic cell death via the glutamatergic N-methy1-D-aspartate receptor.
Anti-inflammatory drugs such as indomethicin can reduce the risk of
developing AD by 50% (Birge, et al., 1997).
Several currently prescribed AD medications stop the actions of
enzymes at the axon terminal from cleaving used neurotransmitter. Two of
the most commonly prescribed medications for AD at this time are donezapi1
(Aricept) (Birge et al., 1997) and tacrin (Cognex) (Henderson et al., 1997).
These medications have been reported to slow the onset ofAD symptoms by
inactivating cho1inesterases (Birge et al., 1997). These enzymes are
responsible for breaking down acetylcholine at cholinergic synapses. The
patients receiving donezapi1 experience an increase in the amount of
acetylcholine at synapses (Birge et al., 1997) between intact cholinergic
6
basal forebrain neurons and efferent neurons such as those located in the
basolateral amygdala, hippocampus, and higher cortical areas.
Estrogen has also been suggested as a treatment for AD (Birge et ai.,
(1997). Neuroprotective effects ofestrogen have been implicated in studies
ofcytotoxicity (Behl et ai, 1995: Goodman. 1996; Green et ai., 1996; Singer
et ai., 1996, 1998), and it has been shown that estrogen increases blood flow
to the brain during clinical trials with AD patients (Ohkura et ai., 1994). In
addition, estrogen replacement therapy has been suggested, through multiple
epidemiological studies, to possibly delay AD onset and slow cognitive
decline in patients experiencing AD symptoms (Brenner et ai., 1994).
Heredity ofAlzheimer's Disease
Genetic research has shown that heredity plays a large role in the
likelihood ofindividuals to develop AD. The chance ofdeveloping AD
increases four times ifa first-degree relative has the disease, and it has been
found that 80% ofall AD occurrence is attributed to heredity (Birge et ai.,
1997). The additional 20% may be caused by environmental factors such as
head injuries and chemical insults that may have occurred at a time as far
back as childhood (Birge et ai., 1997). The inherited trait that represents
7
individual susceptibility to AD development is believed to be the ApoE
epsilon-4 allele (Holtzman et al., 1995). ApoE protein is normally released
in response to the neurodegeneration and other neuritic insults occurring in
the brain (Mahley, 1988; Boyles et al., 1989; Boyles et al., 1990; Poirier et
al., 1991)). AD patients have been tested for the mutated ApoE protein. It
has been found that people who have experienced head trauma with the
"bad" ApoE epsilon-4 allele have been found to develop AD at a greater
frequency than those with the wild-type ApoE gene (Mayeux et al., 1995) .
A malfunctioning ApoE protein is not able to help the neurons recover from
injury. Normal functioning ApoE protein helps the injured neurons by
making cholesterol available for cell membrane repair. Correspondingly,
neurons located in areas ofthe brain affected by AD contain a low amount
ofcholesterol, and the delivery ofcholesterol to these regions is markedly
impaired (Mason et ai, 1992).
Estrogen
Estrogens represent a class ofseveral closely related steroid
hormones, ofwhich 17-~-estradiol (estrogen) is the major form synthesized
8
by the ovary (Johnson and Everitt, 1995). Synthetic pathways for estrogen
are numerous (Fig. 1). Aromatase is an important enzyme in this pathway
because it helps to control local estrogen blood levels (Johnson and Everitt,
1995). This enzyme has a wide distribution throughout different tissues in
the body; including brain tissue (Balthazart and Ball, 1998). The
mammalian limbic and hypothalamic regions contain high levels of
aromatase (Balthazart and Ball, 1998). Activity ofthis enzyme is controlled
in the ovarian cells by local blood levels offollicle stimulating hormone
(Johnson and Everitt, 1995). Follicle stimulating hormone and luteinizing
hormone are secreted from the anterior lobe ofthe pituitary gland, and
control the blood levels ofestrogen in a negative feedback loop with blood
levels ofestrogen (Johnson and Everitt, 1995). Aromatase is responsible for
the aromatization oftestosterone into estrogen and the aromatization of
androstenedione into estrone (Balthazart and Ball, 1998). Estrone is an
estrogen easily converted to l7p-estradiol (Johnson and Everitt, 1995).
Thus, the presence ofaromatase in other tissues gives those tissues the
chance to capitalize on an extra dose ofestrogen ifblood levels of
testosterone are at a concentration to permit aromatization. Luteinizing
hormone participates in the regulation ofestrogen synthesis earlier in the
pathway. The levels ofthis hormone modulate the rate ofpregnenolone,
9
formation from cholesterol (Johnson and Everitt, 1995). Pregnenolone is a
steroid precursor for testosterone/estrogen synthesis (Johnson and Everitt,
1995).
10
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
I
(}Hc;b/OH
tfl)/ctj
16ahydroxytesterone
OIl
-o~~
16a hydroxy androstenediol
? ISOm~'i1!14!
oo:n?? . 1111I'" .
IEstramol I
,,~~
')(1 t...l1uO;rt."
ort?
16a hydroxyandrostenedione
5a dihydrotesterone
.. ~ ~
HoJ.hl
Alomata!l4!t
u
~~
~ ?dPo 0
i I
\111?14
$ (=001<Jo '"
II deoxycortisol
IEstrone I
I U1o~ydroxyla!.f !
I I
(\ hydlOlySlefOl1l I
Ot,OK
\ '
~ ("'O 011 ..rI ""I
Cortisol I
~
(~) C:~J
~o ."~~
Pregnanediol
Pregnenolone
lin'
r-----C EJcetate
.
,~,t,~/()HI .,
lJ-J(TJr--
(Hj I' 16a :::ox~ --'
t=Q {=~H ~Il ~ dehydroepiandrosterone1m
~ ~ c&LJ~ ~ ~ ~
HQ HO' ,>.. 110 HO" >..
i
-
Progesterone 17a hydroprogesterone
1 1
C~J (!,')
H(-OH 11(-011
~ #
I I 20a dihydroprogesterone
The Genomic Pathway ofEstrogen Action
Estrogen has traditionally been described as acting on tissues through
a slowly developing and long-lasting mechanism commonly referred to as
the genomic pathway. In this pathway, estrogen influences the cell by
initially binding to an estrogen receptor (ER) , which is found at highest
concentration in the cell nucleus (Li et at., 1997). After becoming activated,
the following sequence ofevents has been reported to occur (Reviewed by
Tsai and O'Malley, 1994). The estrogen-receptor complex can interact with
transcription factors. These transcription factors regulate specific sequences
ofDNA. In the absence ofestrogen, the ER exists bound to heat shock
proteins and remains inactive. After estrogen binding, the receptor
undergoes a conformational change at its ligand-binding domain, and the
receptor becomes activated at protein activating functions. This leads to
dissociation ofthe receptor from the heat shock protein followed by ER
dimerization, the coupling oftwo ERs. To activate the ER, the receptor
must be in the presence ofestrogen or a receptor agonist. Removal ofthe
heat shock proteins through other means is not sufficient for activation ofthe
ER. After the ER dimerizes, the ER is now able to bind to a specific DNA
complex and interact with transcription factors to activate or inactivate the
target gene. Gene activation leads to mRNA production and increased
12
synthesis ofa specific protein that is then responsible for production ofthe
estrogen-induced cellular response. The period between activation ofER
mediated transcription and post-translational modification ofthe final
protein product may involve hours or days .
The Estrogen Receptor (ER)
There are two well-described estrogen receptors, ERa and ERB,
although some evidence points to the possibility ofa third receptor type
(Kuiper et al., 1997). ERa and ERB belong to a superfamily of
steroid/thyroid receptors. Within this nuclear receptor superfamily,
estrogen receptors belong to the largest subgroup that also includes
glucocorticoid receptors, androgen receptors, progesterone receptors, and
mineralocorticoid receptors. There is a high degree ofconservation
amongst these receptors in their hormone-binding and DNA-binding
domains (Kuiper et al., 1997; Tsai and O'Malley, 1994; Wrenn, 1993).
In rat and human neurons, ER and other androgen receptor proteins
have been detected in neurons (Couse et al., 1997) with distributions from
nuclei to far reaching cytoplasmic extensions such as dendrites and axon
terminals (Greco et al., 1998; Puy et al., 1995). In the rat brain, neurons
13
containing ERB and ERa are distributed together in several cortical
amygdaloid nuclei (Puy et al., 1995) and some areas ofthe hypothalamus
(Shughrue et al., 1998; Weiland 1997). ERB, without the presence ofERa,
is broadly expressed throughout the rat brain. Both isoforms ofER may
have different actions in cells because oftheir different DNA binding
domains (Shughrue, et al., 1998). Complex cellular responses can arise
because both ERa and ERB can dimerize with their homologue or
heterodimerize with the other type ofER (Katzenellenbogen et al., 1996).
When ERs bind to DNA, both DNA recognition sites ofthe dimerized ERs
recognize their own specific sequence. When heterodimerization occurs it is
possible to have differences in function due to the differentiation in binding
characteristics ofERa and ERB (Shughrue, et al., 1998).
The ER protein is large protein with a number offunctionally
important motifs along its chain ofamino acids. cDNA evidence indicates
that ERB consists of485 amino acids with a calculated molecular weight of
54,200 Daltons. Isomer homology at the ligand-binding domain ofERs is
about 90%. Homology between DNA binding motifs is 50% (Kuiper, et al.,
1997). The differences between ERa and ERp have developed to allow
different receptors to activate different areas ofthe genome while being
activated by a common hormone.
14
Antagonism ofthe ER
Estrogen replacement therapy increases the incidence ofdifferent
types ofcancer in female reproductive tissues. Extensive research with
uterus and mammary tissue led to the discovery ofantagonists to inhibit
some ofthese side effects ofestrogens. Drugs such as Tamoxifen, ICI, and
Raloxifene competitively inhibit the estrogen receptor, and thus decrease the
chances ofcancer developing in some peripheral tissues (Gradishar and
Jordan, 1997; Jordan, 1998). However, both Tamoxifen and Raloxifene are
known to also have agonistic actions on the ER (Berry and Metzger. 1993;
Ali et al., 1993; Metzger et al., 1995). In addition, the agonistic actions of
these drugs differ from each other. Cellular responses to estrogen are very
complex, and have yet to be completely defined. Understanding how the
