Genetic Diversity in Oxytocin Receptor Sequence, Neural Expression, and Social Behavior Open Access

King, Lanikea Brackin (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/dr26xz283?locale=en
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Abstract

Oxytocin (OXT) is a neuropeptide with conserved functions in reproductive
physiology and social cognition through signalling with a single receptor, OXTR.
OXTR expression occurs in diverse distributions within the brain and influences
species-typical behavior. The socially monogamous prairie vole has high OXTR
density in the nucleus accumbens (NAcc) compared to non-monogamous vole
species. OXT and dopamine signalling converge in the prairie vole NAcc,
enabling social attachments to distinct individuals. OXTR diversity in the NAcc
also contributes to individual variation in prairie vole social behavior. Thus,
regulatory elements (cis-REs) that influence transcription of the oxtr gene or
synthesis of OXTR must be susceptible to variation. This dissertation examines
the hypothesis that transcription of the prairie vole oxtr is modulated by
polymorphic cis-REs. I found that a molecular signature of polymorphic cis-RE
activity, allelic imbalance, occurs robustly and specifically in the NAcc. The
single nucleotide polymorphism (SNP) marker used for allelic imbalance,
NT204321, predicts OXTR density in the NAcc. NT204321 genotype groups also
exhibit differences in mRNA levels in the NAcc and mRNA and OXTR binding
are correlated in the NAcc. NT204321 also predicts individual differences in
prairie vole social attachment. I next attempted to identify putative locations of
cis-REs using next-generation sequencing and found approximately 3,000 SNPs
within the oxtr locus. I discovered that SNPs in the prairie vole oxtr are in strong
linkage disequilibrium (LD) with each other. One of these SNPs, which is located
in the single intron, outperforms NT204321 as a marker of NAcc OXTR density,
explaining 75% or more density variation in three separate replicates. Future
studies using the intron SNP will investigate gene by environment interactions
and probe more deeply into the mechanisms influence oxtr regulation.

Table of Contents

TABLE OF CONTENTS

CHAPTER 1…………….…………………………………………………………….........1

GENERAL INTRODUCTION…………………………………………..………..1

INTRODUCTION…………..………………………………………….…………..2

OXYTOCIN, VASOPRESSSIN AND

SOCIAL BEHAVIOR………………………………….......……..3

NEUROHYPOPHYSEAL PEPTIDE GENE STRUCTURE, EVOLUTION AND CONSERVED FUNCTION……………………..5

CONSERVATION IN NEURAL EXPRESSION………....….7

OXYTOCIN AND VASOPRESSIN IN THE VERTEBRATE BRAIN......................................................................................8

OXT AND AVP RECEPTORS…………………………………10

PRAIRIE VOLES AND PAIR BONDING……………………….......12

OXT AND AVP INFLUENCE PAIR BONDING………......14

SPECIES DIFFERENCES IN SOCIAL BEHAVIOR ARE PATTERNED BY RECEPTOR DIVERSITY………………….…….16

NEUROPEPTIDE RECEPTOR EXPRESSION CONTRIBUTES TO INDIVIDUAL DIFFERENCES IN BEHAVIOR……………………………………………………....20

HOW DIVERSITY IN RECEPTOR EXPRESSION IS ACHIEVED……………………………………………………....24

TRANSLATIONAL IMPLICATIONS FOR OXTR AND AVPR1A………………………………………………………………..….28 OPPORTUNITIES FOR DISCOVERY……………...............………33

CHAPTER 2……………………………………………………………………...............35

GENETIC VARIATION IN THE OXYTOCIN RECEPTOR CONTRIBUTES TO INDIVIDUAL DIVERSITY IN BRAIN RECEPTOR DISTRIBUTION AND SOCIAL ATTACHMENT IN MONOGAMOUS PRAIRIE VOLES……………………………………………………...………….35

ABSTRACT…………….………………………………………………………….36 INTRODUCTION..……………………………………………………………….37 RESULTS………………………………………………………………………….41

Variation in OXTR density across brain regions…........…..41

Allelic imbalance occurs selectively in the dorsal and ventral striatum...…………………………………………….…42

NT204321 is associated with OXTR density….………….....45

NT204321 genotype predicts individual differences in social behaviors.…………………………………………………………49

