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This is to certify that the
dissertation entitled

NEURAL MECHANISMS OF FEMALE ZEBRA FINCH MATE
CHOICE: THE ROLE OF THE AUDITORY PERCEPTION
SITES, THE SOCIAL BEHAVIOR NETWORK, AND THE

REWARD SYSTEM

presented by

Lace Ann Svec

has been accepted towards fulfillment
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PhD degree in Neuroscience

 

 

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5/08 K./Prolecc&Pres/CIRC/DaleDue.Indd

 

NEURAL MECHANISMS OF FEMALE ZEBRA FINCH MATE
CHOICE: THE ROLE OF THE AUDITORY PERCEPTION SITES,
THE SOCIAL BEHAVIOR NETWORK, AND THE REWARD
SYSTEM

By

Lace Ann Svec

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Neuroscience Program

2009

ABSTRACT
NEURAL MECHANISMS OF FEMALE ZEBRA F INCH MATE CHOICE: THE ROLE
OF THE AUDITORY PERCEPTION SITES, THE SOCIAL BEHAVIOR NETWORK,
AND THE REWARD SYSTEM
By
Lace Ann Svec

Female zebra finches are highly social. Males will court females with song, and
females utilize this song stimuli to make mate choice decisions. After this decision has
been made, the male and female will form a strong pair bond. The unique behaviors and
characteristics associated with zebra finch affiliation and reproduction have provided an
excellent opportunity to examine the intricate and complex interactions among social
behavior, neural systems, and hormones. In the experiments conducted for this
dissertation, I investigated the presentation and regulation of two aspects of social
behaviors: song preferences and pair bonding in the female zebra finch. I used two
methods to examine the neural responses of females to these two types of social stimuli:
immediate early gene analysis and high performance liquid chromatography (HPLC) for
dopamine neurochemistry. Additionally, I examined the effects of estrogen treatment on
song responses.

Females showed preferences for high quality tutored song over untutored song,
regardlessofestrogen treatment. Expressionof the immediateearly gene, ZENK was
decreased in response to estrogen treatment in the ventromedial hypothalamus.
Additionally, a greater induction of ZENK following exposure to tutored versus
untutored song and silence was observed in the auditory perception sites was observed,

but was eliminated when females were treated with estrogen. Females performed higher

levels of associative behaviors when interacting with their mates compared to novel
males. In these females, ZENK expression was correlated with two pairing behaviors,
clumping and preening, in the nucleus taeniae (homologous to the mammalian medial
amygdala), but not in females that were interacting with novel males. In the studies
conducted for this dissertation, the activity of dopaminergic neurons was not affected by
song presentation, estrogen treatment, or pairing status.

The results from this dissertation indicate that female zebra finches show different
behavioral responses to certain social stimuli and these may be separately regulated by
responses in two types of brain regions, auditory perception sites and the nucleus taeniae
(which is part of the Social Behavior Network). In this species, dopamine activity in the
nucleus accumbens or striatum did not appear to relate to responses to social stimuli in
either of the behavioral paradigms. As a result, the present data indicate that these
behaviors may be separately mediated by the particular neural regions examined in this
dissertation, but it remains to be determined how neural systems in the avian brain

integrate to fully coordinate social responses.

ACKNOWLEDGEMENTS

I would like to thank a number of people and organizations for their help and
support in the completion of this dissertation. First, the constant support and guidance
provided by my advisor Dr. Juli Wade has been immeasurable to the completion of this
work. Throughout my dissertation work, she was always willing to take any time that
was needed to discuss my projects and answer questions. I also greatly appreciate her
allowing me to explore my own interests and conduct this line of research in her lab. I
would also like to thank the members of my committee, Dr. Kay Holekamp, Dr. Tony
Nunez, and Dr. Sharleen Sakai for their feedback and suggestions throughout the design
and completion of my dissertation work.

I would also like to thank several other faculty members who supported my work:
Drs. Marc Breedlove and Cindy Jordan for allowing me to rotate in their lab my first
semester, Dr. Keith Lookingland for letting me conduct the high performance liquid
chromatography in his lab and spending a large amount of time teaching me about
dopamine neurochemistry, and Dr. Matt Lovem at Oklahoma State University for
training me and two other lab mates to conduct radioimmunoassays. Finally, I would like
to thank Drs. Patricia Schwagmeyer and Douglas Mock at the University of Oklahoma
for asking me to be a part of their field research program as an undergraduate, which
sparked my interest in avian behavior and encouraged me to continue on to graduate
school.

My time at Michigan State was enhanced by interactions and help from a number
of other graduate students, postdocs, technicians, and undergraduates in the two

laboratories in which I conducted research, who provided a large amount of intellectual,

iv

emotional, and technical support. Thanks to Yu Ping Tang, Camilla Peabody, Michelle
Tomaszycki, Michele Johnson, Melissa Holmes, Jennifer Neal, Laurel Beck, Matt Burke,
Rachel Cohen, Jenny Stynoski, Casey Bartrem, Shannon Jackson, Stephany Latham,
Roya Eshragh, Joe Vandecar, Jessica Caton, and Jenn Yee of the Wade Lab, which has
been my home for the last five years and each of them were a vital part of my laboratory
family. I would like to especially thank Dave Bailey for providing the jumping off point
for my dissertation research and giving me a large amount of advice and support during
my early years in graduate school. Also, Katie Licht, who was a tireless undergraduate
assistant that played a vital role in coding behavioral videos in every project presented in
this dissertation. I also have greatly benefited from the opportunity to serve as mentor in
the completion of her Honors Thesis project. In the Goudreau/Lookingland Lab, I would
like to thank Bahareh Behrouz, Kelly Janis, Sam Pappas, Tyrell Simkins, Chelsea
Tieman, and Matt Biensky for being my neurochemical/HPLC experts and also providing
a happy and inclusive second laboratory home for these parts of my dissertation.

Throughout this dissertation, I have been supported by funding from the National
Institutes of Health, the Neuroscience Program, the Graduate School, and the College of
Natural Sciences.

Finally, I would like to thank my friends and family who have been the best
support system through the completion of this dissertation. I will always appreciate the
time they have been willing to give me at any hour of the day and night. Debbie Soellner
and Laurel Beck have been the best friends I could have asked for and have been there
for me unconditionally throughout the last five years. This work would not have been

possible without all of you.

TABLE OF CONTENTS

LIST OF TABLES .............................................................................................................. x
LIST OF FIGURES .......................................................................................................... xii
CHAPTER ONE: INTRODUCTION ................................................................................. 1
General Introduction to Social Behavior ................................................................ 1
Zebra Finch Biology ............................................................................................... 2
Introduction to Neural Systems .............................................................................. 4
Social Behavior Network ........................................................................................ 5
Ventromedial Hypothalamus ...................................................................... 8
Function .......................................................................................... 8

Anatomy ......................................................................................... 8

Nucleus Taeniae .......................................................................................... 9
Function .......................................................................................... 9

Anatomy ........................................................................................ 11

Bed Nucleus of the Stria Terminalis ......................................................... 12
Function ........................................................................................ l2

Anatomy ....................................................................................... l3

Preoptic area ............................................................................................. 14
Function ........................................................................................ l4

Anatomy ....................................................................................... 15

Estrogen and the social behavior network ................................................ l6
Dopaminergic Systems ......................................................................................... 17
Function .................................................................................................... 17
Anatomy ................................................................................................... 19
Brainstem ...................................................................................... l9

Striatum ........................................................................................ 19

Nucleus Accumbens ..................................................................... 20

Estrogen and the dopaminergic systems ................................................... 21
Auditory Perception .............................................................................................. 22
Auditory perception regions ..................................................................... 23
Function ........................................................................................ 23

Anatomy ....................................................................................... 2S

Estrogen and the auditory perception system ........................................... 26
Conclusions and Hypotheses ................................................................................ 26

CHAPTER TWO: ESTRADIOL INDUCES REGION-SPECIFIC INHIBITION OF
ZENK BUT DOES NOT AFFECT THE BEHAVIORAL PREFERENCE FOR

TUTORED SON IN ADULT FEMALE ZEBRA F INCHES .......................................... 31
Abstract ................................................................................................................ 31
Introduction ........................................................................................................... 32
Materials and Methods ......................................................................................... 35

Animals ..................................................................................................... 35
Hormone Treatment .................................................................................. 36

vi

Song Stimuli ............................................................................................. 36

Choice Test ............................................................................................... 37
Immediate Early Gene Analysis ............................................................... 38
Radioimmunoassay ................................................................................... 44
Results ................................................................................................................... 45
Behavior .................................................................................................... 45

ZENK expression ...................................................................................... 45
Plasma Estradiol ....................................................................................... 50
Discussion ............................................................................................................. 50
Behavioral Response ................................................................................ 50

Neural Response ....................................................................................... 51
Auditory regions ........................................................................... 51

Social Behavior Network .............................................................. 54

Dissociation between behavior and immediate early gene expression ..... 57
Conclusion and Future Directions ............................................................ 58
Acknowledgements ............................................................................................... 59

CHAPTER THREE: ESTRADIOL AND SONG PRESENTATION MEDIATE
BEHAVIOR OF FEMALE ZEBRA FINCHES INDEPENDENT OF DOPAMINE

ACTIVITY IN THE NUCLEUS ACCUMBENS AND MEDIAL STRIATUM ............ 60
Abstract ................................................................................................................. 60
Introduction ........................................................................................................... 61
Materials and Methods ......................................................................................... 64

Animals ..................................................................................................... 64
Raclopride treatment ................................................................................. 65
Hormone treatment ................................................................................... 65

Song stimuli .............................................................................................. 66
Auditory stimulus exposure ...................................................................... 66
Quantification of neurochemicals ............................................................. 69
Statistics ................................................................................................... 70

Results ................................................................................................................... 7O
Estrogen and Song Exposure — Behavior ................................................. 70
Raclopride ................................................................................................. 71
Estrogen and Song Exposure - Neurochemistry ...................................... 71
Discussion ............................................................................................................ 76
Summary ................................................................................................... 76
Behavioral response to estrogen treatment and song exposure ................ 76
Dopaminergic response to raclopride treatment ....................................... 78
Dopaminergic response to estrogen treatment .......................................... 79
Dopaminergic response to song exposure ................................................ 80
Conclusions and future directions ............................................................ 81
Acknowledgements .............................................................................................. 82

CHAPTER FOUR: PAIR BONDING IN THE FEMALE ZEBRA FINCH: A

POTENTIAL ROLE FOR THE NUCLEUS TAENIAE .................................................. 83
Abstract ................................................................................................................. 83

vii

Introduction ........................................................................................................... 84

Methods ................................................................................................................ 87
Animals ..................................................................................................... 87
Behavior ................................................................................................... 88

ZENK ...................................................................................................... 90

Results ................................................................................................................... 92
Behavior .................................................................................................... 92
Principle Components Analysis ................................................................ 92
Female Principle Component ....................................................... 95

Principle Component 2 ................................................................. 95

Neural responses ....................................................................................... 95
Discussion ............................................................................................................. 99
Behavior .................................................................................................. 100
Relationship between behavior and IEG expression in the nucleus

taeniae ....................................................................................... 102

Neural response in other brain regions ................................................... 104
Summary and conclusions ...................................................................... 105
Acknowledgements ............................................................................................. 1 06

CHAPTER FIVE: PAIR BONDING BEHAVIOR IN FEMALE ZEBRA F INCHES IS
DISSOCIATED FROM DOPAMINERGIC ACTIVITY IN THE NUCLEUS

ACCUMBENS AND MEDIAL STRIATUM ................................................................ 107
Abstract ............................................................................................................... 107
Introduction ......................................................................................................... 108
Methods .............................................................................................................. 111

Animals ................................................................................................... 111
Behavior .................................................................................................. 112
HPLC Analysis ....................................................................................... 113
Statistics .................................................................................................. 114
Results ................................................................................................................. 114
Behavior .................................................................................................. 114
HPLC ...................................................................................................... 117
Discussion ........................................................................................................... l 19
Behavior .................................................................................................. 119
Neural correlates ..................................................................................... 121
Conclusions and Future Directions ......................................................... 123
Acknowledgements ............................................................................................. l 24

CHAPTER SIX: DISCUSSION ..................................................................................... 125
Two behavioral paradigms ................................................................................ 125
Neural Correlates ................................................................................................ 128

Methodological Issues ............................................................................ 128
Immediate early genes as indicators of neural responses ........... 128
Neurochemical Analyses ............................................................ 129

Three Neural Systems ......................................................................................... 129
Social Behavior Network ........................................................................ 130

viii

Dopaminergic Systems ........................................................................... 131

Auditory Perception Regions .................................................................. 132

Neural systems summary ........................................................................ 133

Role of Estrogen in Brain and Behavior ............................................................. 135

The uniqueness of the zebra finch model ........................................................... 136
Conclusions and Future Directions ..................................................................... 137
REFERENCES ............................................................................................................... 140

ix

LIST OF TABLES

Table 1. Summary of the functional, anatomical, and neurochemical characteristics of
mammalian and analogous/homologous avian brain regions in the social behavior
network examined in this dissertation. Black letters indicate that the characteristic is
observed in mammals and birds. Green letters indicate it is known to exist in mammals,
and blue letters, indicate it is known to exist in birds. Abbreviations: Preoptic area
(POA), VMH (ventromedial hypothalamus), medial amygdala (MeA), nucleus taeniae
(TnA), bed nucleus of the stria terminalis (BST), lateral septum (LS), ventral tegmental
area (VTA), striatum (ST), substantia nigra (SN), nucleus accumbens -rodent (NAcc),
nucleus accumbens - bird (Ac), prefrontal cortex (PFC), main olfactory bulb (MOB),
accessory olfactory bulb (AOB), globus pallidus (GP), ventral pallidum (VP),
caudolateral nidopallium (N CL), caudomedial nidopallium (N CM), caudomedial
mesopallium (CMM), hippocampus (HP), norepinephrine (NE), dopamine (DA),
serotonin (5-HT), acetylcholine (Ach), glutamate (Glu), vasopressin (VP), vasotocin
(VT), enkephalin (ENK), neurotensin (NT), substance P (SP), neuropeptide Y (N PY),
dynorphin (DYN), Calbindin (CALB), estrogen receptor (ER), aromatase (ARO) ........ 29

Table 2. Summary of the functional, anatomical, and neurochemical characteristics of
mammalian and analogous/homologous avian brain regions (dopaminergic and auditory
perception) examined in this dissertation. Black letters indicate that the characteristic is
observed in mammals and birds. Green letters indicate it is known to exist in mammals,
and blue letters, indicate it is known to exist in birds. Abbreviations: Preoptic area
(POA), VMH (ventromedial hypothalamus), medial amygdala (MeA), nucleus taeniae
(TnA), bed nucleus of the stria terminalis (BST), lateral septum (LS), ventral tegmental
area (V TA), striatum (ST), substantia nigra (SN), nucleus accumbens -rodent (N Acc),
nucleus accumbens - bird (Ac), prefrontal cortex (PFC), main olfactory bulb (MOB),
accessory olfactory bulb (AOB), globus pallidus (GP), ventral pallidum (VP),
caudolateral nidopallium (NCL), caudomedial nidopallium (NCM), caudomedial
mesopallium (CMM), hippocampus (HP), norepinephrine (N E), dopamine (DA),
serotonin (5-HT), acetylcholine (Ach), glutamate (Glu), vasopressin (VP), vasotocin
(VT), enkephalin (ENK), neurotensin (NT), substance P (SP), neuropeptide Y (NPY),
dynorphin (DYN), Calbindin (CALB), estrogen receptor (ER), aromatase (ARO) ........ 30

Table 3. Mean densities of ZENK-IR in various brain regions presented as number of IR
nuclei/mm2 (meanistandard error). Due to histological artifact, only 7 animals were

analyzed in the Ac in the blank, untutored group. No main effects or interactions were
detected, all F< 3.02, p> 0.06 ........................................................................................... 49

Table 4. Descriptions of measured behaviors ................................................................... 67

Table 5. Concentrations of DOPAC, Dopamine and the DOPAC/DA ratio in the nucleus
accumbens and striatum following song presentation and estrogen treatment. No main
effects or interactions were detected, all F< 1.92, p> 0.16 ............................................... 75

Table 6. Descriptions of measured behaviors. Frequencies were assessed for all;
durations were also measured for the behaviors with an asterisk * .................................. 89

Table 7. Principle Components Analysis. Loadings over 0.5 are indicated with
bold type ........................................................................................................................... 93

Table 8. Principal components analysis. Behaviors loading above 0.5 are in bold ....... 115
Table 9. Durations of male and female behaviors displayed following introduction of a
male (meaniS.E.). Results are taken from the 30 min test in the present study and from

the first 30 minutes of the behavior tests in Svec et al. in press. Values from the two
experiments are summed and presented on the right ...................................................... 116

xi

LIST OF FIGURES

Figure 1. Placement of sampling regions within auditory perception and social behavior
areas in sagittal sections. Panel A contains boxes for the caudomedial nidopallium
(NCM; 310nm x 320nm) and caudomedial mesopallium (CMM; 190um x 345 um),
panel B for the bed nucleus of the stria terminalis (BST; 190nm x 345 um) and
ventrolateral subdivision of the caudal lateral septum (LSc. v1; 200um x 200um), panel
C for the nucleus taeniae (TnA; 200um x 320nm), panel D for the ventromedial
hypothalamus (VMH; 190nm x 290nm) and preoptic area (POA; 200nm x 200nm), and
panel E demonstrates for the midbrain central gray (GCt; 190nm x 290um). The
hippocampus (HP), tractus occipito-mesencephalicus (OM), tractus septopallio-
mesencephalicus (TSM), optic chiasm (OC), posterior commisure (CP), and cerebellum
(Cb) were used as landmarks for location of sections and placement of boxes. The more
rostral portion of each photo is towards the right edge. Scale bar (in panel D) =

500nm ............................................................................................................................... 42

Figure 2. Placement of sampling regions within the reward system. Panels A and C
contain sections subjected to tyrosine hydroxylase immunohistochemistry sliced in the
sagittal plane. Panels B and D contain ZENK labeling. Panel B displays the location of
the box for the nucleus accumbens (Ac; l90um x 305 um), and panel D displays the
ventral tegmental area (VTA; 210nm x 368um). The more rostral portion of each photo
is towards the right edge. Scale bar = 300nm ................................................ ‘ ................. 43

Figure 3. Total time spent within the tutored, center and untutored zones in the
behavioral test. Sample sizes are indicated on the graph. * = tutored significantly
greater than untutored and center zones .......................................................................... 46

Figure 4. ZENK-IR nuclei in the ventromedial hypothalamus of female zebra finches.
Panel A displays the quantification of densities of ZENK-IR for each treatment group
and song exposure type. Sample sizes are indicated on the graph. A main effect of
treatment was detected (*); estradiol decreased the expression of ZENK. Panels B and C
depict ZENK in the ventromedial hypothalamus of birds exposed to tutored song. Panel
B = blank treated female, Panel C = estrogen treated female. Scale bar = 100 um ......... 47

Figure 5. Densities of ZENK expression within the caudomedial nidopallium (NCM)
and caudomedial mesopallium (CMM) in female zebra finches. Sample sizes are
indicated on the graphs ..................................................................................................... 48

Figure 6. Examples of microdissections taken from thionin-stained coronal sections of
the zebra finch brain. Panel A depicts punches of (I) the nucleus accumbens and (2)
rostral medial striatum (both 2l-gauge). Panel B depicts punches from the caudal
medial striatum (3; 18-gauge). LV= lateral ventricle, LPS= Lamina pallio-subpallialis.
Scale bar = 1 mm .............................................................................................................. 68

Figure 7. Effects of estradiol on long distance calls (top) and visual scanning behavior
(bottom). Data (meaniS.E.) were pooled across song exposure groups, as the effects of

xii

the auditory stimuli did not differ among them. Sample sizes: Estradiol-treated tutored
song = 6, estradiol-treated untutored song = 8, estradiol-treated silence = 7, blank-treated
tutored song = 5, blank-treated untutored song = 6, blank-treated silence = 6. * p<

0.031 ................................................................................................................................. 72

Figure 8. Effect of auditory stimulus on other calls (meaniS.E.). Data were pooled
across treatment groups, as estradiol did not affect the number of these vocalizations.
Sample sizes: Estradiol-treated tutored song = 6, estradiol-treated untutored song = 8,
estradiol-treated silence = 7, blank-treated tutored song = 5, blank-treated untutored song
= 6, blank-treated silence = 6. "‘ p= 0.006 ....................................................................... 73

Figure 9. Effects of raclopride treatment (meaniS.E.) on the concentration of DOPAC
(top) and DA (middle), and the DOPAC/DA ratio (bottom). Sample sizes are indicated at
the bottom of each bar; * p< 0.008 ................................................................................... 74

Figure 10. Female pairing behavior in the social interaction test. The two left panels
depict the frequency (top) and duration (bottom) of clumping to the male. The right two
panels depict the frequency (top) and duration (bottom) of preening the male. The
bottom center panel depicts attempted mounts by the male. "‘ signifies p< 0.05 ............ 94

Figure 11. ZENK immunoreactivity across the brain regions investigated in the three
groups of females (paired females exposed to novel males or their partner, and unpaired
females exposed to novel males). A main effect of region was detected; the density of
calls was highest in the nucleus accumbens and bed nucleus of the stria terminalis. A
main effect of group and interaction between group and region were not detected.
Abbreviations: caudomedial nidopallium (NCM), caudomedial mesopallium (CMM),
preoptic area (POA), ventromedial hypothalamus (VMH), nucleus accumbens (Ac),
nucleus taeniae (TnA), bed nucleus of the stria terminalis (BST), ventral tegmental area
(VTA) ................................................................................................................... 96

Figure 12. Correlation analyses of the density of ZENK immunoreactive cells in the
nucleus taeniae with female pairing behavior in the social interaction test. The figure
includes only females interacting with their mate. Panel A depicts the frequency and
duration of clumping to the male. Panel B depicts the frequency and duration of
preening the male. Three of the four correlations are statistically significant ................. 97

Figure 13. ZENK immunolabeling in the nucleus taeniae (Panels A and C) in the
nucleus accumbens (Panels B and D) of females interacting with familiar males. The
density of ZENK immunoreactivity was significantly higher in the nucleus accumbens
than nucleus taeniae. Labeling within cells also appeared darker in the nucleus
accumbens. ZENK immunoreactivity was more apparent in the caudal portion of
nucleus taeniae (left side of photos), but was more homogenous in the nucleus
accumbens. The top two panels (A and B) depict labeling from animals that displayed
high levels of pairing behavior. The bottom two panels (C and D) depict labeling in
animals that displayed low levels of pairing behaviors. Scale bar = 50 um .................... 98

xiii

Figure 14. Concentrations of DOPAC (top), DA (middle), and the DOPAC/DA ratio
(bottom) in the nucleus accumbens (left), rostral striatum (center), and caudal striatum
(right). Significant effects of brain region were observed for each measure, indicated by
different letters above the bars. No effects of group or interactions among group and
brain region were observed. Sample sizes: paired with new male, n=6, paired with same
male, n= 10, unpaired, n= 11 .......................................................................................... 118

xiv

CHAPTER 1 : INTRODUCTION

General Introduction to Social Behavior

A variety of species live in groups in which sociality is important. Some classic
examples of social behaviors involve those associated with reproduction, aggression, and
parenting. When interacting with other individuals, communication and recognition are
critical. Investigating reproductive behavior provides numerous opportunities to
examine social communication and the interpretation of social cues. Prior to
reproduction, males and females must locate one another, and often some type of
courtship behavior ensues. Usually courtship involves communication from the male to
the female, which may provide indication of his reproductive fitness. In the studies in
this dissertation, social behavior will be investigated in the context of reproduction in the
female zebra finch.

Females in a number of avian species (including zebra finches) utilize an
auditory signal, male song, to determine the quality of a potential mate. For example, in
song sparrows, females prefer males with a larger repertoire of songs with a species-
specific structure. This preference is indicated by increased copulatory solicitation
behavior in response to the preferred song types (Searcy and Marler 1981). Female
songbirds also show preferences for longer songs (Neubauer 1999; Nolan and Hill
2004), songs presented at higher rates (Nolan and Hill 2004), and songs containing
difficult-to-produce syllables (Ballentine et al. 2004), which all reflect increased male
energy output to produce the song. Repertoire size (Buchanan and Catchpole 2000) and
song rate (Dolby et al. 2005) are both correlated with high paternal effort (increased

feeding of the nestlings). Also, Hasselquist et al. (1996) demonstrated a correlation

between repertoire size of a male and survival of offspring. Low song quality can be an
indicator of developmental stress, and as a result, lower the fitness of a potential mate
(Nowicki et al. 2002). Thus, a female songbird most likely utilizes characteristics of
male song in order to examine both a male’s direct (paternal effort/ general health of
male) and indirect (good genes) benefit to her reproductive fitness and offspring
survival.

Zebra Finch Biology

Zebra finches live in colonies of 20-100, which consist of adult males, females,
and their offspring (Zann 1996). Social interactions occur in many contexts, including
locating food and water, evading predators, and reproducing (Zann 1996). In the colony,
individuals must respond to a variety of social stimuli, which provides a number of
possibilities for examining social communication in this species. There are several other
important aspects of zebra finch behavior and biology that make them excellent models
for these types of investigations.

First, zebra finches are opportunistic breeders; in the wild they reproduce when
there is plentiful water supply. As a result, zebra finches reproduce easily in the
laboratory if fresh water is provided ad libitum. It is unknown whether the presence of
water or the grasses and grass seeds that grow as a result of rainfall in the wild stimulate
the activation of breeding behavior (see Zann 1996 for review). Also, there is
conflicting evidence about how quickly reproduction begins after rain, some report that
courtship begins days after the first rainfall, while others state that it takes months (Zann

et al. 1995; Zann 1996). It has been hypothesized that males may remain in a near-ready

state in terms of reproductive development (F amer and Serventy 1960), but this
phenomenon has not been well characterized in either males or females.

Second, zebra finches, and many other songbird species, form pair bonds prior to
mating. In zebra finches, these bonds can begin to form within 30 minutes of
introduction, but typically take about 2-14 days to consolidate (Silcox and Evans 1982).
In the wild, pairing is characterized by clumping (perching together in physical contact),
allopreening (cleaning and inspecting each other for pests), and synchronized behavior
(eating, drinking, perching etc. at the same time). The display of clumping and preening
are often used in laboratory studies to identify individuals as paired (i.e. Butterfield
1970; Silcox and Evans 1982; Clayton 1990; Zann 1996; Adkins-Regan and Wade 2001;
Adkins-Regan 2002). As the pair bond is consolidated, these behaviors increase in
frequency and the two individuals become largely inseparable. During and after the
formation of the bond, the male and female zebra finch will begin to build a nest
together and eventually reproduce. Some researchers also identify pair bonds by males
and females that enter a nest box together (Mansukhani et al. 1996; Adkins-Regan and
Wade 2001). Tactile contact is required in order to form the pair bond, but it can be
maintained with visual and auditory contact alone (Silcox and Evans 1982).

This pair bond is strong and long lasting (usually throughout life), and both
members of the couple provide care for their young (Zann 1996), which is necessary at
least in part due to the extremely altricial nature of zebra finches at hatching. Success of
broods in another socially monogamous songbird, the reed warbler, decreases when a

male parent is removed, because the female must compensate for the assistance normally

provided by the male (Duckworth 1992). It is unknown whether the same is seen in
zebra finches.

Behaviors indicative of a pair bond, such as clumping, preening, or synchronized
behavior are not typically displayed between unrelated single individuals (Butterfield
1970; Zann 1996). Females that have formed a pair bond react aggressively to new
males, and close contact (clumping and preening) with them is inhibited (Silcox and
Evans 1982). Thus, females both increase pairing behaviors displayed toward their
mate, and also reduce the display of these behaviors toward other males. The pair bond
in this species can be utilized to investigate a more complex social interaction and how a
specific type of social bond can modify female responses to social stimuli.

In sum, the zebra finch is an excellent species in which to examine questions
regarding social responses, because they are highly social, form a strong pair bond prior
to copulation, and have an easily manipulated and analyzed form of auditory
communication. This model provides a perfect opportunity to explore the neural
mediation of female social responses. At this point, the development and characteristics
of the song control system in males has been the focus of neural research in this species.
The neural control of social behaviors in adult females has largely been ignored, and that

is the focus of my dissertation.

Introduction to Neural Systems
Based on their likely functional associations with interpretation of social stimuli
and mediation of social behaviors in mammals and possibly birds, I propose three

categories of neural regions or systems that may be critical for these behaviors in the

female zebra finch: the Social Behavior Network, dopaminergic systems, and regions
involved in auditory perception. These groups of brain areas are not isolated; each
interacts with other systems. Regions in these three nodes may collectively integrate
external and internal environmental cues to influence evaluation of and motor responses
to social stimuli. The areas/systems could work together or influence each other.
Alternatively, individual brain areas or groups of them might act independently to
influence particular aspects of this process.

For regions in each of these categories or systems, I will highlight the existing
comparative information in relation to their functional, anatomical and neurochemical
characteristics. Researchers have documented numerous functional and anatomical ‘
similarities between avian and mammalian neural systems. The comparisons between
birds and mammals have been extensive in some regions, but research in other regions
has been much more limited. In 2004, a forum was conducted to modify the avian brain
nomenclature to reflect these similarities, the results of which have been discussed in
Reiner et al. (2004a, b). For each neural group, I will compare the function and anatomy
of the mammalian and avian regions in relation to the social behaviors investigated in
this dissertation, using the available data. Tables 1-2 will include summaries of this

comparative information.

Social Behavior Network
During social interactions, female behavior is most likely mediated by integrating
hormonal and sensory information to direct a specific motor response. A neural social

behavior network has been proposed to serve this type of function in rodents (Newman

1999) and in other vertebrates (Goodson et al. 2005). Brain areas included in this
interconnected network are responsive to gonadal steroid hormones and mediate
multiple social behaviors, such as reproduction and aggression (Newman 1999). The
network, as described by Newman (1999), includes the medial amygdala, bed nucleus of
the stria terminalis, lateral septum, midbrain, ventromedial hypothalamus, anterior
hypothalamus, and medial preoptic area. At least some of these regions are likely to be
relevant to the reproductive and social behaviors examined in these studies.

In mammals, immediate early gene (IEG) induction is observed following
vaginocervical stimulation or copulation with intromission in the ventromedial
hypothalamus, medial amygdala, preoptic area, and bed nucleus of the stria terminalis
(reviewed in Pfaus and Heeb 1997). The response within this network in rodents has
also been examined following exposure to socially relevant stimuli (for rodents these
stimuli are primarily olfactory, as opposed to auditory, as in songbirds). In female rats,
increased IEG expression in the medial amygdala and bed nucleus of the stria terminalis
is also seen when exposed to bedding with male odors (Bennett et al. 2002).

Several brain regions in this network are also implicated in the mediation of pair
bonding behaviors in prairie voles. Cohabitation with a male results in increased IEG
expression in the bed nucleus of the stria terminalis and preoptic area in females
(Cushing et al. 2003). Following the formation of a pair bond (6 hours of mating), IEG
expression is also seen in the medial amygdala, preoptic area, and bed nucleus of the
stria terminalis (Curtis and Wang 2003). Thus, it appears that regions within the social
behavior network are utilized in the formation/maintenance of pair bonding in another

monogamous/pair bonding species.

The neuropeptides oxytocin and vasopressin, which have been strongly
implicated in rodent social recognition and bonding (see Bielsky and Young 2004; Lim
and Young 2006 for reviews), are present in several of the brain regions in this network.
The rodent bed nucleus of the stria terminalis, septum, and amygdala contain
vasopressin (DeVries et al. 1985; Caffe et al. 1987). Impaired social recognition in the
Brattelboro rat, which is vasopressin deficient (Vandesande and Dierickx 1976), can be
restored with vasopressin infusion directly into the lateral septum (Engelmann and
Landgraf 1994). Antagonism or viral knockdown of the vasopressin 1a receptor in rats
(Landgraf et al. 1995) and knockout of this receptor in mice (Bielsky et al. 2004) also
causes a reduction in social recognition. Vasopressin and oxytocin are also related to
sociality and monogamy in vertebrate species (see Goodson and Bass 2001 for review).
Receptor levels for oxytocin are higher in monogamous than promiscuous species in the
nucleus accumbens and bed nucleus of the stria terminalis, but lower in the lateral
septum (Wang et al. 1996). In contrast, vasopressin receptor levels are higher in the
lateral septum in monogamous species (lnsel et al. 1994; Wang et al. 1996). This pattern
is also observed in estrildid finches, where social species display higher levels of
vasotocin (the avian homologue of vasopressin) receptor la binding than non-gregarious
species in the lateral septum (Goodson et al. 2006).

Due to their involvement in social behaviors and specifically their role in
reproductive behaviors and pair bonding in mammals, several regions in this network
have been selected as the focus of these studies: ventromedial hypothalamus, preoptic

area, amygdala. and bed nucleus of the stria terminalis.

Ventromedial hypothalamus

Function

The ventromedial hypothalamus has long been considered a critical region for the
mediation of female receptive behavior in rodents and also a site for estrogen’s action on
this process (reviewed in Pfaff et al. 1994). Lesions here reduce the occurrence of
lordosis (Pfaff and Sakuma 1979) and also the frequency of coital contact in female rats;
possibly demonstrating reduced motivation for copulation (Emery and Moss 1984). In
addition electrical stimulation of this region can facilitate lordosis in rats (Pfaff and
Sakuma 1979). IEGs are induced in this region following vaginocervical stimulation in
rats (see Pfaus and Heeb 1997). Additionally, the ventromedial hypothalamus plays a
role in the mediation of food intake (see Bray and York 1979; Inoue and Bray 1979 for
review) in mammals.

In birds, the ventromedial hypothalamus appears to mediate similar functions to
mammals. Lesions to this region in ring-doves decrease the display of female courtship
behaviors (Gibson and Cheng 1979). In female quail, interaction with a male induces
IEG expression here, and this expression is positively correlated with the display of
proceptive behaviors by the female (Meddle et al. 1999). In doves, exposure to male and
female nest coos result in inhibitory activity in neurons in the ventromedial
hypothalamus (Cheng et al. 1998), indicating that this region responds to auditory
stimuli. In parallel, song stimuli induce IEG expression in this region in white-throated
sparrows (Maney et al. 2008). Like mammals, the ventromedial hypothalamus is also
implicated in mediation of food intake in bird species, as lesions here can result in

hyperphagia, obesity, and an increase in insulin levels (reviewed in Kuenzel 1994).

Anatomy

Reciprocal connections exist between the ventromedial hypothalamus and the
preoptic area, bed nucleus of the stria terminalis, amygdala, and lateral septum in rodents
(Saper et al. 1976; F ahrbach et al. 1989; Canteras et al. 1994). Although no tract-tracing
studies have been conducted from this region in birds, tracing studies from other regions
have identified projections from the nucleus taeniae (Cheng et al. 1999), preoptic area
(Berk and Butler 1981; Balthazart et al. 1994), auditory thalamus (Cheng and Peng
1997), and the septum (Montagnese et al. 2004) to this region. Studies concerning the
neurochemical characteristics of this region are very limited in birds. However,
examinations of the neural distribution of several neurotransmitters and neurochemicals
have identified similarities between this region in mammals and birds. The avian
ventromedial hypothalamus contains GABA (Domenici et al. 1988; Granda and
Crossland 1989) and enkephalin positive neurons (Cheng and Zuo 1994); in mammals
GABAergic neurons synapse with other GABAergic and enkephalin positive neurons in
this nucleus (Commons et al. 1999; Commons and Pfaff2001). In addition, glutamate
(Comil et al. 2000; Bian et al. 2008) and acetylcholine (Kow and Pfaff 1985; Watson et
al. 1988) are utilized as neurotransmitters in this region in both mammals and birds.
Although the functional, anatomical, and neurochemical research in this region is more
limited in avian species, the available data indicates a strong similarity between the

mammalian and avian ventromedial hypothalamus.

