UNIVERSITY OF CINCINNATI _____________ , 20 _____ I,______________________________________________, hereby submit this as part of the requirements for the degree of: ________________________________________________ in: ________________________________________________ It is entitled: ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ Approved by: ________________________ ________________________ ________________________ ________________________ ________________________ SEXUAL BEHAVIOR CAUSES ACTIVATION AND FUNCTIONAL ALTERATIONS OF MESOLIMBIC SYSTEMS: NEUROBIOLOGY OF MOTIVATION AND REWARD A dissertation submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of DOCTORATE OF PHILOSOPHY (Ph.D.) in the Graduate Program in Neuroscience of the College of Medicine July 25, 2003 by Margaret E. Balfour B.A. Johns Hopkins University, 1997 Committee Chairs: Lique Coolen, Ph.D. Lei Yu, Ph.D. ABSTRACT There is increasing evidence that the various drugs of abuse converge upon common reward pathways in the brain. While it is clear that these circuits are involved in drug-induced reward and reinforcement, less is known about how these systems function when activated by normal motivated behaviors such as sexual behavior. Like drugs of abuse, sexual behavior is a rewarding and reinforcing behavior. Sexual behavior, however, is generally not considered an “addictive” behavior. Thus, understanding how neural circuits are activated by normal motivated behaviors may lead to a better understanding of the pathology of drug addiction. The first set of studies investigated the mechanisms by which sexual behavior activates the mesolimbic dopamine (DA) system – a critical component of the neural circuitry regulating motivation and reward. These studies found that both sexual experience and sex-associated environmental cues cause endogenous opioid release and activation of DA producing neurons in the ventral tegmental area (VTA). In addition, a large population of non-dopaminergic neurons was activated by sexual behavior. Therefore, the second set of studies explored the anatomical relationship between these sex-activation neurons and other components of the limbic system. These studies suggest that the medial prefrontal cortex (mPFC) may contribute to the activation of this cell population. The final set of studies investigated whether repeated endogenous activation of the mesolimbic system causes similar functional changes as repeated administration of drugs of abuse. Indeed, sexually experienced animals displayed a robust sensitization to the locomotor effects of amphetamine. Taken together, these studies suggest that sexual behavior activates the mesolimbic DA pathway and that repeated endogenous activation of this system results in long-term changes in its function. ACKNOWLEDGEMENTS I would like to thank the following: My parents Jimmy and Ellen and my brother Jay, for your love and support through the years, and my fiancé Adam, for your love and support in the years to come. My thesis advisors: Dr. Lique Coolen, for being a both a great friend and advisor, and Dr. Lei Yu, for always being there for me. My thesis committee: Dr. Lique Coolen, Dr. Lei Yu, Dr. Gary Gudelsky, Dr. Neil Richtand, and Dr. Frank Sharp, for your advice and encouragement. The members of the Coolen and Yu labs, past and present, for all of your much appreciated help with this project, and for creating a wonderful environment in which to work, and my fellow students in the Neuroscience Program and PSTP. The Neuroscience Graduate Program, especially Dr. Mike Lehman and Deb Cummins, and the Physician Scientist Training Program, especially Terri Berning, Dr. Les Myatt and Dr. Judy Harmony. The National Institute on Drug Abuse and the PSTP Anonymous Donor, for your financial support of this work. TABLE OF CONTENTS List of Tables ii List of Illustrations iii List of Abbreviations vi Chapter 1: General Introduction 1 References 18 Figures 23 Chapter 2: Sexual behavior and sex-associated environmental cues activate the 25 mesolimbic system in male rats Introduction 26 Methods 28 Results 34 Discussion 38 References 45 Tables 48 Figures 54 Chapter 3: Anatomical relationship between medial prefrontal cortex and 61 nucleus accumbens efferents and sex-activated neurons Introduction 62 Methods 64 Results 73 Discussion 79 References 85 Figures 87 Chapter 4: Sexual behavior causes a sensitized locomotor response to 98 amphetamine in male rats Introduction 99 Methods 101 Results 109 Discussion 119 References 126 Figures 129 Chapter 5: General Discussion 139 References 151 Figures 161 i LIST OF TABLES Chapter 2 Table 1. Summary of sexual behavior during the final pre-test mating session 48 Table 2. Summary of sexual behavior on the test day 49 Table 3. Percentages of TH cells expressing Fos 50 Table 4. Fos expression in non-dopaminergic neurons 51 Table 5a. Fos expression in NAc Core 52 Table 5b. Fos expression in NAc Shell 53 ii LIST OF ILLUSTRATIONS Chapter 1 Figure 1. Schematic diagram of the circuitry involved in motivation and reward. 