Reanalyzing the Role of Estradiol in the Developing Zebra Finch Brain

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REANALYZING THE ROLE OF ESTRADIOL IN THE DEVELOPING ZEBRA FINCH BRAIN

A dissertation submitted

to Kent State University in partial

fulfillment of the requirements for the

degree of Doctor of Philosophy

Andrea Bender Musial

December, 2013

Dissertation written by

Andrea Bender Musial

Associate of Science, Lakeland Community College, 2003

Associate of Arts, Lakeland Community College, 2003

B.S. Notre Dame College, 2006

Masters of Arts, Biological Sciences, Kent State University, 2011

Ph.D., Physiology, Kent State University, 2013

Approved by:

Dr. Sean Veney______, Chair, Doctoral Dissertation Committee

Dr. Eric Mintz______, Member, Doctoral Dissertation Committee

Dr. Dave Glass______, Member, Doctoral Dissertation Committee

Dr. Steve Fountain______, Member, Doctoral Dissertation Committee

Dr. Mary Ann Raghanti______, College of Arts and Sciences, Graduate Representative

Accepted by:

Dr. Laura Leff______, Interim Chair, Department of Biological Sciences

Dr. James Blank ______, Interim Dean, College of Arts and Sciences

TABLE OF CONTENTS

Pages

LIST OF TABLES ………………………………………………………………………..6

LIST OF FIGURES ………………………………………………………………………6

ACKNOWLEDGEMENTS……………………………………………………………….7

OVERALL ABSTRACT……………………………………………………………….....9

CHAPTER I. Introduction ……………………………………………………………...10

The Aromatization Hypothesis………………………………………………………..…10

Zebra Finches…………………………………………………………………………….11

Initial Manipulations with Hormones……………………………………………………13

Estrogen Receptors Antagonists…………………………………………………………15

Plasma Steroid Levels……………………………………………………………………16

Aromatase………………………………….…………………………………………….17

Aromatase Inhibitors……………………………………………………………………..18

Research Inconsistencies………………………………………………………………...20

Support for a Role for Estradiol in Masculinizing the Zebra Finch Brain……………….22

Overall Aims……………………………………………………………………………..23

References………………………………………………………………………………..24

CHAPTER II. Treatment with the specific estrogen receptor antagonists, ICI 182,780 demasculinizes neuron soma size in the developing zebra finch brain………………….32

Abstract…………………………………………………………………………………..32

Introduction……………………………………………………………………………....33

Materials and Methods…………………………………………………………………...35

Treatment………………………………………………………………………………...35 4

Tissue Collection and Histological Preparation………………………………………….36

Measurements and Analysis……………………………………………………………..36

Statistics………………………………………………………………………………….37

Results……………………………………………………………………………………37

Discussion……………………………………………………………………………...... 42

Acknowledgements……………………………………………………………………....48

References………………………………………………………………………………..48

CHAPTER III. Treatment with the specific aromatase inhibitor, Fadrozole, decreases neuron soma size, neuron number, and nuclear volume in the developing zebra finch brain...……………………………………………………………………………………58

Abstract…………………………………………………………………………………..58

Introduction………………………………………………………………………....……59

Materials and Methods…………………………………………………………………...61

Treatments………………………………………………………………………………..61

Tissue Collection and Histological Processing………………………………………..…62

Measurement and Statistics………………………………………………………………62

Results……………………………………………………………………………………63

Discussion………………………………………………………………………………..77

Acknowledgements………………………………………………………………………83

References………………………………………………………………………………..83

CHAPTER IV. An analysis of estrogen receptor beta in the early post-hatching zebra finch brain………………………………………………………………………………..92

Abstract…………………………………………………………………………………..92

Introduction………………………………………………………………………………93

Materials and Methods…………………………………………………………………...95

Subjects…………………………………………………………………………………..95 5

Q-PCR of Zebra Finch ERβ cDNA…………………………………………………..….96

Riboprobe Development…………………………………………………………………97

In Situ Hybridization……………………………………………………………………..98

Semi-Quantitative Analysis……………………………………………………………...99

Results…………………………………………………………………………………....99

Discussion………………………………………………………………………………107

Acknowledgments………………………………………………………………………110

References………………………………………………………………...…………….110

CHAPTER V. Global Discussion………………………………………………………119

Implications of Research……………………………………………………………...... 119

Future Directions……………………………………………………………………….122

References………………………………………………………………………………123

LIST OF TABLES

1. Table 1.0 Relative density of ERβ mRNA in the telencephalon of post-hatching zebra finches after digoxygenin labeled in situ hybridization………………….100

2. Table 2.0 Proposed model for developmental processes and contributing estrogen receptors……………………………………………………………………...…120

LIST OF FIGURES

1. Fig.1.0 Model of sexual differentiation in mammals…………………………….11 2. Fig. 2.0 Schematic of the zebra finch song circuit……………………………….12 3. Fig. 3.0 The effects of ICI 182,780 on neuron soma size in HVC………………38 4. Fig. 4.0 The effects of ICI 182,780 on neuron soma size in RA………………...39 5. Fig. 5.0 The effects of ICI 182,780 on neuron soma size in LMAN…………….40 6. Fig. 6.0 The effects of ICI 182,780 on neuron soma size in Rt………………….41 7. Fig. 7.0 Gross anatomical representation of post-treatment brain…………….…66 8. Fig. 8.0 Fadrozole treatment effects on RA cell size…………………………….67 9. Fig. 9.0 Fadrozole treatment effects on RA volume……………………………..68 10. Fig. 10.0 Fadrozole treatment effects on HVC cell size…………………………69 11. Fig. 11.0 Fadrozole treatment effects on HVC volume………………………….70 12. Fig. 12.0 Fadrozole treatment effects on LMAN cell size……………………….71 13. Fig. 13.0 Fadrozole treatment effects on Area X cell size…………………...... 72 14. Fig. 14.0 Fadrozole treatment effects on Area X volume………………………..73 15. Fig. 15.0 Fadrozole treatment effect on Rt cell size……………………………..74 16. Fig. 16.0 Fadrozole treatment effects on Rt volume……………………………..75 17. Fig. 17.0 Fadrozole treatment effects on cell counts in RA……………………...76 18. Fig. 18.0 Q-PCR analysis of cDNA from post-hatching telencephalon ages P3- P30……………………………………………………………………………...102 19. Fig. 19.0 ERβ mRNA expression in oviduct…………………………………...103 20. Fig. 20.0 In situ hybridization of ERβ mRNA at post-hatching day 15………..104 21. Fig. 21.0 Magnified view of NCM with nuclear labeling of positive cells for ERβ mRNA…………………………………………………………………………..105 22. Fig. 22.0 Stereotaxic atlas of zebra finch brain……………………………...…106

ACKNOWLEDGEMENTS

Many individuals are responsible for guiding me through the doctoral program at

Kent State University. I would first like to thank my advisor, Dr. Sean Veney, for his constant academic support, patience and encouragement. His unique approach to teaching and enthusiasm about the work of our lab was a persistent reminder of our scientific purpose. Additionally, my committee member Dr. Eric Mintz has been someone who guided me as he would his own student, to ensure I fulfilled my goals. He was always my statistical analysis expert. He reminded me of what was most important in each situation and gave great advice. My committee member Dr. David Glass was also always available to teach me something new and guide me to see what I might be missing. He also provided constructive input on my writing, progress, and data reporting tactics. I would also like to thank my committee members Dr. Steve Fountain and Dr.

Mary Ann Raghanti, for their guidance, help and support.

Members of the lab have also been a major part of my success. I want to personally recognize Kalpana Acharya, Ann Dobry, Andrew Curfman, Philip Long, Kim

Eustache, Sierra Kirkland, Josh Meeker, Lo’Rell Martin, Ryan Rundle, Kathleen

Labadie, Khadijah Wilson, and Joshua Thuman for all their love, support, and great memories. I want to especially thank Kalpana for not only her dear friendship, but also her fantastic listening skills, advice, and assistance with in situ, injections, and for so much more.

Lastly, I want to recognize my greatest support, my family. My mother, Kristine

Basiger, has always encouraged me to pursue the most of my education. She always 8 listened and guided me to pursue more than what I ever thought possible. She also gave me the gift of literacy by making me one of the thousands of children she taught to read.

She instilled in me the importance of education and also for that of persistence. My grandmother, Elizabeth Hilston, also encouraged me to pursue the highest degree I could because she was never wealthy enough to attend higher education herself. She started teaching me since I was young and has never stopped. To my uncle, Tom Hilston, who gives the best advice and has always rallied for my success, I cannot thank you enough.

My stepfather, Dane, has also been so supportive of me through this entire process. I especially want to thank, my now, husband, David Musial, who never let me give up no matter how many times I wanted to and always embraced my passion for science as part of who I am. I also thank my role models of academic success, cousins Mary and John, who earned a PhD in their chosen fields, and to my mother Kristine, sister Jennifer, aunt

Candy, uncle Tom and cousin Cari who each earned their masters degrees and demonstrated the worth and value of higher education. Additionally, to all my relatives and friends who always provided an ear to listen and support for this academic endeavor,

I cannot thank you enough.

OVERALL ABSTRACT

The neural dimorphisms in the zebra finch present one of the most unique examples of sexual differentiation observed in vertebrates. Although knowledge of these differences has been established for over 45 years, the exact mechanism by which they arise is not known. This dissertation provides additional support for estrogens’ involvement in brain development. Specifically, blocking of estrogen receptors with ICI 182,780 decreased neuron soma size of song control regions in both sexes during development. These results are distinctive since previous attempts to block estrogen receptors failed to see the large degree of difference my work displayed. I further supported the role of estrogens in neural brain dimorphisms by decreasing the synthesis of aromatase, an enzyme needed for estradiol production, with the administration of Fadrozole. This successfully decreased neuron soma sizes, neuron number, and nuclear volume in song control regions in males and females, which had not been seen in prior attempts from other laboratories.

I have concluded that the route of delivery used in these experiments is likely the largest contributing factor to generating these unique results. I also provide evidence of a potential role for ERβ by displaying its presence at an early post-hatching age in two auditory processing regions. Taken together, my work provides further support for the role of estrogens in the dimorphic development of the brain, and establishes that it is unlikely that ERβ contributes to neural dimorphisms in the zebra finch. 10

CHAPTER 1: INTRODUCTION

The Aromatization Hypothesis

Sexual dimorphisms in neural systems permit the exploration of critical factors responsible for the development and maintenance of the brain. Related to this, differences between males and females can be accounted for, in part, by the organizational effects of hormonal and non-hormonal products acting on the brain early in development. For many animals, gene(s) located on sex chromosomes activates sexual differentiation. For example, in mammals such as rodents, SRY (sex determining region of the Y chromosome) is the gene that directs testes formation in males (Goodfellow and

Lovell-Badge, 1993). There is no equivalent of SRY in genetic (XX) females and as a result, ovarian development occurs. Early in development, the testes of males primarily produce androgens, such as testosterone (T). In contrast, prior to puberty, the female ovaries produce substantially less hormone. The relative lack of hormone exposure in females as compared to males gives rise to feminine brain development. The increased levels of hormones in males give rise to the development of masculine brain structures.

More specifically, T, secreted by the gonads travels through the circulatory system to the brain where it is converted into estradiol (E2) through the action of the enzyme, aromatase. These high levels of E2 permanently masculinize many features of the brain, resulting in masculine behavior (Feder et al., 1981) (Figure 1).

