1. 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

2. ii
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

3. iii
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

4. iv
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

5. v
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

6. vi
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.

7. 1
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

8. 2
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
Chromosomal Sex XY (Sry-TDF) XX
Gonadal Sex Testes Ovaries
Hormonal Sex Testosterone Low estrogen
Aromatized in brain
No aromatization
Masculine neural
development and
behavior
Feminine neural
development and
behavior
Phenotypic
(Behavioral)
Sex
Males Females
(converted into estrogen)
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.

9. 3
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

10. 4
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.,

11. 5
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

12. 6
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 supra-
physiological 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).

13. 7
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.

14. 8
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

15. 9
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

16. 10
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

17. 11
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

18. 12
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.

19. 13
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

20. 14
[Ca2+
]i homeostasis. Although the exact details are not known, according to the Ca2+
set
point hypothesis, proper cell functioning depends on a critical window of intracellular
Ca2+
[Ca2+
]i (Johnson et al., 1992, Hwang et al., 1999). Conditions that result in
prolonged elevated or low [Ca2+
]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

21. 15
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
[Ca2+
]i and/or modulating the communication between Ca2+
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;

22. 16
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.

23. 17
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

24. 18
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.

25. 19
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

26. 20
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).

27. 21
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

28. 22
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.

29. 23
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.

30. 24
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.

31. 25
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-

32. 26
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

33. 27
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).

34. 28
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).

35. 29
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.
RA
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).

36. 30
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.

37. 31
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).

38. 32
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.

39. 33
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.

40. 34
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.

41. 35
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.

42. 36
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).

43. 37
Table 1: Relative amount of neurocalcin immunoreactivity in the
zebra finch brain
Brain
Region
P 10
Male
P10
Female
P18-
22
Male
P18-22
Female
Adult
Male
Adult
Female
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.

44. 38
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
5.
RA
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).

45. 39
Table 2: ΔCT values for Song regions RA and HVC
Brain Region Sex/Time of Treatment
/Treatment
ΔCT
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.

46. 40
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).

47. 41
Table 4: ΔCT values for Song region LMAN
Brain Region Sex/Time of Treatment
/Treatment
ΔCT
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 (↓).

48. 42
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
in [Ca2+
]i regulation is a normal part of the developmental process. For example, a variety of

49. 43
processes such as growth cone behaviors occur as a result of varying [Ca2+
]i levels (Mattson,
1992). In cultures of embryonic rat hippocampal cells, neurite elongation and growth cone
motility are suppressed at very low levels of [Ca2+
]i (< 50 nM). Slightly higher levels of [Ca2+
]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
[Ca2+
]i (70 – 120 nM) permit neurite growth and growth cone motility. Functionally, Ca2+
binding proteins are necessary for regulating [Ca2+
]i for processes such as these. Therefore,
maintaining these critical windows of [Ca2+
]i is crucial for normal cell differentiation, function
and survival. As long as the Ca2+
regulation occurs equally in both sexes, morphological
differences are not likely to arise. However, if a range of [Ca2+
]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

50. 44
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.

51. 45
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

52. 46
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
systems and in the brain, the exact mechanism of how neurocalcin regulates [Ca2+
]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
processes. Through feedback proper levels of [Ca2+
]i are maintained (Kumar et al., 1999;
Krishnan et al., 2004; Braunewell and Szanto, 2009). We propose that when neurocalcin is
dimorphically expressed, such as between P18-23 in our system, [Ca2+
]i regulation is not within
an established set-point and cell death ensues.

53. 47
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

54. 48
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).

55. 49
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

56. 50
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

57. 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.

58. 52
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