DISCUSSION……...…………….……………………………………………….52 METHODS…………………………..…………………………………………….57 Animals…………………………………………………...………57

Sanger sequencing and polymorphism discovery……….....57

Allelic imbalance………….……...…………………..…………58

Genotyping…………………………….…………………………………………..60

Ultrasonic vocalizations………………………....……………..60

Partner preference test……….………………………………..61

Oxytocin receptor autoradiography….……………………….63

In situ hybridization………………………….…………………64

Statistical analysis..…………………………………………….65

CHAPTER 3……………………………………………………………………...............67

DISSECTION OF THE PRAIRIE VOLE OXYTOCIN RECEPTOR GENE TO IDENTIFY ROBUST MARKERS OF BRAIN REGION SPECIFIC TRANSCRIPTIONAL VARIATION…..……………………………………….67

ABSTRACT………………………….…………………………………………….68 INTRODUCTION..……………………………………………………………….69 RESULTS………………………………………………………………………….73

A genetic marker of cis-RE activity selectively predicts OXTR density variation in the NAcc…………………………73

Sample selection and sequencing for polymorphism discovery in the prairie vole oxtr gene……………………….74

Association analyses……............................………………….77

DISCUSSION……...………………………….………………………………….82 METHODS……………………………..………………………………………….84

Long-range PCRs for target enrichment of 70kb surrounding oxtr…………………………………………………84

Amplicon library preparation………….....…….……………..85

Amplicon sequencing analysis…………...……………………86 Genotyping…………………………………………………….…86

Statistical analysis……………………………...………………87

CHAPTER 4……………………………………………………………………...............88

CONCLUDING REMARKS…………………………………………...………..88

SUMMARY OF FINDINGS…………………..………………………..89 FUTURE DIRECTIONS…………..……………………………………93

OXTR in neural networks…………………...…………………93

REFERENCES..………………………………………………………………...............98 TABLE OF FIGURES

Figure 1.1 - Oxytocin and neuropeptide homologs across the animal kingdom…..3

Figure 1.2 - Oxytocin and vasopressin genes: structure and processing…………...6

Figure 1.3 - OXTR binding sites in the forebrain of rat, mouse and prairie vole…………………………………………………………………………………………..11

Figure 1.4 - The monogamous prairie vole………………………………..……….....13

Figure 1.5 - Receptor diversity reveals brain regions underpinning species

specific social behavior………………………………………………………….………..16

Figure 1.6 - A model of a pair bonding network in the prairie vole brain.……….19

Figure 1.7 - Individual diversity in OXTR and AVPR1A.………............................27

Figure 2.1 - Individual variation in OXTR density in the prairie vole

forebrain………………………………………………………………………….…………40

Figure 2.2 - OXTR protein and oxtr mRNA distributions are similar within

Individuals………………………………………………………………………………….41

Figure 2.3 - oxtr mRNA is expressed with allelic imbalance robustly in the NAcc and striatum.………..................................................................................................44

Figure 2.4 - OXTR binding density in striatal and olfactory regions is associated with NT204321…………………………………………………………………………….48

Figure 2.5 - NT204321 predicts isolation induced ultrasonic vocalizations in males…………………................................................................................................49

Figure 2.6 - The effect of NT204321 genotype on partner preference formation in male and female prairie voles.………......................................................................51

Figure 2.7 - OXTR density diversity in the NAcc results from variation in oxtr mRNA transcription.…………………………………………………………………......52

Figure 3.1 - NT204321 is strongly associated with OXTR density in striatal subregions.………......................................................................................................74

Figure 3.2 - Subject selection and sequencing strategy…………………….……….76

Figure 3.3 - SNPs near oxtr are strongly associated with OXTR density in the

NAcc…………………………………………………….…………………………………...78

Figure 3.4 - Identification of NT213739 as a marker of NAcc OXTR density……80

Figure 3.5 - NT213739 genotype robustly predicts oxtr expression across independent replicates……………………………………………………………………82

TABLE OF TABLES

Table 2.1 - Initial polymorphism discovery in 19 individual prairie voles…....….43

Table 2.2 - Two factors encompass the covariation in OXTR binding amongst brain regions in two independent samples…………………………………………….46


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