Effects of Estradiol Deprivation on Striatal-Dependent Learning and Memory in Rat Models of Menopause

A Thesis Submitted in Partial Fulfillment of theRequirements of the Renée Crown University Honors Program

and the Distinction in Biology Program atSyracuse University

Rebekah Schwartz

Candidate for Bachelor of Scienceand Renée Crown University Honors

May 2020

Honors Thesis in Biology

Thesis Advisor: _______________________ Donna Korol, PhD

Thesis Reader: _______________________ Susan Parks, PhD

Honors Director: _______________________ Dr. Danielle Smith, Director

© (Rebekah Schwartz May 2020)

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Abstract

As women enter the climacteric that leads to menopause, ovarian hormone levels begin to drop, leading to widespread physiological changes in the adult organism. It is thought that declines in estrogens mediate many of these changes. Although estrogens are typically considered reproductive hormones they indeed have far-reaching effects on the body beyond just the reproductive system. Specifically, estrogens have numerous impacts on the brain, influencing essentially all regions studied to date, even those that lack classical estrogen receptors. One of those effects is neural protection against age- and disease-related changes, possibly through facilitation of neural plasticity. Estrogens can protect against AD and PD, perhaps via direct actions on hippocampus, striatum, and related structures. However, estrogens can also produce both enhancements and impairments on learning and memory, depending on the type of problem to be solved and treatment regimen. Specifically, acute treatments of estrogens for two days enhance learning and memory that rely on the hippocampus and impair learning and memory that rely on the striatum (Korol and Kolo, 2002; Korol, 2004). Thus, it appears that estrogens have two types of hormone action on the brain, the activating effects on cognition that may be mixed and depend on physiological state of the individual and trophic or long-term effects on the brain that may be largely protective. The time at which hormone therapy is begun may play a role in how estrogens produce these effects on the brain. The critical window hypothesis states hormone replacement therapy is only effective within a certain window of opportunity after menopause begins (McCarrey and Resnick 2015, Maki et al 2013, Sherwin and Henry 2008). Given that these hormones have such far reaching neurobiological effects, the current study was undertaken to assess how learning and memory is altered when the rat is in a state of hormone deprivation modeled after menopausal women. This study evaluated the changes in cognition resulting from increased hormone deprivation during menopausal transition and whether the brain remains sensitive to the action of estradiol with increased hormone deprivation. An impairment in striatal learning from estradiol after three weeks of hormone deprivation, was not found despite a large body of evidence from previous work. Increasing the duration of hormone deprivation also did not lead to impaired performance as was expected due to the proposed loss of neuroprotective effects of estrogens. Several possible explanations for the lack of estradiol effects are explored.

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Executive Summary

Menopause is becoming increasingly salient with the increase in human lifespan. As

lifespan increases, women are spending an increasing proportion of their lives in the

postmenopausal period. As women approach menopause, ovarian hormone levels decline. This

decline, specifically in estrogens, may control the multitude of physiological changes associated

with this period. Estrogens, which we typically consider as one hormone, actually comprise

three different hormones: estrone (E1), estradiol (E2), and estriol (E3) (Cui et al 2013). E2 is

the predominant form of estrogen in the body of reproductively cycling women, and is thus the

focus of this study (Cui et al 2013).

Beyond its role in the reproductive system, E2 mediates a wide array of functions in the

body, most specifically in regards to brain health. E2 plays a protective role in the female brain

against AD and PD, by not only reducing the risk of these neurodegenerative diseases, but also

by ameliorating some of the cognitive deficits associated with them. With the increase in time

spent as a postmenopausal woman, the loss of these protective effects has robust implications

on brain health and progression of neurodegenerative disease.

During menopause, the amount of cycling hormones decreases as a result of the loss of

follicle supply. During the transition into menopause, serum levels of E2 drop by about 85-90%

(Cui et al 2013). This disruption in hormone levels poses implications throughout the body, far

beyond the reproductive system alone. The lack of E2 has implications for learning and

memory, which makes both hormone deprivation and hormone replacement of interest to the

lab.

Previous work in our lab shows that acute E2 given just before maze training impairs

striatum-sensitive learning after three weeks of ovarian hormone deprivation by surgically

induced menopause in rats (Korol and Kolo 2002). Additional findings suggested that the

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impairing effects of E2 were even more robust one week after hormone reduction, primarily

because performance in the hormone-deprived rats was still very strong at this time point

especially when compared to the rats that were hormone deprived for three weeks (Korol and

Kolo, 2002). Together, these findings suggest that although increases in E2 in the circulation

impairs striatum sensitive learning, the long-term effects of E2 loss may also impair striatum-

sensitive learning. Thus, estrogens might have two types of hormone action on the brain, the

activating effects on cognition that may be mixed and depend on the type of problem to be

solved and the physiological state of the individual and also the trophic or long-term effects on

the brain that may be largely be neuroprotective.

This study focuses on elucidating effects of hormone deprivation and replacement in a

model of surgical menopause. In this model, hormone deprivation lasts either three or six weeks

to assess how increasing length of hormone deprivation influences learning. This study extends

the deprivation period while also examining effects of different doses of E2 replacement on

learning. The goal of this project was to assess how differing the length of hormone deprivation

after surgically induced menopause in young adult rats impairs learning and memory.

This project’s first aim is to replicate past work finding an impairment on striatal learning

using this same methodology. This study is novel in its extension of this period of hormone

deprivation to six weeks. E2 treatment, regardless of time point, should produce the same

learning results, whereas the rats deprived of ovarian hormones are expected to have impaired

learning with increased time since surgery. For the rats in the six-week time point, this means

that both groups will have an impairment on learning for different reasons, and thus their

learning behavior may appear to be similar.

In contrast to expected results, impaired striatal learning from E2 replacement after three

weeks of hormone deprivation was not shown in this study. Further, an impairment on striatal

learning in vehicle treated rats with a longer, six-week, hormone deprivation was not found.

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Though this study did not replicate the impairment on striatal learning or identify

significant differences in learning between the two time points and dosages, the work lends itself

to assessing current methodology to make improvements for future studies. The issues

identified in this study provide a basis for a more expansive study looking at the critical window

hypothesis of hormone therapy, as well as new directions investigating the relationships

between stress, E2, and learning.

The cycling of these reproductive hormones, and the confounds they introduce to

experiments has continued to hinder scientific discovery in females. Much of our scientific and

medical knowledge has only been tested in males due to this reason. This has ultimately

resulted in therapies designed for males, as effects on women are difficult to tease out from

inconsistent hormone levels across time. This study works to understand some of the effects of

female reproductive hormones on cognition, working against this bias in the literature towards

male subjects.

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Acknowledgements

First and foremost, I would like to thank Donna Korol for accepting me into her lab,

editing multiple drafts of this project and providing continuous support, both research, and non-

research related for the past three years. I would also like to thank Paul Gold, whose expertise

and guidance have helped me to grow throughout my time here at Syracuse. Additionally, I

would like to thank Susan Parks for agreeing to be my reader and editing this project so

efficiently. Special thank you to Robert Gardner for assisting me with statistical analyses. This

project would not have been possible without the mentorship of Stephen Ajayi, Alesia

Prakapenka, and Robert Gardner, and the assistance and support of my fellow lab-mates.

Specifically, I would like to acknowledge Sarah Riddle and Brooke Rhinesmith for helping with

some of the surgeries. I would also like to acknowledge the LAR staff, for working so hard to

take care of all of the rats and ensure their well-being throughout this project. Lastly, I would

like to thank the Reneé Crown Honors Program and the Biology Department for supporting me

in a multitude of ways throughout my undergraduate career and granting me the opportunity to

conduct my research.

