Neurocalcin Protein Labeling Reveals a Dimorphism Within the Developing Zebra Finch Brain: Possbible Regulation by Estradiol

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NEUROCALCIN PROTEIN LABELING REVEALS A DIMORPHISM WITHIN THE DEVELOPING ZEBRA FINCH BRAIN: POSSBIBLE REGULATION BY ESTRADIOL.

A thesis submitted to Kent State University in partial fulfillment of the requirements for the degree of Master of Science

By Philip S. Long August, 2010

Thesis written by Philip S. Long B.S. Kent State University, 2004 M.S. Kent State University, 2010

Approved by

Sean L. Veney, PhD. ______, Advisor

James Blank, Ph.D.______, Chair, Department of Biological Science

John Stalvey, Ph.D. ______, Dean, College of Art and Sciences

TABLE OF CONTENTS

LIST OF TABLES ...... iv

LIST OF FIGURES ...... v

ACKNOWLEDGMENTS ...... vi

INTRODUCTION ...... 1

METHODS AND EXPERIMENTAL DESIGN ...... 17

RESULTS ...... 28

DISCUSSION ...... 42

FUTURE DIRECTIONS ...... 49

REFERENCES ...... 52

iii

LIST OF TABLES

1. Table 1. Relative amount of neurocalcin immunoreactivity in the zebra

finch brain ...... 36

2. Table 2. ΔCT values for song regions RA and HVC…….……………..38

3. Table 3. Direction of neurocalcin expression with E2 injection in RA and

HVC...... 39

4. Table 4. ΔCT values for Song region LMAN………...………………….40

5. Table 5. Direction of neurocalcin expression with E2 injection in

LMAN………………………………………………………………...40

LIST OF FIGURES

1. Fig. 1. Sexual differentiation in mammals………………………………………2

2. Fig. 2. Zebra finch song system…...………………………………………….....7

3. Fig. 3. Illustration of punched tissue sections………………………………….24

4. Fig.4. Neurocalcin delta gene (NCALD)………………………………………26

5. Fig. 5. Control Western Blot ………………………….…………………….....28

6. Fig. 6. Photomicrograph of neurocalcin-IR labeling of a male and female ...…30

7. Fig. 7. Neurocalcin-IR neurons in RA………………….. …………………….32

8. Fig. 8. Neurocalcin-IR neurons in HVC. ………………………………33

9. Fig. 9. Neurocalcin-IR neurons in LMAN. ………...…………………………34

10. Fig. 10. Neurocalcin-IR neurons in Area X of males. ………………………..35

11. Fig. 11. Neurocalcin-IR neurons in Rt. ………………………………………36

ACKNOWLEDGMENTS

Throughout my graduate studies at Kent State University, I have received a lot of technical and emotional support from my colleagues. Specifically I want to thank my fellow lab mates and friends Kalpana Acharya, Andrea Bender, Khadijah Wilson, Lo’rell C Martin, Joshua Meeker, Ann Dobry, Katie Seipel and Kimberly Eustache. Extra gratitude is given to Kalpana for all of her teaching and training of the qPCR technique, a lot of troubleshooting and mistakes were avoided because of her attention and expertise. I also owe a great deal of thanks to Peter Wickley, Derek Damron and Mike Sulak for aiding and providing materials for many western blots that I performed in their laboratory. Dr Eric Mintz has provided a great deal of statistical clarity as well as the use of equipment to complete experiments, Dr Heather Caldwell has also provided a sanctuary of peace and quiet as well as use of equipment also and I would like to thank both of them. Donna Warner, the best secretary and friend to have in grad school has been an information resource to staying in school and special thanks for her doing such a fantastic job with my situations.

My friends and family have supported me through my emotionally unstable years of graduate school. Most importantly, I need to thank Chiela Long (sister) and Brandon Petelin (brother) for being my best friends and always keeping my mental focus on finishing. I need to thank Dr. Sean Veney for the tough love and constant motivation to be greater than I wanted to be. Karl Baughman has provided unconditional support and guidance and deserves much gratuity. Finally I need to thank my mothers (Kellie McDowell and Candie Gelis) and fathers (Barry McDowell, Steve Gelis, and William Long) for providing me all the love and comfort especially on hard days that I encountered so often during this journey. This work was supported by Kent State University laboratory start-up funds and NINDS 1R15 NS067477-01 to S.V.

vi Introduction

Sexually dimorphic neural systems provide an exceptional model for investigating factors crucial to the development and maintenance of the brain. Much of the neural differentiation that occurs in males and females can be explained by an organizational series of events. Male and female sex chromosomes contain different genes that are assumed to initiate sexual differentiation. The chromosomal sex of an animal determines gonadal sex, which in turn influences hormone directed development of the brain. More specifically in mammals, the gonads of males begin to form when the Y-linked testis determining gene, Sry is expressed within the undifferentiated gonad (Goodfellow,

1993). Sry initiates a cascade of developmental events in males that result in testes formation. In females, there is no Sry gene, so the testis-determining factor’s absence, in part, results in the development of ovaries. We do know of various transcription factors that guide ovarian development (e.g.WT1, SF-1 LHX9, Dax1, WNT4, FOXL2) but to date there is no identified genetic factor that initiates ovarian formation.

Prior to puberty, ovaries secrete very little hormone. In contrast, testes produce significant amounts of androgens (such as testosterone; T) that act to permanently organize the brain and peripheral tissues (Jost, 1973). More specifically, androgens are released into general circulation where they shape the development of male genitalia which is important for copulatory behaviors and permanently masculinize a variety of neural structures necessary for adult courtship behaviors (Wade and Arnold, 2004;

Adkins-Regan, 2009). However, for many regions, it is not T, but estradiol (E2) that is

responsible for masculinizing the male brain. This can be explained by the aromatization hypothesis which essentially states that T can naturally be converted into

E2 by the enzyme, aromatase (Wade, 2001; Arnold, 2004) which is found locally in the brain and other tissues (Fig. 1)

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

Fig. 1. Males with Sry on the Y chromosome develop testes. The testes in turn produce testosterone. Circulating testosterone is converted into estrogens (estradiol being one of the more potent estrogens) resulting in masculine brain development and adult behaviors. Females do not have the Sry gene, and therefore ovaries instead of testes develop. Low levels of estrogens in circulation do not produce the same neural phenotype as in males. Feminine neural development and behavior is the final product. This pathway accurately describes how brain sex differences develop in many mammalian species.

Support Studies for the Aromatization Hypothesis

Historically, rodents have primarily been used as a model for explaining hormonal differentiation of the brain. In particular, the preoptic area of the hypothalamus (POA) is a well-studied dimorphic region that can be hormonally manipulated (Meisel & Sachs,

1994). The POA, which is critical for masculine reproductive behaviors in rodents (e.g. mounting and intromitting) has a sub-division that is larger in males than in females

(Gorski et al., 1978, Gorski et al., 1980; Tobet et al., 1986; Kindon et al., 1996; Ulibarri

& Yahr, 1996). This region is known as the sexually dimorphic nucleus of the preoptic area (SDN-POA) and was originally identified by Roger Gorski (Gorski et al., 1978,

Gorski et al., 1980). In his studies, Gorski experimented on this nucleus in rats and noted that by manipulating hormones perinatally, not only could the volume of this nucleus be altered, but so could the adult behavior. More specifically, when males were castrated shortly after birth (within 1-3 days) the volume of the SDN-POA was reduced and these males did not show typical male reproductive behaviors as adults. This effect was specific to early development. Castration beyond this critical period did not have the same effect on this nucleus. Gorski also experimented on females by administering a single injection of testosterone propionate during the same period that males were castrated. It was discovered that this manipulation caused the SDN-POA to increase in volume (comparable to a control male), and these females exhibited male-like sexual behaviors in adulthood (mounting stimulus females). Surprisingly, E2 caused the same masculinizing effects in females (Gorski, 1983). At the time, it was difficult to make sense out of how a “female hormone” (i.e. E2) could elicit the same effects on the brain as

a “male hormone” (i.e. T). However, the discovery of aromatase enzyme in the brain

explained these seemingly paradoxical effects (Naftolin et al., 1975; Selmanoff et al.,

1977). The aromatization of androgens into estrogens provided the most likely

explanation of how testosterone administration in the earlier studies affected this

dimorphic nucleus.