Nucleus taeniae

Function

The nucleus taeniae is an avian region with homology to the mammalian
amygdala (Thompson et al. 1998; Cheng et al. 1999). In mammals, the amygdala has
numerous functions involving emotional processing of sensory stimuli (reviewed in
Phelps and LeDoux 2005), such as fear conditioning (reviewed in LeDoux 1998; 2003:
2007), determining stimulus-reward value (see Baxter and Murray 2002 for review), and
the emotional modification of memory storage (see McGaugh et al. 1996 for review).
IEGs are induced in this region following copulation or vaginocervical stimulation in
rodents (Pfaus and Heeb 1997), but lesions to this region do not seem to affect sexual
behavior in females (Masco and Carrer 1980). Given its importance in the processing of
sensory information and emotional memory, it seems likely that this region may be
critical for female neural and behavioral responses to social interactions.

Functional similarities exist between the avian nucleus taeniae and the
mammalian amygdala. Cheng et al. (1999) delineated the behavioral effects of lesions to
this region in female doves and starlings. Lesions increase the display of nest coos in
ring doves (a female courtship behavior), possibly due to reduced fear of the aggressive
behavior the male directs toward the female in response to her cooing. In starlings,
lesions result in reduced social feeding, and also increased perch changes and decreased
time spent in proximity to other cage-mates. These behavioral effects may be due to the
cage-mate’s unfavorable response to decreased social inhibition of the lesioned females.
In addition, IEG expression has been detected in this region after exposure to male song
stimuli in white-throated sparrows (Maney et al. 2008). It appears, through these
studies, that the nucleus taeniae plays a role in the mediation of social behavior and

responses to social stimuli in both mammals and birds.

10

 

Anatomy

The mammalian amygdala receives inputs from sensory systems (including
thalarnic and cortical components; Scalia and Winans 1975; Ottersen 1981; Canteras et
al. 1994), the hypothalamus, the bed nucleus of the stria terminalis, and the ventral
tegmental area/ substantia nigra (Ottersen 1980; 1981), sends outputs to several
hypothalamic structures (Caffe et al. 1987; Canteras et al. 1994; Bian et al. 2008), as
well as the bed nucleus of the stria terminalis (Caffe et al. 1987; Canteras et al. 1994;
Dong et al. 2001), prefrontal cortex (Canteras et al. 1994), striatum (Canteras et al.
1994), nucleus accumbens (Canteras et al. 1994) and hippocampus (Caffe et al. 1987).
The nucleus taeniae in birds also has reciprocal connections with the ventromedial
hypothalamus, preoptic area, and bed nucleus of the stria terminalis (Cheng et al. 1999),
striatum, caudolateral neostriatum (homologous to prefrontal cortex; Reiner et al.
2004a), and hippocampus (Cheng et al. 1999). The avian nucleus taeniae also appears to
receive input from the caudomedial mesopallium (Cheng et al. 1999).

In rats, this region has a large amount of vasopressin innervation (DeVries et al.
1985; Sofroniew 1985), with vasopressin fibers that project to the lateral septum and
hippocampus (Caffe et a1. 1987). This innervation is also observed in birds (Voorhuis
and de Kloet 1992; Aste et al. 1996). In addition, GABA (Wong and Moss 1992),
dopamine (Young and Rees 1998), serotonin (Moore et al. 1978), and glutamate (Bian et
al. 2008) act as neurotransmitters in this region in mammals. Similarly in birds, GAD-
65 (a marker for GABA) is present in the nucleus (Yamamoto et al. 2005), and staining
for tyrosine hydroxylase (Bottjer 1993; Appeltants et al. 2001; Roberts et al. 2002) and

DARPP-32 (Roberts et al. 2002) occurs in this region, indicating dopaminergic

11

 

innervation. Serotonin innervation is also present in the nucleus taeniae (Cozzi et al.
1991; Metzger et al. 2002). Calcitonin gene-related peptide (CGRP) is also present in
the amygdala in rodents (Brauth and Reiner 1991; Yasui et al. 1991) and birds (Brauth
and Reiner 1991; Lanuza et al. 2000). In sum, the nucleus taeniae has similar neural
connections, neurochemistry, and also appears to serve a similar function (processing the

emotional context of social stimuli) as the mammalian medial amygdala.

Bed nucleus of the striarterminalis
Function

The bed nucleus of the stria terminalis is implicated in the mediation of social
behaviors including reproduction in mammals. IEG expression is increased in this
region during copulation or vaginocervical stimulation (reviewed in Pfaus and Heeb
1997). As IEG expression is observed here after females are exposed to conditions that
promote the formation of a pair bond in female prairie voles (Curtis and Wang 2003;
Cushing et al. 2003), the bed nucleus of the stria terminalis appears to play a role in pair
bonding. Additionally, this region contains vasopressin innervation (see de Vries and
Miller 1998; De Vries and Panzica 2006 for review).

Avian studies of the bed nucleus of the stria terminalis have been primarily
anatomical, although some functional studies indicate that IEG expression is observed in
this region in females following interaction with a male in quail (Meddle et al. 1999).
The bed nucleus of the stria terminalis may play a role in the mediation of male
reproductive and courtship behaviors in chickens (reviewed in Jurkevich and Grossmann

2003). Sexually motivated singing results in induction of IEG expression in this region

12

in male starlings (Heimovics and Riters 2006). IEG expression is also observed in this
region following exposure to song stimuli in white-throated sparrows (Maney et al.
2008)

Anatomy

The rat bed nucleus of the stria terminalis receives inputs from vomeronasal
system (Scalia and Winans 1975), hippocampus (Weller and Smith 1982), and dorsal
raphe nucleus (Eiden et al. 1985). It also has reciprocal connections with the
ventromedial hypothalamus and medial amygdala (Weller and Smith 1982; Gu et al.
2003; Poulin et al. 2006), and projects to the lateral septum and preoptic area in rats (Gu
et al. 2003). A connection to the nucleus taeniae has been identified from the bed
nucleus of the stria terminalis in birds (Cheng et al. 1999); however, other tract tracing
studies have not been conducted from this region in avian species.

Vasopressin innervation is observed in this region in mammals and birds (rats,
(DeVries et al. 1985; Sofroniew 1985; Veinante and Freund-Mercier 1997); quail, (Aste
et al. 1998; Panzica et al. 1999; Panzica et al. 2001). In addition, this innervation is
sexually dimorphic with higher density in males than females in birds (see Grossmann et
al. 2002 for review, Aste et al. 1998; Jurkevich et al. 1999), as is observed in mammals
(reviewed in De Vries and Panzica 2006). Serotonin and norepinephrine fibers are
present in this region in rats (Phelix et al. 1992; 1992) and birds (Cozzi et al. 1991;
Medina and Reiner 1994; Mello et al. 1998). Aromatase immunoreactive neurons are
also common in this region in birds and mammals (birds: Balthazart et al. 1990; Foidart

et al. 1994; Aste et al. 1998, rat: Sanghera et al. 1991; Jakab et al. 1993).

I3

Preoptic area

Function

The preoptic area is considered a primary neural site for the activation of male
copulatory behaviors in rodents (reviewed in Meisel and Sachs 1994), and also an
important target of the hormones that mediate these behaviors (see Hull and Dominguez
2007). However, the preoptic area may also be involved in the control of aspects of
female rodent reproduction, especially female proceptive behavior (reviewed in Pfaff et
al. 1994; Sakuma 1995). Lesions to this region demonstrate its role in the inhibition of
the lordosis reflex (see Pfaff et al. 1994; Sakuma 1995 for review) and also result in It.
increased display of female proceptive behaviors (Whitney 1986; Hoshina et al. 1994).
This region may also be important for pair bonding in prairie voles, with IEG expression
observed here under conditions when pair bonds are being formed (Curtis and Wang
2003; Cushing et al. 2003).

In birds, as in mammals, the focus of functional studies in this region has been on
the mediation of male copulatory behavior. In parallel with rodent research, the avian
preoptic area is the site of action of testosterone.(Barfield 1969; Balthazart and
Surlemont 1990), and lesions to this region reduce male copulatory behavior in quail
(Balthazart et al. 2001). In addition to its role in male copulatory behavior, the preoptic
area may also be critical to male courtship behavior in songbirds. The display of
sexually motivated song results in the induction of IEGs in male starlings in the preoptic
area (Riters et al. 2004; Heimovics and Riters 2005; 2006; 2007). In starlings, lesions to

this nucleus result in reduced song production in the male (Alger and Riters 2006).

14

The role of this structure in mediating female behaviors is less clear in birds (see
discussion in Gibson and Cheng 1979). Preoptic lesions reduce egg-laying and
incubation behavior in turkeys (Youngren et al. 1989), but do not affect courtship
behavior. This result indicates the preoptic area is involved in reproductive behavior in
birds; however, its role in proceptive and receptive behaviors is unknown at this point.
In addition, neurons in this region are responsive to auditory stimuli in ring-doves and
white-throated sparrows (Cheng et al. 1998; Maney et al. 2008).

Anatomy

The neuroanatomical characteristics of the preoptic area in rodents have been
well described, and avian studies indicate that many of these characteristics apply to
avian species as well. The rat preoptic area has reciprocal connections with the
amygdala, bed nucleus of the stria terminalis, ventromedial hypothalamus, lateral
septum, and ventral tegmental area (Simerly and Swanson 1986; 1988). Afferents to the
POA also exist from the main olfactory bulb (Price et al. 1991). The broad inputs and
outputs of the preoptic area also exist in birds, with connections between the preoptic
area and the nucleus taeniae, lateral septum, ventromedial hypothalamus, and the ventral
tegmental area in birds (Berk and Butler 1981; Kitt and Brauth 1986; Balthazart et al.
1994; Cheng et al. 1999).

This region is characterized by vasopressin/vasotocin innervation in rodents
(DeVries et al. 1985; Caffe et al. 1987; Veinante and Freund-Mercier 1997) and birds
(V iglietti-Panzica et al. 1994; Kimura et al. 1999; Absil et al. 2002), and aromatase
activity/immunoreactivity in both mammals (Wagner and Morrell 1996; 1997; Roselli et

al. 1998) and birds (Balthazart et al. 1990; Balthazart et al. 1996; Foidart et al. 1995;

15

1.. ...__..._I

Shen et al. 1995; Saldanha et al. 2000). The neurotransmitters norepinephrine (rats,
Simerly et al. 1986; Espana and Berridge 2006, birds; Mello et al. 1998; Comil et al.
2004), dopamine (rats, Simerly et al. 1986, reviewed in Hull et al. 1999; Dominguez and
Hull 2005, quail; Comil et al. 2004) and serotonin (rats, Simerly et al. 1986, quail, Cozzi
et al. 1991) are observed in this region in both groups. A wide variety of other
neurochemicals are present in neurons in this region in both groups including enkephalin
(rats, Simerly et al. 1986, chickens, Blahser and Dubois 1980; de Lanerolle et al. 1981),
neurotensin (Simerly et al. 1986; Absil and Balthazart 1994), and Substance P (Simerly

et al. 1986; Aste et al. 1995).

Estrogen and the 5&1?! Behavior Netwogrl;

Neurons in all of these regions are sensitive to estrogen in both mammals and
birds (e. g. Pfaff and Keiner 1973; Vito et al. 1983; Koch and Ehret I989; Simerly et al.
1990; DonCarlos et al. 1991; Lauber et al. 1991; Shughrue et al. 1997). A large
percentage of the neurons in the preoptic area, medial amygdala, bed nucleus of the stria
terminalis, and the ventromedial hypothalamus that express IEGs following
vaginocervical stimulation in the rat also contain estrogen receptors (Tetel et al. 1994).
Estrogen implants into the ventromedial hypothalamus result in activation of lordosis in
female rats (Rubin and Barfield 1980). Specifically, estrogen treatment alone has been
shown to increase IEG expression in the ventromedial hypothalamus, preoptic area, and
the amygdala in rodents (Insel 1990). IEG expression can also be affected by estrogen
treatment after vaginocervical stimulation. Specifically, IEG expression decreases or

remains the same with estrogen treatment in the ventromedial hypothalamus (Tetel et al.

16

1994; Pfaus et al. 1996), decreases in the bed nucleus of the stria terminalis, or increases
in the amygdala (Pfaus et al. 1996).

Similar effects of estrogen may also be observed in birds, although they have not
been extensively studied. IEG responses to song in female white-throated sparrows are
increased by estrogen treatment in the ventromedial hypothalamus, nucleus taeniae. bed

nucleus of the stria terminalis, and preoptic area (Maney et al. 2008).

Dopaminergic Systems
Function

Dopaminergic systems in the mammalian brain include those implicated in the
mediation of motor movements (Rieke 1981; Sanberg and Mark 1983; Albin et al. 1989;
Hauber 1998) and reward and motivation (reviewed in Schultz et al. 1997; Akins et al.
2004; Esch and Stefano 2004; Akins and Geary 2008) in mammals. Neurons projecting
from the ventral tegmental area to the nucleus accumbens (mesolimbic dopaminergic
neurons) are typically considered important for determining the reward value of stimuli
(Schultz et al. 1997; Akins et al. 2004; Esch and Stefano 2004). Dopaminergic neurons
are also a target for addictive drugs in mammals (see Bardo 1998 for review).

These dopaminergic neurons are also activated during natural processes such as
reproductive behavior. IEGs are induced in the ventral tegmental area of female rodents
following reproductive behavior and/or vaginocervical stimulation (reviewed in Pfaus
and Heeb 1997). Dopamine is released in the nucleus accumbens when females are

allowed to pace their mating (Mermelstein and Becker 1995; Pfaus et al. 1995; Becker et

17

al. 2001), as measured through microdialysis. This release also occurs when females are
exposed to reproductive stimuli alone (Mitchell and Gratton 1994).

The mesolimbic dopamine reward system is also critical for the display of pair
bonding behavior in prairie voles. Dopamine levels increase in the nucleus accumbens
in response to mating in female prairie voles (Gingrich et al. 2000). Also, dopamine
antagonists presented either systemically or directly into the nucleus accumbens block
the formation of a pair bond following mating (Aragona et al. 2003). Pair bonds can be
formed between a male and a female without mating by treating systemically or directly
into the nucleus accumbens with a dopamine agonist (Aragona et al. 2003).

The function of dopaminergic neurons appears to be conserved in birds. In avian
species, motor deficits are seen with disruption of the nigrostriatal dopamine system
(Rieke 1981; Sanberg and Mark 1983). In addition, drugs of addiction affect behavior in
birds as they do in rodents (Levens and Akins 2004; Geary and Akins 2007; Akins and
Geary 2008). In songbirds, mesolimbic dopaminergic neurons are responsive during the
display of sexually motivated song (Riters et al. 2004; Heimovics and Riters 2005).
Dopamine is also released in the striatum when male zebra finches are singing directed
song (Sasaki et al. 2006). As a result, dopaminergic activity may play a role in sexual
motivation during courtship behavior in male birds.

Dopaminergic systems are also responsive to social stimuli in birds (song stimuli,
as compared to olfactory stimuli in rodents; see (Mitchell and Gratton 1994).
Phosphorylated tyrosine hydroxylase increases in the ventral tegmental area when

female starlings are exposed to directed song (Riters et al. 2007). Also, dopamine is

18

released in the NCM following exposure to song stimuli in starlings (Sockman and

Salvante 2008).

Anatomy

The anatomical and physiological characteristics of dopaminergic systems are highly
similar in mammalian and avian species (reviewed in Durstewitz et al. 1999; Reiner et
al. 2004a).
Brainstem

The majority of dopaminergic neurons originate in the brainstem in the substantia
nigra and the ventral tegmental area in mammals (Lindvall and Bjorklund 1974;
Swanson 1982; Loughlin and Fallon 1983). These neurons then send projections to the
basal ganglia (including the striatum and nucleus accumbens), but also to the prefrontal
cortex, hippocampus, amygdala, and septum (Lindvall and Bjorklund 1974; Fallon and
Moore 1978; Swanson 1982). In birds, the ventral tegmental area and the substantia
nigra are also the primary origin of the dopaminergic outputs in the brain, and dopamine
neurons project from these regions to the medial striatum (Kitt and Brauth 1986; Szekely
'et al. 1994; Metzger et al. 1996; Mezey and Csillag 2002). The avian ventral tegmental
area also sends afferents to the lateral septum, preoptic area, and nucleus taeniae (Kitt
and Brauth 1986), while the substantia nigra is connected to the hippocampus (Kitt and
Brauth 1986). Catecholaminergic cell bodies are observed in substantia nigra and
ventral tegmental area in both mammals and birds (Bailhache and Balthazart I993;
Bottjer 1993; Appeltants et al. 2001).

Striatum

l9

The neuroanatomical and neurochemical properties of the mammalian and avian
striatum have been extensively studied. Dopaminergic neurons enter the striatum from
the substantia nigra and ventral tegmental area in both mammals and birds (Lindvall and
Bjorklund 1974; Metzger et al. 1996). An input to the striatum also exists from the
cortex in both groups (Donoghue and Kitai 1981; Veenman et al. 1995), which is
primarily glutamatergic (Hassler et al. 1982; Gerfen 1992). The striatum also receives
innervation from the amygdala/nucleus taeniae (Canteras et al. 1995; Cheng et al. 1999).
Intemeurons in this region are cholinergic (Bolarn et al. 1984; Kawaguchi et al. 1995),
and GABAergic (Kawaguchi et al. 1995) in rodents. These neurotransmitters are also
found in the medial striatum in birds (Csillag et al. 1997; Veenman 1997; Roberts et al.
2002; Mezey and Csillag 2003). Both mammals and birds also have efferent projections
from the striatum that are GABAergic (Scheel-Kruger et al. 1981; Csillag et al. 1997;
Mezey and Csillag 2003), and often contain Substance P, dynorphin or enkephalin
(Reiner et al. 1984; Reiner 1986; Reiner and Anderson 1990; Anderson and Reiner
1991)

Nucleus Accumbens

Anatomical and neurochemical characteristics of the nucleus accumbens have been well
established in rodents (see Shirayama and Chaki 2006; Zahm and Brog 1992). This
nucleus receives inputs from the ventral tegmental area and substantia nigra, the
prefrontal cortex, the hippocampus, and the amygdala in the rat (Phillipson and Griffiths
1985). This nucleus also sends projections to the ventral tegmental area and substantia
nigra as well as the bed nucleus of the stria terminalis, the lateral hypothalamus, ventral

pallidum, and the globus pallidus (Usuda et al. 1998). Fibers in the region contain

20

neurotransmitters such as acetylcholine (Meredith et al. 1993), norepinephrine (Delfs et
al. 1998), GABA (Christie et al. 1987; Churchill and Kalivas 1994), and glutamate
(Christie et al. 1987; Fuller et al. 1987), as in the striatum. The neurochemicals
enkephalin, neurotensin, and Substance P are also observed in this nucleus, although in a
regional distribution (Zahm and Brog 1992).

The nucleus accumbens has not been clearly defined in avian species (see Reiner
et al. 2004a), although Balint and Csillag (2007) have proposed its location and
subdivision in chicks. They observed similar cholinergic innervation, DARPP-32 and

calbindin immunoreactivity (Balint and Csillag 2007).

Estrogen and dopaminergic systems

In mammals, estrogen treatment increases the release of dopamine in both the
nigrostriatal (Becker and Beer 1986; Becker and Cha 1989) and mesolimbic dopamine
systems (Saigusa et al. 1997; Thompson and Moss 1997). Estradiol treatment also has
effects on other aspects of dopaminergic neurons (reviewed in Van Hartesveldt and
Joyce I986; Kuppers et al. 2000). For example, estrogen treatment increases the density
of D1 dopamine receptors (Levesque and Di Paolo 1989), D2 dopamine receptor binding
(Bazzett and Becker 1994), and dopamine synthesis in the striatum (Pasqualini et al.
1995).

Estrogen receptors are present in tyrosine hydroxylase positive neurons in canary
ventral tegmental area and substantia nigra (Maney et al. 2001), indicating that they are
sensitive to estrogen. Estradiol does affect the activity of some dopamine neurons in

songbirds, as in mammals. In white-throated sparrows, estrogen treatment increases

21

tyrosine hydroxylase labeling in the ventral tegmental area (LeBlanc et al. 2007). In
starlings, phosphorylated tyrosine hydroxylase in the ventral tegmental area is reduced

by estradiol treatment (Riters et al. 2007).

Auditory Perception

Song is an important signal in social communication in zebra finches. Male
zebra finches produce directed and undirected song (described in Zann 1996). Directed
song is presented during courtship and is oriented toward a nearby female. This song
presentation is typically accompanied by a specific posture that can vary in its degree of
intensity; the male extends his head upward, and his feathers become erect on the head,
as well as the cheeks and breast. Directed song is an integral part of male zebra finch
courtship behavior, and females may use this signal as an indicator in mate choice.
Males may also present song that is not oriented towards another bird (undirected song),
which also occurs when the male is alone. This song may function to attract females,
but is not used in courtship or when males are interacting with females.

Female behavioral responses to these song presentations vary, although the
courtship sequence often involves a copulation solicitation display by the female in
response to directed song. Tail quivers are the primary component of the copulatory
solicitation display in this and other songbird species; this behavior often directly
precedes a mount attempt or successful copulation by the male (Zann 1996). Female
songbirds show preferences for their father’s song (e. g. Miller 1979; Clayton 1988;
Riebel et al. 2002) and can recognize the song of their mate (Vignal et al. 2008).

Females can also discriminate between songs of varying levels of quality using

22

characteristics such as those described in the introduction (i. e. bout length, song

complexity).

Marv Perception Regions
Function

As song is such an important communication tool for males of this species, the
neural systems involved in the perception of song are likely vital to females in a variety
of social contexts. However, this perception is especially important during courtship
displays in reproduction.

Song responses in avian species have been investigated using immediate early
gene (IEG; reviewed in Clayton 1997; Ball and Gentner 1998) and electrophysiology
(reviewed in Theunissen and Shaevitz 2006; Pinaud and Terleph 2008). These studies
have identified the importance of the caudomedial nidopallium and the caudomedial
mesopallium in perception of complex auditory stimuli (Mello et al. 1992; Mello and
Clayton 1994; Grace et al. 2003; Terleph et al. 2006; George et al. 2008). In adult male
zebra finches, the response is selective, with higher IEG expression in these regions
following exposure to conspecific compared to heterospecific song (Mello et al. 1992;
Mello and Clayton 1994). These results have been confirmed with lesion studies, in
which lesions to these regions eliminated the ability of individuals to recognize specific
song types (MacDougall—Shackleton et al. 1998; Gobes and Bolhuis 2007).

Male zebra finches learn song from their fathers by first creating a template of
his song (Nordeen and Nordeen 1992; Kroodsma and Miller 1996; Marler 1997;

Brainard and Doupe 2000); research in male song perception has largely focused on the

23

role of auditory perception during development (when males are forming this template).
Males at 30 days post-hatch, when the song template is being formed (Nordeen and
Nordeen 1992; Brainard and Doupe 2000), show an increased ZENK response in the
NCM and CMM to conspecific song (Bailey and Wade 2003). Female juvenile zebra
finches, however, display increased c-fos expression in the NCM and CMM at day 30 to
conspecific song (Bailey and Wade 2003). At day 45, after template formation, greater
F OS and ZENK responses are observed in males and females following exposure to
conspecific compared to heterospecific song in the NCM (Bailey and Wade 2005).
Males and females at this age also present increased ZENK responses to song of high
quality (from males who had tutors during development compared to those who did not;
Tomaszycki et al. 2006). Thus, selective IEG responses in the auditory perception sites
to song begin to develop while males are learning song from a tutor, and females appear
to develop a selective response to song at this time as well.

Little is known about the neural response to song in female juveniles, and even
less is known about whether or not adult female zebra finches use the auditory
perception system to detect differences in song quality. Since females are required to
make these types of assessments to make mate choice decisions, it is crucial to examine
this process. Adult female zebra finches have an increased FOS response to conspecific
compared to heterospecific song in auditory perception regions (Bailey et al. 2002), but
whether or not they can detect critical differences in the quality of male zebra finch song
is unknown. In other songbird species, higher IEG expression is observed in auditory

perception sites in females following exposure to high compared to low quality song or

24

silence (Gentner et al. 2001; Maney et al. 2003; Leitner et al. 2005; Sockman et al. 2005;
Terpstra et al. 2006).

In addition, the neural response to song can be affected by the context in which it
is presented. For example, a new environment or added visual stimuli can eliminate the
habituation that occurs when exposure to a song is repeated (Kruse et al. 2004). IEG
expression can also be induced in the auditory perception regions with visual courtship
stimuli alone (Avey et al. 2005). In starlings, photostimulation to bring females to an
active reproductive state can increase the selectivity of the IEG response in NCM
(Sockman and Ball 2009). Thus, these regions are responsive to auditory stimuli, but
also take in information about the environment in which they are perceived including

both internal and external cues.

Anatomy

These auditory perception regions are considered comparable to the mammalian
auditory cortex (see Reiner et al. 2004a,b), which are responsive to auditory stimuli
(Merzenich et al. 1975). The neural pathway for auditory processing in songbirds has
largely been described through tract tracing studies. The auditory thalamus sends
projections to Field L (an avian auditory region within the nidopallium) and to a lesser
degree, the NCM and CMM (see Durand et al. 1992; Vates et al. 1996). The NCM and
CMM are reciprocally connected (Vates et al. 1996), and along with the mammalian
auditory cortex are connected to auditory thalamic regions (Huang and Winer 2000). In
addition, morphological features of the neurons within the avian auditory perception

regions in birds are highly comparable to the mammalian auditory cortex (Saini and

25

Leppelsack 1981). Neurons responsive to auditory stimuli are located within Layer IV
of the auditory cortex in mammals, and the avian auditory perception regions contain
microneurons that are similar to stellate calls within layer IV (Saini and Leppelsack
1981). Many neurons within the mammalian auditory cortex are also GABA-
immunoreactive (Prieto et al. 1994), as are neurons in the NCM and CMM (Pinaud and

Mello 2007).

Estrogen and the auditory perceptiog system

Estrogen may act in these regions to mediate song responses. In white-throated
sparrows estrogen treatment reduces IEG expression in the NCM and CMM (although
only in response to tones; (Maney et al. 2006). Estrogen receptors are present along the
caudal edge of the NCM and in the CMM. However, estrogen’s action in these regions
may occur through projections to this system from other estrogen sensitive regions. The
auditory thalamus sends axons to estrogen sensitive regions such as the ventromedial
hypothalamus and the nucleus taeniae (Durand et al. 1992). Sparse projections exist
from the nucleus taeniae to the most lateral portion of the CMM, but apparently not the
NCM (Cheng et al. 1999). It is unknown at this time whether the ventromedial

hypothalamus is directly connected with these auditory regions.

Conclusions and Hypotheses
In sum, the aforementioned regions in the avian social behavior network,
dopaminergic, auditory perception systems are highly comparable both in function and

anatomy to those in mammals. Therefore. we can utilize the zebra finch, which is ideal

26

for investigating questions about social behavior, to examine the role of these three
systems in mediating female behavioral responses to social stimuli. Additionally, zebra
finches are opportunistic breeders, providing the opportunity to examine the hormonal
mediation of behavior in a unique model system. As has been discussed, the three
systems are interconnected and also estrogen sensitive, allowing us to determine how
these systems work together along with estrogen when females are interpreting social
stimuli.

In the following experiments, I will test two hypotheses. First, early detection of
male quality is determined by a varying neural response within the auditory perception
sites, the social behavior network, and the reward system to male song (the primary form
of communication from males to females). Second, once a female has made a mate
choice decision, her altered behavioral response to her mate is due to a change in neural
responses to males after a pair bond is formed. By investigating these systems
simultaneously, the role of each system and/or their interaction with one another in the
mediation of female zebra finch social behavior can be elucidated. To do this, I
examined behavioral and neural responses to male zebra finch song, a simple and easily
controlled social stimulus, and then I utilized a more complex and natural social

interaction and examined the role of pair bonding in social responses.

27

Table 1. Black letters indicate that the characteristic is observed in mammals and birds.
Green letters indicate it is known to exist in mammals, and blue letters, indicate it is
known to exist in birds. Abbreviations: Preoptic area (POA), VMH (ventromedial
hypothalamus), medial amygdala (MeA), nucleus taeniae (TnA), bed nucleus of the stria
terminalis (BST), lateral septum (LS), ventral tegmental area (VTA), striatum (ST),
substantia nigra (SN), nucleus accumbens -rodent (NAcc), nucleus accumbens - bird
(Ac), prefrontal cortex (PFC), main olfactory bulb (MOB), accessory olfactory bulb
(AOB), globus pallidus (GP), ventral pallidum (VP), caudolateral nidopallium (NCL),
caudomedial nidopallium (NCM), caudomedial mesopallium (CMM), hippocampus
(HP), norepinephrine (NE), dopamine (DA), serotonin (5-HT), acetylcholine (Ach),
glutamate (Glu), vasopressin (VP), vasotocin (VT), enkephalin (ENK), neurotensin
(NT), substance P (SP), neuropeptide Y (NPY), dynorphin (DYN), Calbindin (CALB),
estrogen receptor (ER), aromatase (ARO) *Images in this dissertation are presented in
color.

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30

CHAPTER 2: ESTRADIOL INDUCES REGION-SPECIFIC INHIBITION OF ZENK
BUT DOES NOT AFFECT THE BEHAVIORAL PREFERENCE FOR TUTORED
SON IN ADULT FEMALE ZEBRA FINCHES.

Abstract
Female zebra finches display a preference for songs of males raised with tutors
compared to those from males without tutors. To determine how this behavioral
preference may be mediated by auditory perception sites, the social behavior network,
and the dopamine reward system, and whether responses of these regions are affected by
estradiol, females were treated with hormone or blank implants. An auditory choice test
was conducted followed by exposure to tutored or untutored song or silence to examine
induction of the immediate early gene, ZENK. Birds spent significantly more time near
tutored than untutored song, regardless of estrogen treatment, and estradiol significantly
decreased the density of ZENK immunoreactive neurons within the ventromedial
hypothalamus. These results suggest that selective neural and behavioral responses can

be induced by both high quality vocalizations and estradiol, although they are not

necessarily correlated.

31

Introduction

Song is an important signal for reproductive interactions between zebra finches
(Zann 1996); females use male songs to make mate choice decisions. Females are less
likely to pair with males that have been surgically altered to produce low quality song or
have a lower song output (Tomaszycki and Adkins-Regan 2005). They also show
preferences for particular song types, often indicated by increased time spent near a
vocalization (Neubauer I999; Lauay et al. 2004; Nolan and Hill 2004), and/or with
copulatory solicitation displays (Searcy and Marler 1981; Ballentine et al. 2004). In the
zebra finch, females prefer the songs of males that were raised with a male tutor and
produce a normal song, compared to those from males reared without tutors (Lauay et al.
2004), which are similar in pattern and duration to tutored song but contain fewer notes
and more variation in frequency, primarily due to reduced infusion of call notes with
stable frequencies into the song bout (Price 1979).

This preference may be the result of a selective response to songs of higher
quality in a number of neural systems. For example, immediate early genes are induced
within auditory perception regions including the caudomedial nidopallium (NCM) and
the caudomedial mesopallium (CMM) after exposure to song (Mello et al. 1992; Mello
and Clayton 1994). In juveniles, increased immediate early gene expression is observed
in these regions following exposure to conspecific versus heterospecific song (Bailey
and Wade 2003; Bailey and Wade 2005) and tutored versus untutored song (Tomaszycki
et al. 2006). Adult females also display an increased density of immediate early gene
immunoreactive nuclei with conspecific song versus heterospecific song (Bailey et al.

2002), but responses to varying qualities of zebra finch song have not been examined in

32

adult females. In other songbird species, however, immediate early gene expression is
induced in auditory perception regions by high quality songs that elicit a behavioral
preference in females (Gentner et al. 2001; Maney et al. 2003; Leitner et al. 2005;
Sockman et al. 2005).

Brain regions outside the auditory perception system, especially those important
for social behavior, are likely also affected by male song. Newman (1999) describes a
network of interconnected nuclei (including the medial amygdala, bed nucleus of the
stria terminalis, lateral septum, midbrain, ventromedial hypothalamus, anterior
hypothalamus, and medial preoptic area) that is responsive to social stimuli in a number
of species (also reviewed by Goodson et al. 2005 and discussed in Maney et al. 2008).
Under the influence of gonadal steroids, these areas mediate multiple social behaviors.
including reproduction and aggression (Newman 1999). The response of these regions
to male song stimuli has not been investigated in female zebra finches, but some areas,
including the ventromedial hypothalamus and nucleus taeniae (which shares homology
with a portion of the mammalian amygdale, Cheng et al. 1999), have been implicated in
the control of social behaviors in ring doves (Gibson and Cheng 1979; Cheng et al.
1999) and starlings (Gibson and Cheng 1979; Cheng et al. 1999). In addition, in other
avian species some regions within this network are responsive to auditory stimuli
(Cheng and Peng 1997; Cheng et al. 1998), including song (Maney et al. 2008).

The mesolimbic dopamine reward system may also play a role in the response of
female zebra finches to male song. It is conserved among mammals and birds
(Durstewitz et al. 1999), and is activated following exposure to rewarding stimuli. For

example, in female rodents, dopamine is released in this network following reproductive

33

interactions with males (Mermelstein and Becker 1995; Pfaus et al. 1995) and exposure
to male odors (Mitchell and Gratton 1994). In birds, when male starlings are producing
sexually motivated song, increased immediate early gene expression is observed within
the ventral tegmental area (Riters et al. 2004; Heimovics and Riters 2006). One study in
female starlings, however, indicates that the dopaminergic activity may have an
inhibitory effect, which is related to breeding condition (Riters et al. 2007). Breeding
females display decreased phosphorylated tyrosine hydroxylase immunoreactivity in the
ventromedial hypothalamus and lateral septum when they are exposed to song compared
to females exposed to no song. In contrast, non-breeding females displayed the opposite
pattern, with an increased immunoreactivity in these areas in response to song.

