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 Area, dorsal part Ach: Acetylcholine ACAv: Anterior Cingulate Area, ventral part AMPH: Amphetamine aco: Anterior Commissure, olfactory limb BDA: Biotinylated dextran amine BLA: Basolateral Nucleus Amygdala BMAp: Basomedial Nucleus Amygdala, posterior part BST: Bed Nuclei Stria Terminalis CEA: Central Nucleus Amygdala CP: Caudoputamen cpd: Cerebral Peduncle DA: Dopamine DMHa: Dorsalmedial Nucleus fr: Fasciculus Retroflexus [Meynert] GABA: Gamma amino butyric acid GAD: Gamma amino decaorboxylase GPI: Globus Pallidus, Lateral Segment IF: Interfascicular Nucleus Raphe ILA: Infralimbic Area IMD: Intermediodorsal Nucleus Thalamus LA: Later Nucleus Amygdala LH: Lateral Habenula [Nissl] LHA: Lateral Hypothalamic Area [Nissl] LM: Lateral Mammillary Nucleus [Gudden] LPO: Lateral Preoptic Area LS: Lateral Septal Nucleus [Cajal] MA: Magnocellular Preoptic Nucleus [Loo] MD: Mediodorsal Nucleus Thalamus MEApd: Medial Nucleus Amygdala, posterodorsal part MEApv: Medial Nucleus Amygdala, posteroventral part MH: Medial Habenula [Nissl] ml: Medial Lemniscus [Reil] MM: Medial Mammillary Nucleus [Gudden] MOR: Mu opioid receptor mp: Mammillary Peduncle [Meynert] MPN: Medial Preoptic Nucleus MPO: Medial Preoptic Area MRN: Mesencephalic Reticular Nucleus NAc: Nucleus Accumbens NMDA: N-methyl D-aspartate

vi opt: Optic Tract PAG: Periaqueductal Gray PH: Posterior Hypothalamic Nucleus PL: Prelimbic Area pr: Perireuniens Nucleus RE: Nucleus Reuniens [Malone] RL: Rostral Linear Nucleus Raphe RN: Red Nulceus [Burdach] SC: Superior Colliculus SI: Substantia innominata [Reil, Reichert] SNc: Substantia Nigra, compact part SNr: Substantia Nigra, reticular part SPFp: Subparafascicular Nucleus Thalamus, parvicellular part st: Stria Terminalis [Wenzel-Wenzel] TH: Tyrosine hyroxylase TU: Tuberal Nucleus, [Malone] VGLUT: Vesicular glutamate transporter VMH: Ventromedial Nucleus Hypothalamus VTA: Ventral Tegmental Area [Tsai] ZI: Zona Incerta

vii Chapter 1

General Introduction

1 GENERAL INTRODUCTION

Drug addiction is a chronic disorder characterized by compulsive drug use. Addicts display a loss of control over drug seeking and taking despite the adverse health and social consequences resulting from their drug use (APA, 1994). Moreover, addiction is a lifelong disease often consisting of periods of abstinence punctuated by periods of intense craving and relapse. In many cases, environmental stimuli that the addict associates with drug taking (people, places, paraphernalia) trigger bouts of intense craving (Childress et al., 1988; Wallace, 1989), and exposure to these drug-associated cues are believed to play a major role in the high rate of relapse associated with drug addiction. Addiction is a complex disorder in which individual genetic variation, psychological development and environmental and social factors all play a role in making the brain more susceptible to the disease (Lende and Smith, 2002). While much progress has been made towards understanding the pathology of this disorder, the neurobiological basis of the transition from repeated recreational drug use to the compulsive behaviors indicative of addiction remains a mystery. There is increasing evidence that addictive substances converge upon common neural circuitry that evolved for the purpose of mediating natural rewarding and motivated behaviors such as eating, drinking and sexual behavior. The present study explores the mechanisms by which these circuits are activated endogenously, and what, if any, alterations in the function of these circuits arise from repeated exposure to natural rewarding stimuli. A greater understanding of how these systems function in the natural state may provide new insight into the pathology of drug addiction.

2 Neural circuitry

Drug addiction involves a complex set of behaviors, and thus the neural circuitry governing these behaviors is complex and distributed throughout the mammalian brain.

This circuitry includes brain areas associated with diverse functions such as pleasure, fear, emotion, learning, and motor function, all of which interact with each other as an interconnected neural system (Figure 1). Presented below are some of the key components of this system and evidence for their involvement in drug addiction.

The mesolimbic dopamine system

The mesolimbic dopamine system consists of dopamine (DA) producing neurons located in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc).

The NAc is mainly comprised of medium-sized GABAergic projection neurons containing a high density of dendritic spines. The density of these spines is among the highest in the brain, suggesting that the function of these medium spiny neurons is to integrate information from many different sources (Heimer et al., 1997). Indeed, the

NAc has been called a “limbic motor interface” and is believed to be involved in the translation of motivation and emotion into movement and action (Mogenson et al., 1980).

Moreover, the NAc can be subdivided into distinct core and shell subregions which differ in their histochemical characteristics and connectivity with other components of the

3 circuitry mediating motivation and reward (Heimer et al., 1997; Kelley, 1999; Zahm,

1999). In particular, the NAc Core possesses similarities to the dorsal striatum, and sends projections to classic basal ganglia output structures, including the ventral pallidum, subthalamic nucleus and substantia nigra. Conversely, the NAc Shell sends projections mainly to limbic structures such as the VTA, lateral hypothalamus, and brainstem autonomic centers (Heimer et al., 1991; Zahm and Brog, 1992). Thus, the NAc core is in an anatomical position to mediate voluntary motor function, while the NAc Shell is in a position to mediate behaviors related to motivation and emotion.

This mesolimbic circuit is believed to be the point of convergence upon which the addictive drugs exert their effects. This hypothesis is supported by the observation that many of the most commonly abused drugs – including opiates, ethanol, nicotine, amphetamine, cocaine – cause DA release in the NAc (Wise and Bozarth, 1987; Di

Chiara and Imperato, 1988). This accumbal DA release appears to be a critical component of addictive behavior. Numerous studies have shown that selective lesions of mesolimbic DA terminals attenuate self-administration of psychostimulants in rodent models (Roberts et al., 1977; Lyness et al., 1979; Roberts et al., 1980; Roberts and Koob,

1982; Pettit et al., 1984). Moreover, D1 receptor antagonists infused into the NAc inhibit the development of other measures of reward such as conditioned-place preference (Hiroi and White, 1991; Baker et al., 1996). Taken together, these studies suggest that mesolimbic dopamine transmission plays a major role in drug reinforcement.

4 Cortical involvement

Both the NAc and VTA receive robust excitatory glutamatergic input from the medial prefrontal cortex (mPFC). In the NAc, glutamatergic efferents from the mPFC and dopaminergic efferents from the VTA often terminate on the same postsynaptic cell – even on the same dendritic spine (Bouyer et al., 1984; Freund et al., 1984; Sesack and

Pickel, 1992). In the VTA, mPFC efferents terminate predominantly on non- dopaminergic neurons which are hypothesized to contain GABA (Sesack and Pickel,

1992). Moreover, the mPFC receives a robust connection back from the VTA. This mesocortical projection appears to be inhibitory, as the bulk of projection neurons

(~70%) contain GABA. The remaining projection neurons are dopaminergic and terminate primarily on cortical pyramidal cells. The dopaminergic projection to the mPFC is inhibitory as well, and is likely mediated through D2-family receptors (Karler et al., 1998a, b; Zhang et al., 2000). In addition, there appears to be strict specificity of the targets of the mPFC-VTA projection (Carr and Sesack, 2000a). In particular, mPFC efferents synapse on dopaminergic neurons projecting back to the mPFC, but not dopaminergic neurons projecting to the NAc. Conversely, mPFC efferents synapse on

GABAergic neurons projecting to the NAc, but not those projecting back to the mPFC.

The significance of this specificity is unknown.

Compared with the NAc and VTA, the mPFC has received less attention in drug abuse research, and its precise role in addiction is not yet entirely clear. However, the mPFC appears to be important for higher brain processes such as working memory,

5 attention, and executive control (Cardinal et al., 2002). Activation of the PFC has been correlated with cue-induced craving for cocaine in humans (Childress et al., 1988) and for ethanol in rats (Topple et al., 1998), while dopaminergic lesions of the mPFC facilitate the acquisition of cocaine self-administration (Schenk et al., 1991) and impair the extinction of an operant response (Weissenborn et al., 1997). Thus altered PFC function in drug addiction may lead to cognitive deficits in which conditioned responses to environmental stimuli can no longer be inhibited.

Mechanism of Drug Actions

Psychostimulants such as cocaine and amphetamine exert their effects on the DA system via their actions on monoamine axonal terminals. Specifically, cocaine inhibits dopamine and serotonin reuptake transporters, while amphetamine stimulates transporter mediated DA release. As a result, both drugs increase the extracellular concentration of

DA (Feldman et al., 1997). Opiates, however, are believed to stimulate the mesolimbic pathway via an indirect mechanism (Figure 1). Pharmacological and electrophysiological evidence suggests that dopaminergic projection neurons in the VTA are under tonic inhibition by local GABAergic interneurons. Administration of opiates stimulates the

Gi/o-coupled mu opioid receptor (MOR), resulting in the inhibition of these GABAergic neurons. This in turn leads to disinhibition of dopaminergic projection neurons and the subsequent release of DA into NAc. This circuitry model is supported by studies showing that MOR agonists administered into the VTA hyperpolarize GABA-containing

6 interneurons (Johnson and North, 1992), decrease the extracellular GABA concentration

(Klitenick et al., 1992), and increase the firing rate of DA neurons in the VTA (Matthews and German, 1984). Moreover, intra-VTA administration of both MOR agonists (Leone et al., 1991) and GABA antagonists (Ikemoto et al., 1997) result in increased levels of extracellular DA in the NAc.

Functional consequences of repeated drug administration

Repeated stimulation of the mesolimbic pathway by drugs of abuse results in long-term functional and structural changes that may contribute to the pathology of drug addiction. Chronic exposure to morphine, cocaine and amphetamine alter postsynaptic responses to DA signaling. In particular, repeated administration of these drugs affects multiple components of the cAMP/CREB signaling pathway, and results in increased expression of immediate early genes such as ∆FosB, homer1a, narp, and arc (Berke and

Hyman, 2000; Hyman and Malenka, 2001; Nestler, 2001). In addition, changes in synaptic strength are observed following chronic exposure to drugs of abuse: Cocaine, amphetamine, morphine, nicotine and ethanol all cause alterations in the relative ratio of

AMPA- and NMDA-receptor mediated synaptic currents (Thomas and Malenka, 2003).

These events may in turn result in changes in gene expression, leading to more long-term effects on the function of the mesolimbic reward circuitry. In addition, medium spiny neurons in the NAc display increases in dendritic spine density and branching following repeated exposure to cocaine, amphetamine and morphine (Robinson and Kolb, 1997,

1999a, b; Robinson et al., 2001b). These changes are similar to those observed in long-

7 term potentiation and memory formation. Such plasticity may underlie some of the long- term behavioral modifications involved in drug addiction.

Natural motivated behavior

The reward circuitry that is so important for addictive behavior evolved for the purpose of regulating natural behaviors in which reward and motivation are beneficial to the animal (Wise and Rompre, 1989). A variety of natural motivated behaviors activate the mesolimbic system, Feeding, drinking and aggression are associated with increased dopamine transmission in the NAc (Bassareo and Di Chiara, 1999; van Erp and Miczek,

2000; Hajnal and Norgren, 2001). In particular, male rodent sexual behavior appears to be an ideal model to study the activation of mesolimbic reward systems via natural stimuli. Like other motivated behaviors, sexual behavior results in increased DA efflux in the NAc (Pfaus et al., 1990; Robertson et al., 1991; Damsma et al., 1992; Wenkstern et al., 1993; Robinson et al., 2001a). However, unlike feeding and drinking behaviors, sexual motivation is unfettered by the effects of the homeostatic needs of the animal.

Male rodent sexual behavior consists of an appetitive approach phase followed by a consummatory phase in which the animal mounts and intromitts, culminating with ejaculation (Hull et al., 2002). Behavioral studies indicate that male rodent sexual behavior – and in particular ejaculation – is a rewarding and reinforcing behavior. Male rats readily develop a conditioned place preference to copulation (Agmo and Berenfeld,

1990; Agmo and Gomez, 1993; Meisel et al., 1996; Paredes and Alonso, 1997; Lopez et

8 al., 1999; Martinez and Paredes, 2001), and will perform operant tasks to gain access to a sexually receptive female (Everitt et al., 1987; Everitt and Stacey, 1987).

The mesolimbic DA system plays a critical role in the rewarding aspects of sexual behavior. Dopamine is released into the NAc upon presentation of the female, and remains elevated throughout the behavior (Pfaus et al., 1990; Pfaus and Phillips, 1991;

Damsma et al., 1992; Wenkstern et al., 1993). Infusion of DA receptor agonists into the

NAc facilitates the initiation of sexual behavior in male rats (Everitt et al., 1989) while antagonists have inhibitory effects (Pfaus and Phillips, 1989). Moreover, lesions of the medial forebrain bundle and DA terminal lesions in the NAc abolish sexual behavior completely (Hendricks and Scheetz, 1973; Pfaus and Phillips, 1989). In addition, endogenous opioids may be involved in the activation of the mesolimbic system during sexual behavior. Opioid receptor agonists infused into the VTA facilitate sexual behavior

(Band and Hull, 1990; Mitchell and Stewart, 1990) while intra-VTA administration of the

MOR antagonist naloxone impairs performance on tests of sexual motivation (van Furth and van Ree, 1996). Furthermore, antagonists to either DA or opioid receptors inhibit the development of conditioned place preference to sexual behavior (Agmo and Berenfeld,

1990; Meisel et al., 1996).

Role of dopamine in motivation and reward

Although there is clear and convincing evidence that the DA system in involved in motivation and reward, it is not entirely clear what specific role DA plays in mediating

9 the complex behavioral modifications involved in dug addiction. One of the most prominent hypothesis is the hedonia/anhedonia hypotheses, proposed by Wise (1987).

This theory asserts that dopamine systems mediate the pleasure associated with drugs of abuse as well as other rewarding stimuli such as food or sex. Through conditioning, secondary reinforcers also come to elicit pleasure via activation of the DA system. In furtherance of this theory, Koob et al have recently suggested that during drug withdrawal, the decrease in DA transmission results in a negative affect, and therefore addicts seek drugs in order to restore a “hedonic homeostasis.”

However, others have shown that the DA systems are often activated during the anticipatory or approach phases of motivated behavior, before the rewarding stimulus is received (Robinson and Berridge, 1993; Berridge and Robinson, 1998). This is seemingly contradictory to hedonic theories, which would predict that the DA system is maximally activated during the consummatory phase of the behavior, when the pleasurable effects of the stimulus are actually experienced. Shultz and colleagues

(Schultz, 2001) have demonstrated that in primates trained to expect a conditioned stimulus before a palatable food reward, DA neurons in the VTA discharge in response to a conditioned predictor of food reward. Interestingly, the response to these predictive cues is greater than the response to the reward itself. However, in inexperienced monkeys, DA discharge follows the food reward, in agreement with hedonic theories.

Only after multiple trials does the DA response become coupled to the CS. Similarly, during heroin self-administration paradigms. DA neurons in the VTA are activated more during the approach to the lever than when the drug was administered (Kiyatkin and

10 Rebec, 1997). Thus, DA signaling may mediate reward prediction in addition to reward itself.

Robinson and Berridge have proposed that the process of reward can be dissociated into separate components of “wanting” and “liking” (Robinson and Berridge,

1993; Berridge and Robinson, 1998). According to this theory, DA mediates the

“wanting” but not the “liking” or hedonic component of reward. In support of this theory, it has been demonstrated that rats with complete lesions of the medial forebrain bundle lack the motivation to approach food or water, and will die without experimenter intervention. However, when sucrose is administered via an intraoral tube, the animal still displays the facial affects associated with hedonia, suggesting that the DA lesions impair the “wanting” component of motivation and reward while leaving the “liking” component intact. This theory suggests that conditioned stimuli are not only predictors of reward, but that they gain incentive salience, thus they are “wanted” by the animal.

Thus the incentive salience theory provides a model for drug craving in which dopamine systems interact with hedonic reward and associative learning to produce a more complex component process of reward.

Scope of the thesis and discussion of techniques

While natural behaviors can be rewarding and reinforcing, repeated exposure to these behaviors do not result in a pathological state of addiction like drugs of abuse, despite the fact that they share common neural substrates. For example, rats trained to perform an operant task for an addictive drug continue this behavior even after it has

11 been coupled with an aversive stimulus such as lithium chloride (LiCl) injection

(Cardinal et al., 2002). This suggests that drug-related cues elicit “wanting” or craving which persists after the incentive value has been removed. In contrast, male rats will cease to approach a sexually receptive female when copulation has been paired with LiCl

(Peters, 1983; Agmo, 2002). In addition, while both stress and drug priming trigger relapse in rodent self-administration models, sexual behavior fails to do so (Shaham et al., 1997). Thus, activation of the reward circuitry via endogenous DA and opioid agonists during sexual behavior must produce different effects than activation via exogenous administration of addictive drugs. Unfortunately, few studies have addressed the effects of repeated exposure to natural rewarding stimuli. A better understanding of the manner in which these natural behaviors interact with mesolimbic circuitry may provide insight into the pathology of drug addiction.

Chapter 2

The first goal of this study is to explore the mechanisms in which male rodent sexual behavior activates the mesolimbic system. It is hypothesized that endogenous opioid release into the VTA results in disinhibition of dopaminergic neurons that project to the NAc. A second goal of this study is to determine the effects of prior sexual experience and sex-associated environmental cues on activation of the mesolimbic system. One of the hallmark features of drug addiction is the ability of environmental cues associated with prior drug use to elicit intense bouts of craving, and this craving as been correlated to conditioned activation of limbic reward systems, as discussed above.

12 Therefore, a main goal of this study is to determine if environmental cues associated with natural rewarding stimuli can provoke a similar conditioned response.

To address this question, two different functional anatomical techniques are used as markers of activation. First, visualization of MOR internalization is used as a marker for ligand induced activation of the receptor. This technique takes advantage of the fact that G-protein coupled receptors undergo endocytosis following ligand binding. In particular, MORs have been shown to undergo ligand-induced endocytosis both in vitro

(Keith et al., 1998) as well as in vivo (Eckersell et al., 1998; Trafton et al., 2000; Sinchak and Micevych, 2001). Internalized endosome-like particles can be visualized using confocal microscopy, and quantification of these particles provides an indirect measure of endogenous opioid peptide release. In addition, this technique allows cellular resolution in the determination of the targets of endogenous opioid action. Others have used similar techniques to visualize MOR activation in other areas of the brain and spinal cord in response to exogenously applied opioids (Keith et al., 1998; Trafton et al., 2000) and estrogen (Eckersell et al., 1998; Sinchak and Micevych, 2001). In addition, this laboratory has shown that sexual behavior causes MOR internalization in the medial preoptic nucleus of the hypothalamus in male rats (Coolen et al., 2003), marking the first demonstration of this technique to visualize endogenous opioid release in response to a behavior.

Second, Fos – the gene product of the immediate early gene c-fos – is used as a marker for neuronal activation in the VTA. Transcription of the c-fos gene is induced in

13 neurons following stimulation by a variety of physiological, pharmacological and behavioral stimuli, and thus immunostaining for the nuclear gene product Fos is a useful marker for neuronal activation (Curran and Morgan, 1995). A major advantage of this technique is that it allows one to determine which neurons are activated by a particular stimulus with cellular resolution. In addition, immunostaining for the Fos protein can be combined with staining for phenotypic markers for specific populations of neurons.

Thus, this technique not only provides for the determination of the degree of activation

(reflected in numbers of Fos-IR neurons) but also the identification of which neurons being activated. In this study, Fos immunostaining is combined with staining for tyrosine hydroxylase (TH), the rate limiting enzyme in DA synthesis in order to determine if DA producing neurons are activated during sexual behavior.

Chapter 3

The goal of this study is to investigate the anatomical relationship between the sex-activated neurons in the VTA with other components of the limbic reward circuitry.

Specifically, it is hypothesized that the mPFC and NAc provide input to neurons in the

VTA that are activated during sexual behavior. A second goal of this study is to determine the relationship between the mPFC and NAc with sex-activated neurons in other brain areas that have been implicated in sexual behavior and/or motivation. In particular, connections to the NAc, medial preoptic area, bed nucleus of the stria terminalis, basolateral amygdala, and subparafasicular nucleus of the thalamus are investigated.

14 In this study, neuronal tract tracing techniques are used to map the projections of other reward-associated brain regions in relation to activated (Fos-IR) neurons in the

VTA, NAc, and other sex/reward related areas that are activated during sexual behavior.

Specifically, the anterograde tracer biotinylated dextran amine (BDA) is injected into either the mPFC or NAc. This tracer is taken up by dendrites and cell bodies of neurons near the site of injection and subsequently carried via axonal transport through the axon and terminal arborizations, including terminal synaptic boutons. Because the tracer is conjugated to biotin, it can be visualized via standard avidin-biotin-peroxidase staining methods, resulting in a permanent reaction product that allows visualization of the fine morphology of anterogradely labeled axonal terminals and boutons (Brandt and

Apkarian, 1992; Veenman et al., 1992). Moreover, by using iontophoretic injections as opposed to pressure injections, injections can be made that are restricted to discrete subregions of the brain areas of interest. In this study, injections are made in the infralimbic, prelimbic, and anterior cingulate divisions of the mPFC and the core and shell divisions of the NAc.

To determine the relative contributions of the different subdivisions of the mPFC and NAc, a novel quantification method is employed. Brain slices stained for BDA and

Fos are counterstained for the neuron specific protein NeuN, which labels the cytoplasm of neurons (but not glia). This allows for the visualization of BDA labeled synaptic boutons in close apposition to target neurons, and quantitative counts of putative synaptic contacts can be performed. Thus triple labeling for Fos, BDA and NeuN provides a mechanism for determining the amount of synaptic input from a certain brain region

15 relative to the amount of neural activation induced in the target area by a specific behavior.

Chapter 4

The final goal of this study is to determine if repeated exposure to a naturally rewarding stimulus results in functional alterations of the mesolimbic circuitry. Long- term alterations in the function of mesolimbic circuits are believed to underlie many of the behavioral changes involved in drug addiction. However, few studies have investigated whether endogenous activation of this circuit by natural rewarding behaviors can affect similar functional changes.