separate regions ofthe ER function while initiating cellular changes is
integral to understanding the intricacies ofER antagonism/agonism duality.
Antagonism for the ER is an important part ofmodem medicine. To
have an effective ER antagonist is to have the best ofboth worlds; drugs that
can block the cancer associated with estrogen without reducing the
beneficial actions ofestrogen. It is not unusual for ER antagonists to have
15
more than one action in patients. The selective estrogen receptor modulator
(SERM) tamoxifen is an ER antagonist, which is regularly prescribed for
those considered to be at high risk for developing breast cancer. Chances of
developing breast cancer drop by 45% after tamoxifen treatment (Smigel,
1998). At the same time, bone density increases so that fewer fractures
occur, which is an important benefit ofestrogen replacement therapy.
However, there is an unfortunate increase in endometrial cancer incidence
(Smigel, 1998). This phenomenon ofinactivation/activation or
antagonism/agonism is further exemplified by postmenopausal women
experiencing increases in bone density when receiving the SERM raloxifene
for treatment ofosteoporosis (Black et al., 1983). Understanding how
SERMs work in different tissues and considering their actions on the
individual estrogen receptor types (ERa and ERB) will help us to find clues
that will eventually lead to the finding ofa SERM with the ability to mimic
the positive estrogenic influences but block the carcinogenic responses.
Defining the mechanism by which SERMs selectively activate and
inactivate the ER is key in understanding how these chemicals can be both
an asset and a liability. Activating functions (AF-l and AF-2) are regions of
the ER, which are activated following a specific conformational change by
the ER (Jensen EV et al., 1973). It is this conformational change in the ER
16
that is altered or inhibited by tamoxifen, raloxifene, and similar antagonists
(Jordan, 1998). AF-l is located in the N-terminus ofthe ER (Kumar et al.,
1987). AF-2 is located in the ligand-binding domain (Kumar et al., 1987).
The conformational change needed to activate AF-2 is a folding ofhelix 12
ofthe ER to uncover the AF-2 domain (Shiau et al., 1998). This folding of
helix 12 on the ER is inhibited by ER antagonists, and thus the AF-2 domain
remains inactive (Shiau et al., 1998). This does not mean that the other ER
activating function is inactive. Helix 12 is sterically hindered by an alkyl 
amino-ethoxy side chain ofthe antagonist molecule that is otherwise
structurally similar to estrogen (Jordan, 1984).
Although the AF-2 domain ofthe ER is inhibited by ER antagonists,
the AF-1 region may remain active through mitogen-activated protein kinase
pathways (MAPK) (Kato et al., 1995). When the AF-1 region is activated
by SERMs, it initiates some ofthe same cellular changes as estrogen. Itis
ironic that treatment with antiestrogens invokes some estrogenic responses.
Non-Genomic Mechanism ofEstrogen Action
Until recently, estrogen was thought to exert its action only via the
genomic (nucleic acid! protein synthesis) pathway, a process that requires
17
hours or longer to produce a cellular response. However, rapid effects of
estrogens and other steroid hormones were found in frog and fish oocytes in
the 1980's. (Zakon., 1998) Since these initial reports, additional studies have
shown similar rapid cellular responses to estrogen and androgens in several
tissues, including the brain (Balthazart et al., 1998; Gorzynska and
Handelsman., 1995; Woolley., 1999).
These recent findings ofestrogenic and androgenic actions taking
place in minutes or even seconds indicate that the traditional genomic
pathway is not the only mechanism by which steroid hormones may exert
their actions. The problem we now face is discovering a new pathway by
which estrogen can function in a much shorter time frame.
An example ofthe rapid action ofsteroidal hormones is seen in Sertoli
cells ofthe rat testis (Gorzynska and Handelsman., 1995). Testosterone
treatment causes a rapid (20-40 second) rise in cytosolic calcium levels. An
identical rapid cellular response was also seen during the application of
testosterone that had been conjugated to a large protein. (Gorzynska and
Handelsman., 1995). Since the known steroid receptors are found in the
cytosol and the nucleus, their activation depends upon the steroid penetrating
the cell membrane and reaching their ligand-binding domain. With a protein
attached to the steroid hormone, diffusion across the cell membrane is
18
impaired. Actions such as those described in the Sertoli cells ofmale rats
(Gorzynska and Handelsman., 1995) strongly suggest the presence ofa cell
surface steroid receptor. Since androgen receptors are very closely related to
ER in structure and function, the existence ofandrogen membrane receptors
in rat Sertoli cell membranes suggests that cell surface membrane receptors
for estrogen may also exist.
Another possibility for the rapid action ofestrogen is that the known
receptors (ERa and/or ER~) are working in a fashion other than the genomic
pathway. Signal transduction pathways are a common mechanism for
receptors and some evidence suggests that estrogen receptors may evoke
their rapid actions via signal transduction mechanisms. Pyramidal neurons
in the hippocampus are remarkably similar to those found in the BLA. In
CA1 neurons ofthe hippocampus, estrogen activates the cAMP pathway to
induce AMPA/kainate currents, which are normally activated by glutamate
(Gu and Moss., 1996); similarly, in neuroblastoma cells estrogen initiates a
mitogen-activated protein kinase (MAPK) cascade ofevents (Watters et al.,
1997). Growth factors such as epidermal growth factor and insulin-like
growth factor also act through the MAPK pathway. These factors enhance
the genomic response caused by estrogen, suggesting the possibility that the
pathways ofaction for these chemicals may be intersecting.
19
In the MAPK pathway, there is a membrane receptor-associated
tyrosine kinase initially activated by a growth factor, and the resulting
cascade includes activation ofthe proteins ras, raf, and MAPK respectively
(Hill CS and Treisman, 1995; Pelech SL and Sanghera, 1992: Sanghera,
1992). Introduction ofthe activated forms ofany ofthese proteins into the
cytosol ofa cell had the effect ofincreasing ER activity at the AF-1 domain
much like introduction ofthe growth factors (Kato et al., 1995). Further
observation showed that the Ser-118 residue ofthe ER was phosphorylated
following activation ofthe MAPK cascade (Kato et aI, 1995).
Phosphorylation ofthis serine residue is important for the activation ofthe
AF-l domain ofthe ER. Introduction of4-hydroxy-tamoxifen, an active
form oftamoxifen, into the cytosol initiates phosphorylation identical to that
ofestrogen, whereas the stronger antagonist ICI 164,384 does not initiate a
response (Kato et al., 1990). Not only is this a possible pathway ofSERM
agonism, but this information also suggests that a possible method of
blocking estrogen action is through the blocking ofphosphorylation ofthe
estrogen receptor.
The Basal Lateral Amygdala (BLA)
20
The basolateral nucleus ofthe amygdala (BLA) is one ofseveral
nuclei that make up the amygdala, an almond-shaped region located deep
within the temporal lobe ofthe cortex (Martin, , 1996). As part ofthe limbic
system, the amygdala plays important roles in emotion and memory
formation (Martin, , 1996). The BLA consists primarily ofpyramidal type
neurons that are very similar in structural and functional characteristics to
neurons ofthe hippocampus (Washburn and Moises, 1992b), another
important component ofthe limbic system (Martin, , 1996). The amygdala
and the hippocampus receive afferent innervation from the basal forebrain
(Emson et aI., 1979; Woolfand Butcher, 1982; Carlsen et al,. 1985). The
BLA is the amygdaloid region that has been shown to receive the highest
density ofcholinergic inputs in the region (Ben-Ari et al., 1977; Hellendall
et al., 1986). Functionally, this region is thought to be responsible for
giving emotional significance to memories (Martin, , 1996).
Cholinergic inputs from the basal forebrain begin to decrease in
number in AD. The cause ofthe decreased amount ofcholinergic inputs in
AD is the death ofafferent basal forebrain cholinergic neurons (Bartus et al.,
1982). The BLA is a region intimately related to the etiology ofAD, in fact
it is one ofthe first regions ofthe brain to show the evidence of
neurodegeneration upon the onset ofAD. The many psychological
21
dysfunctions associated with AD such as memory loss, emotional instability,
and depression may originate from this region's incapacity to function
properly following AD-inflicted neuronal cell death (Bartus et al., 1982).
A major afferent pathway to the BLA is the external capsule (EC).
This pathway carries both cholinergic and glutamatergic inputs to BLA
neurons (Washburn and Moises, 1992). Briefelectrical stimulation ofthe
EC evokes in BLA neurons a rapid excitatory postsynaptic potential (EPSP)
response, which is often followed by a delayed inhibitory postsynaptic
potential (IPSP) response. Neurons initiate EPSPs from direct innervation
via EC. However, in the case ofan IPSP, neurons receive their signal via an
interneuron. Interneurons receive excitation from a presynaptic source such
as the EC, and generate an IPSP in postsynaptic cells (Rainnie et al., 1991a,
b). A long-lasting depolarization ofBLA neurons is mediated by the
synaptic release ofacetylcholine, but this response is only seen following
high frequency EC stimulations (Washburn and Moises., 1992c), which are
not used in the present study.
Clinical evidence suggests that estrogen may playa protective role
against the onset ofAD (Birge et ai, 1997; Henderson et ai, 1994, 1997;
Kawas et al., 1997). Estrogen has also been reported to have effects on
cognition (Birge et al., 1997; Ripich et al., 1995) and long-term potentiation
22
(Cordoba and Carrer, 1997). Since the amygdala is an early and severely
affected target ofthe neurodegeneration associated with AD, it was of
interest to investigate the actions ofestrogen on neurons ofthis region. This
project was thus undertaken to test whether estrogen altered the functional
properties ofBLA neurons or their synaptic inputs.
23