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Table of Contents

Abstract……………………………………….……………….………………………………………... iii

Executive Summary………………………….……………….………………………………………. iv

Acknowledgements………………………………………………………………………………….. vii

Introduction...…………………………………………………………………………………………… 1

Materials and Methods………………………………………………………………………………... 6

Results…………………………………………………..……………………………………………… 12

Discussion……………………………………………...……………………………………………… 25

Works Cited.…………………………………………………………………………………………… 30

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Introduction

Estrogens are typically thought to play a role in reproduction, however, they have far-

reaching effects on other areas of the body, most notably the brain. Estradiol (E2), the most

potent estrogen in reproductive women, is widely known for its protective effects on the brain. A

number of studies show a protective effect of E2 on the brain especially in relation to

neurodegenerative disorders such as Alzheimer’s Disease (AD) and Parkinson’s Disease (PD).

For example, increased risk of PD has been linked to early reduction in estrogens (Ragonese et

al 2006, Villa et al 2006, Benedetti et al 2001). Similarly, postmenopausal treatments with

estrogens is associated with decreased risk and delayed onset of AD, suggesting the loss of

hormones that comes from menopause exposes the brain to degeneration (Gandy 2003, van

Dujin 1999, Tang et al 1996). Interestingly, one brain region shown to degenerate and show

metabolic dysregulation with AD (Korol and Wang 2018) and with normal aging (Gardner et al.,

2020) is the hippocampus.

The hippocampus is positively affected by the action of estrogens, with increased age

and the concomitant loss of estrogens make this brain area more vulnerable to neural insults.

Administration of estrogens is thus thought to be neuroprotective (Daniel 2013). Due to the

beneficial effects of estrogens on the hippocampus, performance is typically enhanced on tasks

utilizing this brain area, and no effects or impairments on other brain areas, of note the striatum

(Daniel 2013). The presence of estrogens can alter the cognitive strategy rats use to solve a

task, thus differentially affecting tasks of learning and memory depending on brain area required

for that process (Korol and Kolo, 2002; Korol, 2004; Daniel and Lee 2004, Daniel et al 1999,

Quinlan et al 2008). Inactivation of the hippocampus with lidocaine causes a shift in rat

preference for striatal-based learning tasks (Packard and McGaugh 1996). Similarly, rats given

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lidocaine injection into the striatum show a shift in preference for hippocampal based task

(Packard and McGaugh 1996). Infusion of E2 in the hippocampus enhances learning on place

task with no effect on the response task and infusion into the striatum impairs response learning

with no effect on place learning. This double dissociation between infusion site and effect on

performance (Zurkovsky et al 2007) suggests that E2 acts directly on each structure to

modulate learning in the canonical cognitive task.

Estrogens increase dopamine (DA) function particularly within the basal ganglia, a set of

structures including the striatum important for sensory-motor integration, reward value, and

cued-based behaviors. Within the striatum, estrogens and DA treatment increases firing by

neurons connecting the basal ganglia and the striatum (Arnaud et al 1981, Mizumori et al 2004).

Perhaps this is mediated by estrogen-induced DA release (Mizumori et al 2004). E2 treatments

to ovariectomized (OVXd) mice depleted of DA with a neurotoxin to the nigrostriatal DA pathway

lead to increased DA concentration in the striatum (Dluzen and Horstink 2003). Administration

of dopamine receptor antagonists into OVXd rats receiving 17-estradiol benzoate (EB)

treatment shifts strategy preference between hippocampal and striatal based learning tasks,

suggesting the important role of both EB and DA in strategy selection (Quinlin et al 2008).

Benzoate conjugation of E2 to form EB slows down liver metabolism so it lasts longer in

circulation, better matching what is seen during the estrous cycle. EB could mediate choice of

cognitive strategy both by improving the functions of the hippocampus, but also altering that of

the striatum, which is regulated by DA (Quinlin et al 2008). It is thought that a moderate level of

DA signaling produces optimal striatum-sensitive learning; too little or too much would thus

impair striatum-based cognition.

These robust bidirectional effects of estrogens on brain and cognitive function suggest

that endogenous fluctuations in ovarian hormones across the reproductive cycle will lead to

shifts in learning strategy. Hormone-regulated shifts in cognitive ability might also be found with

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transitions to physiological states that lose ovarian cyclicity, such as with pregnancy or

menopause.

Menopause occurs when the ovaries become depleted of follicles and subsequently

produce decreased amounts of ovarian hormones. Clinically, menopause is defined as 12

months after the last menstrual cycle and has an average age of onset of 52.5 years of age

(McCarrey and Resnick 2015). The transition into menopause is associated with a multitude of

symptoms, the most common being hot flashes, but often accompanied by night sweats and

sleep problems (von Mühlen et al 1995). The second most common complaint bringing women

into the clinic relates to cognitive deficits. Post-menopausal women claim they experience the

tip-of-the tongue phenomenon, whereby one struggles to recall a specific word, but can recall

those of similar structure and meaning, and also complain of general cognitive fogginess

accompanied by short attention spans and forgetfulness (Brown and McNeill 1966, Mitchell and

Woods 2001).

Female rats exhibit cycling reproductive hormones translatable to the menstrual cycle.

The hypothalamic-pituitary-gonadal axis is responsible for this cycling through stages in females

and the lack thereof in males (Plant 2015). This cycle in rats is referred to as the estrous cycle.

The estrous cycle consists of the stages proestrus, estrus, and diestrus. During proestrus, there

is a spike in levels of estrogens, followed by a spike in progesterone levels. During estrus, rats

are in heat and ovulation occurs. During diestrus, progesterone levels are intermediate.

Female rats also undergo a shift in reproductive hormones occurring at middle age,

slightly differently than what occurs in menopause (Morrison et al 2006). The estrous cycle

begins to become irregular at about 9—12 months of age (Morrison et al 2006). Specifically,

female rats begin to have irregular cycling that ultimately ceases altogether (Morrison et al

2006). The loss of cycling does not coincide with follicular degeneration and decline in estradiol

concentration, which occurs concurrently in menopause (Morrison et al 2006). Acyclic rats,

rather, are in a state of continuous estrus (Morrison et al 2006). Due to these differences, a

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surgically induced menopause model via OVX is often used for answering research questions

regarding menopause (Morrison et al 2006).

Hormone therapy (HT) was initially developed to abate hot flashes and is still a common

choice for women experiencing menopausal symptoms (Shanafelt et al 2002). The Women’s

Health Initiative (WHI) Studies, however, have produced a sense of uncertainty about the

effectiveness of HT. The WHI began in 1993 and aimed to provide an overview of the risks and

benefits of hormone therapy, yet had major flaws in methodology. The WHI showed

introduction of estrogen therapy did not improve cognitive function in women who were

postmenopausal for 15 years (Rapp et al 2003) In fact, HT was shown to increase risk of mild

cognitive impairment and dementia in postmenopausal women (Shumaker et al 2003,

Shumaker et al 2004). Despite these flaws, the WHI studies informed the scientific, medical,

and lay communities that dosage, timing, and target tissue (brain, colon, breast, cardio-vascular,

etc.) are important variables for HT.

The results of the WHI led to further investigation of HT that showed effects of HT

depend on timing, formulation, mode of delivery, and regimen (Miller and Harman 2017, Zárate

et al 2017). The WHI ultimately led to many future experiments addressing the flaws within that

study, leading to the development of the “critical window hypothesis”, positing there is a time

period after hormone decline with menopause wherein hormone therapy will remain effective

(McCarrey and Resnick 2015, Maki et al 2013, Sherwin and Henry 2008). HT appears to be

most effective when administered at the time of menopause, becoming less effective as time

passes beyond the onset of menopause. The duration of the critical window depends upon the

age of the individual and the target system or property.

The mechanism underlying the critical window hypothesis is unknown, but could be due

do the need for estrogen-sensitive neurons to adapt to estrogen-depleted environments (Yin et

al 2015). Specifically, these neurons lose their signaling integrity with increased age and

deprivation, thus depriving them of the neuroprotective action of estrogens. One way that

xii

estrogens signal in neurons is through estrogen receptors. Interestingly, after long periods of

hormone deprivation, the beneficial effects of estrogens can be reintroduced by increasing the

expression of the gene encoding for ER, one of the two main types of nuclear receptors for

estrogens (Bean et al. 2015), but not ER, the other nuclear estrogen receptor.

We have shown that E2 has differential effects on different areas of the brain.