Changes in mesolimbic function can be measured behaviorally using the locomotor sensitization paradigm (Wise and Bozarth, 1987; Robinson and Berridge,

1993). This paradigm is based on the theory that both goal-directed approach behavior and operant reinforcement are mediated by common mesolimbic circuitry (Glickman and

Schiff, 1967). Wise and Bozarth (1987) extended this theory to include what they termed

“psychomotor stimulants” – i.e. all addictive drugs which induce forward locomotion via activation of the mesolimbic system. As described above, repeated exposure to these drugs causes functional changes in the mesolimbic circuitry, and the resulting hypersensitivity of the DA system is measured as an increased or “sensitized” locomotor response to an equal or lesser dose of the drug. Moreover, repeated administration of one drug can result in a sensitized response to a chemically unrelated drug – e.g. administration of opioids causes sensitization to amphetamine or cocaine, and vice versa

16 (Vezina et al., 1989; Cunningham and Kelley, 1992; Cunningham et al., 1997;

Vanderschuren et al., 1997). Such cross-sensitization provides further evidence that the different addictive drugs activate common reward pathways (Robinson and Berridge,

1993).

In this study, the effect of sexual experience on locomotor sensitization is investigated. If repeated endogenous activation causes functional changes in the mesolimbic system, then one would expect to see an increased locomotor response to a single low dose of amphetamine compared to that of sexually naïve animals. In addition, because environment and conditioning play such as important role in both the behavioral sensitization paradigm and the drug craving, this study investigates the effects of different environments in sex-experience induced locomotor sensitization.

17 REFERENCES

Agmo A (2002) Copulation-contingent aversive conditioning and sexual incentive motivation in male rats: evidence for a two-stage process of sexual behavior. Physiol Behav 77:425-435. Agmo A, Berenfeld R (1990) Reinforcing properties of ejaculation in the male rat: role of opioids and dopamine. Behav Neurosci 104:177-182. Agmo A, Gomez M (1993) Sexual reinforcement is blocked by infusion of naloxone into the medial preoptic area. Behav Neurosci 107:812-818. Association AP (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th Edition. Washington, DC: American Psychiatric Press. Baker DA, Khroyan TV, O'Dell LE, Fuchs RA, Neisewander JL (1996) Differential effects of intra-accumbens sulpiride on cocaine-induced locomotion and conditioned place preference. J Pharmacol Exp Ther 279:392-401. Band LC, Hull EM (1990) Morphine and dynorphin(1-13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats. Brain Res 524:77-84. Bassareo V, Di Chiara G (1999) Differential responsiveness of dopamine transmission to food-stimuli in nucleus accumbens shell/core compartments. Neuroscience 89:637-641. Berke JD, Hyman SE (2000) Addiction, dopamine, and the molecular mechanisms of memory. Neuron 25:515-532. Berridge KC, Robinson TE (1998) What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res Brain Res Rev 28:309- 369. Bouyer JJ, Park DH, Joh TH, Pickel VM (1984) Chemical and structural analysis of the relation between cortical inputs and tyrosine hydroxylase-containing terminals in rat neostriatum. Brain Res 302:267-275. Brandt HM, Apkarian AV (1992) Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey. J Neurosci Methods 45:35-40. Cardinal RN, Parkinson JA, Hall J, Everitt BJ (2002) Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci Biobehav Rev 26:321-352. Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873. Childress A, Ehrman R, McLellan AT, O'Brien C (1988) Conditioned craving and arousal in cocaine addiction: a preliminary report. NIDA Res Monogr 81:74-80. Coolen LM, Fitzgerald ME, Wells AB, Yu L, Lehman MN (2003) Activation of mu opioid receptors in the medial preoptic area following copulation in male rats. Submitted. Cunningham ST, Kelley AE (1992) Evidence for opiate-dopamine cross-sensitization in nucleus accumbens: studies of conditioned reward. Brain Res Bull 29:675-680.

18 Cunningham ST, Finn M, Kelley AE (1997) Sensitization of the locomotor response to psychostimulants after repeated opiate exposure: role of the nucleus accumbens. Neuropsychopharmacology 16:147-155. Curran T, Morgan JI (1995) Fos: an immediate-early transcription factor in neurons. J Neurobiol 26:403-412. Damsma G, Pfaus JG, Wenkstern D, Phillips AG, Fibiger HC (1992) Sexual behavior increases dopamine transmission in the nucleus accumbens and striatum of male rats: comparison with novelty and locomotion. Behav Neurosci 106:181-191. Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274-5278. Eckersell CB, Popper P, Micevych PE (1998) Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala. J Neurosci 18:3967-3976. Everitt BJ, Stacey P (1987) Studies of instrumental behavior with sexual reinforcement in male rats (Rattus norvegicus): II. Effects of preoptic area lesions, castration, and testosterone. J Comp Psychol 101:407-419. Everitt BJ, Cador M, Robbins TW (1989) Interactions between the amygdala and ventral striatum in stimulus-reward associations: studies using a second-order schedule of sexual reinforcement. Neuroscience 30:63-75. Everitt BJ, Fray P, Kostarczyk E, Taylor S, Stacey P (1987) Studies of instrumental behavior with sexual reinforcement in male rats (Rattus norvegicus): I. Control by brief visual stimuli paired with a receptive female. J Comp Psychol 101:395-406. Feldman RS, Meyer JS, Quenzer LF (1997) Principles of Neuropsychopharmacology. Sunderland, Massachusetts: Sinauer Associates. Freund TF, Powell JF, Smith AD (1984) Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 13:1189-1215. Glickman SE, Schiff BB (1967) A biological theory of reinforcement. Psychol Rev 74:81-109. Hajnal A, Norgren R (2001) Accumbens dopamine mechanisms in sucrose intake. Brain Res 904:76-84. Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the projection patterns of accumbal core and shell in the rat. Neuroscience 41:89-125. Heimer L, Alheid GF, de Olmos JS, Groenewegen HJ, Haber SN, Harlan RE, Zahm DS (1997) The accumbens: beyond the core-shell dichotomy. J Neuropsychiatry Clin Neurosci 9:354-381. Hendricks SE, Scheetz HA (1973) Interaction of hypothalamic structures in the mediation of male sexual behavior. Physiol Behav 10:711-716. Hiroi N, White NM (1991) The amphetamine conditioned place preference: differential involvement of dopamine receptor subtypes and two dopaminergic terminal areas. Brain Res 552:141-152. Hull EM, Meisel RL, Sachs BD (2002) Male Sexual Behavior. In: Hormones, Brain and Behavior (Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds), pp 1- 138. San Diego, CA: Elsevier Science (USA).

19 Hyman SE, Malenka RC (2001) Addiction and the brain: the neurobiology of compulsion and its persistence. Nat Rev Neurosci 2:695-703. Ikemoto S, Kohl RR, McBride WJ (1997) GABA(A) receptor blockade in the anterior ventral tegmental area increases extracellular levels of dopamine in the nucleus accumbens of rats. J Neurochem 69:137-143. Johnson SW, North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483-488. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, von Zastrow M (1998) mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Mol Pharmacol 53:377-384. Kelley AE (1999) Functional specificity of ventral striatal compartments in appetitive behaviors. Ann N Y Acad Sci 877:71-90. Kiyatkin EA, Rebec GV (1997) Activity of presumed dopamine neurons in the ventral tegmental area during heroin self-administration. Neuroreport 8:2581-2585. Klitenick MA, DeWitte P, Kalivas PW (1992) Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. J Neurosci 12:2623-2632. Lende DH, Smith EO (2002) Evolution meets biopsychosociality: an analysis of addictive behavior. Addiction 97:447-458. Leone P, Pocock D, Wise RA (1991) Morphine-dopamine interaction: ventral tegmental morphine increases nucleus accumbens dopamine release. Pharmacol Biochem Behav 39:469-472. Lopez HH, Olster DH, Ettenberg A (1999) Sexual motivation in the male rat: the role of primary incentives and copulatory experience. Horm Behav 36:176-185. Lyness WH, Friedle NM, Moore KE (1979) Destruction of dopaminergic nerve terminals in nucleus accumbens: effect on d-amphetamine self-administration. Pharmacol Biochem Behav 11:553-556. Martinez I, Paredes RG (2001) Only self-paced mating is rewarding in rats of both sexes. Horm Behav 40:510-517. Matthews RT, German DC (1984) Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine. Neuroscience 11:617-625. Meisel RL, Joppa MA, Rowe RK (1996) Dopamine receptor antagonists attenuate conditioned place preference following sexual behavior in female Syrian hamsters. Eur J Pharmacol 309:21-24. Mitchell JB, Stewart J (1990) Facilitation of sexual behaviors in the male rat associated with intra-VTA injections of opiates. Pharmacol Biochem Behav 35:643-650. Mogenson GJ, Jones DL, Yim CY (1980) From motivation to action: functional interface between the limbic system and the motor system. Prog Neurobiol 14:69-97. Nestler EJ (2001) Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2:119-128. Paredes RG, Alonso A (1997) Sexual behavior regulated (paced) by the female induces conditioned place preference. Behav Neurosci 111:123-128. Peters RH (1983) Learned aversions to copulatory behaviors in male rats. Behav Neurosci 97:140-145.

20 Pettit HO, Ettenberg A, Bloom FE, Koob GF (1984) Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self- administration in rats. Psychopharmacology (Berl) 84:167-173. Pfaus JG, Phillips AG (1989) Differential effects of dopamine receptor antagonists on the sexual behavior of male rats. Psychopharmacology (Berl) 98:363-368. Pfaus JG, Phillips AG (1991) Role of dopamine in anticipatory and consummatory aspects of sexual behavior in the male rat. Behav Neurosci 105:727-743. Pfaus JG, Damsma G, Nomikos GG, Wenkstern DG, Blaha CD, Phillips AG, Fibiger HC (1990) Sexual behavior enhances central dopamine transmission in the male rat. Brain Res 530:345-348. Roberts DC, Koob GF (1982) Disruption of cocaine self-administration following 6- hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol Biochem Behav 17:901-904. Roberts DC, Corcoran ME, Fibiger HC (1977) On the role of ascending catecholaminergic systems in intravenous self-administration of cocaine. Pharmacol Biochem Behav 6:615-620. Roberts DC, Koob GF, Klonoff P, Fibiger HC (1980) Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 12:781-787. Robertson GS, Pfaus JG, Atkinson LJ, Matsumura H, Phillips AG, Fibiger HC (1991) Sexual behavior increases c-fos expression in the forebrain of the male rat. Brain Res 564:352-357. Robinson DL, Phillips PE, Budygin EA, Trafton BJ, Garris PA, Wightman RM (2001a) Sub-second changes in accumbal dopamine during sexual behavior in male rats. Neuroreport 12:2549-2552. Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Res Brain Res Rev 18:247-291. Robinson TE, Kolb B (1997) Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine. J Neurosci 17:8491-8497. Robinson TE, Kolb B (1999a) Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine. Eur J Neurosci 11:1598-1604. Robinson TE, Kolb B (1999b) Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats. Synapse 33:160-162. Robinson TE, Gorny G, Mitton E, Kolb B (2001b) Cocaine self-administration alters the morphology of dendrites and dendritic spines in the nucleus accumbens and neocortex. Synapse 39:257-266. Schenk S, Horger BA, Peltier R, Shelton K (1991) Supersensitivity to the reinforcing effects of cocaine following 6-hydroxydopamine lesions to the medial prefrontal cortex in rats. Brain Res 543:227-235. Schultz W (2001) Reward signaling by dopamine neurons. Neuroscientist 7:293-302. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320:145-160.

21 Shaham Y, Puddicombe J, Stewart J (1997) Sexually arousing events and relapse to heroin-seeking in sexually experienced male rats. Physiol Behav 61:337-341. Sinchak K, Micevych PE (2001) Progesterone blockade of estrogen activation of mu- opioid receptors regulates reproductive behavior. J Neurosci 21:5723-5729. Thomas MJ, Malenka RC (2003) Synaptic plasticity in the mesolimbic dopamine system. Philos Trans R Soc Lond B Biol Sci 358:815-819. Topple AN, Hunt GE, McGregor IS (1998) Possible neural substrates of beer-craving in rats. Neurosci Lett 252:99-102. Trafton JA, Abbadie C, Marek K, Basbaum AI (2000) Postsynaptic signaling via the [mu]-opioid receptor: responses of dorsal horn neurons to exogenous opioids and noxious stimulation. J Neurosci 20:8578-8584. Tzschentke TM (2001) Pharmacology and behavioral pharmacology of the mesocortical dopamine system. Prog Neurobiol 63:241-320. van Erp AM, Miczek KA (2000) Aggressive behavior, increased accumbal dopamine, and decreased cortical serotonin in rats. J Neurosci 20:9320-9325. van Furth WR, van Ree JM (1996) Sexual motivation: involvement of endogenous opioids in the ventral tegmental area. Brain Res 729:20-28. Vanderschuren LJ, Tjon GH, Nestby P, Mulder AH, Schoffelmeer AN, De Vries TJ (1997) Morphine-induced long-term sensitization to the locomotor effects of morphine and amphetamine depends on the temporal pattern of the pretreatment regimen. Psychopharmacology (Berl) 131:115-122. Veenman CL, Reiner A, Honig MG (1992) Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. J Neurosci Methods 41:239-254. Vezina P, Giovino AA, Wise RA, Stewart J (1989) Environment-specific cross- sensitization between the locomotor activating effects of morphine and amphetamine. Pharmacol Biochem Behav 32:581-584. Wallace BC (1989) Psychological and environmental determinants of relapse in crack cocaine smokers. J Subst Abuse Treat 6:95-106. Weissenborn R, Robbins TW, Everitt BJ (1997) Effects of medial prefrontal or anterior cingulate cortex lesions on responding for cocaine under fixed-ratio and second- order schedules of reinforcement in rats. Psychopharmacology (Berl) 134:242- 257. Wenkstern D, Pfaus JG, Fibiger HC (1993) Dopamine transmission increases in the nucleus accumbens of male rats during their first exposure to sexually receptive female rats. Brain Res 618:41-46. Wise RA, Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469-492. Wise RA, Rompre PP (1989) Brain dopamine and reward. Annu Rev Psychol 40:191- 225. Zahm DS (1999) Functional-anatomical implications of the nucleus accumbens core and shell subterritories. Ann N Y Acad Sci 877:113-128. Zahm DS, Brog JS (1992) On the significance of subterritories in the "accumbens" part of the rat ventral striatum. Neuroscience 50:751-767.

22 Figure 1. Schematic diagram of the circuitry involved in motivation and reward. Dopaminergic projections are depicted in red; glutamatergic projections are depicted in green. Modified after Nestler, 2001.

23 Figure 2. Schematic diagram illustrating the mechanism by which opioid action in the VTA stimulates DA release in the NAc. DA neurons projecting to the VTA are under tonic inhibition by GABA interneurons in the VTA. Opioids bind to MOR receptors, resulting in inhibition of GABA neurons and disinhibition of DA neurons.

24 Chapter 2

Sexual behavior and sex-associated environmental cues activate the mesolimbic system in male rats.

25 INTRODUCTION

The mesolimbic dopamine (DA) system regulates a variety of motivated behaviors, including sexual behavior, and consists of a dopaminergic projection from the ventral tegmental area (VTA) to the nucleus accumbens (NAc). The NAc plays a vital role in the regulation of sexual behavior, and mating has been correlated with increases in both extracellular levels of DA and immediate early gene expression in this area (Pfaus et al., 1990; Robertson et al., 1991). Moreover, administration of amphetamine into the

NAc facilitates the initiation of sexual behavior in male rats (Everitt et al., 1989) while

DA antagonists are inhibitory (Pfaus and Phillips, 1989). In addition, bilateral lesions of the medial forebrain bundle or DA terminal lesions of the NAc abolish sexual behavior

(Hendricks and Scheetz, 1973), suggesting that the dopaminergic projection from the

VTA is essential for the expression of this behavior. Opioids also play a major role, as opioid receptor agonists infused into the VTA or NAc facilitate sexual behavior (Band and Hull, 1990; Mitchell and Stewart, 1990). Conversely, intra-VTA naloxone administration inhibits sexual motivation (van Furth and van Ree, 1996).

Pharmacological and anatomical evidence suggest that activation of mu opioid receptors (MOR) in the VTA results in inhibition of local GABAergic interneurons, leading to disinhibition of DA neurons and subsequent DA release in NAc (Matthews and

German, 1984; Leone et al., 1991; Johnson and North, 1992; Klitenick et al., 1992;

Ikemoto et al., 1997). Despite this wealth of pharmacological data, relatively little is known about the anatomical organization of this circuitry. It has been demonstrated that

26 MOR are primarily present in nondopaminergic neurons in the VTA (Garzon and Pickel,

2001). However, it is unknown if MOR are located on GABA neurons. Therefore, the first goal of the present study is to extend our knowledge of the anatomical distribution of

MOR in the VTA, particularly in respect to GABAergic neurons.

The overall goal of the present study is to investigate activation of the MOR and

DA neurons in the VTA during male sexual behavior. First, to test the hypothesis that opioids are released into the VTA during sexual behavior, we used MOR internalization as a marker for ligand induced activation of the receptor. Receptor internalization has previously been used to visualize MOR activation in response to exogenously applied opioids (Keith et al., 1998; Trafton et al., 2000) and estrogen (Eckersell et al., 1998;

Sinchak and Micevych, 2001). Here we demonstrate the use of this technique to measure the activation of MOR by behavior and environment. Our next goal was to determine if sexual behavior activates DA neurons in the VTA. To address this question, we used Fos immunoreactivity (Fos-IR) as a marker of neuronal activation.

Finally, we investigated the effects of experience and environment on the activation of this circuit. Specifically, we compared animals that were sexually experienced versus naïve animals. In addition, we compared animals that gained their experience in different environments in order to investigate the effect of sex-related environmental cues on the activation of the mesolimbic DA system.

27 MATERIALS AND METHODS

Animals: Adult male Sprague Dawley rats (250-260 grams) were obtained from Harlan

(Indianapolis, IN) and housed individually in plexiglass cages for the duration of the experiment. The colony room was maintained on 12/12 hr reversed light dark cycle

(lights off at 10 AM). Food and water were available ad libitum. Stimulus females for mating behavior tests were bilaterally ovariectomized and received a subcutaneous implant containing 5% estradiol benzoate (EB) and 95% cholesterol. Sexual receptivity was induced by administration of 500 µg progesterone (P; subcutaneous) in 0.1 ml sesame oil 5 hours before testing. All procedures were approved by the Animal Care and

Use Committee of the University of Cincinnati and conformed to NIH guidelines involving vertebrate animals in research.

Sexual Behavior: Rats were randomly divided into four groups differing in sexual experience (naïve versus experienced) and the environment in which sexual behavior testing was performed (home cage versus test cage). Animals in the sexually experienced groups were allowed to mate with a receptive female to one ejaculation or for 60 minutes, whichever came first, during 5 bi-weekly pre-test sessions. During these pre-test sessions, animals either mated in their home cage (Experienced Home Cage: EH); or in a larger test cage (60 x 45 x 50 cm) with clean bedding (Experienced Test Cage: ET). The

ET animals were placed in the test cage for 60 minutes prior to the introduction of the female, then allowed to mate to one ejaculation or 60 minutes. Animals that did not receive sexual experience were either left undisturbed in the home cage (Naïve Home

Cage: NH) or were placed in test cages without a female for one hour for 5 bi-weekly sessions (Naïve Test Cage: NT). One week following the last pre-test mating session, the

28 rats were randomly subdivided into “sex” and “control” groups (total of 8 groups, N=4 each). During the final test, “sex” animals were allowed to mate to one ejaculation in the same environment in which they gained experience. EH Sex (EHS) and NH Sex (NHS) animals mated in their home cages. ET Sex (ETS) and NT Sex (NTS) animals were placed in the test cages for one hour, following which a female was placed in the test cage and the males mated to one ejaculation. Five minutes following ejaculation, the female partner was removed and the male remained in the home cage or test cage for one hour until sacrifice. EH Control (EHC) and NH Control (NHC) animals did not receive a receptive female partner, but instead were taken from the home cage and sacrificed; ET

Control (ETC) and NT Control (NTC) animals were placed in the test cage without a receptive female for 2 hours before sacrifice.

Tissue Preparation: The animals were deeply anesthetized using pentobarbital (200 mg/kg) and perfused transcardially with 100 mL of 0.9% NaCl followed by 500 mL of

4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). The brains were removed and post-fixed for 1 hour at room temperature in the fixative, then placed in 20% sucrose in 0.1 M PB and stored at 4° C. Coronal sections (35 µm) were cut on a freezing microtome (Richard Allen, Kalamazoo, MI), collected in four parallel series in cryoprotectant solution (30% sucrose, 30% ethylene glycol in 0.1 M PB; Watson et al,

1985), and stored at –20°C until further processing.

Immunocytochemistry: All incubations were performed at room temperature with gentle agitation. Free-floating sections were washed extensively with 0.1 M phosphate buffered

29 saline (PBS) between incubations. Sections were incubated for 10 minutes with 1%

H2O2, then blocked for one hour with incubation solution (PBS containing 0.1% bovine serum albumin and 0.4% triton X-100). All primary antibody incubations were performed in the incubation solution, overnight at room temperature. Following staining, the sections were washed thoroughly in 0.1M PB, mounted onto glass slides with 0.3%

gelatin in ddH20 and coverslipped with DPX (Electron Microscopy Sciences, Fort

Washington, PA) or an aqueous mounting medium (Gelvatol) containing an anti fading agent (1,4-diazabicyclo (2,2)octane). Immunocytochemical controls included omission of primary antibodies. VTA and/or NAc series were stained for the following markers:

Fos/TH: VTA and NAc sections were incubated overnight with a rabbit polyclonal antibody to c-Fos (1:7500; SC-52; Santa Cruz Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated donkey anti-rabbit IgG (1:400; Jackson

ImmunoResearch Laboratories, West Grove, PA) and avidin-horseradish peroxidase complex (1:1000; ABC Elite Kit, Vector Laboratories, Burlingame, CA). Finally, the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-

Aldrich, St. Louis, MO) in 0.1M PB containing 0.012% hydrogen peroxide and 0.08% nickel sulfate, resulting in a blue-black reaction product. Next, VTA sections were incubated overnight with mouse monoclonal antibody to tyrosine hydroxylase (TH;

1:400,000; Chemicon International, Temecula, CA), biotinylated donkey anti-mouse IgG secondary antibody (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA) and ABC as described above. Finally, the sections were incubated for 10 minutes in

0.02% DAB in 0.1 M PB containing 0.012% hydrogen peroxide, resulting in a reddish- brown reaction product.