Just as in the rodent studies, the importance of aromatase in masculinizing the

brain has also been demonstrated in Japanese quail. More specifically, the POM (which is

analogous to the rodent SDN-POA), is designated as a steroid target for male typical

behavior and is about 40 % larger in males than in females (Balthazart et al., 2004). This

volumetric difference is seen in adulthood and appears to be the result of increased

plasma T levels. There is substantial evidence that this dimorphism is due to the

aromatization of T into E2. As reviewed in Balthazart (1997) T enhances both aromatase

protein and mRNA in quail which in turn are presumed to be the limiting step necessary

for masculinization of the POM. Males that were castrated show a significant reduction in

the volume of the POM as well as in male typical sex behaviors (e.g. mounting, crowing,

and strutting). This can be restored with aromatizable androgens such as T (Panzica et al.,

1991, Panzica et al., 2001). In gonadectomized females, treatment with T in adulthood

increases the volume of the POM to a size similar to that of a non-castrated male (Panzica et al., 1991). This effect is not just limited to rats and quail. Across several other vertebrates, such as guinea pigs (Hines et al., 1985), ferrets (Tobet et al., 1986), gerbils

(Commins and Yahr, 1984a; 1984b), and humans (Swaab and Fliers, 1985; Allen et al.,

1989) many structures and features of the brain are masculinized because of the

aromatization of T and activation of estrogen receptors.

Zebra Finches

The zebra finch in contrast to the aforementioned models presents some major challenges to the aromatization hypothesis. More specifically, the role of gonadal sex steroids in brain differentiation is not well understood. Similar to rodents and Japanese quail, studies in the zebra finch have focused on factors important for the development of dimorphic nuclei that are responsible for male typical behaviors (reviewed in Adkins-

Regan, 2009). Over 40 years of research has been directed at the structures important for song learning and production (Arnold, 1992; Wade, 2001; Adkins- Regan, 2009). Singing is controlled by a series of interconnected regions within the telencephalon. Area X and the lateral magnocellular nucleus of the anterior nidopallium (LMAN) are necessary for song learning (Bottjer et al., 1984; Scharff and Nottebohm, 1991). HVC (proper name) and the robust nucleus of the arcopallium (RA) form the motor pathway important for song production (Nottebohm et al., 1976; Simpson and Vicario, 1990). Sexual dimorphisms exist at a number of levels within this system (Arnold, 1992; Wade, 1999).

The volumes of HVC and RA as well as soma size and numbers of neurons within these nuclei are greater in males than in females. The projection from HVC to RA is more robust in males than in females. And area X, which is easily identified in males, is not visible in females using standard Nissl stains (Fig. 2). These differences are believed to

be the explanation for the sex dimorphism in song production. Normally, only males sing and females do not. The mechanisms that create these neural sex differences are not completely understood. The manipulations that will be discussed suggest that hormones are most likely not the only factor that contribute to dimorphic development of the brain.

Hormone Manipulations in Female Zebra Finches

Some aspects of zebra finch neural differentiation appear to be somewhat consistent with the aromatization hypothesis. For example, administration of supraphysiological doses of E2 to females during the first two weeks post-hatching (P) causes permanent neural morphological masculinization (Simpson & Vicario, 1991). There is a significant increase in the size and number of neurons in HVC and RA (Gurney and

Konishi, 1980; Gurney 1982). Area X, which is not normally detectable in females, now becomes visible (Grisham and Arnold, 1995; Jacobs et al., 1995). The axonal connection between HVC and RA becomes more robust (Konishi and Akutagwa, 1985; Halloway and Clayton, 2001). And females, who are not normally capable of singing, now produce male-like vocalizations (Simpson & Vicario, 1991). However, these effects are only partial. Unlike in rodents and Japanese quail, there is no evidence to date that supports complete masculine development in females from E2 exposure alone (Nordeen et al.,

1986; Adkins-Regan et al., 1994).

Fig. 2. This is a schematic representation of the male (A) and female (B) zebra finch song system. In males RA, HVC and LMAN are noticeably larger in volume and contain larger and more numerous neurons. They also have a large area X that is not identifiable in a normal female brain. There are robust axonal projections from LMAN and HVC to RA (arrow head points to the postsynaptic cells), from HVC to area X, and from RA to the nXIIts (tracheosyringeal branch of the hypoglossal nucleus, 12th cranial nerve) in male brains. The female brain also contain regions RA, HVC, LMAN, and nXIIts however they consist of fewer neurons and smaller nuclei volumes.

Since the precursor to E2 production is T, then administration of T should also have similar masculinizing effects on the female brain, according to the aromatization hypothesis. However it does not. At most, T had a very weak effect on masculinizing the brain and is specific only to RA (Grisham and Arnold, 1995). Because T can activate androgen (AR) and estrogen receptors (ER), to better clarify how much of a contribution androgens make, additional studies utilized the non-aromatizable androgen, dihydrotestosterone (DHT). In one report, treatment with this hormone resulted in a slight increase in soma size and doubling of RA size in females when treated from the day of hatching up until the third week posthatching (Gurney, 1981), but other studies were not able to reproduce the same conclusions (Jacobs et al., 1995; Schlinger and Arnold, 1991;

Nordeen and Nordeen, 1989). To test the possibility that activation by both AR and ER are important DHT was given in combination with E2. The results were no more effective than E2 alone (Jacobs et al., 1995).

Challenges to the Aromatization Hypothesis in Zebra Finches

Although E2 can significantly masculinize females to a certain degree, many more pieces of evidence are not consistent with the aromatization hypothesis for brain differentiation in this species. For a sex difference to occur naturally, it would be expected that males should by some mechanism have increased exposure to E2. Three

independent studies have investigated whether a consistent sex difference existed in

plasma levels of E2 during early development. In the first, E2, T, androstenedione (AE)

and DHT were measured from P1-P10 (Hutchison et al., 1984). E2 was increased in

males from P2-P4 but declined to basal levels by P10. In females, E2 remained relatively

constant such that a male biased sex difference was present only on P4. The three

androgens declined during this period in males. The only sex difference detected was

female-biased in AE on P2 and P10. A second study (Adkins- Reagan et al., 1990)

measured E2, T, and DHT from embryonic day12 through P54. There was no sex

difference in E2, however there was a trend for levels of T to be increased in females.

And lastly, Schlinger and Arnold (1992) measured various androgens (T, AE and DHT)

and estrogens (estrone; E1 and E2) from P1 until P13. There were no sex differences in any of the estrogens, although levels of AE were below detectability and were not tested.

There was a trend for males to have increased levels of E1 during the first week post- hatching compared to the second week, but there were no sex differences in any of the androgens.

