Exploring the neurogenetics of sociality: creation of models toassess the functional role of V1a receptor diversity 公开
Donaldson, Zoe (2009)
Published
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
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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