30 MOR: VTA sections were incubated overnight with a rabbit polyconal antibody recognizing the C-terminal region of the rat MOR1 (1:10,000; DiaSorin, Saluggia, Italy), biotinylated donkey anti-rabbit IgG and ABC as described above. Next, sections were

incubated for 10 minutes with biotinylated tyramide (BT; 1:250 in PBS + 0.003% H2O2;

Tyramide Signal Amplification Kit, NEN Life Sciences, Boston, MA) and for 30 minute with CY-3 conjugated steptavidin (1:200; Jackson ImmunoResearch Laboratories, West

Grove, PA).

GABA/MOR: VTA sections were incubated overnight with a mouse monoclonal antibody to GABA (1:1,000 in PBS/BSA without TX; Sigma-Aldrich, St. Louis, MO) and for 30 minutes with goat anti-mouse IgG conjugated to Alexa 488 (1:200 in

PBS/BSA; Jackson ImmunoResearch Laboratories, West Grove, PA). The sections were then stained for MOR as described above.

GAD/TH: VTA sections were incubated with a rabbit polyclonal antibody recognizing gamma amino decarboxylase (GAD; 1:1,500, Chemicon International, Temecula, CA) followed by incubations with biotinylated donkey anti-rabbit IgG, ABC, BT and CY-3 conjugated streptavidin as described above. Next, sections were incubated with a mouse monoclonal antibody to tyrosine hydroxylase (TH; 1:40,000; Chemicon International,

Temecula, CA) followed by a 30-minute incubation with goat anti-mouse IgG conjugated to Alexa 488 (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA).

Data Analysis: VTA Anatomy: For anatomical analysis of the VTA, stacks of 1 µm optical sections were captured along the z-axis using a Zeiss LSM-510 laser scanning microscope. CY3-fluorescence was imaged with a 567 nm emission filter and a HeNe

31 laser, and Alexa 488 with 505 nm emission filter and Argon laser. The distribution of

GAD-IR fibers relative to TH neurons was investigated. In addition, the location of MOR in relation to GABA-IR cell bodies was investigated and the rostrocaudal distribution of

MOR-IR neurons co-expressing GABA was quantified in one animal. In addition, the rostrocaudal distribution on MOR-IR cell bodies throughout the VTA was analyzed in 3 animals.

Sexual Behavior: Each pre-test and final test mating session was observed and sexual behavior was recorded: number of mounts (#M), number of intromissions (#I), mount and intromission latencies (ML and IL, i.e the time from presentation of the female to the first mount or intromission), and ejaculation latency (EL; the time from the first intromission to ejaculation). Results of each measure for the last day of the pre-test mating sessions were analyzed using a one-way ANOVA to determine if the mating environment (home cage versus test cage) affected sexual experience. Results from the final test day were analyzed using a two-way ANOVA (factors: experience, environment) and post-hoc comparisons were performed using Fisher’s PLSD, all with 5% significance levels.

MOR internalization: For quantitative analysis of endosome counts in VTA neurons, z- stacks of 1 µm optical sections through 15-30 neurons were captured using a Zeiss laser scanning confocal microscope system (Zeiss LSM-510). Of each stack of images through the neurons, 2 consecutive sections in the middle of the neuron were used for analysis. Numbers of immunoreactive intracellular particles were counted for each cell by an observer blind to experimental group and were averaged per animal. In addition, the percentage of internalized MOR-IR cells was quantified; MOR-IR neurons containing

32 3 or more immunoreactive intracellular particles were considered internalized. Results were analyzed using a three-way ANOVA (factors: mating, experience, and environment) and post-hoc comparisons (Fisher’s PLSD) using 5% significance levels. Images were imported into Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) to compose the figures. Images were not adjusted or altered in any way, except for brightness.

Fos/TH: Using a drawing tube attached to a Leica microscope (Leica Microsystems;

Wetzlar, Germany), camera lucida drawings were made of the analyzed sections from each animal. In the VTA, camera lucida drawings were made of four sections approximately 280 µm apart, representative of four rostral to caudal levels in the VTA

(Figure 1). Using TH staining and the location of the medial lemniscus (ml) and fasciculus retroflexus (fr) as landmarks, standard areas were defined in which to count

Fos-IR nuclei and Fos/TH double labeled cells. Cell counts were performed in standard areas ranging from 2.16 mm2 (rostral and two middle levels) to 1.6 mm2 (most caudal level). In the NAc, camera lucida drawings were made of three sections approximately

600 µm apart, representative of three rostral to caudal levels in the NAc (Figure 2). At each level, standard areas of 0.24 mm2 were defined in which to count Fos-IR nuclei in both the NAc Core and NAc Shell. Group means were calculated for each separate rostral to caudal level in both NAc and VTA. In addition, an average of the 4 (VTA) or 3

(NAc) rostral to caudal levels were calculated per animal and group means were based on animal averages. Results were analyzed using a three-way ANOVA (factors: mating, experience, and environment) and post-hoc comparisons (Fisher’s PLSD) using 5% significance levels. Specifically, comparisons were made between 1) each sex group and the corresponding control group, 2) all control groups, and 3) all sex groups. Digital

33 images of immunostained sections were captured using a digital camera (Magnafire,

Optronics) attached to a Leica microscope (Leica Microsystems; Wetzlar, Germany).

Images were imported into Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) to compose the figures. Images were not adjusted or altered in any way, except for occasional adjustment of brightness.

RESULTS

VTA anatomy

MOR-IR was observed on both axon terminals and cell bodies (Figure 3A), and in fibers in close apposition to GABA-IR neurons (Figure 3B). In addition, MOR-IR was observed to colocalize with GABA-IR in cell bodies. Approximately 79% of MOR-IR neurons co-expressed GABA-IR in the rostral portion of the VTA (Figure 1A-B), where the majority of MOR-IR neurons are located. Few MOR-IR neurons were observed in the caudal VTA, although approximately 50% of these MOR-IR neurons co-express

GABA. In addition, we observed GAD-IR fibers in close apposition to TH-IR neurons throughout the VTA.

Sexual Behavior

Measurements for sexual behavior on the last day of the pre-test mating sessions of the sexually experienced males are illustrated in Table 1. The environment in which the experienced males gained their experience did not affect any of the analyzed parameters of sexual behavior (Table 1). In particular, on the last day of the pre-test mating sessions, there were no differences between the males that mated in the home

34 cage or in the test cage. Measurements for sexual behavior during the test day are illustrated in Table 2. Significant differences were observed between the naïve and

experienced males on the test day (F(1,12)= 8.927; p = 0.0113; Table 2). Naïve males that mated in the test cage (NTS) had higher numbers of mounts and longer latencies to mount, intromission and ejaculation compared to experienced males that mated either in the test cage (ETS) or in the home cage (EHS). We observed a similar trend in the naive males that mated in the home cage (NHS), although the differences did not reach significance in post hoc tests. Statistically significant differences in sexual behavior between NTS and NHS males were only observed in IL, which was higher in NTS males.

MOR internalization

Mating-induced internalization of MOR was analyzed in neurons located in the rostral VTA (Figure 1A-B), where the majority of MOR-IR cell bodies were located.

Sexual behavior significantly increased MOR internalization in the VTA (F(1,23)= 112.382; p < 0.0001). Significant mating-induced increases in the number of MOR-IR endosome- like particles were observed in all males that mated on the test day compared to their controls (Figure 4A, filled vs. open bars; NHC vs. NHS, EHC vs, EHS, NTC vs. NTS,

ETC vs. ETS, p < 0.03). In addition, exposure to the sex-related environment alone also resulted in a significant increase of MOR internalization. In particular, a three-way

interaction between mating, experience and environment was observed (F(123)=16.370; p =

0.0005) and post hoc analysis indicated that MOR internalization was significantly increased in ETC males that had been placed in the environment in which they received prior sexual experience (Figure 4A, open bars; ETC vs. NHC, EHC and NTC; p < 0.05).

35 Sexual behavior also significantly increased the percentage of MOR-IR neurons that

showed internalization (F(1,23)=136.312; p < 0.0001). In particular, a greater proportion of internalized MOR-IR neurons was observed in males that mated on the test day compared to their controls (Figure 4b, filled vs. open bars; NHC vs. NHS, EHC vs, EHS, NTC vs.

NTS, ETC vs. ETS, p < 0.03) and following exposure to the sex-related environment in

ETC males compared to the other controls (Figure 4B, open bars; ETC vs. NHC, EHC and NTC, p < 0.01).

Fos expression in dopamine neurons

Sexual behavior and exposure to the sex-related environment resulted in activation of dopamine neurons throughout the VTA (Figure 5). There was a significant effect of mating on the percentage of TH cells expressing Fos throughout the VTA

(F(1,24)= 99.774; p < 0.0001). Post-hoc analysis indicated significant mating-induced increases in Fos expression in naïve males that mated in either home or test cage, and in experienced males that mated in the home cage (Figure 5, NHC vs. NHS, EHC vs, EHS,

NTC vs. NTS, p < 0.0001). In addition, a two-way interaction between mating and

environment was observed (F(1,24)= 12.479; p = 0.0017). Post hoc analysis indicated that the percentage of activated TH cells was significantly increased in ETC males that were exposed to the environment in which they received prior sexual experience (Figure 5,

ETC vs. NHC, EHC and NTC, p < 0.001). Interestingly, mating did not further increase the percentages of activated TH neurons in ETS males compared to ETC males. Analysis of activated TH neurons in each of the four separate rostrocaudal levels demonstrated that

36 the percentages of activated TH neurons following mating or sex-related environment were higher in the rostral levels of the VTA compared to the caudal levels (Table 3).

Fos expression in nondopaminergic neurons

Although sexual behavior and sex-related cues induced Fos expression in TH neurons, the majority (80-90%) of Fos-IR neurons in the VTA did not express TH.

Analysis of Fos expression in these nondopaminergic neurons revealed a similar induction pattern as in TH neurons (Figure 6). Specifically, a significant effect of mating

on Fos expression was observed (F(1,24) = 40.093, p < 0.0001), and significant mating- induced increases in numbers of Fos-IR neurons were present in the NHS, EHS, and NTS groups compared to their controls (Figure 6, p < 0.006). Moreover, there was a significant

interaction between mating and environment (F(1,24) = 5.288, p = 0.0305), and a significant increase in Fos expression was observed in ETC males that were placed in the environment in which they had received prior sexual experience (Figure 6, ETC vs. NHC,

EHC, p < 0.02). Sexual behavior in ETS males did not further increase the level of Fos expression compared to ETC males. Analysis of Fos expression in each of the separate rostrocaudal levels of the VTA revealed a similar pattern of Fos expression described above for the total VTA; however, the majority of Fos-IR neurons were observed in the rostral levels of the VTA (Table 4).

Fos expression in the NAc

Sexual behavior and exposure to the sex-related environment resulted in neuronal activation in the NAc (Figure 7). There was a significant effect of mating on the number

37 of cells expressing Fos in both the NAc Core (F(1,24)= 457.265; p < 0.0001) and NAc Shell

(F(1,24)= 234.159, p < 0.0001). In the NAc Core, a significant mating-induced increase in the numbers of activated neurons was present in all males that mated on the test day compared to their controls (Figure 7A, NHS vs. NHC, EHS vs. EHC, ETS vs. NTC, ETS vs. ETC, p < 0.0001). In addition, a two-way interaction was observed between mating

and environment (F(1,24)= 3.244; p = 0.0294). Post hoc analysis revealed that the number of activated cells was significantly increased in ETC males that were exposed to the environment in which they received prior sexual experience (Figure 7A, ETC vs. NHC,

EHC and NTC, p < 0.002). Similarly, in the NAc Shell, a significant mating-induced increase in the numbers of activated neurons was present in all males that mated on the test day compared to their controls (Figure 7B, NHS vs. NHC, EHS vs. EHC, ETS vs.

NTC, ETS vs. ETC, p < 0.0001). In addition, a two-way interaction was observed

between mating and environment (F(1,24)= 8.725; p = 0.0069). Post hoc analysis revealed that the number of activated cells was significantly increased in ETC males that were exposed to the environment in which they received prior sexual experience (Figure 7B,

ETC vs. NHC, EHC and NTC, p < 0.005). Results from all three rostrocaudal levels are presented in Table 5.

DISCUSSION

The current study provides clear evidence that the mesolimbic dopamine pathway is activated during male sexual behavior. First, it was demonstrated that mating results in ligand-induced activation of MOR in VTA neurons, suggesting that endogenous opioids are released during sexual behavior. Second, mating was shown to result in the activation

38 of DA neurons in the VTA, potentially contributing to DA release in the NAc. Finally, a similar pattern of mating-induced activation was observed in NAc neurons, possibly due to DA release. Interestingly, all of these effects were also observed in response to environmental cues associated with prior sexual experiences. Taken together, these data support involvement of the mesolimbic DA pathway in male sexual behavior and reward.

The present study marks the first demonstration of MOR activation in the VTA by either a natural behavior or environmental cues. Others have used similar techniques to visualize MOR activation in other areas of the brain and spinal cord in response to exogenously applied opioids (Keith et al., 1998; Trafton et al., 2000) and estrogen

(Eckersell et al., 1998; Sinchak and Micevych, 2001). Recent studies from our laboratory have shown that sexual behavior causes MOR internalization in the medial preoptic nucleus of the hypothalamus in male rats (Coolen et al., 2003). In the present study,

MOR internalization was detected in the VTA following both sexual behavior and sex- associated environmental cues, suggesting that endogenous opioid peptides are released in response to these stimuli. Although this technique provides a useful marker for endogenous activation of MOR, it is an indirect marker for opioid release. In addition, the specific opioid ligand involved in MOR activation in VTA has not been identified.

Anatomical studies show both beta-endorphin (Mansour et al., 1988) and enkephalin

(Johnson et al., 1980; Fallon and Leslie, 1986) terminals in the VTA, and pharmacological evidence shows that both of these peptides can activate the mesolimbic

DA system (Broekkamp et al., 1979; Stinus et al., 1980; Dauge et al., 1992). In addition, recent reports have indicated the presence of the highly MOR-specific ligands

39 endomorphin-1 and -2 in the VTA (Greenwell et al., 2002). Although the present study focused exclusively on MOR, it is possible that other opioid receptors are also involved in the sex-induced activation of the mesolimbic system. Autoradiographic studies show that delta and kappa opioid receptors are present in the VTA, albeit at much lower densities compared to MOR (Mansour et al., 1987, 1988; Dilts and Kalivas, 1990; Xia and Haddad, 1991). Furthermore, delta agonists infused into the VTA are 100-1000 times less potent at stimulating DA release in the NAc, while kappa agonists fail to do so altogether (Devine et al., 1993), suggesting that sex-induced activation of the mesolimbic

DA pathway occurs primarily via MOR activation.

The anatomical data in the present study further support the evidence that MOR indirectly activates mesolimbic dopamine neurons via inhibition of GABA interneurons.

Using confocal microscopy, MOR were observed on both GABAergic cell bodies and fibers in close apposition to GABAergic neurons. These fibers had the appearance of boutons, indicative of axon terminals; hence MOR appear to be located presynaptic to

GABAergic cell bodies. These findings are in agreement with previous observations that

MOR in the VTA are not located on DA neurons (Garzon and Pickel, 2001), suggesting an indirect relationship. Furthermore, pharmacological studies have shown that MOR agonists inhibit GABA neuron firing, stimulate DA neuron firing, and increase DA release into the NAc (Matthews and German, 1984; Leone et al., 1991; Johnson and

North, 1992). In the current study, analysis of mating-induced internalization was restricted to the rostral portion of the VTA, where the majority of MOR-containing cell bodies are located. In this region, 79% of the MOR-containing cell bodies were

40 GABAergic, suggesting that most of the MOR internalization was in GABAergic neurons. However, it is unclear if all GABA/GAD-IR neurons we observed in the VTA are local interneurons, since GABA neurons also send projections to the NAc or prefrontal cortex (Carr and Sesack, 2000a). Nonetheless, these results provide further evidence that during natural motivated behavior, MOR ligands inhibit GABAergic interneurons, resulting in activation of dopaminergic neurons in the VTA and release of

DA in the NAc.

The present study also demonstrates sex-induced activation of DA neurons in the

VTA. Although it was not shown directly that MOR activation caused the activation of

DA neurons, the same pattern of activation was observed in both systems – i.e., activation by both sexual behavior and sex-related environmental cues – suggesting that the neural activation in the MOR and DA systems may be correlated. The percentage of DA cells that was activated appeared small (3-5% when averaged over the entire VTA), with the highest percentage of activation (15%) at the most rostral levels of the VTA. However, it is unclear how much DA neuron stimulation is needed to induce extracellular release of

DA in target areas. It is also important to note that the absence of Fos expression does not exclude the possibility of activation of DA cells via different signal transduction pathways. Moreover, the DA cells are presumably activated via a mechanism of disinhibition, and other markers may be more suitable for detecting this type of activation. Finally, it is currently unknown if the activated DA neurons project to the

NAc. However, anatomical evidence has shown that the NAc is the major target of the

DA projection from the VTA (Swanson, 1982). Furthermore, the pattern of neural

41 activation observed in the NAc was nearly identical to that of DA neuron and MOR activation in the VTA. Previously, it has been shown that morphine-induced Fos-IR in the NAc is a result of opioid action in the VTA (Bontempi and Sharp, 1997). The present data further support this model and indicate that endogenous opioids released into the VTA initiate the activation of this mesolimbic pathway during male sexual behavior.

Although the present results show that key components of the mesolimbic dopamine pathway are activated during male sexual behavior, it is not clear when during the behavior this activation occurs. In fact, it is possible that activation of this pathway may occur at different times during the behavior in sexually naïve versus experienced animals. Specifically, the mesolimbic dopamine pathway was activated in response to sex-related environmental cues, in the absence of interaction with a female partner, which suggests that opioids are released during the appetitive phase of the behavior. This reasoning is in agreement with Shultz’s studies showing dopaminergic activity in VTA of monkeys when reward is anticipated (Schultz, 2001). In the present study, the mating environment appears to act as a conditioned stimulus predicting the sexual reward.

Interestingly, not only were dopamine neurons activated by the predicted reward, but also activation of MOR was observed. Hence, endogenous opioids in the VTA may lead to the activation of this circuit in response to reward predicting environmental cues. In contrast,

Schultz illustrated that when reward was not predicted, dopamine neurons were activated during presentation of the reward (Schultz, 2001). In agreement with this hypothesis, it is possible that in sexually naive animals activation of the mesolimbic dopamine pathway occurs during the unpredicted sexual reward. Previous reports have indicated that

42 ejaculation is the most rewarding component of sexual behavior (Agmo and Berenfeld,

1990; Lopez et al., 1999). However, other components of the behavior may be rewarding as well.

Pharmacological studies suggest that both opioids and dopamine are involved in the motivational aspects of sexual behavior. While systemic opioids inhibit male sexual behavior, MOR agonists infused directly into the VTA facilitate sexual behavior

(Mitchell and Stewart, 1990). Conversely, naloxone decreases the number of anticipatory level changes in a bilevel chamber, a measure of sexual motivation (van Furth and van

Ree, 1996). 6-Hyroxydopamine (6-OHDA) lesions and DA antagonists in the NAc cause similar decreases in performance on this test (van Furth et al., 1995). Furthermore, 6-

OHDA lesions of the NAc impair non-contact erection in male rats, suggesting that DA is involved in sexual arousal in response to external cues (Moses et al., 1995). These manipulations did not alter sexual performance, suggesting that this pathway is involved in the motivational aspects of the behavior rather than the consummatory phase.

Interestingly, we also observed that many non-dopaminergic neurons in the VTA were activated by both sexual behavior and sex-related environmental cues. This suggests that additional pathways may be involved in the activation of the mesolimbic system. Other brain regions associated with reward such as the prefrontal cortex provide excitatory input to the VTA (Sesack and Pickel, 1992; Carr and Sesack, 2000a), and further studies are needed to investigate their role in sexual behavior.

43 In conclusion, the current study demonstrates activation of the mesolimbic system during a natural motivated behavior – male sexual behavior. Specifically, activation of this system is related to mating behavior as well as environmental cues associated with prior sexual experiences. The mesolimbic system is intimately involved in drug abuse – a “non-natural” motivated behavior (Wise, 1996). By studying the function of this system under natural conditions, we may gain a better understanding of its role in drug addiction.

44 REFERENCES

Agmo A, Berenfeld R (1990) Reinforcing properties of ejaculation in the male rat: role of opioids and dopamine. Behav Neurosci 104:177-182. Band LC, Hull EM (1990) Morphine and dynorphin(1-13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats. Brain Res 524:77-84. Bontempi B, Sharp FR (1997) Systemic morphine-induced Fos protein in the rat striatum and nucleus accumbens is regulated by mu opioid receptors in the substantia nigra and ventral tegmental area. J Neurosci 17:8596-8612. Broekkamp CL, Phillips AG, Cools AR (1979) Stimulant effects of enkephalin microinjection into the dopaminergic A10 area. Nature 278:560-562. Carr DB, Sesack SR (2000) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873. Coolen LM, Fitzgerald ME, Wells AB, Yu L, Lehman MN (2003) Activation of mu opioid receptors in the medial preoptic area following copulation in male rats. Submitted. Dauge V, Kalivas PW, Duffy T, Roques BP (1992) Effect of inhibiting enkephalin catabolism in the VTA on motor activity and extracellular dopamine. Brain Res 599:209-214. Devine DP, Leone P, Pocock D, Wise RA (1993) Differential involvement of ventral tegmental mu, delta and kappa opioid receptors in modulation of basal mesolimbic dopamine release: in vivo microdialysis studies. J Pharmacol Exp Ther 266:1236-1246. Dilts RP, Kalivas PW (1990) Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated [2-D- penicillamine, 5-D-penicillamine]enkephalin (125I-DPDPE). Synapse 6:121-132. Eckersell CB, Popper P, Micevych PE (1998) Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala. J Neurosci 18:3967-3976. Everitt BJ, Cador M, Robbins TW (1989) Interactions between the amygdala and ventral striatum in stimulus-reward associations: studies using a second-order schedule of sexual reinforcement. Neuroscience 30:63-75. Fallon JH, Leslie FM (1986) Distribution of dynorphin and enkephalin peptides in the rat brain. J Comp Neurol 249:293-336. Garzon M, Pickel VM (2001) Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area. Synapse 41:311-328. Greenwell TN, Zangen A, Martin-Schild S, Wise RA, Zadina JE (2002) Endomorphin-1 and -2 immunoreactive cells in the hypothalamus are labeled by fluoro-gold injections to the ventral tegmental area. In: International Narcotics Research Conference, p 26. Pacific Grove, CA. Hendricks SE, Scheetz HA (1973) Interaction of hypothalamic structures in the mediation of male sexual behavior. Physiol Behav 10:711-716.