It seems likely that estradiol acts in some or all of these neural systems to
facilitate a reproductive response. All brain areas in the social behavior network express
estrogen receptors in zebra finches (Gahr et al. 1993; Jacobs et al. 1996; Gahr and
Metzdorf 1997). They are also found in parts of the ventral tegmental area (Maney et al.
2001) and in auditory perception regions (Gahr et al. 1993; Jacobs et al. 1996; Gahr and
Metzdorf 1997). In addition, high estradiol levels correlate with periods of reproductive
behavior such as nest building, egg laying, and incubation in female birds (Korenbrot et
al. 1974; Sockman and Schwabl 1999). In many song response studies, females are
treated with estradiol to facilitate reproductive behaviors (Searcy and Marler 1981;
Clayton and Prove 1989; Searcy and Capp 1997; Maney et al. 2003; Ballentine et al.
2004), but studies in canaries have indicated that this manipulation is unnecessary for the
induction of copulatory solicitation displays in response to song (Nagle et al. 1993;

Leboucher et al. 1994). Until now, the effect of estradiol on neural responses to song

34

has only been investigated in seasonal breeding birds; it increases immediate early gene
expression in the auditory perception regions in white crowned sparrows (Maney et al.
2006). The influence of estradiol in neural systems in an opportunistic breeder such as
the zebra finch is unknown. While hormones have not been measured across the
reproductive cycle in zebra finches, presumably fluctuations in estrogen are similar to
those in other avian species, which raises the question of how they alter the neural and
behavioral responses of these females.

In sum, solid data from a variety of sources provide intriguing information on
the neruroendocrine systems involved in perception of and responses to reproductively
relevant auditory cues in female songbirds. A complete picture is still lacking, however.
In an attempt to begin to synthesize and expand information about critical factors, we
used a behavioral assay and immunohistochemistry for the immediate early gene ZENK
in the same birds to simultaneously investigate potential roles of the auditory perception,
social behavior and reward systems in the preference for tutored versus untutored song,

as well as how the neural and behavioral responses may be mediated by estradiol.

Materials and Methods
Animals
Adult female zebra finches were taken from the breeding colony at Michigan
State University. The birds were kept on a 12: 12 light:dark cycle and provided seed and
water ad libitum, with hard-boiled eggs mixed with bread and either spinach or orange
given once a week. Females used in the study were raised in mixed sex breeding

aviaries for at least 100 days, to ensure that they had reached sexual maturity and were

35

exposed to normal song during development. After this period, they were removed and
housed in a single-sex aviary, which allowed auditory, but not visual or tactile contact
with males, for at least two weeks prior to testing. All procedures were approved by the
Michigan State University Animal Use and Care Committee and adhered to the
guidelines of the National Institutes of Health.
Hormone treatment

Estradiol implants were produced by packing 2 mm of 17B-estradiol into 5 mm
of Silastic tubing (i.d. 0.76, o.d. 1.65 mm) and sealing each end with silicone. Blank
capsules were left unpacked. Females were anesthetized with isoflurane and either an
estradiol or a blank capsule was inserted subcutaneously above the breast muscle. The
incision was sealed with collodion adhesive, and the female was placed in an individual
cage for 5 days to recover.
Song Stimuli

Recordings of tutored (males raised with other males present) and untutored
(males isolated from adult males from post-hatch day 18 after hatching) zebra finch
songs were received from Dr. Adkins-Regan at Cornell University (Lauay et a1. 2004).
Two types of sound files were produced. In one, both types of songs were used to
produce ten different 20-minute sound clips for behavioral testing. Each clip was
generated by combining one song of each type from the pool of six untutored and tutored
males into a stereo file. Although there were large differences between the tutored and
untutored songs, pairs were made of one tutored and one untutored song with similar
phrase lengths and relatively high similarity scores (which indicate note type, spectral

characteristics, duration, order, and time between notes; Tchemichovski et al. 1998),

36

determined by comparing phrases from each males song with the freeware Sound
Analysis Pro (httpzofersci.ccnv.cunv.edu/htm/bodv_soundfanalysis.html). Each song
was normalized for amplitude in Adobe Audition (Adobe Systems, Inc., San Jose, CA),
and following the introductory notes, it was repeated for a total of 20 minutes. A file
was then synthesized to simultaneously play a tutored song from one side of the testing
chamber (see below) and an untutored one from the other side, counterbalanced across
tests.

The other type of sound file was used for exposure prior to immediate early gene
analysis. In this case, twelve 30-minute sound clips (six tutored and six untutored) were
created in Adobe Audition. For each file, 30-second song clips from three different
randomly chosen males within the same group (tutored or untutored) were played
sequentially with 30 seconds of silence separating them. This compilation was repeated
for a total of 30 minutes.

Choice Test

Five days after implant surgery, females were taken to a room containing a wood
and plexiglass chamber of (215 cm L x 60 cm W x 60 cm H; Bailey 2006) modeled after
Miller 1979). It consisted of three zones: the center (with one perch), and the left and
right (with three perches each). The bird was placed into the cage through a center door
and allowed to freely move between the zones. After 70 minutes for acclimation, a
randomly chosen song file was broadcast from speakers, one located at each end of the
chamber, for 20 minutes at approximately 60dB. Behaviors were videotaped and the
time spent in each zone was quantified. In addition, two other behavioral measures

(jumps between perches and calls) were taken. To ensure that the females adequately

37

received the song stimulus, animals that were completely unresponsive during the song
presentation (i.e., did not move or call) were not used (20 individuals across the groups
were removed; leaving sample sizes of 20 estradiol-treated and 20 control birds).

Following song presentation, each female was captured and blood was collected
by wing vein puncture, centrifuged at 10,000 rpm for 10 minutes at 4°C and plasma
stored at -80°C until radioimmunoassay to quantify estradiol concentration. Females
were returned to their individual cages and taken back to the colony room.

Behavioral data were compared using a mixed-model ANOVA (treatment
between animals and time spent within each zone within animals). The proportion of
perch jumps and flights in the tutored zone were separately analyzed between treatment
groups using Mann-Whitney U tests, because some individuals did not display these
behaviors and the data were not normally distributed. Statview (SAS Institute; Carey,
NC) was used for all statistical analyses.

Immediate Early Gene Analysis

Two days after the choice test, the forty females were individually taken to a
novel room for stimulus exposure to examine ZENK induction (Bailey et a1. 2002,
Bailey and Wade 2003; Bailey and Wade 2005). They remained in their individual
cages during the test, which were placed within a sound-isolated box. After a 30-minute
acclimation period (as in Bailey et a1. 2002), a randomly chosen 30-minute file of
tutored or untutored song or silence was broadcast from a speaker three inches from the
cage at 60dB. Each group contained 6-8 individuals. In a few cases, a brain region

could not be quantified due to histological artifact; final sample sizes are indicated in the

38

figures and table. To reduce habituation (Chew et a1. 1995), no female was exposed to
song from the same male in the choice test and stimulus exposure for ZENK induction.

The song exposure was videotaped, and calls and receptive behaviors were later
quantified. Following song presentation, the female remained in silence for 1 hour
before being overdosed with 0.12 cc of equithesin and perfusion with 0.1 M phosphate
buffered saline (PBS) and 4% paraformaldehyde. Brains were dissected from the skull
and postfixed for 15 minutes in 4% paraformaldehyde. They were embedded in gelatin,
fixed for another hour in 4% paraformaldehyde and placed in 30% sucrose in 0.1M PBS
at -20°C until sectioning. The embedded tissue was sectioned frozen into four series at
30m in the sagittal plane and stored in cryoprotectant at -20°C until
immunohistochemical processing.

Immunohistochemistry was performed in a method adapted from Bailey et al.
(2002) and Bailey and Wade (2003; 2005). Cryoprotected tissue was rinsed 6x5 minutes
in PBS, reacted for 15 minutes in 0.5% hydrogen peroxide (H202) in PBS and blocked
for 1 hour in 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West
Grove, PA) in PBS with 0.3% Triton-X-100 (PBST). The tissue was then incubated in
primary antibody (Santa Cruz Biotech; catalog #sc-189, 0.1ug/ml) in PBST for two days
at 4°C. It was rinsed 3x5 minutes in PBS and incubated for one hour in Biotin-SP
conjugated donkey anti-rabbit secondary antibody (Jackson ImmunoResearch
Laboratories; 1:500 dilution) in PBST. After a 3x5 minute PBS rinse, tissue was
exposed to an ABC reagent (Elite kit, Vector Laboratories, Burlingame, CA) for 1 hour.

It was then rinsed 3x5 minutes in PBS and incubated in diarninobenzadine (Sigma, St.

39

Louis, M0; 0.5 mg/ml) plus 0.0075% H202 for 7 minutes. After PBS rinses, sections
were mounted, dehydrated, cleared in xylene and coverslipped with DPX_

ZENK immunoreactivity in the auditory regions (NCM and CMM), social
behavior areas (preoptic area [POA], ventromedial hypothalamus [VMH], bed nucleus
of the stria terminalis [BST], nucleus taeniae [TnA], ventrolateral subdivision of the
caudal lateral septum [LS c.vl.], and midbrain central gray [CGt]), and reward system
(nucleus accumbens [Ac] and the ventral tegmental area [VTA]) was assessed using an
Olympus BX60 microscope (Figures 1 and 2). The placement and size of the sampling
regions, which is detailed in the figure captions, was determined after an initial scan of
the tissue to locate the extent of labeling. Immunoreactive cells were hand-counted by
an observer blind to treatment and auditory stimulus within the sampling regions. The
NCM and CMM were analyzed within the same medial sections, which were identified
by the connection of the nidopallium to the rest of the telencephalon and the presence of
the Septopallio-mesencephalic tract (TSM). For the nucleus accumbens and ventral
tegmental area, alternate sections in some animals were subjected to tyrosine
hydroxylase immunohistochemistry to determine the correct placement of the sampling
region (Figure 2). The methods were the same as those described for ZENK
immunohistochemistry, but using tyrosine hydroxylase primary antibody (lmmunostar;
catalog # 22941, 1:10,000 dilution) and donkey anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA). As the precise border of the Ac is not
completely distinguished, the box was placed in the ventromedial portion of the medial

striatum in the most medial sections, as discussed in Reiner et al. (2004a).

40

Figure 1. Placement of sampling regions within auditory perception and social behavior
areas in sagittal sections. Panel A contains boxes for the caudomedial nidopallium
(NCM; 310um x A320um) and caudomedial mesopallium (CMM; 190um x 345 um),
panel B for the bed nucleus of the stria terminalis (BST; 190nm x 345 um) and
ventrolateral subdivision of the caudal lateral septum (LSc. vl; 200nm x 200um), panel
C for the nucleus taeniae (TnA; 200um x 320um), panel D for the ventromedial
hypothalamus (VMH; l90um x 290nm) and preoptic area (POA; 200um x 200um), and
panel E demonstrates for the midbrain central gray (GCt; 190um x 290nm). The
hippocampus (HP), tractus occipito-mesencephalicus (OM), tractus septopallio-
mesencephalicus (TSM), optic chiasm (OC), posterior commisure (CP), and cerebellum
(Cb) were used as landmarks for location of sections and placement of boxes. The more
rostral portion of each photo is towards the right edge. Scale bar (in panel D) = 500um.

41

 

42

 

Figure 2: Placement of sampling regions within the reward system. Panels A and C
contain sections subjected to tyrosine hydroxylase immunohistochemistry sliced in the
sagittal plane. Panels B and D contain ZENK labeling. Panel B displays the location of
the box for the nucleus accumbens (Ac; 190um x 305 um), and panel D displays the
ventral tegmental area (VTA; 210um x 368 pm). The more rostral portion of each photo
is towards the right edge. Scale bar = 300um.

43

The density of ZENK immunoreactive nuclei was determined by dividing the
average number of immunoreactive cells for each animal (at least three sections per
animal were analyzed for each brain region) by the area of the sampling box. Effects of
treatment and song exposure type on ZENK density were analyzed using a two-way
ANOVA followed by post-hoe Tukey-Kramer tests where appropriate.
Radioimmunoassay

A single radioimmunoassay was conducted in a manner adapted from Lovem
and Wade (2003). Parallelism was first demonstrated with recently collected zebra
finch plasma (data not shown). For the full assay, a mean of 3 1 .4 ul of plasma from
each animal was incubated with radioactive tracer [3 H] estradiol (70Ci/mmol; Perkin
Elmer, Boston, MA) at 4°C overnight. Steroids were then extracted twice with diethyl
ether, and samples were dried under nitrogen. They were resuspended in PBS and
stored overnight at 4°C. A competitive binding assay was c0mpleted in duplicate
samples and a serially diluted standard curve (0.98 pg to 250 pg estradiol) in triplicate,
by adding an estradiol antibody (NEG307H; Biogenesis, Kingston, NH) with [3H]
estradiol and incubating overnight at 4°C. Water blanks were added as controls (n=4)
and six aliquots of a known concentration of estradiol were used to determine intra-
assay precision. The next day, dextran-coated charcoal was added and centrifuged at
2200 rpm for 10 min. at 4°C in order to remove unbound tracer. The remaining sample
was combined with scintillation fluid (UltimaGold; Perkin and Elmer, Boston, MA) and
analyzed with a scintillation counter (Beckman 6500, Fullerton, CA). Estradiol levels
were calculated by standardizing samples for individual recovery and the volume

assayed and compared to the standard curve. The intra-assay coefficient of variance

44

was 6%. A Mann-Whitney U test was used to compare the estradiol and blank treated

groups.

Results
Behavior

Females spent significantly more time in the zone near the tutored song than that
neighboring the untutored song or in the center (F= 19.238, p< 0.01 ;Tukey Kramer,
both p< 0.05). However, estrogen did not affect this behavior (F= 0.561 , p= 0.56),
perch jumps (Mann—Whitney U, p= 0.371) or calls (Mann-Whitney U, p= 0.112; Figure
3).

During the ZENK induction, only one female displayed a tail quiver (associated
with receptivity; Zann 1996), so these data were not analyzed statistically. No
significant main effects of hormone, song type or interaction between them were
detected in calling (data not shown).

ZENK expression

The clearest effect of estradiol was detected in the VMH. A main effect of
treatment was observed, such that estradiol reduced the density of ZENK
immunoreactive (IR) nuclei (F = 8.44, p= 0.007; Figure 4). No significant main effects
of hormone or song type or interactions between them were observed in any of the other
seven brain regions with this type of analysis (Table 3). However, the pattern observed
in the auditory regions was striking. While the effect of stimulus exposure was not
statistically significant (NCM: F= 1.72, p= 0.194; CMM: F: 0.98, p=0.385), the mean

density of ZENK-IR nuclei in the NC M and CMM of the blank-treated birds that heard

45

tutored song was three times greater than those exposed to untutored song or silence
(Figure 5). In fact, this increase was observed only in the one group, and a trend for
estradiol treatment to reduce it existed in the CMM (F= 3.61, p= 0.066). A similar,

albeit weaker, pattern was observed in the NCM (F= 1.46, p= 0.236).

 

 

 

 

 

 

 

 

   

 

 

 

 

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E I Estrogen
E 12 -
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o
N
.5 8 ‘
5 6 -
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m 4 ‘
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O 20 20 20

Tutored zone Center Untutored zone

 

 

Figure 3. Total time spent within the tutored, center and untutored zones in the
behavioral test. Sample sizes are indicated on the graph. * = tutored significantly
greater than untutored and center zones.

46

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Figure 4. ZENK-IR nuclei in the ventromedial hypothalamus of female zebra finches.
Panel A displays the quantification of densities of ZENK-IR for each treatment group
and song exposure type. Sample sizes are indicated on the graph. A main effect of
treatment was detected (*); estradiol decreased the expression of ZENK. Panels B and
C depict ZENK in the ventromedial hypothalamus of birds exposed to tutored song.
Panel B = blank treated female, Panel C = estrogen treated female. Scale bar = 100 um

47

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48

Table 3. Mean densities of ZENK-IR in various brain regions presented as number of
IR nuclei/mm2 (meanistandard error). Due to histological artifact, only 7 animals were

analyzed in the Ac in the blank, untutored group. No main effects or interactions were
detected, all F< 3.02, p> 0.06.

 

 

 

 

 

 

 

 

 

 

 

Blank-Treated Estradiol-Treated

Tutored Untutored Silence Tutored Untutored Silence

(n=6) (n=7 or 8) (n=6) @= 6) (n= 6) (n=6)
POA 414.5d:1 17 500.2i 1 26 591.0:t99 467.0i168 279.7i81 389.7:t89
BST 1 05601255 945.3i226 l 106.5:t254 494.8:1:187 9356:5338 694.4:12352
TnA 125.0:t73 67.3i26 30.5i1 1 52.8i23 91.9:t46 216.4:t215
L3“ 591.0i19l 376.621: 1 46 434.2i164 120.8i58 32361207 256.9i254
GCt 61 33:32 54.8i26 132.5i54 23.4i1 l 83.7i45 39.1i19

Ac 552.8i252 837.2:t252 1286.7i224 88841280 1049.6:t223 558.8:tl35

VTA 61.8:t20 52.2:t17 74.2:t30 54.0i40 67.3i25 13.3:1:7

 

 

 

 

 

 

 

Abbreviations: preoptic area (POA), bed nucleus of the stria terminalis (BST), nucleus
taeniae (TnA), ventrolateral subdivision of the caudal lateral septum (LSc. v1), GCt
(midbrain central gray), nucleus accumbens (Ac), ventral tegmental area (VTA)

49

 

Plasma Estradiol

Estrogen treatment reliably increased circulating estradiol levels (Mann-Whitney U:
73, p= 0.002). Estradiol-treated birds had a mean of 5.7 ng/mli1.3 S.E., while the value
for control birds was 2.2 ng/m1i0.46 S.E. However, a number of the samples from
blank-treated females fell below the range of the sensitivity of the assay, so to be
conservative, the minimum detectable value (15.63 pg/sample; equivalent to an average

of 1.84 ng/ml, depending on sample volume and recovery) was assigned.

Discussion
Mavioral Responfl

Female zebra finches spent more time near the tutored song than untutored song
or silence. Lauay et al. (2004) also detected a similar propensity for increased time
spent near tutored over untutored song in female zebra finches, although they did not
examine the effects of estrogen or the neural response to these two types of song.

The tendency to spend increased time nearer this type of stimulus may be related
to reproduction. Females prefer songs with characteristics reflecting increased male
energy input (i.e., long songs, increased song rate, and difficult syllables; Neubauer
1999; Ballentine et al. 2004; Nolan and Hill 2004). In addition, some of these
characteristics correlate with high paternal effort (nestling feeding; Buchanan and
Catchpole 2000; Dolby et a1. 2005) and offspring survival (Hasselquist et al. 1996).
Low song quality may also indicate developmental stress, and as a result, lower the
fitness of a potential mate (N owicki et al. 2002). It therefore seems reasonable to

hypothesize that females spend more time near tutored song because it indicates a

50

higher quality mate than untutored song. It is possible, however, that the females
simply find the untutored song aversive.

The widespread nature of song preference behavior might suggest that its
function is broader than reproduction and mate choice. For example, male birds from a
variety of species prefer song from conspecifics (Calhoun et al. 1993; Braaten and
Reynolds 1999) and their own fathers (Clayton 1988; Riebel et al. 2002; Riebel and
Smallegange 2003). In addition, juvenile zebra finches prefer song from their tutor
(Houx and ten Cate 1999; 1999) prior to the period when they become reproductively
active. The lack of an effect of estradiol on the behavioral response is also consistent
with the idea that the tendency to spend time with a specific song type is dissociated
from mate choice and reproduction in the female zebra finch. Alternatively, it is
possible that the hormone is required to display this behavioral preference, and the
concentration of circulating estradiol in control females was above the threshold

(similar to untreated canaries; Nagle et al. 1993; Leboucher et a1. 1994).

Neural Response

Auditory regions.

Statistically significant differences in ZENK expression due to auditory stimulus
were not detected in the NCM or CMM. However, in control but not estradiol-treated
animals, the average density of these cells was approximately three times greater in
birds exposed to tutored song than the other two stimuli. The effect of estradiol on
ZENK expression in these areas also did not reach statistical significance, and no

interaction was detected between the hormone and auditory stimulus type. One

51

therefore needs to be very careful in suggesting conclusions about potential effects of
the hormone. However, a number of factors suggest that potential biological relevance
might be considered. For example, specific large mean differences in ZENK expression
were seen in just the auditory areas of control birds exposed to high quality song
compared to both low-quality song and silence (see Figure 5). This pattern parallels
previous work in zebra finches in which immediate early gene expression in auditory
regions was increased following exposure to stimuli of greater relevance (Mello et al.
1992; Bailey et a1. 2002; Bailey and Wade 2003; Bailey and Wade 2005; Tomaszycki et
a1. 2006). If a mixed-model 3-way ANOVA is used to consider patterns across brain
regions, a song x treatment x region interaction is detected. This interaction partly
stems from the fact that NCM and CMM are the only regions to show this dramatic
difference between tutored and untutored song and silence and that this effect is only
observed in blank treated individuals. Thus, it is likely that estrogen inhibits this
selective ZENK response in these regions. However, the lack of a significant main
effect of song type in the auditory regions in the present study may be the result of the
reduced requirement to detect variations in song in adulthood in this species. The other
species in which this phenomenon has been investigated are primarily seasonal
breeders, in which the females also sing (Baptista and Petrinovich 1986; Vallet et al.
1996; Pavlova et a1. 2005) and/or male song is variable in adulthood (Smith et a1. 1995;
Riters et a1. 2000; Leitner et a1. 2001). Zebra finches, in contrast, are aseasonal, the
females do not sing, and male zebra song is stable in adulthood (Zann 1996). A
difference in ZENK response to tutored and untutored song was observed in juvenile

female zebra finches (Tomaszycki et al. 2006). However, this plasticity may be lost

52

once females have formed a template and learned the basic characteristics of species-
appropriate song.

From a more technical perspective, we cannot completely eliminate the
possibility that the modest differences in exposure protocols during the behavior test (in
which they were exposed to both song types simultaneously in a large choice cage) and
ZENK induction (in which they were only exposed to one auditory stimulus in a sound
isolated box) affected the animals somewhat differently, such that they showed a
behavioral preference for tutored song, but not increased ZENK expression. However,
in the ZENK induction test they remained within their home cage, which fit inside the
sound-isolated box with the interest of reducing stress from a novel environment.

Regardless of the results in blank treated animals, it is clear that when our
females were treated with estradiol, the response to all auditory stimuli was nearly
identical. This result differs from the findings of Maney et a1. (2006; 2008), in which
estrogen treatment resulted in a greater immediate early gene response to conspecific
song compared to tones. In the present study, however, we examined the difference in
ZENK expression to variations in quality ofsong, whereas Maney et al. (2006; 2008)
compared normal song to a tone stimulus. It is unknown how estradiol may mediate
neural responses in auditory perception sites, but it is plausible that it acts on different
signaling mechanisms. That is, separate pathways might (I) provide information that an
auditory signal is an approximation of song, and (2) assess particular features (i. e.,
relative quality) of the song. If so, it is also possible that estradiol facilitates a neural
response in one case and inhibits it in the other. Some estrogen receptors are located

along the caudal edge of the NCM, as well as within the CMM (Gahr et al. 1993; Jacobs

53

et al. 1996; Gahr and Metzdorf 1997), so regulation may occur directly at these sites or
through connections with other brain regions. LeBlanc et al. (2007) observed an
increase in density of tyrosine hydroxylase immunoreactivity in the NCM following
estrogen treatment, and predicted that estradiol plays a neuromodulatory role on
responses within the auditory perception regions. It will be important to further
investigate all of these ideas in future experiments.

Social Behavior Network.

Although estradiol did not alter behavior in the present study, it did affect the
neural response specifically in the VMH. This region contains a high concentration of
estrogen receptors in a wide range of vertebrates, including rodents (e.g., Pfaff and
Keiner 1973; Vito et al. 1983; Koch and Ehret 1989; Simerly et al. 1990; DonCarlos et
al. 1991; Lauber et a1. 1991; Shughrue et al. 1997) and birds (Balthazart et a1. 1989;
Metzdorf et a1. 1999; Halldin et al. 2006), including zebra finches (Gahr et al. 1993;
Jacobs et al. 1996; Gahr and Metzdorf 1997). In addition, the VMH plays a crucial role
in the control of reproduction in female rodents (Pfaff et a1. 1994) and also appears to
be important in birds (Gibson and Cheng 1979). However, rather than an increase in
immediate early gene expression as has been observed in rodents following exposure to
reproductive stimuli (Pfaus and Heeb 1997), we detected less ZENK-IR in the VMH
with hormone administration. Treatment with estradiol alone can result in increases in
immediate early gene expression (Cattaneo and Maggi 1990; Insel 1990), but similar to
the present data, decreases have also been documented. For example, Tetel et al. (1994)
and Pfaus et al. (1996) observed decreases in the induction of FOS-IR following small

amounts of vaginocervical stimulation, in the VMH when female rats were treated with

54

estradiol. In addition, a decrease in immediate early gene expression in the preoptic
area and amygdala occurs following testosterone administration in male anole lizards
(Neal and Wade 2007).

At least two potential explanations for this estradiol-induced decrease in
immediate early gene expression exist. First, although these genes are often used to
indicate regions that are “activated” by a certain stimulus, expression may also indicate
stimulation of inhibitory neurons such as those expressing GABA. In male gerbils, a
large proportion of cells expressing immediate early genes following mating are
GABAergic (Simmons and Yahr 2003). Estradiol treatment can result in a decrease in
bound GABAA receptor (O'Connor et al. 1988) and responsiveness of GABAB receptors
(Lagrange et al. 1996) possibly resulting in disinhibition. Conversely, estrogen can
cause an increase in GABA levels in the VMH of female rats (Luine et al. 1997). It is
possible that estradiol reduces the activity of GABA neurons in the VMH in the present
study, which is indicated by a decrease in ZENK expression. In zebra finches, song
exposure results in GABA neurons expressing ZENK within the auditory perception
regions (Pinaud et al. 2004; Pinaud and Mello 2007). As GABA neurons are present in
female birds (Domenici et al. 1988; Granda and Crossland 1989), and can be regulated
by social stimuli, as in rodents, it is possible that they may also be affected by estradiol
in zebra finches. Second, it is possible that the decrease in ZENK expression observed
in this study may be partially due to a decrease in activation of dopaminergic neurons in
the VMH as described in Riters et al. (2007). In that study, decreased levels of
phosphorylated tyrosine hydroxylase were observed following exposure to song in only

breeding season female starlings. The authors hypothesize that these neurons may serve

55

an inhibitory function within the VMH during reproductive behavior (Riters et al.
2007)

Maney et al. (2008) conducted a study similar to ours in white-crowned
sparrows; the response to song and how it is modulated by estradiol was examined in
the social behavior network. In that study, increased ZENK responses were observed
within the TnA, VMH, EST, and LSc. v1, and estrogen enhanced this response in all the
examined social behavior network regions, except the medial VMH and the anterior
medial hypothalamus. We did not observe changes in the responses of these regions to
song stimuli, and we did not observe modulation of the response to song in these
regions by estrogen. There are multiple potential explanations for these discrepancies.
For example, housing conditions differed. Our animals were maintained in a colony
room with hundreds of zebra finches, whereas the white crowned females were housed
with several other females in sound-isolated booths for the extent of the study in Maney
et al. (2008). Perhaps more important, however, are biological differences between
zebra finches and white-crowned sparrows that may lead to differences in their
responses within this network to song. Female zebra finches do not sing and adult
males produce a simple and stable song in adulthood (Zann 1996). White-crowned
sparrows, in contrast, have several song dialects (Marler and Tamura 1964) and females
can also produce song (Kern and King 1972). It is possible that if female white-
crowned sparrows produce song when hearing the song stimulus, it could affect the
response of regions in the social behavior network. Some of these regions are activated
in males when they sing (i. e. Heimovics and Riters 2006). In addition, differences in

neural responses may reflect song complexity. For example, because male zebra finch

56

song is so simple and stable, and females do not sing, female zebra finches may not
need to utilize these regions to interpret the meaning of the song stimulus, whereas birds
such as white-crowned sparrows that have more variable song that can be used in more
contexts might require additional regions for processing. Similarly, female zebra
finches may require additional stimulation to activate these regions. Song might be
interpreted by only the auditory perception regions, and other areas of the brain may
only be activated when other stimuli are present, such as courtship displays (visual
input) or physical bonding (tactile input). Differing effects of estrogen between the
species might relate to varying levels of reliance on the hormone for reproduction in
seasonal (white-crowned sparrows) and opportunistic (zebra finches) breeders.
Determining which, if any, of these possibilities led to the differences in ZENK
response between the present study and that of Maney et al. (2008) on white-crowned
sparrows could lead to insights on the specific roles of these brain regions.
Dissociation between behavior and immediate early gene expression

Dissociations between behavior and ZENK expression were observed on two
levels. First, although the females displayed a strong propensity to spend time near the
tutored song, no differences in ZENK expression between auditory stimuli were
observed in the social behavior network or reward system, and more work must be done
on the auditory perception regions before solid conclusions can be drawn. Second, the
reduction of ZENK expression by estrogen in the VMH with the lack of an effect of the
hormone on behavior in the choice test suggests that an increased ZENK response
within the VMH is not necessary for the display of differential behavioral responses to

high quality song.

57

These data suggest a number of possibilities. For example, another immediate
early gene may be involved in the regulation of the behavior. Consistent with that idea,
some differences between the activities of F OS and ZENK have been detected in
juvenile zebra finches (Bailey and Wade 2003), although similar patterns of induction
of the two immediate early genes have also been observed following song presentation
to adult female songbirds (Sockman et al. 2005; Velho et al. 2005). Additionally,
several other immediate early genes (such as c-jun) exist that may also be important to
the changes that occur during the display of this behavior. It is also possible that some
other neural system (in addition to the three networks examined here) is vital to
mediation of this behavior.

Conclusion and Future Directions

In summary, estrogen treatment does not affect the tendency to spend time near
high quality, tutored song as measured in the present study, but it does suppress ZENK
induction in the VMH. The present data suggest that the maintenance of this behavior
does not require higher concentrations of estradiol and may not be associated with the
ZENK expression detected within the examined brain regions. In addition, the fact that
estrogen’s inhibition of the neural response is relatively specific, and does not occur in
most of the brain regions investigated, suggests that it serves some function, although
the nature of it is not clear at this point. Determining the phenotype of the affected cells

will help to elucidate their function(s).

58

Acknowledgements
This research was funded by the National Institutes of Health (R01-MH55488
and K02-MH65907). We thank Elizabeth Adkins-Regan for providing us with the
tutored and untutored song files, Dave Bailey for assistance with the behavioral testing
procedure, Yu Ping Tang for advice with immunohistochemistry, Stephany Latham for

technical assistance, and Katie Licht for coding the song preference tests.

59

CHAPTER 3: ESTRADIOL AND SONG PRESENTATION MEDIATE BEHAVIOR
OF FEMALE ZEBRA FINCHES INDEPENDENT OF DOPAMIN E ACTIVITY IN
THE NUCLEUS ACCUMBENS AND MEDIAL STRIATUM
Abstract

Female songbirds display preferences for certain song characteristics, but the
neural and hormonal mechanisms mediating these preferences are not fully clear. The
present study sought to further explore the role of estradiol, as well as assess potential
roles of dopaminergic systems, on behavioral responses to song. Adult female zebra
finches were treated with estradiol and exposed to tutored or untutored song or silence.
Behavior was quantified and neurochemistry of the nucleus accumbens and striatum
was examined with high performance liquid chromatography. As a control, the
responses of these two systems to treatment with raclopride, a specific D2 receptor
antagonist, were also evaluated. This manipulation did not affect dopamine (DA), but
did increase DOPAC and the DOPAC/DA ratio. Estradiol reduced the display of two
behaviors, long distance calls and visual scanning, but had no effect on dopaminergic
responses. Auditory stimulus exposure affected other vocalizations, but song
presentation did not modulate the levels of DA or its metabolite, DOPAC in the nucleus
accumbens or striatum. Collectively, the results suggest that both estradiol and auditory
stimuli can modify the behavioral responses of adult zebra finches, but they do so
independent of changes in DA concentration and turnover. Thus, these two
dopaminergic regions may not mediate the preference for high versus low quality song

in this species.

60

Introduction

Female songbirds display preferences spending time near song with certain
characteristics, and perhaps as a consequence, vary their behavioral responses to them.
However, the neural mechanisms involved in these processes are not completely clear.
Increased immediate early gene expression in response to male songs of higher quality
are regularly seen in the auditory perception regions in a variety of songbird species
(6. g. Gentner et al. 2001; Bailey et al. 2002; Maney et al. 2003; Leitner et al. 2005;
Sockman et al. 2005; Tomaszycki et al. 2006; Svec and Wade 2009). Information on
responses of other neural regions in songbirds is more limited and not as consistent. For
example, results from the social behavior network (a group of interconnected nuclei that
are implicated in the mediation of social behaviors and sensitive to steroid hormones
(Newman 1999) differ across species. In white-throated sparrows, increased immediate
early gene expression in response to song was observed in the nucleus taeniae, bed
nucleus of the stria terminalis, lateral ventromedial hypothalamus, and lateral septum
(Maney et al. 2008), whereas in zebra finches none of these regions were selectively
responsive to high or low quality song (Svec and Wade 2009).

In the mesolimbic dopamine (DA) system, exposure to song does not induce
immediate early gene expression in zebra finches (Svec and Wade 2009) or white-
throated sparrows (LeBlanc et a1. 2007; Maney et al. 2008), but these results do not
eliminate the possibility that the regions respond to song stimuli. Quantifying the
concentrations of DA and its metabolites might provide more direct assessments of their
responses. This type of involvement is plausible, as song is a critical element of

courtship (Zann 1996), and the mesolimbic reward system is activated in mammals

61

during reproduction in both sexes (reviewed in Mitchell and Gratton 1994; Melis and
Argiolas 1995; Paredes and Agmo 2004), as well as in response to reproductive stimuli
alone (Mitchell and Gratton 1994; Fabre-Nys et al. 1997).

The anatomical and physiological characteristics of the dopaminergic systems
are highly similar in birds and mammals (reviewed in Durstewitz et al. 1999; Reiner et
al. 2004a). In birds, dopaminergic neurons from the substantia nigra and ventral
tegmental area project to the basal ganglia, including the medial striatum (nigrostriatal)
and the nucleus accumbens (mesolimbic; Szekely et a1. 1994; Metzger et al. 1996;
Durstewitz et a1. 1999). In vitro, neurons from the ventral tegmental area and substantia
nigra have similar electrophysiological characteristics to mammalian dopaminergic
neurons, and D2 receptor agonists block their activity (Gale and Perkel 2006). In
addition, in chickens and zebra finches release of DA in striatal slices decreases with
D2 receptor agonist treatment (Gale and Perkel 2005; Jackson et al. 2007). Few avian
studies, however, have simultaneously examined the responses of the nigrostriatal and
mesolimbic dopaminergic systems.

In addition to physiological characteristics, functional similarities exist in these
systems between mammals and birds (see Reiner et al. 1998 for review). In both
groups, the regions are important to the control of motor movements (see Rieke 1981;
Sanberg and Mark 1983; Albin et al. 1989; Hauber 1998), as well as the mediation of
reward (see Schultz et al. 1997; Akins et al. 2004; Esch and Stefano 2004; Akins and
Geary 2008). In particular, these two systems are activated when males sing sexually

motivated song (measured with immediate early gene expression in mesolimbic reward

62

regions; Riters et al. 2004; Heimovics and Riters 2005) and microdialysis in the
striatum (Sasaki et al. 2006).