Specifically, on a hippocampal-dependent learning task, E2 replacement enhances learning,

whereas on a striatal-dependent task, E2 replacement impairs learning (Korol and Kolo 2002).

Female preference of learning task also changes across the estrous cycle. During proestrus,

when ovarian hormones are high, rats prefer to use a spatial, or hippocampal-dependent,

learning task (Korol et al 2004). Rats in estrus, who have lower ovarian hormone levels prefer

to learn using their striatum (Korol et al 2004). These data alone tell us the critical window

remains open at least for 21 days after ovariectomy (OVX) in female rats.

Our preliminary results investigating the critical window found vehicle control or

“placebo” oil-treated rats showed impaired learning behavior three weeks after OVX as

compared to oil-treated rats trained 6 days after OVX (Korol, unpublished). Our data show

decreased performance in the oil-treated rats, with performance of EB-treated rats remaining

the same across these two time points. Thus, we expect the oil-treated rats to continue to

become worse at performance if deprived from ovarian hormones for an additional three weeks.

Without this knowledge, we may expect EB to still impair learning at 6 weeks post OVX. Our

preliminary data suggest, however, that something is also lost in the impairing effect of EB

because oil rats should continue to worsen while performance with EB treatment remains the

same. This means that with increased deprivation, EB would not have the same impairing effect

with increased hormone derivation because oil rats are also getting worse. In this case, the

impairment caused by the deprivation of E2 may match, or even surpass, the impairment

caused by the presence of E2.

xiii

Perhaps this loss of impairing effect of EB is due to a loss in sensitivity to EB in the

brain. If impairment occurs via disrupted cell-cell signaling, then a lack of impairing effect after

six weeks of hormone deprivation would suggest the impaired performance on the response

task is related to a loss of signaling within the brain, perhaps due to a loss of DA or DA neurons,

or a lack of ERs present for EB to increase DA production.

This experiment aims to determine if sensitivity to EBs changes after six weeks of hormone

deprivation in a rat model of surgically-induced menopause. Secondly, this project aims to

determine how quickly the learning behavior in vehicle-treated rats declines following OVX.

Materials and Methods

Subjects

Young-adult, virgin, female Sprague-Dawley rats (3-mo-old) were obtained from Envigo

(N=33). The rats were individually housed in plastic cages and were on a strict 12:12 light/dark

cycle. Each had free access to food and water until the initiation of food-restriction procedures

to motivate rats to perform the appetitive cognitive task. All rats were OVXd to remove

circulating hormones and trained on a striatum-sensitive response maze learning task either 3

weeks (21-22 days) or 6 weeks (38-44 days) after OVX. Rats were randomly assigned to one

of three hormone treatment groups: vehicle control, low dose of EB, high dose of EB. [Note:

sample sizes for the six-week time point and the 4.5g/kg EB treatment are lower than expected

due to unforeseen health issues that interfered with my ability to train the rats.]

All procedures were conducted according to the National Institutes of Health Guide for

the Care and Use of Laboratory Animals and were approved by the Syracuse University

Institutional Animal Care and Use Committee.

xiv

Ovariectomy

Rats were bilaterally OVXd under isoflurane anesthesia. Rats were put in an induction

chamber filled with 5% isoflurane anesthesia for 10 minutes. The rats’ sides were quickly

shaved and the rat was weighed before transfer to an anesthesia nose cone (1.3-5%) on a

surgery table. Isoflurane was delivered at a rate maintaining one breath per second. Eye

ointment was added to lubricate the eyes. The rats were given an intramuscular injection of

penicillin to prevent infection and a subcutaneous injection of flunixon to prevent pain. The

shaved area was cleaned with 70% ethanol and betadine before making the incision ~150mm

from the lowermost rib. The fascia was cut and the muscle blunt-dissected to locate the fat

pads. The fat pad and ovary were pulled through the hole in the muscle and clamped with soft

tissue forceps on the uterine horn. The ovary was tied off and removed. The remainder of the

fat pad was gently put back into the abdominal cavity. The muscle was sutured together and the

skin was secured with tissue adhesive and closed with 2-3 wound clips. The incision was

cleaned throughout the procedure with 70% ethanol and bacitracin was applied after closing.

The same procedure was followed for the other side. Directly following closure of the wound,

bacitracin was applied to further prevent infection and 10mL of saline (s.c.) was given to replace

lost fluids. Children’s Motrin (2.35mL/500mL water) was given in the drinking water for 24

hours following the surgery with free access to the subjects. Post-operative health and wellness

checks were conducted for the week following surgery to ensure proper recovery.

Estradiol Administration

Rats from each time point were randomly assigned to be in either the EB group or the

vehicle control group. A single injection was given to rats both 48hrs and 24hrs prior to

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behavioral training. Injections consisted of either a sesame oil vehicle, a low (4.5µg/kg) dose of

EB in sesame oil, or a high (45 µg/kg) dose of EB in sesame oil. The low dose of EB treatment

was only used for the 3-week group. Final rat numbers for each group were as follows: 3-week

high dose OIL, N = 6; 3-week high dose EB, N = 8; 6-week high dose OIL, N = 4; 6-week high

dose EB, N = 4; 3-week low dose OIL, N = 5; 3-week low dose EB, N = 4. Rats received two

days of a single injection given at the same time as behavioral training would take place. This

regimen controlled for possible interactions in hormone levels with shifting circadian rhythms,

thus ensuring circulating titers are as similar across rats as possible.

Food Restriction

During food restriction, the rats were limited to a certain amount of food each day,

between 6 to 13 grams, supplemented by 3 pieces of Frosted Cheerios®, the food reward used

during behavioral training. Food restriction lasted between 7-10 days until the animal reached

80-85% of their starting weight.

Training

For response learning, rats were trained to make a body turn to the right or left to find

food on a simple T-shaped maze (Figure 1). The training maze was a symmetrical 4-arm maze,

constructed from black Plexiglas®, that had one arm blocked off each trial to make it a T shape,

and was located in a room. The wall cues were curtained to reduce the use of spatial cues

instead of a body turn to guide navigation. The arms of the maze were each 45cm long, 14cm

wide and 7.5cm tall that extended from a 14x14cm central square. At the end of each maze

arm was a cup that contained inaccessible crumbs of the food reward to prevent the rat from

using olfactory cues to guide its navigation.

xvi

The arm that was rewarded had the food reward, half of one frosted Cheerio®, placed

on top of this cup. The direction of the turn was held constant for each rat over the course of

the training session. The correct turn (right or left) for a specific training session was

counterbalanced among the rats. Rats were place in a clean holding cage prior to training to

further prevent them from using olfactory cues. The subjects were put in the training room to

get accustomed to the training environment prior to training. During training, the rat was placed

on the start arm (stem of the “T”) and allowed to enter a single goal arm in attempt to find the

food reward. A choice was defined as entrance of all four paws out of the central square into

one of the choice arms. The rat was removed from the maze approximately 3 seconds following

consumption of the frosted Cheerio®. If the rat did not eat the Cheerio®, the rat was left in the

arm for approximately 10 seconds, or until it just turns around to leave the arm to allow them

time to think.

Time to choose and correct vs incorrect choices were recorded on the data sheet. Any

unusual behaviors were also recorded. There was a 30 second interval between trials, when

the maze was also rotated to prevent using cues from within the maze. If the rat took longer

than two minutes to make a decision, the rat was removed from the maze and placed back into

the cage to start a new trial. This was recorded as no decision on the data collection sheet. All

rats were trained for a total of 75 trials.

Figure 1 Graphic depicting the maze used for response behavioral training.

xvii

Determination of Estrogen Status

Vaginal smears of the rats were taken each day of food-deprivation to track the stage of

the cycle of the rats, to ensure that the OVX was complete and to verify that rats in the EB

groups showed change in vaginal cytology indicating circulating estrogens. Smears were taken

daily at the same time for 7-10 days prior to behavioral testing. Vaginal smears were taken by

soaking a small sterile cotton swab in sterile saline and gently swabbing the vaginal wall. The

slides were fixed with EtOH, and then stained using toluidine blue, coverslipped, and observed

under a light microscope to determine the estrous stage of each rat based on the type of cells

present on each slide. After OVX, rats should stop cycling; thus, smears from oil controls

should resemble those from cycling rats in diestrus (Auletta 1994). The smears taken from the

EB-treated rats after injection should indicate the return of cycling and appear as a proestrous-

like smear.