Studies have also investigated aromatase. Microdissected regions of HVC, RA and area X were analyzed in P20 animals (Vockel et al., 1990), as well is in whole brain lysates at P4-6 and P10-12 (Schlinger and Arnold, 1992). In no case was a sex difference detected. Similarly, high levels of aromatase activity were detected in two week old cultures of homogenized zebra finch telencephalon, but again without a sex difference

(Wade et al., 1995). Additional studies that investigated aromatase mRNA did not detect labeling in area X or HVC (Jacobs et al., 1999). There was little expression in RA and slightly more in LMAN, but in no case was a sex difference evident. Examining the protein distribution of aromatase has also given no evidence to support greater aromatization in the male brain (Saldanha et al., 2000). The most intense labeling was

detected outside of song regions. Taken together, these data indicate that although many

regions within the telencephalon are rich in aromatase enzyme, minimal to no expression

in song control regions and the lack of a sex difference suggests that aromatase does not

account for the dimorphism in the zebra finch song system.

There is also little evidence to support the idea that males can even respond to

more E2. There have been several studies that investigated the distribution of estrogen

receptor alpha (Nordeen et al., 1987; Gahr and Konishi, 1988; Jacobs et al., 1999). In

general, few neurons containing this receptor type were detected in song regions,

however more were found in HVC than in any other song areas. None of these studies

could find a sex difference. A second form of estrogen receptor (beta) has never been

described in zebra finches but it is present in a closely related songbird, the European

starling (Bernard et al., 1999). However, in this species it is not dimorphic and has not

been localized to any song areas.

Although significant masculine development in females can be induced by E2,

most researchers have not yet succeeded in completely preventing masculinization of the

male brain by blocking the production of estrogens. At best, the vast majority of studies

only minimally opposed masculine patterns of development, had no effect at all, or

hypermasculinized males. For example, Fadrozole is a powerful aromatase inhibitor in

vitro (Wade et al., 1994). However when this drug was administered in vivo it had no effect on preventing masculine development of the song system (Wade and Arnold, 1996;

Gong et al., 1999). One of the few studies that even hinted at success with this

manipulation was Merten and Stocker-Bruschina (1995). They administered Fadrozole to juvenile males at P10. Their results indicated no significant change in neuron soma size or volume of song control regions when brains were examined at P35. However, a subset of animals from this same study were examined at P135. Soma sizes in RA had a

tendency to be smaller however, this effect was not significant. In contrast, cell size in

HVC was significantly decreased as a result of early Fadrozole treatment. Overall, one of

the most convincing pieces of evidence that inhibiting estrogen production was not

important for dimorphic brain development came from a study in which zebra finch eggs

were dipped daily in Fadrozole (Gong et al., 1999). In birds, estrogens are necessary for

normal ovarian development. In females, this treatment resulted in animals that

developed functional testicular tissue. Despite this the brain was unaffected and still

developed in a feminine fashion suggesting that gonadal hormones do not contribute to

the process.

Furthermore, attempts to block the action of estrogens (using antagonists) have

also not been effective and depending on the compound, have actually hypermasculinzied

males and/or masculinized females. Tamoxifen, LY117018 and CI628 have all been

used. Tamoxifen treatment for the first 20 days after hatching increased neuron soma size

in RA, HVC, and MAN of both males and females, and it increased the volume of HVC,

RA, MAN, and area X in males (Mathews et al., 1988). In another report, song system

morphology was assessed immediately following treatment with LY117018 and CI628

for the first 25 days after hatching. Both compounds induced an area X in females (a

result that only occurs when females are treated with estrogens). One or both antagonists

also masculinized the volume of RA in both sexes and the volume and neuron soma size

in HVC of males. Although these results are contradictory to the proposed role of E2

according to the aromatization hypothesis, they can be explained by the fact that these

drugs are known to have partial agonistic actions (Mathews and Arnold, 1990). Because

of this, recent studies in our lab utilized a more pure estrogen receptor antagonist (ICI

182,780). Males and females were given intracerebral injections for the first 25 days after

hatching. When soma size was measured, we found a significant decrease in both sexes.

To date, this study is one of the very few in which an anti-estrogen has been shown to

significantly demasculinize the male brain (Bender and Veney, 2008).

In summary, substantial masculinization of females occur with E2 treatment in the

first few weeks after hatching. However, females do not completely develop masculine

song regions comparable to normal males. Whereas some specific components of

masculine development appear to be responsive to steroid action, attempts to block the

synthesis or action of gonadal hormones (particularly estrogens) have largely proven

unsuccessful. In only a very few cases has it been possible to significantly sex-reverse the male's song system development by interfering with estrogenic activity. Therefore, much of the data does not support a major role for gonadal sex steroids in the differentiation process. These findings can be taken as evidence that additional non-hormonal factors might participate in sexual differentiation. This thesis investigates the possibility that a calcium binding protein might significantly contribute to dimorphic neural development.

Neurocalcin

Because steroid hormones did not appear to play a major role in dimorphic brain

development, it was believed that non-hormonal factors contributed more. To test this

hypothesis, Veney et al. (2003) utilized differential display PCR to identify gene products

that were expressed differently in males and females. Based on this, they detected a

single product in juvenile females that was not present in juvenile males or adults. This

gene was isolated, cloned, sequenced and identified as the calcium binding protein,

neurocalcin. In order to examine the expression of neurocalcin during development, a

probe was synthesized and Northern blot analyses of telencephalic tissue were performed.

Using a specifically designed probe, three RNA species were detected. A large product

(10.6 kb) was present in both sexes at all ages examined (early post-hatching through

adulthood). Two other products (6.2 kb and 3.3 kb) were expressed almost exclusively in females and primarily between P18-25.

Given this information they next wanted to know if neurocalcin was localized to song control regions and therefore may possibly play a role in dimorphic brain development. To examine this, in situ hybridization was performed on P22 males and females using a probe that recognized all three transcripts. This age was chosen because it fell right in the middle of a time when neurocalcin RNA was expressed maximally in the female brain. Results from those studies indicated a very broad but enhanced expression of neurocalcin mRNA in females as compared to males. This was significant because they initially reasoned that the increased neurocalcin expression in females contributed to sexually dimorphic neural development by upsetting intracellular calcium

2+ 2+ [Ca ]i homeostasis. Although the exact details are not known, according to the Ca set

point hypothesis, proper cell functioning depends on a critical window of intracellular

2+ 2+ Ca [Ca ]i (Johnson et al., 1992, Hwang et al., 1999). Conditions that result in

2+ prolonged elevated or low [Ca ]i outside of an established range can lead to cell death

(Franklin and Johnson, 1992; Mattson, 1992; Yu et al., 2001; Case et al., 2007; Ringler et

al., 2008). Consistent with this idea, it is known that many of the morphological

differences in the song circuit of males and females zebra finches are the result of

enhanced cell death in females (Kirn and DeVoogd, 1989).

There are a number of mechanisms describing how changes in Ca2+ can lead to

cell death. Considerable evidence for different, cross-amplifying cascades has been obtained. First, Ca2+activates (either directly or indirectly) cysteine proteases called

calpains and caspases that degrade a variety of substrates, including cytoskeletal proteins,

membrane receptors and metabolic enzymes (Chan & Mattson, 1999; Nixon, 2003).

Calpains also play an important role in the triggering of apoptotic cascades by virtue of

their ability to ensue activation of caspases (Leist et al., 1997; Stefanis, 2005). Induction

of oxidative stress is another way that Ca2+ can contribute to cell death (Lafon-Cazal et

al., 1993; Mattson, 2003). This occurs through several different mechanisms, including

activation of oxygenases such as those in the arachidonic acid metabolism cascade,

perturbation of mitochondrial Ca2+ and energy metabolism, and induction of

mitochondrial oxidases. The reactive oxygen species generated in response to glutamate-

induced Ca2+ influx include superoxide anion radical, hydrogen peroxide, hydroxyl radical, nitric oxide and peroxynitrite (Lipton et al., 1993; Mattson, 1998) which all can

be toxic to cellular processes yielding DNA defragmentation. Third, Ca2+ triggers

apoptosis, a form of programmed cell death (Ankarcrona et al., 1995). This might occur

by Ca2+ - mediated induction/activation of pro-apoptotic proteins such as Bax, Par-4, and p53 leading to mitochondrial membrane permeability changes, release of cytochrome c and caspase activation (Duan et al., 1999; Dargusch et al., 2001; Culmsee and Mattson,

2005).