The response of neurons in the mesolimbic DA system to song exposure has
only been examined using immediate early genes, which are either expressed to a
greater degree in response to song (Maney et al. 2008) or are unchanged (LeBlanc et al.
2007; Svec and Wade 2009) in these regions. In components of the social behavior
network, Riters et al. (2007) observed increased phosphorylated tyrosine hydroxylase
(the rate limiting enzyme in DA synthesis) in response to male starling song. Also, DA
release is increased in the auditory perception system (caudomedial nidopallium) in
female starlings exposed to song (Sockman and Salvante 2008). It remains to be seen if
exposure to song stimuli results in increased dopaminergic activity in the striatum and
nucleus accumbens of female songbirds.

Although song is a critical component of reproduction, and estrogen is often
used to prime females in song response studies (i. e. Searcy and Marler 1981; Clayton
and Prove 1989; Searcy and Capp 1997; Maney et al. 2003; Ballentine et al. 2004), the
role of steroid hormones in neural responses to song has only recently been evaluated.
The effects are not completely consistent (see LeBlanc et al. 2007; Riters et al. 2007;
Maney et al. 2008; Svec and Wade 2009). Zebra finch females present more distance
calls in response to complex song versus other song types when they are estrogen-
treated, but not control-treated (Vyas et al. 2008; 2009). In our previous study in zebra
finches (Svec and Wade 2009), estradiol reduced expression of the immediate early
gene ZENK in the ventromedial hypothalamus but did not affect neural responses to

song, behavioral preference for tutored versus untutored male song, or general calling

63

behavior in response to these auditory stimuli. In contrast, a selective increase in ZENK
expression in response to male song was observed following estrogen treatment in
several regions in the social behavior network in white-throated sparrows (Maney et al.
2008)

Estrogen may also affect the response of dopaminergic systems. The ventral
tegmental area contains estrogen receptors (LeBlanc et al. 2007). In female starlings,
the activity of dopaminergic neurons in response to male song in the ventral tegmental
area, as well as regions in the social behavior network, is affected by breeding condition
(Riters et al. 2007). These results suggest potential for dopaminergic systems to be
modified by estradiol.

The present experiments had two primary goals. First, we examined the specific
behavioral responses of female zebra finches to tutored versus untutored song, and in
particular how they might be affected by estradiol treatment. Second, we characterized
the response of the nucleus accumbens and striatum of estrogen-treated and control
females to song stimuli using high performance liquid chromatography (HPLC) to
measure the levels of DA and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC).
Prior to this main experiment, a separate group of adult females was used to verify the
technique. For this, we determined that predictable changes in dopaminergic neural
activity after raclopride treatment are reflected in changes in DA metabolism in the

nucleus accumbens and striatum.

Methods

Animals

64

Adult female zebra finches were raised in mixed-sex aviaries in our breeding
colony at Michigan State University. They were kept on a 12:12 light:dark cycle, and
seed and water were provided ad libitum. Orange and spinach as well as a hard-boiled
egg/bread mixture were given once per week. After reaching adulthood (at least 100
days after hatching), female birds were housed in a unisex aviary, in acoustic (but not
visual) contact with adult males for at least three weeks prior to testing. All procedures
were approved by the Michigan State IACUC and adhered to the guidelines of the
National Institutes of Health.

Raclopride treatment

Females were removed from the all-female aviary, taken to a separate room in
individual cages, and given an intramuscular injection of either 1mg/kg raclopride (1
mg/kg; Sigma-Aldrich Corp., St. Louis, MO) or a vehicle (lml/kg). They remained in
cages in the room for 70 min., after which they were rapidly decapitated and brain
tissue was collected, flash—frozen, and stored at -80°C until processing.

Hormone treatment

Estradiol treatment was conducted as in Svec and Wade (2009). Briefly,
females were implanted subcutaneously with 5 mm long Silastic capsules (i.d. 0.076
mm, o.d. 1.65 mm) under isoflurane anesthesia. Implants were made from Silastic
tubing, which was packed with 2 mm of 17B-estradiol and sealed with silicone.
Implants of this size result in hormone levels slightly above the natural range of adult
female zebra finches (Adkins-Regan et al. 1990; Svec and Wade 2009). Females were
not gonadectomized, as ovariectomy in this species results in an increased level of

circulating estradiol (Adkins-Regan et al. 1990). Control females received empty

65

implants. Females were housed in individual cages in a room with males and females in
group aviaries for 5 days following the surgery.
Song Stimuli

The same auditory stimuli were used as in Svec and Wade (2009), recorded by
Dr. Adkins-Regan at Cornell University (also used in Lauay et al. 2004). They came
from males that had been tutored (raised with adult males present) or untutored (raised
in the absence of other males after 18 days of age) during development. Twelve 30-min
song clips (six tutored and six untutored) were created in Adobe Audition (Adobe
Systems Inc., San Jose, CA), each containing a unique combination of 30 sec of song
from three randomly chosen males in each category that were separated by 30 sec of
silence and repeated for 30 min.
Auditogy stimulus exposure

Five days after implant surgery, females were taken to a novel room for auditory
stimulus exposure. The procedure was conducted as described in Svec and Wade
(2009), except that tissue was collected 10 min rather than 1 hour after the conclusion of
the song presentation. Briefly, females were placed in a sound isolated box in their
individual cages, allowed 30 min for acclimation, and then exposed to 30 min of one of
the two types of song. A speaker with a taxidermic model of an adult male zebra finch
on top broadcast the song at 60 dB three inches from the cage. As a control, some birds
were not exposed to song and remained in silence during the test. The stimulus
exposure was videotaped, and behaviors displayed were quantified by an observer blind

to treatment condition (see Table 4 for descriptions of behaviors, largely generated from

66

Zann (1996). Brain tissue was collected following rapid decapitation, flash frozen, and

stored at -80 °C until processing.

Table 4. Descriptions of measured behaviors

Behavior

Distance calls

‘Other calls’

Duration of
movement

Flights/J umps

Beak wipes

Allopreening

Visual Scanning

Beak open

Tail quiver

Description

Long, loud sounds emitted by the female, often
presented after song begins and shortly after song
stops, often used for localizing other individuals

Shorter calls, at a lower volume, including both
5tets’ and ‘stacks’, presented frequently throughout
the test in many cases

The amount of time an individual bird was moving
throughout the cage, excluding time in which the
bird was sitting still either on the perch or on the
ground for more than 10 seconds

Movement to or from the perch or up to the sides
of the cage

Rhythmic movement of the beak back and forth
along the perch

Cleaning feathers with beak

Turning head to one side, including craning the
neck and/or moving one of the eyes in the
direction of the speaker and bird model

Top and bottom beak are separated slightly for
more than 2 seconds

Rapid movement of the tail, often associated with
a bowed position on the perch

67

 

 

Figure 6. Examples of microdissections taken from thionin-stained coronal sections of
the zebra finch brain. Panel A depicts punches of (1) the nucleus accumbens and (2)
rostral medial striatum (both 21-gauge). Panel B depicts punches from the caudal
medial striatum (3; 18-gauge). LV= lateral ventricle, LPS= Lamina pallio-subpallialis.
Scale bar = 1 mm.

68

Quantification of neurochemicals

Tissue from both the preliminary study including raclopride-treated birds and
those in the main experiment that were exposed to the auditory stimuli was coronally
sectioned frozen (500 pm thick). Samples of the nucleus accumbens, rostral medial
striatum and caudal medial striatum were microdissected from these sections using the
Palkovits method (Palkovits 1973). A 21-gauge micropunch was utilized for the
nucleus accumbens and rostral medial striatum, and an 18-gauge punch was used for the
caudal medial striatum (Figure 6). Punches were placed in 0.1 M phosphate-citric buffer
in 20% methanol (pH 2.5; 50 pl for nucleus accumbens and rostral medial striatum, and
100 [.11 for caudal medial striatum) and stored at -20 °C.

Samples were thawed, sonicated with three l-sec bursts (Sonicator Cell
Disruptor, Heat Systems-Ultrasonic, Plainview, NY, USA) and centrifuged (12000 rpm)
for 1 min. The supernatant was removed and stored at -20 °C. HPLC coupled with ’
electrochemical detection was conducted to determine the levels of DA and DOPAC as
described in Lindley et a1. (1990) and Behrouz et al. (2007). Briefly, the supernatant
was injected into a C-18 reverse phase analytical column and using a mobile phase of
1.0M phosphate citrate buffer with 0.1 M EDTA, 0.35% sodium octylsulfate and 20%
methanol. DA and DOPAC content were determined by comparing the heights of peaks
generated by a Hewlett Packard Integrator (Model 3393A) with those generated the
same day with standards. To determine the protein content, the pellet was dissolved in
l N NaOH, sonicated, and assayed using the method developed by Lowry et al. (1951).
Briefly, samples were compared to a standard curve serially diluted from 12.5 ug to 50

ug of protein. Samples and the standard curve were reacted with a reagent of sodium

69

carbonate, cupric sulfate, and KNA tartrate for 10 min and then reacted with Folin
reagent (phenol in ddH20) for 30 min. Absorbance was measured with a microplate
reader (MicroQuant, Biotek Instruments, Winooski, VT).

To control for variation in size of each sample, concentrations of DA and
DOPAC were determined by dividing the absolute quantity of each neurochemical by
the protein content within the sample. The DOPAC/DA ratio was also calculated as a
measure of the ratio of metabolized to stored DA. No differences in the values were
observed between the two portions of the striatum, so they were pooled to form a total
striatum value.

Statistics

Two-way ANOVAs were conducted to examine effects of estrogen and auditory
stimulus type on behaviors. These were followed by Tukey-Kramer post- hoc tests for
pairwise comparisons as appropriate. The HPLC results were analyzed with mixed-
model ANOVAs to compare DA and DOPAC concentrations and the DOPAC/DA ratio
across treatments and song exposure (between animals) and brain regions (within
animals) for both the raclopride and song exposure studies. All statistical analyses were
conducted using StatView (SAS Institute; Carey, NC). Sample sizes for each group are

indicated in the legends to Figures 7-9 and Table 5.

Results

Estrogen and Song Exposure - Behavior
A main effect of treatment was observed, such that estradiol decreased the

frequency of long distance calls (F= 5.12, p= 0.031) and visual scanning (F= 6.26, p=

0.018), regardless of type of stimulus exposure (Figure 7, data pooled across the groups

70

of auditory stimuli since they did not differ). In addition, a main effect of auditory
stimulus was observed in the frequency of ‘other calls’ (F= 5.93, p= 0.006); females
produced more of them when exposed to untutored song than silence (Tukey Kramer,
p< 0.05; Figure 8). No main effects of song or treatment, or interactions between these
variables, were observed for any of the other behaviors (all F< 3.99, p> 0.055).
Raclopride
A main effect of treatment was observed in the concentration of DOPAC (F= 23.35, p=
0.001) and the DOPAC/DA ratio (F= 25.95, p= 0.001); raclopride increased both
compared to the control manipulation (Figure 9). An interaction between brain region
and treatment indicated that the effect of raclopride on DOPAC concentration was
greater in the striatum than in the nucleus accumbens (F= 10.14, p= 0.008). In addition,
the concentrations of DOPAC and DA were higher in the striatum than in the nucleus
accumbens (F= 39.70, p< 0.001; F: 40.48, p< 0.001, respectively). As expected, no
effect of raclopride treatment on DA concentration was observed (F= 2.43, p= 0.145).
Estrogen and Song Exposure - Neurochemistry

A main effect of brain region was observed such that concentrations of DA (F =
250.314, p< 0.001) and DOPAC (F= 82.24, p<0.001) were greater in the striatum than
in the nucleus accumbens. In addition, the DOPAC/DA ratio was higher in nucleus
accumbens than in the striatum (F= 58.69, p< 0.001). A trend for an interaction among
treatment and song type was detected in concentration of DA (F= 03.26, p= 0.051), but
one-way ANOVAs between treatments or among song exposure groups revealed no
significant differences (all F< 4.44, p> 0.059). No other effects of song or treatment on

neurochemistry approached statistical significance (all F< 1.92, p> 0.16; Table 5).

71

 

 

I Estrogen
60 1 I [:1 Blank

 

40-

Number of Distance Calls

17

 

 

 

 

 

 

 

 

A
N
———t

 

on

A

Frequency of
visual scanning

 

 

 

 

 

17
Estrogen Blank

 

 

 

 

Figure 7. Effects of estradiol on long distance calls (top) and visual seaming behavior
(bottom). Data (meaniS.E.) were pooled across song exposure groups, as the effects of
the auditory stimuli did not differ among them. Sample sizes: Estradiol-treated tutored
song = 6, estradiol-treated untutored song = 8, estradiol-treated silence = 7, blank-

treated tutored song = 5, blank-treated untutored song = 6, blank-treated silence = 6. *
p< 0.031.

72

 

 

I Tutored Song

0)

O

O
X-

  

 

 

g I Untutored Song
(U
S [:1 Silence
2 400
O
“5
g 200
E
3
z

Tutored Untutored Silence

song song

 

 

 

Figure 8. Effect of auditory stimulus on ‘other calls’ (meaniS.E.). Other calls include
“tets” and “stacks”, which are shorter calls presented throughout the test. Data were
pooled across treatment groups, as estradiol did not affect the number of these
vocalizations. Sample sizes: Estradiol-treated tutored song = 6, estradiol-treated
untutored song = 8, estradiol-treated silence = 7, blank-treated tutored song = 5, blank-
treated untutored song = 6, blank-treated silence = 6. * p= 0.006

73

 

 

80

 

 

 

 

 

 

 

 

 

 

 

 

 

5 * I Raclopride
"é A
E .25, 60- 1:] Water
8 §
8 O.
o g’ 40
0 B:
E 5
o 20-
o

0-
c 200
.9
(U
3: A 160-

0

g a 120‘
0 a)
0 E 80-
: \
.E g
g 40-
o

O
(I)
.E
E
(U
8.9
o 16
G o:
E
O
o

Nucleus Striatum
Accumbens

 

 

 

Figure 9. Effects of raclopride treatment (meaniS.E.) on the concentration of DOPAC
(top) and DA (middle), and the DOPAC/DA ratio (bottom). Sample sizes are indicated
at the bottom of each bar; * p< 0.008.

74

Table 5. Concentrations of DOPAC, Dopamine and the DOPAC/DA ratio in the
nucleus accumbens and striatum following song presentation and estrogen
treatment. No main effects or interactions were detected, all F < 1.92, p> 0.16.

 

 

 

 

 

 

. DOPAC Dopamine DOPAC/
Bram . .
Region Treatment Song concentration concentration DA
(ng/mg)d:SE (ng/mg)1SE ratioiSE
Nucleus Estrogen T“‘_°r°d 119511.85 20.631315 0.610.10
Accumbens n—7
“if?“ 12.261237 258316.25 05110.06
82:“? 146113.88 21.641406 0.671013
Tutored
Blank n= 6 12.511238 298515.43 04610.09
“2:?“ 9.701196 17.121191 05510.80
8:37“ 12.61286 230415.93 0.741016
Striatum* Estrogen Tutored 228814.51 85.07112.37 02910.06
Untutored 30.241570 l3 7.8912094 02210.02
Silence 37.401485 113.43110.63 0.221002
Blank Tutored 294914.92 1255811883 02610.05
Untutored 329815.34 1 10.151888 02910.04
Silence 388119.52 131.95116.58 02410.05

 

 

* Sample sizes same as in Nucleus Accumbens

75

 

Discussion

Summag

Estradiol decreased long-distance calls and visual scanning behavior, and across
hormone manipulations, females presented a greater number of ‘other calls’ when
hearing untutored song compared to silence. Through the raclopride study, we verified
our measures of dopaminergic activity in the nucleus accumbens and striatum.
However, neither estrogen treatment nor exposure to either tutored or untutored song
affected these measures in the two measured neural centers.
I_3e_haviora_l resporyse to estrogen treatment and song exposure

The present results provide a more detailed analysis of the efi’ect of estradiol on
behavior than our previous work on female zebra finches (Svec and Wade 2009). In the
earlier study, we did not observe an effect of these manipulations on a general call
measure. However, vocalizations were not separated into long distance and ‘other calls’
in that study, and visual scanning behavior was not quantified. Distance calls and ‘other
calls’ have two very distinct fimctions and by examining them separately, we were able
to determine whether the song stimuli or treatment affected these two types of behaviors
separately. In the current study, the estrogen-induced decrease in long distance calls
and visual scanning behavior clearly indicates that behavioral effects of the hormone do
occur. These behaviors may be associated with the same function, involving a reduced
need or desire for locating other individuals. Visual scanning behavior would be
consistent with this idea, and distance calls are typically used for localization of other
birds and often uttered when an individual is isolated (Zann 1996). It is interesting to

note that this reduction in behavior parallels a general reduction of ZENK expression in

76

the VMH following estrogen treatment in a previous study (Svec and Wade 2009).

At least three potential explanations exist for the observed reductions in
behaviors. First, it is possible that estrogen treatment results in decreased social
interest. Perhaps the visual cues received from the model zebra finch were sufficient
for estrogen-treated females and increased attempts to contact or find an individual are
not required. Second, estradiol facilitates receptive behaviors in a variety of vertebrate
species (see Pfaff et al. 1994; Ball and Balthazart 2002; Godwin and Crews 2002 for
reviews). Perhaps an increased focus on mating results in a decrease in the amount of
time the female spends on other behaviors such as calling or scanning their
environment. Third, it is possible that estrogen-treated females have decreased anxiety,
as has been observed in mammals (reviewed in Walf and Frye 2006). This reduction in
anxiety may then result in a diminished response to the novelty of the testing
environment, which is manifested in fewer long distance calls and visual scanning.
Although modulated by estradiol treatment, these behaviors were unaffected by
auditory stimulus type. This result suggests that these behaviors may be displayed in
response to some other aspect of the testing situation, perhaps isolation or one or more
features of the novel environment.

Our results differ from those of Vyas et al. (2008; 2009) who showed that
estrogen treatment resulted in increased selectivity of the behavioral response to song
(i.e. increased number of distance calls in response to high versus lower quality song).
However, several important differences exist between their studies and ours. First,
Vyas et al. (2008; 2009) utilized three song stimuli: prototypical song (normal song),

long bout song (which had additional motifs), and complex song (which included

77

acoustically different song motifs and high numbers of unique syllables). Thus, female
responses to normal song were compared to those of artificially higher quality. In our
study, we compared behavior following exposure to normal versus lower quality song.
It is possible that females employ distinct mechanisms to distinguish between the
features, and estradiol mediates these mechanisms in different ways. Second, we
exposed females to only one song type during the behavioral test, whereas in the other
studies, females were exposed to all three song types during each test, and proportional
responses to them were compared across the groups. Third, no silence group was
utilized by Vyas et al. (2008; 2009).

In contrast, other types of calls (tets and stacks collectively) were not affected by
estradiol treatment in our study, but were increased in response to untutored song
compared to silence. This result is consistent with the idea that females are responding
to the unfamiliarity of this song type, which they would not have previously heard. In
parallel, exposure to novel stimuli can result in increased locomotion. Tets are used as
contact calls and are often associated with movement, and stacks are frequently
presented when taking off in flight (Zann 1996). Although we did not detect an effect
of song type on movement, it may have been undetected due to the small size of the
cage in which the test was conducted. However, it is also possible that hearing this
unusual and novel stimulus, untutored song, induces females to increase their attempts
to contact other birds.

Dopamineggic response to raclopride treatment
The increased levels of DOPAC and DOPAC/DA ratio in both the striatum and

nucleus accumbens of raclopride-treated birds indicates that activity of these neurons

'78

was increased by blocking D2 dopaminergic receptors. In mammals, D2 receptors
serve as autoreceptors, and raclopride treatment results in an increase in DOPAC in
both of the regions (Ogren et al. 1986; Eaton et al. 1992). Based on the results of
several in vitro studies, it appears that this also holds true in birds. For example, DA
release decreases when D2 receptor agonists are administered to chicken hyperstriatal
slices (Jackson et al. 2007), and the activity of striatal dopaminergic neurons is
decreased with activation of the D2 receptor (Ding and Perkel 2002). The results here
confirm that this effect is also observed following in vivo treatment with raclopride,
validating the use of DOPAC concentration and the DOPAC/DA ratio as reliable
indicators of dopaminergic activity in birds, as in mammals.
Dopaminergic response to estrogen treatment

The lack of effects of estrogen treatment on DA, DOPAC, and the DOPAC/DA
ratio was surprising, as estradiol increases DA release in both the striatum (Becker and
Beer 1986; Becker and Cha 1989) and the nucleus accumbens (Saigusa et al. 1997;
Thompson and Moss 1997) in mammals. Estradiol also increases labeling for tyrosine
hydroxylase, the rate-limiting enzyme for DA synthesis, in white-throated sparrows in
the ventral tegmental area, the location of dopaminergic cell bodies (LeBlanc et al.
2007). However, as zebra finches are opportunistic breeders, hormones may not play
the same role as in seasonal breeders like sparrows. The level of estrogen in adult
female zebra finches may remain above threshold, reducing the effect of changes in
circulating estradiol. It is unlikely that the treatment itself was ineffective, since we
observed changes in behavior due to the estrogen implants.

Parallel to the lack of DA response, we did not detect an effect of estrogen on

79

ZENK expression in response to song in either the social behavior network or
mesolimbic dopaminergic system (Svec and Wade 2009). Similarly, ZENK expression
in catecholamine neurons (tyrosine hydroxylase positive neurons which may be
dopaminergic or noradrenergic) following song exposure was unaffected by estrogen
treatment in white-throated sparrows (LeBlanc et a1. 2007). Perhaps in this system
estradiol is involved with slower or more long-term modulation, but short-term
neurochemical responses within these regions are less sensitive to the hormone.
Dopaminergic respofies to song exposure

To our knowledge, the present study is the first to concurrently examine the
effects of song perception on dopaminergic neurons in the striatum and nucleus
accumbens in songbirds. Concentrations of DA, DOPAC and the DOPAC/DA ratio
were not affected by song exposure in the nucleus accumbens or striatum. This result
corresponds with the fact that presentation of the same auditory stimulus did not
increase the density of ZENK immunoreactive nuclei in the nucleus accumbens or the
ventral tegmental area in zebra finches (Svec and Wade 2009), and ZENK expression in
other catecholiminergic brain regions was also unaffected by song presentation in
white-throated sparrows (LeBlanc et al. 2007). Similarly, in female starlings, male song
had no effect on immunoreactivity of phosphorylated tyrosine hydroxylase (an indicator
of dopaminergic activity) in the ventral tegmental area (Riters et a1. 2007) in starlings.
In contrast, dopaminergic neurons in some brain regions do appear to be involved in
song perception. For example, the level of DOPAC in the auditory perception regions
increases following exposure to high quality song stimuli (Sockman and Salvante

2008), and phosphorylated tyrosine hydroxylase increases after exposure to male song

80

in the breeding season in the lateral septum and ventromedial hypothalamus of starlings
(Riters et al. 2007).

Perception of zebra finch song may differ from what is observed in other
songbirds in part because adult male zebra finch song is simple and highly stable in
adulthood (Zann 1996). Therefore, it may not be as critical for female zebra finches to
utilize additional neuronal processing (such as the activity of these dopaminergic
systems) to assess the relative value of or distinguish among song types. Alternatively,
females may utilize auditory perception systems to determine whether or not song is
present and then process other cues such as visual courtship or tactile stimuli to
determine the relative value of those signals via the dopaminergic system.

We also cannot completely rule out the possibility that the time point at which
we collected the tissue may have affected our results. However, DA levels rapidly
increase and remain high for 30-40 minutes in Area X (in the medial striatum) of male
zebra finches after singing (Sasaki et al. 2006). As tissue for the present experiment
was collected 10 min after the conclusion of the song presentation, it seems likely that
the DA levels would still be elevated at this time point.

Conclusions and future directions

The results from this study, along with otherrecently collected data from this
lab, reveal that estrogen has specific effects on both behavior and neural activity in
adult female zebra finches. Estrogen decreased the display of long distance calls and
visual seaming (present study) and inhibited the expression of ZENK in the
ventromedial hypothalamus (Svec and Wade 2009). These effects, however, appear

independent of auditory stimulus presentation, and therefore may result from some

81

other aspect of the behavior testing environment. As estradiol in the ventromedial
hypothalamus in other model systems affects both anxiety and sexual receptivity (see
above), either of these factors could have influenced the present behavioral results.
Other vocalizations were increased in response to untutored song (present study), and
female zebra finches spend more time near tutored than untutored song (Svec and Wade
2009). Neither the effects of estradiol nor those of song exposure appear related to
dopaminergic responses in the nucleus accumbens or the medial striatum. However,
DA neurons are also present in cortical regions in songbirds (i. e. Bottjer 1993;
Appeltants et al. 2001), and it is possible that their activity plays a role in mediating

responses to song.

Acknowledgements
Research was supported through grants from the National Institutes of Health to
Juli Wade (R01-MH55488 and K02-MH65907). We would like to thank Katie Licht for
behavioral coding and Elizabeth Adkins-Regan for providing the tutored and untutored

song stimuli.

82

CHAPTER 4: PAIR BONDING IN THE FEMALE ZEBRA FINCH: A POTENTIAL
ROLE FOR THE NUCLEUS TAENIAE

Abstract

Male and female zebra finches are highly social and form pair bonds typically
associated with reproduction. To determine how these bonds affect a female’s
behavioral response to future interactions, females were paired with a male for two
weeks, separated for 48 hours, and then exposed to the same or a novel male. Control
females were left unpaired and introduced to a novel male. Behaviors, as well as neural
ZENK expression, were quantified. Females displayed higher levels of behaviors
associated with pair bonds (clumping and preening) toward their mates than novel
males, and display of these behaviors was correlated with expression of the immediate
early gene ZENK in the nucleus taeniae of one group of females, those interacting with
their mates. Behaviors of the stimulus males were largely unaffected, but those
interacting with an unpaired female attempted to mount more than those interacting
with their mates. The results indicate that the nucleus taeniae may play some role in the
maintenance of pair bonds in this species. Additionally, females may provide some

signal to influence elements of the behavior of males.

83

Introduction

The formation of social bonds is important to an individual’s health and success
in many social species, including humans (Lucas et al. 2003). In monogamous species,
the reproductive sequence commonly involves formation of a pair bond. The neural
mediation of these bonds has only been examined in a few model systems, including
monkeys and prairie voles (see Mason and Mendoza 1998; Wang and Aragona 2004 for
reviews). Zebra finches form strong pair bonds prior to reproduction. They are formed
in 2-14 days, can be maintained through auditory contact alone (Silcox and Evans,
1982), and are characterized by close contact, allopreening, and synchronized behaviors
(Zann 1996). Appearance of these behaviors typically occurs shortly after the male and
female are introduced, and they increase in frequency by the second day (Silcox and
Evans 1982). These behaviors are not typically displayed between unrelated single
individuals (Zann 1996; Butterfield 1970). Females that have formed a pair bond react
aggressively to new males, and close contact (clumping and preening) with them is
inhibited (Silcox and Evans 1982). Pair bonding seems to affect female behavior in two
ways. It increases the incidence of pairing behaviors directed toward her mate and
reduces the display of them toward other males. Studies designed to examine the
responses of females to pairing manipulations are limited. Variations in female
responses toward males that are observed following pair bonding are likely mediated by
multiple neural systems, including those related to auditory perception, social behavior,
and reward.

Responses to interactions with males have been investigated in several species

using immediate early genes (IEGs; see Pfaus and Heeb 1997; Meddle et al. 1999),

84

although research on the neural correlates of behaviors associated with pairing in avian
species is limited. Increased IEG expression is observed in female rodents following
sexual interactions with males in regions in the social behavior network (defined by
Newman 1999) as brain areas involved in the mediation of social behaviors which are
responsive to steroid hormones), including the ventromedial hypothalamus, bed nucleus
of the stria terminalis, and amygdala (Pfaus and Heeb 1997). In female Japanese quail,
interaction with a male results in increased F 05 expression in the ventromedial
hypothalamus, intercollicular nucleus, and mesopallium (Meddle et al. 1999), at least
some of which are involved in the control of reproductive and social behavior in birds
(Gibson and Cheng 1979; Cheng et al. 1999). The mesolimbic dopamine reward
system is also implicated in the regulation of female reproductive behaviors in rodents
(see Melis and Argiolas 1995; Paredes and Agmo 2004 for reviews). Dopamine levels
increase following paced copulation in female rats (i.e. Becker et al. 2001; Merrnelstein
and Becker 1995; Pfaus et al. 1995). Although work on the reward system in female
birds is limited, singing behavior by male starlings results in increased IEG expression
in the ventral tegmental area during the breeding season (Riters et al. 2004; Heimovics
and Riters 2005; 2006).

In the monogamous rodent, the prairie vole, IEG expression is seen in the bed
nucleus of the stria terminalis and preoptic area when females cohabitate with a novel
male (Cushing et al. 2003), and in the medial amygdala, preoptic area, and bed nucleus
of the stria terminalis after 6 hours of mating, which is typically sufficient for the
formation of a pair bond in this species (Curtis and Wang 2003). The reward system is

also vital to the regulation and formation of the pair bond in these animals. Dopamine

85

levels increase in the nucleus accumbens in response to mating in female prairie voles
(Gingrich et al. 2000). Dopamine antagonists presented either systemically or directly
into the nucleus accumbens block the formation of a pair bond following mating
(Aragona et al. 2003). Similarly, pair bonds can be formed between a male and a
female without mating by treating systemically or directly into the nucleus accumbens
with a dopamine agonist (Aragona et al. 2003). Thus, regions in both the social
behavior and mesolimbic reward system mediate pairing behaviors in the prairie vole.

Studies in birds have tended to focus on the role of auditory perception in female
mate choice and pair formation. Females behaviorally discriminate between mate and
stranger calls (Vignal et al. 2008), and auditory cues from males are critical for pair
bond formation (Silcox and Evans 1982; Tomaszycki and Adkins-Regan 2005).
Exposure to male song results in increased IEG expression in the auditory perception
regions in numerous songbird species including zebra finches (Mello et al. 1992; Mello
and Clayton 1994; Bailey et al. 2002), and these regions display differential levels of
IEG expression based on variations in male song (cg. Gentner et al. 2001; Maney et al.
2003; Leitner et al. 2005; Sockman et al. 2005).

The present study was designed to examine the mediation of pair bonding
behavior in the female zebra finch. Although studies have confirmed that females
change their behavior as pair bonds are formed, the only study to examine how pairing
affects responses to new individuals was conducted with male zebra finches (Caryl
1976). To investigate whether forming a pair bond changes how a female behaves
during subsequent social interactions with males, we evaluated behavioral responses in

three groups of birds — females re-exposed to their partners after a brief break, and

86

previously paired and unpaired females exposed to novel males. More importantly, to
assess potential roles of the auditory perception, social behavior, and mesolimbic
reward systems in the mediation of these behavioral responses, we examined neural

correlates by measuring expression of the IEG, ZENK after these social interactions.

Methods
Animals
Adult female and male zebra finches were raised in our breeding colony at

Michigan State University. Animals were kept on a 12: 12 light:dark cycle, and
provided seed and water ad libitum. Their diets were enriched with orange and spinach
and a hard-boiled egg/bread mixture provided once a week. Adults were raised in
mixed-sex aviaries until they had reached adulthood (at least 100 days of age). They
were then housed in unisex aviaries, in acoustic but not visual contact with the opposite
sex, for at least three months prior to testing.
Behavior

Females were placed in an individual cage (30 cm x 23 cm x 38 cm) either alone
or with a sexually mature male zebra finch for two weeks. This time period is sufficient
for the formation of a pair bond in this species (Silcox and Evans 1982). A test was
then conducted to confirm the formation of a pair bond. The cage containing two birds
was taken to a separate testing room, and after one half hour for acclimation, behaviors
of both individuals were video-recorded for one hour. Male and female behaviors
(Table 6) were quantified by an observer blind to experimental group based on

descriptions in Zann (1996) and Adkins-Regan and Ascenzi (1987). They were

87

classified as bonded if they displayed either clumping or preening during the test.
These criteria are routinely used to identify paired birds in this species (Butterfield
1970; Silcox and Evans 1982; Clayton 1990; Zann 1996; Adkins-Regan and Wade
2001; Adkins-Regan 2002), and are uncommon among unrelated, unpaired adult birds
(Zann, 1996). Females housed alone were also taken to the testing room for the same
time period, although their behavior was not videotaped. Following the test, cages were
returned to the colony room, and males were removed and housed individually within
visual and acoustic contact with the female for 48 hours. Novel stimulus males were
also placed in individual cages at this time, but they were housed out of visual contact
with the experimental females. Birds that were not classified as bonded in this first test
were not included in the study.

After the 48-hour separation, a second behavior test was conducted. Paired females
were taken into the testing room in their individual cages. After 30 minutes of
acclimation, they were exposed to either a familiar male (n=7) or a novel male (n=10).
Individually housed (unpaired) females were exposed to a novel male (n=10).
Behaviors were videotaped for one hour, and those of both individuals were evaluated.
A principle components analysis was conducted to determine associations among the
behavioral variables. Several of the behaviors were infrequently displayed. If fewer
than 35% of the individuals displayed a specific behavior (e. g., males preening the
female, copulation, and producing undirected song), we excluded it from the principle
components analysis. This procedure allowed us to create groups of related variables

that could then be examined as a whole to determine whether they were affected by our

88

Table 6. Descriptions of measured behaviors. Frequencies were assessed for all;
durations were also measured for the behaviors with an asterisk *.

Behavior
Pairing

Clumping *

Individual in proximity

of another *

Preening *

Reproductive

Tail quiver

Attempted Mount

Copulation

Beak Wipe
Other

Directed Singing *

Undirected singing *

Beak fencing

Approach

Description

Male and female perch in physical contact, facing the
same direction

Male or female moves to perch less than a body width
apart; they face the same direction

One individual cleans another’s feathers with beak

Rapid back and movement of the tail. In the female often
associated with a bowed position on the perch, but also
may be performed by males following copulation

Male perches at least briefly on the back of the female, but
no cloacal contact is observed

Male perches on the back of the female and cloacal
contact is observed

Beak is moved back and forth in a rhythmic fashion along
the perch

Male produces song facing the female
Male produces the song, but is not facing the female

Male and female swipe beaks with one another, can be an
aggressive behavior

Direct movement toward the other individual with an
upright posture (often followed by clumping or courtship
behavior)

89

experimental manipulations. To do this, we compared the principle components scores
using one-way ANOVAs across exposure groups, followed by post-hoe Tukey/Kramer
tests where appropriate. Further analyses focused only on behaviors with high loadings
for the principle components which were affected by group. Kruskal-Wallis tests were
then used to compare the frequencies and durations of these specific behaviors among
the three exposure groups. Pair-wise comparisons were conducted, as appropriate, with
Mann-Whitney U tests.

Z_ETSE

The female was removed from the cage one half hour after the conclusion of the
second test, overdosed in the testing room with 0.12 cc equithesin, and perfused
intracardially with 0.1 M phosphate-buffered saline (PBS) and 4% paraformaldehyde.
The brain was removed, fixed in 4% paraformaldehyde for 15 minutes, embedded in
gelatin, fixed for one hour in 4% paraformaldehyde, and then placed in 30% sucrose
overnight. It was then sagittally sectioned into four series at 30pm on a freezing
microtome. Tissue was stored in cryoprotectant at -20°C until immunohistochemistry
was performed.