45 Ikemoto S, Kohl RR, McBride WJ (1997) GABA(A) receptor blockade in the anterior ventral tegmental area increases extracellular levels of dopamine in the nucleus accumbens of rats. J Neurochem 69:137-143. Johnson RP, Sar M, Stumpf WE (1980) A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry. Brain Res 194:566-571. Johnson SW, North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483-488. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, von Zastrow M (1998) mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Mol Pharmacol 53:377-384. Klitenick MA, DeWitte P, Kalivas PW (1992) Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. J Neurosci 12:2623-2632. Leone P, Pocock D, Wise RA (1991) Morphine-dopamine interaction: ventral tegmental morphine increases nucleus accumbens dopamine release. Pharmacol Biochem Behav 39:469-472. Lopez HH, Olster DH, Ettenberg A (1999) Sexual motivation in the male rat: the role of primary incentives and copulatory experience. Horm Behav 36:176-185. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1987) Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7:2445-2464. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1988) Anatomy of CNS opioid receptors. Trends Neurosci 11:308-314. Matthews RT, German DC (1984) Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine. Neuroscience 11:617-625. Mitchell JB, Stewart J (1990) Facilitation of sexual behaviors in the male rat associated with intra-VTA injections of opiates. Pharmacol Biochem Behav 35:643-650. Moses J, Loucks JA, Watson HL, Matuszewich L, Hull EM (1995) Dopaminergic drugs in the medial preoptic area and nucleus accumbens: effects on motor activity, sexual motivation, and sexual performance. Pharmacol Biochem Behav 51:681- 686. Pfaus JG, Phillips AG (1989) Differential effects of dopamine receptor antagonists on the sexual behavior of male rats. Psychopharmacology (Berl) 98:363-368. Pfaus JG, Damsma G, Nomikos GG, Wenkstern DG, Blaha CD, Phillips AG, Fibiger HC (1990) Sexual behavior enhances central dopamine transmission in the male rat. Brain Res 530:345-348. Robertson GS, Pfaus JG, Atkinson LJ, Matsumura H, Phillips AG, Fibiger HC (1991) Sexual behavior increases c-fos expression in the forebrain of the male rat. Brain Res 564:352-357. Schultz W (2001) Reward signaling by dopamine neurons. Neuroscientist 7:293-302. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320:145-160.

46 Sinchak K, Micevych PE (2001) Progesterone blockade of estrogen activation of mu- opioid receptors regulates reproductive behavior. J Neurosci 21:5723-5729. Stinus L, Koob GF, Ling N, Bloom FE, Le Moal M (1980) Locomotor activation induced by infusion of endorphins into the ventral tegmental area: evidence for opiate- dopamine interactions. Proc Natl Acad Sci U S A 77:2323-2327. Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull 9:321-353. Trafton JA, Abbadie C, Marek K, Basbaum AI (2000) Postsynaptic signaling via the [mu]-opioid receptor: responses of dorsal horn neurons to exogenous opioids and noxious stimulation. J Neurosci 20:8578-8584. van Furth WR, van Ree JM (1996) Sexual motivation: involvement of endogenous opioids in the ventral tegmental area. Brain Res 729:20-28. van Furth WR, Wolterink G, van Ree JM (1995) Regulation of masculine sexual behavior: involvement of brain opioids and dopamine. Brain Res Brain Res Rev 21:162-184. Wise RA (1996) Neurobiology of addiction. Curr Opin Neurobiol 6:243-251. Xia Y, Haddad GG (1991) Ontogeny and distribution of opioid receptors in the rat brainstem. Brain Res 549:181-193.

47 Table 1. Summary of sexual behavior during the final pre-test mating session

ML IL EL #M #I #E

Home 27.9 ± 8.4 43.0 ± 8.1 744.9 ± 15.1 ± 3.0 13.8 ± 2.8 4.5 ± 0.3

Cage 108.0

Test 29.8 ± 36.1 ± 717.4 ± 13.6 ± 1.3 11.8 ± 0.5 4.8 ± 0.2

Cage 11.2 12.9 101.2

Presented are the mean ± SEM for latencies (in seconds) to mount (ML), intromission

(IL) and ejaculation (EL), the numbers of mounts (#M) and intromissions (#IM) during the final pre-test mating session, as well as the total number of ejaculations over the course of the entire training period (#E). No significant differences were observed between groups.

48 Table 2. Summary of sexual behavior on the test day

ML IL EL #M #I

NHS 133.3 ± 3 237.8 ± 66.1 2245.0 ± 51.5 ± 14.1 15.0 ± 1.5

469.4 *

EHS 34.8 ± 22.9 47.8 ± 18.7 1048.8 ± 15.3 ± 3.3 14.5 ± 1.6

296.5

NTS 290.3 ± 158.2 748.8 ± 289.6 2702.5 ± 66.0 ± 18.3 † 10.8 ± 1.1

* # 594.6 #

ETS 9.3 ± 3.8 34.8 ± 12.5 521.0 ± 166.9 26.0 ± 10.4 13.0 ± 1.7

Presented are the mean ± SEM (in seconds) for latencies to mount (ML), intromission

(IL), and ejaculation (EL) and the numbers of mounts (#M) and intromissions (#IM).

Indicated are significant differences from *ETS (p < 0.05), † ETS and EHS (p < 0.05),

#ETS, EHS and NHS (p < 0.05).

49 Table 3. Percentages of TH cells expressing Fos

Rostral Middle-1 Middle-2 Caudal

NHC 2.73 ± 0.76 1.03 ± 0.36 0.68 ± 0.30 0.20 ± 0.12

NHS 14.54 ± 2.07 *# 5.35 ± 0.10 * 3.19 ± 0.90 * 1.94 ± 0.36 *

EHC 5.76 ± 1.21 1.12 ± 0.45 0.42 ± 0.42 0.27 ± 0.17

EHS 12.64 ± 1.46 *$ 5.80 ± 0.66 * 2.16 ± 0.41 * 0.79 ± 0.27

NTC 2.91 ± 0.39 0.74 ± 0.11 0.20 ± 0.20 0.00 ± 0.00

NTS 9.09 ± 1.27 * 5.29 ± 0.37 * 1.86 ± 0.38 * 0.65 ± 0.18 $

ETC 11.27 ± 2.66 † 2.24 ± 0.58 § 2.71 ± 0.55 † 1.38 ± 0.57 †

ETS 7.14 ± 1.20 4.56 ± 0.78 2.78 ± 0.45 1.98 ± 0.58 #

Presented are the mean percentages of TH cells expressing Fos at four levels through the

VTA (mean ± SEM). Indicated are significant differences from: * corresponding control group (p < 0.03), † NHC, EHC, NTC (p < 0.04), # NTS (p < 0.0188), $ ETS (p <

0.0179), § NTC (p = 0.0396).

50 Table 4. Fos expression in nondopaminergic neurons

Rostral Middle-1 Middle-2 Caudal

NHC 68.50 ± 8.31 48.25 ± 16.90 15.5 ± 5.55 3.5 ± 1.85

NHS 313.75 ± 33.51 * 170.75 ± 61.89 * 78.75 ± 11.51 * 27.00 ± 8.27 *

EHC 90.75 ± 32.37 54.50 ± 36.93 22.50 ± 17.56 14.75 ± 7.82

EHS 296.75 ± 37.95 * 173.00 ± 8.81 * 67.00 ± 12.28 * 25.00 ± 5.45

NTC 115.00 ± 17.44 92.50 ± 17.49 49.00 ± 4.81 $ 7.25 ± 0.75

NTS 244.25 ± 42.14 * 174.75 ± 16.91 104.25 ± 15.00 * 57.25 ± 9.50 *#

ETC 220.50 ± 9.24 † 124.75 ± 25.89 70.00 ± 19.51 14.75 ± 4.84

ETS 232.00 ± 28.27 156.25 ± 32.17 84.25 ± 18.53 30.25 ± 5.22

Presented are the numbers of Fos-IR cells not expressing TH at four levels through the

VTA (mean ± SEM). Indicated are significant differences from: * corresponding control group (p < 0.05), † NHC, EHC, NTC (p < 0.05), # NHS, EHS, ETS (p < 0.05), $ NTC (p

< 0.05).

51 Table 5a. Fos expression in NAc Core

Rostral Middle Caudal

NHC 29.00 ± 11.50 28.00 ± 8.95 30.00 ± 7.71

NHS 176.75 ± 21.81 * 183.00 ± 9.33 * 181.75 ± 6.41 *

EHC 51.75 ± 8.34 41.50 ± 0.87 46.00 ± 6.41

EHS 195.00 ± 21.41 * 189.50 ± 18.41 * 195.50 ± 8.91 *

NTC 59.25 ± 5.75 52.00 ± 4.98 55.00 ± 8.16

NTS 207.00 ± 16.39 * 176.75 ± 17.10 * 194.25 ± 10.53 *

ETC 106.25 ± 5.31 # 99.75 ± 6.25 † 94.25 ± 6.93 †

ETS 223.75 ± 29.31 * 184.50 ± 8.01 * 237.25 ± 11.44 *$

Presented are the numbers of Fos-IR cells at three levels through the NAc Core (mean ±

SEM). Indicated are significant differences from: * corresponding control group (p <

0.0001), † NHC, EHC, NTC (p < 0.04), # NHS, EHS, ETS (p < 0.05), $ NHS, EHS, NTS

(p < 0.02).

52 Table 5b. Fos expression in NAc Shell

Rostral Middle Caudal

NHC 35.75 ± 11.02 45.00 ± 9.19 54.50 ± 5.28

NHS 179.50 ± 9.35 * 171.25 ± 17.76 * 226.75 ± 16.42 *

EHC 45.25 ± 9.53 62.75 ± 8.72 85.00 ± 3.31

EHS 154.25 ± 10.21 * 161.00 ± 11.51 * 206.50 ± 24.46 *

NTC 62.75 ± 6.09 45.75 ± 8.86 51.75 ± 9.54

NTS 127.00 ± 15.62 *$ 145.00 ± 9.34 *$ 178.25 ± 10.34 *§

ETC 88.00 ± 1.87 # 107.00 ± 9.51 † 131.25 ± 10.36 †

ETS 188.50 ± 32.99 * 170.00 ± 7.82 * 199.25 ± 19.14 *

Presented are the numbers of Fos-IR cells not expressing TH at four levels through the

VTA. Indicated are significant differences from: * corresponding control group (p <

0.006), † NHC, EHC, NTC (p < 0.03), # NHC (p = 0.0206), $ NHS, ETS (p < 0.025), §

NHS (p = 0.229).

53 Figure 1

Figure 1. Schematic drawings illustrating the areas of analysis of Fos/TH-IR, indicated by the boxes, in the VTA at four rostral to caudal levels. A, Rostral, area of analysis = 1.8mm x 1.2 mm. B, Middle-1, area of analysis = 1.8 mm x 1.2 mm. C, Middle-2, area of analysis = 1.8 mm x 1.2 mm. D, Caudal, area of analysis = 1.6 mm x 1.0 mm. Abbreviations: AQ, cerebral aqueduct; PAG, periaquaductal gray; fr, fasiculus retroflexus; ml, medial lemniscus; cpd, cerebral peduncle; SN, substantia nigra; LG, lateral geniculate complex; SPFp, subparafasicular nucleus thalamus parvicellular part; VPM, ventral posteriomedial nucleus thalamus; PH, posterior hupothalamic nucleus; MRN, mesencephalic reticular nucleus; MGv, medial geniculate complex ventral part; APN, anterior pretectal nucleus; SC, superior colliculus; MM, medial mammillary nucleus.

54 Figure 2

Figure 2. Schematic drawings illustrating the areas of analysis (indicated by the boxes; 400 µm x 600 µm) of Fos-IR in the NAc Core and Shell at three rostral to caudal levels (A-C). Abbreviations: C, NAc Core; S, NAc Shell; VL, lateral ventricle; ac, anterior commissure; cc, corpus callosum; ec, external capsule; CP, caudate putamen.

55 Figure 3

Figure 3. A, Confocal image illustrating MOR (red) located on GABA-IR (green) cell bodies (arrow) and MOR-IR fibers in close apposition to GABA-IR cells (triangle) in the VTA. B, Confocal image illustrating GAD-IR fibers (red) in close apposition to TH-IR (green) cells in the VTA. C. Confocal image illustrating MOR-IR in the VTA of a non- mated control animal. D, Confocal image illustrating MOR-IR in the VTA of a mated animals. Arrows indicate MOR-IR endosome like particles. Cconfocal images in B, C, and D are 1 µm optical sections, and image A is a 5 µm optical section. E, D. Photomicrographs illustrating Fos-IR (black, filled triangle) and TH-IR (brown, open triangle) in the VTA. Double-labeled cells are indicated by arrows. Scale bars indicate 10 µm.

56 Figure 4

Figure 4. MOR internalization in VTA neurons. A, Numbers of MOR-IR endosome-like particles per cell. Mean numbers ± SEM of MOR-IR endosome-like particles per cell per behavioral group (NHC, Naïve Home Cage Control; NHS, Naïve Home Cage Sex; EHC, Experienced Home Cage Control; EHS, Experienced Home Cage Sex; NTC, Naïve Text Cage Control; NTS, Naïve Test Cage Sex; ETC, Experienced Test Cage Control; ETS, Experienced Test Cage Sex). B, Percentages of cells that show MOR internalization. Mean percentages ± SEM of VTA MOR-IR cells that contain 3 or more endosomes per behavioral group. Solid bars represent groups that mated on the test day, and open bars represent control groups that did not mate on the test day. The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

57 Figure 5

Figure 5. Percentage of TH-IR cells that are Fos-IR in the VTA. Mean percentages ± SEM of TH-IR cells that are Fos-IR averaged over four rostral-to-caudal levels. (NHC, Naïve Home Cage Control; NHS, Naïve Home Cage Sex; EHC, Experienced Home Cage Control; EHS, Experienced Home Cage Sex; NTC, Naïve Text Cage Control; NTS, Naïve Test Cage Sex; ETC, Experienced Test Cage Control; ETS, Experienced Test Cage Sex) Solid bars represent groups that mated on the test day, and open bars represent control groups that did not mate on the test day. The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

58 Figure 6

Figure 6. Numbers of nondopaminergic cells that are Fos-IR in the VTA. Mean numbers ± SEM of Fos-IR cells that are not TH-IR averaged over four rostral-to-caudal levels. (NHC, Naïve Home Cage Control; NHS, Naïve Home Cage Sex; EHC, Experienced Home Cage Control; EHS, Experienced Home Cage Sex; NTC, Naïve Text Cage Control; NTS, Naïve Test Cage Sex; ETC, Experienced Test Cage Control; ETS, Experienced Test Cage Sex). Solid bars represent groups that mated on the test day, and open bars represent control groups that did not mate on the test day. Solid bars represent groups that mated on the test day, and open bars represent control groups that did not mate on the test day. The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

59 Figure 7

Figure 7. Numbers of Fos-IR cells in the NAc. Mean numbers ± SEM of Fos-IR cells in the NAc Core (A) or NAc Shell (B) averaged over three rostral-to-caudal levels. (NHC, Naïve Home Cage Control; NHS, Naïve Home Cage Sex; EHC, Experienced Home Cage Control; EHS, Experienced Home Cage Sex; NTC, Naïve Text Cage Control; NTS, Naïve Test Cage Sex; ETC, Experienced Test Cage Control; ETS, Experienced Test Cage Sex) Solid bars represent groups that mated on the test day, and open bars represent control groups that did not mate on the test day. The statistical relationship between the groups is

60 Chapter 3

Anatomical relationship between mPFC and NAc efferents and sex-activated neurons

61 INTRODUCTION

The mesolimbic system plays an important role in the regulation of the motivating aspects of sexual behavior. A critical component of this system is the ventral tegmental area (VTA). Indeed, pharmacological manipulations of dopamine and opioid systems in the VTA have been shown to affect performance on tests of sexual motivation (Hull et al., 1990; van Furth et al., 1995; van Furth and van Ree, 1996). Moreover, studies from our laboratory have shown increased immediate early gene expression in this region both during and in anticipation of sexual behavior (Balfour et al., 2003). Interestingly, both dopaminergic and non-dopaminergic neurons were activated during sexual behavior or anticipation. Activation of dopaminergic neurons is hypothesized to be a result of endogenous opioid peptide action, via activation of mu opioid receptors, in turn resulting in inhibition of GABA interneurons, finally leading to disinhibition of dopamine neurons.

However, the afferents involved in activation of non-dopaminergic neurons in VTA are currently unknown. Therefore, in the current study, the anatomical relationships between

VTA afferents and mating-induced neural activation was investigated. VTA receives major inputs from two other components of the mesolimbic system: the nucleus accumbens (NAc) and medial prefrontal cortex (mPFC) (Sesack et al., 1989; Heimer et al., 1991; Berendse et al., 1992a). These brain areas receive dopaminergic inputs from the VTA and in turn provide projections back to the VTA. Thus, afferents of these two brain regions may be in the position to influence neural activity in the VTA. Moreover, the NAc and PFC are involved in sexual behavior. In particular, the NAc and PFC are activated during sexual behavior and in anticipation of sexual reward (Balfour et al.,

62 2003), and lesions of these areas have negative effects on sexual behavior and motivation

(Hendricks and Scheetz, 1973; Pfaus and Phillips, 1991). Therefore, the main goal of the present study was to determine the anatomical relationship between PFC and NAc efferents and the sex-activated neurons in the VTA. Efferent projections were identified using the anterograde tract tracer biotinylated dextrane amine (BDA) injected into discrete subregions of either the NAc or PFC. In particular, the NAc has been shown to consist of functional and anatomical subregions, i.e. core and shell. These subregions have distinct afferent and efferent connections, as well as neurotransmitter content

(Heimer et al., 1997; Kelley, 1999; Zahm, 1999). Similarly, the PFC consists of three subregions, i.e. the anterior cingulated area (ACA), prelimbic area (PL), and infralimbic area (ILA). These PFC subregions appear to have distinct connections, but less is known about their anatomical as well as functional differences (Tzschentke, 2001). Visualization of the efferent connections of the NAc and PFC subregions was combined with visualization of copulation-induced activation of VTA neurons using the immediate early gene product c-Fos as a marker for neuronal activation. Finally, a quantitative analysis was performed to determine the relative amount of inputs each subregion of the NAc or

PFC provides to the sex-activated neurons in the VTA.

A secondary goal of the study was to determine if PFC and NAC send inputs to sex-activated neurons in other brain regions that have been shown to be important for sexual behavior and motivation (Hull et al., 2002). Specifically, we focused on the medial preoptic area/nucleus (MPOA/MPN), principal nucleus of the bed nucleus of the

63 stria terminalis (BST), posterodorsal medial amygdala (MeA), basolateral amygdala

(BLA), and parvocellular subparafasicular nucleus of the thalamus (SPFp).

Finally, we investigated the role of glutamate in sex-induced activation of the

VTA via the PFC. Specifically, we verified whether the PFC projection to the VTA is glutamateregic, and furthermore, we investigated whether sex-activated cells in the VTA contain glutamate receptors.

MATERIALS AND METHODS

Subjects

Adult male Sprague Dawley rats (250-260 grams) were obtained from Harlan

(Indianapolis, IN) and housed in pairs in plexiglass cages for the duration of the experiment. The colony room was maintained on 12/12 hr reversed light dark cycle

(lights off at 10 AM). Food and water were available ad libitum. Stimulus females for mating behavior tests were bilaterally ovariectomized and received a subcutaneous implant containing 5% estradiol benzoate (EB) and 95% cholesterol. Sexual receptivity was induced by administration of 500 µg progesterone (P; subcutaneous) in 0.1 ml sesame oil 5 hours before testing. All procedures were approved by the Animal Care and

Use Committee of the University of Cincinnati and conformed to NIH guidelines involving vertebrate animals in research.

64 Tract Tracing

BDA injections into the NAc or PFC: Animals were anesthetized using a mixture of ketamine (13%) and xylazine (87%). Heads were shaved and rats were placed into a stereotaxic apparatus (Kopf, California), with Lambda and Bregma at the same level. A single midline scalp incision was made. A small hole (approximately 3 mm in diameter) was drilled in the skull. A glass micropipette (tip diameter 32 µm) filled with biotinylated dextran amine (BDA; 10,000 MW; lysine fixable; 5% dissolved in 0.1M phosphate buffer pH 7.4; Sigma) was lowered into the brain. After a period of 3 minutes, the tracer was applied by iontophoresis (positive pulse of 5 µA, 8 seconds on/of) for a period of 25 minutes. After an additional period of 3 minutes, the micropipette was slowly removed under negative current?, and the scalp incision closed with wound clips.

Animals were sacrificed 5-7 days following tracer injections to allow sufficient transport of the tracer.

Stereotaxic coordinates: The following coordinates were used for tracer injections into the NAc Shell: AP 1.2, ML 0.8, DV 7.4; AP 1.7, ML 0.9, DV 6.8; AP 1.8, ML 0.9, DV

6.8; AP 1.2, ML 0.9, DV 6.7. The following coordinates were used for tracer injections into the NAc Core: AP 1.2, ML 2.0, DV 7.4; AP 1.7, ML 1.5, DV 6.7; AP 1.8, ML 1.5,

DV 6.7. The following coordinates were used for tracer injections into the PFC: AP 3.0,

ML 0.5, DV 5.0; AP 3.0, ML 0.5, DV 4.8; AP 3.0, ML 0.4, DV 4.2; AP 2.7, ML 0.4, DV

5.2; AP 2.0, ML 0.4, DV 2.4. All AP and ML coordinates are expressed as mm from

Bregma, and all DV coordinates are expressed as mm from skull.

65 Sexual Behavior

In order to investigate mating-induced Fos-IR, all male rats for this study were sacrificed following sexual behavior. Male rats gained sexual experience before tract tracing surgery during four or five pre-test mating sessions, during which the males copulated with a receptive female for 60 minutes. Sexual behavior testing was performed four hours after onset of the dark period, in a rectangular mating arena (60 x 45 x 50 cm), under dim red illumination. Five-to-seven days following tract tracing surgery, males were placed in the test arena with a receptive female and were allowed to copulate until one ejaculation, and sacrificed 60 minutes later.

Immunocytochemistry

Tissue Preparation: The animals were deeply anesthetized using pentobarbital (200 mg/kg) and perfused transcardially with 100 mL of 0.9% NaCl followed by 500 mL of

4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3). The brains were removed and post-fixed for 1 hour at room temperature in the fixative, then placed in 20% sucrose in 0.1 M PB and stored at 4° C. Coronal sections (35 µm) were cut on a freezing microtome (Richard Allen, Kalamazoo, MI), collected in four parallel series in cryoprotectant solution (30% sucrose, 30% ethylene glycol, 0.01% sodium azide in 0.1 M

PB in 0.1 M PB), and stored at –20°C until further processing.