Unfortunately, very little is known about neurocalcin or exactly how it functions.

This Ca2+ binding protein is a member of the visinin-like (VSNL) sub-family within the

neuronal Ca2+ sensor proteins (Vijay-Kumar and Kumar, 2002; Burgoyne, 2007;

Braunewell and Klein-Szanto, 2009). VSNLs share a common four EF-hand motif, however only three are functional (Braunewell and Gundelfinger, 1999; Burgoyne et al.,

2004). As a whole, family members are believed to be involved in binding, buffering

2+ 2+ [Ca ]i and/or modulating the communication between Ca and other cellular systems

(Braunewell and Gundelfinger, 1999; Burgoyne and Weiss, 2001; Burgoyne et al., 2004;

Burgoyne, 2007). What is known about neurocalcin suggests that it activates guanylyl

cyclase (Krishnan et al., 2004), and may induce Ca2+ release into the cytosol.

Neurocalcin has been identified in several species, cell types (neurons,

interneurons and glia) and in various tissues. For example, in bovine it has been detected

in several regions of the brain, adrenal glands and in cells of the retina (Nakano et al.,

1992; Terasawa et al., 1992; Hidaka and Okazaki, 1993; Nakano et al., 1993; Kato et al.,

1998; Braunewell and Gundelfinger, 1999). In rats, it is in the brain and cells of the

auditory, visual and olfactory systems (Bastianelli et al., 1993, 1995a; Iino et al., 1995;

Braunewell and Gundelfinger, 1999). In humans, neurocalcin is present in the brain, ovaries and testes (Wang et al., 2001).

Generally, in order for a product to be important for sexually dimorphic development, it is assumed that expression would be greater in one sex compared to the other during the critical period(s). Although neurocalcin mRNA was enhanced in females, it was not clear how much of that expression would be represented by protein.

Expression levels of mRNA and protein exhibit a wide range of correlations for different genes (Pascal et al., 2008). Studies indicate that these correlations can range anywhere from 20-80% (Tian et al., 2004; Groves et al., 2006; Nie et al., 2006a, b). These data in combination with a recent study that demonstrated a sex difference in neurocalcin protein expression in the mouse olfactory bulb (Murias et al., 2007) lead us to hypothesize that neurocalcin protein is sexually dimorphic in the zebra finch brain.

In this thesis, my first aim was to describe the distribution of neurocalcin in male and female zebra finch brain, and report a quantitative analysis of the protein expression in song control nuclei at various ages. For areas outside of song areas, a semi-qualitative analysis will be reported. In the second aim I wanted to address the hypothesis that neurocalcin is regulated by estrogens. Changes in the neurocalcin gene within song control regions will be quantified 2 and 24 hours after a single E2 injection.

Methods and Experimental Design

Experiment 1: Neurocalcin Protein Labeling In the Zebra Finch Developing Brain

Aim 1: Describe the distribution of neurocalcin protein in the developing zebra finch brain

Hypothesis 1: Neurocalcin is expressed significantly more in females and will be detected within dimorphic song control regions

1.A Animals

Subjects were obtained from our facility at Kent State University. The animals were housed in communal aviaries containing 5-7 pairs of breeders on a 14:10 L:D cycle.

Adults were fed a finch bird seed diet that was supplemented weekly with hard boiled chicken eggs mixed with bread and fresh oranges or spinach. Water and seed were provided ad libitum. 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.

1.B Specification of the Primary Antibody by Western Blot Analysis

A primary polyclonal antibody made in rabbit against neurocalcin (Biomol,

Plymouth Meeting, PA) was utilized to examine whether neurocalcin is present in the zebra finch brain. Because it was important to test its usefulness in our avian model it was necessary to first confirm the specificity of the antibody. To do this fresh brain was

removed from P18-23 day old juveniles. The telencephalic lobes were homogenized in 10

ml of RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl , 1% Triton x-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA) with protease inhibitor for 1 hour on ice.

Centrifugation at 10,000 rpm separated the lysate and pellet. The lysate was assayed and analyzed on a plate reader to determine protein concentration. 2 mg of the protein sample was loaded onto a 10-15 % SDS-Page ready gel (Bio-Rad; Hercules, CA) with 5X running buffer (1x: 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3) and PBS. 25µL total volume was run at 100V for 1 hr to partition the protein bands. Afterwards, the proteins were transferred onto a nitrocellulose membrane (Bio-Rad) at 100V for 1 hour in transfer buffer (1x: 25 mM Tris, 192 mM glycine, pH 8.3, 20% methanol, and ddH2O to a

total volume of 1000ml).

The membrane was blocked with 5% milk (7.5 g dry milk and 50ml of TBS {50

mM Tris HCl, pH 7.4 and 150 mM NaCl })for 1hr. Immediately the membrane was washed 3 times in TBS-T (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) and

incubated overnight at 4˚C in the neurocalcin primary antibody (1:5,000, in 5% milk).

After rinsing 3 times with TBS-T, the secondary HRP conjugated goat anti-rabbit

(1:1,000; Santa Cruz; Santa Cruz, CA) was added over the membrane and was allowed to

incubate for 1hr. Neurocalcin protein was identified with the addition of 1:1 luminol and

peroxide solution and imaged using LAS-3000 (Fujifilm; Tokyo, Japan). A single

product that corresponded to the expected molecular weight of neurocalcin protein was

detected, 22 kDa (Fig. 4).

1.C Histology: Immunohistochemistry in Brain Slices.

Males (n = 8) and females (n = 8) were collected. We targeted animals at ages

P18-23. However, to investigate if neurocalcin might affect brain development at other

times, we also included animals from a time point earlier in development (P10) and

adults (greater than P100). In all cases subjects were injected with an overdose (0.10 mL)

of equithesin anesthesia and transcardially perfused with 0.75% phosphate buffered

saline (PBS ; 137 mM NaCl; 2.7 mM KCl; 4.3 mM Na2HPO4; 1.47 mM KH2PO4) followed by 150-200 mL of phosphate buffered formalin (PBF). Visual inspection of the gonads was used to determine the animal’s sex. The brain was extracted from the skull, post-fixed in PBF at room temperature for 2 hrs, and cryoprotected in 20% sucrose at 4°C overnight. Brains were then quickly frozen in dry ice and coronally sectioned at 30µm onto gelatin-coated slides. Consecutive slices were mounted onto alternate slides. The tissue was stored at -20°C until processing.

For immunohistochemical labeling, sections were first rinsed in 0.1M PBS. To remove endogenous peroxidases, the tissue was then placed in 0.5% H2O2 in 0.1 M PBS for 15 min followed by 3, 5 min rinses in 0.1 M PBS. To minimize non-specific binding,

100 µL of 10% donkey serum was added to the sections for 1 hr. Neurocalcin delta primary antibody (Biomol) was diluted 1:10,000 in 0.1M PBS, added to the slides, and incubated at 4°C on a shaker for at least 48 hrs. After exposure to the primary antibody, slides were rinsed for 5 min in PBS-T (0.4% triton-X-100). Biotinylated goat anti-rabbit secondary antibody (Vector Labs; Burlingame, CA) diluted 1:2,000 was then incubated over the slides at room temperature for 1 hr. Following the secondary, tissue was briefly washed 3 times in PBS-T. Avidin-Biotin Complex (Vector Labs) was added to the slides

at room temperature for 1 hr and then washed for 3 min in PBS-T. Neurocalcin protein

was visualized after reaction with brown DAB (Sigma-Aldrich). A negative control was performed by omitting the primary antibody.