/
External
Capsule
~-- Stimulator
Presynaptic
Stimulating
Electrode----
(
BLA Inhibitory
Interneuron
Amplifier, computer,
and oscilloscope
Ground
Electrode
BLA
Nerve cell
Materials and Methods
Long-Evans rats ofrandom sex and aged 3 weeks to 1 year were used
in this study. Rats were housed on 12 hour light/dark schedule and received
food and water ad libitum. All procedures involving live animals were
approved by the Animal Care and Use Committee, Youngstown State
University. Procedures were modified from Washburn and Moises (1992b).
Animals were sacrificed by decapitation, their brains removed promptly, and
placed in oxygenated and ice-cold artificial cerebrospinal fluid (ACSF). The
ACSF was made in 1.5 liter batches daily including the following
constituents with concentrations in millimolars 124 NaCl, 3.5 KC1, 1.5
MgS04, 1.0 anhydrous monobasic NaH2P04, 26.2 NaHC03, 11.0 glucose,
and 3.0 CaCho Three to five slices ofventral forebrain containing the
amygdala were cut on the horizontal plane at a thickness of400 Jlm using a
Vibroslicer (Campden Instruments). The slices were held at room
temperature (23?C) in ACSF bubbled continuously with 95% O2/ 5% CO2
for one hour before any recordings were attempted. This assured time for
the cells to recover from the vibrotome. Individual slices were placed the
recording chamber as needed. The slice was held between two layers of
nylon mesh submerged in continuously flowing ACSF at room temperature.
25
Intracellular recordings were obtained from neurons ofthe BLA using
glass microelectrodes filled with 2.7M KCII OAM K acetate (pH 7.0) and
having resistances of70-120 MOhms. Recordings were amplified with the
Axoclamp 2B in Bridge mode (Axon Instruments) and displayed on a
Tektronix 2213A oscilloscope and Cole Parmer chart recorder. Signals were
also fed to a computer interface (INDEC, Sunnyvale California) that
digitized the analog signal for storage and analysis by a microcomputer 
based program (pCLAMP, Axon Instruments).
Only cells with membrane potentials greater that -55 mV and over
shooting action potentials were included in this study. Under our recording
conditions, the vast majority ofBLA cells were not spontaneously active but
cells could be made to fire by passage ofdirect depolarizing circuit through
the electrode. Neuronal input resistance was determined by passing an
incremental series ofcurrent pulses (0.2 nA increments; range, -1.0 nA to
+0.4 nA; 450 msec duration) through the recording electrode and measuring
the resultant voltage deflections.
EPSPs were elicited in BLA neurons by delivering voltage pulses of
0.1 msec duration originating from an A31 0 Accupulser (World Precision
Instruments), isolated via an A360 Stimulus Isolator (World Precision
Instruments), and introduced through a bipolar stimulating electrode placed
26
on the surface ofthe slice over the external capsule (EC). While the EC
could be easily identified visually, we relied on anatomical landmarks in the
slice and diagrams ofstereotaxically defined sections (Paxinos and Watson,
Academic Press, 1998) for guiding placement ofthe recording electrode in
the BLA. Typically, the stimulus intensity delivered was that which evoked
an EPSP whose amplitude was just below threshold for generating an action
potential when recordings were made at the resting membrane potential.
Membrane potential was manually adjusted by intracellular injection of
direct current through the recording electrode, and was held at -65 to -75 mV
when characterizing EPSPs elicited by EC stimulation.
Estrogen (17- ~-estradiol) (Research Biochemical International)
and/or 4-0H-tamoxifen (Calbiochem) were applied to the slice by switching
the bath superfusate from normal ACSF to ACSF containing known
concentrations ofthe drugs. A 2 IlM Estrogen concentration in ACSF was
prepared by the addition of 1.2 ml ofdaily-made 100 IlM stock solution to
60 ml saline. A 60 nM 4-0H-tamoxifen solution was prepared by the
addition of12 ilL ofO.061lM stock solution stored at O?C to 60 ml saline.
All stock solutions were prepared with 95% ETOH as a solvent.
Tests were performed on neurons that passed criteria listed above for
membrane health. Tests were conducted in a series, which included current
27
stimulation, single-pulse presynaptic stimulation, and train presynaptic
stimulations. Voltage recordings were taken for all tests in the series, and
some recordings were used in determining resting membrane potential. To
decipher the early and late membrane resistances, the injected current and
voltage responses were used in accordance with Ohm's Law. EPSP
amplitudes were measured from resting membrane potential to the maximum
point on the voltage traces resultant ofsingle-pulse stimulation. The number
ofaction potentials fired was counted during the 450 ms current stimulation
at 800 pA ofdepolarizing current. Accommodation was analyzed by
comparing experimental recordings to control. The results ofthe experiment
were analyzed for statistical significance using a two-tailed t-test.
During the course ofthe experiments involving estrogen, the bath
superfusate was changed from control saline to estrogen and back to control
saline for washout. During the course ofexperiments involving 4 OH 
tamoxifen, the bath superfusate was changed in the following manner:
control, 4-0H-tamoxifen, control for washout, saline containing estrogen
and 4-0HT (to test blocking), control saline for washout, and saline
containing estrogen. The series oftests were conducted for 20 minutes after
switching the bath to the next solution in sequence. New slices were placed
in the recording chamber at the conclusion ofan experiment.
28
Results
The experiments presented here were designed to investigate the
actions ofestrogen on synaptic transmission in the BLA. Perfusion ofBLA
neurons with ACSF containing estrogen (2 f.lM) resulted in a significant
decrease in excitatory transmission and EPSP amplitude (Fig. 3). The mean
EPSP amplitude in control ACSF was 9.9 ? 2.8 mV (? SD; n=7).
Approximately 20 minutes after the bathing saline was changed to ACSF
containing estrogen, mean EPSP amplitude was reduced to 0.8 ? 1.2 mV
(n=7, p <0.001 from control). Switching the saline flow back to control
ACSF was followed by a complete recovery ofevoked EPSP amplitude,
which returned to a mean amplitude of9.6 ? 3.4 mV (n=7), a value not
significantly different from the control amplitude.
When observing EPSP recordings (Fig. 4b), the change in EPSP
amplitude upon addition ofestrogen is seen as a gradual decrease ofEPSP
amplitude within 20 to 30 minutes. Frequently (4 out of7 cells), estrogen
completely eliminated an evoked EPSP. Upon wash, the evoked EPSP
amplitude recovered and the cell once again showed normal synaptic
responses within 30 minutes. Spontaneous synaptic activity was also
reduced in the presence ofestrogen (Fig. 5a-c). The level ofspontaneous
29
activity in control cells (Fig. 5a) is higher than that observed for cells in the
presence ofestrogen (Fig. 5B). Typically, cells with lower EPSP amplitudes
had correspondingly lower levels ofspontaneous activity.
Effects ofethanol, the vehicle used to dissolve estrogen and
tamoxifen, on EPSP amplitude were studied as a control against the
possibility ofethanol playing a role in the effects ofestrogen on EPSP
amplitude (Fig. 8). Mean EPSP amplitude in the presence ofACSF
containing 2% ethanol was 9.0 ? 1.4 mY compared to mean control EPSP
amplitude of9.9 ? 2.9 mY (n=2). These findings indicate that alcohol alone
has no effect on EPSP amplitude.
Effects ofestrogen on synaptic transmission were also evident during
multiple (train) stimulations ofthe presynaptic pathway (n=9). When the
recordings oftrain stimulation are superimposed, the effects ofestrogen in
comparison to control ACSF are visible as a lower amplitude response to the
stimulation (Fig. 6a). The reversibility ofthe response to estrogen is seen as
a return ofthe control train response to normal (Fig. 6b). Same cell traces of
train stimulation responses in the presence ofcontrol ACSF (Fig. 7a),
estrogen (Fig. 7b), and wash (Fig. 7c) confirm the effect ofestrogen on
responses to train stimulation.
30
Preliminary data suggested that estrogen did not alter IPSP amplitude.
When comparing IPSP amplitudes in control ACSF and ACSF plus
estrogen (Fig. lOA), there appeared to be no noticeable difference.
Individual traces in figures 11 a-c further emphasize the similarity ofIPSPs
in the presence ofnormal ACSF, estrogen, and wash.
Estrogen appeared to have no effect on early membrane resistance
(Fig. 12), late membrane resistance (Fig. 13), resting membrane potential
(Fig. 15), number ofaction potentials (Fig. 16), or accommodation response
(Fig. 17).
Membrane resistance was calculated from the size ofthe voltage
deflection produced during the intracellular injection ofa 450 ms, 400 pA
hyperpolarizing current pulse. BLA pyramidal neurons normally respond to
a hyperpolarizing current with an initial peak negative voltage response,
followed by depolarizing (upward) voltage sag (Fig. 12) This is consistent
with studies ofneurons characterized in this region (Washburn and Moises,
1992b) The depolarizing sag is due to the hyperpolarization-induced
activation ofoutward current (Womble and Moises, 1992). The input
resistance ofBLA neurons was thus determined at 2 time points: at the
initial peak ofthe voltage response (early resistance) and just prior to the end
ofthe current pulse (late resistance). Estrogen had no significant effect on
31
either the early or late membrane resistances. Mean early membrane
resistance for control neurons in the presence ofcontrol estrogen and after
estrogen washout were 61.5 ? 15.6 MO, 59.0 ? 13.0 MO, and 70.2 ? 9.9
MO respectively. These values are consistent with previous reports
(Washburn and Moises, 1992b; Womble and Moises, 1992; Gean and
Shinnick-Gallagher, 1988, 1989) Mean late membrane resistances for
neurons in the presence ofcontrol ACSF, estrogen, and wash were 53.5 ?
13.6 MO, 52.0 ? 14.4 MO, and 63.4 ? 11.7 MO respectively.
Estrogen appeared to have no effect on the resting membrane potential
ofBLA neurons. Mean values ofresting membrane potentials recorded in
control ACSF, estrogen, and wash were not significantly different. Mean
resting membrane potentials recorded in the presence ofACSF, estrogen,
and wash are -62.6 ? 6.3 mY, -64.8 ? 7.5 mY, and -62.5 ? 9.3 mY
respectively.
Estrogen also appeared to have no effect on action potential frequency
in BLA neurons. Action potentials are easily distinguished by a sharp
depolarization followed by a sharp hyperpolarization. Action potential
frequency was measured by the number ofaction potentials generated during
450 ms pulse of800 pA depolarizing current. In control ACSF, the mean
action potential number was 5.9 ?2.5 APs. Cells in the presence ofestrogen
32
and wash had mean AP numbers of4.6 ? 3.0 APs and 7.8 ? 2.2 APs
respectively. In addition, the recordings were examined for any change in
accommodation response (fig. 17). Accommodation response is seen as a
relative change in the frequency ofaction potential firing over time. Upon
examination, no significant differences were found in the accommodation
response ofneurons studied under saline flow containing control ACSF and
estrogen. Additionally, no changes were found when the bath was switched
from ACSF containing estrogen back to normal ACSF.
To test the specificity ofthe estrogen response, an estrogen receptor
antagonist was used to block estrogen receptors. The chosen antagonist, 4 
OHT was used at 60 nM concentration. This concentration was used
because 4-0HT's binding affinity for ERa is over 300 times that ofestrogen
(Kuiper et al., 1997) The effects ofthis drug on BLA neurons (n=2) were
examined to study the drug's blocking and direct effects on the cell. These
preliminary results may point to possible roles estrogen has in the brain.
4-0HT has the effect ofsignificantly increasing the EPSP amplitude
(p < 0.05)(Fig. 16). Mean EPSP amplitude for recordings made in the
presence of4-0HT are 15 ? 0 mY. This is significantly greater than the
mean ofEPSP amplitudes recorded in control ACSF (9.9 ? 2.9 mY, p <
0.05). 4-0HT prevented the inhibitory effect ofestrogen on EPSP
33
amplitude. Concurrent 4-0HT and estrogen perfusion gave no significant
change in the EPSP amplitude (mean= 9.8 ? 4.6 mY).
4-0HT alone or concurrent perfusion of4-0HT and estrogen
appeared to have no effect on early membrane resistance (Fig. 17), late
membrane resistance (Fig. 18), resting membrane potential (Fig. 19),
frequency ofaction potential number (Fig. 20), or the accommodation
response (not shown).
4-0HT and concurrent perfusion of4-0HT and estrogen had no
significant effect on the early or late membrane resistances. In control
ACSF, the mean value ofearly membrane resistances was 63.8 ? 14.8 MO.
Cells in the presence of4-0HT and concurrent perfusion of4-0HT and
estrogen had mean early membrane resistances of54.0 ? 8.5 MO and 62.5 ?
16.3 MO respectively. The mean value oflate membrane resistances in
control ACSF was 62.6 ? 6.3 MO Cells in the presence of4-0HT and
concurrent perfusion of4-0HT and estrogen had mean late membrane
resistances of62.5 ? 7.2 MO, and 72.5 ? 3.5 MO respectively.
4-0HT and concurrent perfusion of4-0HT and estrogen also appear
to have no effect on the resting membrane potential ofBLA neurons. In
control ACSF, the mean value ofresting membrane potentials was -62.6 ?
6.3 mY. Cells in the presence of4-0HT and concurrent perfusion of4-0HT
34
and estrogen had mean resting membrane potentials of-62.5 ? 3.5 mY and 
72.5 ? 3.5 mY respectively. These values were not significantly different.
4-0HT and concurrent perfusion of4-0HT and estrogen appeared to
have no effect on action potential number. In control ACSF, the mean
number ofaction potentials was 5.9 ? 2.8 APs. Recordings in the presence
of4-0HT and concurrent perfusion of4-0HT and estrogen had mean action
potential numbers of5.0 ? 2.8 APs, and 3.0 ? 1.5 APs respectively. In
addition to action potential frequency, the recordings were examined for any
change in accommodation response. Upon examination, no noticeable
differences were found in accommodation response ofneurons studied in
control ACSF, ACSF plus 4-0HT, or concurrent perfusion of4-0HT and
estrogen (Fig. 17a, b).
35
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
l
Effect of Estrogen on EPSP Amplitude
14 T -----_..~ ~ IllllJ Control
J
T ACSF
12 -j rn Estrogen
9.9mV
10 I 9.6mV III Wash
:;-.s
8
CD
"0:e
0..E
? 6a..
(f)a..
w
4
2
o+---------
I
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
>E
o
T"""
(/)E
oo
T"""