The methods for visualizing ZENK were as described in Svec and Wade (2009),
modified from Bailey et al., (2002) and Bailey and Wade (2003; 2005). Briefly,
sections were rinsed in PBS, incubated in 0.5% hydrogen peroxide in PBS, rinsed,
blocked in 5% normal donkey serum in PBS with 0.3% Triton-X-100 (PBST) for 1
hour, and then incubated with primary antibody (Santa Cruz biotech; catalog #sc-189,
0.1 ug/ml) in PBST overnight at 4°C. After primary incubation, sections were rinsed,

incubated in Biotin-SP conjugated donkey-anti-rabbit antibody (Jackson

90

ImmunoResearch laboratories; 1:500 dilution) in PBST for 1 hour, exposed to ABC
reagent (Elite kit, Vector Laboratories, Burlingame, CA), and reacted with
diaminobenzadine with 0.0075% hydrogen peroxide for 3 minutes. Tissue was then
rinsed, mounted, dehydrated, cleared with xylenes, and coverslipped with DPX.

ZENK immunoreactivity was assessed using brightfield microscopy. Sampling
regions were placed in brain areas within three neural systems: the auditory perception
regions (caudomedial nidopallium and caudomedial mesopallium), the social behavior
network (ventromedial hypothalamus, preoptic area, nucleus taeniae, and bed nucleus of
the stria terminalis), and the reward system (ventral tegmental area and nucleus
accumbens) as described and pictured in (Svec and Wade 2009). A number of
anatomical landmarks were utilized to identify these regions and place the boxes within
the same portion of the region in each animal. Box sizes were as follows: caudomedial
nidopallium (310um x 320um), caudomedial mesopallium (190nm x 345 um),
ventromedial hypothalamus (190um x 290nm), preoptic area (200nm x 200nm),
nucleus taeniae (200nm x 320nm), bed nucleus of the stria terminalis (l90um x
345 um), nucleus accumbens (190um x 305 um), and ventral tegmental area (210nm x
368nm). Immunoreactive cells were manually counted within each sampling region.
The average number of immunoreactive cells was calculated from at least three sections
per animal, and the density of ZENK immunoreactivity was calculated by dividing this
average by the area of the sampling box. A mixed-model ANOVA was conducted to
compare the density of ZENK immunoreactivity across brain regions (within animals)
and groups (between animals), followed by Tukey post-hoc tests where appropriate. To

further investigate the relationship between behavior and ZENK expression, correlation

91

analyses within each exposure group were used to determine associations between the
densities of immunoreactive cells in each brain region with behaviors that differed
among the groups.

An outlier was detected among the paired females interacting with their mates in
the caudomedial mesopallium and one was also detected in the paired females
interacting with novel males in the bed nucleus of the stria terminalis and ventral
tegmental area using Dixon’s test (Rohlf and Sokal 1981); data reported exclude them.

The principle components (PC) analysis and ANOVA of the PC scores was
conducted in SPSS, while other statistical analyses were conducted with Statview (SAS
Institute; Carey, NC).

Results

Behavior
Principle Components Analysis

Six PCs were revealed (Table 7). The first two explained almost 50% of the
variation in behavior. The first component (female PC) had high loadings for behaviors
in which females increased proximity to or initiated contact with males. The second
(PC2) involved reproductive behaviors and female beak wipes. ANOVAs revealed a
main effect of group on scores for the female PC (F= 3.56, p= 0.044), such that paired
females interacting with their mates displayed higher principle component scores than
paired females exposed to novel males (Tukey HSD, p= 0.037). A main effect of group
on scores for the second PC was also detected (F= 3.92, p= 0.034), but post-hoe tests

revealed no significant differences between pairs of groups (all p> 0.059). Scores for

92

Table 7. Principle Components Analysis. Loadings over 0.5 are indicated with

 

 

bold type.
Female PC PC 2 PC3 PC4 PC5 PC6
Directed Singing
Frequency 040] 0.759 -0.331 -0. l 87 0.187 0038
Duration -0.395 0.693 -0.412 -O.186 0.071 0.067
Beak wipe to female -0.172 0.395 0.552 0.409 0.243 0.391
Beak wipe away from
female -0.140 0.309 0.724 0.160 -0.027 0.464
Beak wipe toward male 0.146 0.573 0.637 -0.225 0.020 -0.348
Beak wipe away from
male 0.259 0.545 0.604 -0.269 0.1 19 -0.367
Male approaching female -0. 192 0.745 -0.386 -0.030 -0.170 0.142
Female approaching male 0.844 0.292 -0.1 19 -0.013 -0.043 0.017
Male in proximity of
female
Frequency 0.067 0.427 -0. 142 0.792 0.219 -0.159
Duration -0.102 0.082 -0.155 0.830 0.200 -0.360
Female-imitated
clumping
Frequency 0.866 0.052 -0. 1 5 1 0.062 -0.069 0.1 83
Duration 0.560 -0.066 -0.105 0.238 -0.098 0.296
Female in proximity of
male
Frequency 0.725 0.267 0.020 0.123 -0.3 75 -0.126
Duration 0.722 0.142 -0.068 0.053 -0.358 -0. 124
Female preening male
Frequency 0.819 0.120 -0.034 -0.157 0.361 0.002
Duration 0.852 0.185 -0.1 19 -0.091 0.297 0.184
Attempted Mount -0.263 0.780 -0.242 -0.072 -0.277 0.128
Beak Fencing 0.178 -0.075 -0.220 -0.252 0.790 0.040
Eigen values 4.828 3.525 2.247 1.881 1.435 1.009

93

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
   

 

 

 

 

 

 

 

Clumping Preening
1 .
84 2
5‘
g 6- 3.
3
E 4.
LL l 4_
24
0- o.
200-
A 600‘
8 150-
L”,
c 400-
.g 100'
9
3 200« 50. l
Ndvel Familiar Novel Novel Familiar Novel
I Male Male II male I I Male Male ll male I
Paired Unpaired Paired Unpaired
Attempted Mounts
4
a 3‘ *
5 ' '
3 2
o-
“.3
u.
1 4
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Novel Familiar Novel
I Male Male ll male I
Paired Unpaired

Figure 10. Female pairing behavior in the social interaction test. The two left panels
depict the frequency (top) and duration (bottom) of clumping to the male. The right two
panels depict the frequency (top) and duration (bottom) of preening the male. The
bottom center panel depicts attempted mounts by the male. * signifies p< 0.05.

94

the other four principle components did not differ among the groups (all F< 1.78, p>
0.190).
Female Principle Component

Among the individual behaviors with high loadings for the female PC, a
significant effect of group was detected in the frequency of female-initiated clumping
(Kruskall-Wallis: H= 6.49, p= 0.039) and the duration of females preening males (H=
5.43, p= 0.043). Both were higher in paired females interacting with their mates
compared to novel males (Mann-Whitney U: 12, p= 0.012, Mann-Whitney U: l l, p=
0.015, respectively; Figure 1). A trend also existed for paired females to spend more
time preening their mates than unpaired females preened novel males (Mann-Whitney
U= 17, p= 0.064). Finally, Kruskall-Wallis tests revealed trends for an effect of group
on the duration of female-initiated clumping (H= 4.74, p= 0.057) and the frequency of
females preening males (H= 4.92, p= 0.086; Figure 10).
Principle Component 2

From the analyses of behaviors with high loadings for the second principle
component, the only statistically significant effect detected was a difference among the
groups on attempted mounts (H= 5.429, p= 0.027), such that novel males attempted to
mount unpaired females more frequently than familiar males did their mates (Mann-
Whitney U: 14, p= 0.016).
Neural responses
A main effect of brain region existed such that density of ZENK immunoreactive cells
was higher in the nucleus accumbens and the bed nucleus of the stria terminalis than all

of the other areas investigated (F= 15.22, p< 0.00], Figure 11). No main effect of group

95

 

500
I Novel Male ,
> Paired

I Familiar Male
[:I Novel Male —- Unpaired

.h
C
‘1’

   
   

OJ

C

O
m

N
O
‘13

Density (cells/mm2)

_x
O
‘13

.‘x,
......

b A

 

 

 

 

 

 

fl

NCM CMM POA VMH Ac TnA BST VTA

Figure 11. ZENK immunoreactivity across the brain regions investigated in the three
groups of females (paired females exposed to novel males or their partner, and unpaired
females exposed to novel males). A main effect of region was detected; the density of
calls was highest in the nucleus accumbens and bed nucleusof the stria terminalis. A
main effect of group and interaction between group and region were not detected.
Abbreviations: caudomedial nidopallium (NCM), caudomedial mesopallium (CMM),
preoptic area (POA), ventromedial hypothalamus (VMH), nucleus accumbens (Ac),
nucleus taeniae (TnA), bed nucleus of the stria terminalis (BST), ventral tegmental area

(VTA).

96

 

>

 

Frequency

 

0“1”

r= 0.94, p= 0.005

 

 

 

 

rvvvrrfifi rrrrr

Duration (sec)

0 51015202530354045

Density (cells/mmz)

 

1200
1000‘
800*
600‘
400‘
200‘

6 '5' '101'5‘2'0'2'5'50'55'40‘45
Density (cells/mmz)

r= 0.67, p= 0.103

 

 

 

 

 

 

 

 

F
B

 

25
201
015-1

en

:10.

Freq

00!

 

r= 0.83, p= 0.019

 

 

 

 

 

 

Duration (sec)

0 5 10 15 202530354045

Density (cells/mmz)

 

40° r= 0.91, p= 0.002

300
200

100

 

 

 

 

 

 

0 5101520 25 3035 4045
Density (cells/mmz)

 

Figure 12. Correlation analyses of the density of ZENK immunoreactive cells in the
nucleus taeniae with female pairing behavior in the social interaction test. The figure
includes only females interacting with their mate. Panel A depicts the frequency and
duration of clumping to the male. Panel B depicts the frequency and duration of
preening the male. Three of the four correlations are statistically significant.

97

Nucleus taeniae Nucleus accu

mbens

r

       

Figure 13. ZENK immunolabeling in the nucleus taeniae (Panels A and C) in the
nucleus accumbens (Panels B and D) of females interacting with familiar males. The
density of ZENK immunoreactivity was significantly higher in the nucleus accumbens
than nucleus taeniae. Labeling within cells also appeared darker in the nucleus
accumbens. ZENK immunoreactivity was more apparent in the caudal portion of
nucleus taeniae (left side of photos), but was more homogenous in the nucleus
accumbens. The top two panels (A and B) depict labeling from animals that displayed
high levels of pairing behavior. The bottom two panels (C and D) depict labeling in
animals that displayed low levels of pairing behaviors. Scale bar = 50 um.

98

or interaction between group and brain region was observed (F< 0.73, p> 0.700).
Correlation analyses on the female behaviors that differed among the groups revealed
associations in only the nucleus taeniae within only the group of paired females exposed
to their mates. Positive correlations in this group existed in the nucleus taeniae between
the density of ZENK immunoreactive nuclei and the frequency (r= 0.94, p= 0.005) of
clumping, as well as the frequency (r= 0.83, p= 0.019) and duration (r= 0.91 , p= 0.002)
of preening (Figures 12 and 13). The correlation between ZENK expression and
duration of clumping was not statistically significant (r= 0.67, p= 0.103). No significant
correlations were observed for these behaviors in any of the other groups (unpaired

females or paired females interacting with novel males) or brain regions.

Discussion

In this study, we detected higher levels of behaviors indicative of a pair bond in
females that interacted with their mates compared to novel males. While that is not
particularly surprising, we were excited to observe specific correlations between those
behaviors and ZENK immunoreactivity in the nucleus taeniae only in females
interacting with their mates. These selective results suggest that the relationship
between neural activity in nucleus taeniae and behavior differs among the groups, and
that this particular brain region may play a role in maintaining pair bonds in zebra
finches. Immunoreactivity was substantially higher in the bed nucleus of the stria
terminalis and nucleus accumbens than nucleus taeniae, but the responses in those areas

occurred with the introduction of any male stimuli, indicating that they were not

99

specific to pair bonding; they might be important in more general responses to social
interaction, however.
Behavior

The increased levels of female preening and clumping we observed in response
to familiar males parallels the results from other studies examining pairing in zebra
finches (Butterfield 1970; Caryl 1976; Silcox and Evans 1982). Most of our data
confirmed that females change their behaviors toward their partners after pairing, as
occurs with male zebra finches (Caryl 1976). We defined birds as partners if they
presented clumping and/or preening behavior in after an initial period of co-habitation.
Other studies generally utilize one or both of these criteria when defining a bonded pair
(Butterfield 1970; Silcox and Evans 1982; Clayton 1990; Zann 1996; Adkins-Regan
and Wade 2001; Adkins-Regan 2002). However, if a more stringent method for
classifying males and females as paired is used following the initial test (criteria
involving a certain number of instances of proximity, clumping and preening, as in
Ikebuchi and Okanoya 2006), the sample sizes in the present study are reduced limiting
statistical power, but the same pattern of results is detected. This fact suggests that the
criteria commonly employed are appropriate for defining paired animals, and that the
behavioral results we observed in the paired groups likely reflected the effects of a pair
bond.

It is intriguing that the amount of clumping overall did not differ between
unpaired and paired females in the final behavioral test. When the behaviors are more
closely examined, the average latency for unpaired females to clump was two times

greater than for the females interacting with their mates. In addition, the pattern

100

throughout the course of the test differed. If the data from the hour-long test are
examined in ten minute bins, it is clear that duration of clumping in females interacting
with their mates is high in the first 20 minutes, but decreases over the next 40 minutes.
In contrast, in unpaired females, the duration of clumping increases after 10 minutes
and then remains consistent. The duration of preening was consistently higher in
females interacting with their mates than that of unpaired females throughout the test
period. Most pairing behaviors were initiated by females in all three groups, but males
did display low levels of both clumping and preening. However, only males interacting
with their mates clumped and preened for long durations.

All of these data lead to some interesting possibilities that need to be further
explored. For example, the shorter latency compared to unpaired females and decrease
in clumping over time in females interacting with their mates may indicate that initially
the display of the behavior is important for re-establishment of the bond after a short
absence, but is not required for continual maintenance. In contrast, previously unpaired
females might be more gradually attempting to form new bonds when males are 9
introduced. It is also plausible that the unpaired females displayed clumping behavior
they might not otherwise simply due to a heightened need for social contact. This group
was isolated 12 days longer prior to the test than the paired females. In addition, the
fact that the pattern of clumping, but not preening, differs through the test seems to
indicate that two behaviors may serve different firnctions. For example, clumping
might be more important in the social aspects associated with re-establishing or forming

a new bond, whereas preening may be vital to the maintenance of an established pair

bond.

10]

Pairing itself did not have a strong effect on a female’s response to novel males,
given that both clumping and preening were similar between paired and unpaired
females interacting with novel males. This result corresponds with observations by
Caryl (Caryl 1976) that males courted novel females the same amount after pairing as
they did before forming a bond. Finally, although clumping and preening are
consistently used as indicators of pair bonding in this species (Butterfield 1970; Silcox
and Evans 1982; Clayton 1990; Zann 1996; Adkins-Regan and Wade 2001; Adkins-
Regan 2002), it is unknown whether additional levels or functions of these behaviors
might exist.

Although the present experiment was designed to examine female behavior, we
were also able to observe how manipulations of a female’s pairing status affected male
behavior. Females play a vital role in mate choice decisions in this species (Butterfield
1970; Clayton 1990; Zann 1996), and in the present study they initiated 66% of the
instances of clumping and 71% of the preening. This result is consistent with the idea
that females control a large amount of the behavior associated with pair bonds. Male
actions are likely influenced by signals from the females. This possibility is supported
by the fact that unpaired males attempted more mounts with novel unpaired females
than paired males did with their mates. Unpaired females may have provided different
cues to the males. Of course, as the pairing status also differed between the males in
these two groups, either the fact that they had formed a bond or had not been in contact
with females recently could have contributed to the differential response.

Relationship between behavior and IEG expression in the nucleus taeniae

102

The relationship observed between clumping and preening and ZENK-IR was
highly specific. Correlations were observed only in one group, females interacting with
their mates, and in only one brain region, the nucleus taeniae. This region is
comparable to the mammalian amygdala in several ways. It contains some sensory
inputs and sends projections to the hypothalamus and hippocampus (Cheng et al. 1999).
It also expresses GAD 65, LAMP, and COUP-TF II like the mammalian amygdala
(Yamamoto et al. 2005). Functionally, nucleus taeniae appears to mediate similar
behaviors in birds as the amygdala does in mammals, such as social (Kollack-Walker
and Newman 1995; Numan and Sheehan 1997; Cheng et al. 1999; Reiner et al. 2004a)
and reproductive behaviors (Pfaff et al. 1994; Thompson et al. 1998; Newman 1999;
Reiner et al. 2004a). The mammalian amygdala has also been implicated in the
mediation of interpretation of social cues, especially the reward and emotional
significance of these cues (for review see Baxter and Murray 2002; Phelps and LeDoux
2005; Phelps 2006; Murray 2007).

In animals that form pair bonds, this region shows either increases (prairie vole;
Curtis and Wang 2003; Cushing et al. 2003) or decreases in activity (9. g. human;
Bartels and Zeki 2000, monkey; Bales et al. 2007) with regards to pairing. In the prairie
vole, formation of a pair bond is correlated with an increase in fos expression in the
amygdala (Curtis and Wang 2003), and lesions to the region decrease affiliative
behaviors (Kirkpatrick et al. 1994). Neurochemical mediation of formation and
maintenance of pair bonds differs, however. Formation involves D2 receptor binding
(Wang et al. 1999; Gingrich et al. 2000), whereas bond maintenance involves altered

dopamine D1 receptor density and binding in the nucleus accumbens (Aragona et al.

103

2006). It is possible that in the zebra finch, neurons within the nucleus taeniae are
involved in the maintenance of a pair bond, whereas another system may be vital to the
mediation of initial formation of a bond. It remains to be investigated whether similar
relationships between neural activity in nucleus taeniae and behavior occur in other
social situations.

Several potential explanations exist for the function of the relationship between
bonding behaviors and ZENK induction in the nucleus taeniae of female zebra finches
observed in the present study. First, this association between ZENK expression with
clumping and preening may indicate that nucleus taeniae actively facilitates the
production of these pairing behaviors or inhibits the presentation of other behaviors that
are not related to bonding. The context of a specific behavior can affect IEG expression
(as in singing behavior in starlings in breeding conditions vs. non-breeding conditions;
i. e. Heimovics and Riters, 2005, 2006, Riters et al., 2004), so it is reasonable that this
correlation was only detected when the female was interacting with her mate. However,
it is also possible that the correlation might reflect sensory stimulation related to the
behavior, perhaps affecting how sensory information is perceived when the female is in
contact with an individual she remembers as her mate (see above references on
mammals), or it might represent activity in cells that inhibit responses to less salient
sensory stimuli. In either case, the increased neuronal response could serve to increase
the focus of the female to the relevant cues and aid in maintaining the pair bond. These
possibilities need to be explored further and could be partly elucidated by determining

the neuronal subtypes of the responsive neurons within this brain region.

Neural response in other brain regions

104

Although no effects of our treatment groups were detected in any of the other
brain regions, it is intriguing that we observed higher levels of ZENK immunoreactivity
in the nucleus accumbens and bed nucleus of the stria terminalis compared to the other
measured regions. There are at least two potential explanations for the higher level of
immunoreactivity in these regions. First, it may have functional relevance, as females
in all the groups interacted with a male during the behavior test. The relatively high
neural activity in these brain regions may be associated in social interactions, whereas
the nucleus taeniae might respond more specifically depending on whether or not a pair
bond has been formed between the male and female that are interacting. Second, it is
possible that these regions have a higher general level of activity than the other
examined regions, resulting in greater baseline immediate early gene expression. These
ideas and the specific roles of the nucleus accumbens and bed nucleus of the stria
terminalis in social situations warrant further investigation. '

Summary and conclusions

The present data provide information about both behavioral interactions and
their neural mechanisms. Although pair bonding is generally identified and defined by
the presence of specific behaviors (clumping and preening) in zebra finches, their
functions appear to be more complex. Our results indicate that clumping and preening
might serve different roles and can be displayed differentially. In addition, a region in
the social behavior network (nucleus taeniae) was identified in which immediate early
gene expression is strongly correlated with the behaviors only in females interacting
with their mates. Thus, the neural signal associated with the behaviors depends on the

contexts in which they occur. Finally, the bed nucleus of the stria terminalis, a region in

105

the social behavior network, and the nucleus accumbens, a region in the mesolimbic
dopamine reward system, may be implicated in responses to female-male (or perhaps
even other) social interactions in general. In contrast, the auditory perception regions
investigated in this study appear to play little if any role in mediating the formation or

maintenance of social bonds under the conditions tested here.

Acknowledgements
Research was supported through grants from the National Institutes of Health to
Juli Wade (R01-MH55488 and K02-MH65907). We thank Michele Johnson and
Deborah Kashy for advice and assistance with statistics. All procedures were approved
by the Michigan State University Animal Use and Care Committee and adhered to the

guidelines of the National Institutes of Health.

106

CHAPTER 5: PAIR BONDING BEHAVIOR IN FEMALE ZEBRA FINCHES IS
DISSOCIATED FROM DOPAMINERGIC ACTIVITY IN THE NUCLEUS
ACCUMBENS AND MEDIAL STRIATUM

Abstract
Female zebra finches display higher levels of pairing behavior towards their mates than
to novel males. To determine the role of dopaminergic systems in this behavior, we
paired females with males for two weeks, and then they were separated from their mates
for two days. These birds were then separated for two days and either their mates or a
novel male was introduced to the female for 30 minutes. Novel males were introduced
to additional unpaired females as controls. The content of dopamine and its metabolite,
DOPAC, in the nucleus accumbens and striatum was determined using high
performance liquid chromatography. Although females appeared to demonstrate higher
levels of clumping and preening when interacting with their partners, dopaminergic
activity was not affected by our behavioral manipulations in either of these regions.
These results indicate dissociation between the activity of the mesolimbic dopaminergic

reward system and the behaviors potentially involved in the maintenance of pair bonds

in the zebra finch.

107

Introduction

Social bonds are formed in many species, although their neural mediation has
been investigated in relatively few animals (reviewed in Mason and Mendoza 1998;
Wang and Aragona 2004; Adkins-Regan 2009). Bonds are particularly common among
songbirds (see Black 1996). Zebra finches are opportunistic breeders, and form pair
bonds prior to mating (Zann 1996). Extra pair fertilizations are relatively low in this
species (Birkhead et al. 1989; Birkhead and Hunter 1990), suggesting that copulations
are uncommon among unpaired birds. Pair bonds in this avian species are characterized
by clumping (sitting in close proximity), preening (cleaning another individuals
feathers), and synchronized behavior. Laboratory investigations of pair bonding
typically utilize the presence of clumping and preening to identify bonded individuals
(Butterfield 1970; Silcox and Evans 1982; Clayton 1990; Zann 1996; Adkins-Regan
and Wade 2001; Adkins-Regan 2002), as they are rarely displayed among unpaired
individuals (Butterfield 1970; Zann 1996).

The formation of a pair bond affects the display of both male and female
behaviors. Courtship display decreases with the formation of a bond, but increases
upon reunion following a period of separation (Butterfield 1970). Males and females
are also more likely to clump and preen as pair bonds are formed (Caryl 1976; Silcox
and Evans 1982; Svec et al. 2009). An unpaired female is unlikely to preen novel
individuals, but may display clumping if she interacts with a male for a longer amount
of time (Svec et al. 2009). These results indicate that while clumping and preening are

both associated with pair bonds, they may represent different aspects of the bond.

l08

The mesolimbic dopaminergic reward system may be critical for mediating the
display of clumping and preening in paired individuals. Dopaminergic neurons are
involved in the interpretation of rewarding stimuli (see reviews in Schultz et a1. 1997;
Schultz 2000; Ikemoto 2007), including those associated with reproduction (reviewed in
Paredes and Agmo 2004; Mitchell and Gratton 1994; Melis and Argiolas 1995). In
monogamous species, including humans, this system is active when individuals are
exposed to their partners, or representations of them (Bartels and Zeki 2000; Gingrich et
al. 2000; Bartels and Zeki 2004; Aron et al. 2005).

In the prairie vole, one of the relatively few monogamous mammals, pair bonds
can be formed after a day of cohabitation with copulation. This bond is typically
characterized in laboratory tests by a preference for spending time with the partner in a
choice test (see Williams et al. 1992; Young et al. 1998). When a male and female are
housed together and begin to copulate, dopamine (DA) is released in the nucleus
accumbens in female prairie voles (Gingrich et al. 2000). Preference for the mating
partner can be eliminated in these females by infusing DA D2 receptor antagonists into
the cerebrospinal fluid or directly into the nucleus accumbens prior to the mating period
(Aragona et al. 2003). Conversely, treatment with D2 agonists can induce a female’s
preference for a male even if they did not copulate (Aragona et al. 2003).

A variety of similarities exist between the reward systems of birds and
mammals, suggesting similar dopaminergic mechanisms may be involved in the pair
bonding of zebra finches and mammals (prairie voles). For example, the majority of
dopaminergic neurons emerge from the brainstem in the substantia nigra and the ventral

tegmental areas and project to the basal ganglia including the striatum and the nucleus

109

accumbens in both avian and mammalian species (reviewed in Veenman 1997; Ikemoto
2007)). The activity of dopaminergic neurons in the striatum is mediated through D2
autoreceptors in birds (Gale and Perkel 2005; Jackson et al. 2007), as in mammals. In
parallel, we demonstrated that the regulation of dopaminergic activity through D2
autoreceptors can be reliably measured using a micro-punch sampling technique and
high-performance liquid chromatography (HPLC) in the nucleus accumbens and
striatum (Svec et al. submitted). Functionally, addictive drugs known to affect
dopaminergic systems in mammals have similar behavioral effects in birds (Levens and
Akins 2004; Geary and Akins 2007; Akins and Geary 2008). In addition, both the
striatal and mesolimbic DA systems respond during the production of sexually
motivated song in male birds (Riters et al. 2004; Heimovics and Riters 2005; Sasaki et
al. 2006), indicating that their importance for motivated behaviors.

While immediate early gene expression in the nucleus taeniae (homologous to
the mammalian amygdala; Cheng et al. 1999) correlates with clumping and preening in
female zebra finch females interacting with their partners, no relationship with pairing
behaviors existed in the nucleus accumbens or ventral tegmental area 2009). Similarly,
in female prairie voles the mating bouts that occur during formation of a pair bond do
not result in a corresponding change in immediate early gene expression in the nucleus
accumbens (Curtis and Wang 2003; Cushing et al. 2003). However, dopamine release
occurs in this region following copulation in female prairie voles (Gingrich et al. 2000),
indicating that immediate early gene expression may not be the best indicator of

dopamine release. Based on these results and the limitations of immediate early genes

110

in their use as markers for neural activity (see Pfaus and Heeb 1997), a more direct
examination of the response of the dopaminergic systems seemed prudent.

The current study was designed to investigate whether differences in female
responses to males after the formation of a pair bond are related to changes in activity of
the dopaminergic systems. Females were paired with individual males, and control
individuals were unpaired. After a period of isolation, each female was exposed to
either her partner (if one existed) or a novel male. Dopaminergic activity was measured
by determining the concentrations of DA and DOPAC in samples from the nucleus

accumbens and striatum using high performance liquid chromatography (HPLC).

Methods

Animals

Adult male and female zebra finches were raised in the breeding colony at
Michigan State University. They were kept on a 12:12 light dark cycle and were fed
seed and water ad libitum supplemented with a mixture of hard-boiled eggs and bread,
as well as spinach and orange once a week. Birds were raised in mixed-sex aviaries
with about seven adult males and females, and their offspring. After they reached
sexual maturity, the birds were housed in single-sex aviaries for at least one month prior
to testing. They were in acoustic, but not visual, contact with members of the opposite
sex. All procedures were approved by the Michigan State University Animal Use and

Care Committee and adhered to the guidelines of the National Institutes of Health.

111

Behavior

Stimulus exposure and behavior testing were conducted as in Svec et al. (in
press). Briefly, females were housed alone or with a sexually mature male for two
weeks in an individual cage (30 cm x 23 cm x 38 cm) in the room with the group
aviaries. To confirm formation of a bond between the cohabitating birds, they Were
taken to a sound-isolated room, given 30 min for acclimation, and then videotaped for
one hour. A pair was classified as bonded if the individuals displayed clumping,
preening, or copulation during this hour. Those that had not bonded were excluded
from analyses. Each cage was then returned to the aviary room, and the male was
removed and housed individually in acoustic and visual contact with the female.
Females housed alone were taken to the testing room for the same period of time, but
not videotaped. At this time, additional stimulus males were placed in individual cages
and housed in the same aviary room in acoustic, but not visual, contact of the females.

Two days later, the female was re-acclimated to the testing room for 30min, and
then either the same male or a new male was introduced to their cage for 30 min.
Behavior was videotaped and later analyzed by an observer blind to group (paired
female with partner, paired female with novel male, or unpaired female with novel
male). Measured behaviors are described in detail in Svec et al. (in press), and include
those associated with pairing (clumping, preening, approach) and courtship (directed
singing and beak wipes), as well as those more directly related to reproduction (tail
quivers, attempted mounts, and copulation). Additional analyzed behaviors included

undirected song and beak fencing. After the conclusion of the test, the female was

112

rapidly decapitated. The brain tissue was flash-frozen in methyl-butane, and stored at -
80 °C until processing.
HPLC Analysis

As in Svec et al. (submitted), tissue was sectioned coronally at 500 um, and
portions of the nucleus accumbens (21-gauge), and rostral (21-gauge) and caudal (18-
gauge) medial striatum were microdissected using the Palkovits punch method
(Palkovits 1973). Tissue was placed in 50 ul of 0.1M phosphate-citric buffer in 20%
methanol (pH 2.5) and stored at -20 °C. Prior to HPLC, samples were centrifuged,
sonicated with three l-sec bursts, centrifuged for 1 min, and the supernatant was
removed and stored at -20 oC. HPLC coupled with electrochemical detection was
conducted as in Lindley et al. (1990), Behrouz et al (2007), and Svec et al. (submitted).
Supernatant was first run through a c-l8 reverse phase analytical column and then
electrodes with 1.0M phosphate citrate buffer with 0.1M EDTA, 0.3 5% sodium
octylsulfate and 20% methanol. Peaks were generated for each sample with a Hewlett
Packard Integrator (Model 3393A). Standards containing known amounts of DA and
DOPAC were run on the same day and were used to determine the content of DA and
DOPAC within each sample.

A Lowry assay (Lowry et al. 1951) was used to determine protein content. The
pellet from each sample was dissolved in 0.1M NaOH and sonicated. Two reactions
were then conducted on the samples and a standard curve of protein serially diluted
from 12.5 ug to 50 ug. First, they were exposed to sodium carbonate, cupric sulfate,
and KNA tartrate for 10 minutes, and then F olin reagent (phenol in ddH20) was added

for 30 minutes. A microplate reader (MicroQuant, Biotek Instruments, Winooski, VT)

113

was used to determine absorbance of the samples and standard curve, and these values
were used to determine the protein content of each sample. DA and DOPAC values
were divided by the protein content for each sample to determine the concentration of
the neurochemicals. The DOPAC/DA ratio was also calculated using the absolute
quantity of DA and DOPAC for each sample.
Statistics

A principal components (PC) analysis was conducted to determine relationships
among the behaviors (see Svec et al. 2009). Behaviors which were presented by less
than 25% of individuals were not included in this analysis. The scores for each PC
were then compared among the three groups with one-way ANOVAs. As in Svec et al.
(in press), the frequency and duration of female-initiated clumping and preening and the
frequency of attempted mounts were also compared with non-parametric Kruskall-
Wallis tests. HPLC data for each metabolite were analyzed with mixed-model
ANOVAs to compare data across brain regions (within individuals) and across the
groups (between individuals). Tukey-Kramer post-hoe tests were conducted for pair-

wise comparisons, as appropriate.

Results

Behavior
Six PCs were revealed in the PC analysis (Table 8). Collectively, the first two
explained 42 percent of the variance in the data. The first principal component (PCl)

included female-initiated clumping (frequency and duration), preening (both male and

114

Table 8. Principal components analysis. Behaviors loading above 0.5 are in bold.

PC 1 PC 2 PC3 PC4 PCS PC6

 

 

Directed Singing
Frequency -0.278 0.748 0.353 -0.3 57 -0.12 0028
Duration -0.335 0.728 0.382 -0.332 -0.06 0.036
Beak wipe to
female* -0.015 0.009 0.670 0.105 0.413 0.371
Beak wipe away
from female -0.180 -0.28 0.156 0.128 0.061 0.739
Beak wipe toward
male -0.005 0.208 0.156 0.727 -0.28 0.026
Beak wipe away
from male -0.062 0.070 0.243 0.709 -0.07 0.047
Male approaching
female 0.563 -0.28 0.700 -0.04 0. 148 -0.04
Female approaching
male -0.225 0.553 0.221 -0.305 -0.28 -0.196
Male-initiated Clumping
Frequency 0.314 0.769 -0.17 -0.168 -0.28 0.288
Duration 0.222 0.03 7 -0.25 -0. 166 -0.37 0.707
Male in proximity of female 1
Frequency 0.279 0.501 -0.020 0.521 0.492 0.065
Duration 0.170 0.307 -0.22 0.261 0.573 -0.026
Female-imitated clumping
Frequency 0.840 -0.25 0.389 -0.177 -0.01 -0.138
Duration 0.922 -0.03 -0.17 -0.191 -0.06 0.230
Female in proximity of male
Frequency 0.642 0.35 5 0.104 0.314 -0.45 -0.129
Duration 0.388 0.236 -0.02 0.553 -0.47 -0. l 27
Male preening female
Frequency 0.590 0.409 -0.52 -0.091 0.292 -0.010
Duration 0.611 0.329 -0.54 -0.042 0.306 -0.012
Female preening male
Frequency 0.908 -0.19 0.300 -0.136 -0.05 0.028
Duration 0.900 -0.18 0.239 -0.192 -0.01 -0. 127
Beak Fencing 0.012 0.643 0.457 0.005 0.279 0.057
Eigenvalues 5.321 3.538 2.613 2.325 1.792 1.441

*Unless otherwise indicated, values represent frequencies.

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female-initiated, frequency and duration), female approaching the male (frequency), and
female in proximity of male (frequency and duration). The second principal component
(PC2) included primarily male behaviors: directed singing (frequency and duration),
male approaching the female (frequency), male-initiated clumping (frequency and
duration), male in proximity of female (frequency and duration), and beak fencing
(frequency). No group effects were detected with one-way ANOVAs conducted on any
of the PC scores (all F< 2.30, p> 0.123). However, a trend for an effect of group on
PCI (F= 2.79, p= 0.083) was detected, levels appeared increased in females interacting
with their mates. Kruskall-Wallis tests revealed no significant effects of group on the
five behaviors analyzed in Svec et al. (in press); all H< 3.66, all p> 0.16). Selected
behavioral data (durations) are presented in Table 9, along with a portion of the data

from our previous study (Svec et al. 2009) for comparison.

H1141;

A main effect of brain region on DOPAC concentration was detected (F= 7.91,
p= 0.001; Figure 14). Across the groups, it was higher in the nucleus accumbens than
the rostral striatum (Tukey-Kramer, p< 0.05). In contrast, the concentration of DA was
higher in both the rostral and caudal striatum than the nucleus accumbens (F= 22.43, p=
0.42; Tukey Kramer p< 0.05). In addition, a main effect of brain region was observed
in the DOPAC/DA ratio (F= 72.09, p< 0.001), such that it was higher in the nucleus
accumbens than both the rostral and caudal striatum, and higher in the caudal than the
rostral striatum (Tukey-Kramer, p< 0.05). A trend for an interaction between group and

brain region was also observed within the concentration of DOPAC (F = 2.32, p< 0.07).