General Methods: All incubations were performed at room temperature with gentle agitation. Free-floating sections were washed extensively with 0.05 M Tris-buffered

66 saline (TBS, pH 7.6) between incubations. Sections were incubated for 10 minutes with

1% H2O2, then blocked for one hour with incubation solution (TBS containing 0.1% bovine serum albumin and 0.2-0.4% triton X-100). All primary antibody incubations were performed in the incubation solution, overnight at room temperature. Each

subsequent overnight incubation was preceded by a 10 minute incubation with 1% H2O2.

Following staining, the sections were washed thoroughly in 0.05M TB, mounted onto

glass slides with 0.3% gelatin in ddH20 and coverslipped with DPX (Electron Microscopy

Sciences, Fort Washington, PA) or an aqueous mounting medium (Gelvatol) containing an anti fading agent (1,4-diazabicyclo (2,2)octane). Immunocytochemical controls included omission of primary antibodies.. Immunocytochemical controls included omission of primary antibodies.

BDA/Fos: To investigate the location of injection sites and general distribution of fiber projections, one series of brain sections (140 µm apart) was stained for BDA and Fos.

The sections were incubated overnight with a rabbit polyclonal antibody to c-Fos

(1:7500; SC-52; Santa Cruz Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated donkey anti-rabbit IgG (1:500; Vector Laboratories,

Burlingame, CA) and avidin-horseradish peroxidase complex (1:500; ABC Elite Kit,

Vector Laboratories, Burlingame, CA). Finally, the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing 0.012% hydrogen peroxide and 0.4% nickel sulfate, resulting in a blue-black reaction product.

67 BDA/Fos/NeuN: For quantification of putative contacts, one series of sections of selected brains were stained for BDA, Fos, and the neuronal specific protein NeuN. The sections were incubated overnight in incubation solution, followed by a one-hour incubation in avidin-horseradish peroxidase complex (1:1000; ABC Elite Kit, Vector Laboratories,

Burlingame, CA). Then the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing

0.012% hydrogen peroxide and 0.4% nickel sulfate, resulting in a blue-black reaction product. Next, the sections were incubated overnight with a rabbit polyclonal antibody to c-Fos (1:7500; SC-52; Santa Cruz Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated donkey anti-rabbit IgG (1:500; Vector Laboratories,

Burlingame, CA) and avidin-horseradish peroxidase complex (1:5000; ABC Elite Kit,

Vector Laboratories, Burlingame, CA). Subsequently, the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing 0.012% hydrogen peroxide and 0.4% nickel sulfate, resulting in a blue-black reaction product. Next, the sections were incubated overnight with mouse monoclonal antibody to the neuronal specific protein NeuN (1:10,000; Chemicon International,

Temecula, CA), followed by incubation with biotinylated donkey anti-mouse IgG secondary antibody (1:500; Vector Laboratories, Burlingame, CA) and ABC as described above. Finally, the sections were incubated for 10 minutes in 0.02% DAB in 0.05 M TB containing 0.012% hydrogen peroxide, resulting in a reddish-brown reaction product.

68 BDA/VGLUT1 and BDA/VGLUT2: To determine whether projection fibers from ILA or

PL were glutamatergic, one series of VTA sections of selected brains was stained for

BDA and the vesicular glutamate transporters VGLUT1 or VGLUT2. The sections were incubated overnight with a guinea pig polyclonal antibody to either VLGUT1 or

VLGUT2 (1:500; Chemicon International, Temecula, CA) followed by 30 minutes incubations with Alexa-488 conjugated goat anti-guinea pig IgG (1:100; Jackson

ImmunoResearch Laboratories, West Grove, PA). Next, the sections were incubated in avidin-horseradish peroxidase complex (1:1000; ABC Elite Kit, Vector Laboratories,

Burlingame, CA) for one hour followed by a 10-minute incubation with biotinylated

tyramine (BT; 1:250 in PBS + 0.003% H2O2; Tyramide Signal Amplification Kit, NEN

Life Sciences, Boston, MA). Finally, the sections were incubated for 30 minutes with

CY3-conjugated streptavidin (1:200; Jackson ImmunoResearch Laboratories, West

Grove, PA)

Fos/NMDAR1: To determine if sex-activated neurons contain the glutamate receptors, sections were stained from mated animals sacrificed 60 minute after ejaculation and unmated controls. The sections were incubated overnight with a goat polyclonal antibody to the NMDAR1 subunit of the NDMDA receptor (1:600; SC-1467; Santa Cruz

Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated rabbit anti-goat IgG (1:500; Vector Laboratories, Burlingame, CA) and avidin- horseradish peroxidase complex (1:500; ABC Elite Kit, Vector Laboratories, Burlingame,

CA). Finally, the sections were incubated for 10 minutes in 0.02% diaminobenzidine

(DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing 0.012% hydrogen

69 peroxide and 0.4% nickel sulfate, resulting in a blue-black reaction product. The sections were then incubated overnight with a rabbit polyclonal antibody to c-Fos (1:7500; SC-52;

Santa Cruz Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated donkey anti-rabbit IgG (1:500; Vector Laboratories, Burlingame, CA) and avidin-horseradish peroxidase complex (1:500; ABC Elite Kit, Vector Laboratories,

Burlingame, CA). Finally, the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing

0.012% hydrogen peroxide, resulting in a brown reaction product.

Data Analysis

Injection sites: Cameral lucida drawings were made to indicate the location of the injection sites in sections stained for BDA and Fos, using a Leica microscope (Leica

Microsystems; Wetzlar, Germany) with a drawing tube attached. Injection sites were defined as the area of densely stained neuropil and BDA-labeled neurons. Drawings of the injection sites were mapped onto digital brain maps as described below.

General distribution of projection fibers: Three representative animals for each brain area in which injections were made were chosen for analysis. The location of BDA- containing fibers was mapped using camera lucida drawings of ipsilateral sections stained for BDA and Fos through the NAc, MPN, BST, BLA, SPFp, and VTA (introduce abbreviations). Drawings of fibers were made using a Leica microscope with a drawing tube attached at 5-10X magnification. The drawings were imported into the computer

70 (Apple MacIntosh G4) using a Wacom Graphic Tablet (Intuos 2) and Adobe Illustrator

10 software. Subsequently, the camera lucida drawings were mapped onto digital brain maps (Swanson 1998). The sections corresponded to the following plates in the Swanson

Brain Maps Atlas (Swanson, 1998): 10, 12, 14, 21, 29, 35, 36, 37, 38. Comparisons of the digital maps and sections stained for BDA/Fos were used to ensure the accuracy of the data presentation, using landmarks including lateral and third ventricles, optic chiasm, optic tract, anterior commissure, fornix, and stria terminalis.

Quantification of putative contacts: To determine if projection fibers terminate on sex- activated neurons, counts of BDA-labeled boutons in close apposition to neurons were performed in sections stained for BDA, Fos and NeuN. Three animals for each brain area of injection (NAc Shell, NAc Core, ILA, PL, ACA) were chosen for quantitative analysis, and counts were performed in four rostral-to-caudal sections through the VTA, three rostral-to-caudal sections through both the NAc Core and Shell, and one section each through the MPN, BST, BLA, and SPFp. Standard areas measuring 400 x 300 µm were defined in which to count for each section through the NAc, MPN, BST, BLA, and

VTA, and a 400 x 150 µm area was defined for the SPFp (Figure 1). Neurons and BDA- labeled fibers were examined under 40X magnification, and putative contacts were defined as a BDA-labeled bouton in close apposition to a NeuN labeled cell body, at the same focal plane (Figure 2). Camera lucida drawings were made at 40X magnification, and counts were performed of the following: numbers of cells dual labeled for Fos and

NeuN (Fos/NeuN), numbers of cells labeled for NeuN only (NeuN-only), numbers of

Fos/NeuN labeled cells receiving one or more contacts, and numbers of NeuN-only

71 labeled cells receiving one or more putative contacts. For each section, the following percentages were calculated: percentage of all cells that receive contacts, percentage of contacted cells that are Fos-IR, and percentage of Fos-IR cells that receive contacts.

Group means were calculated for the MPN, BST, BLA and SPFp, and each separate rostral to caudal level in both NAc and VTA. In addition, an average of the 4 VTA or 3

NAc rostral to caudal levels were calculated per animal and group means were based on animal averages. Results were analyzed using a one-way ANOVA and post-hoc comparisons (Fisher’s PLSD) using 5% significance levels.

BDA/VGLUT colocalization: Co-expression of BDA and VGLUT1 or VGLUT2 in VTA sections was analyzed using confocal microscopy. Stacks of 0.6 µm optical sections were captured along the z-axis using a Zeiss LSM-510 laser scanning microscope. CY3- fluorescence was imaged with a 567 nm emission filter and a HeNe laser, and Alexa 488 with 505 nm emission filter and Argon laser.

Digital photographs: Digital images of immunostained sections were captured using a digital camera (Magnafire, Optronics) attached to a Leica microscope (Leica

Microsystems; Wetzlar, Germany). Images were imported into Adobe Photoshop 7.0

(Adobe Systems, San Jose, CA) to compose the figures. Images were not adjusted or altered in any way, except for occasional adjustment of brightness.

72 RESULTS

Injection Sites:

Injection sites consisted of an area of densely stained neuropil and BDA-labeled neurons.

Twenty-four animals had injections located in either the NAc or mPFC and were included in the analysis. Fifteen animals had either missed injections or extensive tissue damage and were excluded from analysis. Injections into the NAc were restricted to either the Core or Shell subregions, and injections into the PFC were located in either the

ILA, PL, or ACA subregions. In both NAc and PFC, injections were distributed along the rostral-caudal extend of these brain regions. Of these injections, three representative animals for each subregion within PFC and NAc were selected for the graphic illustrations and quantitative analysis of putative contacts (total 12 injections) presented in the current paper. The location and sizes of these injections are shown in Figure 3.

General Distribution of Projection Fibers:

Figures 4-9 illustrate the general distribution of BDA-lebaled fibers following the 12 representative injections shown in Figure 3. However, qualitative analysis of the general distribution of BDA-labeled fibers was based on all 24 injections restricted to NAc Core or Shell, ILA, PL, or ACA. The distribution of BDA-labeled fibers is described with emphasis on target brain regions involved in regulation of motivation, reward, or male sexual behavior. In particular, BDA fiber labeling following PFC or NAc injections is described for VTA, PFC, Nac, MPOA, BST, BLA, and SPFp.

73 To VTA: Projections from the NAc and PFC were analyzed in four rostral-to-caudal levels in the VTA. In animals with PFC injections, the most robust projection to the

VTA was from the ILA and PL (Figure 4). These projections were bilateral, but most robust on the ipsilateral side. No differences in the distribution of BDA-labeled fibers was observed between animals with injections in rostral versus caudal ILA, PL, or ACA.

Therefore Figure 4 illustrates BDA labeling following one representative injection in ILA

(I2), PLA (P2), and ACA (A2). BDA-labeled fibers originating from ILA and PL were observed in all four rostral-caudal levels of the VTA, and were varicose in appearance with numerous bouton-like swellings. BDA labeling following ACA injections was less robust, but fibers did appear varicose with numerous boutons.

BDA labeling in VTA following injections in NAc Shell (Figure 5) and Core (Figure 6) is illustrated for all three represenative injections distributed along the rostral-caudal extent of either core (C1-3) or shell (S1-3), since previous reports have demonstrated a topographical distribution in connections between VTA and NAc (Berendse et al.,

1992a). BDA-labeled fibers originating from NAc Core or Shell were found at all rostral- caudal levels of the VTA. However, there were no apparent differences in distribution of

BDA labeling in VTA between NAc injections that were located rostral versus caudal within the NAc Core or Shell (Figure 5). Projections from both the NAc Shell and Core were restricted to the ipsilateral side and appeared less robust than the ILA and PL projections (Figures 5 and 6). NAc Core fibers projected primarily to the lateral VTA, along the border with SNc, while BDA labeling following NAc shell injections was located medially within the VTA. BDA-labeled fibers in VTA originating from core or

74 shell were varicose and many boutons were observed. In addition, many of the fibers bordering the lateral SNc were smooth and had the appearance of passing fibers.

To other areas related to sexual behavior or reward:

From PFC: The NAc Core and Shell receive a robust projection from ILA and PL subregions of the PFC (Figure 7). These projections were bilateral In addition, ILA and

PL project to the ventral portion of the caudate putamen (CP). Bilateral ACA projections were mostly restricted to the caudate putamen (CP), and few projections to the NAc were observed (Figure 7). The ILA and PL also sent robust projections to MPN, MPO, principal nucleus of the BST, substantia innominata, zona incerta (ZI) and lateral hypothalamic area (LHA), basolateral nucleus of the amygdala (BLA), central nucleus of the amygdala (CeA), and areas within the limbic thalamus (lateral habenula, dorsomedial nucleus of the thalamus, and interomedialdorsal nucleus of the thalamus) (Figure 8),.

Projections from the ACA were not observed in these areas except for lateral habenula, mediadorsal nucleus of the thalamus, and zona incerta (Figure 8). The projections from the PFC were bilateral, with more robust fiber labeling on the side ipsilateral to the injection.

From NAc: Projections from NAc to areas involved in regulation of motivation, reward, and sexual behavior were more restricted compared to the projections from PFC. In particular, no projections were observed from the NAc to the PFC, BLA, CeA or thalamus. A robust projection was observed in the substantia inominata and lateral

75 preoptic area, with the projections from Shell located medial to the projections from

Core.

Quantitative Analysis of Putative Contacts

Quantitative Analysis: Several measures were performed in order to characterize the relationship between projection fibers and sex-activated neurons in brain areas involved in sexual behavior and motivation. First, the percentage of all the cells within the area of analysis in the target area that received one or more putative contacts was used to compare the amount of afferent input each brain area received from either the PFC or

NAc. Second, the percentage of contacted cells that were Fos-IR indicated whether the putative targets of these projection fibers were activated during sexual behavior. Finally, the percentage of Fos-IR cells within the area of analysis in the target area that received contacts was used in order to determine to what extent the NAc or PFC projections may contribute to sex-induced activation of these brain areas.

Fos expression: Fos expression was noted in all areas analyzed as described previously

(Coolen et al., 1996; Veening and Coolen, 1998), indicating that BDA labeling did not prevent induction of Fos during mating. Moreover, BDA labeling did not cause Fos induction, since five animals in the present study that received BDA injections, but did not mate on the sacrifice day, displayed low numbers of Fos-IR cells in sex-relevant brain areas equivalent to the levels of Fos previously described in non-mated animals (data not shown).

76 PFC and Nac projections to VTA: Figure 10 illustrates the quantitative analysis of putative contacts from PFC and NAc subregions to the VTA. Data shown are averaged over the four rostral-caudal levels, since differences were not detected between the rostral-caudal levels. VTA neurons received putative inputs from ILA, PL, ACA, and

NAc Shell and Core, with a significantly greater percentage of cells receiving contacts from ILA and PL (Figure 10: left block of bars). The majority of neurons contacted by

ILA or PL axons were activated (Fos-positive) during sexual behavior (Figure 10, middle block of bars), while significantly fewer percentages of the neurons receiving afferents from ACA, Nac shell or core were activated during mating. Moreover, 40-50% of all sex-induced Fos cells in the VTA received inputs from ILA, PL or ACA. In contrast, inputs from the NAc Core or Shell contact significantly fewer (less than 10%) of sex- induced Fos neurons in the VTA. Thus, ILA, PL and ACA provide substantial input to the sex-activated neurons in the VTA and are ideally situated to contribute to the activation of the VTA during sexual behavior.

PFC projections to other areas related to sexual behavior or reward:

To NAc: Figure 11A-B illustrate the quantitative analysis of putative contacts from PFC subregions to the NAc. Data shown are averaged over the three rostral-caudal levels of the Nac shell and core, since differences were not detected between the rostral-caudal levels. NAc neurons in shell and core receive the most robust input from ILA and PL

(Figure 11A-B, left block of graphs). In contrast, only a small percentage (less than 10%) of cells received inputs from ACA. In the NAc Shell, 30-50% of neurons contacted by

ILA, PL, or ACA were activated during sexual behavior (Figure 11A; middle block).

77 Moreover, the majority of sex-activated neurons received contacts from ILA or PL, while the ACA contacted fewer than 10% of the activated neuron in the NAc Shell (Figure

11A: right block). In the NAc Core, a significantly greater percentage of cells received contacts from the PL compared to the ILA and ACA (Figure 11B; left block). In addition, 35-45% of the cells contacted by either the ILA, PL or ACA were Fos-positive

(Fgure 11B; middle block). However, the majority of sex-activated cells in the NAc Core received contacts from ILA and PL, while the ACA contacted fewer than 10% of the activated neurons (Figure 11B; right block). Thus, the ILA and PL – and to a lesser extent the ACA – may contribute to the activation of the NAc during sexual behavior.

To MPN, BST, BLA, SPFp: Only the ILA and PL sent projections to the MPN, BSTpr,

BLA, and SPFp; the ACA did not sent any projections to these areas and was not included in the analysis. In the MPN, BSTpr and SPFp, 20-30% of the neurons received contacts from either the ILA or PL (Figures 11C, D, F: left block). In the BLA, this number was slightly larger, with 40-60% of the cells receiving contacts (Figure 11E; left block). In the MPN and SPFp, a majority of the neurons contacted by the ILA and PL were activated during sexual behavior (Figures 11C and F: middle block). In particular, in the SPFp, a significantly higher percentage of cells contacted by the ILA was activated compared to those contacted by the PL. In MPN, BSTpr, and SPFp, 30-50% of sex- activated cells received putative contacts from ILA or PL (Figures 11C, D, F; right block), while a majority of the sex-activated neurons in the BLA received contacts from the ILA or PL (Figure 11E; right block). These data suggest that the ILA and PL may contribute to the sex-induced activation of these brain areas.

78 Localization of glutamate in PFC afferents in VTA:

Since PFC afferents are primarily glutamatergic, dual labeling was performed for BDA labeling in VTA and vescicular glutamate transporters 1 or 2. Qualitative analysis using confocal microscopy revealed that the majority of BDA-labeled boutons in VTA following injections in the ILA and PL indeed contained VGlut 1 or 2 (Figure 12).

Localization of NMDAR1 receptors in VTA

To determine if sex-activated neurons in the VTA contain glutamate receptors, VTA sections from mated animals sacrificed 60 minutes following ejacutation were stained for

Fos and NMDAR1. Qualitative analysis revealed that a large majority of the Fos-IR neurons in the VTA express NMDAR1.

DISCUSSION

The present study demonstrates that the PFC – and in particular the ILA and PL subregions – sends robust inputs to the VTA. In addition, the majority of sex-activated neurons in the VTA receive inputs from the ILA and PL. Thus, inputs from the ILA and

PL are in an anatomical position to provide a major source of stimulatory inputs during sexual behavior, In contrast, the NAc sends few projections to the VTA, and likely provides little contribution to sex-induced activation of the VTA. Moreover, the ILA and

79 PL send robust projections to other areas involved in sexual behavior and motivation, including the NAc, MPN, BST, BLA and SPFp, while the NAc sends no projections to these areas. Thus, the ILA and PL subregions of the PFC may also influence sexual behavior and motivation via brain regions other than the VTA.

Overall, the anatomical connections described in this study are consistent with prior reports describing afferent input to the VTA. Robust projections from the ILA and

PL subregions of the PFC were observed, as has been described previously (Sesack et al.,

1989). Moreover, fewer afferents were observed originating from the ACA and NAc compared to the ILA and PL. The majority of these projections originated in the more medial core subregion, consistent with previous reports (Heimer et al., 1991; Berendse et al., 1992a). Expression of the immediate early gene c-Fos was observed in all brain areas in which copulation-induced Fos expression has previously been described (Coolen et al.,

1996; Balfour et al., 2003). Moreover, qualitative analysis revealed that Fos expression was not induced in five unmated control animals, indicating that injection of the tracer

BDA does not alter Fos expression in response to mating.

One major finding of the present study is that the PFC – and in particular the ILA and PL – send massive projections to the sex-activated neurons in the VTA. The majority of cells in the VTA that received inputs from the ILA or PL were Fos-IR following sexual behavior. Furthermore, the majority of the Fos-IR neurons in the VTA received contacts from either the ILA or PL. It is important to note that the inputs observed in this study are putative contacts resolved at the light microscopic level. Thus

80 to determine if these are indeed synaptic contacts requires further electron microscopic analysis. Nevertheless, the present study demonstrates that PFC efferents are in an ideal anatomical position to provide stimulatory input to the VTA neurons that are activated during sexual behavior. Numerous studies have shown that the PFC-to-VTA projection is largely glutamatergic (Tzschentke, 2001), and the present study demonstrates the presence of vescicular glutamatergic transporters in PFC efferent fibers, as well as the presence of glutamate receptors on sex-activated VTA neurons. Thus, it is possible that

PFC inputs contribute to excitation of VTA neurons via release of endogenous glutamate during sexual behavior. In support of this hypothesis, electrical stimulation of the PFC both increases extracellular glutamate in the VTA and induces Fos expression in VTA neurons (Rossetti et al., 1998). However, further studies are needed to determine if the

PFC efferents are important for glutamate release and Fos induction in the VTA during sexual behavior. To further address this question, future studies may include investigation of expression of sexual behavior or sex-induced neural activation following ablation of PFC inputs or pharmacological manipulations of glutamate receptors in the

VTA.

In addition to the VTA, efferent projections from the PFC were observed in other brain areas involved in sexual behavior and motivation. Robust projections from ILA and

PL to the NAc Core and Shell were observed. In contrast, the ACA projections to the

NAc were less robust, and with the majority of labeling in the dorsal striatum. Overall, these projections were in agreement with prior reports (Sesack et al., 1989; Berendse et al., 1992b). However, PFC projections to areas specifically related to sexual behavior

81 have not been well characterized. The present study demonstrates robust connections from the ILA and PL – but none from the ACA - to the MPN, BNSTpr, and SPFp. These areas are important for expression of sexual behavior, evidenced by effects of lesions

(Hull et al, bookchapter) and induction of Fos in MPN and BNSTpr following all stages of mating, and in BNSTpr and SPFp following ejaculation. (Coolen et al., 1996; Coolen et al., 1997b; Coolen et al., 1997a). A substantial portion of the cells in these areas that displayed copulation-induced Fos-IR cells also received contacts from the ILA or PL.

Thus, the PFC may contribute to the activation of these areas during different elements of sexual behavior. In contrast, the NAc appears to play no direct role in the mating- induced activation of these structures. In the current study, no direct projections from either the NAc core or shell were observed in any of the areas analyzed.