Fig. 1.0 A model of sexual differentiation in mammals

Sexual Differentiation in Mammals

Males Females

Chromosomal Sex XY (Sry-TDF) XX

Gonadal Sex Testes Ovaries

Hormonal Sex Testosterone Low estrogen

Aromatized in brain (converted into estrogen) No aromatization

Phenotypic Masculine neural Feminine neural (Behavioral) development and development and Sex behavior behavior

Zebra Finches

Despite support for the aromatization hypothesis in brain sexual differentiation (primarily generated from work in rodents), there is evidence that it does not equally fit all vertebrates. One such example is the zebra finch. In this passerine bird there is a series of interconnected telencephalic nuclei that project to the vocal organ, or syrinx. This circuit, which is known as the song system, is responsible for singing behavior (Nottebohm and Arnold, 1976; Fig. 2). More specifically, connections between

HVC and the robust nucleus of the arcopallium (RA) are crucial for song production

(Nottebohm et al., 1976; Simpson and Vicario, 1990). Neurons within RA project axons to a population of motor neurons known as nXIIts (tracheosyringeal branch of the 12th cranial nerve) located in the brain stem. These motor neurons then innervate muscles of

12 the syrinx. Connections between the lateral magnocellular nucleus of the anterior nidopallium (LMAN) and Area X are important for song learning (Bottjer et al., 1984;

Scharff and Nottebohm, 1991). Many aspects of this circuit are enhanced in males as compared to females. More specifically, the volumes of HVC and RA and the size of cells in LMAN, RA, and HVC are larger in males than in females (Nottebohm and

Arnold, 1976; Gurney, 1981; Gurney, 1982). The axonal projections from HVC to RA are more robust in males (Konishi and Akutagawa, 1985). Additionally, Area X, which is clearly defined in males with a Nissl stain, is not visible in females using this same technique (Nordeen and Nordeen, 1989). These male-biased dimorphisms are first evident around post-hatching day 8 and continue to develop through approximately post- hatching day 60. They completely parallel the adult behavior. Males learn and sing a courtship and nest defense song that females do not normally produce.

Fig. 2.0 Schematic drawing of the zebra finch song system illustrating brain regions involved in vocal control and some of their anatomical connections (modified from

Bottjer and Johnson, 1997).

Initial Manipulations with Hormones

Although we have a firm understanding of the neural connections necessary for singing behavior, the mechanism(s) responsible for directing sexual differentiation of the song system are not completely understood. Using the aromatization hypothesis as a basis, early studies in zebra finches used various hormone manipulations in an attempt to masculinize females. Three studies specifically looked at E2 and its influence on neural morphology, however, the methodologies and results were somewhat inconsistent. For example, two experiments detected increases in the volumes of the telencephalic nuclei following androgen administration to adult females that had received E2 as hatchlings

(Gurney, 1982; Gurney and Konishi, 1980). However, two other studies could not replicate these results with the same dose (Jacobs et al., 1995) or even higher doses of E2 and androgen (Simpson and Vicario, 1991). Each of these studies used an implant containing the E2. However, a separate study alone administered the hormone as an implant packed inside of Silastic tubing. The Silastic implants of T and E2 administered to females on the day of hatching masculinized the volumes of RA and HVC, induced an

Area X, and increased cell sizes in RA, HVC, and LMAN when measured after two months of administration (Grisham and Arnold, 1995). The E2 was administered to females on the day of hatching via implants, and with the exception of the size of neurons in RA, none of the measures in females equaled that of normal male brain morphology. 14

Although, putting together these data, it is profound that even when females were given high levels of E2, partial masculinization of the brain was seen and song was induced, and yet the effects were never as great as was demonstrated in males (Simpson and Vicario,

1991; Adkins-Regan et. al., 1994). Taken together, these data demonstrate that E2 masculinized many features of the zebra finch song system, but not one study was able to induce complete masculinization with E2 alone.

In agreement with what is predicted by the aromatization hypothesis, E2 by far has the most potent effect on masculinizing the female brain (Gurney and Konishi, 1980;

Simpson, 1991; Grisham et al., 1994; Adkins-Regan et al., 1994; Jacobs et al., 1995). As a result of supra-physiological doses of E2 only, the volumes of RA and HVC increase.

Neuron size and number in these regions also increase. Even more captivating is the fact that E2 administration to females induces a visible Area X, which is not normally detectable in this sex. Interestingly, these females also develop the ability to produce male-like song, which is enhanced with continued T treatment into adulthood (Gurney

1982, Gurney and Konishi, 1980, Pohl-Apel and Sossinka 1984; Simpson and Vicario).

Notably, the effects on the brain are only partial. That is, no amount of E2 has been able to induce full and complete neural masculinzation in a female comparable to a normal male, suggesting that other factor(s) are likely involved.

Since E2 was so effective at masculinizing the female brain (at least partially) researchers expected that administration of T would have the same effect since it is the precursor hormone. Interestingly, it did not. Instead, T was slightly less effective than E2

(Grisham and Arnold, 1995). More specifically, T did masculinize the volumes of HVC, 15

RA and LMAN, as well as the size of cells within this region, but failed to increase neuron number and spacing between cells in RA.

If E2 were the primary masculinizing hormone, then there should be a greater number of estrogen receptors in males as compared to females. However, the quantity and distribution of classical nuclear estrogen receptors (ERα and ERβ) does not directly support this idea. ERα mRNA was examined in male and female zebra finches during post-hatching days 5-25 (Jacobs, et al., 1999). HVC was the only song control region that showed substantial labeling, however there were no sex differences observed. ERβ is yet to be described within the zebra finch model, but in a closely related species, the

European starling, expression of ERβ mRNA is not localized to song regions but is concentrated in auditory processing regions (Bernard et al., 1999). More recently, a G- protein coupled estrogen receptor, GPR30, localized to the plasma membrane has been described in zebra finches (Acharya and Veney, 2012). GPR30 receptor expression was seen in RA, and was dimorphically expressed at post-hatching (P) day 21 in HVC.

Estrogen Receptor Antagonists

Although a sex difference in nuclear estrogen receptors was not detected, there is still the potential for estrogens to dimorphically influence the system. More specifically, through the action of co-regulators (co-repressors and co-activators), the numbers of receptors does not necessarily have to be different in order to have dimorphic effects on development (Duncan and Carruth, 2007; Duncan, Jimenez and Carruth 2009). To address this possibility, subsequent studies attempted to block activation of the entire receptor with various pharmacological agents. Surprisingly, these compounds did not 16 always demasculinize the brain, but rather in some instances hypermasculinized males, masculinized females, or had no effect in either sex. For example, treatment with

Tamoxifen for the first 20 days after hatching increased cell size in RA, HVC and the magnocellular nucleus of the anterior nidopallium (MAN) of both sexes when the brains were examined at day 60. It also increased the volume of RA, HVC, MAN and Area X in males (Mathews et al., 1988). Similar effects of Tamoxifen on cell size and volume were found in a follow-up study, which also included females that formed a visible Area

X after treatment (Mathews, 1991). At the time, these results were somewhat puzzling, but years later it was determined that Tamoxifen is a partial agonist for estrogens, known as a selective estrogen receptor modulator (SERM), when administered in vivo (Mathews and Arnold, 1991). In contrast, Bottjer and Hewer (1992) administered Tamoxifen in vivo and determined that males exposed to the drug from approximately day 20 post- hatching until days 90-100 had no changes in the size of song control nuclei. In addition to Tamoxifen, a few other antiestrogens have been tested. LY117018 and CI628 were administered to zebra finches for the first 20 days after hatching and brains were examined at day 60. Neither compounds prevented neural masculinization. Instead, both increased soma area in male MAN and in HVC of both sexes. In females, both compounds induced the formation of an Area X (Mathews and Arnold, 1990).

Plasma Steroid Levels

In addition to the above data, our understanding of how estrogens act to affect dimorphic neural development has further been clouded by the lack of evidence supporting a sex difference in plasma levels of steroid hormones. If estrogens are in fact a major masculinizing agent, one would expect higher levels of them in males than in 17 females around the time when sex differences in the brain are first detected. On the contrary, however, research does not support this conclusion at all. A radioimmunoassay was used in three separate studies to evaluate whether a sex difference exists in plasma hormones during early development, particularly when dimorphisms in the song circuit are first becoming detectable. One study evaluated differences in E2, T, dihydrotestosterone (DHT), and androstenedione (AE) at post-hatching ages 1-10

(Hutchinson et al., 1984). It was noted that E2 concentrations in males increased from post-hatching days 2-4, but by day 10 these levels decreased to the amount present on day

1. Levels in females stayed constant during this time, which generated a male bias by day 4. All three androgens (T, DHT, and AE) decreased from days 1-10 in males, however in females, AE was increased on days 2 and 10. Another study measured E2, T, and DHT from embryonic day 12 until P54 (Adkins-Regan et al., 1990). A significant main effect of sex was seen in T, but with a female bias. There was no significant difference in E2 or DHT levels between males and females at any age. A third study measured circulating E2, estrone (E1), T, AE, and DHT from P1-13 (Schlinger and

Arnold, 1992). E2 was only detected in two samples and E1 was not detected at all.

There were no significant effects of sex or age on T or DHT. In the samples where E2 was detected, there was a similar level in females across the various age groups, but in males, it was higher in the first week after hatching as compared to the second. Although it is difficult to directly compare all three studies because of variations in the time periods of evaluation, one conclusion is very clear. None of these studies found a consistent male bias in T, E2 or AE during development.

Aromatase

Equally puzzling, no study has been able to document a sex difference in telencephalic aromatase activity. One study evaluated the enzyme in micro-dissected tissue punches of song control regions HVC, RA and Area X from P20 birds (Vockel et al., 1990). Results indicated that RA and HVC samples contained significantly high levels of aromatase, but there was no difference between the sexes. A second study measured aromatase activity in whole brain homogenates from 4-6 and 10-12 day olds and again found no sex difference (Schlinger and Arnold, 1992). Dissociated cell cultures of individual male and female zebra finches were also assessed for aromatase activity (Wade et al., 1995). After 14 days in culture, high levels of aromatase activity were detected, but there was no sex difference.

In addition to enzyme activity, further analyses have investigated aromatase mRNA and protein. In situ hybridization was conducted on P5, 10, 18 and 25 day old juveniles (Jacobs et al., 1999). Moderate mRNA expression was detected in LMAN at these ages and minimal expression was noted in RA, but only at day 18. However, at none of these ages was aromatase mRNA detected in Area X or HVC, or was there a sex difference. Immunohistochemistry for the distribution of aromatase protein revealed information that paralleled mRNA data; there were no examined juvenile or adult ages where a sex difference in aromatase protein was found (Saldanha et al., 2000).

Interestingly, this study also revealed that there was minimal expression of aromatase protein within song control regions, and that the majority is outside of these regions.

These data demonstrated a disconnect between aromatase mRNA and protein. LMAN and HVC demonstrate mRNA signal but no protein. The question becomes, how exactly 19 are estrogens getting into song regions to masculinize this circuit, when the enzyme, aromatase, that makes estrogens from androgen precursors is outside of the nucleus?

Clearly, more research is needed to fully understand by which mechanisms (receptors, gene products, or possibly compensatory mechanisms) estrogens are not only reaching their targets but the mechanism by which they are masculinizing this species song circuit.

Aromatase inhibitors

Despite the fact that the majority of aromatase protein in the zebra finch brain resides outside of song control nuclei, it is still possible for estrogens to have influences in song regions. So to further understand their importance, additional work has focused on minimizing the production of estrogens. Historically, this has been addressed two ways: through gonadectomy and treatment with aromatase inhibitors. When males were gonadectomized shortly after hatching, as adults they were still able to produce song, although the rate and tempo was slowed. The masculine brain morphology, however, appeared unaffected (Adkins-Regan et al., 1990). Attempts to prevent masculine brain development with the use of aromatase inhibitors have yielded conflicting results. More specifically, administration of Fadrozole, in vitro, significantly inhibited enzyme activity and demasculinzed male-biased dimorphisms in the projections between HVC and RA

(Wade et al., 1994; Holloway and Clayton, 2001). However, when treatment occurred with a closely related aromatase inhibitor (Vorozole) in vivo, effects on dimorphic neural morphology were non-existent to minimal. For example, in one study, vorozole was administered to hatchling zebra finches through subcutaneously implanted Silastic capsules until 45 days of age. The animals were then gonadectomized and treated with T for three weeks beginning at 105 days of age. Males were assessed for changes in the 20 song circuit and singing ability. Vorozole significantly decreased singing rate, but did not alter the volumes of any song control nuclei (Balthazart et al., 1995). In a second study, Wade and Arnold (1994a) peripherally injected zebra finches with Fadrozole on

P1-P30 and examined brains on P31. Treatment had no effect on the volumes of RA or area X. It also did not alter neuron soma size in RA or HVC. In an additional study by

Wade and Arnold (1994b) the effects of Fadrozole were investigated in adults after five days of peripheral injections, and in P4-P6 day old juveniles after one injection. At neither age group did Fadrozole significantly alter brain morphology. In contrast to the above, Merten and Stocker-Buschina (1995) did find a significant effect with Fadrozole.

They treated males from P10-P30 with peripheral injections, and then sacrificed them either at day 35 or 135. Neuron soma size and volumes of song control regions were assessed. There was no effect of the compound on the brain when measured at day 35, but at day 135 there was a slight, but significant decrease only in HVC cell number.