117

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Nucleus Rostral Caudal
Accumbens Striatum Striatum

Figure 14. Concentrations of DOPAC (top), DA (middle), and the DOPAC/DA ratio
(bottom) in the nucleus accumbens (left), rostral striatum (center), and caudal striatum
(right). Significant effects of brain region were observed for each measure, indicated by
different letters above the bars. No effects of group or interactions among group and
brain region were observed. Sample sizes: paired with new male, n=6, paired with
same male, n= 10, unpaired, n= 11.

118

While the groups responded similarly in each region, the DOPAC seemed greater in the
nucleus accumbens than both portions of the striatum in females exposed to their
partners (F = 13.83, p= 0.0002; both Tukey-Kramer, p< 0.05), and greater than the
rostral striatum in unpaired females (F = 4.64, p= 0.022; Tukey-Kramer, p< 0.05). No
other effects of group or interactions among the groups and brain regions were detected

for any of the neurochemical measures (all F < 0.994, p> 0.42).

Discussion

The present study was designed to investigate the response of the dopaminergic
reward system in female zebra finches associated with pairing behaviors and/or pairing
status. Upon introduction, paired females behave differently toward their partners than
toward novel males. However, we did not detect statistically significant changes in
activity of this system with pairing and interactions with males. These results parallel
those in a previous study conducted in our lab (Svec et al. submitted), documenting a
lack of relationship between DA neurochemistry and female responses to high quality
song, which induced a preference behavior. Together, these studies suggest that the role
of this system in mediating behavioral preferences for specific social stimuli may be

limited in this species.

Behavior
Unlike our previous study (Svec et al. 2009), we did not detect a significant
effect of pairing on female clumping and preening with males. The only difference

between the two experiments was the duration of the behavioral test (30 min after male

119

introduction in the present study, which was required for neurochemical analysis,
compared to 60 min in the previous study). Group differences in the display of some
behaviors may not have been detected in the current study because of the shorter testing
period. In fact, our earlier work suggested that pairing behaviors emerged later in the
test, particularly in unpaired individuals (Svec et al. 2009). Still, the general patterns of
behavior seemed similar between the two studies, with higher levels of clumping and
preening in paired females interacting with their mates.

A comparison of the durations of each behavior between the first 30 min of the
two studies indicated that in each case they were statistically equivalent (Table 9). In a
collective analysis, paired females clumped longer with their partners than with novel
males (Kruskall-Wallis H: 7.3, p= 0.03, Mann-Whitney U, p= 0.01), but males did not
differ among groups in the duration of clumping (Kruskall-Wallis H= 1.3, p= 0.53).
Overall, males initiated very few episodes of clumping. HoWever, both males and
females preened longer when interacting with their mates then they did when the male
was novel (with both paired and unpaired females; both Mann-Whitney U, p< 0.05).

The combined data, which allowed a more powerful analysis of the effect of
pairing condition on the presentation of these behaviors, suggest the idea that clumping
and preening may serve quite different functions. As clumping appears more frequently
in unpaired individuals than preening, it is possible that this behavior indicates a more
general level of social interaction. Clumping can be observed between sick individuals
or juvenile siblings (Zann 1996), indicating it may represent something more social than

primarily reproductive. Preening, in contrast, was not displayed between unpaired

120

individuals, but only after formation of a pair bond, suggesting it is more specific to
pairing.

It is intriguing that no effect of group was observed in male-initiated clumping;
indicating pairing status may affect males and females differently. However, males and
females in the unpaired group experienced different situations prior to the test. It is
possible that the longer durations of female-initiated clumping displayed by females
that were unpaired prior to this interaction may have resulted from the reduced social
interaction these females experienced during the two weeks prior to the test. The fact
that the females differed from males in clumping but not preening, lends more support
to the possibility that clumping might serve a more general social function than

preening.

Neural correlates

The neurochemical results indicate that dopaminergic systems likely do not
play a role in mediating the behavioral responses associated with pairing. Although
females appeared to display higher levels of pairing behaviors when interacting with
their mates, this effect did not correspond with a difference in dopaminergic activity in
the striatum or nucleus accumbens. Several biological, as well as methodological
explanations can be proposed for the lack of relationship between these behaviors and
the activity of the dopaminergic systems.

First, zebra finches are highly social (Zann 1996), and separation from mates has
both behavioral and physiological effects (Butterfield 1970; Remage-Healey et al.

2003). The effect of social isolation alone on stress and corticosterone levels has not

121

been examined in this species. However, the intense sociality of this species may
modify how females respond to individuals following isolation, suggested by the
behavioral differences between unpaired females and stimulus males in this study. It is
possible that interaction with another individual following the short social isolation
prior to the behavior test resulted in a dopaminergic response in all of the groups of
females.

Second, dopamine responses may relate more to copulation than other aspects of
bond formation or maintenance. In mammals, increased mesolimbic dopaminergic
responses are typically observed after copulation (Mermelstein and Becker 1995; Pfaus
et al. 1995; Gingrich et al. 2000). Studies in the prairie vole have examined the
response of dopaminergic neurons following pair formation resulting from a period of
copulation. Female prairie voles can form bonds in the absence of mating, if they
cohabitate with an individual for long periods (24-48 hours; Williams et al. 1992) or
have decreased levels of corticosterone (DeVries et al. 1995). However, the neural
mechanisms mediating the formation of pair bonds in these cases have not been
elucidated. In zebra finches, copulation does not appear to play an important role in
bond formation, as it is rarely observed prior to the establishment of a pair bond (Zann
1996). As a result, the importance of the response of mesolimbic dopamine neurons
may be reduced compared to what has been previously observed in prairie vole studies.

Third, the present study investigated the role of this system in mediating
responses to individuals after formation of a pair bond, rather than during the formation
of these bonds, which is commonly investigated in prairie voles. The formation of a

pair bond results in increased levels of D-l receptor binding in male prairie voles

122

(Aragona et al. 2006). A parallel change in receptors might occur in the zebra finch
with bond formation, and if so, it would provide for selective dopamine response in the
absence of a change in neurotransmitter release.

Finally, we cannot rule out the possibility that the timing of tissue collection
might have affected the present results. We based the duration of behavioral exposure
on a study by Sasaki et a1 (Sasaki et al. 2006), who observed increased DA levels in the
striatum for 30 min when males produced directed song. Females interacted with the
introduced male throughout the 30 min period in the present study. Thus, while we
cannot be sure, it seems unlikely that any DA responses would have returned to

baseline.

Conclusions and Future Directians

Female zebra finches exhibit specific changes in behavioral responses to males
after they have formed a pair bond. The results indicate that preening and clumping
may be separately mediated or indicate different motivational states — bonding for social
purposes rather than bonding specifically for the purpose of reproduction, perhaps.
Modifications of dopaminergic activity or turnover do not appear to be involved in
these varied behavioral responses, however. That does not mean that DA plays no role;
changes in receptor levels may be associated with pair bonds, which would increase
responsiveness to DA release. This possibility should be explored in future studies.
Another potential mechanism for the mediation of these behaviors involves other
neurotransmitters or neuromodulators, such as arginine vasotocin, the avian homologue

of vasopressin. This neuropeptide is implicated in social recognition (see Bielsky and

123

Young 2004) for review) and pair bonding (reviewed in Lim and Young 2006) in
mammals, and may well regulate social responses in zebra finches. Vasotocin mediates
aggressive behaviors (Goodson et a1. 2004), and vasotocin-positive neurons in the bed
nucleus of the stria terminalis respond when male zebra finches interact with females
(Goodson et al. 2009). To fully elucidate mechanisms associated with formation and
maintenance of pair bonds, it will be vital to examine the role of a variety of
neurochemicals and associated molecules in bonding in. both sexes and across diverse

species.

Acknowledgements
This work was funded by a grant from the National Institutes of Health (ROl-

MH55488) to J .W. We thank Sam Pappas and Tyrell Simkins for assistance with the

HPLC procedure and Katie Licht for assistance with video analysis.

124

CHAPTER 6: DISCUSSION

The unique behaviors and characteristics of the zebra finch have provided an
excellent opportunity to examine the intricate and complex interactions among social
behavior, neural systems, and hormones. In the experiments conducted for this
dissertation, I have investigated the presentation and regulation of two types of social
behavior: song preferences and pair bonding in the female zebra finch. Females showed
behavioral preferences for high value stimuli in both paradigms (exposure to tutored
song or their partners). Also, I observed relationships between IEG expression and
behaviors: (1) increased ZENK expression in response to tutored song with a parallel
preference for that song in blank treated birds; and (2) a positive correlation between
ZENK expression and clumping and preening behaviors in the nucleus taeniae.
Estradiol treatment also reduced the induction of ZENK in the ventromedial
hypothalamus. However, the neural correlates for song preference and pairing differed
in that song preference corresponded with changes in the auditory perception regions,
while pairing behaviors related to a region in the social behavior network, the nucleus
taeniae. In this species, dopamine activity in the nucleus accumbens or striatum did not

appear to relate to behavioral responses in either of the paradigms.

Two behavioral paradigms
I have verified that female zebra finches display behavioral preferences for
certain social stimuli using two different behavioral paradigms. First, females spend
more time near tutored than untutored song, and also display higher levels of clumping

and preening towards their partners after a pair bond has been formed. These

125

preferences were strong and repeatable and fit with previous research on both song
preference (N eubauer 1999; Lauay et al. 2004; Nolan and Hill 2004) and pair bonding
(Silcox and Evans 1982; Clayton 1990; Zann 1996). To fully understand the regulation
and mediation of these two behaviors, it is important to consider how they relate to one
another.

In a zebra finch colony, single males court single females. This courtship
includes directed singing, some dancing, and perhaps eventually copulation attempts by
the male (Zann 1996). Early in this period, it is important for the female to acquire
information from the male as to whether the courtship should continue to the formation
of a pair bond. In the studies conducted for this dissertation, approximately 25% of the
pairs housed together for two weeks did not form a pair bond (according to our criteria,
defined in Chapters 4 and 5). One characteristic the female may utilize to make this
decision is the quality of the male song, which can indicate reproductive fitness
(Hasselquist et al. 1996; Buchanan and Catchpole 2000; Nowicki et al. 2002; Dolby et
al. 2005). Females show preferences for high quality song (Neubauer 1999; Lauay et
al. 2004; Searcy and Marler 1981; Ballentine et al. 2004; Nolan and Hill 2004), and
male zebra finches producing poor quality songs are less likely to form pair bonds with
females (e. g., Tomaszycki and Adkins-Regan 2005).

After the male and female begin courtship, a pair bond can begin to form (Zann
1996), and the value of social stimuli may begin to change. Once a bond is formed a
female will recognize her partner and present different behaviors towards him than to
other males (Chapter 3 and 5, Silcox and Evans 1982). Females remain bonded with

males surgically altered males that do not producing song or produce low quality song,

126

indicating that the importance of the quality of song is diminished after a pair bond has
developed (Tomaszycki and Adkins-Regan 2006). Females can identify their mate’s
song (Vignal et al. 2008), but they may also utilize additional visual or tactile stimuli to
recognize their mate. Thus, song preferences likely represent the first stage of courtship
and pairing responses represent a later stage of reproduction, in which decisions about
relative male quality have already been made.

Some important similarities between the two paradigms must be noted. The
presentation of both responses to courtship song and pairing behavior is most likely
mediated by the female making a determination of the value of the social stimulus. To
make this determination, females in both situations may compare the new stimulus to a
memory stored as a template. Female zebra finches isolated during a sensitive period
do not display preferences for tutored song (Lauay et al. 2004), and juvenile zebra
finches can memorize both conspecific and heterospecific song (Braaten et al. 2006;
Braaten et al. 2008), indicating that templates are formed in development in these birds.
The role of memory has been unexplored in terms of pair bonding in birds, but it is
logical that a memory of one or more aspects (including visual, tactile and or behavioral
cues) of the partner is formed and stored in the brain of the female and other males are
compared to this template when females interact with them.

However, the types of stimuli differ. Song stimuli are simple auditory cues,
whereas interactions with males involve a number of sensory stimuli that the female
must interpret. Increased IEG responses to a variety of types of preferred song stimuli
are observed in males, females, and juveniles (Bolhuis et al. 2001; Bailey and Wade

2003; Stripling et al. 2003; Bailey and Wade 2005; Tomaszycki et al. 2006), indicating

127

that these neural responses may be primarily sensory and may not involve high levels of
integration or be uniquely associated with reproduction. A greater amount of
integration is likely required to determine value of a stimulus in a social interaction with
a variety of sensory inputs, such as that observed in the pair bonding paradigm. Thus,
females may utilize different mechanisms to mediate the behavioral responses in these

two paradigms.

Neural Correlates

Methodological Issues
Immediate early genes as indicators of neural response

IEG expression results can be somewhat difficult to interpret (see Pfaus and
Heeb 1997 for discussion). At a basic level, IEGs indicate that a signal transduction
event has occurred (Morgan and Curran 1989). The induction of IEGs can represent
either excitation or inhibition in a specific brain region. IEG expression may indicate a
role for a brain region in either the presentation of a behavior or the interpretation of
sensory information. By coupling examination of IEG expression with the expression
of a specific neurotransmitter within a brain region, researchers may better be able to
determine the precise meaning of the IEG expression. In addition, more direct studies
such as those using lesions, microdialysis or pharmacological treatments could be
utilized in the implicated brain regions to verify their precise role in a behavior.
Finally, lack of IEG expression does not completely rule out the possibility that a brain
region may be involved in a behavior (see discussions in Hoffman et al. 1993; Pfaus and

Heeb 1997; Hoffman and Lyo 2002; Kovacs 2008). IEG expression may be dissociated

128

from neuronal stimulation (i. e. F igueiredo et al. 2003). Additionally, specific IEGs (e. g.
c-jun, c-fox, Arc) may be upregulated in different contexts (i.e. Guzowski et al. 2001;
Bailey and Wade 2005).

In sum, IEG analyses provide excellent starting points for examining neural
correlates of behavior, given that an investigator can efficiently examine a wide variety
of regions and determine which, if any, might be involved in certain behaviors.
However, due to the limitations in interpretation of IEG results, other methods are
required to verify the role of certain brain regions and also to more closely investigate
responses identified by immediate early gene induction.

Neurochemical analyses

In this dissertation, I followed up IEG studies with neurochemical investigations
of the dopaminergic reward system. Dopamine was predicted to be important for the
mediation of these behaviors, because of its role in motivation and reward. By
examining neurochemistry in micropunches of specific brain regions, I was able to
clearly investigate the response of dopaminergic regions in these systems and determine
if dopaminergic activity in these regions played a role in song preference and pair
bonding in this species. Neurochemical analyses, unlike IEG analyses, can provide
direct measures of the activity of neurons in a specific region and allow predictions
about neurotransmitter release. However, unlike microdialysis, these analyses only
allow for the quantification at a specific time point, and if the neuronal activity is

transient, then it is possible that rapid responses may be undetected.

Three Neural Systems

129

Social Behavior Network

Although some of the regions in this network displayed high constitutive levels
of ZENK during the behavior tests, most did not exhibit selective ZENK responses
during song presentation or interactions with males. Only in one region of this network,
the nucleus taeniae (homologous to the mammalian medial amygdala, see Chapter I),
was a correlation with behavioral responses displayed towards female’s partners. No
regions in this network were associated with song preference.

There are several reasons why the social behavior network may be involved in
mediating responses to interactions with male partners and not song preferences. First,
song stimuli are much less complex than the multiple stimuli received during a full
social interaction. Male zebra finches produce a fairly simple and stable song, and only
one sensory modality is needed to interpret this stimulus. In addition, a strong
connection may not exist between song preference and pair bonding. Male and juvenile
songbirds also display song preferences (Clayton 1988; Calhoun et al. 1993; Braaten
and Reynolds 1999; Houx and ten Cate 1999; 1999; Riebel et al. 2002; Riebel and
Smallegange 2003), indicating they may be dissociated from reproduction and might
instead simply reflect a general social response to the song stimulus. If song playback
does not involve a clear reproductive response from the female, then the activation of
the social behavior network during the presentation of this preference behavior may be
more limited.

In the pairing paradigm, the situation is much more complex. Females are
interpreting a much larger suite of social cues, including auditory, tactile, and visual

stimuli. In addition, the behavioral response to the stimulus is varied between the two

130

paradigms used in this dissertation. In the song response studies, females presented a
very limited repertoire of behaviors that primarily consisted of presentation of calls. In
contrast, in the pairing paradigm females presented behaviors to the males including
beak wipes, calls, and even copulatory solicitation displays. Perhaps the social behavior
network is primarily utilized to mediate interpretation of more complex social stimuli,
which require or reflect more complex behavioral responses.

The identification of the nucleus taeniae as an important node in the behavioral
preference for partners in the zebra finch is novel. This idea parallels data from the
mammalian homologue (medial amygdala), which is utilized in interpreting the
emotional context of sensory stimuli (for review see Baxter and Murray 2002; Phelps
and LeDoux 2005; Phelps 2006; Murray 2007). As the neural mediation of these types
of behaviors in zebra finches has been largely unexplored, the nucleus taeniae will be an
important region to further examine the neural mediation of reproduction and other
social interactions in females.

Dopaminergic Systems

Responses to the social stimuli in the two paradigms investigated in this
dissertation were not reflected by changes in dopaminergic responses, using either IEGs
or neurochemistry. These results may indicate that dopaminergic systems are not
important for determining the value of social stimuli or that they do not play a role in
the behavioral responses I observed. Although dopaminergic systems have been
implicated in reproductive behavior (reviewed in Mitchell and Gratton 1994; Melis and

Argiolas 1995; Paredes and Agmo 2004), responses to reproductive stimuli (Mitchell

131

and Gratton 1994), and pair bonding (Gingrich et al. 2000) in mammals, there are some
notable differences between mammalian model systems and zebra finches.

In mammal studies, increased dopaminergic activity is observed following
copulatory behavior in males and female rodents (Pfaus et al. 1990; Pleim et al. 1990;
Damsma et al. 1992; Pfaus et al. 1995; Kohlert and Meisel 1999). Sexually naive
individuals will demonstrate a lower level of dopamine release, which will increase
with more sexual experience (Kohlert and Meisel 1999). In some cases, these responses
can be observed following exposure to sensory stimuli alone without reproduction (i.e.
Louilot et a1. 1991; Damsma et al. 1992; Mitchell and Gratton 1994; Fabre-Nys et al.
1997). In the medial preoptic area, dopamine release in response to reproductive odors
is not observed in prepubertal male hamsters (Schulz et al. 2003), indicating that a
connection with copulatory behavior may be present for this release to occur. In
contrast, both behavioral paradigms used in the studies in this dissertation have limited
relationships with copulation. Song preferences may not relate to reproduction, but
instead might reflect a general sensory preference for familiarity, which could occur at a
number of levels including recognition of a member of the same species, a particular
type of conspecific song previously heard (tutored vs. untutored), or one’s father or
mate. Similarly, pair bonding has a strong social component, perhaps indicated by the
presentation of clumping behavior, and this bond may be somewhat dissociated from
copulatory behavior, as well. As a result, these behaviors (song preference and pair
bonding) may have reduced association with activity of the mesolimbic dopaminergic
systems in female zebra finches.

Auditory Perception Regions

132

In the song preference paradigm, IEG results in blank-treated birds replicated a
number of studies in which increased IEG expression is observed in the auditory
perception regions in response to high quality or preferred song stimuli (e. g. Gentner et
al. 2001; Bailey et al. 2002; Maney et al. 2003; Leitner et al. 2005; Sockman et al. 2005;
Tomaszycki et al. 2006). Electrophysiological studies have verified that neurons within
these regions display selective responses to high quality song (reviewed in Theunissen
and Shaevitz 2006; Pinaud and Terleph 2008).

However, dissociation between IEG expression in these regions and behavioral
preferences was also observed (Chapter 2). When females were estrogen treated, the
selective neural response was eliminated without affecting the behavior preference for
tutored song. In addition, although females identify and respond differently to their
mate’s song (Vignal et al. 2008), IEG expression in the auditory regions did not differ
between exposures to song produced by different males in the pair bonding study
(Chapter 4). Both of these studies (Chapter 2 and 4) demonstrate that a selective IEG
response may not be required for the presentation of a behavioral preference for tutored
song or a familiar mate. It may be that these preferences are so pervasive and critical
that redundancy in their neural mediation exists. Thus, a preference may be displayed
regardless of a differential neural response in the auditory perception regions
specifically.

Neural systems summary

For this dissertation, I proposed that components in three neural systems might

interact in a number of ways to mediate behavioral responses to social stimuli. The

results of the four studies indicate that components of the social behavior network, the

133

nucleus taeniae in particular, and auditory perception sites may act independently to
mediate behavioral responses in both of the behavioral paradigms. It is likely, however,
that additional brain areas that were not investigated play some role. Presumably, the
presentation of complex behavioral responses requires the coordination of several brain
regions and systems, although that was not detected in these studies. In addition to
evaluating more sites, further work using other techniques or modifications to these
paradigms may help in elucidating some of these more complex relationships between
neural systems.

Both the auditory perception sites and the nucleus taeniae/amygdala have been
implicated in memory processes using immediate gene induction as a marker
(McGaugh 2004; Richter-Levin 2004; Gobes and Bolhuis 2007). Thus, they may be
utilized to determine the salience of a stimulus, perhaps through accessing some type of
memory. Immediate early genes have been implicated in the formation of memories
(reviewed in Tischmeyer and Grimm 1999). IEG expression is observed during
learning processes (Anokhin and Rose 1991; Brennan et al. 1992), and antagonism of
immediate early gene with antisense-oligonucleotides can block learning and
association in rodents (Tischmeyer et al. 1994; Lamprecht and Dudai 1996; Swank et al.
1996). The connection with memory formation fits with the hypothesis that the
behavioral responses I observed result from the connection of a stimulus with a memory
template. To more fully investigate the neural mediation of these behaviors, it would be
prudent to utilize the expression of several immediate early genes such as c-fos or c-jun,
or take a more broad approach by measuring responses of additional neurochemicals

within several brain regions.

134

Role of Estrogen in Brain and Behavior

Estrogen did not affect preference for tutored song, but treatment resulted in
decreased long distance calls and visual scanning behavior. These data may be
connected with the estradiol-induced decrease of ZENK in the ventromedial
hypothalamus. The ventromedial hypothalamus contains estrogen receptors in zebra
finches (Gahr et al. 1993; Jacobs et al. 1996), suggesting that the hormone can act there
directly. This change in ZENK expression may be associated with increased
receptivity, perhaps due to disinhibition in this brain region (see Commons and Pfaff
2001). This function might enable the female to focus more on reproductive behaviors
or stimuli than song. Alternatively, the reduction in ZENK expression in the
ventromedial hypothalamus could be due to the novel conditions in which the testing
occurred. In mammals, estradiol can modulate the binding of oxytocin in the
ventromedial hypothalamus (reviewed in Johnson 1992), which has been implicated in
the reduction of anxiety (see McCarthy 1995; Neumann 2008 for review). However,
estradiol-treated female rats infused with oxytocin into the ventromedial hypothalamus
display reduced sexual behaviors, without a decrease in anxiety—related behaviors (Bale
et a1. 2001). At this point, the role of estradiol and the ventromedial hypothalamus in
the mediation of anxiety behavior in songbirds is unknown. It will be important to test
whether their interaction contributes directly to behavioral changes.

Additionally, estrogen reduced the selective ZENK response in the auditory
perception regions. This result parallels the estrogen induced decrease of ZENK in the
ventromedial hypothalamus (see above). Perhaps these neural responses are linked and

reflect a shift from a focus on the details of song to receptive behavior once a mate has

135

been selected. Although NCM and CMM have very few estrogen receptors, the
auditory system is connected to the ventromedial hypothalamus (Cheng and Peng

1997), and mediation by estradiol may occur through this connection.

The uniqueness of the zebra finch model

The work presented here highlights some of the unique qualities of the zebra
finch model and provide a number of directions to be explored in future research in this
model.

First, as zebra finches are opportunistic breeders, it is possible that the role of
estrogen in mediating their behaviors may be reduced. The song preference studies
indicate that this may be this case, as behavior was completely unaffected by estrogen
treatment. It is unknown at this time how estrogen levels fluctuate in female zebra
finches. Regardless of reproductive state, measures of GnRH activity appear to remain
at a relatively high level in male zebra finches (Perfito et a1. 2006). A similar
phenomenon may also be observed in females.

The regulation of song preference behavior by estradiol may also be reduced by
the fact that zebra finches form pair bonds prior to reproduction. If estradiol levels
fluctuate in this species, they are likely to be highest just prior to egg-laying. As song
preference does not appear to be critical during this time period, it is unlikely that these
behaviors are mediated by changes in estradiol.

The pair bond in this species also has some unique characteristics that are
important to investigate further. While bonds are typically formed for reproduction,

they also may serve other purposes related more generally to sociality. The experiments

136

conducted in this dissertation shed light onto this distinction, indicating that clumping
behavior may be a less specific social behavior, whereas preening is likely more

specific to pair bonded individuals.

Conclusions and Future Directions

Using IEGs, brain regions (the auditory perception regions and the nucleus
taeniae) were identified that may play a role in the presentation of song preference and
pair bonding behaviors. Since IEG results can be difficult to interpret, a more direct
investigation must be conducted in these brain regions to determine the meaning of the
IEG response. Co-localization studies with specific neuropeptides and/or
neurotransmitters in these regions should eventually be conducted in these regions to
identify the specific factors regulating these neural responses and behaviors. Some
brain regions that were not examined in this dissertation may also be investigated,
including the lateral septum (which has been implicated in social behaviors in mammals
and birds; Insel et al. 1994; Wang et a1. 1996; Goodson et al. 2005) and the prefrontal
cortex (which is a site of dopamine release; Bannon and Roth 1983). The caudolateral
nidopallium (NCL) in birds is considered analogous to the prefrontal cortex, based on
function and anatomy (Mogensen and Divac 1982; Reiner et al. 2004a). Perhaps
some of these other neural systems are vital to the overall neural coordination of these
types of social responses. They, and their potential interactions with the three circuits
investigated, should be considered further.

Although dopamine activity in the reward system does not appear to play a role

in either of these behaviors, there are some other neurochemicals on which future

137

research can focus. For example, vasopressin has been implicated in pairing behaviors
in mammals (see Lim et a1. 2004), and its avian homologue, arginine vasotocin, also
plays a role in mediating social behaviors in avian species (Goodson et al. 2004;
Goodson et al. 2009). It is also critical to social recognition (Bielsky and Young 2004),
and this behavior may be important to both song preference and pair bonding in the
zebra finch. Vasotocin receptor knockout mice have difficulty recognizing conspecifics
(Bielsky et al. 2004). These receptors are found in the nucleus taeniae in birds
(Voorhuis and de Kloet 1992; Aste et al. 1996), and action of vasotocin in this region
may be critical for the recognition of partners by paired females. Oxytocin is also
critical for social recognition (see Bielsky and Young 2004), and recognition of mate
quality as oxytocin knockout mice cannot distinguish between healthy and parasitized
males (Kavaliers et al. 2003). Oxytocin has also been implicated in mediating pair
bonding behaviors in prairie voles (Insel and Hulihan 1995), although its role has only
been examined in the context of mating-induced pair formation. Mesotocin, the avian
homologue of oxytocin, may function as oxytocin does in mammals. Goodson et al.
(2004) found no role for vasotocin and mesotocin in individual recognition in male and
female zebra finches, but the cohabitation time used in their study was much shorter
than is usually required for the formation of a bond in this species.

One other molecule, norepinephrine, might be also be critical in regulation of
these behaviors. Norepinephrine is important for attention and focusing on salient
information (reviewed in Berridge and Waterhouse 2003), which is critical to the
presentation of preference in both of the behavioral paradigms examined in this

dissertation. Mammalian studies have demonstrated a role for norepinephrine in

138

increasing the magnitude and latency of responses to specific stimuli and perhaps
reducing spontaneous neuronal firing (reviewed in Berridge and Waterhouse 2003),
which can result in the ability to distinguish salient information from noise. In addition,
norepinephrine can enhance long-term potentiation (see Berridge and Waterhouse 2003)
and mediates emotional memory formation in the amygdala (Cahill and McGaugh
1996). In birds, norepinephrine mediates the ability of females to respond to song when
auditory distracters are present (Appeltants et al. 2002), and its release and innervation
is increased after long-term exposure to high quality song (Sockman and Salvante
2008)

In conclusion, in these studies I have noted several unique behavioral
characteristics of zebra finches. 1n addition, I have identified a brain region (the
nucleus taeniae) that may be critical for the maintenance or response to pairing in
female zebra finches. Finally, I observed an effect of estrogen on calling and visual
scanning behavior and response of the auditory perception systems and the
ventromedial hypothalamus. All of these results are novel, and warrant future
investigation to more fully elucidate the mechanisms associated with mate choice and

subsequent responses once a partnership has been formed.

139

REFERENCES

Absil, P. and J. Balthazart (1994). Testosterone effects on neurotensin-immunoreactive
cells in the quail preoptic area. Neuroreport 5(9): 1129-32.

Absil, P., M. Papello, et al. (2002). The medial preoptic nucleus receives vasotocinergic
inputs in male quail: a tract-tracing and immunocytochemical study. J Chem
Neuroanat 24(1): 27-39.

Adkins-Regan, E. (2002). Development of sexual partner preference in the zebra finch:
a socially monogamous, pair-bonding animal. Arch Sex Behav 31(1): 27-33.

Adkins-Regan, E. (2009). Neuroendocrinology of social behavior. ILAR J 50(1): 5-14.

Adkins-Regan, E., M. Abdelnabi, et al. (1990). Sex Steroid Levels in Developing and
Adult Male and Female Zebra Finches (Poephila guttata). Gen Comp
Endocrinol 78: 93-109.

Adkins-Regan, E. and M. Ascenzi (1987). Social and sexual behaviour of male and
female zebra finches treated with oestradiol during the nestling period. Anim
Behav 35: 1100-1112.

Adkins-Regan, E. and J. Wade (2001). Masculinized sexual partner preference in
female zebra finches with sex-reversed gonads. Horm Behav 39(1): 22-8.

Akins, C. K. and E. H. Geary (2008). Cocaine-induced behavioral sensitization and
conditioning in male Japanese quail. Pharmacol Biochem Behav 88(4): 432-7.

Akins, C. K., N. Levens, et al. (2004). Dose-dependent cocaine place conditioning and
D1 dopamine antagonist effects in male Japanese quail. Physiol Behav 82(2-3):
309-15.

Albin, R. L., A. B. Young, et al. (1989). The functional anatomy of basal ganglia
disorders. Trends Neurosci 12(10): 366-75.

Alger, S. J. and L. V. Riters (2006). Lesions to the medial preoptic nucleus
differentially affect singing and nest box-directed behaviors within and outside
of the breeding season in European starlings (Sturnus vulgaris). Behav Neurosci
120(6): 1326-36.

Anderson, K. D. and A. Reiner (1991). Striatonigral projection neurons: a retrograde
labeling study of the percentages that contain substance P or enkephalin in
pigeons. J Comp Neurol 303(4): 658-73.

Anokhin, K. V. and S. P. Rose (1991). Leaming-induced Increase of Immediate Early
Gene Messenger RNA in the Chick Forebrain. Eur J Neurosci 3(2): 162-167.

140

Appeltants, D., G. F. Ball, et al. (2001). The distribution of tyrosine hydroxylase in the
canary brain: demonstration of a specific and sexually dimorphic

catecholaminergic innervation of the telencephalic song control nulcei. Cell
Tissue Res 304(2): 237-59.

Appeltants, D., C. Del Negro, et al. (2002). Noradrenergic control of auditory
information processing in female canaries. Behav Brain Res 133(2): 221-35.

Aragona, B. J ., Y. Liu, et al. (2003). A critical role for nucleus accumbens dopamine in
partner-preference formation in male prairie voles. J Neurosci 23(8): 3483-90.

Aragona, B. J ., Y. Liu, et al. (2006). Nucleus accumbens dopamine differentially
mediates the formation and maintenance of monogamous pair bonds. Nat
Neurosci 9(1): 133-9.

Aron, A., H. Fisher, et al. (2005). Reward, motivation, and emotion systems associated
with early-stage intense romantic love. J Neurophysiol 94(1): 327-37.

Aste, N., J. Balthazart, et al. (1998). Anatomical and neurochemical definition of the
nucleus of the stria terminalis in Japanese quail (Cotumix japonica). J Comp
Neurol 396(2): 141-57.

Aste, N., E. Muhlbauer, et al. (1996). Distribution of AVT gene expressing neurons in
the prosencephalon of japanese quail and chicken. Cell Tissue Res 286(3): 365-
73.

Aste, N., C. Viglietti-Panzica, et al. (1995). Mapping of neurochemical markers in quail
central nervous system: VIP- and SP-like immunoreactivity. J Chem Neuroanat
8(2): 87-102.

Avey, M. T., L. S. Phillmore, et al. (2005). Immediate early gene expression following
exposure to acoustic and visual components of courtship in zebra finches. Behav
Brain Res 165(2): 247-53.

Bailey, D. J. (2006). Neurobiology of song learning and perception in the zebra finch
(T aeniopygia guttata), with a focus on the role of the hippocampus.
Dissertation. Department of Psychology. East Lansing, Michigan State
University.

Bailey, D. J ., J. C. Rosebush, et al. (2002). The Hippocampus and Caudomedial
Neostriatum Show Selective Responsiveness to Conspecific Song in the Female
Zebra Finch. J Neurobiol 52: 43-51.

Bailey, D. J. and J. Wade (2003). Differential expression of the immediate early genes
F OS and ZENK following auditory stimulation in the juvenile male and female
zebra finch. Mol Brain Res 116: 147-154.

141

Bailey, D. J. and J. Wade (2005). FOS and ZENK responses in 45-day-old zebra finches
vary with auditory stimulus and brain region, but not sex. Behav Brain Res
162(1): 108-15.

Bailhache, T. and J. Balthazart (1993). The catecholaminergic system of the quail brain:

immunocytochemical studies of dopamine beta-hydroxylase and tyrosine
hydroxylase. J Comp Neurol 329(2): 230-56.

Bale, T. L., A. M. Davis, et al. (2001). CNS region-specific oxytocin receptor

expression: importance in regulation of anxiety and sex behavior. J Neurosci
21(7): 2546-52.

Bales, K. L., W. A. Mason, et al. (2007). Neural correlates of pair-bonding in a
monogamous primate. Brain Res 1184: 245-53.

Balint, E. and A. Csillag (2007). Nucleus accumbens subregions: hodological and
immunohistochemical study in the domestic chick (Gallus domesticus). Cell
Tissue Res 327(2): 221-30.

Ball, G. F. and J. Balthazart (2002). Neuroendocrine Mechanisms Regulating
Reproductive Cycles and Reproductive Behavior in Birds. Non-Mammalian
Hormone-Behavior Systems. San Diego, Elsevier B.V. 2: 649-788.

Ball, G. F. and T. Q. Gentner (1998). They're Playing Our Song: Gene Expression and
Birdsong Perception. Neuron 21: 271-274.

Ballentine, B., J. Hyman, et a1. (2004). Vocal performance influences female response
to male bird song: an experimental test. Behav Ecol 15(1): 163-168.

Balthazart, J ., P. Absil, et al. (1996). Distribution of aromatase-immunoreactive cells in
the forebrain of zebra finches (Taeniopygia guttata): implications for the neural

action of steroids and nuclear definition in the avian hypothalamus. J Neurobiol
31(2): 129-48.

Balthazart, J ., V. Dupiereux, et al. (1994). Afferent and efferent connections of the
sexually dimorphic medial preoptic nucleus of the male quail revealed by in
vitro transport of DiI. Cell Tissue Res 276(3): 455-75.

Balthazart, J ., A. Foidart, et al. (1990). Distribution of aromatase in the brain of the
Japanese quail, ring dove, and zebra finch: an immunocytochemical study. J
Comp Neurol 301(2): 276-88.

Balthazart, J ., M. Gahr, et al. (1989). Distribution of estrogen receptors in the brain of
the Japanese quail: an immunocytochemical study. Brain Res 501(2): 205-14.

Balthazart, J ., A. Stamatakis, et al. (2001). Effects of lesions of the medial preoptic
nucleus on the testosterone-induced metabolic changes in specific brain areas in
male quail. Neuroscience 108(3): 447-66.

142

Balthazart, J. and C. Surlemont (1990). Androgen and estrogen action in the preoptic
area and activation of copulatory behavior in quail. Physiol Behav 48(5): 599-
609.

Bannon, M. J. and R. H. Roth (1983). Pharmacology of mesocortical dopamine
neurons. Pharmacol Rev 35(1): 53-68.

Baptista, L. F. and L. Petrinovich (1986). Song development in the white-crowned
sparrow - social-factors and sex-differences. Anim Behav 34(5): 1359-1371.

Bardo, M. T. (1998). Neuropharrnacological mechanisms of drug reward: beyond
dopamine in the nucleus accumbens. Crit Rev Neurobiol 12(1-2): 37-67.

Barfield, R. J. (1969). Activation of copulatory behavior by androgen implanted into the
preoptic area of the male fowl. Horm Behav 1: 37-52.

Bartels, A. and S. Zeki (2000). The neural basis of romantic love. Neuroreport 11(17):
3829-34.

Bartels, A. and S. Zeki (2004). The neural correlates of maternal and romantic love.
Neuroimage 21(3): 1155-66.

Baxter, M. G. and E. A. Murray (2002). The amygdala and reward. Nat Rev Neurosci
3(7): 563-73.

Bazzett, T. J. and J. B. Becker (1994). Sex differences in the rapid and acute effects of
estrogen on striatal D2 dopamine receptor binding. Brain Res 637(1-2): 163-72.

Becker, J. B. and M. E. Beer (1986). The influence of estrogen on nigrostriatal
dopamine activity: behavioral and neurochemical evidence for both pre- and
postsynaptic components. Behav Brain Res 19(1): 27-33.

Becker, J. B. and J. H. Cha (1989). Estrous cycle-dependent variation in amphetamine-
induced behaviors and striatal dopamine release assessed with microdialysis.
Behav Brain Res 35(2): 1 17-25.

Becker, J. B., C. N. Rudick, et al. (2001). The role of dopamine in the nucleus
accmnbens and striatum during sexual behavior in the female rat. J Neurosci
21(9): 3236-41.

Behrouz, B., R. E. Drolet, et al. (2007). Unique responses to mitochondrial complex I
inhibition in tuberoinfundibular dopamine neurons may impart resistance to
toxic insult. Neuroscience 147(3): 592-8.

Bennett, A. L., B. Greco, et al. (2002). Response to male odours in progestin receptor-
and oestrogen receptor-containing cells in female rat brain. J Neuroendocrinol
14(6): 442-9.

143

Berk, M. L. and A. B. Butler (1981). Efferent projections of the medial preoptic nucleus
and medial hypothalamus in the pigeon. J Comp Neurol 203(3): 379-99.

Berridge, C. W. and B. D. Waterhouse (2003). The locus coeruleus-noradrenergic
system: modulation of behavioral state and state-dependent cognitive processes.
Brain Res Brain Res Rev 42(1): 33-84.

Bian, X., Y. Yanagawa, et al. (2008). Cortical-like functional organization of the
pheromone-processing circuits in the medial amygdala. J Neurophysiol 99(1):
77-86.

Bielsky, I. F ., S. B. Hu, et al. (2004). Profound impairment in social recognition and
reduction in anxiety-like behavior in vasopressin Vla receptor knockout mice.
Neuropsychopharmacology 29(3): 483-93.

Bielsky, I. F. and L. J. Young (2004). Oxytocin, vasopressin, and social recognition in
mammals. Peptides 25(9): 1565-74.

Birkhead, T. R. and F. M. Hunter (1990). Numbers of Sperm-Storage Tubules in the
Zebra Finch (Poephila-Guttata) and Bengalese Finch (Lonchura-Striata). Auk
107(1): 193-197.

Birkhead, T. R., F. M. Hunter, et al. (1989). Sperm competition in the Zebra Finch
Taeniopygia guttata. Anim Behav 38: 935-950.

Black, J. M., Ed. (1996). Partnerships in birds: the study of monogamy. Oxford, UK,
Oxford University Press.

Blahser, S. and M. P. Dubois (1980). Immunocytochemical demonstration of met-
enkephalin in the central nervous system of the domestic fowl. Cell Tissue Res
213(1): 53-68.

Bolam, J. P., B. H. Wainer, et al. (1984). Characterization of cholinergic neurons in the
rat neostriatum. A combination of choline acetyltransferase
immunocytochemistry, Golgi-impregnation and electron microscopy.
Neuroscience 12(3): 711-8.

Bolhuis, J. J ., E. Hetebrij, et al. (2001). Localized immediate early gene expression
related to the strength of song learning in socially reared zebra finches.
European Journal of Neuroscience 13: 2165-2170.

Bottjer, S. W. (1993). The distribution of tyrosine hydroxylase immunoreactivity in the
brains of male and female zebra finches. J Neurobiol 24(1): 51-69.

Braaten, R. F ., S. S. Miner, et al. (2008). Song recognition memory in juvenile zebra
finches: effects of varying the number of presentations of heterospecific and
conspecific songs. Behav Processes 77(2): 177-83.

144

Braaten, R. F., M. Petzoldt, et al. (2006). Song perception during the sensitive period of
song learning in zebra finches (Taeniopygia guttata). J Comp Psycho] 120(2):
79-88.

Braaten, R. F. and K. Reynolds (1999). Auditory preference for conspecific song in
isolation-reared zebra finches. Anim Behav 58(1): 105-111.

Brainard, M. S. and A. J. Doupe (2000). Auditory feedback in learning and maintenance
of vocal behaviour. Nat Rev Neurosci 1(1): 31-40.

Brauth, S. E. and A. Reiner (1991). Calcitonin-gene related peptide is an evolutionarily
conserved marker within the amniote thalamo-telencephalic auditory pathway. J
Comp Neurol 313(2): 227-39.

Bray, G. A. and D. A. York (1979). Hypothalamic and genetic obesity in experimental
animals: an autonomic and endocrine hypothesis. Physio] Rev 59(3): 719-809.

Brennan, P. A., D. Hancock, et al. (1992). The expression of the immediate-early genes
c-fos, egr-l and c-jun in the accessory olfactory bulb during the formation of an
olfactory memory in mice. Neuroscience 49(2): 277-84.

Buchanan, K. L. and C. K. Catchpole (2000). Song as an indicator of male parental
' effort in the sedge warbler. Proc Biol Sci 267(1441): 321-6.

Butterfield, P. A. (1970). The Pair Bond in the Zebra Finch. Social Behaviour in Birds
and Mammals. J. H. Crook. New York, Academic Press Inc.: 249-278.

Caffe, A. R., F. W. van Leeuwen, et al. (1987). Vasopressin cells in the medial

amygdala of the rat project to the lateral septum and ventral hippocampus. J
Comp Neurol 261(2): 237-52.

Cahill, L. and J. L. McGaugh (1996). Modulation of memory storage. Curr Opin
Neurobiol 6(2): 237-42.

Calhoun, 8., S. H. Hulse, et al. (1993). Responsiveness to Conspecific and Alien Song
by Canaries (Serinus canaria) and European Starlings (Sturnus vulgaris) As a
Function of Photoperiod. J Comp Psycholy 107(3): 235-241.

Canteras, N. S., R. B. Simerly, et al. (1994). Organization of projections from the
ventromedial nucleus of the hypothalamus: a Phaseolus vulgaris-leucoagglutinin
study in the rat. J Comp Neurol 348(1): 41-79.

Canteras, N. S., R. B. Simerly, et al. (1995). Organization of projections from the
medial nucleus of the amygdala: a PHAL study in the rat. J Comp Neurol
360(2): 213-45.

Caryl, P. G. (1976). Sexual-Behavior in Zebra Finch Taeniopygia-Guttata - Response to
Familiar and Novel Partners. Anim Behav 24(Feb): 93-107.

145

Cattaneo, E. and A. Maggi (1990). c-fos induction by estrogen in specific rat brain
areas. Eur J Pharmacol 188(2-3): 153-9.

Cheng, M., M. Chaiken, et al. (1999). Nucleus taenia of the amygdala of birds:
anatomical and functional studies in ring doves (Streptopelia risoria) and
European starlings (Sturnus vulgaris). Brain Behav Evol 53(5-6): 243-70.

Cheng, M. F. and J. P. Peng (1997). Reciprocal talk between the auditory thalamus and
the hypothalamus: an antidromic study. Neuroreport 8(3): 653-8.

Cheng, M. F., J. P. Peng, et al. (1998). Hypothalamic neurons preferentially respond to
female nest coo stimulation: demonstration of direct acoustic stimulation of
luteinizing hormone release. J Neurosci 18(14): 5477-89.

Cheng, M. F. and M. Zuo (1994). Proposed pathways for vocal self-stimulation: met-
enkephalinergic projections linking the midbrain vocal nucleus, auditory-
responsive thalamic regions and neurosecretory hypothalamus. J Neurobiol
25(4): 361-79.

Chew, S. J ., C. V. Mello, et a1. (1995). Decrements in Auditory Responses to a
Repeated Conspecific Song are Long-Lasting and Require Two Periods of
Protein Synthesis in the Songbird F orebrain. Proc Nat] Acad Sci USA 92(8):
3406-3410.

Christie, M. J ., R. J. Summers, et al. (1987). Excitatory amino acid projections to the
nucleus accumbens septi in the rat: a retrograde transport study utilizing
D[3H]aspartate and [3H]GABA. Neuroscience 22(2): 425-39.

Churchill, L. and P. W. Kalivas (1994). A topographically organized garnma-
arninobutyric acid projection from the ventral pallidum to the nucleus
accumbens in the rat. J Comp Neurol 345(4): 579-95.

Clayton, D. F. (1997). Role of gene regulation in song circuit development and song
learning. J Neurobiol 33(5): 549-71.

Clayton, N. (1988). Song discrimination learning in zebra finches. Anim Behav 36:
1016-1024.

Clayton, N. and E. Prove (1989). Song Discrimination in Female Zebra Finches and
Bengalese Finches. Anim Behav 38: 352-362.

Clayton, N. S. (1990). Mate Choice and Pair Formation in Timor and Australian
Mainland Zebra Finches. Anim Behav 39: 474-480.

Commons, K. G., L. M. Kow, et al. (1999). In the ventromedial nucleus of the rat
hypothalamus, GABA-immunolabeled neurons are abundant and are innervated
by both enkephalin- and GABA-immunolabeled axon terminals. Brain Res
816(1): 58-67.

146

Commons, K. G. and D. W. Pfaff (2001). Ultrastructural evidence for enkephalin
mediated disinhibition in the ventromedial nucleus of the hypothalamus. J Chem
Neuroanat 21(1): 53-62.

Cornil, C., A. Foidart, et al. (2000). Immunocytochemical localization of ionotropic
glutamate receptors subunits in the adult quail forebrain. J Comp Neurol 428(4):
577-608.

Comil, C. A., V. Seutin, et a1. (2004). Electrophysiological and neurochemical
characterization of neurons of the medial preoptic area in Japanese quail
(Cotumix japonica). Brain Res 1029(2): 224-40.

Cozzi, B., C. Viglietti-Panzica, et al. (1991). The serotoninergic system in the brain of
the Japanese quail. An immunohistochemical study. Cell Tissue Res 263(2):
271-84.

Csillag, A., A. D. Szekely, et al. (1997). Synaptic terminals immunolabelled against
glutamate in the lobus parolfactorius of domestic chicks (Gallus domesticus) in
relation to afferents from the archistriatum. Brain Res 750(1-2): 171-9.

Curtis, J. T. and Z. Wang (2003). Forebrain c-fos expression under conditions
conducive to pair bonding in female prairie voles (Microtus ochrogaster).
Physiol Behav 80(1): 95-101.

Cushing, B. S., N. Mogekwu, et al. (2003). Cohabitation induced Fos immunoreactivity
in the monogamous prairie vole. Brain Res 965(1-2): 203-11.

Damsma, G., J. G. Pfaus, et a1. (1992). Sexual behavior increases dopamine
transmission in the nucleus accumbens and striatum of male rats: comparison
with novelty and locomotion. Behav Neurosci 106(1): 181-91.

de Lanerolle, N. C., R. P. Elde, et al. (1981). Distribution of methionine-enkephalin
immunoreactivity in the chick brain: an immunohistochemical study. J Comp
Neurol 199(4): 513-33.

de Vries, G. J. and M. A. Miller (1998). Anatomy and function of extrahypothalamic
vasopressin systems in the brain. Prog Brain Res 119: 3-20.

De Vries, G. J. and G. C. Panzica (2006). Sexual differentiation of central vasopressin
and vasotocin systems in vertebrates: different mechanisms, similar endpoints.

Neuroscience 138(3): 947-55.

Delfs, J. M., Y. Zhu, et al. (1998). Origin of noradrenergic afferents to the shell
subregion of the nucleus accumbens: anterograde and retrograde tract-tracing
studies in the rat. Brain Res 806(2): 127-40.

147

DeVries, A. C., M. B. DeVries, et a1. (1995). Modulation of pair bonding in female
prairie voles (Microtus ochrogaster) by corticosterone. Proc Nat] Acad Sci U S A
92(17): 7744-8.

DeVries, G. J ., R. M. Buijs, et al. (1985). The vasopressinergic innervation of the brain
in normal and castrated rats. J Comp Neurol 233(2): 236-54.

Ding, L. and D. J. Perkel (2002). Dopamine modulates excitability of spiny neurons in
the avian basal ganglia. J Neurosci 22(12): 5210-8.

Dolby, A. S., C. E. Clarkson, et al. (2005). Do song-phrase production rate and song
versatility honestly communicate male parental quality in the Gray Catbird?
Journal of Field Ornithology 76(3): 287-292.

Domenici, L., H. J. Waldvogel, et al. (1988). Distribution of GABA-like
immunoreactivity in the pigeon brain. Neuroscience 25(3): 931-50.

Dominguez, J. M. and E. M. Hull (2005). Dopamine, the medial preoptic area, and male
sexual behavior. Physio] Behav 86(3): 356-68.

DonCarlos, L. L., E. Monroy, et al. (1991). Distribution of estrogen receptor-
immunoreactive cells in the forebrain of the female guinea pig. J Comp Neurol
305(4): 591-612.

Dong, H. W., G. D. Petrovich, et a1. (2001). Topography of projections from amygdala
to bed nuclei of the stria terminalis. Brain Res Brain Res Rev 38(1-2): 192-246.

Donoghue, J. P. and S. T. Kitai (1981). A collateral pathway to the neostriatum from

corticofugal neurons of the rat sensory-motor cortex: an intracellular HRP study.
JComp Neuro] 201(1): 1-13.

Duckworth, J. W. (1992). Effects of mate removal on the behaviour and reproductive
success of reed warblers Acrocephalus scirpaceus. Ibis 134: 164-170.

Durand, S. E., J. M. Tepper, et al. (1992). The shell region of the nucleus ovoidalis: a
subdivision of the avian auditory thalamus. J Comp Neurol 323(4): 495-518.

Durstewitz, D., S. Kroner, et al. (1999). The dopaminergic innervation of the avian
telencephalon. Prog Neurobiol 59(2): 161-95.

Eaton, M. J ., Y. Tian, et al. (1992). Comparison of the effects of remoxipride and
raclopride on nigrostriatal and mesolimbic dopaminergic neuronal activity and
on the secretion of prolactin and a1pha-melanocyte-stimulating hormone.
Neuropsychopharmacologv 7(3): 205-1 1.

Eiden, L. E., T. Hokfelt, et al. (1985). Vasoactive intestinal polypeptide afferents to the
bed nucleus of the stria terminalis in the rat: an immunohistochemical and
biochemical study. Neuroscience 15(4): 999-1013.

148

Emery, D. E. and R. L. Moss (1984). Lesions confined to the ventromedial
hypothalamus decrease the frequency of coital contacts in female rats. Horm
Behav 18(3): 313-29.

Engelmann, M. and R. Landgraf (1994). Microdialysis Administration of Vasopressin
into the Septum Improves Social Recognition in Brattleboro Rats. Physiology &
Behavior 55(1): 145-149.

Esch, T. and G. B. Stefano (2004). The neurobiology of pleasure, reward processes,
addiction and their health implications. Neuro Endocrinol Lett 25(4): 235-51.

Espana, R. A. and C. W. Berridge (2006). Organization of noradrenergic efferents to
arousal-related basal forebrain structures. J Comp Neurol 496(5): 668-83.

F abre-Nys, C., S. Ohkura, et al. (1997). Male faces and odours evoke differential
patterns of neurochemical release in the mediobasal hypothalamus of the ewe
during oestrus: an insight into sexual motivation? Eur J Neurosci 9(8): 1666-77.

F ahrbach, S. E., J. I. Morrell, et al. (1989). Studies of ventromedial hypothalamic
afferents in the rat using three methods of HRP application. Exp Brain Res
77(2): 221-33.

Fallon, J. H. and R. Y. Moore (1978). Catecholamine innervation of the basal forebrain.
IV. Topography of the dopamine projection to the basal forebrain and
neostriatum. J Comp Neurol 180(3): 545-80.

Famer, D. S. and D. L. Serventy (1960). The Timing of Reproduction in Birds in the
Arid Regions of Australia. Anatom Rec 137(3): 354-354.

Figueiredo, H. F., B. L. Bodie, et al. (2003). Stress integration after acute and chronic
predator stress: differential activation of central stress circuitry and sensitization
of the hypothalamo-pituitary-adrenocortical axis. Endocrinology 144(12): 5249-
58.

Foidart, A., A. de Clerck, et al. (1994). Aromatase-immunoreactive cells in the quail
brain: effects of testosterone and sex dimorphism. Physiol Behav 55(3): 453-64.

Foidart, A., J. Reid, et al. (1995). Critical re-examination of the distribution of
aromatase-immunoreactive cells in the quail forebrain using antibodies raised
against human placental aromatase and against the recombinant quail, mouse or
human enzyme. J Chem Neuroanat 8(4): 267-82.

Fuller, T. A., F. T. Russchen, et a1. (1987). Sources of presumptive

glutamergic/aspartergic afferents to the rat ventral striatopallidal region. J Comp
Neuro] 258(3): 317-38.

149

Gahr, M., H. R. Guttinger, et al. (1993). Estrogen receptors in the avian brain: survey
reveals general distribution and forebrain areas unique to songbirds. J Comp
Neurol 327(1): 112-22.

Gahr, M. and R. Metzdorf (1997). Distribution and dynamics in the expression of
androgen and estrogen receptors in vocal control systems of songbirds. Brain
Res Bull 44(4): 509-17.

Gale, S. D. and D. J. Perkel (2005). Properties of dopamine release and uptake in the
songbird basal ganglia. J Neurophysiol 93(4): 1871-9.

Gale, S. D. and D. J. Perkel (2006). Physiological properties of zebra finch ventral
tegmental area and substantia nigra pars compacta neurons. J Neurophysiol
96(5): 2295-306.

Geary, E. H. and C. K. Akins (2007). Cocaine sensitization in male quail: temporal,
conditioning, and dose-dependent characteristics. Physio] Behav 90(5): 818-24.

Gentner, T. Q., S. H. Hulse, et a1. (2001). Response Biases in Auditory F orebrain
Regions of Female Songbirds Following Exposure to Sexually Relevant
Variation in Male Song. J Neurobiol 46: 48-58.

George, 1., H. Cousillas, et al. (2008). A potential neural substrate for processing
functional classes of complex acoustic signals. PLoS ONE 3(5): e2203.

Gerfen, C. R. (1992). The neostriatal mosaic: multiple levels of compartmental
organization in the basal ganglia. Annu Rev Neurosci 15: 285-320.

Gibson, M. J. and M. F. Cheng (1979). Neural mediation of estrogen-dependent
courtship behavior in female ring doves. Journal of Comparative and
Physiological Psychology 93: 855-867.

Gingrich, B., Y. Liu, et al. (2000). Dopamine D2 receptors in the nucleus accumbens
are important for social attachment in female prairie voles (Microtus
ochrogaster). Behav Neurosci 114(1): 173-83.

Gobes, S. M. and J. J. Bolhuis (2007). Birdsong memory: a neural dissociation between
song recognition and production. Curr Biol 17(9): 789-93.

Godwin, J. and D. Crews (2002). Hormones, Brain, and Behavior in Reptiles. Non-
Mammalian Hormone-Behavior Systems. D. Pfaff, A. P. Arnold, A. M. Etgen, S.
Fahrbach and R. Rubin. San Diego, Elsevier B.V. 2: 545-585.

Goodson, J. L. and A. H. Bass (200]). Social behavior functions and related anatomical

characteristics of vasotocin/vasopressin systems in vertebrates. Brain Res Brain
Res Rev 35(3): 246-65.

150

Goodson, J. L., A. K. Evans, et al. (2005). Neuro-evolutionary patterning of sociality.
Proc Biol Sci 272(1560): 227-35.

Goodson, J. L. (2005). The vertebrate social behavior network: evolutionary themes and
variations. Horm Behav 48(1): 11-22.

Goodson, J. L., A. K. Evans, et al. (2006). Neuropeptide binding reflects convergent
and divergent evolution in species-typical group sizes. Horm Behav 50(2): 223-
36.

Goodson, J. L., L. Lindberg, et al. (2004). Effects of central vasotocin and mesotocin
manipulations on social behavior in male and female zebra finches. Horm Behav
45(2): 136-43.

Goodson, J. L., J. Rinaldi, et a1. (2009). Vasotocin neurons in the bed nucleus of the
stria terminalis preferentially process social information and exhibit properties
that dichotomize courting and non-courting phenotypes. Horm Behav 55(1):
197-202.

Grace, J. A., N. Amin, et al. (2003). Selectivity for conspecific song in the zebra finch
auditory forebrain. J Neurophysiol 89(1): 472-87.

Granda, R. H. and W. J. Crossland (1989). GABA-like immunoreactivity of neurons in
the chicken diencephalon and mesencephalon. J Comp Neuro] 287(4): 455-69.

Grossmann, R., A. Jurkevich, et al. (2002). Sex dimorphism in the avian arginine
vasotocin system with special emphasis to the bed nucleus of the stria
terminalis. Comp Biochem Physio] A -Mol Integ Physio] 131(4): 833-837.

Gu, G., A. Cornea, et al. (2003). Sexual differentiation of projections from the principal
nucleus of the bed nuclei of the stria terminalis. J Comp Neuro] 460(4): 542-62.

Guzowski, J. F., B. Setlow, et al. (2001). Experience-Dependent Gene Expression in the
Rat Hippocampus after Spatial Learning: A Comparison of the Immediate-Early
Genes Arc, c-fos, and zif268. J Neuroscience 21(14): 5089-5098.

Halldin, K., J. Axelsson, et a1. (2006). Localization of estrogen receptor-alpha and -
betamRNA in brain areas controlling sexual behavior in Japanese quail. J
Neurobiol 66(2): 148-54.

Hasselquist, D., S. Bensch, et al. (1996). Correlation between male song repertoire,
extra-pair paternity and offspring survival in the great reed warbler. Nature 381:
229-232.

Hassler, R., P. Haug, et al. (1982). Effect of motor and premotor cortex ablation on
concentrations of amino acids, monoamines, and acetylcholine and on the
ultrastructure in rat striatum. A confirmation of glutamate as the specific cortico-
striatal transmitter. J Neurochem 38(4): 1087-98.

151

Hauber, W. (1998). Involvement of basal ganglia transmitter systems in movement
initiation. Prog Neurobiol 56(5): 507-40.

Heimovics, S. A. and L. V. Riters (2005). Immediate early gene activity in song control
nuclei and brain areas regulating motivation relates positively to singing
behavior during, but not outside of, a breeding context. J Neurobiol 65(3): 207-
24.

Heimovics, S. A. and L. V. Riters (2006). Breeding-context-dependent relationships
between song and cFOS labeling within social behavior brain regions in male
European starlings (Sturnus vulgaris). Horm Behav 50(5): 726-35.

Heimovics, S. A. and L. V. Riters (2007). ZENK labeling within social behavior brain
regions reveals breeding context-dependent patterns of neural activity associated
with song in male European starlings (Sturnus vulgaris). Behav Brain Res
176(2): 333-43.

Hoffman, G. E. and D. Lyo (2002). Anatomical markers of activity in neuroendocrine
systems: are we all 'fos-ed out"? J Neuroendocrinol 14(4): 259-68.

Hoffman, G. E., M. S. Smith, et al. (1993). c-Fos and related immediate early gene

products as markers of activity in neuroendocrine systems. Front
Neuroendocrinol 14(3): 1 73-21 3.

Hoshina, Y., T. Takeo, et al. (1994). Axon-sparing lesion of the preoptic area enhances
receptivity and diminishes proceptivity among components of female rat sexual
behavior. Behav Brain Res 61(2): 197-204.

Houx, B. B. and C. ten Cate (1999). Do Stimulus-Stimulus Contingencies Affect Song
Learning in Zebra Finches (T aeniopygia guttata)? J Comp Psycho] 113(3): 23 5-
242.

Houx, B. B. and C. ten Cate (1999). Song learning from playback in zebra finches: is
there an effect of operant contingency? Anim Behav 57(4): 83 7-845.

Huang, C. L. and J. A. Winer (2000). Auditory thalamocortical projections in the cat:
laminar and area] patterns of input. J Comp Neuro] 427(2): 302-31.

Hull, E. M. and J. M. Dominguez (2007). Sexual behavior in male rodents. Horm Behav
52(1): 45-55.

Hull, E. M., D. S. Lorrain, et al. (1999). Hormone-neurotransmitter interactions in the
control of sexual behavior. Behav Brain Res 105(1): 105-16.

Ikebuchi, M. and K. Okanoya (2006). Growth of pair bonding in Zebra Finches:
physical and social factors. Ornitho] Sci 5: 65-75.

152

Ikemoto, S. (2007). Dopamine reward circuitry: two projection systems from the ventral
midbrain to the nucleus accumbens-olfactory tubercle complex. Brain Res Rev
56(1): 27-78.

Inoue, S. and G. A. Bray (1979). An autonomic hypothesis for hypothalamic obesity.
Life Sci 25(7): 561-6.

Insel, T. R. (1990). Regional induction of c-fos-like protein in rat brain after estradiol
administration. Endocrinology 126(4): 1849-53.

Insel, T. R. and T. J. Hulihan (1995). A gender-specific mechanism for pair bonding:
oxytocin and partner preference formation in monogamous voles. Behav
Neurosci 109(4): 782-9.

Insel, T. R., Z. X. Wang, et al. (1994). Patterns of brain vasopressin receptor
distribution associated with social organization in microtine rodents. J Neurosci
14(9): 5381-92.

Jackson, G., A. L. Hudson, et a1. (2007). Pharmacological characterisation of the
electrically evoked release of monoamines from chicken brain in vitro. Br Poult
Sci 48(1): 76-83.

Jacobs, E. C., A. P. Arnold, et a1. (1996). Zebra finch estrogen receptor cDNA: cloning
and mRNA expression. J Steroid Biochem Mol Bio] 59(2): 135-45.

Jakab, R. L., T. L. Horvath, et al. (1993). Aromatase immunoreactivity in the rat brain:
gonadectomy-sensitive hypothalamic neurons and an unresponsive "limbic ring'
of the lateral septum-bed nucleus-amygdala complex. J Steroid Biochem Mol
Bio] 44(4-6): 481 -98.

Johnson, A. E. (1992). The regulation of oxytocin receptor binding in the ventromedial
hypothalamic nucleus by gonadal steroids. Ann N Y Acad Sci 652: 357-73.

Jurkevich, A., S. W. Barth, et a1. (1999). Development of sexually dimorphic
vasotocinergic system in the bed nucleus of stria terminalis in chickens. J Comp
Neurol 408(1): 46-60.

J urkevich, A. and R. Grossmann (2003). Vasotocin and reproductive functions of the
domestic chicken. Domest Anim Endocrinol 25(1): 93-9.

Kavaliers, M., D. D. Colwell, et al. (2003). Impaired discrimination of and aversion to
parasitized male odors by female oxytocin knockout mice. Genes Brain Behav
2(4): 220-30.

Kawaguchi, Y., C. J. Wilson, et al. (1995). Striatal intemeurones: chemical,

physiological and morphological characterization. Trends Neurosci 18(12): 527-
35.

153

Kern, M. D. and J. R. King (1972). Testosterone-Induced Singing in Female White-
Crowned Sparrows. Condor 74(2): 204-209.

Kimura, T., K. Okanoya, et al. (1999). Effect of testosterone on the distribution of
vasotocin immunoreactivity in the brain of the zebra finch, Taeniopygia guttata
castanotis. Life Sci 65(16): 1663-70.

Kirkpatrick, B., C. S. Carter, et a1. (1994). Axon-sparing lesions of the medial nucleus
of the amygdala decrease affiliative behaviors in the prairie vole (Microtus
ochrogaster): behavioral and anatomical specificity. Behav Neurosci 108(3):
501-13.

Kitt, C. A. and S. E. Brauth (1986). Telencephalic projections from midbrain and
isthmal cell groups in the pigeon. II. The nigral complex. J Comp Neuro] 247(1):
92-110.

Koch, M. and G. Ehret (1989). Immunocytochemical localization and quantitation of
estrogen-binding cells in the male and female (virgin, pregnant, lactating) mouse
brain. Brain Res 489(1): 101-12.

Kohlert, J. G. and R. L. Meisel (1999). Sexual experience sensitizes mating-related
nucleus accumbens dopamine responses of female Syrian hamsters. Behav Brain
Res 99(1): 45-52.

Kollack-Walker, S. and S. W. Newman (1995). Mating and agonistic behavior produce
different patterns of F os immunolabeling in the male Syrian hamster brain.
Neuroscience 66(3): 721-36.

Korenbrot, C. C., D. W. Schomberg, et al. (1974). Radioimmunoassay of Plasma
Estradiol During the Breeding Cycle of Ring Doves (Streptopelia risoria).
Endocrinology 94: 1 126-1 132.

Kovacs, K. J. (2008). Measurement of immediate-early gene activation- c-fos and
beyond. J Neuroendocrinol 20(6): 665-72.

Kow, L. M. and D. W. Pfaff (1985). Estrogen effects on neuronal responsiveness to
electrical and neurotransmitter stimulation: an in vitro study on the ventromedial
nucleus of the hypothalamus. Brain Res 347(1): 1-10.

Kroodsma, D. E. and E. H. Miller (1996). Ecology and evolution of acoustic
communication in birds. Ithaca, New York, Cornell University Press.

Kruse, A. A., R. Stripling, et a1. (2004). Context-specific habituation of the zenk gene
response to song in adult zebra finches. Neurobiol Learn Mem 82: 99-108.

Kuenzel, W. J. (1994). Central neuroanatomical systems involved in the regulation of
food intake in birds and mammals. J Nutr 124(8 Suppl): 1355S-137OS.

154

Kuppers, E., T. Ivanova, et a1. (2000). Estrogen: a multifunctional messenger to
nigrostriatal dopaminergic neurons. J Neurocytol 29(5-6): 375-85.

Lagrange, A. H., E. J. Wagner, et al. (1996). Estrogen rapidly attenuates a GABAB
response in hypothalamic neurons. Neuroendocrinology 64(2): 114-23.

Lamprecht, R. and Y. Dudai (1996). Transient expression of c-Fos in rat amygdala
during training is required for encoding conditioned taste aversion memory.
Learn Mem 3(1): 31-41.

Landgraf, R., R. Gerstberger, et a1. (1995). V1 Vasopressin Receptor Antisense
Oligodeoxynucleotide into Septum Reduces Vasopressin Binding, Social
Discrimination Abilities, and Anxiety-Related Behavior in Rats. J Neurosci
15(6): 4250-4258.

Lanuza, E., D. C. Davies, et a1. (2000). Distribution of CGRP-like immunoreactivity in
the chick and quail brain. J Comp Neurol 421(4): 515-32.

Lauay, C., N. M. Gerlach, et al. (2004). Female zebra finches require early song
exposure to prefer high-quality song as adults. Animal Behaviour 68: 1249-
1255.

Lauber, A. H., C. V. Mobbs, et al. (1991). Estrogen receptor messenger RNA
expression in rat hypothalamus as a function of genetic sex and estrogen dose.
Endocrinology 129(6): 3180-6.

LeBlanc, M. M., C. T. Goode, et a1. (2007). Estradiol modulates brainstem
catecholaminergic cell groups and projections to the auditory forebrain in a
female songbird. Brain Res 1171: 93-103.

Leboucher, D., M. Kreutzer, et al. (1994). Copulation-solicitation Displays in Female
Canaries (Serinus canaria): are Oestradiol Implants Necessary? Ethology 97:
190-197.

LeDoux, J. (1998). Fear and the brain: where have we been, and where are we going?
Biol Psychiatry 44(12): 1229-38.

LeDoux, J. (2003). The emotional brain, fear, and the amygdala. Cell Mol Neurobiol
23(4-5): 727-38.

LeDoux, J. (2007). The amygdala. Curr Biol 17(20): R868-74.

Leitner, S., C. Voigt, et al. (2001). Seasonal activation and inactivation of song motor
memories in wild canaries is not reflected in neuroanatomical changes of
forebrain song areas. Horm Behav 40(2): 160-8.

155

Leitner, S., C. Voigt, et a1. (2005). Immediate early gene (ZENK, Arc) expression in the
auditory forebrain of female canaries varies in response to male song quality. J
Neurobiol 64(3): 275-84.

Levens, N. and C. K. Akins (2004). Chronic cocaine pretreatment facilitates Pavlovian
sexual conditioning in male Japanese quail. Pharmacol Biochem Behav 79(3):
451-7.

Levesque, D. and T. Di Paolo (1989). Chronic estradiol treatment increases
ovariectomized rat striatal D-1 dopamine receptors. Life Sci 45(19): 1813-20.

Lim, M. M., E. A. Hammock, et a1. (2004). The role of vasopressin in the genetic and
neural regulation of monogamy. J Neuroendocrinol 16(4): 325-32.