1.D Quantitative Analysis of Neurocalcin Protein Expression in Song Control

Nuclei.

Based on previous work (Veney et al., 2003) there was precedence to believe that neurocalcin protein would heavily overlap with song control nuclei, but we did not know if the borders would be clearly defined. To aid in the identification of regions we utilized thionin-stained tissue. We selected a single section that corresponded to the approximate middle of HVC, RA, LMAN and area X (where each nucleus would be represented in its fullest cross-sectional extent). We also investigated labeling in a control monomorphic region, nucleus rotundus (Rt). Using MicroSuites Image Analysis (Olympus), a digital image of the selected thionin-stained section was captured at 40X and overlaid with a digital image of neurocalcin-IR labeling from an adjacent section at the same magnification. This technique allowed us to clearly identify the borders of all nuclei. An observer who was blind to the conditions of the experiment counted the total number of neurocalcin immunoreactive (-IR) neurons in the single representative tissue section based on our established criteria (cytoplasmic perinuclear staining that extended into processes).

1.E Semi-Quantitative Analysis of Neurocalcin Protein Expression Outside of

Song Nuclei.

In addition to song nuclei, neurocalcin mRNA was broadly distributed in many other regions spanning the entire rostral-caudal continuum of the brain (Veney et al.,

2003). We were interested in knowing how closely this reported expression would be

represented by protein as well. To get an impression of the overall distribution and

relative amount of neurocalcin labeling, specific neural regions were ranked. The

assigned scores were based on a visual inspection of a single section from each region as

described above. A “---” represented an area with no detectable immunolabeled product,

and at the opposite end of the spectrum, “***” was assigned to areas with the highest amount of labeled cells. As in Veney et al. (2003), the following areas were analyzed; however, the names were revised to reflect the updated avian brain nomenclature (Reiner et al., 2004): arcopallium, dorsal arcopallium, cerebellum, hippocampus, mesopallium, nidopallium, caudal nidopallium, lateral striatum, preoptic/hypothalamic area, and optic tectum.

1.F Statistics.

Two-way analyses of variance (sex x age) were employed to determine statistical

significance for measurements within song control regions and Rt. Data from area X were

analyzed by one-way analysis of variance. These were followed by Tukey-Kramer post-

hoc tests for pairwise comparisons when significant main effects were observed. The

level of significance was determined at P<0.05. All statistical analyses were conducted using Sigma Stat. For the semi-quantitative rankings, data are presented descriptively.

Experiment 2: Estradiol’s Influence on Neurocalcin

Aim 2: Determine if estradiol can regulate neurocalcin gene expression.

Hypothesis 2: Administration of estradiol will reduce the expression of neurocalcin

in both sexes.

2.A Time dependent injection of 17 β-Estradiol

20mg of 17 β-Estradiol (Mann Research Laboratories, New York, NY) was injected subcutaneously 24 hours and 2 hours prior to the animals’ sacrifice (24hours, n=4 males and n=7 females; 2 hours, n=6 males and n=4 females ). For controls, an equal volume of propylene glycol vehicle (3 µL) was substituted in the injection needle (24 hours, n=4 males and n=4 females; 2 hours, n=4 males and n=7 females. At the time of sacrifice, the brain was removed and rapidly frozen in cold methyl butane (Sigma-

Aldrich) and stored in -80˚C until sectioning.

2.B Punch sections of Song control Nuclei

Frozen brains were mounted with OTC tissue tech and cut on a cryostat (Leica

1850 CM) 240µm thick. These thick sections were mounted on cleaned slides and warmed by hand to adhere the slice of brain. Alternate sections were cut 60µm thick and mounted on gelatin coated slides for thionin staining. The thionin stained sections were used as a guide to locate song regions RA, HVC, and LMAN.

Using a 0.5 mm stainless steel punch (Stoelting), song control nuclei were quickly dissected out of the thick sections on a dry ice to prevent RNA degradation (fig. 3). Each section was stored in a 0.5 microcentrifuge tube until RNA purification.

Fig. 3. Illustrates where the Punch section were extracted. A Nissl stained image of RA (left), HVC (middle), and LMAN (right) shows where the 0.5 mm punch location was taken from. This is denoted by the arrow head.

2.C RNA Purification

In order to achieve sufficient concentration, punched tissue was pooled 4-7

animals per sample and was purified for RNA using the RNeasy mini kit (Qiagen

sciences; Maryland, US). The tissue punches were homogenized using 350µL of buffer

RLT and vortexed for 45 seconds on ice. Lysate was centrifuged at 15,000 rpm for 3

minutes. The supernatant was transferred into a clean 1.5µL microcentrifuge tube with

350µL of 70% ethanol and mixed with a pipette. This mixture was then transferred into the RNeasy spin column and centrifuged into a collection tube for 15 seconds at 15,000 rpm, 4˚C. 700 µL of buffer RW1 was centrifuged through the spin columns at 15,000 rpm, 4˚C, for 15 seconds. Next, 500µL of buffer RPE was filtered though the columns for

15seconds at 15,000 rpm, 4˚C, followed by another RPE wash for 2 minutes. DNase

(15u) was incubated on each spin column for 15 minutes to ensure pure RNA extraction without genomic DNA contamination. RNA was extracted from the column and collected

in a new sterile RNase free 1.5 µL microcentrifuge tube by adding 30µL of RNase free

water, then centrifugation at 4˚C, 15,000 rpm.

The RNA concentration was obtained using a Nanodrop spectrophotometer (ND-

1000, software; Wilmington, DE). Furthermore the integrity of the RNA was visualized

after running samples on an agarose gel. 10µL of RNA elute was mixed with 5µL of 2X

RNA loading buffer (Fermentas; 95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 0.025% ethidium bromide, 0.5 mM EDTA), forward and reverse primers to the zebra finch neurocalcin gene (fig. 4) and run for 45 minutes at 90-

94Volts. The presence of two distinct bands (5S rRNAand 18S rRNA) were evaluated after UV exposure to confirm RNA quality.

Fig. 4. Neurocalcin delta gene (NCALD, gene name; NCBI gene bank) is a 684 Base pair amino acid sequence.

2.D Quantitative PCR

cDNA was made from pooled punch sections using the High Capacity cDNA

reverse transcriptase kit (Applied Biosystems, Foster City, CA). cDNA assays without

Reverse Transcriptase(-RT) was made as a control. Briefly 300 ng, 150 ng or 75 ng of cDNA was synthesized in a 20 µL total reaction (RT buffer, 10X; dNTP mix, 25X, RT

random primers, 10X; Reverse transcriptase[MultiScribeTM]. The cycle parameter for

cDNA was as follows: 25°C for 10 min, 37°C for 120 min, 85°C for 5 sec and then held

at 4°C using the Eppendorf (Germany) mastercycler gradient .

For quantitative PCR, SYBR Green Master Mix (Applied Biosystems, Foster

City, CA) was used according to manufacturer’s instructions and the reactions were run with the default program on the ABI Prism 7000 (Applied Biosystems, Foster City CA;

50˚C for 2 min, 95˚C for 10 min, then 40 repetitions of 95˚C for 15 min and 60˚C for 1

min). No template controls were included to verify the specificity of each primer, for

which the dissociation curve was carefully inspected to confirm the absence of primer

dimers and other unwanted products. For each set of primers, 200 nM produced clean,

detectable amplification, so this concentration was used in a standard curve under the

conditions above, with duplicate samples containing the cDNA produced from75 to 300

ng total RNA (along with no template controls). Each of the primer sets of interest was

run in parallel with GAPDH, and the efficiency of amplification was in all cases close to

100% and equivalent for the target primer pair and the GAPDH. Analysis of relative gene

expression was conducted using the ΔCT method (Livak and Schmittgen, 2001).