Research Inconsistencies

Despite these data and the inconsistencies concerning the role of E2 in zebra finch brain masculinization, I strongly feel there is still reason to believe that estrogens are important for this process. More specifically, failed attempts by many of the previous studies can be explained, in part, by methodological limitations as well as choice in compounds that were administered. For example, in the Arnold et al. (1980), Mathews and Arnold (1988) and Mathews, Brenowitz and Arnold, (1991) the antagonists that were used may not have completely blocked receptors as expected, and in at least one proven case, partially activated them. Also, the manner in which the compounds were administered may have diminished their efficacy in the brain. The majority of studies 21 administered compounds via a somewhat uncontrolled release through Silastic implants, or peripheral subcutaneous injections that may not have effectively reached the brain.

In mammals the blood brain barrier (BBB) is formed by epithelial cells that create tight junctions within the endothelium of capillaries that have access to the brain.

The function is to act as a filter and selectively block substances from gaining entrance.

This barrier limits the brain’s exposure to larger molecules in systemic circulation

(Pardridge, 1999). To date there is no information available about the size or composition of molecules that can pass through the zebra finch BBB. However, in humans, anything larger than about 500 Daltons generally cannot gain access to the brain

(Fischer et al., 1998). If we consider the aromatase inhibitor experiments specifically, the

Fadrozole molecule is 259.74 g/Mol, which is the equivalent of about 1.5 x 1026 Daltons, suggesting that it would theoretically be too large to access the human brain.

Using positron emission tomography, differences in the permeability of the BBB were evaluated in several species including humans, rats, and monkeys (Syvanen et al.,

2009). It was determined that passive diffusion through the BBB of larger animals such as humans and monkeys was more prevalent than in smaller organisms. Based on these findings, it is reasonable to deduce that molecules the size of Fadrozole and Vorozole would have extreme difficultly readily passing through the BBB of a small organism, like zebra finches. The indirect approaches of drug administration could indicate that the compounds were never reaching the target tissue.

In addition to the BBB, metabolism by the liver is another possible explanation for why systemic injections of compounds may not have been extremely effective in zebra finches. The liver is a major organ that breaks down compounds in the blood and is 22 most commonly the first metabolizing organ for substances in the body (Yagami et al.,

1993; Rowland, 1972). Because of this, even as early as the 1970’s it was known that in order to by-pass the effects of the liver it was necessary to increase the dose of drugs administered peripherally or orally so that they would effectively reach the desired target

(Rowland, 1972). In support of this, a more recent study conducted in rats determined that approximately 90% of all orally-administered Fadrozole was taken up by the liver within 2 hours of administration (Yamagami et al., 1993). The fact that previously administered compounds or injections were given such that they were released into systemic circulation the dose reaching the target tissue was likely to be substantially less than the dose administered. Therefore, based on these reasons outlined, we proposed that there are several ways to approach this question with greater detail and efficacy.

Support for a Role for Estradiol in Masculinizing the Zebra Finch Brain

Despite the conflicting evidence and inconsistencies, there is still reason to hypothesize that estrogens are involved at some level in dimorphic neural development in zebra finches. More specifically, brain-derived estrogens may be the critical factor important for masculine brain development in this model (Schlinger and Arnold, 1992;

Holloway and Clayton, 2001). Not only does the brain synthesize estrogens, but compared to the gonads it does so in vastly large quantities. This may explain why attempts to remove hormone production through gonadectomy had no effect on the brain.

In addition, some in vitro work further supports the importance of estrogens on masculine development (Holloway and Clayton, 2001). Brain slices from 25-day-old male and female zebra finches were manipulated in terms of length of time in culture, arrangement of co-culturing (either single sex cultures or co-cultures containing brain 23 slices from both sexes together), and/or culturing with estrogen antagonists. One specific interesting finding was that the pathway from HVC to RA was masculinized in female tissue that was co-cultured with male sections. Additionally, the effects of estrogen antagonists were evaluated in single sex cultures and in the cultures containing both sexes co-cultured together. In male cultures, administration of Fadrozole, in low doses, significantly inhibited normal masculine brain development within song control regions.

Specifically, the axonal projections from HVC to RA were diminished, and the neuron soma size in RA and HVC was decreased significantly. Female slices cultured with

Fadrozole or Tamoxifen underwent normal female development. However the female slices cultured together with male slices, that had originally masculine features induced, no longer underwent the masculine development seen if they were in the presence of either estrogen antagonist.

The results of this work highly suggest the integral role of estrogen in masculinization of the brain. Also, the effects seen in female cultures housed with male cultures directly suggests that brain derived estrogens in males are sufficient enough to induce masculinization in females.

Overall Aims

Despite data indicating the lack of a sex differences in plasma hormones, aromatase, and estrogen receptor alpha, there may be a role for estrogens in zebra finch development. This is mostly because one cannot ignore the significant effects that E2 administration had in females in vivo as well as the effects of estradiol manipulation in vitro. The method of administration and the dose of treatment may have been extenuating variables that altered the effectiveness of previous attempts to fully understand estrogenic action in zebra finches. 24

The purpose of the experiments in this work was to revisit the role of estrogens during early development and to re-assess the potential of the aromatization hypothesis in zebra finches. The first project’s objective was to evaluate the effects of a more potent and specific estrogen receptor antagonist, ICI 182,780 on masculine brain development when administered in higher concentration to the brain. The second aim was to re- evaluate the effects of Fadrozole on masculine brain development by more directly exposing the brain to the drug. The third aim was to further examine the possibility that a second nuclear estrogen receptor (ERβ) might contribute to dimorphic brain development by analyzing its gene expression and mRNA distribution during early post-hatching ages.

References

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CHAPTER II. TREATMENT WITH THE SPECIFIC ESTROGEN RECEPTOR

ANTAGONIST, ICI 182,780 DEMASCULINIZES NEURON SOMA SIZE IN THE

DEVELOPING ZEBRA FINCH BRAIN.

Bender, A.T. and Veney, S.L., 2008. Brain Res 1246, 47-53.

Aim: Evaluate the effects of intracranial injections of the pure estrogen receptor antagonist, ICI, on dimorphic development of the song circuit of zebra finches.

Hypothesis: Intracranial injections of ICI 182,780 administered to juvenile zebra finches will prevent masculinization of the brain in both sexes.

Abstract

In zebra finches, many features of the neural song system are more pronounced in males compared to females. The exact mechanism(s) responsible for these differences are not known, but potentially involve steroid hormones. For instance, estrogens are most effective in masculinizing the avian female brain. Efforts to prevent masculine development through several estrogen receptor antagonists have been for the most part, unsuccessful, perhaps due to partial agonistic activity of the compounds. To further investigate the role of estrogens in dimorphic development we used a more potent estrogen receptor blocker, ICI 182,780. For the first 25 days post-hatching, daily intracranial injections were administered. This treatment significantly decreased neuron

33 soma size in RA and HVC of both sexes. A similar effect was noted in LMAN.

Treatment also visibly decreased the volume of several song control nuclei. Together, these data support the hypothesis that ICI is an effective estrogen receptor antagonist in the zebra finch brain and that estrogens may have a role in sexually dimorphic development of the song circuit.

Introduction

The mechanism(s) responsible for sexual differentiation of the song system are, for the most part, unknown. According to a model established in rats, male biased dimorphisms occur when circulating testosterone (T) from the testes is converted locally in the brain by aromatase to estrogens. Activation of estrogen receptors (ER) masculinizes the brain. Females lack comparable levels of T, and therefore are not normally masculinized (aromatization hypothesis) (Feder, 1981; Naftolin and MacLusky

1984). Although this hypothesis has been used as a basis for understanding neural dimorphisms across a number of species, the extent to which it fits all model systems remains unclear. For example, in zebra finches, many attempts have been made to alter dimorphic brain development solely by manipulating the production and/or action of gonadal hormones such as estrogens and/or androgens (reviewed in Wade, 2001). For the most part, estrogens have had by far the most potent effects of masculinizing the female brain, although not completely (Gurney and Konishi, 1980; Simpson, 1991;

Grisham et al., 1994; Adkins-Regan et al., 1994; Jacobs et al., 1995). Attempts to significantly demasculinize the male brain by removing estrogens have been substantially less successful (Arnold, 1980; Wade and Arnold, 1994; Balthazart et al., 1994). It is not known why such manipulations in males did not yield the expected results. Despite

34 discrepancies in these data the effects that estrogens had in females gives precedence to hypothesize that if they are important for masculine development of the song system, then inhibiting their actions should prevent these sex differences.

In an effort to better understand the role of estrogens in dimorphic brain differentiation, several studies attempted to block their action with the use of various pharmacological agents. Surprisingly, these compounds did not always demasculinize the brain, but rather in some instances hypermasculinized males, masculinized females, or had no effect in either sex. For example, treatment with Tamoxifen for the first 20 days after hatching increased cell size in RA, HVC and MAN of both sexes when the brains were examined at day 60. It also increased the volume of RA, HVC, MAN and

Area X in males (Mathews et al., 1988). Similar effects of Tamoxifen on cell size and volume were found in a follow up study, which also included females that formed a visible Area X after treatment (Mathews, 1991). In contrast, Bottjer and Hewer (1992) determined that males exposed to Tamoxifen from approximately day 20 post-hatching until days 90–100 had no changes in the size of song control nuclei.

In addition to Tamoxifen, a few other antiestrogens have been used in an attempt to block masculine brain development. LY117018 and CI628 were administered to zebra finches for the first 20 days after hatching and brains were examined at day 60. Neither compound prevented neural masculinization; instead, both increased soma area in male

MAN and in HVC of both sexes. In females, both compounds induced the formation of an Area X (Mathews and Arnold, 1990). It is unclear why these compounds were not completely effective in demasculinizing the brain. One likely possibility is that rather than being complete antagonists, these drugs may have partial agonistic actions 35

(Anderson et al., 1975a; Jordan and Gosden, 1982; Mathews and Arnold, 1990; Mathews,

1991; McMurray et al., 2003).

With this idea in mind, we attempted to further address the role of estrogens in masculine brain development by using a more potent ER blocking agent, ICI 182,780

(ICI). This drug is considered a pure ER antagonist that binds to both ERα and ERβ

(Wade et al., 1993; Van Den Bemd et al., 1999; Sun et al., 2008) with minimal agonistic properties (Wijayaratne et al., 1999; Howell et al., 2000; Alfinito et al., 2008). It acts by lowering the overall number of available ER (Howell et al., 2000). Given its potential as a pure antagonist, our hypothesis is that if estrogens are important for masculinizing the neural song circuit, then ICI treatment should prevent masculinization.

Materials and Methods

Treatments

Subjects were obtained from our breeding facility at Kent State University. The experimental zebra finches were taken from nest boxes inside of aviaries containing 6–7 pairs of adults. There is conflicting evidence as to whether ICI readily crosses BBB from the periphery (Wade et al., 1993; Howell et al., 2000; Alfinito et al., 2008). To increase the likelihood that the drug would reach the CNS, within 24 h of hatching, each subject received daily intracranial (IC) injections just below the skull of either 25 μg of ICI

182,780 (n=6 males, 8 females) or propylene glycol vehicle (n=6 males, 6 females). The injections were performed using a sterile 26 gauge stainless steel needle inserted at the midline of the brain at an approximate 45° angle. This technique is feasible because at this young age the zebra finch skull is thin, flexible, and easily penetrable by a needle. 36

Injections were administered for 25 days. During this time, experimental animals received parental care by adult birds that were provided with ad libitum access to finch seed, cuttle bone and water. Their diet was also supplemented weekly with fresh greens or fruit, and hard-boiled chicken eggs mixed with bread. Animals were housed on a 14:10 light/dark cycle. Adequate measures were taken to minimize pain and discomfort to subjects. All procedures conformed to national guidelines and were approved by the

Kent State University Animal Care and Use Committee.

Tissue collection and histological preparation

On day 25, 30 min following their last injection, animals were administered an overdose of Equithesin anesthesia (0.15 cc) and transcardially perfused with 0.75% saline followed by 200 ml of 10% phosphate buffered formalin (PBF). Visual inspection of the gonads confirmed the sex of the animals. Intact whole brains were collected and post-fixed in

10% PBF (phosphate buffered formalin) for 7 days, and then cryoprotected overnight in

20% sucrose solution. Tissue was quickly frozen in cold methyl butane, coronally sectioned at 20μm onto gelatin-coated slides and stained with Thionin.