Lim, M. M. and L. J. Young (2006). Neuropeptidergic regulation of affiliative behavior
and social bonding in animals. Horm Behav 50(4): 506-17.

Lindley, S. E., J. W. Gunnet, et al. (1990). 3,4-Dihydroxyphenylacetic acid
concentrations in the intermediate lobe and neural lobe of the posterior pituitary

gland as an index of tuberohypophysial dopaminergic neuronal activity. Brain
Res 506(1): 133-8.

Lindvall, O. and A. Bjorklund (1974). The organization of the ascending catecholamine
neuron systems in the rat brain as revealed by the glyoxylic acid fluorescence
method. Acta Physiol Scand Suppl 412: 1-48.

Loughlin, S. E. and J. H. Fallon (1983). Dopaminergic and non-dopaminergic
projections to amygdala from substantia nigra and ventral tegmental area. Brain
Res 262(2): 334-8.

Louilot, A., J. L. Gonzalez-Mora, et al. (1991). Sex-related olfactory stimuli induce a
selective increase in dopamine release in the nucleus accumbens of male rats. A
voltarnmetric study. Brain Res 553(2): 313-7.

Lovem, M. B. and J. Wade (2003). Sex steroids in green anoles (Anolis carolinensis):
uncoupled maternal plasma and yolking follicle concentrations, potential
embryonic steroidogenesis, and evolutionary implications. Gen Comp
Endocrinol 134(2): 109- l 5.

Lowry, O. H., N. J. Rosebrough, et al. (1951). Protein measurement with the F olin
phenol reagent. Journal of Biological Chemistry 193: 265-275.

Lucas, R. E., A. E. Clark, et a1. (2003). Re-examining adaptation and the set point
model of happiness: reactions to changes in marital status. J Pers Soc Psycho]
84(3): 527-39.

Luine, V. N., D. R. Grattan, et al. (1997). Gonadal hormones alter hypothalamic GABA
and glutamate levels. Brain Res 747(1): 165-8.

156

MacDougall-Shackleton, S. A., S. H. Hulse, et al. (1998). Neural bases of song
preferences in female zebra finches (Taeniopygia guttata). Neuroreport 9(13):
3047-52.

Maney, D. L., D. J. Bernard, et a1. (2001). Gonadal steroid receptor mRN A in
catecholaminergic nuclei of the canary brainstem. Neurosci Lett 311(3): 189-92.

Maney, D. L., E. Cho, et al. (2006). Estrogen-dependent selectivity of genomic
responses to birdsong. Eur J Neurosci 23(6): 1523-9.

Maney, D. L., C. T. Goode, et al. (2008). Estradiol modulates neural responses to song
in a seasonal songbird. J Comp Neurol 511(2): 173-86.

Maney, D. L., E. A. MacDougall-Shackleton, et a1. (2003). Immediate early'gene

response to hearing song correlates with receptive behavior and depends on
dialect in a female songbird. J Comp Physio] A 189: 667-674.

Mansukhani, V., E. Adkins-Regan, et al. (1996). Sexual partner preference in female
zebra finches: the role of early hormones and social environment. Horm Behav
30(4): 506-13. ‘

Marler, P. (1997). Three models of song learning: evidence from behavior. J Neurobiol
33(5): 501-16.

Marler, P. and M. Tamura (1964). Culturally TransmittedPattems of Vocal Behavior in
Sparrows. Science 146: 1483-6.

Masco, D. H. and H. F. Carrer (1980). Sexual receptivity in female rats after lesion or
stimulation in different amygdaloid nuclei. Physio] Behav 24(6): 1073-80.

Mason, W. A. and S. P. Mendoza (1998). Generic aspects of primate attachments:
parents, offspring and mates. Psychoneuroendocrinologv 23(8): 765-78.

McCarthy, M. M. (1995). Estrogen modulation of oxytocin and its relation to behavior.
Adv Exp Med Biol 395: 235-45.

McGaugh, J. L. (2004). The amygdala modulates the consolidation of memories of
emotionally arousing experiences. Annu Rev Neurosci 27: 1-28.

McGaugh, J. L., L. Cahill, et a1. (1996). Involvement of the amygdala in memory
storage: interaction with other brain systems. Proc Nat] Acad Sci U S A 93(24):
13508-14.

Meddle, S. L., A. Foidart, et al. (1999). Effects of sexual interactions with a male on

fos-like immunoreactivity in the female quail brain. J Neuroendocrinol 11(10):
771-84.

157

Medina, L. and A. Reiner (1994). Distribution of choline acetyltransferase
immunoreactivity in the pigeon brain. J Comp Neuro] 342(4): 497-537.

Meisel, R. L. and B. D. Sachs (1994). The Physiology of Male Sexual Behavior. The
Physiology of Reproduction. E. Knobil and J. D. Neill. New York, Raven Press,
Ltd. 2: 3-105.

Melis, M. R. and A. Argiolas (1995). Dopamine and sexual behavior. Neurosci
Biobehav Rev 19(1): 19-38.

Mello, C. V. and D. F. Clayton (1994). Song-induced ZENK gene expression in
auditory pathways of songbird brain and its relation to the song control system. J
Neurosci 14(11 Pt 1): 6652-66.

Mello, C. V., R. Pinaud, et al. (1998). Noradrenergic system of the zebra finch brain:
immunocytochemical study of dopamine-beta-hydroxylase. J Comp Neuro]
400(2): 207-28.

Mello, C. V., D. S. Vicario, et al. (1992). Song presentation induces gene expression in
the songbird forebrain. Proc Nat] Acad Sci USA 89: 6818-6822.

Meredith, G. E., C. M. Pennartz, et al. (1993). The cellular framework for chemical
signalling in the nucleus accumbens. Prog Brain Res 99: 3-24.

Mermelstein, P. G. and J. B. Becker (1995). Increased extracellular dopamine in the
nucleus accumbens and striattun of the female rat during paced copulatory
behavior. Behav Neurosci 109(2): 354-65.

Merzenich, M. M., P. L. Knight, et al. (1975). Representation of cochlea within primary
auditory cortex in the cat. J Neurophysiol 38(2): 231-49.

Metzdorf, R., M. Gahr, et al. (1999). Distribution of aromatase, estrogen receptor, and
androgen receptor mRNA in the forebrain of songbirds and nonsongbirds. J
Comp Neuro] 407(1): 1 15-29.

Metzger, M., S. Jiang, et al. (1996). Organization of the dopaminergic innervation of
forebrain areas relevant to learning: a combined
immunohistochemical/retrograde tracing study in the domestic chick. J Comp
Neurol 376(1): 1-27.

Metzger, M., C. Toledo, et al. (2002). Serotonergic innervation of the telencephalon in
the domestic chick. Brain Res Bull 57(3-4): 547-51.

Mezey, S. and A. Csillag (2002). Selective striatal connections of midbrain
dopaminergic nuclei in the chick (Gallus domesticus). Cell Tissue Res 308(1):
35-46.

158

Mezey, S. E. and A. Csillag (2003). The light and electron microscopic characterisation
of identified striato-ventrotegmental projection neurons in the domestic chick
(Gallus domesticus). Neurosci Res 47(3): 299-308.

Miller, D. B. (1979). The acoustic basis of mate recognition by female Zebra finches
(T aeniopygia guttata). Anim Behav 27(2): 376-380.

Miller, D. B. (1979). Long-terrn recognition of father's song by female zebra finches.
Nature 280: 389-391.

Mitchell, J. B. and A. Gratton (1994). Involvement of mesolimbic dopamine neurons in
sexual behaviors: implications for the neurobiology of motivation. Rev Neurosci
5(4): 317-29.

Mogensen, J. and I. Divac (1982). The prefrontal 'cortex' in the pigeon. Behavioral
evidence. Brain Behav Evol 21(2-3): 60-6.

Montagnese, C. M., A. D. Szekely, et al. (2004). Efferent connections of septal nuclei
of the domestic chick (Gallus domesticus): an anterograde pathway tracing study
with a bearing on functional circuits. J Comp Neurol 469(3): 43 7-56.

Moore, R. Y., A. E. Halaris, et al. (1978). Serotonin neurons of the midbrain raphe:
ascending projections. J Comp Neurol 180(3): 417-38.

Morgan, J. I. and T. Curran (1989). Stimulus-transcription coupling in neurons: role of
cellular immediate-early genes. Trends Neurosci 12(11): 459-62.

Murray, E. A. (2007). The amygdala, reward and emotion. Trends C ogn Sci 11(11):
489-97.

Nagle, L., M. L. Kreutzer, et al. (1993). Obtaining copulation solicitation displays in
female canaries without estradiol implants. Experientia 49: 1022-1023.

Neal, J. K. and J. Wade (2007). Effects of season, testosterone and female exposure on
c-fos expression in the preoptic area and amygdala of male green anoles. Brain
Res 1166: 124-31.

Neubauer, R. L. (1999). Super-normal length song preferences of female zebra finches
(Taeniopygia guttata) and a theory of the evolution of bird song. Evolutionary
Ecology 13: 365-380.

Neumann, I. D. (2008). Brain oxytocin: a key regulator of emotional and social
behaviours in both females and males. J Neuroendocrinol 20(6): 858-65.

Newman, S. W. (1999). The medial extended amygdala in male reproductive behavior.
A node in the mammalian social behavior network. Ann N Y Acad Sci 877: 242-
57.

159

Nolan, P. M. and G. E. Hill (2004). Female choice for song characteristics in the house
finch. Anim Behav 67: 403-410.

Nordeen, K. W. and E. J. Nordeen (1992). Auditory feedback is necessary for the
maintenance of stereotyped song in adult zebra finches. Behav Neural Bio]
57(1): 58-66.

Nowicki, S., W. A. Searcy, et al. (2002). Brain development, song learning and mate
choice in birds: 3 review and experimental test of the "nutritional stress
hypothesis". J Comp Physio] A 188: 1003-1014.

Numan, M. and T. P. Sheehan (1997). Neuroanatomical circuitry for mammalian
maternal behavior. Ann N Y Acad Sci 807: 101-25.

O'Connor, L. H., B. Nock, et al. (1988). Regional specificity of gamma-arninobutyric
acid receptor regulation by estradiol. Neuroendocrinology 47(6): 473-81.

Ogren, S. O., H. Hall, et al. (1986). The selective dopamine D2 receptor antagonist
raclopride discriminates between dopamine-mediated motor functions.
Psychopharmacology (Berl) 90(3): 287-94.

Ottersen, O. P. (1980). Afferent connections to the amygdaloid complex of the rat and
cat: 11. Afferents from the hypothalamus and the basal telencephalon. J Comp
Neuro] 194(1): 267-89.

Ottersen, O. P. (1981). Afferent connections to the amygdaloid complex of the rat with
some observations in the cat. III. Afferents from the lower brain stem. J Comp
Neurol 202(3): 335-56.

Palkovits, M. (1973). Isolated removal of hypothalamic or other brain nuclei of the rat.
Brain Res 59: 449-50.

Panzica, G., M. Pessatti, et al. (1999). Effects of testosterone on sexually dimorphic
parvocellular neurons expressing vasotocin mRNA in the male quail brain.
Brain Res 850(1-2): 55-62.

Panzica, G., C. Viglietti-Panzica, et al. (2001). Sexual dimorphism in the neuronal
circuits of the quail preoptic and limbic regions. Microsc Res Tech 54(6): 364-
74.

Paredes, R. G. and A. Agmo (2004). Has dopamine a physiological role in the control of
sexual behavior? A critical review of the evidence. Prog Neurobiol 73(3): 179-
226.

Pasqualini, C., V. Olivier, et al. (1995). Acute stimulatory effect of estradiol on striatal
dopamine synthesis. J Neurochem 65(4): 1651-7.

160

 

Pavlova, D., R. Pinxten, et al. (2005). Female song in European Starlings: Sex
differences, complexity, and composition. Condor 107: 559-569.

Perfito, N., G. Bentley, et al. (2006). Tonic activation of brain GnRH immunoreactivity
despite reduction of peripheral reproductive parameters in opportunistically
breeding zebra finches. Brain Behav Evol 67(3): 123-34.

Pfaff, D. and M. Keiner (1973). Atlas of estradiol-concentrating cells in the central
nervous system of the female rat. J Comp Neuro] 151(2): 121-58.

Pfaff, D. W. and Y. Sakuma (1979). Deficit in the lordosis reflex of female rats caused
by lesions in the ventromedial nucleus of the hypothalamus. J Physio] 288: 203-
10.

Pfaff, D. W., S. Schwartz-Giblin, et al. (1994). Cellular and Molecular Mechanisms of
Female Reproductive Behaviors. The Physiology of Reproduction. E. Knobil and
J. D. Neill. New York, Raven Press, Ltd.: 107-220.

Pfaus, J. G., G. Damsma, et al. (1990). Sexual behavior enhances central dopamine
transmission in the male rat. Brain Res 530(2): 345-8.

Pfaus, J. G., G. Damsma, et a1. (1995). Sexual activity increases dopamine transmission
in the nucleus accumbens and striatum of female rats. Brain Res 693(1-2): 21-
30.

Pfaus, J. G. and M. M. Heeb (1997). Implications of immediate-early gene induction in
the brain following sexual stimulation of female and male rodents. Brain Res
Bull 44(4): 397-407.

Pfaus, J. G., C. Marcangione, et al. (1996). Differential induction of Fos in the female
rat brain following different amounts of vaginocervical stimulation: modulation
by steroid hormones. Brain Res 741(1-2): 314-30.

Phelix, C. F., Z. Liposits, et al. (1992). Monoamine innervation of bed nucleus of stria
terminalis: an electron microscopic investigation. Brain Res Bull 28(6): 949-65.

Phelix, C. F., Z. Liposits, et a1. (1992). Serotonin-CRF interaction in the bed nucleus of

the stria terminalis: a light microscopic double-label immunocytochemical
analysis. Brain Res Bull 28(6): 943-8.

Phelps, E. A. (2006). Emotion and cognition: insights from studies of the human
amygdala. Annu Rev Psychol 57: 27-53.

Phelps, E. A. and J. E. LeDoux (2005). Contributions of the amygdala to emotion
processing: from animal models to human behavior. Neuron 48(2): 175-87.

Phillipson, O. T. and A. C. Griffiths (1985). The topographic order of inputs to nucleus
accumbens in the rat. Neuroscience 16(2): 275-96.

161

Pinaud, R. and C. V. Mello (2007). GABA immunoreactivity in auditory and song
control brain areas of zebra finches. J Chem Neuroanat 34(1-2): 1-21.

Pinaud, R. and T. A. Terleph (2008). A songbird forebrain area potentially involved in
auditory discrimination and memory formation. J Biosci 33(1): 145-55.

Pinaud, R., T. A. Velho, et al. (2004). GABAergic neurons participate in the brain's
response to birdsong auditory stimulation. Eur J Neurosci 20(5): 1318-30.

Pleim, E. T., J. A. Matochik, et al. (1990). Correlation of dopamine release in the
nucleus accumbens with masculine sexual behavior in rats. Brain Res 524(1):
160-3.

Poulin, J. F ., B. Chevalier, et al. (2006). Enkephalinergic afferents of the centromedial
amygdala in the rat. J Comp Neuro] 496(6): 859-76.

Price, J. L., B. M. Slotnick, et al. (1991). Olfactory projections to the hypothalamus. J
Comp Neurol 306(3): 447-61.

Price, P. (1979). Developmental Determinants of structure in zebra finch song. J Comp
Physiol Psychol 93: 260-277.

Prieto, J. J ., B. A. Peterson, et al. (1994). Morphology and spatial distribution of
GABAergic neurons in cat primary auditory cortex (A1). J Comp Neuro] 344(3):
349-82.

Reiner, A. (1986). The co-occurrence of substance P-like immunoreactivity and
dynorphin-like immunoreactivity in striatopallidal and striatonigral projection
neurons in birds and reptiles. Brain Res 371(1): 155-61.

Reiner, A. and K. D. Anderson (1990). The patterns of neurotransmitter and
neuropeptide co-occurrence among striatal projection neurons: conclusions
based on recent findings. Brain Res Brain Res Rev 15(3): 251-65.

Reiner, A., B. M. Davis, et al. (1984). The distribution of enkephalinlike
immunoreactivity in the telencephalon of the adult and developing domestic
chicken. J Comp Neuro] 228(2): 245-62.

Reiner, A., L. Medina, et al. (1998). Structural and functional evolution of the basal
ganglia in vertebrates. Brain Res Brain Res Rev 28(3): 235-85.

Reiner, A., D. J. Perkel, et al. (2004a). Revised nomenclature for avian telencephalon
and some related brainstem nuclei. J Comp Neuro] 473(3): 377-414.

Reiner, A., D. J. Perkel, et al. (2004b). Songbirds and the revised avian brain
nomenclature. Ann N Y Acad Sci 1016: 77-108.

162

Remage-Healey, L., E. Adkins-Regan, et al. (2003). Behavioral and adrenocortical
responses to mate separation and reunion in the zebra finch. Horm Behav 43(1):
108-14.

Richter-Levin, G. (2004). The amygdala, the hippocampus, and emotional modulation
of memory. Neuroscientist 10(1): 31-9.

Riebel, K. and I. M. Smallegange (2003). Does Zebra Finch (T aeniopygia guttata)
Preference for the (Familiar) Father's Song Generalize to the Songs of
Unfamiliar Brothers. J Comp Psycho] 117(1): 61-66.

Riebel, K., I. M. Smallegange, et al. (2002). Sexual equality in zebra finch song
preference: evidence for a dissociation between song recognition and production
learning. Proc Roy Soc Lon B 269: 729-733.

Rieke, G. K. (1981). Movement disorders and lesions of pigeon brain stem analogues of
basal ganglia. Physiol Behav 26(3): 379-84.

Riters, L. V., M. Eens, et al. (2000). Seasonal changes in courtship song and the medial
preoptic area in male European starlings (Sturnus vulgaris). Horm Behav 38(4):
250-61.

Riters, L. V., K. M. Olesen, et al. (2007). Evidence that female endocrine state
influences catecholamine responses to male courtship song in European
starlings. Gen Comp Endocrinol 154(1-3): 137-49.

Riters, L. V., D. P. Teague, et al. (2004). Vocal production in different social contexts
relates to variation in immediate early gene immunoreactivity within and outside
of the song control system. Behav Brain Res 155(2): 307-18.

Roberts, T. F ., W. S. Hall, et al. (2002). Organization of the avian basal forebrain:
chemical anatomy in the parrot (Melopsittacus undulatus). J Comp Neurol
454(4): 383-408.

Rohlf, F. J. and R. R. Sokal (1981). Statistical Tables Stony Brook, New York, W.H.
Freeman and Co.

Roselli, C. E., S. E. Abdelgadir, et al. (1998). Anatomic distribution and regulation of
aromatase gene expression in the rat brain. Biol Reprod 58(1): 79-87.

Rubin, B. S. and R. J. Barfield (1980). Priming of Estrous Responsiveness by Implants
of l7-Beta-Estradiol in the Ventromedial Hypothalamic Nucleus of Female
Rats. Endocrinology 106(2): 504-509.

Saigusa, T., K. Takada, et al. (1997). Dopamine efflux in the rat nucleus accumbens

evoked by dopamine receptor stimulation in the entorhinal cortex is modulated
by oestradiol and progesterone. Synapse 25(1): 37-43.

163

Saini, K. D. and H. J. Leppelsack (1981). Cell types of the auditory caudomedial
neostriatum of the starling (Sturnus vulgaris). J Comp Neurol 198(2): 209-29.

Sakuma, Y. (1995). Differential control of proceptive and receptive components of
female rat sexual behavior by the preoptic area. Jpn J Physio] 45(2): 211-28.

Saldanha, C. J ., M. J. Tuerk, et al. (2000). Distribution and Regulation of Telencephalic
Aromatase Expression in the Zebra Finch Revealed with a Specific Antibody.
The J Comp Neurol 423: 619-630.

Sanberg, P. R. and R. F. Mark (1983). The effect of striatal lesions in the chick on
haloperidol-potentiated tonic immobility. Neuropharmacology 22(2): 253-7.

Sanghera, M. K., E. R. Simpson, et al. (1991). Immunocytochemical distribution of
aromatase cytochrome P450 in the rat brain using peptide-generated polyclonal
antibodies. Endocrinology 129(6): 2834-44.

Saper, C. B., L. W. Swanson, et al. (1976). The efferent connections of the
ventromedial nucleus of the hypothalamus of the rat. J Comp Neuro] 169(4):
409-42.

Sasaki, A., T. D. Sotnikova, et al. (2006). Social context-dependent singing-regulated
dopamine. J Neurosci 26(35): 9010-4.

Scalia, F. and S. S. Winans (1975). The differential projections of the olfactory bulb and
accessory olfactory bulb in mammals. J Comp Neuro] 161(1): 31-55.

Scheel-Kruger, J ., G. Magelund, et al. (1981). Role of GABA in the striatal output
system: globus pallidus, nucleus entopeduncularis, substantia nigra and nucleus
subthalamicus. Adv Biochem Psychopharmaco] 30: 165-86.

Schultz, W. (2000). Multiple reward signals in the brain. Nat Rev Neurosci 1(3): 199-
207.

Schultz, W., P. Dayan, et al. (1997). A neural substrate of prediction and reward.
Science 275(5306): 1593-9.

Schulz, K. M., H. N. Richardson, et al. (2003). Medial preoptic area dopaminergic
responses to female pheromones develop during puberty in the male Syrian
hamster. Brain Res 988(1-2): 139-45.

Searcy, W. A. and M. S. Capp (1997). Estradiol dosage and the solicitation display
assay in red-winged blackbirds. The Condor 99: 826-828.

Searcy, W. A. and P. Marler (1981). A Test for Responsiveness to Song Structure and
Programming in Female Sparrows. Science 213(4510): 926-928.

164

Shen, P., B. A. Schlinger, et al. (1995). An atlas of aromatase mRNA expression in the
zebra finch brain. J Comp Neuro] 360(1): 172-84.

Shirayama, Y. and S. Chaki (2006). Neurochemistry of the nucleus accumbens and its
relevance to depression and antidepressant action in rodents. Curr

Neuropharmacol 4(4): 277-91.

Shughrue, P., P. Scrimo, et al. (1997). The distribution of estrogen receptor-beta mRNA
in forebrain regions of the estrogen receptor-alpha knockout mouse.
Endocrinology 138(12): 5649-52.

Silcox, A. P. and S. M. Evans (1982). Factors affecting the formation and maintenance
of pair bonds in the zebra finch, Taeniopygia guttata. Anim Behav 30: 123 7-
1243.

Simerly, R. B., C. Chang, et a1. (1990). Distribution of androgen and estrogen receptor
mRNA-containing cells in the rat brain: an in situ hybridization study. J Comp
Neuro] 294(1): 76-95.

Simerly, R. B., R. A. Gorski, et al. (1986). Neurotransmitter specificity of cells and
fibers in the medial preoptic nucleus: an immunohistochemical study in the rat. J
Comp Neurol 246(3): 343-63.

Simerly, R. B. and L. W. Swanson (1986). The organization of neural inputs to the
medial preoptic nucleus of the rat. J Comp Neuro] 246(3): 312-42.

Simerly, R. B. and L. W. Swanson (1988). Projections of the medial preoptic nucleus: a
Phaseolus vulgaris leucoagglutinin anterograde tract-tracing study in the rat. J
Comp Neurol 270(2): 209-42.

Simmons, D. A. and P. Yahr (2003). GABA and glutamate in mating-activated cells in
the preoptic area and medial amygdala of male gerbils. J Comp Neuro] 459(3):
290-3 00.

Smith, G. T., E. A. Brenowitz, et al. (1995). Seasonal changes in song nuclei and song
behavior in Garnbel's white-crowned sparrows. J Neurobiol 28(1): 114-25.

Sockman, K. W. and G. F. Ball (2009). Independent effects of song quality and
experience with photostimulation on expression of the immediate, early gene
ZENK (EGR-l) in the auditory telencephalon of female European starlings. Dev
Neurobiol 69(6): 339-349.

Sockman, K. W., T. Q. Gentner, et a1. (2005). Complementary Neural Systems for the
Experience-Dependent Integration of Mate-Choice Cues in European Starlings.
J Neurobiol 62: 72-81.

165

Sockman, K. W. and K. G. Salvante (2008). The integration of song environment by
catecholaminergic systems innervating the auditory telencephalon of adult
female European starlings. Dev Neurobiol 68(5): 656-68.

Sockman, K. W. and H. Schwabl (1999). Daily Estradiol and Progesterone Levels
Relative to Laying and Onset of Incubation in Canaries. General and
Comparative Endocrinology 114: 257-268.

Sofroniew, M. V. (1985). Vasopressin- and neurophysin-immunoreactive neurons in the
septal region, medial amygdala and locus coeruleus in colchicine-treated rats.
Neuroscience 15(2): 347-58.

Stripling, R., L. Milewski, et al. (2003). Rapidly learned song-discrimination without
behavioral reinforcement in adult male zebra finches (Taeniopygia guttata).
Neurobiol Learn Mem 79: 41-50.

Svec, L., K. M. Licht, et al. (2009). Pair bonding in the female zebra finch: a potential
role for the nucleus taeniae. Neuroscience. 160(2): 275-283.

Svec, L. A., K. J. Lookingland, et al. (submitted). Estradiol and song presentation
mediate behavior of female zebra finches independent of dopamine activity in
the nucleus accumbens and medial striatum. Brain Behav Evol.

Svec, L. A. and J. Wade (2009). Estradiol induces region-specific inhibition of ZENK
but does not affect the behavioral preference for tutored song in adult female
zebra finches. Behav Brain Res 199(2): 298-306.

Swank, M. W., A. E. Ellis, et al. (1996). c-Fos antisense blocks acquisition and
extinction of conditioned taste aversion in mice. Neuroreport 7(11): 1866-70.

Swanson, L. W. (1982). The projections of the ventral tegmental area and adjacent
regions: a combined fluorescent retrograde tracer and immunofluorescence
study in the rat. Brain Res Bull 9(1-6): 321-53.

Szekely, A. D., M. I. Boxer, et al. (1994). Connectivity of the lobus parolfactorius of the
domestic chicken (Gallus domesticus): an anterograde and retrograde pathway
tracing study. J Comp Neuro] 348(3): 374-93.

Tchemichovski, O., H. Schwabl, et al. (1998). Context determines the sex appeal of
male zebra finch song. Anim Behav 55: 1003-1010.

Terleph, T. A., C. V. Mello, et al. (2006). Auditory topography and temporal response
dynamics of canary caudal telencephalon. J Neurobiol 66(3): 281-92.

Terpstra, N. J ., J. J. Bolhuis, et al. (2006). Localized brain activation specific to auditory
memory in a female songbird. J Comp Neuro] 494(5): 784-91.

166

Tetel, M. J ., D. C. Celentano, et al. (1994). Intraneuronal convergence of tactile and
hormonal stimuli associated with female reproduction in rats. J Neuroendocrinol
6(2): 211-6.

Tetel, M. J ., M. J. Getzinger, et al. (1994). Estradiol and progesterone influence the
response of ventromedial hypothalamic neurons to tactile stimuli associated with
female reproduction. Brain Res 646(2): 267-72.

Theunissen, F. E. and S. S. Shaevitz (2006). Auditory processing of vocal sounds in
birds. Curr Opin Neurobiol 16(4): 400-7.

Thompson, R. R., J. L. Goodson, et al. (1998). Role of the archistriatal nucleus taeniae
in the sexual behavior of male Japanese quail (Cotumix japonica): a comparison
of function with the medial nucleus of the amygdala in mammals. Brain Behav
Evol 51(4): 215-29.

Thompson, T. L. and R. L. Moss (1997). Modulation of mesolimbic dopaminergic
activity over the rat estrous cycle. Neurosci Lett 229(3): 145-8.

Tischmeyer, W. and R. Grimm (1999). Activation of immediate early genes and
memory formation. Cell Mo] Life Sci 55(4): 564-74.

Tischmeyer, W., R. Grimm, et al. (1994). Sequence-specific impairment of learning by
c-jun antisense oligonucleotides. Neuroreport 5(12): 1501-4.

Tomaszycki, M. L. and E. Adkins-Regan (2005). Experimental alteration of male song
quality and output affects female mate choice and pair bond formation in zebra
finches. Anim Behav 70(4): 785-794.

Tomaszycki, M. L. and E. Adkins-Regan (2006). Is male song quality important in
maintaining pair bonds? Behaviour 143: 549-567.

Tomaszycki, M. L., E. M. Sluzas, et al. (2006). Immediate early gene (ZENK)
responses to song in juvenile female and male zebra finches: effects of rearing
environment. J Neurobiol 66( 1 1): 1 175-82.

Usuda, 1., K. Tanaka, et al. (1998). Efferent projections of the nucleus accumbens in the
rat with special reference to subdivision of the nucleus: biotinylated dextran
amine study. Brain Res 797(1): 73-93.

Vallet, E., M. Kreutzer, et al. (1996). Testosterone induces sexual release quality in the
song of female canaries. Ethology 102: 617-628.

Van Hartesveldt, C. and J. N. Joyce (1986). Effects of estrogen on the basal ganglia.
Neurosci Biobehav Rev 10(1): 1-14.

167

Vandesande, F. and K. Dierickx (1976). Immuno-Cytochemical Demonstration of
Inability of Homozygous Brattleboro Rat to Synthesize Vasopressin and
Vasopressin-Associated Neurophysin. Cell Tiss Res 165(3): 307-316.

Vates, G. E., B. M. Broome, et al. (1996). Auditory pathways of caudal telencephalon
and their relation to the song system of adult male zebra finches. J Comp Neurol
366(4): 613-42.

Veenman, C. L. (1997). Pigeon basal ganglia: insights into the neuroanatomy
underlying telencephalic sensorimotor processes in birds. Eur J Morphol 35(4):
220-33.

Veenman, C. L., J. M. Wild, et al. (1995). Organization of the avian "corticostriatal"

projection system: a retrograde and anterograde pathway tracing study in
pigeons. J Comp Neuro] 354(1): 87-126.

Veinante, P. and M. J. Freund-Mercier (1997). Distribution of oxytocin- and

vasopressin-binding sites in the rat extended amygdala: a histoautoradiographic
study. J Comp Neuro] 383(3): 305-25.

Velho, T. A., R. Pinaud, et al. (2005). Co-induction of activity-dependent genes in
songbirds. Eur J Neurosci 22(7): 1667-78.

Viglietti-Panzica, C., N. Aste, et al. (1994). Vasotocinergic innervation of sexually
dimorphic medial preoptic nucleus of the male Japanese quail: influence of
testosterone. Brain Res 657(1-2): 171-84.

Vignal, C., N. Mathevon, et a1. (2008). Mate recognition by female zebra finch:
Analysis of individuality in male call and first investigations on female decoding
process. Behav Process 77(2): 191-198.

Vito, C. C., J. F. DeBold, et al. (1983). Androgen and estrogen receptors in adult
hamster brain. Brain Res 264(1): 132-7.

Voorhuis, T. A. and E. R. de Kloet (1992). Immunoreactive vasotocin in the zebra finch
brain (Taeniopygia guttata). Brain Res Dev Brain Res 69(1): 1-10.

Vyas, A., C. Harding, et al. (2008). Noradrenergic neurotoxin, N-(2-chloroethyl)-N-
ethyl-2-bromobenzylamine hydrochloride (DSP-4), treatment eliminates

estrogenic effects on song responsiveness in female zebra finches (Taeniopygia
guttata). Behav Neurosci 122(5): 1148-57.

Vyas, A., C. Harding, et al. (2009). Acoustic characteristics, early experience, and
endocrine status interact to modulate female zebra finches' behavioral responses
to songs. Horm Behav 55(1): 50-9.

168

Wagner, C. K. and J. I. Morrell (1996). Distribution and steroid hormone regulation of
aromatase mRNA expression in the forebrain of adult male and female rats: a
cellular-level analysis using in situ hybridization. J Comp Neurol 370(1): 71-84.

Wagner, C. K. and J. I. Morrell (1997). Neuroanatomical distribution of aromatase
MRNA in the rat brain: indications of regional regulation. J Steroid Biochem
Mo] Bio] 61(3-6): 307-14.

Walf, A. A. and C. A. Frye (2006). A review and update of mechanisms of estrogen in
the hippocampus and amygdala for anxiety and depression behavior.
Neuropsychopharmacology 31(6): 1097-1 1 1.

Wang, Z. and B. J. Aragona (2004). Neurochemical regulation of pair bonding in male
prairie voles. Physio] Behav 83(2): 319-28.

Wang, Z., G. Yu, et al. (1999). Dopamine D2 receptor-mediated regulation of partner
preferences in female prairie voles (Microtus ochrogaster): a mechanism for pair
bonding? Behav Neurosci 113(3): 602-11.

Wang, Z., L. Zhou, et al. (1996). Immunoreactivity of central vasopressin and oxytocin
pathways in microtine rodents: a quantitative comparative study. J Comp Neurol
366(4): 726-37.

Watson, J. T., E. Adkins-Regan, et al. (1988). Autoradiographic localization of nicotinic
acetylcholine receptors in the brain of the zebra, finch (Poephila guttata). J Comp
Neuro] 274(2): 255-64.

Weller, K. L. and D. A. Smith (1982). Afferent connections to the bed nucleus of the
stria terminalis. Brain Res 232(2): 255-70.

Whitney, J. F. (1986). Effect of medial preoptic lesions on sexual behavior of female
rats is determined by test situation. Behav Neurosci 100(2): 230-5.

Williams, J. R., K. C. Catania, et al. (1992). Development of partner preferences in
female prairie voles (Microtus ochrogaster): the role of social and sexual
experience. Horm Behav 26(3): 339-49.

Wong, M. and R. L. Moss (1992). Modulation of single-unit activity in the rat medial
amygdala by neurotransmitters, estrogen priming, and synaptic inputs from the
hypothalamus and midbrain. Synapse 10(2): 94-102.

Yamamoto, K., Z. Sun, et al. (2005). Subpallial amygdala and nucleus taeniae in birds
resemble extended amygdala and medial amygdala in mammals in their
expression of markers of regional identity. Brain Res Bull 66(4-6): 341-7.

Yasui, Y., C. B. Saper, et al. (1991). Calcitonin gene-related peptide (CGRP)
immunoreactive projections from the thalamus to the striatum and amygdala in
the rat. J Comp Neurol 308(2): 293-310.

169

Young, A. M. and K. R. Rees (1998). Dopamine release in the amygdaloid complex of
the rat, studied by brain microdialysis. Neurosci Lett 249(1): 49-52.

Young, L. J ., Z. Wang, et al. (1998). Neuroendocrine bases of monogamy. Trends
Neurosci 21(2): 71-5.

Youngren, O. M., M. E. el Halawani, et al. (1989). Effects of preoptic and hypothalamic
lesions in female turkeys during a photoinduced reproductive cycle. Biol Reprod
41(4): 610-7.

Zahm, D. S. and J. S. Brog (1992). On the significance of subterritories in the
"accumbens" part of the rat ventral striatum. Neuroscience 50(4): 751-67.

Zann, R. A. (1996). The Zebra Finch: A Synthesis of Field and Laboratory Studies.
New York, Oxford University Press.

Zann, R. A., S. R. Morton, et al. (1995). The Timing of Breeding by Zebra Finches in
Relation to Rainfall in Central Australia. Emu 95: 208-222.

170

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