RESULTS

Western blot

A single, 22 kDa, band was detected using the specific antibody for neurocalcin

(fig. 4). This result is consistent with prior knowledge of neurocalcin delta antibody in purified protein samples (Okazaki et al., 1992; Ivings et al., 2002., O’Callaghan et al.,

2002).

Fig. 5. A single protein product was detected at 22 kDa, which corresponds to the molecular weight of neurocalcin (Hidaka and Okazaki, 1993).

Neurocalcin labeling in song control regions

Immunohistochemical labeling with a specific antibody revealed that neurocalcin

protein was broadly distributed throughout the brain. Cells of multiple shapes (fusiform,

round and pyramidal) with an average soma size of 10-25 µm were identified as

immunopositive by cytoplasmic perinuclear staining that extended into processes (Fig. 6).

For the quantitative analyses, neurocalcin-IR cells were counted in a single representative

section from each region.

In RA there was a main effect of age (F = 3.97, p = 0.031) and a significant

interaction between sex and age (F = 6.04, p = 0.007). Females had more neurocalcin-IR neurons than males at P18-23. In males, neurocalcin positive cells decreased from P10 to

P18-23, but increased again by adulthood (Fig. 7).

Fig. 6. Photomicrograph of neurocalcin-IR labeling in HVC of a male (A) and female (B) at P18-23. Arrows represent a sample of immunopositive neurons that were identified by a transparent nucleolus and cytoplasmic staining that extended into processes. Scale bar = 100 µm.

HVC

In HVC, data revealed a marginally significant effect of sex (F = 4.01, p = 0.052),

a significant effect of age (F = 4.76, p = 0.018), but no interaction (F = 2.20, p = 0.132).

Females had more neurocalcin-IR neurons than males at P18-23. There was also a progressive increase in the number of immunopositive cells detected in females, such that there were significantly more in adulthood than at P10 (Fig. 8).

LMAN

For LMAN there was a significant effect of age (F = 71.48, p < 0.001) but no

main effect of sex (p = 0.37) or an interaction (p = 0.33). In both sexes, the number of

cells that contained neurocalcin increased from P10 to P18-23, but decreased by

adulthood (Fig. 9).

Area X

In area X, statistical analysis also revealed a significant effect of age (F = 6.29, p

= 0.023). The number of neurocalcin positive neurons significantly decreased from P10

to P18-23 and remained relatively unchanged through adulthood (Fig. 10).

Fig. 7. Mean (+ SEM) of neurocalcin-IR neurons in RA (n=8 males and n=8 females). An * represents a significant sex difference at P18-23. In males, different letters denote a significant effect of age between groups. In females there was no significant effect of age.

Fig. 8. Mean (+ SEM) of neurocalcin-IR neurons in HVC (n=8 males and n=8 females). A * represents a significant sex difference at P18-23. In females, different letters denote a significant effect of age between groups. In males there was no significant effect of age.

Fig. 9. Mean (+ SEM) of neurocalcin-IR neurons in LMAN (n=8 males and n=8 females). Within each sex different letters denote a significant effect of age.

Fig. 10. Mean (+ SEM) of neurocalcin-IR neurons in area X of males (n=8). Different letters denote a significant effect of age

We also investigated neurocalcin protein expression in a single representative section of Rt, a monomorphic visual nucleus. As expected, there was no main effect of sex in this region, but there was a significant effect of age (F = 23.06, p < 0.001). In males and females, the number of cells that contained neurocalcin remained relatively consistent at the two juvenile ages that were investigated, but significantly decreased by adulthood (Fig. 11). Interestingly we also noticed that out of all the regions that were quantitatively analyzed, Rt contained the fewest number of neurocalcin-IR cells.

Fig. 11. Mean (+ SEM) of neurocalcin-IR neurons in Rt (n=8 males and n=8 females). Within each sex different letters denotes a significant effect of age.

For the neural regions that were semi-quantitatively analyzed, data indicated that the relative number of immunopositive cells varied across specific regions. Based on visual observations, no obvious sex differences were apparent, with the possible

exception of the lateral striatum where the amount of labeling in males and females

appeared different at each of the three ages that were examined (Table 1).

Table 1: Relative amount of neurocalcin immunoreactivity in the zebra finch brain

Brain P 10 P10 P18- P18-22 Adult Adult Region 22 Male Female Female Male Female Male

A *** *** *** *** *** ***

AD *** *** *** *** *** ***

Cb *** *** *** *** *** ***

Hp ** ** ** ** *** ***

LSt * --- ** * ** *

M *** *** ** ** ** **

N *** *** *** *** *** ***

NC *** *** ** ** *** ***

POA/Hyp * * *** *** ** **

TeO *** *** *** *** *** ***

--- = no detectable labeling, * = low labeling, ** = moderate labeling, *** = high labeling. Abbreviations. A = arcopallium; AD = dorsal arcopallium; Cb = cerebellum; Hp = hippocampus; LSt = lateral striatum; M = mesopallium; N = nidopallium; NC = caudal nidopallium; POA/Hyp = preoptic/hypothalamic area; TeO = optic tectum.

RT- PCR after Estradiol Injection

RT-PCR was used to examine if E2 administration had any effect on neurocalcin

gene expression. Two time points after E2 injections were reported in table 2 and table 4.

The direction of expression change due to E2 injection was illustrated in table 3 and table

E2 injected males at 2 hrs and at 24 hrs had lower ΔCT values than the vehicle treated animals. The same trend is evident with females treated with E2 2 hrs prior to euthanasia. Interestingly, the 24 hr treatment group displayed an increase in ΔCT value

(Table 2).

HVC

E2 injected males and females had lower ΔCT values than the vehicle treated animals however, this was only apparent after 2hrs of treatment. Both sexes showed an increase in ΔCT value 24 hrs following the E2 injection (Table 2).

Table 2: ΔCT values for Song regions RA and HVC

Brain Region Sex/Time of Treatment ΔCT /Treatment GAPDH - NCALD RA M 2 HR OIL 1.59 F 2 HR OIL 2.07 M 2HR E2 -0.65 F 2 HR E2 0.68 M 24 HR OIL 2.49 F 24 HR OIL -0.7 M 24 HR E2 1.44 F 24 HR E2 1.01 HVC M 2 HR OIL 1.22 F 2 HR OIL 1.65 M 2 HR E2 -0.22 F 2 HR E2 0.63 M 24 HR OIL 2.86 F 24 HR OIL 3.08 M 24 HR E2 3.48 F 24 HR E2 4.08 Song regions RA and HVC have ΔCT values reported. Higher ΔCT values represent lower expression of neurocalcin gene product when comparing oil injected animals (24 hours, n=4 males and n=4 females; 2 hours, n=4 males and n=7 females) to E2 injected animals (24hours, n=4 males and n=7 females; 2 hours, n=6 males and n=4 females) of the same sex. Male and female ΔCT values in HVC increased at the 24 hr time point and an increase was also detected at the 24 hr time point of females in RA. Unfortunately, the sample size (n= 1) was not large enough to perform a statistical analysis.

Table 3: General direction of neurocalcin gene change in response to an E2 injection. Brain Region Time point Neurocalcin Expression

RA Male 2 HR ↑

Female 2 HR ↑

Male 24 HR ↑

Female 24 HR ↓

HVC Male 2 HR ↑

Female 2 HR ↑

Male 24 HR ↓

Female 24 HR ↓

Relative neurocalcin gene expression change after a single E2 injection. Table indicates the direction of change, either an increase in neurocalcin expression (↑) or decrease (↓).