23 Figure 2. Schematic diagram illustrating the mechanism by which opioid action 24 in the VTA stimulates DA release in the NAc. Chapter 2 Figure 1. Schematic drawings illustrating the areas of analysis of Fos/TH-IR, 54 indicated by the boxes, in the VTA at four rostral to caudal levels. Figure 2. Schematic drawings illustrating the areas of analysis (indicated by the 55 boxes; 400 µm x 600 µm) of Fos-IR in the NAc Core and Shell at three rostral to caudal levels Figure 3. Color plate 56 Figure 4. MOR internalization in VTA neurons. A, Numbers of MOR-IR 57 endosome-like particles per cell. Figure 5. Percentage of TH-IR cells that are Fos-IR in the VTA 58 Figure 6. Numbers of nondopaminergic cells that are Fos-IR in the VTA 59 Figure 7. Numbers of Fos-IR cells in the NAc. Mean numbers ± SEM of Fos-IR 60 cells in the NAc Core (A) or NAc Shell (B) averaged over three rostral-to-caudal levels. Chapter 3 Figure 1. Schematic diagram illustrating areas of analysis for Fos/BDA/NeuN 87 counts in the NAc Core and Shell at three rostrocaudal levels (A-C), BST (D), BLA (E), VTA at four rostrocaudal levels (F-I) and SPFp (G). Figure 2. Color plate 88 Figure 3. Schematic illustration of location and sizes of injections sites 89 Figure 4. Camera lucida drawings illustrating the distribution of fibers projecting 90 from the ILA, PL and ACA to four rostrocaudal levels of the VTA iii Figure 5. Camera lucida drawings illustrating the distribution of fibers projecting 91 from the ILA, PL and ACA to three rostrocaudal levels of the NAc Figure 6. Camera lucida drawings illustrating the distribution of fibers projecting 92 from the ILA, PL and ACA to the MPN, BST, and MEA Figure 7. Camera lucida drawings illustrating the distribution of fibers projecting 93 from the NAc Shell to four rostrocaudal levels of the VTA Figure 8. Camera lucida drawings illustrating the distribution of fibers projecting 94 from the NAc Core to four rostrocaudal levels of the VTA Figure 9. Camera lucida drawings illustrating the distribution of fibers projecting 95 from the NAc Core and Shell to the MPN Figure 10. Quantification of the relative contribution of the mPFC and NAc to 96 sex-induced activation of VTA neurons Figure 11. Quantification of the relative contribution of ILA and PL to sex- 97 induced activation of neurons in other areas related to sexual behavior and motivation Chapter 4 Figure 1. Schematic diagram of the zone map used to measure locomotor activity 129 Figure 2. Schematic drawings illustrating the areas of analysis of Fos/TH-IR, 130 indicated by the boxes, in the PFC, NAc, and VTA Figure 3. Experiment 1: Locomotor response of sexually experienced and naïve 131 animals in response to saline or amphetamine administered in a novel environment Figure 4. Experiment 2: Locomotor response of sexually experienced and naïve 132 animals in response to saline or amphetamine administered in a novel or non- novel environment Figure 5. Experiment 3: Locomotor response to the mating environment 133 Figure 6. Experiment 3: Locomotor response of sexually experienced and naïve 134 animals in response to saline or amphetamine administered in the same environment in which they received sexual experience, one day following the final pre-test mating session (Day 8). iv Figure 7. Experiment 3: Locomotor response of sexually experienced and naïve 135 animals in response to saline or amphetamine administered in the same environment in which they received sexual experience, one week following the final pre-test mating session (Day 14) Figure 8. Experiment 3: Locomotor response of sexually experienced and naïve 136 animals in response to saline or amphetamine administered in the same environment in which they received sexual experience, one month following the final pre-test mating session (Day 35). Figure 9. Experiment 1: Numbers of Fos-IR cells in the NAc of sexually 137 experienced and naïve animals in response to saline or amphetamine administered in a novel environment Figure 10. Experiment 1: Numbers of Fos-IR cells in the VTA of sexually 138 experienced and naïve animals in response to saline or amphetamine administered in a novel environment Chapter 5 Figure 1. Proposed circuitry model 161 v LIST OF ABBREVIATIONS AAA: Anterior Amygdaloid Area ACAd: Anterior Cingulate