Measurements and analysis

All measurements were taken without knowledge of sex or treatment. Cross- sectional area of neurons within song control regions, as well as nucleus rotundus (a sexually monomorphic thalamic visual nucleus), was measured using MicroSuite FIVE

(Olympus Imaging Systems) under 40× magnification. Within each nucleus, analysis was performed on 25 randomly selected cells from the left and 25 randomly selected cells 37 from the right side of the brain. Measurements for each side were taken from the same section of tissue, approximately at the middle of the nucleus where the volume of the specific region was most robust. Initial results did not indicate an effect of laterality.

Therefore, area measurements from both sides of the brain were summed together and averaged for statistical analysis. Due to minor tissue damage that occurred during processing, there was not a large enough sample size to statistically evaluate possible effects of ICI on Area X or to measure volumes for all song control nuclei.

Statistics

For cross-sectional neuron area, effects of treatment and sex were evaluated by two-way analysis of variance. Statistics were conducted using SPSS (version 15.0). The level of significant effects was at a P<0.05.

Results

Twenty-five days of ICI treatment significantly decreased neuron soma size in both sexes. An examination of song control region HVC revealed a significant effect of sex [F(1, 27)=9.21; P=0.006] and treatment [F(1, 27)=7.24; P=0.013] on neuron soma size (Fig. 3.0). We did not detect a significant interaction between the two variables [F(1,

27)=0.480; P=0.495]. For RA there was a significant effect of sex [F(1, 27)=31.93;

P<0.0001] and treatment [F(1, 27)=7.42; P=0.012] (Fig. 4.0). Again, there was no significant interaction [F(1, 27)= 0.991; P=0.330). Measurements of neuron soma size in

LMAN revealed a significant effect of sex [F(1,25)=5.38; P=0.030), but not treatment

[F(1, 25)=0.753; P=0.395], or an interaction [F(1,25)=1.24; P=0.278] (Fig. 5.0). As a comparison, we also measured neuron soma size in nucleus rotundus, a monomorphic 38 visual nucleus. Results indicated no significant effect of sex [F(1,25)=1.739; P=0.201], treatment [F(1,25)=0.688; P=0.416], or an interaction [F(1,25)=0.408; P=0.530] (Fig.

6.0). Unfortunately during histological processing, some tissue damage occurred and we were unable to obtain sufficient cell size measurements for Area X. However, visual inspection of available brain sections from females treated with ICI did not indicate any evidence of an induction of Area X. Additionally, we were unable to obtain sufficient numbers to statistically analyze the volume of all song regions, but it was visually apparent that ICI treatment did result in a decrease in at least RA and HVC of both sexes.

C B D

Fig. 3.0. The effects of ICI on neuron soma size in HVC. Bars represent means ± SEM.

There was a significant effect of sex. Twenty-five days of ICI treatment significantly decreased soma size in males and females (P<0.05). Different letters denote significant effects of sex, treatment and the interaction.

A B

C D

Fig. 4.0. The effects of ICI on neuron soma size in RA. Bars represent means±SEM.

A A B

Fig. 5.0. The effects of ICI on neuron soma size in LMAN. Bars represent means±SEM.

There was a significant effect of sex (P<0.05). Daily ICI treatment showed a trend towards a significant demasculinization in females, but this was less pronounced in males. Different letters denote significant effects of sex, but not for treatment.

Fig. 6.0. In the monomorphic nucleus rotundus, ICI treatment had no effect in either sex on cell size (P>0.05). Bars represent means ± SEM.

Discussion

To our knowledge, this is the first study to document a significant demasculinization of the zebra finch brain as a result of estrogen antagonism in vivo.

Twenty-five days of treatment with the ER blocker ICI demasculinized neuron soma size in several song control regions of both sexes. More specifically, the size of cells in RA and HVC were significantly decreased. In LMAN, the direction of the effect was similar although it was not significant. For Area X, unfortunately minor tissue damage prevented analysis of a significant number of subjects. However, in females, ICI treatment did not result in the induction of a visible Area X as was seen in previous zebra finch studies that attempted to block estrogenic action (Mathews and Arnold, 1990, Mathews, 1991). It also appeared that ICI was effective in demasculinizing the volume of at least HVC and

RA in both sexes. These neural changes were most likely not an effect of general development, but rather related specifically to the process of cell differentiation in song control regions. Nucleus rotundus, which to our knowledge has not been reported to contain ER in zebra finches, was not significantly altered by ICI.

Our data are in agreement with a previous in vitro study indicating that brain- derived estrogens are necessary and sufficient in establishing a male-biased synaptic connection between two song control nuclei (Holloway and Clayton, 2001). In parallel, both studies find that inhibiting the action of estrogens, with an ER antagonist, prevents masculinization of specific dimorphic features. Both studies also demonstrate that the demasculinizing effects of blocking estrogens occur in both sexes. Taken together these data provide additional evidence supporting the hypothesis that the action of estrogens in both sexes may play a role in establishing dimorphic brain development. 43

The significantly decreased soma size in females due to ICI suggests that an estrogen dependent process may be involved in female song system development.

Although an interesting hypothesis, it definitely complicates our attempts to understand how the male–female differences are created. This is particularly evident when considering the lack of consistent sex differences in ER and plasma E2. We must also acknowledge the possibility that our results are related to the dose of ICI used (25 μg).

This concentration was chosen to be comparable to antagonists used in previous studies

(Mathews et al., 1988; Mathews and Arnold, 1990; Mathews, 1991). Had we used different doses, possibly lower, we may have seen a more selective response. For instance, a lower dose could have resulted in a decrease in the soma size of males, but have had no effect on female cells. Such findings would suggest that the functional selectivity of ICI may depend on the dosage (Alfinito et al., 2008). It has also been proposed that a range of sensitivities to ICI could account for variations across tissues and even within the same tissue (Steyn et al., 2006).

The effectiveness of ICI in completely antagonizing the actions of estrogens has been demonstrated in a number of studies. For example, administration of this drug to rats and pigtail monkeys suppressed tumors and reduced the size of breast cancer in nude mice (Wakeling et al., 1991). In ovariectomized rats, ICI completely blocked the effects of estradiol (E2) on uterine weight and disrupted the actions of E2 on sexual receptivity, fat pad weight and carcass fat content (Wade et al., 1993). In human clinical trials, ICI inhibited the growth of cancerous breast and endometrial tissues; it also down-regulated

ER actions to inhibit the growth of breast carcinomas in cases where Tamoxifen failed to do so (Howell et al., 2000). In a more recent study in rats, ICI blocked estrogenic actions 44 on naloxone-induced tail-skin temperature elevations in the morphine-dependent model of hot flush and on body weight change (Alfinito et al., 2008). In the zebra finch, ICI blocked the effects of E2 on syrinx development (Martin and Veney, 2008). It is unknown if ICI acts as an antagonist on reproductive tissues in finches, but in rats, it has no estrogenic-like effects on uterine tissue (Lundeen et al., 1997; Alfinito et al., 2008).

Previous studies in zebra finches attempted to demasculinizes the song system with Tamoxifen, LY117018 and CI628, although the results were considerably less successful. The reasons for the effects of these compounds are still unclear. One likely possibility is that rather than being complete antagonists, these drugs had partial agonistic actions. For example in humans, Tamoxifen has known estrogenic actions in bone and endometrial tissues, but is anti-estrogenic in most cancerous breast tissues because of its ability to inhibit the proliferation of breast epithelial cells (Howell et al., 2000). In zebra finches, Tamoxifen in combination with estradiol benzoate (EB) acted as an anti-estrogen to suppress oviduct weight in adult females (Mathews et al., 1988), but mimicked the effects of estrogen treatment in brain when given alone (Mathews and Arnold, 1990).

LY117018 also has reported estrogenic activity. It potentially activated ER in ovariectomized nude mice as measured by an enhancement in regeneration of the sciatic nerve (McMurray et al., 2003). In the rat fetus it acted as a partial ER agonist by enhancing development of uterine tissues (Henry and Miller, 1986). Additionally, administration of CI628 to ovariectomized rats revealed estrogenic actions by promoting the hypertrophy of endometrial cells and the production of apical microvilli in the uterus

(Anderson et al., 1975a). These same investigators determined that CI628 also stimulated peroxidase secretion in epithelial cells of the vagina and cervix, a known 45 estrogen dependent process (Anderson et al., 1975b). Together, these data suggest that the varying biological activity of antiestrogens (antagonist versus partial agonist) can depend on the species studied, tissue examined, dosage used and the measured endpoint

(Howell et al., 2000; Steyn et al., 2006).

Another factor that could possibly explain differences in the efficacy of antiestrogens is the downstream processes activated as a result of binding to the receptor.

For instance, Tamoxifen has a weaker binding affinity for ER than does ICI (Howell et al., 2000). Once bound, Tamoxifen will yield a different conformational change of the receptor-ligand complex than that of ICI. As a result, one might reasonably predict differences in the activation of downstream target proteins. Additionally, the composition of the functional side chains of antiestrogens can also affect the final conformation of the complex. Large and bulky side chains may impede processes such as physically preventing coactivator binding onto specific sites (Dayan et al., 2006). Side chains composition is also important because full antiestrogenicity depends on precise regions lining up and establishing bonds with specific residues on the ER (Aspartate-

351), although some exceptions are known (Dayan et al., 2006).

To put all of this into perspective, during normal activation of ER, E2 binds and initiates a number of events. These include dissociation of heat shock proteins, ligand receptor dimerization, interaction of the N-terminal receptor activation function, AF-1, with the C-terminal receptor activation function, AF-2, and the recruitment of coregulators. In total, these actions determine the rate of gene transcription (Beato, 1989;

MacGregor and Jordan, 1998). In comparison, Tamoxifen binds weakly to ER and initiates similar downstream events, but activates AF-1 and not AF-2. The activation of 46

AF-1 has been suggested to be critical for the partial agonistic activity of antiestrogens

(Berry et al., 1990; Tzukerman et al., 1994). By this occurrence, there is continued recruitment of specific coregulators that interact with the DNA (Howell et al., 2000). In contrast, ICI does not activate AF1 or AF2 (Tolon et al., 2000) and it blocks nucleocytoplasmic shuttling of the receptor ligand complex (Dauvois et al., 1993), thus preventing dimerization, coactivator recruitment and transcription (Howell et al., 2000).

To date, there is no exact mechanism proposed to explain why LY117018 and CI628 have partial agonistic properties, but it is possible that they act similarly to Tamoxifen.

Although this study demonstrated the effectiveness of ICI in demasculinizing the song system in both sexes, a number of inconsistencies throughout the literature make it difficult to conclude that estrogens are entirely responsible for masculine development.

For this idea to be true, males would have to be exposed to more E2 during development than females. Three independent studies have used radioimmunoassays to determine whether sex differences in plasma hormone levels exist. In two of the studies, hormone levels were evaluated in animals during approximately the first 1.5–2 weeks post- hatching (Hutchison et al., 1984; Schlinger and Arnold, 1992). The third examined males and females from embryonic day 12 through post-hatch day 54 (Adkins-Regan and

Ascenzi, 1990). None of these experiments detected a consistent male or female biased sex difference.

Studies have also evaluated possible sex differences in telencephalic aromatase using a number of methods. One of the earliest, Vockel et al., (1990) used microdissected tissue punches that included song control regions, but found no main effect of sex. A second study measured aromatase activity in homegenates of brain and also found 47 no sex difference (Schlinger and Arnold, 1992). Wade et al., (1995) measured aromatase activity in dissociated cell cultures made from telencephalon and detected high levels of aromatase activity, but no sex difference. Aromatase message was evaluated by in situ hybridization during early development and although there was some expression throughout song control nuclei, no sex differences were detected (Jacobs et al., 1999).

Finally, aromatase protein was described in zebra finches using a specific antibody and again no sex differences were found (Saldanha et al., 2000).

The distribution of ER also does not strongly support a role for estrogens normally during development. Autoradiography, immunohistochemistry and in situ hybridization were used to evaluate possible differences in ERα expression. Relatively few ERα have been detected in song control regions. Overall, HVC is the only region to show significant labeling, but without any sex differences (Nordeen et al., 1987; Gahr and Konishi, 1988; Jacobs et al., 1999). Given the lack of detectable ERα in the zebra finch song system, the exact receptor subtype upon which ICI acts is unclear. It is possible that these effects are occurring through binding to ERβ, but to date, no published study is available in zebra finches describing the distribution of this receptor beyond post-hatch day 1 (Perlman and Arnold, 2003). We are currently investigating expression of ERβ beyond this age. However, in the European starling, expression of ERβ mRNA has been detected in a few neural regions including medial preoptic area, nucleus taeniae, and caudomedial nidopallium, but not within song control regions (Bernard et al.,

1999).

In sum, these data confirm our hypothesis that ICI is an effective compound that prevents masculinization of the zebra finch brain in vivo. Our data also support the long- 48 standing hypothesis that estrogens may be important for masculinizing the neural song system, but their exact role during normal development still remains uncertain. Clearly, further investigations are needed to more completely evaluate and understand the mechanisms responsible for sexual differentiation in this important model system.

Acknowledgements

I would like to thank Kalpana Acharya and Andrew Curfman for the assistance with the injections for this study. Additionally I want to thank Dr. Eric Mintz for his help pertaining to the statistics of this study. This work was supported by NSF DBI0001973 to S.V

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CHAPTER III: TREATMENT WITH THE SPECIFIC AROMATASE INHIBITOR,

FADROZOLE, DECREASES NEURON SOMA SIZE, NEURON NUMBER, AND

NUCLEAR VOLUME IN THE DEVELOPING ZEBRA FINCH BRAIN.

Stated Aim: Determine if a more direct application of Fadrozole to the brain will effectively demasculinize the neural song system.

Hypothesis: Intracranial administration of the aromatase inhibitor, Fadrozole, will significantly demasculinize the finch brain.

Abstract

The neural song system in zebra finches is more robust and developed in males than in females. The mechanism(s) responsible are not completely known but may involve hormones. In particular, estradiol partially masculinizes females; however, blocking production of this hormone (with aromatase inhibitors) has generally resulted in minimal to no effect on brain morphology in males. One explanation for this discrepancy may be related to drug administration. Previous studies primarily utilized subcutaneously administered compounds, which may have had a reduced impact on the brain because of the blood brain barrier and/or metabolizing organs. To investigate this possibility, in the present study we utilized a more direct method of drug delivery. The aromatase inhibitor,

Fadrozole, was intracranially injected into juvenile zebra finches. After 25 days of daily treatment, neuron soma size was significantly decreased in HVC of both sexes and in female RA. There was no effect on cell size in LMAN or Area X. Inhibiting aromatase also significantly decreased the volumes of RA, HVC, and Area X in treated groups.

Data further revealed that Fadrozole exposure had differential effects on measures within males and females. Across song regions affected by Fadrozole treatment, the percentage of volume decrease was greater in males as compared to females. In comparison, the percentage of cell size decrease was greater in females than in males. Taken together, these data provide evidence that in this species estrogens do play an important role in dimorphic development of both sexes and choice of how drugs are administered is important.

Introduction

Zebra finches contain a sexually dimorphic circuit of interconnected nuclei in the telencephalon that project to the syrinx (vocal organ; Nottebohm and Arnold, 1976).

Many features of this circuit, such as neuron number, size, and nuclear volume, are more developed and prominent in males as compared to females (reviewed in Wade et al.,

2001; Wade and Arnold, 2004). The total factors responsible for creating these dimorphisms are not known.

Significant efforts have been made to modify dimorphic brain development in this species by altering exposure to gonadal hormones, particularly estrogens. Somewhat consistent with the aromatization hypothesis of sexual differentiation established in rodents, administration of supra-physiological doses of E2 to females during the first two

60 to three weeks after hatching has the most potent effect of partially masculinzing the brain (Gurney and Konishi, 1980; Pohl-Apel, 1985; Konishi and Akutagawa, 1988;

Simpson and Vicario, 1991). Paradoxically, attempts to minimize E2 production in males by inhibiting aromatase have not resulted in significant demasculinization. At least two compounds have been evaluated. Fadrozole, which strongly inhibits aromatase enzyme in vitro (Wade et al., 1994) failed to alter sexual differentiation of the song system when administered in vivo for the first 30 days after hatching in 20μg doses (Wade and Arnold,

1994). Another study obtained similar results with two different doses of Fadrozole

(16μg and 150 μg) (Merten and Stocker-Buschina, 1995). Daily subcutaneous injections between post-hatching days 10 and 30 did not result in any significant effects on brain morphology when investigated at day 35. The use of a second chemically related compound, Vorozole, also did not have the predicted effect of demasculinizing the brain

(Balthazart et al., 1995). It was given to hatchlings through Silastic implants until 45 days of age. Animals were then gonadectomized and treated with T at 105 days, for three weeks. Vorozole decreased singing rate, but did not alter the volumes of any song control nuclei.

Overall, it is unclear why inhibiting aromatase did not significantly affect brain morphology in males. However, given the potency of E2 in females, manipulations of aromatase in vivo should have had a more robust effect on the male brain. The idea that estrogens are important for brain masculinization in both sexes is supported by a study in which the estrogen receptor (ER) antagonist, ICI 182,780, was intracranially injected for

25 consecutive days, resulting in significantly decreased neuron soma size in RA and

HVC (Bender and Veney, 2008). 61

Why prior attempts to demasculinize the brain in vivo with aromatase inhibitors did not mimic the results from our ICI 182,780 study are not known. One possible reason may be related to the method of drug administration. Previous studies utilizing aromatase inhibitors gave systemic injections or used Silastic implants. Because of the blood brain barrier (BBB), it is possible that the amount of inhibitor that actually reached the brain from the periphery was reduced and not enzymatically effective enough to induce morphological changes in the brain (Misra et al., 2003). It is also likely that compounds delivered systemically are first metabolized by organs such as the liver, and are therefore significantly less potent (or completely inactive) once they reach the brain (Lukas et al.,

1971). With this in mind, we reasoned that a more direct delivery of drugs to the brain might be more effective. To test this, we hypothesized that intracranial injections of

Fadrozole during early development would result in significant demasculinization of the zebra finch brain.

Materials and Methods

Treatments

All subjects originated from the animal breeding facility at Kent State University.

The experimental subjects (n=7 male Fadrozole; n=7 male controls; n=6 female

Fadrozole; n = 7 female controls) were obtained from aviaries containing 7 pairs of breeding adults. Because the goal was to flood the BBB, in an attempt to by-pass this semi-permeable membrane, intracranial injections just below the skull (but not into the brain) were administered using a Hamilton 26 gauge stainless steel needle. This technique is possible because the zebra finch skull in young animals is thin, flexible, and 62 easily penetrated by a needle (Bender and Veney, 2008). The injections were given within 24 hours of hatching at a 450 angle at the midline of the brain in approximately the same location each day. The injections resulted in no blood loss, but in rare cases there was minimal edema, which lasted no more than 2 hours. The 5µL daily doses were of either a 10% solution of sterile saline (control vehicle) or 10 µg/µL Fadrozole, which continued for 25 days. Normal parental care continued during this time. Birds were provided finch seed, cuttle bone, and water ad libitum. Their diet was supplemented weekly with spinach, oranges, and hard boiled chicken eggs mixed with bread. Animals were maintained on a 14:10 light/dark cycle. All experimental procedures were approved by the Kent State University Animal Care and Use Committee and were in compliance with national guidelines regarding the ethical use and treatment of animals.

Tissue Collection and Histological Processing

Thirty minutes following their last injection, at 25 days of age, animals were given an overdose of equithesin anesthesia and transcardially perfused with 0.75% saline and 200 mL of 10% phosphate buffered formalin (PBF). The animal’s sex was determined at the time of perfusion by visual inspection of their gonads, which appeared normal in all cases. Whole, intact brains were collected, post-fixed in 10% PBF, and then placed in 20% sucrose overnight for cryoprotection. Tissue was then snap-frozen in cold methyl butane. Coronal brain sections were obtained at 20µm increments and mounted onto gelatin-coated slides. Sections were counterstained with Thionin.

Measurements and Statistics 63

All measurements were taken without knowledge of treatment condition or sex.

In both sexes the cross sectional area of neuron soma sizes were measured (40x magnification) in RA, HVC, LMAN, and nucleus rotundus (Rt), a monomorphic nucleus.

Neuron soma size was also measured in Area X of males, but this region is not visible in females in either treatment group. Measurements were taken from 25 randomly selected cells in a single histological section. The chosen section was from the approximate middle of each nucleus where the cross sectional area was greatest. Initial results did not indicate an effect of laterality; therefore, the randomly selected cells were measured on one consistent side of the telencephalon for all subjects. The volume of each song region was also measured (20x magnification) by adding the cross sectional area in every third tissue section and multiplying by the sampling interval (60 µm). All measurements were acquired using MicroSuite FIVE (Olympus Imaging Systems). Cross sectional neuron area and volume were analyzed by two-way analyses of variance to determine the effect of treatment and sex. The statistical analyses for measurements in Area X were conducted using a one-way analysis of variance. All statistics were performed using

SPSS (version 15.0). The level of significance was at a P<0.05.

Results

Analysis of brain condition after 25 days of injections revealed no obvious signs of trauma or tissue damage (Fig. 7.0). Preliminary measurements were taken to evaluate any potential effects of laterality and no statistical differences were found between the two hemispheres (F=7.71; p=0.541). Therefore, data were collected consistently on one side of the brain. 64

In song control region RA, there was a significant effect of sex (F=11.32;

P=0.0001) and treatment (F=10.49; p=0.003) on neuron soma size, but no interaction between the two variables (F=6.51; P=0.23). More specifically, 25 days of a more direct application of Fadrozole to the brain decreased neuron soma size in females, but not in males (Fig. 8.0). There was also a significant effect of sex (F=9.92, P<0.001), treatment

(F=9.85; P=0.003), and an interaction (F=12.22, P<0.001) on RA volume. This measure was more significantly decreased in treated animals of both sexes as compared to controls

(Fig. 9.0). In HVC, there was a significant effect of sex (F= 13.31; P<0.001) and treatment (F=14.57; P<0.001), but no interaction (F= 21.83; P=0.84) on neuron soma size

(Fig. 10.0). Fadrozole significantly decreased neuron soma size in both sexes. Results also revealed that within this nucleus there was a significant effect of sex (F=22.91;

P<0.001), treatment (F=4.55; P=0.003), and an interaction (F=24.48; P<0.0001) on volume (Fig. 11.0). Measurements of neuron soma size in LMAN revealed a significant effect of sex (F=16.65; P<0.001), but not treatment (F=8.88; P=0.142) or an interaction

(F=19.71; P=0.748) (Fig 12.0). We were unable to measure the volume of LMAN given the lack of a clearly visible border at this age. In Area X, there was no significant effect of treatment (F= 7.73; P=0.81) on neuron soma size (Fig. 13.0). However, there was a significant effect of treatment (F=38.92; P<0.001) on the volume of this nucleus (Fig.

14.0).

To determine if the effects of Fadrozole were specific to song nuclei we evaluated nucleus rotundus (Rt), a monomorphic visual nucleus. Results indicated no effect of sex

(F=21.76; P=0.51) or treatment on neuron soma size. There was also no interaction (Fig.

15.0). Volume of Rt was also measured, and as expected, there was no effect of sex 65

(F=15.34; P=0.484), treatment (F=8.61; P=0.96), or an interaction (F=18.67; P=0.62)

(Fig. 16.0).

After the initial data analysis, we noted that across treatment groups, Fadrozole more significantly decreased the volume, of at least RA and HVC, in males as compared to females (average 7% more). To determine what accounted for this effect we used RA as a representative nucleus. In both sexes, the total numbers of Thionin stained cells were counted (20 X magnification) within a 4.0 mm2 area from a single representative section. The boxed area was centered within the nucleus, and the single section was from the approximate middle of RA where the cross sectional area was greatest. After counting cells, it was determined that nuclear volume was reduced most likely because

Fadrozole treatment significantly decreased the number of cells in males (F=8.93;

P<0.001), but this same effect was not seen in females (F=5.79; P=0.43) (Fig. 17.0).

Figure 7.0. Gross anatomical representation of post-treatment. The brain is absent of any insult or damage that could have been from dose delivery.

Figure 8.0 Mean (+ SEM) of neuron soma size in RA. Different letters denote significant effects of sex and treatment.

Figure 9.0 Mean (+ SEM) of RA volume. Different letters denote significant effects of sex, treatment and the interaction.

Figure 10.0 Mean (+ SEM) of neuron soma size in HVC. Different letters denote significant effects of sex and treatment.