N
....lIo.o
o
3en
....lIo.o
3<

U1o
3en

(]'Io
3en
(J'l
~ 0"o -
3<

U)
Eo
L{)
>E
-. 0U
T"""
LO

-<0
>E
o-r--
enE
oo
L()
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
*
CJ'1o
o
3
CJ)
....Ji,.o
3<
0>
C'"-

I\.)
CJ1o
3en
-lo.o
3<

*
NCJ1
o
3en
...lIo.o
3<

*
N01
o
3en
~o
3<
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
Effect of Ethanol Vehicle on EPSP Amplitude
14 -"-----------------
12 I ---------tl-------------------
11IIII Control
ACSF
9.9mV
10 +1-------------,
:;-.s all- _
CD"0
:::J:!:
"i5..E
~ 6 1--------(f)
a..w
4 +1------------j
2 +I---------~
o+1---------
9.8mV
rn Ethanol
and
ACSF
I
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
....ll.o
o
3en
....ll.o
3<
CD
Q)-
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
...Jo.o
o
3(J)
CD0'"
'--"

~o
3<
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
~o
3<
~o
0"---

~o
3<

~o
3<

-J.o
3<

Effect of Estrogen on Early Membrane Resistance
90 '",----.
80
70
60
Ii)
E.r:.
o 50
~
Q)
(,)c:
.l9 401Il
"iii
Q)0::
30 -I .
20
10
o-'-,----------
61.5 MOhms 59.0 MOhms
70.2 MOhms
IIIllJ Control
ACSF
rn Estrogen
[]]I Wash
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
I
80TI~~~~~
Effect of Estrogen on Late Membrane Resistance
IlJIl) Control
ACSF
70
60
50
40
30
20
10
O-'--I~~~~~
53.5 MOhms
l
52.6 MOhms
63.4 MOhms mEstrogen
m1Wash
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
-<C'D
<
I0)
o
3<
* '"
oo
3en
'"o3
<
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
J
Effect of Estrogen on Resting Membrane Potential
o"------------,
-10
-20
~ -30
]i
"E2
~ -40
OJc:
~.0
E
~ -50
-60
-70
-64.8 mV -62.5 mV
II1lIJControl
ACSF
rn Estrogen
mWash
~I ~
I
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
Effect of Estrogen on Number of Action Potentials
12 I ~ IlIill Control
ACSF
10
8
6
4
2
O+I-----~
1
5.9
4.6
7.8
rn Estrogen
mWash

*
?0)o
3<
No
o
3en
No
3<
*
*
*
*
*
*

*
I0)
o
3<
'"oo
3en
'"o
3<
*
*
*
*
*
*
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
Effect of 4-0HT and Estrogen Blocking Effect of 4-0HT on EPSP Amplitude
16 I Modulation IIlIlD Control
15.0mV ACSF
14 +1------------------
m4-0HT
12 1 ----+1-----
9.9mV:>
10 1 ---
E-
Q)
"0:E
c.. 8 1
~
0 C/)
0-
W 6 I
4+1-------
2 +1--------
o+1--------
9.8mV
rn4-0HT
and
Estrogen
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
I
Effect of 4-0HT and Estrogen Blocking Effect of 4-0HT on Early Membrane
Resistance
90 1 IIlIIIJ Control
ACSF
80
70
Ii) 60E
.co
~
~ 50c
~
"iii
CDc:: 40
CDc
e!.0
~ 30::2:
20
10
o.."-1 _
1
63.8 MOhms
T
54.0 MOhms
I
62.5 MOhms
m4-0HT
rn4-0HT
and
Estrogen

Effect of 4-0HT and Estrogen Blocking Effect of 4-0HT on Late Membrane
Resistance
80,,~~~~~~
70 1 "T" I
IllllJ Control
ACSF
rnJ4-0HT
60 1-?---?__?-55.3 MOhms
lilE
.s:::.o 50
6
~c:
~.~ 40
130:::
Q)c:
~~ 30
rl~-~~~~-
Q)
~
20 +1~~~~~~~-
10+1~~~~~~
O..LI~~ ~
55.0 MOhms 56.3 MOhms rn4-0HT
and
Estrogen