LMAN

E2 injection in males at both 2 hrs and 24 hrs showed and reduction in ΔCT values. This same region showed an increase in ΔCT value at the 2 hr time point in females however at the 24 hr time point there was a reduction of ΔCT value. This same trend for reduced expression was also detected in males at both time points (Table 4).

Table 4: ΔCT values for Song region LMAN

Brain Region Sex/Time of Treatment ΔCT /Treatment GAPDH - NCALD

LMAN M 2 HR OIL 2.76

F 2 HR OIL 0.56

M 2 HR E2 1.69

F 2 HR E2 0.75

M 24 HR OIL 1.91

F 24 HR OIL 2.14

M 24 HR E2 0.47

F 24 HR E2 0.47

A small increase in ΔCT value was detected in females in LMAN at the 2hr time point when comparing oil injected animals (24 hours, n=4 males and n=4 females; 2 hours, n=4 males and n=7 females) to E2 injected animals (24hours, n=4 males and n=7 females; 2 hours, n=6 males and n=4 females).

Table 5: General direction of neurocalcin gene change in response to an E2 injection. Brain Region Time Point Neurocalcin Expression

LMAN Male 2 HR ↑

Female 2 HR ↓

Male 24 HR ↑

Female 24 HR ↑

Relative neurocalcin gene expression change after a single E2 injection. This table indicates the direction of change either an increase in neurocalcin expression (↑) or decrease (↓).

DISCUSSION

The first study documents a sex difference in the number of neurocalcin IR-cells in two song production nuclei, RA and HVC, at P18–23. Based on counts taken from a single representative section, the female brain contained more immunopositive neurons than males.

These data suggest that within these areas the dimorphic expression of neurocalcin protein may influence physiological processes that significantly contribute to the development of neural sex differences. The extent of the sex dimorphism was limited only to the neural regions that control song production. A statistically significant sex difference was not detected in LMAN or the monomorphic visual nucleus Rt. This is interesting because at this age range, males are forming mental templates of zebra finch song and have not yet entered the song vocalization phase

(Immelmann, 1969). Based on visual observations there were also no apparent sex differences in neurocalcin outside of song nuclei with the possible exception of the lateral striatum. Although not statistically confirmed, the significance of a sex difference in this region is not immediately clear since to our knowledge it does not have sexually dimorphic characteristics associated with it. Furthermore, the lack of an obvious protein sex difference in other monomorphic regions of the brain (Table 1), served as a good control further strengthening the link between sex differences in neurocalcin expression and developmental processes that lead to dimorphic neural structures.

Across the brain regions that were quantitatively analyzed, data also indicated significant effects of age. Between P10 and adulthood the number of neurocalcin-IR cells fluctuated in both sexes. This finding is consistent with the idea that during aging, region-specific dynamic changes

2+ in [Ca ]i regulation is a normal part of the developmental process. For example, a variety of

2+ processes such as growth cone behaviors occur as a result of varying [Ca ]i levels (Mattson,

1992). In cultures of embryonic rat hippocampal cells, neurite elongation and growth cone

2+ 2+ motility are suppressed at very low levels of [Ca ]i (< 50 nM). Slightly higher levels of [Ca ]i

(50 – 70 nM) will promote the formation of microtubules but not microfilaments. The

developing axon can elongate but there will be little growth cone motility. Further increases in

2+ 2+ [Ca ]i (70 – 120 nM) permit neurite growth and growth cone motility. Functionally, Ca

2+ binding proteins are necessary for regulating [Ca ]i for processes such as these. Therefore,

2+ maintaining these critical windows of [Ca ]i is crucial for normal cell differentiation, function

and survival. As long as the Ca2+ regulation occurs equally in both sexes, morphological

2+ differences are not likely to arise. However, if a range of [Ca ]i is established but not regulated equally in both sexes, dimorphisms in various processes can result. More specifically, when an established set point is exceeded or held below normal levels for an extended period of time,

Ca2+ homeostasis is disturbed and cellular structures and functions are compromised, leading

directly to cell death or an increased susceptibility to it (Johnson et al., 1992; Mattson, 1992;

Braunewell and Gundelfinger, 1999; Hwang et al., 1999; Case et al., 2007; Hara and Snyder,

2007). For example, over-expression of calcineurin promotes neuronal death in apoptosis-

resistant cells (Jayaraman and Marks, 1997; 2000). Enhanced levels of calsenilin increase

apoptosis in stable H4 neuroglioma cells (Lilliehook et al., 2002). In the rat brain, dimorphisms

in the expression of calbindin-D28k and calretinin during an early critical period affect cell

death/survival and may be involved in sexual differentiation of hypothalamic structures (Stuart

and Lephardt, 1999; Brager et al., 2000). Taken together, these data support the idea that

neurocalcin may play a role in establishing sexual dimorphisms in the neural song system by

altering Ca2+ homeostasis beyond an established set point during a specific period of

development.

In addition to the above, several lines of evidence also point to disturbances in Ca2+

binding proteins which may specifically contribute to cell death associated with

neurodegenerative disorders. For example, in Alzheimer’s disease (AD), neurocalcin, VILIP-1

and VILIP-3 are reduced in the temporal and entorhinal cortices (Shimohama et al., 1996;

Braunewell et al., 2001). In contrast, another study found that calsenilin was increased in the

cortex of Alzheimer’s patients and in the neocortex and hippocampus of β-amyloid precursor

protein transgenic mice (Dong-Gyu et al., 2004).

The fact that neurocalcin protein was significantly increased in females compared to

males only at P18-23 is also consistent with the idea that enhanced expression may promote cell

death. Previous work has demonstrated that when compared to males, the volume of female

HVC begins to decrease as early as P10 and is statistically different by P20. In RA, the

developmental divergence begins at P20, and is significantly different from males by P30

(Nixdorf-Bergweiler, 1996). Differential cell death is the primary mechanism responsible for creating neural sex differences within these regions (Kirn and DeVoogd, 1989). Not only are the

numbers of dying cells significantly increased at ages when a dimorphism in the volume of these

nuclei are detected, but there is also an overlap with when we find neurocalcin protein to be

significantly increased in females. Interestingly, the sex difference in neurocalcin does not

extend to earlier ages (P10) even though dimorphic neural events are taking place. Based on this

we conclude that neurocalcin is most likely not the trigger that initiates dimorphic neural

development, but rather may play an important role during a limited specified period.

We did not detect a significant sex difference in neurocalcin protein expression in

LMAN, which parallels the morphological changes that are known to occur in this nucleus during early development. In both males and females, the volume and number of neurons in

LMAN increases until approximately P20 and P35, respectively (Nixdorf-Bergweiler, 1996;

Nixdorf-Bergweiler and von Bohlen und Halbach, 2005). At P20 the volume of LMAN begins to first show a decrease in males. Surprisingly we did not detect a sex difference in neurocalcin protein in this region during this period (P18-23). We also did not see a decrease in gene expression for the most part in LMAN as well. This result can be explained if we consider the following scenario. The maximum age of subjects within this grouping of animals was P23.

Beyond this, we only investigated adults. Thus, there was about a 3-day window of overlap between when dying cells in LMAN are first detected in males and when we investigated neurocalcin protein. Depending on the developmental age of the bird (as compared to the chronological age) it is possible that we may have just missed the change in neurocalcin expression. Perhaps if we had investigated animals at ages just beyond P23, for example at P25-

P35 a sex difference in neurocalcin expression in this region would have become evident.