The PFC plays an important role in many of the cognitive functions involved in appetitive behaviors. In particular, the PFC has been implicated in executive brain processes important for learning, conditioning, and stimulus-reward associations (for review see (Tzschentke, 2000, 2001; Cardinal et al., 2002). During sexual arousal, activation of the PFC is increased both in freely moving rats (Hernandez-Gonzalez et al.,

1998) and in human subjects (Bocher et al., 2001). The effects of PFC lesions on expression of sexual behavior have been controversial. PFC lesions have been shown to inhibit male rodent sexual behavior (Fernandez-Guasti et al., 1994; Agmo et al., 1995).

In contrast, studies from our laboratory (Davis et al., 2003) and others (Michal, 1973) failed to demonstrate significant effects of PFC lesions on sexual behavior. Interestingly,

PFC lesions that did inhibit sexual behavior included the ACA, while leaving ILA and PL

82 intact. In contrast, lesions that include ILA and PL do not disrupt sexual behavior. These data are in contrast to our hypothesis that ILA and PL via their direct connections to sex- related areas may influence sexual behavior. One possible explanation is that lesions in

ACA also affected adjacent motor cortex, exerting their effects on motor patterns essential for sexual behavior, rather than sexual motivation.

The present study does not distinguish between the connections to dopaminergic versus non-dopaminergic neurons in the VTA. However, 85% of the sex-activated neurons in the VTA are non-dopaminergic (Balfour et al., 2003). Furthermore, the majority of excitatory inputs from the PFC synapse onto non-dopaminergic neurons in the VTA (Sesack and Pickel, 1992; Wang and French, 1995). Thus, it is likely that the majority of sex-activated neurons that receive input from the PFC are non-dopaminergic.

In fact, Sesack and Carr have shown that the majority of the non-dopaminergic neurons in the VTA receiving PFC innervation are GABAergic (Carr and Sesack, 2000a). While

GABA neurons in VTA project to both the NAc (Van Bockstaele and Pickel, 1995) and the PFC (Carr and Sesack, 2000b), Carr and Sesack have shown that a strict target specificity exists with respect to PFC innervation of VTA DA and GABA neurons.

Specifically, the PFC contacts DA cells that project back to the PFC, but not to the NAc, and conversely, the PFC contacts GABA cells that project to the NAc but not to the PFC

(Carr and Sesack, 2000a). This is consistent with electrophysiological studies demonstrating two populations of DA cells in the VTA that respond differently to PFC stimulation (Tong et al., 1998).

83 Based on these findings, one would expect PFC stimulation to inhibit the activity of the NAc. Indeed, electrical stimulation of the PFC at physiologically relevant frequencies inhibits DA transmission in the NAc (Jackson et al., 2001), and blockade of glutamate antagonists administered into the PFC increases DA transmission in the NAc

(Takahata and Moghaddam, 2000). Furthermore, 6-hydroxydopamine (6-OHDA) lesions of PFC increase NAc electrical activity in response to sex-related olfactory cues (Mitchell and Gratton, 1992), suggesting that the PFC may be exert an inhibitory influence on the mesolimbic systems involved in conditioning and reward. Together these data suggest that the PFC inhibit DA release and activity in the NAc in a feed-forward inhibitory manner via activation of mesoaccumbens GABA cells. Furthermore, the PFC may be involved in stimulation of other brain areas involved in sexual performance and sexual motivation, though the phenotype of these target neurons is currently unknown. A schematic diagram of this putative circuit is presented in Figure 12.

In conclusion, the present study demonstrated that ILA and PL subregions of the

PFC send robust inputs to VTA and other areas involved in sexual motivation and performance. Moreover, ILA and PL efferents to these areas indeed form putative contacts with neurons that are activated during sexual behavior. In contrast, few inputs to sex-activated neurons in VTA or other brain areas derive from the NAc core or shell.

Together, these results suggest that ILA and PL are in the anatomical position to influence sexual motivation and performance.

84 REFERENCES

Agmo A, Villalpando A, Picker Z, Fernandez H (1995) Lesions of the medial prefrontal cortex and sexual behavior in the male rat. Brain Res 696:177-186. Balfour ME, Yu L, Coolen LM (2003) Sexual behavior and a sex-associated environment cause activation of the mesolimbic system. Submitted. Berendse HW, Groenewegen HJ, Lohman AH (1992a) Compartmental distribution of ventral striatal neurons projecting to the mesencephalon in the rat. J Neurosci 12:2079-2103. Berendse HW, Galis-de Graaf Y, Groenewegen HJ (1992b) Topographical organization and relationship with ventral striatal compartments of prefrontal corticostriatal projections in the rat. J Comp Neurol 316:314-347. Bocher M, Chisin R, Parag Y, Freedman N, Meir Weil Y, Lester H, Mishani E, Bonne O (2001) Cerebral activation associated with sexual arousal in response to a pornographic clip: A 15O-H2O PET study in heterosexual men. Neuroimage 14:105-117. Cardinal RN, Parkinson JA, Hall J, Everitt BJ (2002) Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci Biobehav Rev 26:321-352. Carr DB, Sesack SR (2000a) GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex. Synapse 38:114-123. Carr DB, Sesack SR (2000b) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873. Coolen LM, Peters HJ, Veening JG (1996) Fos immunoreactivity in the rat brain following consummatory elements of sexual behavior: a sex comparison. Brain Res 738:67-82. Coolen LM, Peters HJ, Veening JG (1997a) Distribution of Fos immunoreactivity following mating versus anogenital investigation in the male rat brain. Neuroscience 77:1151-1161. Coolen LM, Olivier B, Peters HJ, Veening JG (1997b) Demonstration of ejaculation- induced neural activity in the male rat brain using 5-HT1A agonist 8-OH-DPAT. Physiol Behav 62:881-891. Davis JF, Loos M, Coolen LM (2003) Lesions of the medial prefrontal cortex do not disrupt sexual behavior in male rats. In: Society for Behavioral Neurendocrinology, p 45. Cincinanti, OH: Hormones and Behavior. Fernandez-Guasti A, Omana-Zapata I, Lujan M, Condes-Lara M (1994) Actions of sciatic nerve ligature on sexual behavior of sexually experienced and inexperienced male rats: effects of frontal pole decortication. Physiol Behav 55:577-581. Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the projection patterns of accumbal core and shell in the rat. Neuroscience 41:89-125. Hernandez-Gonzalez M, Guevara MA, Cervantes M, Morali G, Corsi-Cabrera M (1998) Characteristic frequency bands of the cortico-frontal EEG during the sexual interaction of the male rat as a result of factorial analysis. J Physiol Paris 92:43- 50.

85 Jackson ME, Frost AS, Moghaddam B (2001) Stimulation of prefrontal cortex at physiologically relevant frequencies inhibits dopamine release in the nucleus accumbens. J Neurochem 78:920-923. Michal EK (1973) Effects of limbic lesions on behavior sequences and courtship behavior of male rats (Rattus norvegicus). Behaviour 44:264-285. Mitchell JB, Gratton A (1992) Partial dopamine depletion of the prefrontal cortex leads to enhanced mesolimbic dopamine release elicited by repeated exposure to naturally reinforcing stimuli. J Neurosci 12:3609-3618. Rossetti ZL, Marcangione C, Wise RA (1998) Increase of extracellular glutamate and expression of Fos-like immunoreactivity in the ventral tegmental area in response to electrical stimulation of the prefrontal cortex. J Neurochem 70:1503-1512. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320:145-160. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract- tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290:213- 242. Takahata R, Moghaddam B (2000) Target-specific glutamatergic regulation of dopamine neurons in the ventral tegmental area. J Neurochem 75:1775-1778. Tong ZY, Overton PG, Martinez-Cue C, Clark D (1998) Do non-dopaminergic neurons in the ventral tegmental area play a role in the responses elicited in A10 dopaminergic neurons by electrical stimulation of the prefrontal cortex? Exp Brain Res 118:466-476. Tzschentke TM (2000) The medial prefrontal cortex as a part of the brain reward system. Amino Acids 19:211-219. Tzschentke TM (2001) Pharmacology and behavioral pharmacology of the mesocortical dopamine system. Prog Neurobiol 63:241-320. Van Bockstaele EJ, Pickel VM (1995) GABA-containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain. Brain Res 682:215- 221. Wang T, French ED (1995) NMDA, kainate, and AMPA depolarize nondopamine neurons in the rat ventral tegmentum. Brain Res Bull 36:39-43.

86 Figure 1. Schematic diagram illustrating areas of analysis for Fos/BDA/NeuN 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). Each area of analysis measured 400 µm x 300 µm, except for that of the SPFp, which measured 400 µm x 150 µm.

87 Figure 2. Color plate. A. BDA labeled fibers contacting NeuN (brown) + Fos (black) labeled neurons in the VTA. B. Higher power magnification illustrating BDA synaptic boutons in close apposition to a Fos/NeuN labeled neuron in the VTA. C. VTA neurons stained for Fos (brown) and NMDAR1 (black). D-E. Confocal images showing colocalization of BDA (red) and VGLUT1 (green) in synaptic terminals projecting from the ILA to the VTA. Optical slice = 0.6 µm. For all panels, scale bar = 10 µm.

88 Figure 3. Schematic illustration of location and sizes of injections sites in animal with injections restricted to ILA (animals I1-3), PL (animals P1-3), ACA (animals (A1-3), NAc Shell (animals S1-3), or NAc Core (animals C1-3). Scale bar indicates 1 mm.

89 Figure 4. Camera lucida drawings illustrating the distribution of fibers projecting from the ILA, PL and ACA to four rostrocaudal levels of the VTA.

90 Figure 5. Camera lucida drawings illustrating the distribution of fibers projecting from the ILA, PL and ACA to three rostrocaudal levels of the NAc.

91 Figure 6. Camera lucida drawings illustrating the distribution of fibers projecting from the ILA, PL and ACA to the MPN, BST, and MEA.

92 Figure 7. Camera lucida drawings illustrating the distribution of fibers projecting from the NAc Shell to four rostrocaudal levels of the VTA.

93 Figure 8. Camera lucida drawings illustrating the distribution of fibers projecting from the NAc Core to four rostrocaudal levels of the VTA.

94 95 Figure 9 (previous page). Camera lucida drawings illustrating the distribution of fibers projecting from the NAc Core and Shell to the MPN.

Figure 10. Quantification of the relative contribution of the mPFC and NAc to sex- induced activation of VTA neurons. Presented are mean percentages ± SEM of all cells receiving contacts, percentages of contacted cells that express Fos, and percentages of For-IR cells that receive contacts. The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

Figure 11 (next page). Quantification of the relative contribution of ILA and PL to sex- induced activation of neurons in other areas related to sexual behavior and motivation. Presented are mean percentages ± SEM of all cells receiving contacts, percentages of contacted cells that express Fos, and percentages of For-IR cells that receive contacts. The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

96 Figure 11

97 Chapter 4

Sexual experience causes a sensitized response to amphetamine in male rats

98 INTRODUCTION

There is increasing evidence that drugs of abuse converge upon common reward pathways. A key component of these reward pathways is the mesolimbic dopamine system, and indeed, multiple addictive drugs have been shown to activate the dopamine system (Wise and Bozarth, 1987; Di Chiara and Imperato, 1988). Repeated drug administration can cause functional and morphological changes in these pathways, and this phenomenon may contribute to the transition from drug use to drug addiction. In vivo exposure to a variety of abused drugs increases dopamine release in the nucleus accumbens (Di Chiara and Imperato, 1988) and enhances synaptic strength at excitatory synapses on midbrain dopamine neurons (Saal et al., 2003). In addition, repeated drug administration results in long-lasting changes in dendritic spine density and morphology in the nucleus accumbens and prefrontal cortex (Robinson and Kolb, 1997, 1999a; Li et al., 2003).

One useful behavioral measure for functional changes in the mesolimbic dopamine system in rodent models is locomotor sensitization. In this paradigm, repeated administration of all major drugs of abuse results in an increased or “sensitized” locomotor response that can persist for many months. Moreover, administration of one drug can result in a sensitized response to a chemically unrelated drug. Such “cross- sensitization” provides further evidence that the different addictive drugs activate common reward pathways (Robinson and Berridge, 1993).

99 The reward pathways implicated in drug addiction mediate natural rewarding behaviors, such as sexual behavior. In rodents, male sexual behavior results in dopamine efflux in the nucleus accumbens (Pfaus et al., 1990; Robertson et al., 1991; Damsma et al., 1992; Wenkstern et al., 1993; Robinson et al., 2001a). Other natural behaviors, such as sucrose intake, feeding, and aggression are also associated with increased dopamine transmission in the nucleus accumbens (Bassareo and Di Chiara, 1999; van Erp and

Miczek, 2000; Hajnal and Norgren, 2001). Moreover, there is increasing evidence that natural behaviors can cause functional changes in the mesolimbic systems involved in drug addiction. Stressful stimuli activate dopamine systems (Thierry et al., 1976) and have been shown to cause locomotor sensitization to psychostimulants (Robinson and

Becker, 1986; Kalivas and Stewart, 1991; Robinson and Berridge, 1993). However, stress is an aversive stimulus, and very few studies have investigated the effects of repeated exposure to a natural rewarding stimulus. Recently, Bradley and Meisel showed that sexually experienced female hamsters display a more rapid onset to the locomotor effects of amphetamine than naïve hamsters (Bradley and Meisel, 2001). However, it has been shown that rodents display sexual dimorphic responses to psychostimulants. In particular, females develop greater sensitization than males and display greater psychostimulant-induced dopamine release in the NAc (Castner et al., 1993; Becker et al., 2001).

Therefore, the main goal of the present study is to determine if a natural rewarding stimulus (sexual experience) causes functional changes in the reward systems associated with drug addiction in male rats. First, we used a locomotor sensitization

100 paradigm in order to determine if sexual experience causes changes in the sensitivity to the behavioral effects of drugs of abuse. In addition, because environment plays an important role in drug addiction, we investigated the effects of novelty and sex-associated environments. Second, we used immediate early gene expression in order to determine which components of the mesolimbic reward systems are differentially activated by both sexual experience and acute administration of drugs of abuse.

METHODS

Animals: Adult male Sprague Dawley rats (250-260 grams) were obtained from Harlan

(Indianapolis, IN) and housed individually in plexiglass cages. The colony room was maintained on 12/12 hr light dark cycle. Food and water were available ad libitum.

Stimulus females (210-220 grams) for mating behavior sessions were bilaterally ovariectomized and received a subcutaneous implant containing 5% estradiol benzoate

(EB) and 95% cholesterol. Sexual receptivity was induced by administration of 500 µg progesterone (P) in 0.1 ml sesame oil 4 hours before testing. All procedures were approved by the Animal Care and Use Committee of the University of Cincinnati and conformed to NIH guidelines involving vertebrate animals in research.

Drugs: D-Amphetamine sulfate (Sigma, St. Louis, MO) was dissolved in 0.9% sterile saline. Animals received 0.5 mg/kg body weight, described as the free base. Control

101 animals received saline. All injections were given subcutaneously during the first half of the light phase in a volume of 1 mL/kg body weight.

Locomotor Activity Testing: Locomotor activity was measured using a group of 30 custom-designed residential activity chambers (RACs), modeled on chambers designed by Segal and Kuczenski (Segal and Kuczenski, 1987). Each chamber consists of a 40 x

40 x 38 cm Plexiglas enclosure located inside of a lighted, ventilated, sound-attenuated cabinet (Cline Builders, Covington, KY). The floor of each chamber is covered in clean cob bedding. Locomotor activity was measured using a 16 x 16 photo beam array (San

Diego Instruments, San Diego, CA) located 3.5 cm above the floor of the enclosure.

Locomotor activity is expressed as crossovers per unit time. A crossover is recorded each time the animal enters any of the “active zones” of the chamber, depicted as shaded areas in Figure 1. These zones are mapped by the data acquisition software; there are no markings on the floor of the chamber.

Experimental Design:

Experiment 1: The purpose of Experiment 1 was to determine if sexual experience results in an increased locomotor response to amphetamine. Male rats were randomly divided into sexually experienced and naïve groups. Animals in the sexually experienced groups received 5 pre-test mating sessions spaced 3-4 days apart, during which they were allowed to mate in their home cage with a receptive female for 3 copulatory series or 60 minutes, whichever came first. Each copulatory series consists of mounts and intromissions followed by ejaculation. Animals that completed less than five cumulative

102 copulatory series were excluded from the study. All mating sessions took place during the first half of the dark phase under dim red illumination. Sexually naïve animals did not receive female partners, but were present in the same room during the pre-test mating sessions of the sexually experienced males. One week following the last pre-test mating session, the sexually experienced and naïve animals were subdivided into groups receiving amphetamine or saline for a total of four groups (Naïve Amphetamine: NA;

Experienced Amphetamine: EA; Naïve Saline: NS; and Experienced Saline: ES; n=6 each). The experienced animals were divided such that the EA and ES groups received the same amount of sexual experience. Specifically, the groups did not differ in total numbers of ejaculations (ES: 7.6 ± 0.9; EA: 7.0 ± 0.7), total numbers of mounts (ES:

239.3 ± 29.8; EA: 233.6 ± 33.5) or total numbers of intromissions (ES: 82.5 ± 7.7; EA:

79.8 ± 7.7). The animals were placed in the RACs immediately following injection, and locomotor activity was recorded for 90 minutes. At the end of the test, the animals were removed from the RACs and sacrificed. Their brains were processed for immunocytochemistry as described below.

Experiment 2: The purpose of Experiment 2 was to investigate the role of a novel environment in sex-induced locomotor sensitization to amphetamine. The pre-test mating paradigm was identical to that used in Experiment 1. The key difference is that in

Experiment 2, half of the sexually naïve and experienced animals were allowed to acclimate to the RACs prior to the final test day. Five days following the last mating session, these animals were placed inside the RACs, and were housed inside the RACs for the remainder of the experiment. The animals were injected with saline immediately

103 prior to being placed in the RACs and again 24 hours later. Locomotor activity was recorded after each injection to determine the locomotor response to saline in both a novel and non-novel environment (Naïve Saline Novel: NS+ (n=6); Experienced Saline

Novel: ES+ (n=9); Naïve Saline Non-Novel: NS- (n=6); Experienced Saline Non-Novel:

ES- (n=9)). The following day (seven days after the last mating session) these acclimated animals were injected with amphetamine (Naïve Amphetamine Non-Novel: NA-;

Experienced Amphetamine Non-Novel: EA-). The novel groups were treated as in

Experiment 1 and were placed inside the RACs immediately following amphetamine injection (Naïve Amphetamine Novel: NA+ (n=6); Experienced Amphetamine Novel:

EA+ (n=9)). The experienced animals were divided such that the EA+ and EA- groups received the same amount of sexual experience. Specifically, the groups did not differ in total numbers of ejaculations (EA+: 9.4 ± 0.7, EA-: 10.1 ± 0.9), total numbers of mounts

(EA+: 256.0 ± 17.1; EA-: 242.7 ± 22.6) or total numbers of intromissions (EA+: 114.2 ±

15.7; EA-: 119.6 ± 11.6). Locomotor activity was recorded for 90 minutes. At the end of the test, the animals were removed from the RACs and sacrificed. Their brains were processed for immunocytochemistry as described below.

Experiment 3: Experiment 3 investigated sex-induced locomotor sensitization to amphetamine when the animals were challenged with amphetamine in the same environment in which they gained their sexual experience. Male rats were randomly divided into sexually experienced and naïve groups. The sexually experienced animals received seven consecutive daily pre-test mating sessions. During each mating session, the animals were placed in the RACs, and locomotor activity was recorded for 15

104 minutes. The animals were then given a receptive female partner and allowed to mate to one ejaculation. The sexually naïve animals were placed in the RACs for seven consecutive sessions without mating, and locomotor activity was recorded for 30 minutes. Cages were cleaned with 70% ethanol and filled with fresh bedding before each mating session. The animals were returned to their home cage following each pre-test mating session, and all pre-test sessions took place in the first half of the dark phase under dim red illumination. The morning (first half of the light phase) following the final pre-test session (Day 8 of the experiment), the animals were subdivided into 4 groups receiving amphetamine or saline (Naïve Amphetamine: NA; Experienced Amphetamine:

EA; Naïve Saline: NS; and Experienced Saline: ES; n=8-9 each). The experienced animals were divided such that the EA and ES groups received the same amount of sexual experience. Specifically, the groups did not differ in total numbers of ejaculations

(ES: 6.4 ± 0.3; EA: 6.6 ± 0.2), total numbers of mounts (ES: 54.0 ± 7.6; EA: 61.6 ± 11.1) or total numbers of intromissions (ES: 74.7 6.6± ; EA: 79.2 ± 7.2). The animals were placed in the RACs immediately following injection, and locomotor activity was recorded for 90 minutes, after which the animals were returned to their home cages. The animals were tested in the RACs again one week following the final pre-test mating session (Day 14). Animals that received amphetamine on Day 8 received saline on Day

14, and animals that received saline on Day 8 received amphetamine on Day 14. Half of the naïve and experienced animals were sacrificed one day later for RNA extraction (data not shown in this report). One month following the final pre-test mating session (Day 35), the remaining half of the animals was given amphetamine and locomotor activity was recorded for 90 minutes. All animals received saline injections during the first half of the

105 light phase of the three days prior to each test day (Day 8, 14, and 35), for acclimation to the injection procedure. Following these saline injections, males were returned to their home cages.

Tissue Preparation: Subjects from Experiments 1 and 2 were sacrificed 90 minutes following amphetamine or saline injection. The animals were deeply anesthetized using pentobarbital (200 mg/kg) and perfused transcardially with 100 mL of 0.9% NaCl followed by 500 mL of 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.3).

The brains were removed and post-fixed for 1 hour at room temperature in the fixative, then placed in 20% sucrose in 0.1 M PB and stored at 4° C. Coronal sections (35 µm) were cut on a freezing microtome (Richard Allen, Kalamazoo, MI), collected in four parallel series in cryoprotectant solution (30% sucrose, 30% ethylene glycol, 0.01% sodium azide in 0.1 M PB; Watson et al, 1985), and stored at –20°C until further processing.

