Characterization of the Oxytocin Receptor in the Nonhuman Primate Brain Pubblico

Abstract

Oxytocin (OT) and vasopressin (AVP) are two structurally related neuropeptides that act in the brain to modulate the expression of species-specific social behaviors. Based on decades of extensive work in rodent systems, research in this field has recently expanded to explore the effects of OT on social cognition in humans, including clinical populations. However, this expansion has proceeded without a fundamental understanding of the neurophysiology of the OT system in the primate brain. Studying the brains of nonhuman primates (NHP) provides an opportunity to elucidate the neural mechanisms by which OT and AVP modulate social cognition. However, the lack of highly selective radioligands for the primate oxytocin receptor (OXTR) and vasopressin 1a receptor (AVPR1a) has prevented the reliable mapping of central OXTR and AVPR1a distributions in NHP tissue. To ameliorate this issue, we developed a pharmacologically informed, competitive binding protocol for receptor autoradiography to selectively reveal OXTR and AVPR1a binding in NHP tissue. We then characterized OXTR binding in the brain of a common NHP model organism for biomedical research, the rhesus macaque (Macaca mulatta). We also characterized OXTR and AVPR1a binding in the brain of the coppery titi monkey (Callicebus cupreus), a socially monogamous New World monkey. Our results demonstrate that while there are species differences in OXTR distribution in the primate brain, OXTR is consistently found in brain regions that modulate visual attention and control orienting responses to visual stimuli, such as the nucleus basalis of Meynert and the superior colliculus. These results should inform future studies in NHP and ultimately facilitate the development of optimal pharmacological strategies to target the OT system for the improvement of social function in psychiatric disorders such as autism spectrum disorder.

Table of Contents

CHAPTER 1: Introduction..................................................................... 1

General Introduction........................................................................... 2

Oxytocin, vasopressin, and the evolution of monogamy in mammals.............. 4

Vasopressin........................................................................................ 8

Antidiuretic hormone.............................................................................. 8 Territoriality and aggression in hamsters................................................... 9

From territoriality to pair bonding: vasopressin and the male prairie vole...... 10

Vasopressin and social behavior in male voles.......................................... 10

Neuroanatomy and pharmacology of vasopressin and pair-bond

formation in male prairie voles.............................................................. 13

Genetic variation and pair bonding in male voles....................................... 15

Vasopressin receptors & fitness in the wandering male prairie vole.............. 19

Oxytocin........................................................................................... 20

A maternal hormone............................................................................ 20

Oxytocin and maternal behavior............................................................. 20

Oxytocin and pair bonding in female voles................................................ 22

Social recognition and reward: the pathway to bonding......................... 25

Beyond monogamy: comparative work in other species......................... 28

Oxytocin and African mole-rats............................................................... 28 Vasopressin and monogamy in deer mice................................................. 31

Oxytocin, vasopressin, and primate neuroanatomy and social behavior.......... 32

Variation in oxytocin and social behavior in macaques................................ 32

Monogamous primates: marmosets, titi monkeys, & tamarins...................... 34

Conclusions and future directions........................................................ 36

General conclusions.............................................................................. 36

Challenges in identifying OXTR and AVPR1a in primate brain tissue............... 38

Specific aims of this dissertation............................................................. 40

CHAPTER 2: Pharmacological Characterization of the Oxytocin

Receptor and Vasopressin 1a Receptor in Primates:

Design of a Competitive Binding Receptor Autoradiography Protocol...... 43

Abstract............................................................................................ 44

Introduction...................................................................................... 46

Methods............................................................................................ 49

Cell culture......................................................................................... 49

Membrane Preparation & Protein Quantification......................................... 50

Ligand Binding Assay Optimization.......................................................... 51

Determination of best pretreatment of filter paper.................................... 51

Determination of non-specific binding to untransfected CHO cells................. 52

Determination of optimal amount (ug) of protein to prevent ligand

depletion........................................................................................... 53

Saturation Binding Assay...................................................................... 54

Competition Binding Assay.................................................................... 54

Results............................................................................................. 56

Ligand Binding Assay Optimization.......................................................... 56

Determination of best pretreatment of filter paper.................................... 56

Determination of non-specific binding to untransfected CHO cells................. 58

Determination of optimal amount (ug) of protein to prevent ligand

depletion........................................................................................... 60

Saturation Binding Assay...................................................................... 61

Competition Binding Assay.................................................................... 66

Discussion........................................................................................ 69

CHAPTER 3: Characterization of the Distribution of the

Oxytocin Receptor in the Brain of the Rhesus

Macaque (Macaca mulatta)............................................................... 72

Abstract........................................................................................... 73

Introduction..................................................................................... 75

Methods........................................................................................... 79

Tissue preparation and sectioning.......................................................... 79

Receptor autoradiography..................................................................... 80

In situ hybridization............................................................................. 81 Acetylcholinesterase staining................................................................. 82

Results............................................................................................. 83

Radioligands alone............................................................................... 83

Radioligand binding in the presence of unlabeled competitors...................... 85

In situ hybridization reveals OXTR expression........................................... 85

Comparison of OXTR mRNA with ¹²⁵I-OVTA binding in the

presence of SR49059............................................................................ 87

Detailed neuroanatomical distribution of OXTR in the rhesus

macaque brain.................................................................................... 89

Discussion........................................................................................ 92

Summary of findings and potential caveats.............................................. 92

Functions of OXTR-expressing regions of the macaque brain....................... 94

Ventromedial hypothalamus: sexual behavior........................................... 94

Trapezoid body: auditory input from the cochlea....................................... 96

Superior colliculus and oculomotor nucleus: visual processing

and eye movement.............................................................................. 97

Nucleus basalis of Meynert and pedunculopontine tegmental

nucleus: visual attention, reinforcement learning, and

cholinergic innervation of the brain........................................................ 98

Comparison to OXTR in other primates.................................................. 101 Conclusion........................................................................................ 104 CHAPTER 4: Distribution of Oxytocin and Vasopressin 1a

Receptors in the Brain of the Socially Monogamous Coppery

Titi Monkey (Callicebus cupreus)....................................................... 105

Abstract.......................................................................................... 106 Introduction.................................................................................... 108 Methods.......................................................................................... 112 Animals............................................................................................ 112

Tissue preparation.............................................................................. 113

Receptor autoradiography.................................................................... 113

Quantification and statistical analysis..................................................... 114

Acetylcholinesterase staining................................................................ 115 Results............................................................................................ 116

Selectivity of radioligands.................................................................... 116

Efficacy of competitors........................................................................ 119

Vasopressin 1a receptor distribution ..................................................... 119

Oxytocin receptor distribution............................................................... 125 Discussion....................................................................................... 126

Comparison to OXTR in other nonhuman primates.................................... 126

Visual processing centers of the brains of primates.................................. 128

OXTR, AVPR1a, and brain regions important for reinforcement learning....... 131

Assessments of primate-selective antagonists......................................... 133

CHAPTER 5: Discussion..................................................................... 135

Abstract.......................................................................................... 136 Introduction.................................................................................... 138

Methodological comparisons between rodent and primate

OT research..................................................................................... 138

Intracerebroventricular injections of drugs into the brain........................... 138

Receptor autoradiography.................................................................... 139

Measuring concentrations of OT in the brain and periphery......................... 142

Site-specific injections of OT-related compounds into the brain................... 144

Peripheral administration of OT............................................................. 146

Genetic studies of the OT system in rodents and primates.......................... 149

Behavioral paradigms.......................................................................... 151

Future directions: selective non-peptide ligands for behavioral

neuropharmacology in primates............................................................ 153

Mixed affinities of OT and AVP: relevance to future research................ 155

Evolutionary perspectives on central OXTR distributions...................... 157

OXTR in rodents is concentrated in brain regions involved in

olfactory processing............................................................................ 157

OXTR in primates is concentrated in brain regions involved in

visual processing............................................................................... 159

Conclusions and future directions...................................................... 164

Informing the design of experiments in NHP........................................... 164

Clinical relevance of this dissertation..................................................... 165

REFERENCES.................................................................................... 167

APPENDIX: List of Publications.......................................................... 213

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Psychology, Physiological Biology, Molecular
Parola chiave	rhesus macaque titi monkey autoradiography neuroanatomy oxytocin receptor pharmacology
Committee Chair / Thesis Advisor	Young, Larry J, Emory University
Committee Members	Hall, Randy A, Emory University Rilling, James K, Emory University Parr, Lisa, Emory University Goodman, Sherryl H, Emory University Sanchez, Mar, Emory University

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