Effect of4-0HT and Estrogen Blocking Effect of 4-0HT on Resting
Membrane Potentialo
-10 +1-------
-20 +I------~
>5 -30
;m
c::2
~ -40 1
Q)c::
e!.0
E~ -50 I I
-60 +I------~
-62.5 mV
-70 I -
-72.5 mV
-80 -'-1 _
IIIllI Control
ACSF
rn4-0HT
rnI4-0HT
and
Estrogen
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
Effect of 4-0HT and Estrogen Blocking Effect of 4-0HT on Action Potential
Frequency
10 I ------- ~ Ililll Control
ACSF
9 -t-I----------------------------------------------j
m4-0HT
8 I -----+1-----------------------------1
rn4-0HT
and
Estrogen
5.0
5.9
- 1111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111111 3.00L-
Q) 3
.0E
::JZ
2
1
0
I
Q)
IJ)
~ 71
IJ)
Eo
o
'<t 61
~
IJ)ro
'E 51
~a..
c:
.Q 4 I1:5
?
Discussion
The experiments described here investigated the actions ofestrogen in
the rat BLA, utilizing intracellular recordings from pyramidal neurons ofthe
BLA. This is the major neuronal cell type found in the BLA, where it
accounts for over 95% ofneurons (McDonald, 1982, 1984: Millhouse and
DeOlmos, 1983). Intracellular recordings obtained in this study also closely
match properties ofpyramidal BLA neurons described in previous studies
(Gean and Shinnick-Gallagher, 1988, 1989: Washburn and Moises, 1992a,
b, c; Womble and Moises, 1992). In addition, the properties ofBLA
neurons closely resemble the properties ofpyramidal neurons in the rat
hippocampus (Wong and Prince, 1981; Madison and Nicoll, 1984) and
cerebral cortex (Connors et al., 1982; McCormick et al., 1985; Changac 
Amatai et al., 1990).
Direct electrical stimulation ofpresynaptic afferents to the BLA
produces a sequence ofsynaptic responses in the postsynaptic neuron
consisting ofan EPSP, IPSP, and a delayed cholinergic response. EPSPs
are elicited in the postsynaptic cell by the release ofglutamate at the
synapse, and subsequent activation ofits receptors (Rainnie et al., 1991a;
Washburn and Moises, 1992b). There are three known glutamate receptor
67
types in mammalian neurons: NMDA ionotropic, non-NMDA kainate/ u 
amino-3-hydroxy-5-methyl-4-isoxaso1e propionic acid (AMPA) ionotropic,
and glutamate metabotropic (Recasens et al., 1991). When activated by
glutamate, ionotropic receptors directly activate their associated ion channels
and metabotropic receptors activate their associated ion channels through
second messenger system (Rainnie et al., 1991a). Stimulation ofpresynaptic
afferents contained in the external capsule (EC) induced the release of
glutamate (Rainnie et al., 1991a; Washburn and Moises, 1992b) and evoked
EPSPs in the postsynaptic BLA neuron. The EPSPs in figures 4 and 5 are
sample recordings showing the effects ofstimulated release ofglutamate via
the EC.
In the present study, estrogen was found to markedly decrease the
amplitude ofEPSPs recorded from BLA neurons. This finding contrasts
other studies in the hippocampus that show estrogen increases EPSPs
(Barraclough et al., 1999; Foy et ai, 1999; SanMartin et al., 1999; Wong and
Moss, 1994). However, reductions ofEPSP (Valentino and Dingledine,
1981; Dodt and Misge1d, 1986; Dutar and Nicoll, 1988) have been recorded
previously in brain regions with properties similar to the BLA upon
activation ofcholinergic receptors.
68
Other electrophysiological reports have revealed that field potentials
(extracellular) increase upon estrogen treatment in the hippocampus (Foy et
al., 1980, 1982, 1983). Field potentials are extracellular recordings ofEPSP.
Pretreatment with 17 a estradiol was found to block the effects ofestrogen
treatment, and this suggests involvement ofan ER (Foy et al., 1982, 1983).
Additional investigations into these effects have uncovered estrogen-induced
excitatory effects on NMDA receptors, enhancement ofLTP, and increased
EPSP amplitude (Foy et al., 1999).
San Martin et al found that diethylstilbestrol (DES), a synthetic
estrogen, increased EPSP amplitude, but did not effect field EPSPs in the
pyramidal layer ofthe hippocampus. These results may suggest a difference
in action between synthetic estrogens and naturally occurring estrogens, or it
may be a functional difference between the pyramidal layer and CAl region.
Foy et al (1999) showed that estrogen increased both the field EPSP, and
the amplitude ofEPSPs (intracellular) in the CAl region ofthe
hippocampus.
In vitro recordings from CAl hippocampal neurons after 14 days of
estrogen treatment has been shown to have no effect on LTP, EPSP
amplitude, or neuron excitability (Barraclough et al., 1999). This could
suggests that the period ofestrogen action ends within days.
69
Wong and Moss have shown that application ofestrogen to CA1
hippocampal neurons causes a rapid and reversible depolarization, increased
input resistance, and increased excitability. Similar effects ofestrogen on
excitability in the hippocampus have been reported by Vardaris et at.,
(1990). These effects are blocked by 4-0HT. Further investigations by Gu
and Moss have shown that kainate currents are enhanced in the CAl region
ofthe hippocampus by estrogen via a G-protein-linked pathway and show
reversibility (1996, 1998). Interestingly, estrogen must be present on both
sides ofthe membrane for this action to occur (Wong and Moss, 1998).
Wong and Moss have shown that estrogen treatment for 2 days
increased excitability, prolonged EPSPs, and increased spontaneous activity
ofCA1 hippocampal neurons (1992). Instant effects ofestrogen treatment
on ovarectomized female rats showed a rapid (1 min) increase in EPSP
amplitude (Wong and Moss, 1992). These actions were reversible and
inhibited by a non-NMDA receptor antagonist (Wong and Moss. 1992). The
estrogen-induced effect on EPSP amplitude was also seen when glutamate
was directly applied to the cell (Wong and Moss, 1992), suggesting that the
actions ofestrogen were postsynaptic. These findings further suggest that
the synaptic alterations seen in BLA neurons by this study may be caused by
changes in the glutamate system.
70
Additional evidence supporting the modulation ofneuronal and
synaptic functioning in the mammalian brain by estrogen exists. In
hippocampal pYramidal cells and extrahypothalamic neurons, estrogen
treatment increases the excitatory effects ofNMDA and glutamate (Teyler et
ai., 1990, Smith et ai., 1987). The number ofNMDA receptors in the
hippocampus CAl region is reduced after treatment with estrogen (Weiland,
1992). Ovariectomy has been found to decrease dendritic spine density on
CAl pYramidal cells in vivo and in vitro (Woolley and McEwen, 1994;
Murphy and Segal, 1996), while estrogen replacement reverses this effect
(Woolley and McEwen, 1992). Estrogen increases cell survival rates
following glutamate excitotoxic insults, oxidative stress, and beta-amyloid
treatment (Behl et ai., 1995; Goodman et ai., 1996; Green et ai., 1996;
Singer et ai., 1996, 1998). Estrogen antagonists and MAPK inhibitors have
been shown to block estrogen's neuroprotective functions (Singh et ai.,
1999).
IPSPs are elicited in the postsynaptic cell by the release ofGABA
(gamma-amino-butYric acid) by local inhibitory intemeurons. Ithas been
shown that IPSPs in the BLA are initiated by activation ofGABAA and
GABAB (Rainnie et ai., 1991b). GABA is the major inhibitory
neurotransmitter in the BLA (Rainnie et ai., 1991b). Stimulation ofBLA
71
afferents can elicit both an early IPSP and a late IPSP in BLA neurons
(Rainnie et al., 1991b, Washburn and Moises, 1992a, b). Early IPSPs occur
through the activation ofthe ionotropic GABAA receptor, and late IPSPs
occur through activation ofthe metabotropic GABAB receptor (Rainnie et
al., 1991b, Washburn and Moises, 1992a, b). Figures 10 and 11 are sample
recordings showing IPSPs occurring through direct stimulation ofan
inhibitory interneuron.
Preliminary data shows the possibility that IPSP amplitude is not
affected by estrogen (Figs. 10, 11). Decreases in IPSP amplitude have been
recorded previously upon activation ofcholinergic receptors in brain regions
with properties similar to the BLA (Haas., 1992).
Wong and Moss (1992) found no significant estrogen-induced
difference in stimulated IPSPs or those initiated by GABA treatment.
However, Some changes ofIPSP amplitude have been reportedly induced by
estrogen treatment. Canonaro et al found that in vivo estrogen treatment
reduced the number ofchloride channels associated with GABAA receptors
(1993). However, changes in chloride channels were only found after
treating with estrogen over a relatively long period (>24 hours), and were
not found in the BLA (Canonar et al., 1993). Therefore these results
72
(Canonaro et ai., 1993) should have few implication on the synaptic
alteration found in BLA neurons.
Cholinergic responses are elicited in the postsynaptic cell by the
release ofacetylcholine from presynaptic cholinergic neurons. Their axons
run within the EC and are activated following EC stimulation (Washburn ad
Moises, 1992a, b). Cholinergic receptors on BLA neurons are activated by
acetylcholine release following high frequency stimulation ofthe
presynaptic external capsule pathway (Washburn and Moises., 1992b, c;
Rainnie et ai., 1991a). Cholinergic input to cells in the BLA arises, for the
most part, from regions outside ofthe BLA (Emson et ai., 1979; Woolfand
Butcher., 1982; Carlsen et ai., 1985), although there is evidence showing
small populations ofcholinergic neurons in the BLA (Carlsen and Heimer,
1986). The cholinergic axons found in the EC have been traced back to
cholinergic neurons residing across a broad region that encompasses the
diagonal band ofBroca to the ventral palladium (Emson et ai, 1979; Woolf
and Butcher, 1982; Carlsen et ai., 1985). Acetylcholine can elicit excitatory
responses in BLA neurons via the muscarinic type ofcholinergic receptor
(Washburn and Moises., 1992a). Cholinergic responses consist of a long,
sustained depolarization ofthe postsynaptic BLA neuron (Washburn and
Moises, 1992a). The effect ofestrogen on the acetylcholine pathway was
73
not a focus ofthis study. However, pursuit ofinformation regarding how
estrogen interacts with the acetylcholine pathway remains an area ofgreat
interest.
Acetylcholine and glutamate pathways are the two known and
described excitatory pathways to the BLA. In the present study, EPSP
amplitude and spontaneous activity decreased upon addition ofestrogen
(Figs. 3,4,5). Glutamate is the neurotransmitter associated with EPSP
elicitation. Thus, the cause ofsynaptic activity alteration in BLA neurons is
likely associated with the glutamate system.
The receptors involved in the estrogenic alterations ofsynaptic
activity were examined with the use ofthe ER antagonist 4-hydroxy 
tamoxifen (4-0HT). Non-steroidal ER antagonists such as 4-0HT are
structurally similar to estrogen with the exception ofan alkyl-amino-ethoxy
side chain being present (Jordan., 1998). Estrogen and 4-0HT share the
same binding site on the ER (Brzozowski et al., 1997). Since they compete
for the same binding domain on the ER, 4-0HT was used to test the
involvement ofthe ER in alteration ofsynaptic transmission. 4-0HT has a
binding affinity 339 greater than that ofestrogen for the binding site on