Immunocytochemistry: All incubations were performed at room temperature with gentle agitation. Free-floating sections were washed extensively with 0.05 M Tris-buffered saline (TBS, pH 7.6) between incubations. Sections were incubated for 10 minutes with

1% H2O2, then blocked for one hour with incubation solution (TBS containing 0.1% bovine serum albumin and 0.4% triton X-100). All primary antibody incubations were performed in the incubation solution, overnight at room temperature. The sections were incubated overnight with a rabbit polyclonal antibody to c-Fos (1:7500; SC-52; Santa

Cruz Biotechnology, Santa Cruz, CA) followed by one hour incubations with biotinylated

106 donkey anti-rabbit IgG (1:500; Vector Laboratories, Burlingame, CA) and avidin- horseradish peroxidase complex (1:1000; ABC Elite Kit, Vector Laboratories,

Burlingame, CA). Finally, the sections were incubated for 10 minutes in 0.02% diaminobenzidine (DAB; Sigma-Aldrich, St. Louis, MO) in 0.05M TB containing

0.012% hydrogen peroxide and 0.4% nickel sulfate, resulting in a blue-black reaction product. Next, VTA sections were incubated overnight with mouse monoclonal antibody to tyrosine hydroxylase (TH; 1:400,000; Chemicon International, Temecula, CA), biotinylated donkey anti-mouse IgG secondary antibody (1:500; Vector Laboratories,

Burlingame, CA) and ABC as described above. Finally, the sections were incubated for

10 minutes in 0.02% DAB in 0.05 M TB containing 0.012% hydrogen peroxide, resulting in a reddish-brown reaction product. Following staining, the sections were washed

thoroughly in 0.05M TB, mounted onto glass slides with 0.3% gelatin in ddH20 and coverslipped with DPX (Electron Microscopy Sciences, Fort Washington, PA).

Immunocytochemical controls included omission of primary antibodies.

Data Analysis:

Sexual Behavior: Each mating session was observed and sexual behavior was recorded: number of mounts, number of intromissions, and number of ejaculations. Results of each measure were summed over the entire pre-test mating period for each animal and group means were calculated. For Experiments 1 and 3, these results were analyzed using a one- way ANOVA to determine if the treatment groups (amphetamine vs. saline) differed from each other. For Experiment 2, these results were analyzed using a one-way ANOVA to determine if the amphetamine treated sexually experienced novel (EA+) and non-novel

107 (EA-) groups differed from each other. For all experiments, post-hoc comparisons were performed using Fisher’s PLSD with significance set at 5%.

Locomotor Activity: Data were collected in 3-minute bins following amphetamine or saline injection. Results are shown as the mean ± SEM for each group. In Experiment 1, results were analyzed using a two-way ANOVA (factors: sexual experience, drug treatment), and post-hoc comparisons were performed using a two-tailed t-test. In

Experiment 2, results were analyzed using a three-way ANOVA (factors: sexual experience, drug treatment, environment) with repeated measures, and post-hoc comparisons were made using a two-tailed t-test. In Experiment 3, results were analyzed for each test day using a two-way ANOVA (factors: sexual experience, drug treatment) with repeated measures, and post-hoc comparisons were made using a two-tailed t-test.

Locomotor activity during the pretest mating sessions was analyzed using a one-way

ANOVA (factor: sexual experience) with repeated measures, and post-hoc comparisons were made using a two-tailed t-test. For all experiments, significance was set at p < 0.05.

Immunocytochemistry Analysis: Using a drawing tube attached to a Leica microscope

(Leica Microsystems; Wetzlar, Germany), camera lucida drawings were made of the analyzed sections from each animal. Schematic drawings illustrating the areas of analysis are shown in Figure 2. In the VTA, camera lucida drawings were made of four sections approximately 280 µm apart, representative of four rostral to caudal levels in the

VTA (Figure 2, panels E-F). Using TH staining and the location of the medial lemniscus

(ml) and fasciculus retroflexus (fr) as landmarks, standard areas were defined in which to

108 count Fos-IR nuclei and Fos/TH double labeled cells. Cell counts were performed in standard areas ranging from 2.16 mm2 (rostral and two middle levels) to 1.6 mm2 (most caudal level). In the NAc, camera lucida drawings were made of three sections approximately 600 µm apart, representative of three rostral to caudal levels in the NAc

(Figure 2, panels B-C). At each level, standard areas of 0.24 mm2 were defined in which to count Fos-IR nuclei in both the NAc Core and NAc Shell. In the mPFC, camera lucida drawings were made in the anterior cingulate (AC), prelimbic, (PL) and infralimbic (IL) regions in stand areas of 0.64 mm2 (Figure 2A). Group means were calculated for each separate rostral to caudal level in both NAc and VTA. In Experiment 1, results were analyzed using a two-way ANOVA (factors: sexual experience, drug treatment), and post-hoc comparisons were made using Fisher’s PLSD. In Experiment 2, results were analyzed using a one-way ANOVA (factor: sexual experience), and post-hoc comparisons were made using Fisher’s PLSD. For all experiments, significance was set at p < 0.05.

RESULTS

Locomotor activity

Experiment 1

The goal of Experiment 1 was to determine if sexual experience affects the locomotor response to amphetamine in male rats. Locomotor activity during a 90-minute period was measured in sexually experienced and naïve rats following treatment with 0.5 mg/kg amphetamine or saline. Results from Experiment 1 are illustrated in Figure 3.

109 Both sex experience (F1,22=15.88; p =0.0006) and drug treatment (F1,22=45.00; p < 0.0001) had significant effects on locomotor activity, and a two-way interaction between sex

experience and drug treatment was observed (F1,1,22=14.27; p=0.0010). Specifically, both naïve and experienced animals showed a significantly increased locomotor response to amphetamine compared to the appropriate saline controls (Figure 3A; NA vs. NS, p=0.0422; EA vs. ES, p < 0.0001). Moreover, sexually experienced rats displayed an increased locomotor response to amphetamine compared to naïve animals (Figure 3A;

EA vs. NA; p < 0.0001). Sexually experienced and naïve rats did not differ in their response to saline.

Analysis of the locomotor response to amphetamine in smaller time intervals of

30 minutes and 3 minutes is illustrated in Figure 3, panels B-D. Sexually experienced males displayed an increased locomotor response to amphetamine compared to naïve rats throughout the 90-minute test period (EA vs. NA; p < 0.001). Moreover, sexually experienced rats showed an increased locomotor response to amphetamine compared to their saline controls throughout the 90-minute test period (EA vs. ES; p < 0.002), while naïve animals only displayed a significantly higher locomotor response during the last

30-minute interval (Figure 3D; NA vs. NS; p=0.0024).

Experiment 2

Experiment 2 investigated the role of a novel environment in sex experience- induced locomotor sensitization to amphetamine. As in Experiment 1, sexually experienced and naïve rats were challenged with amphetamine in a novel environment.

110 In addition, separate groups of sexually experienced and naïve rats were allowed to acclimate to the testing chamber for several days prior to amphetamine treatment.

Results from Experiment 2 for the total 90-minute testing period are illustrated in Figure

4. Both sex experience (F1,26=8.86; p=0.0062) and drug treatment (F1,26=145.13; p <

0.0001) had significant effects on locomotor activity, and a two-way interaction between

sex experience and drug treatment was observed (F1,1,26=15.32; p=0.0006). When challenged with amphetamine in a novel environment, sexually experienced rats displayed an increased locomotor response compared to naïve animals (Figure 4A; EA+ vs. NA+; p < 0.0001), replicating Experiment 1. This sex experience-induced locomotor sensitization to amphetamine was also observed in animals that were allowed to acclimate to the testing chamber prior to amphetamine treatment (Figure 4A; EA- vs.

NA-; p=0.03). However, the testing environment did have a significant effect on

locomotor activity (F1,26=19.40; p=0.0002), and a two-way interaction between environment and drug treatment was observed (F1,1,26=11.56; p=0.0022). In particular, sexually experienced animals receiving amphetamine in a novel environment had a greater locomotor response to amphetamine than animals that were allowed to acclimate to the testing environment (Figure 4A; EA+ vs. EA-; p < 0.0001). In contrast, naïve animals receiving amphetamine in a novel environment did not display significantly higher locomotion than naïve animals receiving amphetamine in a non-novel environment, although a trend was present (Figure 4A; NA+ vs. NA-; p=0.0620).

Although the overall response to amphetamine was increased in the novel environment, sexual experience resulted in a sensitized locomotor response to amphetamine in both environments. In particular, the fold difference between the experienced and naive

111 animals (EA/NA) was similar in both cases: 1.8x in the novel environment vs. 1.6x in the non-novel environment. Finally, animals treated with amphetamine displayed significantly higher locomotor activity than their saline controls in both environments

(Figure 4A; NA+ vs. NS+; EA+ vs. ES+; NA- vs. NS-; EA- vs. ES-; p < 0.0025).

Furthermore, naïve and experienced animals did not differ in their response to saline in either environment, and animals receiving saline in a novel environment did not differ from those receiving saline in a non-novel environment.

Analysis of the locomotor response to amphetamine in smaller time intervals of

30 minutes is illustrated in Figure 4, panels B-D. Sexually experienced males challenged with amphetamine in a novel environment displayed an increased locomotor response to amphetamine compared to naïve rats throughout the 90-minute test period (Figure 4-B-D;

EA+ vs. NA+; p < 0.006). Sexually experienced animals that were allowed to acclimate to the testing environment displayed an increased locomotor response to amphetamine compared to naïve animals in the second and third 30-minute intervals (Figure 4B-C; EA- vs. NA-; p < 0.035). Moreover, both sexually experienced and naïve rats showed an increased locomotor response to amphetamine compared to their saline controls throughout the 90-minute test period when tested in a novel environment (Figure 4-B-D ;

EA+ vs. ES+, NA+ vs. NS+; p < 0.02). When challenged with amphetamine in a non- novel environment, sexually experienced animals also displayed a significantly higher locomotor response throughout the 90-minute test period (Figure 4-B-D; EA- vs. ES-; p <

0.0003), but naïve animals tested in a non-novel environment displayed a significantly higher locomotor response only during the first and second 30-minute intervals (Figure

112 4B-C; NA- vs. NS-; p < 0.015). Sexually experienced animals receiving amphetamine in a novel environment displayed a significantly higher locomotor response than experienced animals receiving amphetamine in a non-novel environment throughout the

90-minute test period (Figure 4-B-D; EA+ vs. EA-; p < 0.007). Sexually naive animals receiving amphetamine in a novel environment displayed a significantly higher locomotor response than naïve animals receiving amphetamine in a non-novel environment only during the second and third 30-minute intervals (Figure 4-B-C; NA+ vs. A-; p < 0.05). Furthermore, naïve and experienced animals did not differ in their response to saline in either environment, and animals receiving saline in a novel environment did not differ from those receiving saline in a non-novel environment.

Experiment 3

Experiment 3 tested the locomotor response to amphetamine when administered in the same environment in which the animals gained sexual experience. Sexually experienced animals gained their experience in the locomotor activity testing chambers, while sexually naïve animals were placed in the chambers a corresponding number of times without mating. To determine if exposure to the sex-paired environment itself causes increased locomotion, locomotor activity was measured for 15 minutes prior to each mating session. Sexually experienced and naïve animals did not differ before the first mating session. By the third mating session, hence following two experiences, sexually experienced animals displayed an increased locomotor response to the mating environment (Figure 5; p=0.02). Due to computer error, data is missing for the

113 experienced animals on Day 4. However, this difference did not persist when the animals were placed in the testing environment during the light phase. In particular, naïve and experienced animals challenged with saline on the morning of Day 8 did not differ in their locomotor responses during the first 15 minutes of testing (NS vs. ES; data not shown).

The locomotor response to amphetamine was measured one day (Day 8, Figure

6), one week (Day 14, Figure 7), and one month (Day 35, Figure 8) following the last mating session. Results from Experiment 3 for each of the 90-minute testing periods are illustrated in panel A in each figure. Sexually experienced rats displayed a greater locomotor response to amphetamine compared to naïve animals on all three testing days

(EA vs. NA; Day 8: p=0.313; Day 35: p=0.0172), although at Day 14 this effect did not reach significance when behavior was analyzed for the 90 minute period (Figure 7A; p=0.1549), but was only statistically significant during the second 30-minute interval (see below). Moreover, naïve and experienced animals did not differ in their response to saline on any of the testing days, and rats that received amphetamine displayed increased locomotor activity when compared to their saline controls NA vs. NS; EA vs. ES; p <

0.0125).

Analysis of the locomotor response to amphetamine in smaller time intervals of

30 minutes is illustrated in panels B-D of Figures 6-8. On Day 8, experienced rats displayed an increased locomotor response to amphetamine compared to their saline controls throughout the 90-minute test period (Figure 6B-D; EA vs. ES; p < 0.0004),

114 while naïve animals only displayed a significantly higher locomotor response during the second 30-minute interval (Figure 6C; NA vs. NS; p=0.0029). Sexually experienced animals displayed an increased locomotor response to amphetamine compared to naïve animals in the second and third intervals (Figure 6C-D; EA vs. NA; p < 0.035). On Day

14, both experienced and naïve animals displayed an increased locomotor response to amphetamine compared to their saline controls throughout the 90-minute test period

(Figure 7B-D; EA vs. ES; p < 0.006). However, sexually experienced animals showed an increased response to amphetamine compared to naïve animals during only during the second 30-minute interval (Figure 7C; p= 0.05). On Day 35, sexually experienced rats showed an increased locomotor response to amphetamine compared to naïve animals during the second 30-minute interval (Figure 8C; EA vs. NA; p < 0.0028). The locomotor response to saline did not differ between naïve and experienced animals during any of the testing days.

Neural activation

Experiment 1

Activation of NAc, VTA, and mPFC was investigated in the sexually experienced and naïve rats of Experiment 1 following either amphetamine or saline treatment in a novel environment. In the NAc, Fos expression was analyzed in both the Core and Shell regions at three rostrocaudal levels. A significant effect of drug treatment was observed

in the caudal portion of the NAc Shell (F(1,17)=8.629, p=0.0092) and throughout the NAc

Core (rostral: F(1,17)=25.985, p < 0.0001; middle NAc: F(1,17)=15.529, p=0.0011; caudal:

F(1,17)=10.823, p=0.0043). In particular, sexually experienced animals that received

115 amphetamine had increased numbers of Fos-IR cells compared to their saline controls throughout the NAc Core (Figure 9A-C; EA vs. ES; p < 0.035). Naïve animals that received amphetamine had increased numbers of Fos-IR cells compared to their saline controls in the rostral and middle portions of the NAc Core and the caudal portion of the

NAc Shell (Figure 9A-D; NA vs. NS; p < 0.02). In addition, there was a significant

effect of sexual experience in the rostral NAc Core (F(1,17)=5.069; p=0.0379).

Specifically, sexually experienced rats receiving saline had significantly more Fos-IR cells than naïve animals receiving saline (Figure 9A; ES vs. NS; p=0.01). No differences in Fos expression were detected between sexually experienced and naïve males receiving amphetamine.

In the VTA, Fos expression was analyzed in both dopaminergic and non- dopaminergic cells at four rostrocaudal levels (rostral, middle-1, middle-2, and caudal).

There were no significant differences among groups in Fos expression in dopaminergic neurons. However, when Fos expression was analyzed in non-dopaminergic cells,

significant effects of drug treatment were observed in the middle-1 (F(1,19=10.787;

p=0.0039), middle-2 (F(1,19)=4.80; p=0.0411) and caudal (F(1,19)=24.560; p < 0.0001) portions of the VTA. In particular, naïve animals that received amphetamine had increased numbers of Fos-IR cells compared to their saline controls in the middle 1 and caudal portions of the VTA (Figure 10A-B; NA vs. NS; p < 0.02), and sexually experienced animals that received amphetamine had increased numbers of Fos-IR cells compared to their saline controls in the caudal portion of the VTA (Figure 10B; EA vs.

116 ES; p=0.0069). However, no differences were detected between sexually experienced and naive animals in Fos-induction following saline or amphetamine.

In the mPFC, Fos expression was analyzed in the infralimbic (IL), prelimbic (PL) and anterior cingulate (AC) areas. In each of these areas, no differences were detected between groups.

Experiment 2

Activation of NAc, VTA, and mPFC was investigated in sexually experienced and naïve rats following amphetamine treatment in a non-novel environment, as described above. In each of these areas, no significant differences were detected between naïve and experienced animals in response to amphetamine.

There were, however, significant differences were observed between the locomotor response to amphetamine administered in a novel environment (Experiment 1;

NA and EA groups) compared to amphetamine administered in a non-novel environment

(Experiment 2; NA- and EA- groups ). In the VTA, there was a significant effect of

novelty on Fos expression in non-dopaminergic cells in the rostral (F1,18=29.064; p <

0.0001) and middle-1 (F1,18=33.00; p < 0.0001) levels. Specifically, naïve animals receiving amphetamine in a novel environment had significantly higher numbers of Fos-

IR cells than naïve animals receiving amphetamine in a non-novel environment (NA vs.

NA-; p <0.0001). Similarly, sexually experienced animal receiving amphetamine in a novel environment had significantly higher numbers of Fos-IR cells than experienced

117 animals receiving amphetamine in a non-novel environment (EA vs. EA-; p < 0.005).

There was also a significant effect of novelty on the number of Fos/TH double-labeled

cells in the rostral (F1,18=5.623; p=0.0291) and middle-1 (F1,18=6.540; p=0.0198) levels of the VTA. Post-hoc analysis did not reveal any significant differences between the individual groups (NA vs. NA- and EA vs EA-) groups, although a trend was present in the experienced animals at the middle-1 level (EA vs. EA-; p=0.053).

In the NAc, a significant effect of novelty on Fos expression throughout both the

Core (rostral: F1,18=100.049, p < 0.0001; middle: F1,18=46.067, p < 0.0001; caudal:

F1,18=46.152, p < 0.0001) and Shell (rostral: F1,18=12.249, p=0.026; middle: F1,18=70.051,

p < 0.0001; caudal: F1,18=53.220, p < 0.0001). Specifically, naïve animals receiving amphetamine in a novel environment had significantly higher numbers of Fos-IR cells than naïve animals receiving amphetamine in a non-novel environment (NA vs. NA-; p

<0.025). Similarly, sexually experienced animal receiving amphetamine in a novel environment had significantly higher numbers of Fos-IR cells than experienced animals receiving amphetamine in a non-novel environment (EA vs. EA-; p < 0.025).

In the PFC, there was a significant effect of novelty on Fos expression in the

anterior cingulate (F1,19=6.660, p=0.0183), prelimbic (F1,19=16.826, p=0.0006), and infralimbic (F1,19=30.925, p < 0.0001) areas. Specifically, naïve animals receiving amphetamine in a novel environment had significantly higher numbers of Fos-IR cells in the PL than naïve animals receiving amphetamine in a non-novel environment (NA vs.

NA-; p=0.017). Sexually experienced animal receiving amphetamine in a novel

118 environment had significantly higher numbers of Fos-IR cells in the PL and IL than experienced animals receiving amphetamine in a non-novel environment (EA vs. EA-; p

< 0.002).

DISCUSSION

The present study demonstrates that sexual experience can cause locomotor sensitization to amphetamine in male rats, persisting for at least one month. Moreover, this sex experience-induced sensitization is not dependant on the animals receiving drug and sex experience in different environments. In addition, sexual experience is correlated with increased neural activity in the NAc during baseline conditions, but not in the VTA or PFC.

In the present study, sexual experience was shown to cause a robust sensitized response to the locomotor stimulating effects of amphetamine. This suggests that repeated exposure to a natural rewarding behavior results in functional changes in limbic systems associated with drug addiction. Sensitization has been proposed as a model of drug craving, in which hyperresponsiveness of the dopamine system results in increased

“wanting” or craving of the drug (Robinson and Berridge, 1993). However, there is no evidence that sexual behavior results in craving or addiction. For example, rats trained to perform an operant task for an addictive drug continue this behavior even after it has been coupled with an aversive stimulus such as lithium chloride (LiCl) injection

(Cardinal et al., 2002). This suggests that drug-related cues elicit “wanting” or craving which persists after the incentive value has been removed. In contrast, male rats will

119 cease to approach a sexually receptive female when copulation has been paired with LiCl

(Peters, 1983; Agmo, 2002). In addition, while both stress and drug priming trigger relapse in rodent self-administration models, sexual behavior fails to do so (Shaham et al., 1997).

Other natural behaviors interact with the dopamine reward systems. Exposure to stressful stimuli can activate dopamine systems (Thierry et al., 1976) and can cause both psychomotor stimulant sensitization (Kalivas and Stewart, 1991) and relapse in self- administration models (Robinson and Berridge, 1993). However, stress in an aversive stimulus, and few studies have investigated whether natural rewarding behaviors can cause functional changes in the dopamine system as well. Recently, Bradley and Meisel showed that sexually experienced female hamsters display a more rapid onset to the locomotor effects of amphetamine than naïve hamsters (Bradley and Meisel, 2001).

However, rodents display sexual dimorphic responses to psychostimulants (Castner et al.,

1993; Becker et al., 2001), so it is important to determine whether this phenomenon occurs in males as well. The present study marks the first demonstration of sex experience-induced sensitization in male rodents. Moreover, there is increasing evidence that natural rewarding behaviors interact with the same neural pathways mediating sensitization. Male rats that have been sensitized to amphetamine display both increased sucrose craving and facilitated sexual behavior (Fiorino and Phillips, 1999; Wyvell and

Berridge, 2001; Avena and Hoebel, 2003), lending further evidence that natural rewarding behavior and drug addiction are mediated by common neural circuitry.

120 Male rodent sexual behavior consists of an appetitive approach phase followed by a consummatory phase in which the animal mounts and intromitts, culminating with ejaculation. Dopamine is released into the NAc upon presentation of the female, and remains elevated throughout the behavior (Pfaus et al., 1990; Pfaus and Phillips, 1991;

Damsma et al., 1992; Wenkstern et al., 1993). Behavioral studies suggest that ejaculation is the most rewarding component of male sexual behavior in rats (Agmo and Berenfeld,

1990; Lopez et al., 1999). It is unknown if ejaculation is the critical component of sex experience induced-sensitization in male rats. However, in the present study, sensitization to amphetamine was not observed in males that performed less than the requisite number of ejaculations to be included in the Experienced groups (Experiment 1, data not shown). Despite their low numbers of ejaculations, these animals had similar numbers of mounts and intromissions as the sensitized Experienced group (EA), suggesting that ejaculation plays a major role in sex experience-induced sensitization.

Alternatively, it is possible that sex experience induced sensitization is dependent on gonadal steroids, though unlikely. Specifically, testosterone (T) is released during sexual behavior (Hull et al., 2002), although it is unknown whether basal levels remain elevated in sexually experienced males. While castrated males have lower basal levels of

DA in the NAc (Alderson and Baum, 1981); castration does not affect DA levels in NAc in response to a sexually receptive female (Baum et al., 1986), suggesting testosterone- independent mechanisms of dopamine stimulation. In females, estrogen (E) stimulates

DA release in the NAc, but gender differences in acute locomotor responses and sensitization to psychostimulants persist in ovariectomized females, suggesting that there

121 are sexual dimorphisms in neural circuitry mediating these behaviors (Becker et al., 2001;

Hu and Becker, 2003).