In general we find that much of the mRNA expression described in Veney et al., (2003) was represented by functional protein. With the additional analysis of protein, a sex difference in two song control regions emerged. Within cells there is a dynamic relationship between mRNA and protein. At one time a linear stoichiometric relationship was believed to exist. However, more recent studies indicate that this correlation can range anywhere between 20 – 80% indicating that mRNA and protein expression does not always correspond (Gygi et al., 1999;

Chen et al., 2002; Tian et al., 2004; Nie et al., 2006a,b). There are several likely explanations that could account for this range of correlations. For example, translational activities can be

partially or totally inhibited by microRNAs (Farh et al., 2005; Lim et al., 2005; Baek et al., 2008;

Bartel, 2009). Alternatively, the half-life of proteins and mRNA can vary as a result of

differences in the rate of degradation (Pratt et al., 2002; Carpousis, 2009). Based on these

explanations, a comprehensive study of a gene should include investigations on transcription,

translation, degradation, posttranslational modification and function (Kasinath et al., 2008;

Kozak, 2007).

To our knowledge only one other study has reported a sex difference in neurocalcin

protein labeling (Murias et al., 2007). This paper examined expression across divisions of the

olfactory bulb in mice and found a split between male- and female-biased dimorphic labeling

which may contribute to differences in olfactory processing related to sexual behavior. In

addition to olfaction, neurocalcin has also been reported (although not dimorphically) in

specialized cells within the visual and auditory sensory systems (Nakano et al., 1992; Bastianelli

et al., 1995b; Iino et al., 1995; Iino et al., 1998; Braunewell and Gundelfinger, 1999). In these

2+ systems and in the brain, the exact mechanism of how neurocalcin regulates [Ca ]i remains unclear, but there is likely some level of commonality. One possibility is that across systems, neurocalcin affects ion channels, receptor function or membrane trafficking (Burgoyne et al.,

2004). Under normal circumstances Ca2+ binds neurocalcin and the complex activates cell specific membrane-bound cyclases, which in turn, regulates activity of various cellular

2+ processes. Through feedback proper levels of [Ca ]i are maintained (Kumar et al., 1999;

Krishnan et al., 2004; Braunewell and Szanto, 2009). We propose that when neurocalcin is

2+ dimorphically expressed, such as between P18-23 in our system, [Ca ]i regulation is not within

an established set-point and cell death ensues.

Steroids, mainly E2, play a significant role in the brain to protect against cell death (Cho et al., 2003; Merchenthaler et al., 2003). Furthermore, early administration of E2 shows a neuroprotective effect of cells in the female HVC (Burek et al., 1995), promoting cell survival where cell death is typical. The second aim of this thesis was an attempt to investigate if a single injection of E2 would alter neurocalcin gene expression. This study was based on the assumption

that if females are undergoing more cell death in RA and HVC because of increased neurocalcin expression, then E2 treatment (which partially masculinizes the female brain), would be expected to decrease neurocalcin, to a level closer to male-like expression. Although data from the second

aim has no statistical significance due to a small sample size (n=1) per pooled group, it does

suggest the possibility that E2 treatment decreases neurocalcin gene expression. However, we

cannot make definite conclusions until the sample size is increased. If increased sample sizes

confirm these results to be real, it can be concluded that E2, through its reduction of neurocalcin,

contributes to dimorphic brain development.

In addition to some decreases in neurocalcin expression (which agree with our

hypothesis), there is also a hint from the data that E2 may be able to upregulate neurocalcin. If true, contrary to our hypothesis, this would imply that increased neurocalcin may be attempting to act as a protective agent against apoptosis. More specifically, around P18-25 females are undergoing more cell death than males. Because this process may not be desired, neurocalcin protein is elevated in females in an attempt to rescue cells. Under this scenario, when E2 is

administered (which attenuates cell death) neurocalcin levels might be actually expected to

increase. Alternatively, data might demonstrate that neurocalcin levels in females do not change,

and stay elevated as compared to males after E2 treatment. This would imply that either E2 does

not affect neurocalcin, or that the timing of E2 treatments was not optimal. There could be a time

point that falls between 2 hours and 24 hours, or even beyond 24 hours that may be the critical

time of hormone exposure to act on neurocalcin. Another possibility is that there is a split in the direction of neurocalcin change. For example, after E2 injection, neurocalcin could be up regulated at 2 hours but down regulated at 24 hours or vice versa. Having results such as this would provide information related to how quickly E2 can act on neurocalcin. More specifically,

once E2 is in systemic circulation, it may cause a change in neurocalcin gene expression rather

rapidly (within 2 hours) or have slower actions (up to 24 hours).

Future Directions

In order to completely understand neurocalcin and the role that estrogens may play on the protein, it will be necessary to add more samples to the second aim. From the data collected thus far, we do not know with certainty how E2 affects the regulation of neurocalcin. Further data

collection will confirm if E2 causes a down regulation of neurocalcin specifically by the 24 hour

time point in females. It is not known exactly when neurocalcin is likely to be affected by E2 or

other hormonal manipulations. Based on our data there appears to be a crucial period of time

between 2 and 24 hours where supraphysiological E2 exposure results in a gene response.

Expanding the time points to successive intervals would tell us if or how long it takes to affect this gene. For example, we can examine 3 hour time intervals up to 24 hours and pool these

samples to run a statistical analysis.

It might be equally important to perform the same type of q-PCR analysis with DHT treatment as well. Although it has been shown that E2 has the most potent masculinizing effect in

female zebra finches (reviewed in Wade and Arnold, 2004), and neuroprotective effect on brains

(Sawada et al., 2002; Baum, 2005; Zhao and Brinton, 2006; Nunez et al., 2007) other hormones

may have the potential to regulate neurocalcin as well. For example, DHT has some

masculinizing effects on RA in females (Gurney, 1981). It is possible that DHT acting through

neurocalcin may have been responsible for this effect on cell survival.

Another way to test the effectiveness of E2 on neurocalcin in the song control system

would be to block the synthesis or the action of the hormone. Experiments utilizing aromatase

inhibitors (i.e. fadrozole) would serve as an exceptional model to indicate that exposure to

elevated E2 alters neurocalcin. As an extension, ICI could be used to provide mechanistic

information about the type of receptor that E2 binds to affect neurocalcin gene expression. Either treatment could utilize q-PCR or a Western blot analysis to study changes in gene response and protein, respectively.

A quantitative approach, similar to that of the first aim using histological techniques could be used to compare changes in neurocalcin protein distribution and quantity in dimorphic regions using hormone treatments, specifically E2 and DHT. This would provide a lot of detail on where and how much increase/decrease these proposed treatments actually have on the protein. We would expect that neurocalcin is reduced in at least RA for both hormone applications. Furthermore, antagonist to E2 synthesis and activity would be expected to increase

neurocalcin protein and decrease sex differences in dimorphic regions. An elaborate comparison

of hormone interactions could possibly be the best developments in present neurocalcin research

in our songbird model.

New to songbird research, transgenic protocols and other molecular tools are being

developed to allow us to look deeper into functional genes and products (Adgate et al., 2009).

Knock out studies illustrate the importance of gene products. Although the survival rate of the

zebra finch offspring is not optimal, development of this research may lead to a more complete

understanding of neurocalcin and its importance in the zebra finch brain. More practically,

knocking down the function with siRNA, specific to neurocalcin, would reveal its function.

Inhibiting neurocalcin would directly illustrate the importance of this particular calcium binding

protein within the brain, specifically in the dimorphic nuclei. The reduction of neurocalcin in

females would be expected to cause song control regions to become more male-like, if

neurocalcin is truly a cell death agent. Alternatively, this type of experiment might result in an

increase in compensatory mechanisms that would regulate other calcium sensor proteins to

51 promote the natural death in females. In sum, the role of neurocalcin is still very unclear. There is a lot left to explore with this protein as well as other mechanisms that guide sexual dimorphism of the song control system.

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