Sacrifice and Tissue Collection

Immediately following behavioral training, the rat was injected with a lethal overdose of

1mL sodium pentobarbital (50mg/mL, i.p.). Once the rat was no longer responsive to both a toe

pinch and blink test, it was decapitated using a laboratory rodent guillotine. Following

decapitation, trunk blood was harvested for later analysis of serum hormone levels. Given its

high estrogen sensitivity, one uterine horn was dissected out and 1.0 cm weighed to assess

hormone state. The brain was removed from the skull for collection of the striatum, frontal

cortex, hippocampus, cerebellum, and medulla for biochemical analyses used in a different

experiment.

Quantification of Serum Estradiol Levels

xviii

Trunk blood was centrifuged at 3500rpm for 15 minutes (2013Gs) and the serum

supernatant collected and stored at -20C until processing. An ELISA kit (Cayman Chemical)

was used to quantify the E2 levels in the serum, following the manufacturer’s protocol.

Data analysis

Measures

The main cognitive measures were trials to criterion and percent correct choices per trial

block. Learning criterion was set at 9/10 trials with 6 trials in a row correct. Percent correct arm

choices were blocked into groups of 15 trials of training, reflecting accuracy of learning

throughout behavior. Other physiological measures included weight prior to behavioral training,

uterine horn weight, and serum E2 concentration.

Statistical Analysis

T-tests were used to assess differences in uterine horn weight and trials to criterion.

Because of the low sample size, non-parametric analyses using the Mann Whitney U test were

run for trials to criterion to avoid type II errors (false negative). Mann Whitney U tests were not

performed for the time of day analysis due to low sample sizes. Repeated measures ANOVAs

were used to assess differences in treatment condition and learning behavior. 2-way factorial

ANOVAs (Analysis of Variance) were used to compare three-week and six-week data.

Exclusions

Two of the rats in the six-week group were excluded from the data because they failed to

perform on the maze. This was determined by failure to move on the maze for two minutes for

five consecutive trials. One of these rats was treated with oil and the other with 45g/kg EB.

xix

Results

Theoretical Results

The expected behavioral results for the main aim of this experiment are shown in Figure

2 for comparing behavior of rats treated with 45g/kg EB after either 3 weeks or 6 weeks

between OVX and behavioral training. Figure 2A shows an impairment on learning with EB

treatment. Looking between figures 2A and 2B, the oil rats do progressively worse with

increased time between OVX and response training. If sensitivity to estrogens is maintained

despite the loss of function, then replacement would still impair an already deficient response

learning. If the effects of estrogens are lost along with the neural protection, then replacement

of estradiol would produce the same behavior as replacement with oil. Because it appears that

sensitivity to EBs is reduced from 1 to 3 weeks, we predicted that EB would have no effect after

6 weeks. This is why in Figure 2B, EB does not have an impairing effect on response training

because the behavior of oil rats was reduced compared to oil rats 3 weeks after OVX with no

change in EB-treated rats. Essentially, the impairment brought about from increased hormone

deprivation will match or could even exceed the impairment of EB on striatal learning.

xx

1 2 3 4 540

60

80

1003 Week Oil 3 Week EB

% C

orre

ct

A

1 2 3 4 540

60

80

1006 Week Oil6 Week EB

% C

orre

ct

B

Figure 2 Expected performance on the response learning task three weeks (A) and six weeks (B) after OVX and treatment with either 45g/kg EB or sesame oil vehicle. EB impairs striatal learning when administered 3 weeks after OVX (A). Similar performance is expected from both EB and oil treated rats six weeks after OVX (B).NOTE that the oil-treated, hormone deprived rats show significant decline in performance from 3 to 6 week time points.

Task Acquisition

Three-week time point – high dose EB:

All of the rats in this group started around chance performance (50%) and then reached

learning criterion within the 75-trial limit. Evaluation of percent accuracy shows a significant

increase in learning across trial blocks (F(,4,48) = 25.64; p<0.0001). There was no main effect

of treatment for rats on the three-week time point, (F(1,12) = 0.0001; p>0.99). There was no

interaction between performance across trial block and treatment in rats on the three-week time

point (F(4,48) = 0.343; p>0.84) (Figure 3A). There was no significant difference between trials

to reach criterion between these same groups (t(7) = 0.017, p > 0.05; MW U(6,8) = 23.5; p >

0.05) (Figure 3B).

xxi

1 2 3 4 540

60

80

100

Oil (n=6)Estradiol (n=8)

Trial Block

Perc

ent C

orre

ct (

Mea

n ±

S.E.

M)

A

chance

10

20

40

60

80

100

Oil (n=6)Estradiol (n=8)

Tria

ls to

Crit

erio

n

(Mea

n ±

S.E.

M)

B

Figure 3 Peripheral treatment of a high dose of EB (45g/kg) did not show a significant effect on the learning curve (A) nor the trials to reach learning criterion (B) after 3 weeks of hormone deprivation as compared to vehicle-treated rats.

xxii

Time of Day Assessment in 3-week high EB groups:

One possibility for our lack of robust striatum-sensitive learning impairments by EB was

that most rats in this study were trained at earlier times in the day compared to our previous

reports. All of the rats in each of the groups were trained ~ 9:00-10:00AM, except for some of

the rats in the three-week time point treated with the 45g/kg EB were trained at ~12:30PM.

These rats were placed into groups depending on both treatment, oil or EB, and time of training,

9:00AM or 12:30PM (Figure 4; note, splitting up the data is the reason for the smaller sample

size in making the time of day comparison). There were no main effects of treatment or time of

day regardless of trial block (all p’s > 0.5). There is a significant difference in performance

across trial blocks, without regard to treatment or time of day tested in rats on the three-week

time point, excluding the low dose cohort (F(4,40) = 25.357; p<0.0001). There is no interaction

between performance across trial block and time of day tested, regardless of treatment in rats

on the three-week time (F(4,40) = 1.081; p>0.37). There is no interaction between performance

across trial block and treatment, regardless of time of day tested in rats on the three-week time,

excluding the low dose cohort (F(4,40) = 0.345; p>0.84). There is no interaction between

performance across trial block, treatment and time of day tested in rats on the three-week time

point, excluding the low dose cohort (F(4,40) = 0.333; p>0.85). Visual inspection of the data

suggests there is a subtle benefit of being trained in the morning; rats trained in the morning

started out slightly better than those trained in the afternoon (Figure 4A). However, time of day

the rats were trained did not have a significant effect on the trials to reach the learning criterion

in oil-treated rats (t(3) = -0.64, p >0.57) or EB-treated rats (t(6) = 0.56, p>0.59), as shown in

Figure 4B.

xxiii

1 2 3 4 5

20

40

60

80

100

MORNING OIL (n=3)

MORNING EB (n=5)

AFTERNOON OIL (n=3)

AFTERNOON EB (n=3)

Perc

ent C

orre

ct (M

ean

± S.

E.M

)

A

chance

10

20

40

60

80

100MORNING OIL (n=3)

MORNING EB (n=5)

AFTERNOON OIL (n=3)

AFTERNOON EB (n=3)

Tria

ls to

Crit

erio

n

(Mea

n ±

S.E.

M)

B

Figure 4 Comparison of behavior based on treatment of 45g/kg EB or oil vehicle three weeks after OVX and time of day behavioral training took place. The learning curve (A) and the trials to criterion (B) reveal time of day did not play a significant role on behavior.

xxiv

Six-week time point:

Most (8/10) of the rats in this group reached learning criterion within 75 trials without

regard to treatment. Evaluation of percent accuracy shows a significant increase in learning

across trial blocks (F(4,40) = 10.283, p<0.0001). As with the 3-week deprivation, 45 g/kg EB

treatment did not increase trials to criterion when administered 6 weeks after OVX (t(4) = 0.972,

p > 0.05; MW U(4,4) = 4; p > 0.05). There is no main effect of treatment in rats on the six-week

time point (F(1,6) = 0.06; p>0.81). There is no interaction between performance across trial

block and treatment in rats on the six-week time point (F(4,24) = 0.526; p>0.71) (Figure 5A-B).

In comparing the rats in the three-week group to those in the six-week group, the oil rats

did not have higher trials to criterion with increased hormone deprivation on the response task,

as was expected (t(6) = -0.089, p > 0.05; MW U(4,4) = 12, p>0.05). There is no main effect of

testing time on response learning in oil or EB-treated rats from three weeks to six weeks ((F(1,8)

=.204; p>0.66); (F(1,10) = 2.069; p>0.18)). There is no interaction between performance across

trial block and testing time in oil (F(4,32) = 0.968, p>0.43) or EB-treated rats (F(4,40) = 0.216;

p>0.92).

There is a significant difference in performance across trial blocks, without regard to

treatment or time after OVX in oil rats (F(4,72) = 31.411; p<.0001). There is no main effect of

weeks since OVX (F(1,18) = 1.127; p>0.3), or of treatment (F(1,18) = 0.038; p>0.84) on

performance. There is no interaction between performance across trial block and testing time

regardless of treatment (F(4,72) = 0.571, p>0.68), between performance across trial block and

treatment regardless of testing time (F(4,72) = 0.146, p>0.96), or between treatment and testing

time regardless of trial block (F(1,18) = 0.38; p>0.84). There is no interaction between

performance across trial block, treatment and testing time (F(4,72) = 0.787, p>0.53).

xxv

1 2 3 4 540

60

80

100

Oil (n=4)Estradiol (n=4)

Perc

ent C

orre

ct (M

ean

± S.

E.M

)

A

chance

10

20

40

60

80

100

Oil (n=4)EB (n=4)

Tria

ls T

o C

riter

ion

(Mea

n ±

S.E.

M)

Oil (n=4)Estradiol (n=4)

B

Figure 5 45g/kg EB treatment did not impair learning when administered 6 weeks after OVX. The learning curve (A) nor the trials to reach learning criterion (B) differed between EB-treated groups and oil vehicle treated groups.

Three-week time point – lower EB dose:

xxvi

After collecting and analyzing the data for the two time points using the high dose and

noting the lack of impairing effects of EB on learning, a different dose was used to determine if

indeed a lower dose was effective. Although the original findings showing the striatum-sensitive

impairments used the physiologically high dose of EB, recent work from our lab showed that a

lower dose of 4.5g/kg was more effective at impairing response learning and at enhancing

hippocampus sensitive cognition (Pisani et al., 2012). All rats reached the learning criterion

within the 75 trials, with learning curves showing significant improvement across trial blocks

(F(4,28) = 9.429, p < 0.0001). The impairment of EB was still not observed. In fact, the EB-

treated rats were slightly better at solving the maze than the oil-treated rats were, however this

difference was not significant as there was no main effect of treatment on learning curves

(F(1,7) = 0.596; p>0.46) or interaction between performance across trial block and treatment in

rats on the three-week time point with low EB doses (F(4,28) = 0.430; p>0.78) (Figure 6A).

These rats also showed no difference in trials to learning criterion (Figure 6B) when evaluated

parametrically (T(7) = -0.29, p > 0.05) or non-parametrically (MW U(4,5) = 8; p > 0.05).

xxvii

1 2 3 4 520

40

60

80

100

OIL (n=5)EB (n=4)

Perc

ent C

orre

ct (M

ean

± S.

E.M

)

A

Oil (n=5)

Estradiol (n=4)

chance

10

20

40

60

80

100

OIL (n=4)EB (n=4)

Tria

ls to

Crit

erio

n (M

ean

± S.

E.M

)

BOil (n=5)

Estradiol (n=4)

Figure 6 Administration of 4.5g/kg EB did not impair striatal learning when administered 3 weeks following OVX. Learning behavior between the EB-treated rats and the oil vehicle treated rats (A). The trials to reach learning criterion was not significantly different between groups (B).

xxviii

Verification of Circulating Estradiol

Uterine horn weights:

Uterine horn weights (Figure 7) were significantly higher in EB-treated rats than in their

vehicle-treated counterparts, regardless of dose and length of hormone deprivation (Effect of

4.5µg/kg in 3 week time point: (t(3) = 8.60, p = < 0.005; MW U (4,5) = 0, p<0.05); Effect of

45µg/kg in 3 week time point: (t(9)= 3.37, p = 0.001; MW U (6,8) = 5, p<0.05); of Effect 45µg/kg

in 6 week time point: (t(4) = 3.65, p = <0.05; MW U (4,4) = 0, p<0.05)). Rats administered

4.5g/kg EB did not have a lighter uterine horn weight than rats given 45g/kg EB (t(4) = -0.55,

p = 0.61; MW U (4,4) = 7, p>0.05). Uterine horn weights verified ovariectomies were successful

and EB treatments were effective.

3 Week - Low Dose 3 Week - High Dose 6 Week - High Dose0

20

40

60

80

100

5 6 44 8 4

OILEB

Ure

rine

Hor

n W

eigh

t (m

g)

(Mea

n ±

S.E.

M)

*** **

*

OilEstradiol

Figure 7 Assessment of uterine horn weight to verify levels of circulating E2 levels and thus the success of subcutaneous injections prior to training. *p<0.05 **p<0.01 ***p<0.001

xxix

Serum Estradiol Levels

As shown in Figure 8, circulating E2 levels were significantly higher in the 45g/kg EB-

treated rats than the vehicle-treated in the three-week time point (t(8) = -4.20, p<0.01; MW U

(6,8) = 0, p<0.05 and the six-week time point (t(6) = -7.60, p<0.001; MW U (4,4) = 0, p<0.05).

Circulating E2 levels, however, were not significantly different between the 4.5g/kg EB-treated

rats and their vehicle-treated counterparts (t(7) = 1.06, p>0.3; MW U (4,5) = 5, p>0.05). These

data suggest that the higher dose was more effective in circulating through the body. On the

other hand, the uterine horn weight data does suggest a difference between 4.5g/kg EB-

treated rats and their vehicle-treated counterparts. Serum E2 levels were significantly higher in

the 45g/kg EB- treated rats in the three-week group compared to the six-week group treated

with the same dose when evaluated parametrically (t(8) = -2.80, p<0.05) but not non-

parametrically (MW U(4,8) = 6, p>0.05). The serum E2 levels of the rats on the three-week time

point treated with 45g/kg EB were also significantly higher than the rats on the same time point

but treated with 4.5g/kg EB (t(9) = 4.839, p<0.0005; MW U(4,8) = 0, p<0.05). Unexpectedly,

the concentrations of E2 in the serum of oil-treated rats from the six-week time point and the oil

rats used as a control to the three-week rats who received 45g/kg of EB were significantly

different (t(8)=-3.41, p<0.005; MW U(4,6) = 0,p<0.05).

xxx

3 Week - Low Dose 3 Week - High Dose 6 Week - High Dose0

20

40

60

80

100

120

140

160

180

200Oil

Estradiol

[Est

radi

ol] p

g/m

L (M

ean

± S.

E.M

)

**

**

**

***

Figure 8 Serum E2 concentration collected from trunk blood following behavioral training. Each group of rats receiving 45g/kg EB had significanty higher E2 concentrations in the blood than their oil vehicle counterpart. The rats on the six-week time point had significantly higher serum E2 levels than both other groups treated with EB. Interestingly, there was a significant increase in E2 levels in the groups of oil rats that were controlling for groups receiving a 45g/kg EB. *p<0.05 **p<0.01 ***p<0.001

Correlation Plots – Serum Estradiol Level and Behavior

The E2 levels in the trunk blood of the rats, measured by the ELISA, suggested one

reason for the lack of impairment on learning behavior could be due to abnormally high levels of

E2 in the vehicle treated rats. If so, learning scores should covary with serum E2 levels. To

determine if this was observed, the total percent correct arm choices throughout all training

(Figure 9A) or trials to reach criterion (Figure 9B) was plotted against serum E2 concentration.

There was no correlation between serum E2 levels and total percent correct (R2 = 0.033, p >

0.3) or trials to criterion (R2 = 0.014, p > 0.5).

xxxi

0 50 100 150 200 25040

60

80

100

R² = 0.0333595179242643

[Estradiol] (pg/mL) (Mean ± S.E.M)

Tota

l % C

orre

ct

A

0 50 100 150 200 2500

20

40

60

80

100

R² = 0.0142193013278415

[Estradiol] (pg/mL) (Mean ± S.E.M)

Tria

ls to

Crit

erio

n

BFigure 9 Plotting serum E2 concentration against total percent correct arm choices in behavior (A) or trials to reach learning criterion (B). The R2 value indicates that it is not just serum E2 level that is influencing behavior. In this case, there could be something else going on that is controlling behavior.

xxxii

Discussion

The first aim of this study was to validate past work in the lab showing impaired

response learning three weeks after OVX with subsequent EB administration 24 and 48 hours

prior to behavior. Although all rats learned the striatum-sensitive response task quite well, there

was not an impairing effect of EB like was shown previously (Korol and Kolo, 2002; Zurkovsky

et al., 2006; Pisani et al., 2012). The six-week time point was conducted next, to see if there

was any difference between these two time points, specifically looking at whether oil rats

perform worse with increased deprivation. When there was neither a learning impairment in the

six-week time point nor an impairment in performance of vehicle treated rats, a lower dose was

utilized in attempt to replicate our past work. After replicating this experiment with this lower

dosage of EB treatment in exclusively the three-week time point, an impairing effect of EB on

learning was still not found.

Perhaps the reason for the lack of impairment on response learning was due to poor

injections. Circulating levels of E2 validated the efficacy of EB injections. High uterine horn

weight and serum E2 concentration show the injections, however, were effective. The uterine

horn weight and serum E2 concentrations suggest that despite not getting the expected

behavioral results, the injections of EB were effective. However, despite significant differences

in uterine horn weight and serum E2 levels between EB- and oil-treated rats, the vehicle treated

groups nevertheless had unusually high E2 levels. Other work shows vehicle rats should have

serum E2 concentrations <8pg/mL (Pisani et al 2012, Korol and Kolo 2002). These values

greatly differ from the values in this study, where vehicle-treated rats had serum E2

concentrations that ranged from ~27-51pg/mL. Uterine horn weights, even in vehicle-treated

animals were also larger than expected.

xxxiii

A significant difference existed between serum E2 levels between the vehicle treated

rats in the six-week time point and the vehicle treated rats controlling for the high dose treated

rats on the three-week time point. The oil groups corresponding to each EB treatment group

should not differ, as they received no E2 replacement. If anything, perhaps this difference is

related to having more time for E2 to clear from the blood after OVX, but by three weeks, this

should have already occurred. Nonetheless, perhaps this high E2 concentration in vehicle-

treated rats could be the reason for the lack of impairment on behavior in E2 treated rats

compared to their oil counterparts and the lack of impairment in oil-treated rats over time.

Perhaps there was still enough E2 in the body to have an effect on learning and thus the

behavior of those with E2 replacement did not look much different than oil rats. Serum E2

concentration of ~15pg/mL is enough to produce the learning impairment in rats. Thus, oil

animals might have had serum E2 concentrations well above this critical level for impairments,

which could be the reason for the lack of treatment effects (Pisani et al 2012). This would

suggest that despite having circulating E2, there may not have been enough additional E2

above levels seen in vehicle controls to produce impairments in the EB-treated rats. However,

this possibility is less likely due to the high concentration of serum E2. There seems to be a

threshold level of E2 to produce behavioral effects and changes in vaginal cytology (Davidson et

al 1968). The correlations suggest that concentration of E2 did not influence trials to criterion or

total percent correct (Figure 9), perhaps suggesting a threshold effect of E2 in learning behavior

as well.

The oil-treated rats could have higher levels of circulating EB, as shown in uterine horn

weight and serum E2 concentration, due to contamination of the sesame oil used for injection.

Contamination could happen in a number of ways, such as using a pipette that had previously

been used with E2, or putting the sesame oil into E2 glassware. Another study currently being

conducted in the lab has also shown similar high levels of circulating E2 in vehicle treated rats.

Additionally, sesame oil has been shown to be a phytoestrogen, which may in turn influence

xxxiv

results (Wu et al 2006, El Wakf et al 2013). To address this issue within the lab, perhaps we

should begin inclusion of a group with no injection at all. If we included a vehicle injection

group, a no injection group, and the experimental group, we can control for both effects of

injection and whether the vehicle was contaminated.

The XY plots comparing serum EB concentration to both the total percent correct arm

choices (Figure 9A) and trials to criterion (Figure 9B) were created to see if serum EB

concentration was correlated to performance on the maze. If there was a significant correlation

between these variables, it might suggest high EB levels in oil rats prevented an impairment on

behavior, which may indicate a contamination issue. The lack of significance comparing these

relationships in Figure 9, however, decrease the likelihood that sesame oil contamination is the

reason for the lack of E2 impairment, yet the high uterine horn weights suggest it still could play

a role.

The lack of effect of E2 on learning and the lack of change in learning in oil rats with a

longer period of hormone deprivation left many questions unanswered regarding the way the

brain’s sensitivity to E2 over time. One reason underlying the lack of effect of E2 on striatal

learning could related to circadian rhythms and corticosterone (CORT). Corticosterone is the

rodent equivalent of the human hormone cortisol, which plays a role in the stress response

(Feodora et al 2014). CORT, like female reproductive hormones, is also synthesized from

cholesterol, passing through progesterone intermediates during synthesis (Lemaire et al 2005;

Feodora et al 2014). Within the brain, actions of CORT are mediated by glucocorticoid and

mineralocorticoid receptors (Lemaire et al 205). CORT levels are known to fluctuate with

circadian rhythms, specifically, CORT levels are highest midday, at about 2-4 PM and are

highest during proestrus than during the other estrous stages in the F344 rat model (Haim et al

2003).

Male rats who are stressed during behavioral training are known to shift to using

response behavior (Sadowski et al 2009). Therefore, we know stress may also have some

xxxv

implications on learning in female rats. One initial thought explaining the results from this study

was that perhaps rats happened to be less stressed during behavior in the morning, shifting

their performance to slower response learning, and rats trained in the afternoon were more

stressed, potentially masking any hormone effect. If rats trained in the afternoon do learn

quicker because of stress, EB may be less likely to impair learning because stress forces a

cognitive shift that instead favors the striatum. On the other hand, an impairment may be easier

to detect if rats learn quickly because there is more room for impairment, unless the effect of

stress blocks the EB effect. The rats trained in the morning do seem to exhibit lower scores

through the training, yet this difference is small and unlikely to be important, especially

considering the small sample size. Although we did not see a difference in performance when

binning rats based on the time of day, it remains likely that circulating CORT and circadian

rhythms could play a role in learning due to their changes based on estrous cycle stage and

relation to stress. Indicators of stress on the maze include number of fecal boli, time spent

grooming, and time spent rearing vs. hovering. These could have been assayed through video

recording to look at stress as a variable, although this was not done in this study.

Other future work focuses on assessing the effects of differing lengths of ovarian

hormone depletion across ages on both the hippocampus- and striatum-sensitive learning tasks.

This approach will account for any differences in how hormone depletion modulates learning in

multiple brain areas. Within this aim, two tasks per brain area may be used to examine whether

there is a difference in hormone effects based on the task, even if both are associated with the

same brain area. Striatum-sensitive object recognition and/or striatum-sensitive response

learning may impair learning like was previously reported in the literature but not shown in this

study (Kundu et al 2018). Similarly, utilizing hippocampus-sensitive object location and/or

hippocampal place learning may also reveal previously reported effects that were not able to be

reproduced in this study. This approach might lead to the development of batteries of cognitive

xxxvi

tests that tap a number of brain regions, which in turn would be useful for evaluating the effects

of hormone deprivation, interventions, and the critical window hypothesis across the lifespan.

Moving forward, the work of this study has identified multiple future directions and new

interests building upon the findings from this work. The neuroscience field, especially

behavioral neuroscience, has mostly focused on the use of male subjects to avoid any

confounds of cycling hormones in females. As a result, much of what we know about the brain

and behavior is only applicable to males. Because of these hormone fluctuations, we typically

find that results in males cannot be comparable to females. Some therapies, especially those for

neurodegenerative disease, may even be designed with men in mind. Avoiding the confound of

cycling hormones leaves half the population out of the question when addressing behavioral

neuroscience knowledge. The field is beginning to work against this bias with increasing

vigilance, due to the emerging relationships between estrogens, cognition, and

neurodegenerative disease.

xxxvii

Works Cited

Ábrahám, István M., et al. “Action of Estrogen on Survival of Basal Forebrain Cholinergic Neurons: Promoting Amelioration.” Psychoneuroendocrinology, vol. 34, no. SUPPL. 1, Pergamon, Dec. 2009, pp. S104–12, doi:10.1016/j.psyneuen.2009.05.024.

Armstrong, Katrina, et al. “Hormone Replacement Therapy and Life Expectancy After Prophylactic Oophorectomy in Women With BRCA1/2 Mutations: A Decision Analysis.” Journal of Clinical Oncology, vol. 22, no. 6, American Society of Clinical Oncology, Mar. 2004, pp. 1045–54, doi:10.1200/JCO.2004.06.090.

Arnauld, E., et al. “Effects of Estrogens on the Responses of Caudate Neurons to Microiontophoretically Applied Dopamine.” Neuroscience Letters, vol. 21, no. 3, Elsevier, Feb. 1981, pp. 325–31, doi:10.1016/0304-3940(81)90225-1.

Auletta, Carol S. “Vaginal and Rectal Administration.” Journal of the American College of Toxicology, vol. 13, no. 1, 1994, pp. 48–63, doi:10.3109/10915819409140655.

Azcoitia, I., et al. “Estradiol Synthesis within the Human Brain.” Neuroscience, vol. 191, Pergamon, 15 Sept. 2011, pp. 139–47, doi:10.1016/j.neuroscience.2011.02.012.

Bean, Linda A., et al. “Re-Opening the Critical Window for Estrogen Therapy.” The Journal of Neuroscience, vol. 35, no. 49, Dec. 2015, p. 16077 LP-16093, http://www.jneurosci.org/content/35/49/16077.abstract.

Benedetti, Maria D., et al. “Hysterectomy, Menopause, and Estrogen Use Preceding Parkinson’s Disease: An Exploratory Case-Control Study.” Movement Disorders, vol. 16, no. 5, John Wiley & Sons, Ltd, Sept. 2001, pp. 830–37, doi:10.1002/mds.1170.

Brown, Roger, and David McNeill. “The ‘Tip of the Tongue’ Phenomenon.” Journal of Verbal Learning and Verbal Behavior, vol. 5, no. 4, Academic Press, Aug. 1966, pp. 325–37, doi:10.1016/S0022-5371(66)80040-3.

Conrad, Cheryl D., et al. “Acute Stress Impairs Spatial Memory in Male but Not Female Rats: Influence of Estrous Cycle.” Pharmacology Biochemistry and Behavior, vol. 78, no. 3, Elsevier, 2004, pp. 569–79, doi:10.1016/j.pbb.2004.04.025.

Cora, Michelle C., et al. “Vaginal Cytology of the Laboratory Rat and Mouse: Review and Criteria for the Staging of the Estrous Cycle Using Stained Vaginal Smears.” Toxicologic Pathology, vol. 43, no. 6, SAGE PublicationsSage CA: Los Angeles, CA, Aug. 2015, pp. 776–93, doi:10.1177/0192623315570339.

Cui, Jie, et al. “Estrogen Synthesis and Signaling Pathways during Aging: From Periphery to Brain.” Trends in Molecular Medicine, vol. 19, no. 3, Mar. 2013, pp. 197–209, doi:10.1016/j.molmed.2012.12.007.

Daniel, Jill M., and Christopher D. Lee. “Estrogen Replacement in Ovariectomized Rats Affects Strategy Selection in the Morris Water Maze.” Neurobiology of Learning and Memory, vol.

xxxviii

82, no. 2, Academic Press, Sept. 2004, pp. 142–49, doi:10.1016/j.nlm.2004.06.001.

Daniel, Jill M. “Estrogens, Estrogen Receptors, and Female Cognitive Aging: The Impact of Timing.” Hormones and Behavior, vol. 63, no. 2, Academic Press, 1 Feb. 2013, pp. 231–37, doi:10.1016/j.yhbeh.2012.05.003.

Daniel, Jill M., et al. “Effects of Ovarian Hormones and Environment on Radial Maze and Water Maze Performance of Female Rats.” Physiology and Behavior, vol. 66, no. 1, Elsevier Inc., Mar. 1999, pp. 11–20, doi:10.1016/S0031-9384(98)00272-8.

Davidson, Julian M., et al. “Relative Thresholds of Behavioral and Somatic Responses to Estrogen.” Physiology and Behavior, vol. 3, no. 2, Elsevier, Mar. 1968, pp. 227–29, doi:10.1016/0031-9384(68)90090-5.

Dluzen, Dean E., and Martin W. I. M. Horstink. “Estrogen as Neuroprotectant of Nigrostriatal Dopaminergic System Laboratory and Clinical Studies.” Endocrine, vol. 21, no. 1, 2003.

El Wakf, Azza M., et al. “Osteoprotective Effect of Soybean and Sesame Oils in Ovariectomized Rats via Estrogen-like Mechanism.” Cytotechnology, vol. 66, no. 2, Springer, Mar. 2014, pp. 335–43, doi:10.1007/s10616-013-9580-4.

Finch, Amy P. M., et al. Impact of Oophorectomy on Cancer Incidence and Mortality in Women With a BRCA1 or BRCA2 Mutation. 2014, doi:10.1200/JCO.2013.53.2820.

Gandy, Sam. “Estrogen and Neurodegeneration*.” Neurochemical Research, vol. 28, no. 7, 2003.

Gould, Elizabeth, et al. “Gonadal Steroids Regulate Dendritic Spine Density in Hippocampal Pyramidal Cells in Adulthood.” The Journal of Neuroscience, no. 4, 1990, http://www.jneurosci.org/content/jneuro/10/4/1286.full.pdf.

Haim, S., et al. “Serum Levels of Sex Hormones and Corticosterone throughout 4- and 5-Day Estrous Cycles in Fischer 344 Rats and Their Stimulation in Ovariectomized Females.” Journal of Endocrinological Investigation, vol. 26, no. 10, Editrice Kurtis s.r.l., 2003, pp. 1013–22, doi:10.1007/BF03348201.

Hendrix, Susan L. “Bilateral Oophorectomy and Premature Menopause.” American Journal of Medicine, vol. 118, no. 12 SUPPL. 2, Elsevier Inc., 2005, pp. 131–35, doi:10.1016/j.amjmed.2005.09.056.

Kostadinova, Feodora, et al. “Why Does the Gut Synthesize Glucocorticoids?” Annals of Medicine, vol. 46, no. 7, Informa Healthcare, 1 Nov. 2014, pp. 490–97, doi:10.3109/07853890.2014.932920.

Korol, Donna L., and Lacy L. Kolo. “Estrogen-Induced Changes in Place and Response Learning in Young Adult Female Rats.” Behavioral Neuroscience, 2002, doi:10.1037/0735-7044.116.3.411.

xxxix

Korol, Donna L., and Wei Wang. “Using a Memory Systems Lens to View the Effects of Estrogens on Cognition: Implications for Human Health.” Physiology and Behavior, 2018, doi:10.1016/j.physbeh.2017.11.022.

Korol, Donna L., et al. “Shifts in Preferred Learning Strategy across the Estrous Cycle in Female Rats.” Hormones and Behavior, vol. 45, no. 5, Academic Press, May 2004, pp. 330–38, doi:10.1016/j.yhbeh.2004.01.005.

Kundu, Payel, et al. “Licorice Root Components Mimic Estrogens in an Object Location Task but Not an Object Recognition Task.” Hormones and Behavior, vol. 103, Academic Press Inc., July 2018, pp. 97–106, doi:10.1016/j.yhbeh.2018.06.002.

Lemaire, V., et al. “Chapter 3.5 Glucocorticoids and Motivated Behaviour.” Techniques in the Behavioral and Neural Sciences, vol. 15, no. PART 1, Academic Press, 2005, pp. 341–58, doi:10.1016/S0921-0709(05)80020-3.

McCarrey, Anna C., and Susan M. Resnick. “Postmenopausal Hormone Therapy and Cognition.” Hormones and Behavior, vol. 74, Academic Press, Aug. 2015, pp. 167–72, doi:10.1016/J.YHBEH.2015.04.018.

Miller, Virginia M., and S. Mitchell Harman. “An Update on Hormone Therapy in Postmenopausal Women: Mini-Review for the Basic Scientist.” American Journal of Physiology - Heart and Circulatory Physiology, vol. 313, no. 5, American Physiological Society, 6 Nov. 2017, pp. H1013–21, doi:10.1152/ajpheart.00383.2017.

Mitchell, Ellen Sullivan, and Nancy Fugate Woods. Midlife Women’s Attributions about Perceived Memory Changes: Observations from the Seattle Midlife Women’s Health Study. Vol. 10, no. 4, Mary Ann Liebert, Inc, 2001, www.liebertpub.com.

Mizumori, Sheri J. Y., et al. “Parallel Processing across Neural Systems: Implications for a Multiple Memory System Hypothesis.” Neurobiology of Learning and Memory, vol. 82, no. 3, Academic Press, 2004, pp. 278–98, doi:10.1016/j.nlm.2004.07.007.

Packard, Mark G., and James L. Mcgaugh. “Inactivation of the Hippocampus or Caudate Nucleus with Lidocaine Differentially Affects Expression of Place and Response Learning Inactivation of Hippocampus or Caudate Nucleus with Lidocaine Differentially Affects Expression of Place and Response Learning.” Article in Neurobiology of Learning and Memory, vol. 65, 1996, https://www.researchgate.net/publication/312750029.

Pisani, Samantha L., et al. “Acute Genistein Treatment Mimics the Effects of Estradiol by Enhancing Place Learning and Impairing Response Learning in Young Adult Female Rats.” Hormones and Behavior, vol. 62, no. 4, Academic Press, Sept. 2012, pp. 491–99, doi:10.1016/j.yhbeh.2012.08.006.

Plant, Tony M. “The Hypothalamo-Pituitary-Gonadal Axis.” Journal of Endocrinology, vol. 226, no. 2, BioScientifica Ltd., 2015, pp. T41–54, doi:10.1530/JOE-15-0113.

Quinlan, Matthew G., et al. “Use of Cognitive Strategies in Rats: The Role of Estradiol and Its Interaction with Dopamine.” Hormones and Behavior, vol. 53, no. 1, Academic Press, Jan. 2008, pp. 185–91, doi:10.1016/j.yhbeh.2007.09.015.

xl

Ragonese, P., et al. “Implications for Estrogens in Parkinson’s Disease: An Epidemiological Approach.” Annals of the New York Academy of Sciences, vol. 1089, no. 1, Blackwell Publishing Inc., Nov. 2006, pp. 373–82, doi:10.1196/annals.1386.004.

Rapp, Stephen R., et al. “Effect of Estrogen Plus Progestin on Global Cognitive Function in Postmenopausal Women - The Women’s Health Initiative Memory Study: A Randomized Controlled Trial.” Journal of the American Medical Association, vol. 289, no. 20, American Medical Association, May 2003, pp. 2663–72, doi:10.1001/jama.289.20.2663.

Roebuck, Andrew J., et al. “Acute Stress, but Not Corticosterone, Facilitates Acquisition of Paired Associates Learning in Rats Using Touchscreen-Equipped Operant Conditioning Chambers.” Behavioural Brain Research, vol. 348, Elsevier B.V., Aug. 2018, pp. 139–49, doi:10.1016/j.bbr.2018.04.027.

Sadowski, Renee N., et al. “Effects of Stress, Corticosterone, and Epinephrine Administration on Learning in Place and Response Tasks.” Behavioural Brain Research, vol. 205, no. 1, Elsevier, Dec. 2009, pp. 19–25, doi:10.1016/J.BBR.2009.06.027.

Shanafelt, Tait D, et al. “Pathophysiology and Treatment of Hot Flashes.” Mayo Clinic Proceedings, vol. 77, 2002.

Sherwin, Barbara B., and Jessica F. Henry. “Brain Aging Modulates the Neuroprotective Effects of Estrogen on Selective Aspects of Cognition in Women: A Critical Review.” Frontiers in Neuroendocrinology, vol. 29, no. 1, Academic Press, 1 Jan. 2008, pp. 88–113, doi:10.1016/j.yfrne.2007.08.002.

Shumaker, Sally A., et al. “Conjugated Equine Estrogens and Incidence of Probable Dementia and Mild Cognitive Impairment in Postmenopausal Women: Women’s Health Initiative Memory Study.” Journal of the American Medical Association, vol. 291, no. 24, American Medical Association, June 2004, pp. 2947–58, doi:10.1001/jama.291.24.2947.

Shumaker, Sally A., et al. “Estrogen Plus Progestin and the Incidence of Dementia and Mild Cognitive Impairment in Postmenopausal Women - The Women’s Health Initiative Memory Study: A Randomized Controlled Trial.” Journal of the American Medical Association, vol. 289, no. 20, American Medical Association, May 2003, pp. 2651–62, doi:10.1001/jama.289.20.2651.

Tang, Ming Xin, et al. “Effect of Oestrogen during Menopause on Risk and Age at Onset of Alzheimer’s Disease.” Lancet, vol. 348, no. 9025, Lancet Publishing Group, Aug. 1996, pp. 429–32, doi:10.1016/S0140-6736(96)03356-9.

Van Duijn, Cornelia M. “Hormone Replacement Therapy and Alzheimer’s Disease.” Maturitas, vol. 31, no. 3, Elsevier, Mar. 1999, pp. 201–05, doi:10.1016/S0378-5122(99)00005-5.

Villa, Alessandro, et al. “Estrogens, Neuroinflammation, and Neurodegeneration.” Endocrine Reviews, vol. 37, no. 4, Oxford University Press, 1 Aug. 2016, pp. 372–402, doi:10.1210/er.2016-1007.

von Mühlen, Denise Goldani, et al. “A Community-Based Study of Menopause Symptoms and Estrogen Replacement in Older Women.” Maturitas, 1995, doi:10.1016/0378-

xli

5122(95)00917-A.

Wu, Wen-Huey, et al. “Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions Sesame Ingestion Affects Sex Hormones, Antioxidant Status, and Blood Lipids in Postmenopausal Women 1.” J. Nutr, vol. 136, 2006, https://academic.oup.com/jn/article-abstract/136/5/1270/4669984.

Yin, Weiling, et al. “Testing the Critical Window Hypothesis of Timing and Duration of Estradiol Treatment on Hypothalamic Gene Networks in Reproductively Mature and Aging Female Rats.” Endocrinology, vol. 156, no. 8, Endocrine Society, Aug. 2015, pp. 2918–33, doi:10.1210/en.2015-1032.

Zárate, Sandra, et al. “Role of Estrogen and Other Sex Hormones in Brain Aging. Neuroprotection and DNA Repair.” Frontiers in Aging Neuroscience, vol. 9, Frontiers Media SA, 2017, doi:10.3389/FNAGI.2017.00430.

Zurkovsky, L., et al. “Estrogen Modulates Learning in Female Rats by Acting Directly at Distinct Memory Systems.” Neuroscience, vol. 144, no. 1, Pergamon, Jan. 2007, pp. 26–37, doi:10.1016/j.neuroscience.2006.09.002.

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