The environment in which drugs are administered plays an important role in both the development and expression of sensitization (Robinson et al., 1998). In the present study, sex experience-induced sensitization to amphetamine was investigated in three different contexts. In the first experiment, sexually experienced animals gained their experience in the home cage, and were challenged with amphetamine in a novel environment. The challenge environment in this case was a truly novel, non-reward paired environment, as the animals had never been exposed to it until the challenge day.

In the second experiment, sexually experienced animals gained their experience in the home cage, and one group was challenged with amphetamine in a novel environment, as in the first experiment. Another group of animals was allowed to acclimate to the challenge environment and injection regimen for 48 hours prior to amphetamine challenge, and thus received amphetamine in a non-novel, non-reward paired environment. Robust sensitization to amphetamine was observed in both of these environments. Moreover, animals receiving amphetamine in the novel environment displayed a greater locomotor activity than animals receiving amphetamine in the non- novel environment. This is consistent with reports that a novel environment enhances the locomotor response to acute amphetamine administration (Badiani et al., 1995; Uslaner et al., 2001). However, in the present study, the environment did not influence the degree of sensitization, i.e., following amphetamine challenge, the ratio of the locomotor response of experienced animals to that of naive animals was the same in both

122 environments. This suggests that although novelty affects acute locomotor response to amphetamine, it does not affect expression of sensitization. In the third experiment, the sexually experienced animals were challenged with amphetamine in the same environment in which they gained their sexual experience (different from the home cage).

After a single sexual experience, experienced animals display increased locomotor activity after being placed in the mating environment compared to naïve animals.

However, this difference is not present when the animals are challenged with saline in the mating environment during the daytime. Thus, at the time of amphetamine challenge, the animals may not perceive the testing environment as a reward-paired environment.

Nonetheless, sensitization to amphetamine was observed at one day, one week, and one month following the last sexual experience. These results suggest that context may play a different role in sex experience-induced sensitization compared to drug-induced sensitization. Robinson and colleagues have reported that the expression of amphetamine sensitization is most robust when the challenge is given in a drug-paired environment that is distinct from the home environment (Badiani et al., 1995; Anagnostaras and Robinson,

1996; Badiani et al., 1997; Robinson et al., 1998). In particular, Anagnostaras and

Robinson observed that sensitization was not expressed when the animals were challenged in an environment other than that in which they received the sensitizing drug regimen (Anagnostaras and Robinson, 1996). In contrast, our data clearly demonstrates that sex experience-induces sensitization to amphetamine is expressed in a variety of contexts.

123 Amphetamine-induced sensitization is a long lasting phenomenon which can persist for many months (Paulson et al., 1991). Similarly, sex-experience-induced sensitization was observed one month following the last sexual encounter. Such long- term behavioral effects are believed to be mediated by functional alterations in the mesolimbic dopamine system (Kalivas and Stewart, 1991; Robinson and Berridge, 1993), and indeed, morphological changes have been observed in the NAc of amphetamine sensitized animals up to one month following their last drug exposure (Robinson and

Kolb, 1997). In the present study, neural activation was investigated in several components of the mesolimbic pathway that are by sexual behavior – the NAc, VTA, and mPFC (Balfour et al., 2003). Amphetamine induced Fos expression was observed in both the NAc and VTA. Interestingly, in the rostral NAc Core, sexually experienced animals challenged with saline had increased numbers of Fos-IR cells compared to sexually naïve animals, suggesting a basal change in neural activity. The NAc Core has been implicated in instrumental learning behaviors and may be involved in the development of drug sensitization (Di Chiara, 2002). In particular, drug-induced sensitization is associated with increased dopamine transmission in the NAc Core but not the Shell (Cadoni et al.,

2000), suggesting that these compartments play a distinct roles in this behavior.

However, sexually experienced animals did not exhibit increased Fos expression in the

NAc compared to naïve animals is response to amphetamine, whereas drug sensitized animals challenged with amphetamine do show an increase in Fos expression in the NAc compared to drug-naïve animals (Hedou et al., 2002). This suggests that the mechanisms governing sex experience-induced sensitization and drug-induced sensitization are not entirely the same.

124 Although previous studies have reported that amphetamine administration induces

Fos expression in the mPFC (Uslaner et al., 2001), amphetamine-induced Fos expression was not observed in the mPFC in the present study. However, Fos expression in the mPFC, NAc, and VTA of animals challenged with amphetamine in a novel environment was significantly greater than that of animals receiving amphetamine in a non-novel environment. This effect has been previously reported in the mPFC and NAc (Uslaner et al., 2001); the present study demonstrates that novelty enhances amphetamine-induced

Fos expression in the VTA as well.

In summary, the present study demonstrates that sexual behavior – a natural rewarding stimulus – can induce long-lasting sensitization to the locomotor effects of amphetamine, and this sensitization can be expressed in a variety of different contexts.

Furthermore, repeated sexual experience is associated with basal changes in neural activity in the NAc Core, and neural activation of the mesolimbic system in sex experience-sensitized animals follows a similar (but not identical) profile as is seen in drug-sensitized animals. These results suggest that stimulation of reward pathways by natural rewarding behaviors can cause functional changes in the mesolimbic dopamine system. Understanding how both natural behaviors and drugs of abuse activate these systems may provide further insight into the mechanisms of drug addiction.

125 REFERENCES

Agmo A (2002) Copulation-contingent aversive conditioning and sexual incentive motivation in male rats: evidence for a two-stage process of sexual behavior. Physiol Behav 77:425-435. Agmo A, Berenfeld R (1990) Reinforcing properties of ejaculation in the male rat: role of opioids and dopamine. Behav Neurosci 104:177-182. Alderson LM, Baum MJ (1981) Differential effects of gonadal steroids on dopamine metabolism in mesolimbic and nigro-striatal pathways of male rat brain. Brain Res 218:189-206. Anagnostaras SG, Robinson TE (1996) Sensitization to the psychomotor stimulant effects of amphetamine: modulation by associative learning. Behav Neurosci 110:1397- 1414. Avena NM, Hoebel BG (2003) Amphetamine-sensitized rats show sugar-induced hyperactivity (cross-sensitization) and sugar hyperphagia. Pharmacol Biochem Behav 74:635-639. Badiani A, Anagnostaras SG, Robinson TE (1995) The development of sensitization to the psychomotor stimulant effects of amphetamine is enhanced in a novel environment. Psychopharmacology (Berl) 117:443-452. Badiani A, Camp DM, Robinson TE (1997) Enduring enhancement of amphetamine sensitization by drug-associated environmental stimuli. J Pharmacol Exp Ther 282:787-794. Balfour ME, Yu L, Coolen LM (2003) Sexual behavior and a sex-associated environment cause activation of the mesolimbic system. Submitted. Bassareo V, Di Chiara G (1999) Differential responsiveness of dopamine transmission to food-stimuli in nucleus accumbens shell/core compartments. Neuroscience 89:637-641. Baum MJ, Melamed E, Globus M (1986) Dissociation of the effects of castration and testosterone replacement on sexual behavior and neural metabolism of dopamine in the male rat. Brain Res Bull 16:145-148. Becker JB, Molenda H, Hummer DL (2001) Gender differences in the behavioral responses to cocaine and amphetamine. Implications for mechanisms mediating gender differences in drug abuse. Ann N Y Acad Sci 937:172-187. Bradley KC, Meisel RL (2001) Sexual behavior induction of c-Fos in the nucleus accumbens and amphetamine-stimulated locomotor activity are sensitized by previous sexual experience in female Syrian hamsters. J Neurosci 21:2123-2130. Cadoni C, Solinas M, Di Chiara G (2000) Psychostimulant sensitization: differential changes in accumbal shell and core dopamine. Eur J Pharmacol 388:69-76. Cardinal RN, Parkinson JA, Hall J, Everitt BJ (2002) Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci Biobehav Rev 26:321-352. Castner SA, Xiao L, Becker JB (1993) Sex differences in striatal dopamine: in vivo microdialysis and behavioral studies. Brain Res 610:127-134. Damsma G, Pfaus JG, Wenkstern D, Phillips AG, Fibiger HC (1992) Sexual behavior increases dopamine transmission in the nucleus accumbens and striatum of male rats: comparison with novelty and locomotion. Behav Neurosci 106:181-191.

126 Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 137:75-114. Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274-5278. Fiorino DF, Phillips AG (1999) Facilitation of sexual behavior and enhanced dopamine efflux in the nucleus accumbens of male rats after D-amphetamine-induced behavioral sensitization. J Neurosci 19:456-463. Hajnal A, Norgren R (2001) Accumbens dopamine mechanisms in sucrose intake. Brain Res 904:76-84. Hedou G, Jongen-Relo AL, Murphy CA, Heidbreder CA, Feldon J (2002) Sensitized Fos expression in subterritories of the rat medial prefrontal cortex and nucleus accumbens following amphetamine sensitization as revealed by stereology. Brain Res 950:165-179. Hu M, Becker JB (2003) Effects of sex and estrogen on behavioral sensitization to cocaine in rats. J Neurosci 23:693-699. Kalivas PW, Stewart J (1991) Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res Brain Res Rev 16:223-244. Li Y, Kolb B, Robinson TE (2003) The Location of Persistent Amphetamine-Induced Changes in the Density of Dendritic Spines on Medium Spiny Neurons in the Nucleus Accumbens and Caudate-Putamen. Neuropsychopharmacology. Lopez HH, Olster DH, Ettenberg A (1999) Sexual motivation in the male rat: the role of primary incentives and copulatory experience. Horm Behav 36:176-185. Paulson PE, Camp DM, Robinson TE (1991) Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentrations during amphetamine withdrawal in rats. Psychopharmacology (Berl) 103:480-492. Peters RH (1983) Learned aversions to copulatory behaviors in male rats. Behav Neurosci 97:140-145. Pfaus JG, Phillips AG (1991) Role of dopamine in anticipatory and consummatory aspects of sexual behavior in the male rat. Behav Neurosci 105:727-743. Pfaus JG, Damsma G, Nomikos GG, Wenkstern DG, Blaha CD, Phillips AG, Fibiger HC (1990) Sexual behavior enhances central dopamine transmission in the male rat. Brain Res 530:345-348. Robertson GS, Pfaus JG, Atkinson LJ, Matsumura H, Phillips AG, Fibiger HC (1991) Sexual behavior increases c-fos expression in the forebrain of the male rat. Brain Res 564:352-357. Robinson DL, Phillips PE, Budygin EA, Trafton BJ, Garris PA, Wightman RM (2001) Sub-second changes in accumbal dopamine during sexual behavior in male rats. Neuroreport 12:2549-2552. Robinson TE, Becker JB (1986) Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis. Brain Res 396:157-198. Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Res Brain Res Rev 18:247-291.

127 Robinson TE, Kolb B (1997) Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine. J Neurosci 17:8491-8497. Robinson TE, Kolb B (1999) Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine. Eur J Neurosci 11:1598-1604. Robinson TE, Browman KE, Crombag HS, Badiani A (1998) Modulation of the induction or expression of psychostimulant sensitization by the circumstances surrounding drug administration. Neurosci Biobehav Rev 22:347-354. Saal D, Dong Y, Bonci A, Malenka RC (2003) Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37:577-582. Segal DS, Kuczenski R (1987) Individual differences in responsiveness to single and repeated amphetamine administration: behavioral characteristics and neurochemical correlates. J Pharmacol Exp Ther 242:917-926. Shaham Y, Puddicombe J, Stewart J (1997) Sexually arousing events and relapse to heroin-seeking in sexually experienced male rats. Physiol Behav 61:337-341. Thierry AM, Tassin JP, Blanc G, Glowinski J (1976) Selective activation of mesocortical DA system by stress. Nature 263:242-244. Uslaner J, Badiani A, Day HE, Watson SJ, Akil H, Robinson TE (2001) Environmental context modulates the ability of cocaine and amphetamine to induce c-fos mRNA expression in the neocortex, caudate nucleus, and nucleus accumbens. Brain Res 920:106-116. van Erp AM, Miczek KA (2000) Aggressive behavior, increased accumbal dopamine, and decreased cortical serotonin in rats. J Neurosci 20:9320-9325. Wenkstern D, Pfaus JG, Fibiger HC (1993) Dopamine transmission increases in the nucleus accumbens of male rats during their first exposure to sexually receptive female rats. Brain Res 618:41-46. Wise RA, Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469-492. Wyvell CL, Berridge KC (2001) Incentive sensitization by previous amphetamine exposure: increased cue-triggered "wanting" for sucrose reward. J Neurosci 21:7831-7840.

128 Figure 1. Schematic diagram of the zone map used to measure locomotor activity. A crossover is recorded each time the animal enters one of the shaded zones.

129 Figure 2. Schematic drawings illustrating the areas of analysis of Fos/TH-IR, indicated by the boxes, in the PFC, NAc, and VTA. A, Areas of analysis of Fos-IR for the IL, PL, and AC regions of the PFC (indicated by the boxes; 800 µm x 800 µm). B-D, Areas of analysis (indicated by boxes; 400 µm x 600 µm) of Fos-IR in the NAc Core and Shell at three rostral to caudal levels. E-H, Areas of analysis of Fos/TH-IR for the VTA at four rostral to caudal levels (E, Rostral, area of analysis = 1.8mm x 1.2 mm. F, Middle-1, area of analysis = 1.8 mm x 1.2 mm. G, Middle-2, area of analysis = 1.8 mm x 1.2 mm. H, Caudal, area of analysis = 1.6 mm x 1.0 mm). Abbreviations: AC, anterior cingulate area; PL, prelimbic area; IL, infralimbic area; C, NAc Core; S, NAc Shell; VL, lateral ventricle; ac, anterior commissure; cc, corpus callosum; ec, external capsule; CP, caudate putamen; AQ, cerebral aqueduct; PAG, periaquaductal gray; fr, fasiculus retroflexus; ml, medial lemniscus; cpd, cerebral peduncle; SN, substantia nigra; LG, lateral geniculate complex; SPFp, subparafasicular nucleus thalamus parvicellular part; VPM, ventral posteriomedial nucleus thalamus; PH, posterior hupothalamic nucleus; MRN, mesencephalic reticular nucleus; MGv, medial geniculate complex ventral part; APN, anterior pretectal nucleus; SC, superior colliculus; MM, medial mammillary nucleus.

130 Figure 3. Experiment 1: Locomotor response of sexually experienced and naïve animals in response to saline or amphetamine administered in a novel environment. Mean ± SEM of total numbers of crossovers over 90 minutes (A) and in 30-minute intervals (B-D). B, 0-30 minutes. C, 31-60 minutes. D, 61-90 minutes. E illustrates crossovers per 3-minute interval over 90 minutes. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

131 Figure 4. Experiment 2: Locomotor response of sexually experienced and naïve animals in response to saline or amphetamine administered in a novel or non-novel environment. Mean ± SEM of total numbers of crossovers over 90 minutes (A) and in 30-minute intervals (B-D). B, 0-30 minutes. C, 31-60 minutes. D, 61-90 minutes. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

132 Figure 5. Experiment 3: Locomotor response to the mating environment. Mean ± SEM of numbers of crossovers over 15-minutes following exposure to the mating environment. * = significant difference between Naïve and Experienced groups (p <0.05).

133 Figure 6. Experiment 3: Locomotor response of sexually experienced and naïve 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). Mean ± SEM of total numbers of crossovers over 90 minutes (A) and in 30-minute intervals (B-D). B, 0-30 minutes. C, 31-60 minutes. D, 61-90 minutes. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

134 Figure 7. Experiment 3: Locomotor response of sexually experienced and naïve 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). Mean ± SEM of total numbers of crossovers over 90 minutes (A) and in 30- minute intervals (B-D). B, 0-30 minutes. C, 31-60 minutes. D, 61-90 minutes. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

135 Figure 8. Experiment 3: Locomotor response of sexually experienced and naïve 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). Mean ± SEM of total numbers of crossovers over 90 minutes (A) and in 30- minute intervals (B-D). B, 0-30 minutes. C, 31-60 minutes. D, 61-90 minutes. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

136 Figure 9. Experiment 1: Numbers of Fos-IR cells in the NAc of sexually experienced and naïve animals in response to saline or amphetamine administered in a novel environment. Mean numbers ± SEM of Fos-IR cells in the Rostral NAc Core (A), Middle NAc Core (B), Caudal NAc Core (C) or Caudal NAc Shell (D). (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

137 Figure 10. Experiment 1: Numbers of Fos-IR cells in the VTA of sexually experienced and naïve animals in response to saline or amphetamine administered in a novel environment. Mean numbers ± SEM of Fos-IR cells in the Middle-1 (A) and Caudal (B) levels of the VTA. (NS, Naïve Saline; ES, Experienced Saline; NA, Naïve Amphetamine; EA, Experienced Amphetamine) The statistical relationship between the groups is indicated by lowercase letters; groups that share a common letter do not differ significantly.

138 Chapter 5

General discussion

139 GENERAL DISCUSSION

The overall goal of this thesis was to investigate the mechanisms by which natural motivated behaviors interact with the neural circuitry of motivation and reward. These studies have provided new insights into the endogenous activation of these pathways during male rodent sexual behavior, and the functional changes in these circuits that can arise as a result. More importantly, these studies have laid the groundwork for future studies that will further investigate the differences between and consequences of endogenous versus exogenous stimulation of the mesolimbic system.

The data presented in Chapter 2 demonstrated that the mesolimbic system is activated during sexual behavior. Specifically, this study showed that endogenous opioids are released into the VTA during sexual behavior, and that this release is correlated with the activation of dopaminergic cells in the VTA as well as neurons in the

NAc, which is the main target field of the VTA. Moreover, environmental cues associated with sexual behavior caused the same degree of activation of this system.

Interestingly, the majority of neurons activated in the VTA following either sexual behavior or exposure to sex-associated cues were non-dopaminergic. The study outlined in Chapter 3 explored the relationship between these sex-activated cells and other components of the limbic system. In particular, the ILA and PL regions of the mPFC were observed to have the most robust input to the VTA. Moreover, the majority of the sex-activated neurons in the VTA receive inputs from either the ILA or PL, and thus the mPFC is in an anatomical position to contribute to the activation of the VTA during

140 sexual behavior. In addition, the ILA and PL provide robust input to other brain areas associated with sexual behavior and reward, and significant portions of the sex-activated neurons receive contacts from ILA and PL. Thus, the mPFC may play a role in the activation of these areas as well. The data presented in Chapter 4 showed that the repeated endogenous activation of this system during sexual behavior causes the same types of functional changes that are observed after repeated exposure to drugs of abuse.

Specifically, sexually experienced animals displayed a long-lasting, sensitized locomotor response to amphetamine, suggesting that endogenous activation of the circuitry governing motivation and reward causes functional alternations in the responsiveness of these circuits to DA.

The studies presented in Chapter 2 confirm the hypotheses that during sexual behavior, endogenous opioids are released into the VTA and dopaminergic cells are activated. Interestingly, the same amount of activation was observed in sexually experienced animals following exposure to sex-associated environmental cues. This suggests that in experienced males, opioids are released during the appetitive phase of the behavior rather than the consummatory phase. These results are in agreement with pharmacological studies demonstrating that opioid antagonists delivered into the VTA of sexually experienced males impair performance on tests of sexual motivation, but have no effect on sexual performance during the consummatory phase of the behavior (van

Furth and van Ree, 1996). In addition, exposure to either an estrous female behind a mesh screen or soiled bedding from the cage of an estrous female stimulates DA release in the NAc of sexually experienced males (Pfaus et al., 1990; Damsma et al., 1992),

141 indicating that sex-associated olfactory cues can activate the mesolimbic system. In the present study, conditioned activation of the mesolimbic DA system was elicited by pairing the sexual behavior with a specific environment, which was cleaned thoroughly between each trial in order to remove any odors that may be associated with copulation.

Therefore this study marks the first demonstration of sex-induced conditioned activation of the mesolimbic system in response to spatial and tactile cues, in the absence of olfactory cues. This distinction is important to consider when making comparisons between human and rodent behavior, because rodents have a much more developed olfactory system than humans, and olfaction in the rat plays an important role in learning, memory and other complex behaviors.

Although it appears that the DA system is activated during the appetitive phase in sexually experienced males, the present study does not allow one to determine if this is also the case with sexually naïve males. Microdialysis studies showed that DA is released in the NAc of naïve males when an estrous females is introduced, but these studies do not make the distinction between appetitive and consummatory behavior

(Wenkstern et al., 1993). In electrophysiological studies performed by Shultz et al.

(2001) in primates, DA neurons in the VTA fire in response to the first presentation of a palatable food reward only when the animal is allowed to touch or consume it. In contrast, animals trained to associate a conditioned stimulus with the presentation of the food reward exhibit DA neuron activation only in response to the conditioned stimulus; no DA firing is observed during the consumption of the reward. Thus Shultz and coworkers hypothesized that in this paradigm, DA acts as a predictor of reward.

142 Because the c-Fos gene product can be detected up to 90 minutes following the initial stimulus, the present study was unable to determine exactly when DA neurons in the VTA were activated. However, other markers of neuronal activation have much shorter windows of activation and thus allow for more a precise determination of the timing of the activating stimulus. One such marker is phosphorylation of the MAP kinases ERK1 and 2, which are rapidly phosphorylated and subsequently dephosphorylated following neuronal activation. Immunostaining for the phosphorylated form (pERK) can detect neuronal activation with a temporal window of 5-10 minutes

(Valjent et al., 2000). Therefore, it would be interesting to repeat the experiment in

Chapter 3 using pERK as an alternative marker for neuronal activation. If DA acts as a predictor for sexual reward, one would expect that naïve animals would display the highest levels pERK following ejaculation, whereas experienced animals would display the highest levels of pERK upon exposure to the test environment, but not following ejaculation.

Surprisingly, the majority of sex-activated neurons in the VTA are non- dopaminergic. While the DA producing cells have received much attention in the literature, there is much less known about the non-dopaminergic population. Most of these non-dopaminergic neurons are believed to be GABAergic (Tzschentke, 2001).

While some of these neurons are likely GABAergic interneurons, a considerable number of them project to the same terminal fields as the DA population. In particular, 15% of the VTA-NAc projection and ~70% of the VTA-PFC projection is non-dopaminergic

143 (Swanson, 1982). Thus, these cells are anatomically positioned such that they may play an important role in the circuitry mediating motivation and reward. In addition, while sexual behavior causes robust activation of this population, systemic morphine administration does not (unpublished observation). Therefore, these cells may play different roles in natural reward compared to morphine-induced reward, and should be further investigated.

The phenotype of these non-dopaminergic neurons is currently unclear, but electrophysiological evidence suggests that they contain ionotropic glutamate receptors, as AMPA, NMDA and KA receptor agonists cause depolarization of non-dopaminergic cells in VTA slice preparations (Wang and French, 1995). Moreover, the majority of the mPFC input to the VTA synapses on non-dopaminergic neurons (Sesack and Pickel,

1992; