Exploring the neurogenetics of sociality: creation of models to assess the functional role of V1a receptor diversity Open Access

Donaldson, Zoe (2009)

Permanent URL: https://etd.library.emory.edu/concern/etds/jq085k35k?locale=en
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Abstract

ABSTRACT

Understanding the biological mechanisms regulating individual and species differences in behavior has implications for both evolutionary biology and human mental health. The vasopressin V1a receptor (V1aR) system provides an ideal model for exploring the relationship between genetic sequence diversity, protein expression, and behavioral variation. Activation of V1aR modulates a wide array of behaviors including social memory, anxiety, and many species-specific affiliative and aggressive behaviors. In both humans and rodents, diversity in these behaviors is hypothesized to result from polymorphic repetitive DNA elements located upstream of the V1a receptor gene (AVPR1A). These elements are thought to influence gene expression, thereby altering neural V1aR expression patterns. However, despite significant interest in this system, there remain a number of unanswered questions, and studies to date have not been able to establish causality with respect to AVPR1A genetic diversity, V1aR expression, and behavior. Therefore, the goal of this dissertation is to establish various models that will allow us to directly investigate the V1aR gene-brain-behavior relationship. In order to do so, I first explore evolution and novel genetic diversity within the primate AVPR1A locus. I then establish genetically modified rodent models which will be used to explore the causal relationship between genetic diversity, protein expression, and social behavior. Specifically, within three congenic mouse lines, the relationship between genetic polymorphsims and V1aR expression will be directly examined through targeted introduction of variable repetitive elements upstream of the avpr1a transcription start site. In voles, I establish transgenic and RNAi technologies to generate voles with reduced V1aR expression which will be used to directly investigate the behavioral role of V1aR and V1aR variability. These varied models will build on previous correlational studies and lay the foundation for understanding the role of genetic and protein diversity in determining individual and species differences in behavior.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1 1

A general introduction: Oxytocin, vasopressin, and the neurogenetics of sociality _ 1

ABSTRACT _ 2

Conservation of neuropeptide systems regulating social behavior 4

Oxytocin, Nurturing, and Social Attachment 7

Vasopressin and the genetic bases for variation in social behavior 9

Neurogenetics of variation in human social behavior 12

Neuropeptides, human social cognition and trust 14

Neuropeptides, neurogenetics and society 16

Remaining questions and objectives 18

CHAPTER 2 23

Evolution of a behavior-linked microsatellite-containing element in the 5' flanking region of the primate AVPR1A gene _ 23

INTRODUCTION _ 26

METHODS _ 29

DNA extraction, amplification, and sequencing 29

Sequence and phylogenetic analysis 31

Microsatellite variation 31

Macaque sequence diversity 32

Determination of allele frequency in wild chimpanzees 32

Analysis of chimpanzee alleles for potential non-neutral evolution 33

Tests of neutrality 35

RESULTS _ 35

Primate AVPR1A evolution 35

Microsatellite variation 40

Macaque sequence diversity 41

Chimpanzee sequence diversity 43

Tests of neutrality at the chimp AVPR1A locus 44

DISCUSSION _ 46

CONCLUSIONS _ 52

CHAPTER 3 53

Development of mouse models to directly examine the relationship between microsatellite diversity and gene expression in the V1a receptor system _ 53

ABSTRACT _ 54

INTRODUCTION _ 55

METHODS AND RESULTS _ 60

Methodological overview _ 60

Experiment 3.1 Methods 61

Experiment 3.1 Results 67

Experiment 3.2 Methods 70

Experiment 3.2a Results 75

Experiment 3.2b Results 78

DISCUSSION _ 81

FUTURE DIRECTIONS _ 87

CHAPTER 4 89

Production of germline transgenic prairie voles (Microtus ochrogaster) using lentiviral vectors: Implications for rapid transgenesis in non-traditional rodent model species 89

ABSTRACT _ 90

INTRODUCTION _ 91

METHODS _ 93

Production of lentivirus 93

Generation of transgenic prairie voles 94

Genotyping by PCR _ 96

Southern blot confirmation and determination of integration number 96

Western blot assessment of GFP expression 97

Immunohistochemical investigation of transgene expression 98

RESULTS _ 99

Generation of transgenic prairie voles 99

Verification of GFP expression in F0 and F1 transgenic prairie voles 101

Verification of GFP expression in F0 and F1 transgenic prairie voles 102

DISCUSSION _ 103

Applications of Lentiviral Transgenics 105

CHAPTER 5 108

Development of RNAi technologies in prairie voles: creation of vasopressin V1a receptor knockdown voles 108

ABSTRACT _ 109

INTRODUCTION _ 110

METHODS _ 113

Tandem sh-RNA construct generation 113

Packaging of lentiviral vector 116

In vivo efficacy of the viral vector 117

Production of shRNA-containing transgenic voles 117

PCR genotyping of offspring for transgene presence 118

RESULTS _ 119

In vivo investigation of lentiviral efficacy 119

Generation of siRNA-containing transgenic prairie voles 120

PCR verification of transgene integration and progress on germline transmission 121

DISCUSSION _ 121

FUTURE DIRECTIONS _ 124

CHAPTER 6 126

Implications of vasopressin V1a receptor research for understanding sociobehavioral diversity: general conclusions and future directions 126

ABSTRACT _ 127

The V1a receptor as a model system for exploring the gene-brain-behavior axis 128

Specific future directions 130

Behavioral dissection the role of V1aR in social bonding 136

Behaviorally relevant sources of protein expression diversity 138

Complex mechanisms governing complex behaviors 140

Advantages of diverse animal models 142

APPENDIX _ 144

Central vasopressin receptor 1a activation is independently necessary for both pair bond formation and expression _ 144

ABSTRACT _ 145

INTRODUCTION _ 147

METHODS _ 149

Experimental Timecourse 149

Subjects 149

Cannulation 150

Injections 150

Behavioral testing 151

RESULTS _ 152

DISCUSSION _ 153

Proposed model for V1aR modulation of pair bonding 154

Behavioral dissection of behavior 155

REFERENCES _ 156

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