Figure 11.0 Mean (+ SEM) of HVC volume. Different letters denote significant effects of sex, treatment and the interaction.

Figure 12.0 Mean (+ SEM) of neuron soma size in LMAN. Different letters denote the significant effect of sex. There was no effect of treatment.

Figure 13.0 Mean (+ SEM) of neuron soma size in Area X. There was no effect of treatment.

Figure 14.0 Mean (+ SEM) of Area X volume. Different letters denote the significant effect of treatment.

Figure 15.0 Mean (+ SEM) of neuron soma size in Rt. There was no significant effect of sex or treatment.

Figure 16.0 Mean (+ SEM) of Rt volume. There was no significant effect of sex or treatment.

Figure 17.0 Mean (+ SEM) of cell counts in RA. Different letters denote significant effects of sex, treatment and the interaction.

Discussion

To our knowledge, this is one of a very few studies to demonstrate a significant effect of an aromatase inhibitor on the zebra finch brain in vivo. Twenty-five days of daily intracranial Fadrozole injections effectively decreased several measures. More specifically, neuron soma size was significantly smaller in female RA and in HVC of both sexes. The volumes of these regions were also significantly decreased in both sexes as a result of treatment. Interestingly, Fadrozole did not affect neuron cell size, in LMAN or Area X; however, treatment did significantly decrease Area X volume.

Our data also revealed that Fadrozole did not affect measures in males and females equally. When comparing across treatment groups (male control vs. male

Fadrozole and female control vs. female Fadrozole) we found that in at least RA and

HVC the average magnitude of volume decrease was greater in males (7%) than in females (3%). Even in Area X (which was not visible in females in any treatment condition) the volume was reduced by a similar percentage as compared to controls. One explanation accounting for reduced volumes in males was that there were fewer cells after Fadrozole treatment. This is consistent with what is known about the importance of estrogens for cell protection and survival. The relationship has been demonstrated in a number of species (reviewed in Brann et al., 2007). For example, in rats injected with E2 there was only minimal brain damage from induced ischemia as compared to controls not provided with E2 (Liao et al., 2001). In a related study, it was emphasized that aromatase inhibitors and estrogen receptor blockers caused the degree of brain insult in rats to increase in ischemia-induced subjects (Sawada et al., 2000). The neuroprotective effects of estrogens have also been evaluated in mice expressing autoimmune encephalomyelitis 78

(Tiwari-Woodruff et al., 2007). In this particular study, ovariectomized mice were treated with an ERβ or ERα agonist for 10 days. Administration of these compounds was neuroprotective as measured by a reduction in demyelination of white matter as well as axonal numbers. The neuroprotective effects of estrogens are paralleled in avians. For example, in zebra finches that underwent uni-lateral, chemically induced neural injury, there was increased cell death in the hemisphere of the subject if given in combination with Fadrozole. In the opposite brain hemisphere that received saline vehicle and chemically–induced injury there was minimal apoptosis (Wynne and Saldanha, 2004).

This was most likely due to locally synthesized estrogens (Schlinger and Arnold, 1992a;

Holloway and Clayton 2001).

In contrast to Fadrozole having a greater impact on the number of cells in males

(which affected volume) in females, treatment seemed to have more influence on reducing cell size. Within HVC and RA the cross-sectional area of cells was decreased more in females after treatment (average 5.2%) as compared to males. Overall, it is not clear why Fadrozole preferentially affected cell number in males and cell size in females.

Clearly both sexes respond to treatment (suggesting feedback from an estrogen receptor).

And clearly, estrogens are important for cell survival (number) and cell size. But perhaps our data is suggestive that the mechanisms by which estrogens affect these two measures are inherently different in males and females.

In zebra finches Fadrozole is an effective aromatase inhibitor in vitro (Wade et al., 1994, Holloway and Clayton, 2001). However, attempts to reverse masculine characteristics of the brain by minimizing estradiol production in vivo have not been extremely effective. For example, Wade and Arnold (1994) injected zebra finches with 79

Fadrozole on post-hatching days 1-30 and examined brains on day 31. Treatment had no effect on the volumes of RA or Area X. It also did not alter neuron soma size in RA or

HVC. In a separate study, the effects of Fadrozole were investigated in adults after five days of peripheral injections and in post-hatching 4-6 day old juveniles after one injection

(Wade et al., 1994). At neither age group did Fadrozole significantly alter brain morphology. In contrast to the above, to our knowledge only one other study has been able to demonstrate a significant effect on brain morphology with Fadrozole. Merten and

Stocker-Buschina (1995) treated males from post-hatching days 10-30 with systemic injections and then sacrificed them either at day 35 or 135. There was no change in neuron soma size or song region volume at day 35. The volume of HVC however did show a modest, but significant decrease when measured in animals 135 days of age.

Although it is not entirely clear why previous treatments with Fadrozole in vivo have been so limited in zebra finches, one possibility may be related to how the compound was administered. Whereas previous works primarily relied on subcutaneous injections or peripheral systemic applications, in the present report, we utilized intracranial injections as a way to more directly expose the brain to the compound. Part of the reasoning for this approach stemmed from an in vitro study (Clayton and

Holloway, 2001) in which Fadrozole added to media containing male brain slices significantly demasculinized the neuronal projections from HVC to RA. Based on this we postulated that structures such as the BBB, or metabolizing organs such as the liver perhaps limited the amount and/or efficacy of compounds that were administered systemically. Thus, by using intracranial injections, our goal was to by-pass or “flood” the BBB. 80

In at least one study conducted in Japanese quail, (Cornhil et. al., 2006) there is indication that systemic injections of Vorozole do reach the brain. However, there is plenty of other evidence suggesting that large molecules, comparable to the size of

Fadrozole, are more likely to be stopped by the BBB or metabolized elsewhere, when administered systemically. In mammals the BBB is formed by epithelial cells that create tight junctions within the endothelium of capillaries that have access to the brain. The function is to act as a filter and selectively block substances from gaining entrance. This barrier limits the brain’s exposure to larger molecules in systemic circulation (Pardridge,

1999). To date there is no information available about the size or composition of molecules that can pass through the zebra finch BBB. However, in humans, anything larger than about 500 Daltons generally cannot gain access to the brain (Fischer et al.,

1998). The Fadrozole molecule is 259.74 g/Mol, which is the equivalent of about 1.5 x

1026 Daltons, suggesting that it would theoretically be too large to access the human brain.

Using positron emission tomography, differences in the permeability of the BBB were evaluated in several species including humans, rats, and monkeys (Syvanen et al.,

2009). It was determined that passive diffusion through the BBB of larger animals such as humans and monkeys was more prevalent than in smaller organisms. Based on these findings, it is reasonable to deduce that molecules the size of Fadrozole would have extreme difficultly readily passing through the BBB of a small organism, like zebra finches. Thus our methodology may have surpassed previous attempts by allowing

Fadrozole to reach the brain at a slightly higher concentration than was possible through 81 other less direct applications, making the compound more effective at altering brain morphology.

In addition to the BBB, metabolism by the liver is another possible explanation for why systemic injections of aromatase inhibitors may not have been extremely effective in zebra finches. The liver is a major organ that breaks down compounds in the blood and is most commonly the first metabolizing organ for substances in the body

(Yagami et al., 1993; Rowland, 1972). Because of this, even as early as the 1970’s it was known that in order to by-pass the effects of the liver it was necessary to increase the dose of drugs administered peripherally or orally so that they would effectively reach the desired target (Rowland, 1972). In support of this, a more recent study conducted in rats determined that approximately 90% of all orally-administered Fadrozole was taken up by the liver within 2 hours of administration (Yamagami et al., 1993). Our method of delivery was likely to have by-passed this rapid absorption and more directly influenced the tissue of interest.

Overall, results from the current study as well as others (Bender and Veney 2008;

Holloway and Clayton 2001) all provide support for the hypothesis that estrogens are important for masculine development of the song system. However, how these effects naturally occur is still not clear since there have been no reported sex differences in aromatase enzyme, aromatase receptors, or aromatase mRNA (Vockel et al.,1990;

Schlinger and Arnold 1992b; Wade et al., 1995; Jacobs et al., 1999; Saldanha et al.,

2000). There have also been no demonstrated sex differences in estrogen receptor alpha

(Jacobs, et al. 1999) or beta, which appears to be completely absent from song control nuclei of zebra finches (Perlman and Arnold, 2003; Bender and Veney, unpublished data) 82 and in European starlings (Bernard et. al., 1999). Instead, it is possible that dimorphic responses to estrogens may exist because of coregulators, such as coactivators and corepressors, which increase or decrease the transcriptional activity of nuclear steroid receptors (Duncan et al., 2009). Under the influence of co-regulators, plasma levels of hormones or the number of steroid receptors do not need to be dimorphic in order for differences in estrogen responses to occur.

Another explanation for how estrogens can influence development of the song system is if there are other dimorphically expressed forms of estrogen receptors. One possible candidate is GPR-30, a membrane bound receptor, that has been investigated in mammals (Canonico et al., 2008, Maggiolini et al., 2010) and is sexually dimorphic in hamsters and rats (Sakamoto et al., 2007, Canonaco et al., 2008). In zebra finches, the

GPR-30 gene is dimorphic (more in males than in females) during early periods and may contribute to dimorphic development of the song system (Acharya and Veney, 2012).

In summary, results of this study support the hypothesis that intracranial injections of Fadrozole during early development effectively demasculinize the zebra finch brain. Thus, these results provide additional support that estrogens are important for masculinizing the neural song system. In addition, the method of drug delivery also appears to make a significant difference on the effectiveness of administered compounds.

Still, the precise role of estrogens during normal development is yet to be completely understood. A greater understanding of coregulators and an increased knowledge of the location and distribution of other forms of ER may shed some light on the exact mechanism(s) through which this process is occurring.

Acknowledgements

The authors would like to thank Novartis Pharmaceuticals for the provision of Fadrozole.

Additionally we would like to thank Kalpana Acharya, Shandilya Ramdas, and Ann

Dobry for their assistance with animal care, injections and research protocols.

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683.

CHAPTER IV: ESTROGEN RECEPTOR BETA IN THE EARLY POST-

HATCHING ZEBRA FINCH BRAIN

Aim 1: Examine telencephalic expression of the ERβ gene and the pattern of ERβ mRNA distribution during early post-hatching development

Hypothesis: During the first four weeks after hatching, when the brain is most sensitive to the effect of estrogens, ERβ is present throughout the developing telencephalon and its expression overlaps with song control regions.

Abstract:

The zebra finch song system represents an extreme dimorphism with correlating behavioral differences between the sexes. The neural differences are believed to be due, at least in part, to the action of estrogens but how this occurs is not known since there have been no reported sex differences in the “classic” nuclear estrogen receptor (ERα) and overall expression is limited. Alternatively, it is possible that a second nuclear estrogen receptor (ERβ) may be involved. Quantitative Polymerase Chain Reaction (Q-

PCR) was used to examine the expression of the ERβ gene in the telencephalon of animals at post-hatching (P) ages 3, 5, 8, 11, 15, 20, and 30. The gene was detectable in both sexes at all ages examined, but there were no significant differences between the sexes or across any of the ages. Additionally we wanted to investigate specifically where the gene expression was localized. Analysis of ERβ mRNA expression revealed specific labeling only in two brain regions, nucleus teniae (Tn) and the caudomedial nucleus

(NCM), which are associated with song interpretation and auditory processing,

93 respectively. Signal was not detected in any song control regions at any of the investigated ages. Taken together, these data suggest a specific role for estrogens acting through ERβ for auditory processing in both sexes, but does not support the hypothesis that ERβ is present in song control regions, and therefore is not likely to regulate dimorphisms in this circuit.

Introduction:

Zebra finches are a widely studied model for understanding sex differences in brain development and how they control reproductive behaviors (Gurney and Konishi,

1980; Wade, 2001; Wade and Arnold, 2004). In this species males produce a courtship vocalization (song), that females normally cannot produce. This singing behavior is controlled by a sexually dimorphic group of interconnected nuclei within the telencephalon that are larger in volume and neuron number in males as compared to females (Nottebohm and Arnold, 1976; Gurney, 1982; Konishi and Akutagawa, 1985).

The factor(s) that direct dimorphic development of these brain regions are not completely understood. However, evidence suggests they may partially depend on steroid hormones.