estrogen receptor B(ERB) and 178 times greater for ERa (Kuiper et al.,
1997). Preliminary data shows that the addition of4-0HT(60 nM) to the
74
bathing saline was followed by an increase in EPSP amplitude (Fig. 16).
This may be due to the displacement ofchemicals previously attached to the
estrogen ligand-binding site, suggesting that there may be a certain level of
endogenous tonic inhibition. Preliminary data also shows that 4-0HT
prevented the inhibition ofexcitatory synaptic activity by estrogen (Fig. 16).
This suggests the possibility that ER activation is necessary for the
alterations ofsynaptic activity seen in this study.
Defining the non-genomic mechanism by which estrogen-induced
alterations in synaptic activity are occurring is speculative. Mainly, this is
due to the limited knowledge ofER function. Genomic mechanisms of
estrogen action are the most clearly understood. However, the genomic
mechanism takes hours from estrogen binding and initiation to completion
and release ofpost-translationally modified products (Zakon., 1997). In the
case ofalteration ofsynaptic activity by estrogen, the period required to see
effects (20 -30 minutes) is too short to be a product ofthe genomic
mechanism. Alteration ofsynaptic activity by estrogen is most likely
occurring via a rapid, non-genomic mechanism. A possible mechanism
through which estrogen may be altering synaptic function in the BLA is via
various protein kinase pathways. Mitogen activated protein kinase (MAPK),
protein kinase A (PKA), and protein kinase C (PKC) are protein kinase
75
pathways that may serve as potential alternative pathways for estrogen
function.
Studies ofprimary cultured cortical neurons have shown that estrogen
induces an increase in MAPK activity within 30 minutes (Singer, et al
1999). Estrogen has also been shown to initiate activation ofMAPK in
neuroblastoma cells (Watters et al., 1997), and non-neuronal cells via non 
receptor kinase src (Migliaccio et al., 1996). Ifthe estrogen receptor
antagonist leI 182,780 or a MAPK pathway inhibitor is used in conjunction
with estrogen, there is no increased kinase activity in primary cortical
neurons (Singer et al., 1999). This implies that estrogen activates MAPK
through the activation ofan ER in cultured cortical neurons.
MAPK is activated by many intracellular signals, including nerve
growth factor (NGF) (Seger and Krebs, 1995). NGF has been suggested to
promote survival ofneurons in several cases (Kromer, 1987; Hefti, 1986).
Estrogen is also believed to modulate neurotrophins (such as NGF) levels
(Gibbs et al., 1994; Singh et al., 1995), and neurotrophin receptors (Sorabji
et al., 1994a, b; McMillan et al., 1996). This suggests the possibility that
estrogen and neurotrophins may participate in a negative feedback
mechanism to control the amount ofkinase activity mediated by the estrogen
receptor, NGF, and other factors.
76
Another pathway through which estrogen may be inducing its actions
is the protein kinase A (PKA) pathway. In guinea pig hypothalamic slices,
estrogen weakens the ability ofJ,!-opioids to hyperpolarize beta-endorphin
neurons. This action ofestrogen takes place within 20 minutes, and it was
blocked by the ER antagonists ICI 164,384 and diethylstilbestrol.
Furthermore, this action was not prevented by the protein synthesis inhibitor
cyclohexamide but was blocked by selective PKA antagonists (Lagrange et
al., 1997).
Another possible pathway for rapid actions ofestrogen in the BLA is
by protein kinase C (PKC). In normal and tumor tissue, estrogen has been
shown to increase PKC levels (Drouva et al., 1990, Maeda et al., 1993,
Maizels et aI., 1993).
Although the actions ofthe kinases in relation to estrogen action have
not been investigated in the BLA, the demonstration ofrapid (30 min)
estrogen action, with inhibition by an ER antagonist, make them plausible
mechanisms for the synaptic activity alteration found in BLA neurons, and
potential targets for future research.
Some ofthe effects ofestrogen occurring in the brain have been
shown to be associated with NMDA receptors, due to the recent findings of
Woolley and McEwen (1994), who showed that the dendritic spine density
77
changes occurring in the CA1 region ofthe hippocampus following estrogen
treatment rely upon the activation ofNMDA receptors. In addition, the
competitive NMDA antagonist MK 801 prevents estrogen-induced changes
in spine density. Metabotropic and ionotropic NMDA receptor were
reportedly affected (Woolley and McEwen 1994). This has implications on
our study because BLA neuronal EPSPs rely in part upon the activation of
NMDA receptors (Rainnie et al., 1991a).
Blocking ofER by tamoxifen shows that alteration ofsynaptic
behavior may be dependent upon on a specific activating function (AF) of
the ER. AF-l is located in the N terminus and AF-2 is located in the ligand 
binding domain ofERa. These are two areas suggested to mediate
transcriptional activation ofERa (Tsai and O'Malley, 1994). ERa
antagonists inhibit and agonists activate AF-2, but some antagonists have
been reported to have agonistic actions (Berry et al., 1990). AF-lofERa
has been shown to be regulated by growth factors associated with the MAPK
pathway, and also may be the cause ofagonistic effects ofER antagonists
(McEwen, 1997; Kato et al., 1995).
While analyzing the preliminary blocking effect oftamoxifen, it is
plausible to hypothesize that the actions ofestrogen seen in BLA pyramidal
neurons are mediated by the AF-2 region on the ER. Distribution ofERs in
78
the amygdala (Shughrue et al., 1998; Weiland, 1997) suggests that both ERa
and ER~ may be present in the BLA. The ligand binding domains ofERa
and ER~ show a 50% homology (Kuiper, et al., 1997), thus the nearby AF-2
regions ofthe ERs may have similar homologies. Therefore, defining which
ofthe ER subtypes is involved the alteration ofsynaptic activity in the BLA
can help find a more specific location for the actions described here.
Passive and intracellu1ar1y stimulated membrane activity recordings
did not show significant alteration upon estrogen treatment. Resting
membrane potential, early and late membrane resistance, action potential
frequency, and action potential accommodation response showed no
significant change upon switching the bathing solution to estrogen. This
data suggests that the passive currents analyzed in resting membrane
potential measurements show no conductance change, and the voltage-gated
currents analyzed in the early membrane resistance, late membrane
resistance, and action potential accommodation response also show no
conductance change. The accommodation response results from the action
ofa slowly decaying after hyperpo10rization current (Womble and Moises,
1993). This Ca++-activated K+ current was not significantly changed.
Action potential frequency allows us to analyze the voltage-gated Na+ and
K+ currents associated with action potential formation (Kandel et al., 1995).
79
These currents had no significant change. Therefore, no changes were found
in the Na+ and K+ ion currents, which are known to underlie, active neuronal
responses, such as action potential firing patterns (Kandel et aI, 1995).
Voltage recordings from BLA neurons presented in this study
revealed significant decreases in EPSP amplitude and spontaneous synaptic
activity upon estrogen treatment. This is a rapid (20-30 min.) response. 4 
OHT appeared to block this effect ofestrogen, therefore suggesting an ER 
mediated response. The speed ofthe response suggests that the ER is not
acting by the classical genomic mechanism ofER action.
Voltage recordings analyzing passive and voltage-gated ion channels
show no significant change. With the evidence ofprotein kinase pathways
associated with estrogen and ER phosphorylation, it would seem likely that
some modulation ofion channels should be occurring. Kinases have been
found to effect ion channels in past studies ofestrogen action in
hippocampal neurons (Gu and Moss, 1996; Kawasaki et aI, 1994; Wyneken
et aI, 1997; Potier and Rovira, 1999; Ghetti and Heinemann, 2000). The
decreases in EPSP amplitude seen in this study are attributed to changes of
the glutamate system. Preliminary evidence showed estrogen does not affect
IPSP amplitude. Thus, the GABA system does not appear to be affected.
Estrogen does not appear to affect any ofthe membrane properties ofthe
80
BLA neurons studied; therefore, ion channels ofthe studied neurons do not
appear to be affected. In conclusion, the accumulated evidence appeared to
suggest that estrogen is acting to inhibit the glutamate system. Reduction in
glutamate function at BLA synapses supports previous studies that suggest
estrogen protects from the glutamate-associated excitotoxicity associated
with cerebral ischemia (Hum PD, Macrae, 2000; Sawada et al., 2000; Rusa
et ai., 1999; Fung et al., 1999).
81
References
Ali S, Metzger D, Bomert JM, Chambon P (1993). Modulation of
transcriptional activation by ligand-dependent phosphorylation ofthe
human oestrogen receptor AlB region. EMBO Journal. 12:1153.
Balthazart J, Ball GF. (1998). New insights into the Regulation and Function
ofBrain Estrogen Synthase (aromatase). Trends in Neuroscience.
21:243-249.
Barraclough DJ, Ingram CD, Brown MW. (1999). Chronic treatment with
oestradiol does not alter in vitro LTP in subfield CAl ofthe female rat
hippocampus. Neuropharmacology. Jan;38(1):65-71.
Bartus RT, Dean RL, Beer B, and Lippa AS. (1982). The cholinergic
hypothesis ofgeriatric memory dysfunction. Science. 217:408-417.
Bedard P, Langelier P, Villeneuve A. (1977). Oestrogens and extrapyramidal
system. Lance.t Dec 24-31;2(8052-8053):1367-1368
Ben-Ari Y, Zigmond RE, Shute CCD, and Lewis PR. (1977). Regional
distribution ofcholine acetyltransferase and acetylcholinesterase within
the amygdaloid complex and stria terminalis system. Brain Research.
120:435-445.
Berry M, Metzger P, Chambon (1990). Role ofthe two activating domains of
the oestrogen receptor in the cell-type and promoter-context dependent
agonistic activity ofthe anti-oestrogen 4-hydroxy tamoxifen.. EMBO
Journal. 9:2811.
Bi R, Broutman G, Foy MR, Thompson RF, Baudry M. (2000). The tyrosine
kinase and mitogen-activated protein kinase pathways mediate multiple
82
effects ofestrogen in hippocampus. Proceedings ofthe National
Academy ofScience ofthe USA. Mar 28;97(7):3602-3607.
Birge SJ, Mortel KF. (1997). Estrogen and the Treatment ofAlzheimer's
Disease. American Journal ofMedicine. 103(3A):36S-45S.
Black LJ, Jones CD, Falcone JF. (1983). Antagonism ofestrogen action with a
new benzothiophene derived antiestrogen. Life Science. 32:1031-1036.
Bondi MW and Lange KL. (1998). Alzheimer's Disease. Encyclopedia of
Mental Health, Volume 1. San Diego, California: Academic Press.
Boyles JK, Notterpek LM, Anderson LJ. (1990). Accumulation of
apolipoproteins in the regenerating and remyelinating mammalian
peripheral nerve. Journal ofBiological Chemistry. 265:17805-17815.
Boyles JK, Zoeliner CD, Anderson LJ, Kosik LM, Pitas RE, Weisgraber KH,
Hui DY, Mahley RW, Gebicke-Haerter PH, Ignatius MJ, Shooter EM.
(1989). A role for apolipoprotein E, apolipoprotein E, apolipoprotein A 
1, and low density lipoprotein receptors in cholesterol transport during
regeneration and remyelination ofthe rat sciatic nerve. Journal of
Clinical Investigation. 83: 1015-1031.
Brenner DE, Kukull WA, Stergachis A, Van Belle G, Bowen JD, McCormick
WC, Teri L, Larson EB. (1994). Postmenopausal estrogen replacement
therapy and the risk ofAlzheimer's disease: a population based case 
control study. American Journal ofEpidemiology. 140:262-267.
Brzozowski AM, Pike ACW, Dauter Z, Hubbard RE, Bonn T, Engstrom 0,
Ohman L, Greene GL, Gustafsson J, Carlquist M. (1997). Molecular
Basis ofAgonism and antagonism in the Oestrogen Receptor. Nature.
389: 753-758.
Canonaco M, Tavolaro R, Maggi A. (1993). Steroid hormones and receptors
ofthe GABAA supramolecular complex. ILProgesterone and estrogen
inhibitory effects on the chloride ion channel receptor in different
83
forebrain areas ofthe female rat. Neuroendocrinology. May;57(5):974 
84.
Carlsen J and Heimer L. (1986). A correlated light and electron microscopic
immunocytochemical study ofcholinergic terminals and neurones in the
ra1 amygdaloid body with special emphasis on the baso1atera1
amygdaloid nucleus. Journal ofComparative Neurology. 244:121-136.
Carlsen J, Zaborsky L, and Heimer L. (1985). Cholinergic projections from the
basal forebrain to the baso1atera1 amygdaloid complex: a combined
retrograde fluorescent and immuno-histochemica1 study. Journal of
Comparative Neurology. 234:155-167.
Chao HM, Ma LY, McEwen BS, Sakai RR. (1998). Regulation of
Glucocorticoid Receptor and Mineralocorticoid Receptor Messenger
Ribonucleic Acids by Selective Agonists in the Rat Hippocampus.
Endocrinology. 139:1810-1814.
Chao HM, Sakai RR, Ma LY, McEwen BS. (1998). Adrenal Steroid
Regulation ofNeurotrophic Factor Expression in the Rat Hippocampus.
Endocrinology. 139: 3112-3118.
Chao HM, Spencer RL, Frankfurt M, McEwen BS. (1994). The Effects of
Aging and hormonal Manipulation on Amyloid Precursor Protein
APP695 mRNA expression in the Rat Hippocampus. Journal of
Neuroendocrinology. 6:517-521.
Chiaia N, Foy M, Tey1er TJ. (1983). The hamster hippocampal slice: II.
Neuroendocrine modulation. Behavioral Neuroscience. Dec;97(6):839 
843
Clarke WP, Goldfarb J. (1998). Estrogen Enhances a 5-HT1a Response in
Hippocampal Slices from Female Rats. European Journal of
Pharmacology. 160: 195-197.
84
Connors BW, Gutnick JA, Prince DA. (1982) Electrophysiological properties
ofneocortical neurons in vitro. Journal ofNeurophysiology. 48: 1302 
1320.
Cordoba Montoya DA, Carrer HF. (1977). Estrogen Facilitates Induction of
Long Term Potentiation in the Hippocampus ofawake rats. Brain
Research. 778:430-438.
Cotman CW, Pike CJ, Copani A. (1992). ~-amyloid neurotoxicity: a
discussion ofin vitro findings. Neurobiology Aging. 13:587-590.
Couse JF, Lindzey J, Grandien K, Gustafsson J, Korach KS. (1997). Tissue
Distribution and Quantitative Analysis ofEstrogen Receptor-a (ERa)
and Estrogen Receptor-~ (ER~) Messenger Ribonucleic Acid in the
Wild-Type and ERa-Knockout Mouse. Endocrinology. 138: 4613-4621.
Dodt and Misgeld U. (1986). Muscarinic slow excitation and muscarinic
inhibition ofsynaptic transmission in the rat neostriatum. Journal of
Physiology. 280:593-608.
Drouva SV, Gorenne I, Laplavte E, Rerat E, Enjalbert A, and Kordon C.
(1990). Estradiol modulates protein kinase C activity in the rat pituitary
in vivo and in vitro. Endocrinology 126, 536-544.
Dutar P and Nicoll RA. (1986). Classification ofmuscarinic responses in
hippocampus in terms ofreceptor subtypes and second messenger
systems electrophysiological studies in vitro. Journal ofNeuroscience.
8:4214-4224.
Emson PC, Paninos G, Le Gal La Salle G, Ben Ari Y, and Silver A. (1979).
Choline acetyltransferase and acetylcholinesterase containing projections
from the basal forebrain to the amygdaloid complex ofthe rat. Brain
Research. 165:271-282.
Emson PC, Paninos G, Le Val La Salle G, Ben-Ari Y, and Silver A. (1979).
Choline acetyltransferase and acetylcholinesterase containing projections
85
from the basal forebrain to the amygdaloid complex ofthe rat. Brain
Research. 165:271-282.
Feng W, Ribeiro RC, Wagner, RL, Nguyen H, Apriletti JW, Fletterick RJ,
Baxter JD, Kushner PJ, West BL. (1998). Hormone-Dependent
Coactivator Binding to a Hydrophobic Cleft on Nuclear Receptors.
Science. 280: 1747-1749.
Foy MR, Teyler TJ, Vardaris RM. (1982). Effects ofdelta 9-THC and 17-P 
estradiol in hippocampus. Brain Research Bulletin. Apr;8(4):341-345.
Foy MR, Teyler TJ. (1983). 17-a-Estradiol and 17-p-estradiol in
hippocampus. Brain Research Bulletin. Jun;10(6):735-739.
Foy MR, XU J, Xie X, Brinton RD, Thompson RF, Berger TW. (1999). 17P 
Estradiol Enhances NMDA Receptor-Mediated EPSPs and Long-Term
Potentiation. Journal ofNeurophysiology. 81: 925-929.
Fung MM, Barrett-Connor E, Bettencourt RR. (1999). Hormone replacement
therapy and stroke risk in older women. Journal of Womens Health.
8(3):359-364.
Gean PW, Shinnick-Gallagher P (1988) Characterization ofthe epileptiform
activity induced by magnesium-free solution in rat amygdala slices: an
intracellular study. Experimental Neurology. 101:248-255.
Gean PW, Shinnick-Gallagher P (1989) The transient outward current in a
mammalian central neurone blocked by 4-aminopyradine. Nature.
299:252-254.
Ghetti A, Heinemann SF. (2000). NMDA-Dependent modulation of
hippocampal kainate receptors by calcineurin and Ca(2+)/calmodulin 
dependent protein kinase. Journal ofNeuroscience. Apr 15;20(8):2766 
2773.
86
Goodman Y, Bruce AJ, Cheng B, Mattson MP. (1996). Estrogens Attenuate
and Corticosterone Exacerbates Excitotoxicity, Oxidative Injury, and
Amyloid ~ Peptide Toxicity in Hippocampal Neurons. Journal of
Neurochemistry. 66: 1836-1844.
Gorczynska E, Handelsman DJ. (1995). Androgens Rapidly Increase the
Cytosolic Calcium Concentration in Sertoli Cells. Endocrinology.
136:2052-2059.
Gradishar WJ and Jordan VC (1997). Clinical potential ofnew antiestrogens.
Journal ofClinical Oncology. 15:840-852.
Greco B, Edwards DA, Michael RP, Clancy AN. (1998). Androgen Receptors
and Estrogen Receptors are Co-localized in Male Rat Hypothalamic and
Limbic Neurons that Express Fos Immunoreactivity Induced by mating.
Neuroendocrinology. 67:18-28.
Gu Q, Moss RL J. (1996). 17 beta-Estradiol potentiates kainate-induced
currents via activation ofthecAMP cascade. Neuroscience. Jun
;16(11):3620-3629.
Gu Q, Moss RL. (1996). 17~-Estradiol Potentiates Kainate-Induced Currents
via Activation ofthe cAMP Cascade. Journal ofNeuroscience.
16(11):3620-3629.
Gu Q, Moss RL. (1998). Novel mechanism for non-genomic action of17 beta 
oestradiol on kainate-induced currents in isolated rat CA1 hippocampal
neurones. Journal Physiology (London). Feb 1;506:745-754
Haas HL. (1982). Cholinergic disinhibition in hippocampal slices ofthe rat.
Brain Research. 233:200-204.
Hefti T. (1986). Nerve Growth Factor Promotes Survival ofSeptal Cholinergic
Neurons After Fimbrial Transections. Journal ofNeuroscience.
6(8):2155-2162.
87
Hellendall RP, Godfrey DA, Ross CD, Armstrong DM, and Prive JL. (1996).
The distribution ofcholine acetyltransferase in the rat amygdaloid
complex and adjacent cortical areas as determined by quantitative micro 
assay and immunohistochemistry. Journal ofComparative Neurology.
249:486-498.
Henderson Y W. (1997). Estrogen, Cognition, and a Woman's Risk of
Alzheimer's Disease. American Journal ofMedicine. 103(3A): 11 S-18S.
Hill CS, and Treisman R. (1995). Transcriptional regulation by extracellular
signals: mechanisms and specificity.. Cell. 80: 199-211.
Holtzman DM, Pitas RE, Kilbridge J, Nathan B, Mahley RW, Bu G, Schwartz
AL. (1995). Low density lipoprotein receptor related protein mediates
apolipoprotein E-dependent neurite outgrowth in a central nervous
system derived neuronal cell line. Neurobiology. 92:9480-9484.
Hum PD, Macrae 1M Estrogen as a neuroprotectant in stroke. (2000). J
Cerebral Blood Flow and Metabolism.. 20(4):631-652.
Jaffe AB, Toran-Allerand CD, Greengard P, Gandy SE. (1994). Estrogen
Regulates Metabolism ofAlzheimer Amyloid ~ Precursor Protein. The
Journal ofBiochemistry andMolecular Biology. 269: 13065-13068,
1994.
Jensen EY, Mohla S, Brecher PI, DeSombre ER, (1973). Estrogen receptor
transformation and nuclear RNA synthesis. Advanced Experimental
Medical Biology. 36:60-79.
Johnson MG and Everitt BJ (1995). Essential Reproduction, Fourth Edition.
Cambridge, Massachusetts: Blackwell Science.
Jordan YC. (1984). Biochemical pharmacology ofantiestrogen action.
Pharmacology Reviews. 36:245-256.
88
Jordan VC. (1998). Antiestrogenic Action ofRaloxifene and Tamoxifen:
Today and Tomorrow. Journal ofthe National Cancer Institute. 90:
967-971.
Kandel ER, Schwartz JH, Jessell TM (1995). Essentials ofNeural Science and
Behavior. Norwalk, Connecticut: Appleton and Lange.
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama s, Sasaki H, Masushige
S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P. (1995).
Activation ofthe Estrogen Receptor Through Phosphorylation by
Mitogen-Activated Protein Kinase. Science. 270:1491-1494.
Katzenellenbogen JA, O'Malley BW, and Katzenellenbogen BS (1996).
Molecular Endocrinology. 10: 119-131.
Kawas C, Resnick S, Morrison A, Brookmeyer R, Corrada M, Zonderman A,
Baca1 C,Lingle DD, Metter E. (1997). A prospective study ofestrogen
replacement therapy and the risk ofdevelopingAlzheimer's disease: the
Baltimore Longitudinal Study ofAging. Neurology. Jun;48(6):1517-21
Kawasaki M, Uchida S, Monkawa T, Miyawaki A, Mikoshiba K, Mammo F,
Sasaki S. (1994). Protein, Nucleotide Cloning and expression ofa
protein kinase C-regu1ated chloride channel abundantly expressed in rat
brain neuronal cells. Neuron. Mar;12(3):597-604.
Korach KS. (1994). Insights from the Study ofAnimals Lacking Functional
Estrogen Receptor. Science. 266: 1524-1527.
Kow LM, PfaffDW. (1984). Suprachiasmatic neurons in tissue slices from
ovariectomized rats: e1ectrophysio1ogical and neuropharmacological
characterization and the effects ofestrogen treatment. Brain Research.
Apr 16;297(2):275-286.
Kromer LF. (1987). Nerve Growth Factor Treatment After Brain Injury
Prevents Neuronal Death. Science. 235:214-216.
89
Kubo K, Gorski RA, Kawakami M. (1975). Effects ofestrogen on neuronal
excitability in the hippocampa1-septa1-hypotha1amic system.
Neuroendocrinology. 18(2):176-91.
Kuiper G, Carlsson B, Grandien K, Enmark E, Haggb1ad J, Nilsson S,
Gustafsson J. (1997). Comparison ofthe Ligand Binding Specificity and
Transcript Tissue Distribution ofEstrogen Receptors a and p.
Endocrinology. 138: 863-870.
Kumar V, Green S, Stack G, Berry M, Jin J, Chambon P. (1987). Functional
Domains ofthe Human Estrogen Receptor. Cell. 51: 941-951.
Lagrange AH, Ronnek1eiv OK, Kelly MJ. (1994). The Potency ofJl-Opioid
Hyperpolarization ofHypothalamic Arcuate Neurons is Rapidly
Attenuated by 17B-Estradiol. The Journal ofNeuroscience. 14(10):
6196-6204.
Lagrange AH, Ronnek1eiv OK, Kelly MJ. (1997). Modulation ofG Protein 
Coupled Receptors by an Estrogen Receptor that Activates Protein
Kinase A. Molecular Pharmacology. 51: 605-612.
Li X, Schwartz PE, Rissman EF. (1997). Distribution ofEstrogen Receptor B 
like Immunoreactivity in Rat Forebrain. Neuroendocrinology. 66:63-67.
Madison DV, Nicoll RA. (1984). Control ofthe repetitive discharge ofrat
CAl pyramidal neurons in vitro. Journal ofPhysiology (London).
354:319-331.
Maeda T, and L10ud RV. (1993) Protein kinase C activity and messenger RNA
modulation by estrogen in normal and neoplastic rat pituitary tissue..
Laboratory Investigations. 68,472-480.
Mah1ey RW (1988). Apo1ipoprotein E: cholesterol transport role with
expanding role in cell biology. Science. 240:622-630.
90
Maizels, E. T., Miller, J. B., Cutler, f. J., Jackiw, V., Carney, E. M., Mizuno, K.,
0000, S., and Hunzicker, D. M. (1992). Journal ofBiological
Chemistry. 267, 17061-17068.
Martin JH (1996). Neuroanatomy: Text andAtlas, Second Edition. Stamford,
Connecticut: Appleton and Lange.
Mason RP, Shoemaker WJ, Shajenko L, Chambers TE, Herbette LG. (1991).
Evidence for changes in the Alzheimer's disease brain cortical membrane
structure mediated by cholesterol. Neurobiology ofAging. 13:143-419.
Maury APJ. (1995). Biology ofdisease: molecular pathogenesis ofp 
amyloidoses. Laboratory Investigation. 72:4-16.
Mayeux R, Ottman R, Maestre G, Ngai C, Tang MX, Ginsberg H, Chun M,
Tycko B, Shelanski M.. (1995). Synergistic effects oftraumatic head
injury and apolipoprotein-epsilon 4 in patients with Alzheimer's disease.
Neurology. 45:555-557.
McCormick DA, Connors BW, Lightahall JW, Prince DA (1985). Comparative
electrophysiology ofpyramidal and sparsely spiny stellate neurons ofthe
neocortex. Journal ofNeurophysiology. 54:782-806.
McCormick DA, Connors BW, Lighthall JW, Prince DA. (1985). Comparative
electrophysiology ofpyramidal and sparsely spiny stellate neurons ofthe
neocortex. Journal ofNeurophysiology. 54:782-806.
McDonald AJ (1982). Neurones ofthe lateral and basolateral amygdaloid
nuclei: a Golgi study in the rat. Journal ofComparative Neurology.
212:293-312.
McDonald AJ (1984). Neuronal organization ofthe lateral and basolateral
amygdaloid nuclei in the rat. Journal ofComparative Neurology.
222:589-606.
91
McEwen BS, Alves SE, Bullock K, Weiland NG. (1997). Ovarian Steroids
and the Brain: Implications for cognition and aging. Neurology. 48:S8 
S15.
McEwen BS, Tanapat P, Weiland NG. (1997). Inhibition ofDendritic Spine
Induction on Hippocampal CAl Pyramidal Neurons by a Non-steroidal
Estrogen Antagonist in Female Rats. Endocrinology. 140(3):1044-1047.
Metzger D, Berry M, Ali S, Chambon (1995). Effect ofantagonists on DNA
binding properties ofthe human estrogen receptor in vitro and in vivo.
Molecular Endocrinology. 9:579-591.
Millhouse OE and DeOlmos J (1983). Neuronal configuration in lateral and
basolateral amygdala. Neuroscience. 10: 1269-1300.
Murphy DD, Segal M (1996). Regulation ofdendritic spine density in cultured
rat hippocampal neurons by steroid hormones. Journal ofNeuroscience.
16:4059-4068.
Ohkura T, Isse K, Akazawa K, Hamamoto M, Yaoi Y, Hagino N. (1994).
Related Articles Evaluation ofestrogen treatment in female patients with
dementia ofthe Alzheimer type. Journal ofEndocrinology. 41(4):361 
371.
Paxinos GT, Watson C. (1997). The Rat Brain in Stereotaxic Coordinates.
Academic Press, Incorporated.
Pelech SL and Sanghera (1992). MAP kinases: charting the regulatory
pathways.. Science. 257: 1355-1356.
Pike CJ, Walencewicz AJ, Glabe CG, Cotman CWo (1991). In vitro aging of
~-amyloid protein causes peptide aggregation and neurotoxicity. Brain
Research. 563:311-314.
92
Poirier J, Hess M, May PC, Finch CE (1991). Astrocytic apolipoprotein E
mRNA and GFAP mRNA in hippocampus after entorhinal cortex
lesioning. Molecular Brain Research. 11:97-106.
Potier B and Rovira C. (1999). Related Protein tyrosine kinase inhibitors
reduce high-voltage activating calcium currents in CA1 pyramidal
neurones from rat hippocampal slices. Brain Research. Jan
23;816(2):587-597.
Puy L, MacLusky NJ, Becher L, Karsan N, Trachtenberg J, Brown TJ. (1995).
Immunocytochemical Detection ofAndrogen Receptor in Human
Temporal Cortex: Characterization and Application ofPolyclonal
Androgen Receptor Antibodies in Frozen and Paraffin-embedded
Tissues. Journal ofSteroid Biochemistry and Molecular Biology.
55:197-209.
Rainnie DG Asprodini EK, Shinnick-Gallagher P (1991a). Excitatory
transmission in the basolateral amygdala. Journal ofNeurophysiology.
66:986-998.
Rainnie DG Asprodini EK, Shinnick-Gallagher P (1991b). Inhibitory
transmission in the basolateral amygdala. Journal ofNeurophysiology.
66:999-1009.
Recasens M, Guiramand J, Vignes M. (1991). The putative molecular
mechanism(s) responsible for the enhanced inositol phosphate synthesis
by excitatory amino acids: an overview. Neurochemistry Research.
16:659-668.
Reibel S, Andre V, Chassagnon S, Andre G, Marescaux C, Nehlig A, Depaulis
A. (2000). Neuroprotective effects ofchronic estradiol benzoate
treatment on hippocampal cell loss induced by status epilepticus in the
female rat. Neuroscience Letters. Mar 10;281(2-3):79-82.
93
Ripich DN, Petrill SA, Whitehouse PJ, Ziol EW. (1995). Gender Differences
in Language ofAD Patients: A Longitudinal Study. Neurology. 45: 299 
302.
Rusa R, Alkayed NJ, Crain BJ, Traystman RJ, Kimes AS, London ED, Klaus
JA, Hum PD. (1999). 17~-estradiol reduces stroke injury in estrogen 
deficient female animals. Stroke. 30(8):1665-1670.
SanMartin S, Gutierrez M, Menendez L, Hidalgo A, Baamonde A. (1999).
Effects ofdiethylstilbestrol on mouse hippocampal evoked potentials in
vitro. Neurobiology. 19(6):691-703.
Sawada M, A1kayed NJ, Goto S, Crain BJ, Traystman RJ, Shaivitz A, Nelson
RJ, Hum PD. (2000). Estrogen receptor antagonist ICI182,780
exacerbates ischemic injury in female mouse. Journal Cerebral Blood
Flow andMetabolism. Jan;20(1):112-118.
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, Greene GL.
(1998). The Structural Basis ofEstrogen Receptor/Coactivator
Recognition and the Antagonism ofthis Interaction by Tamoxifen. Cell.
95:927-937.
Shughrue PJ, Scrimo PJ, Merchenthaler. (1998). Evidence for the Co 
localization ofestrogen Receptor B mRNA and Estrogen Receptor a
Immunoreactivity in Neurons ofthe Rat Forebrain. Endocrinology.
139(12): 5267-5270.
Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM. (1999). The
Mitogen-activated Protein Kinase Pathway Mediates Estrogen
Neuroprotection after Glutamate Toxicity in Primary Cortical Neurons.
Journal ofNeuroscience. 19(7):2455-2463.
Smigel K (1998). Breast cancer prevention trial shows major benefit some
risk. Journal ofthe National Cancer Institute. 90:647-648.
94
Smith SS, Water house BD, Woodward DJ. (1987). Sex steroid effects on
extrahypothalamic CNS. 1. Estrogen augments neuronal responsiveness
to iontophoretically applied glutamate in the cerebellum. Brain
Research. 422: 50-51.
Smith SS. (1989). Estrogen administration increases neuronal responses to
excitatory amino acids as a long-term effect. Brain Research. 503:354 
357.
Teyler TJ, Vardaris RM, Lewis D, Rawitch AB. (1980). Gonadal steroids:
effects on excitability ofhippocampal pyramidal cells. Science. Aug
29;209(4460): 1017-8.
Toran-Allerand CD, Miranda RC, Bentham W, Sohrabji F, Brown TJ,
Hochberg RB, MacLusky NJ. (1992). Estrogen Receptors Colocalize
with Low-Affinity Nerve Growth Factor Receptors in Cholinergic
Neurons ofthe Basal Forebrain. Neurobiology. 89: 4668-4672.
Tsai, M and O'Malley BW. (1994). Molecular Mechanisms ofAction of
Steroid/Thyroid Receptor Superfamily Members. Annual Review of
Biochemistry. 63 :451-486.
Valentino, RJ, and Dingledine R. (1981). Presynaptic inhibitory effect of
acetylcholine in the hippocampus. Journal ofNeuroscience. 1:784-792.
Vardaris RM, Lewis D, Rawitch AB. (1990) Gonadal steroids: effects on
excitability ofhippocampal pyramidal cells. Science. 209: 1017-1019.
Washburn MS, Moises HC (1992a) Inhibitory responses ofrat basolateral
amygdaloid neurons recorded in vitro. Neuroscience. 50:811-830.
Washburn MS, Moises HC. (1992b) Electrophysiological and Morphological
Properties ofRat Basolateral Amygdaloid Neurons in vitro. The Journal
ofNeuroscience. 12(10): 4066-4079.
95
Washburn MS, Moises HC. (1992c) Muscarinic Responses ofRat Basolateral
Amygdaloid Neurons Recorded in vitro. Journal ofPhysiology.
449: 121-154.
Weiland NG, Orikasa C, Hayashi S, McEwen BS. (1997). Distribution and
Hormone Regulation ofEstrogen Receptor Immunoreactive Cells in the
Hippocampus ofMale and Female Rats. The Journal ofComparative
Neurology. 388:603-612.
Weiland NG. (1992). Estradiol Selectively Regulates Agonist Binding Sits on
the N-Methyl-D-Aspartate Receptor Complex in the CAl Region ofthe
Hippocampus. Endocrinology. Vol. 131, no. 2: 662-668.
Womble MD, Moises HC (1992). Voltage-clamp analysis ofcholinergic
action in the basolateral amygdala. Journal ofPhysiology. 457:93-114.
Womble MD, Moises HC. (1993). Muscarinic modulation ofconductances
underlying the afterhyperpolarization in neurons ofthe rat basolateral
amygdala. Brain Research. Sep 3;621(1):87-96.
Wong M, Moss RL. (1991). Electrophysiological evidence for a rapid
membrane action ofthe gonadal steroid, 17 beta-estradiol, on CAl
pyramidal neurons ofthe rat hippocampus. Brain Research. Mar
8;543(1):148-152.
Wong M, Moss RL. (1992). Long-term and short-term electrophysiological
effects ofestrogen on the synaptic properties ofhippocampal CAl
neurons.. Journal ofNeuroscience. 12(8):3217-3225.
Wong M, Moss RL. (1992). Modulation ofsingle-unit activity in the rat
medial amygdala by neurotransmitters, estrogen priming, and synaptic
inputs from the hypothalamus and midbrain. Synapse. Feb;10(2):94 
102.
Wong RK, Prince DA (1981). Afterpotential generation in hippocampal
pyramidal cells. Journal ofNeurophysiology. 45:86-97.
96
WoolfNJ and Butcher LL. (1982). Cholinergic projections to the basolateral
amygdala: a combined Evans Blue and acetylcholinesterase analysis.
Brain Research Bulletin. 8:751-763.
Woolley CS, McEwen BS. (1994). Estradiol Regulates Hippocampal Dendritic
Spine Density via an N-Methyl-D-Aspartate Receptor-Dependent
Mechanism. Journal ofNeuroscience. 14(12): 7680-7687.
Woolley CS, Weiland NG, McEwen BS, Schwartzkroin PA. (1997). Estradiol
increases the sensitivity ofhippocampal CAl pyramidal cells to NMDA
receptor-mediated synaptic input: correlation with dendritic spine
density. Journal ofNeuroscience. Mar 1;17(5):1848-1859.
Woolley CS. (1998). Estrogen-mediated structural and functional synaptic
plasticity in the female rat hippocampus. Hormones and Behavior.
Oct;34(2):140-148.
Woolley CS. (1999). Effects ofEstrogen in the CNS. Current Opinion in
Neurobiology. 9: 349-354.
Wrenn CK, Katzenellenbogen BS. (1993). Structure-Function Analysis ofthe
hormone Binding Domain ofthe Human Estrogen Receptor by Region 
specific Autogenesis and Phenotypic Screening in Yeast. The Journal of
Biochemistry andMolecular Biology. 268: 24089-24098.
Wyneken U, Riquelme G, Villanueva S, Orrego F. (1997). Effect ofglutamate
receptor phosphorylation by endogenous protein kinases on electrical
activity ofisolated postsynaptic densities ofcortex and hippocampus.
Neuroscience Letters. Mar4;224(2):131-135.
Zakon Harold H. (1998) The Effects ofSteroid Hormones on the Electrical
Activity ofExcitable Cells. Trends in Neuroscience. 21:202-207.
97