Manipulation of E2 levels during development results in changes in the normal development of the brain. For example, females that were treated with E2 within the first few weeks after hatching developed a partially masculinized song circuit (Gurney and

Konishi, 1980; Gurney, 1981; Simpson and Vicario, 1991; Grisham et al., 1994). This was characterized in part by an increase in the volume of song regions, an increase in the number and size of neurons within these areas as well as the formation of Area X, which is normally not detectable in female tissue stained for Nissl.

Administration of the estrogen receptor antagonist ICI 182,780 resulted in a significant reduction of neuron soma size in select song control regions (Bender and

Veney, 2008). These results were novel compared to previous studies that had used other

ER antagonists, which resulted in minimal to no effect on brain morphology (Mathews and Arnold, 1995, Mathews, Brenowitz and Arnold, 1998). Despite the inconsistencies across the studies, the overall potency and effect of E2 manipulations on this system demonstrate a role for E2 in development. What is not yet understood is how estrogens are influencing this circuit.

In zebra finches, the expression of ERα has been thoroughly investigated. The distribution of its mRNA and protein has been described at various embryonic (E), post- hatching and adult ages (Gahr and Konishi, 1988; Jacobs and Arnold, 1996; Jacobs et al.,

1999; Perlman and Arnold, 2003). Signal was detected throughout several telencephalic regions including song control nucleus HVC, which contains a moderate amount of labeling from P10 into adulthood. Labeling in RA was only detectable at P25, and was considerably less than what was seen in HVC (Jacobs and Arnold, 1996; Jacobs et al.,

1999). Neither song region had a documented sex difference. LMAN and Area X did not contain any ERα positive cells at any age. In contrast, ERβ has not been as extensively investigated in zebra finches. In one study, the mRNA was examined at embryonic day (E34) and P1 (Perlman and Arnold, 2003). Detectable signal was present in a number of telencephalic regions, however song control nuclei are not yet formed this early in development, therefore, it is unclear if the expression overlapped. To our knowledge, ERβ has never been described at later ages in the zebra finch. But in

European Starlings, a closely related species of songbird, ERβ mRNA is not found in 95 song control nuclei, but instead in auditory processing regions of birds at a variety of ages

(Bernard et al., 1999). To determine what functional role ERβ has in zebra finches, in this study the goal was to more completely examine the distribution and localization of

ERβ in the developing brain.

Materials and Methods:

Subjects

Subjects were obtained from the breeding facility at Kent State University. The experimental zebra finches (Taeniopygia guttata) were taken from nest boxes inside of aviaries containing 6-7 pairs of adults who had ad libitum access to finch seed, water, and cuttle bone. Their diets were supplemented with spinach, hard-boiled chicken eggs, and oranges. Animals were housed on a 14:10 light/dark cycle. Experimental subjects (n=6 males, n=6 females at each age) at P3, P5, P8, P11, P15, P21, and P30 were rapidly decapitated and their telencephalon was removed and snap-frozen in methyl butane for analysis of ER beta gene expression. These ages were chosen based on the sensitivity of the brain to estrogens during the first four weeks post-hatching (Simpson and Vicario,

1991) as well as for the fact that ERβ has never been described beyond P1 in the zebra finch. These ages are also significant because they correspond to a period before and during song nuclei development, which is a critical time to evaluate the role of estrogens for this process (Perlman and Arnold, 1999). We reasoned that if ERβ were important to this system, it would be abundantly expressed during these “snap shot” ages (Gurney

1980; Bottjer et al., 1985; Nordeen et al., 1986; Simpson and Vicario, 1990). At the time the brain was removed, the gonads were inspected to confirm the sex of the animals. 96

Additional subjects (n= 4 males, n=4 females) were collected at P15 for in situ hybridization (ISH). P15 was chosen because at this age the boundaries of song control nuclei are just becoming clearly definable and the nuclei are rapidly developing. Animals were deeply anesthetized with carbon dioxide and then transcardially perfused with saline and 10% phosphate buffered formalin solution (PBF). Brains were post-fixed overnight in 10% PBF, cryo-protected overnight in 20% sucrose and then snap frozen in methyl butane. Brains were sectioned (20µm) onto alternate slides (a series of three) and stored at -800C until use. Two series were used for ISH (sense and antisense), the third was stained with thionin to aid in the identification of brain regions.

Q- PCR of zebra finch ERβ cDNA

Total RNA was extracted from the telencephalon of zebra finches at post-hatching ages

3,5,8,11,15,21,and 30 using the RNeasy Mini kit (Qiagen). The quantity and quality of

RNA was confirmed to be within valid ranges and visualized on a gel. The RNA was converted into cDNA using the High Capacity cDNA Reverse Transcription kit (ABI) and the product cleaned using the Qiaquick PCR Purification kit (Qiagen). Q-PCR analysis was performed (ABI Prism 7000) using the SYBR Green PCR master mix kit with 20 ng of cDNA and 200 nM final primer concentration, in a total volume of 20 μl.

IDT Primer Quest was used to design the ERβ forward primer: 5’ ATC TCA GCC TCT

ATG ACC AAG TAA G 3’ and the ERβ reverse primer: 5’ CAG TTT CCC AAG ATG

GTC AAT T 3’ from zebra finch. The reactions were run in triplicate for each sample and primer combination. Negative RT controls were made for each sample by omitting the reverse transcriptase from the reaction. Amplification of GAPDH (GAPDH forward primer: 5’-TGTGGACCTGACCTGCCGTCTG-3’ and GAPDH reverse primer: 5’- 97

TGAAGTCACAGGAGACAACCTG-3’) both synthesized from the zebra finch genome was used as a loading control. Cycling conditions were 50˚C for 2 min, 95˚C for 10 min, and 40 cycles at 95˚C for 15 sec, and 60˚C for 1 min. The dissociation curves of the amplified PCR products were examined to confirm the absence of DNA contamination and any unwanted products. The average δCt was obtained by subtracting the Ct value of

GAPDH from the Ct value of ERβ. The relative expressions were calculated using the equation 2^- δCt values. Statistical analyses were performed using Sigma Stat. A Two

Way ANOVA was conducted on expression values to examine the effect of age and sex on gene expression. The level of significance was determined at P<0.05.

Riboprobe Development

Template for ERβ was provided by Dr. Arthur Arnold (UCLA). The linearized 636 bp cDNA encoded for a portion of the zebra finch ERβ 3’ UTR, which was generated using primers designed against the ERβ 3’ UTR of the European starling (Bernard et al., 1999).

The amplified fragment was subcloned into the pGEM T-easy vector (Promega, Madison,

WI) and sequenced using an ABI Prism DNA sequencer (ABI, Foster City, CA). The sequence shared the following overall nucleic acid percentage identities with ERβ of other avian species: European starling, 97% (GenBank accession no. AF113513);

Japanese quail, 91% (AF045149); chicken, 91% (AB036415). Plasmids containing the

ERβ insert were streaked onto agar plates and grown overnight in a 370C incubator.

Individual colonies were picked with sterile toothpicks and grown for 16 hours in luria broth with ampicillin. Qiagen maxi prep kit was used to extract the DNA template from the bacteria. The template was linearized using NCO I and Sal I restriction enzymes.

Riboprobe was made using this template and a transcription reaction kit (ABI) with the 98 incorporation of digoxygenin (Roche). Confirmation of successful transcription was verified by running the products on an RNA gel. Single bands of equal size

(approximately 836 bp) for both sense and antisense generated probes were detected.

In Situ Hybridization

Day 1:

Slides were removed from -80oC and warmed to room temperature for 10-15 minutes.

Next, a pap pen (Vector) was used to outline the perimeter of each slide. One hundred

µL of 4% paraformaldehyde made with 1x PBS was applied for 7 minutes. After rinsing this off, 100-150µL of pre-hybridization buffer (2mL of 4x saline sodium citrate (SSC)

1g of dextran sulphate, 0.2mL of 1x Denhardts, 40µL 0.5mM EDTA, 5mL of 50% deionized formamide, 40µL of Herring Sperm DNA, and 2.72mL of DEPC treated water) was applied to each slide for 90 minutes at 550C. The digoxygenin probe was denatured at 800C for 5 minutes and diluted in hybridization buffer (same recipe as pre- hybridization buffer) at a concentration of 200ng/mL. Next, 200µL of hybridization buffer was applied to each section of tissue and hybridized for 16 hours at 500C in a humid chamber. For each step of the protocol, solutions were applied directly onto each tissue section and the slide on which it was mounted (no coverslip) was placed inside a plastic box and covered with a lid. To keep the chamber humidified, moist filter paper soaked in 1x PBS was added.

Day 2: 99

Slides were washed in 1 x SSC at 370C for 5 minutes. They were then placed in 2xSSC and 50% formamide at 500C for 7 minutes. Next the tissue was washed with 0.5xSSC at

370C for 5 minutes. Buffer 1 (100mM Tris-HCl and 150mM NaCl) was then applied.

Sections were then equilibrated to pH of 7.0 with blocking buffer (5% solution made with

Buffer 1 and Roche Blocking Reagent) for one hour. A 1:500 dilution of anti-dig antibody (Roche) in blocking buffer was applied for 2 hours and then the sections were washed with Buffer 1 for 5 minutes. Sections were then washed using Buffer 2 (100mM

Tris-HCL and 100mM NaCl) to bring the pH back to neutral. Visualization of the reaction product occurred in detection buffer made from a combination of 20 µL of

NBT/BCIP stock solution (Sigma-Aldrich), 20 µL of levamisole and 960µL of DEPC treated water. The reaction was stopped with Buffer 3 (0.79 grams of Tris-HCl and

0.5mL of 1mM EDTA in a total volume of 500mL DEPC treated water. Sections were then counter-stained with thionin, and the slides covered in Eukitt aquamount (Electron

Microspcoy Sciences) and a coverslip.

Semi-quantitative analysis

To get an overall impression of ERβ labeling at P15, we opted to perform a semi quantitative analysis. Positively labeled cells were identified by the visual identification of dense staining in the nucleus of the cell and a non-labeled cytoplasm. The amount of labeling was recorded as follows: little to no labeling=10 or less positively labeled cells, moderate =10-19 positively labeled cells, high level of labeling=20 or more positively labeled cells. The positively labeled cells were examined throughout the rostral to caudal extent of each song control nucleus. 100

Results

Q-PCR analysis revealed no significant effect of sex (F1,84= 4.621, p=0.114) or age

(F1,84=2.315, P=0.084) on ERβ gene expression (Figure 18.0). At P15 analysis of the mRNA revealed positive labeling in control oviduct tissue (Figure 19.0), but no detectable labeling was found in song control regions (Figure 20.0) or in many other regions of the brain (Table 1.0). Positive labeling was detectable in nucleus teniae (Tn) and the caudomedial nicopallium (NCM). The relative amount of cells that contained

ERβ mRNA was moderate and there was no obvious sex difference based on a visual observation (Figure 21.0). Compared to these two regions, fewer cells were detected lining the lateral ventricles and spanning the apical hyperpallium (Table 1.0). The locations of the respective nuclei investigated and documented for the presence and absence of labeling were based on the zebra finch sterotaxic atlas (Nixdorf-Bergweiler and Bischof, 2007) (Figure 22.0).

101

Table 1.0 Relative amount of ERβ labeling in regions outside of song control areas at

P15.

Brain Male Female Region Tn ** ** NCM ** ** Nif -- -- HP -- -- Rt -- -- PV -- -- TrSM -- -- mPOA -- --

Abbreviations:

Tn (nucleus taeniae), NCM (medial caudale nidopallium), Nif (Nucleus inferfascialis),

HP (hippocampus), Rt (nucleus rotundus), PV (nucleus posteroventralis thalami) TrSM

(Tractus Septopallio-Mesencephalicus), mPOA (medial preoptic area)

-- = No labeling

* = low labeling

** = moderate labeling

*** = high labeling

102

Figure 18.0 Average delta Ct values at post-hatching ages. Bars represent means + SEM.

There was no significant effect of age (F1, 84=2.315, P=0.084) or sex (F1,84= 4.621, p=0.114) on ERβ gene expression.

103

Figure 19.0 ERβ mRNA expression in oviduct labeled with the antisense (top) and sense

(bottom) probes. 104

Figure 20.0 Note the lack of ERβ mRNA in RA at P15 with the antisense probe (Top).

A Thionin counterstain outlines the borders of RA (Bottom). Scale bar refers to all panels.

105

Figure 21.0 Magnified view of the NCM with nuclear labeling of positive cells for ERβ mRNA.

106

Figure 22.0 The images provided are collected from the stereotaxic atlas of the zebra finch brain (Nixdorf-Bergweiler and Bischof, 2007). The regions of interest for ERβ are shown in the provided panels: Tn (Panel A), NCM (Panel B), mPOA (Panel C), PV

(Panel C), TrSM (Panel E), Nif (Panel C and D). Also song control nuclei are displayed 107 for RA (Panel A), HVC (Panel A and B), LMAN and Area X (Panel E), and the monomorphic nucleus Rt (Panel C and D). The highlighted areas represent the nuclei where analysis occurred and is not indicative to the amount of labeling or the location within each nucleus.

Discussion

This study is the first to demonstrate labeling of ERβ in the zebra finch brain after P1. These results support and extend previous work that has evaluated the presence of ERβ in the songbird brain. At embryonic ages in the zebra finch, labeled

ERβ cells are present in Tn, NCM, PV, TrSM, and mPOA. In partial agreement with these data, our work revealed labeling in Tn and NCM at P15, but not in the other regions. These differences suggest that ERβ may have a significant organizational role during embryonic development that is not apparent at P15. For example, in Japanese quail and chicken, PV is a thalamic nucleus that integrates sensory information (Cheng and Zuo, 1994). TrSM is a region of the hypothalamus directly involved in feeding responses and behaviors in Japenese quail (Voigt, Ball, and Balthazart, 2007).

Additionally, the mPOA is a hypothalamic nucleus in many species, including the finch, that is involved in sexual behavior, courting and mating (Arnold and Gorski, 1984). The presence of ERβ in these regions at embryonic ages, but not at P15, suggests that estrogens may be involved in the organization of the circuitry that is important for these behaviors. The European starling is an additional species in which labeling of ERβ has been detected (Bernard et al., 1999). In adults of various ages, labeling was demonstrated in Tn, NCM, and throughout the hypothalamus. We similarly detected labeling in Tn and 108

NCM, but not in hypothalamic regions. The absence of labeling in the hypothalmus of zebra finch, specifically in mPOA, may be due to the early age we investigated. In particular, zebra finch do not display courtship or mating behaviors until adulthood (after

P90) (Burley, Parker and Lundy, 1996) and therefore, one would not expect to see ERβ activated for these behaviors at this time.

ERβ mRNA at P15 provides only a snapshot during development of the expression of this receptor. There is a possibility that examining later ages may reveal an increased level of receptor when additional processes, such as song learning occur. Song learning in passerine birds is a process that involves a tutor or parent and requires auditory feedback for learning. In particular, the neural connections between NCM, Area

X, and HVC are important. Studies involving, the immeadiate early gene, zenk, in finches reveals activation in NCM and Area X during song listening and in HVC during the production of song as memories are being consolidated (Jin and Clayton, 1997).

Additionally, the interpretation of conspecific sounds during early development can influence an emotional memory or evoke a behavioral response in which Tn is activated;

Tn is considered to be the avian homolouge to the mammalian amygdala (Remage-

Healey et al., 2010). We were able to detect labeling in both Tn and NCM. However, in contrast to these regions, which are more developed at P15, additional nuclei involved in the song learning pathway, Nif, Area X, and LMAN, are not as greatly developed at this time (Bohner, 1990; Morrison and Nottebohm, 1993). The development at P15 is not fully established for HVC either, (a nucleus responsible for song production). Therefore, the lack of labeling in these regions may be partially due to the early age in which we investigated. In fact, song memories which involve the activation of Nif, Area X and 109

LMAN, are not established until approximately P28-P35 (Bohner, 1990) and finches do not begin to practice singing until around P30-P40 (Arnold, 1975; Bottjer et al., 1984).

Although we detected limited ERβ labeling in the auditory processing and song learning pathways at P15, there is evidence that estrogens acting later are essential to their development (Coleman et al., 1994; Tremere et al., 2009; Remage-Healey et al.,

2010). Estradiol (E2) is directly involved in the MAP kinase pathway of song plasticity and memorization (Tremere, Jeong, and Pinaud, 2009). Removal of this hormone during development prevents the activation of MAPK-induced gene expression and results in abnormal song production. A second study, demonstrated that disruption of E2 production and availability, during the first three weeks after hatching, prevents zebra finches from responding to song stimuli (Remage-Healey, et al. 2010). This study also suggested the possibility that estrogens may be involved in species-specific song recognition, because when estrogens are removed, zebra finches respond to general sounds, but do not recognize conspecific song.

If estrogens are importmant for brain dimporhisms, then there should be expression demonstrated using ISH for some type of estrogen receptor at P15. The fact that neither ERα nor ERβ show a sex difference in dimoprhic brain regions and are minimally expressed at this age, indicates that estrogens are working through some other receptor or mechanism during development. One potential candidate is GPR 30, a membrane bound estrogen receptor, which is present in RA and dimorphically expressed in HVC at P21 (Acharya and Veney, 2012). Given this, it is possible that GPR 30 by itself, may contribue to the majority of brain dimoprhisms. Alternatively, at later ages, when the expression of ER may increase, both receptors may functionally contribute to 110 neural sex differences. Additional work will be necessary to determine which of these two receptors, if either, are more important. However, as a result of these data, we know it is not ERβ at least through P15.

Acknowledgements

The authors would like to thank Kalpana Acharya for her assistance with PCR and cDNA samples. They also would like to thank Jacque Balthazart, Sylvia Bardet, Nilam Sinha,

Andrew Curfman, Ann Dobry, Eric Mintz, and Shandilya Ramdas, for their assistance or expert advice regarding in situ hybridization, gene expression, and statistical analyses.

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CHAPTER V: GLOBAL DISCUSSION

Implications of Research

For years, mammalian studies investigating brain sex differences have provided models indicating the importance of hormones, particularly estrogens, for brain masculinization. How widely this applies to all vertebrates is a question that has been investigated in many species. For example, in zebra finches, estradiol (E2) does affect development of the dimorphic neural song system, but several inconsistencies in results across labs have made it difficult to understand if this hormone is primarily important, and if so, how it normally acts. Using different approaches compared to ones in the past, in this dissertation, I set out to re-analyze the role of E2 in this avian model.

In my first study, I used a potent antagonist to block estrogen receptors. My results clearly demonstrated support for E2 in brain masculinization. Specifically, in both sexes, when ICI 182,780 was administered, neuron soma sizes significantly decreased in song control nuclei. These results were a clear extension of previous knowledge. They demonstrated a complete demasculinization in males by blocking hormones. No study prior to this was ever able to achieve these same results using similar compounds. In addition, blocking estrogen receptors also resulted in a significant decrease in female measures as well. Again, no prior study had ever reported a single antagonist affecting both males and females in the same direction.

My second study limited the production of estrogens by blocking aromatase, the enzyme needed for E2 synthesis. This study also supported a significant role for

119

120 estrogens in brain masculinization because in the presence of Fadrozole, I detected decreased neuron soma sizes, neuron number, and nuclear volumes within the song circuit. Similar to the first study, the results of the second also provided new information regarding estrogens actions in the zebra finch. Prior work, which had attempted to block aromatase, did not detect any significant effects in the brain. The fact that I did successfully block aromatase was novel, and indicated that the choice in how I administered the drug may have played an important role. This concept that has never been explored in the zebra finch, but should be further investigated because my work suggests that systemic injections may not be as effective in this model as they are in mammals. Additionally, the time frame was unique for the onset and extent of drug delivery during development. Previous studies were not consistent with days of drug administration or length of treatment.

In my third experiment, I attempted to determine which receptor estrogens were primarily acting on to initiate the above effects. Since ER has already been thoroughly described in the zebra finch and does not likely contribute to brain development, I sought to address a missing gap by investigating ER . My data demonstrates that this receptor is unlikely to play a major role in dimorphic brain development. Instead, the results show that ERβ plays a role for this receptor in early development of auditory processing regions outside of song control nuclei. Given the unlikely possibility that ER is mediating the effects of estrogens on the neural song system, other receptors must be involved. One possibility is GPR30 (Acharya and Veney, 2012) an estrogen receptor that is dimorphic at a select age during early development.

120

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Below is a timeline of major events in zebra finches (Table 2.0). It outlines processes during development and in adulthood that are affected by estrogens. The table suggests likely receptor(s) through which estrogens are binding to contribute to the various processes.

Table 2.0 Proposed model for developmental processes and contributing estrogen receptors.

RESPONSIBLE RECEPTOR AGES PROCESS ERβ E30-P1 Early formation of auditory processing regions and hypothalamic nuclei GPR30? P3-P8 Early formation of song control nuclei prior to the appearance of dimorphisms GPR30? P15 Onset of neural morphology differences Nuclei responsible for auditory processing ERβ P15 are activated. ERβ? P28-P35 Song learning pathway is functional ERβ Adults Adult behaviors such as courtship and mating

This table highlights some of the contributions of my work and integrates it with results from other research conducted in the same and similar species. Earlier work with zebra finches demonstrated a potential role for estrogens, acting through ER , during development in the organization of the auditory processing pathways of the hypothalamus (Perlman and Arnold, 2003). Additionally, I demonstrated the presence of

ERβ receptors in NCM and Tn at P15, which reveal its contribution to audition and potentially song learning. Work in the European starling examined ERβ functionality into adulthood for auditory processing, courtship behaviors and mating (Bernard et. al., 1999). 122

The activation of song learning nuclei, such as Nif, LMAN, and Area X occurs between

P28-P35 (Bohner 1990). Since these nuclei are involved in the song learning pathway and the auditory processing of song is so closely correlated, estrogens may be mediating this process through ERβ, however there is no data to date addressing this possibility at these specific ages. The early formation of song control nuclei occurring between P3-P8, and the visible dimorphisms in these regions at P15, are likely controlled by estrogens binding to GPR 30. To date, the only dimorphically expressed estrogen receptor is GPR

30 (Acharya and Veney, 2012). Therefore, visible brain dimorphisms revealed early on have the greatest possibility to be working through GPR 30. However, the potential neural morphology changes after blocking this receptor have not been reported yet in the zebra finch. Additional information involving ERβ and GPR 30 need to be gathered evaluating these potential roles for estrogens.

Future Directions

Although the presence of ERβ has been demonstrated in the zebra finch at embryonic ages and now at P15, a more complete model needs to be established in which this receptor is described throughout development and into adulthood. To fully understand the role of estrogens in auditory processing, dimorphic brain development, song learning and song production, more work is needed that evaluates effects within this circuit when all known estrogen receptors in the finch (GPR30, ERα and ERβ) are blocked simultaneously. This may likely be achieved with a combination of known ER antagonists such as G15 (for GPR 30 receptors, Dennis et al., 2009) along with ICI 182,

780 (for ERα and ERβ). Evidence has also suggested the possibility of an additional ER, 123

ER-X, through work in the mammalian brain (Toran-Allerand et al., 2002) but it is not known if this receptor is present in the zebra finch.

My work with Fadrozole supported a role for estrogens in the zebra finch, and suggested that the blood brain barrier is more protective in birds than in mammals. It would be interesting to investigate the overall size and consistency of substances entering the zebra finch blood brain barrier to determine if the method of delivery was the confounding factor that produced these unique outcomes. A study conducted with the release of dyes of various molecular weights and consistencies, for example, water vs. lipid soluble substances, could quickly determine the likelihood of previously used compounds ever reaching the brain.

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Bohner, J., 1990. Early acquisition of song in the zebra finch, Taeniopygia guttata. Anim.

Behav. 39, 369–374.

124

Dennis, M.K., Burai, R., Ramesh, C., Petrie, W.K., 2009. In vivo effects of a GPR 30 antagonist. Nat Chem. Biol., 5, 421-427.

Jacobs, E.C., Arnold, A.P., Campagnoni, A.T., 1999. Developmental regulation of the distribution of aromatase- and estrogen-receptor mRNA- expressing cells in the zebra finch brain. Dev. Neurosci. 21, 453–472.

Perlman, W.R., Arnold, A.P., 2003. Expression of estrogen receptor and aromatase mRNAs in embryonic and posthatch zebra finch brain. J. Neurobiol. 55, 513-530.

Toran-Allerand, C.D., Guan, X., MacLusky, N.J., Horvath, T.L., Diano, S., Singh, M.,

Connolly, E.S., Jr, Nethrapalli, I.S., Tinnikov, A.A., 2002. ER-X: a novel, plasma membrane-associated, putative estrogen receptor that is regulated during development and after ischemic brain injury. J, Neurosci. 1, 8391-8401.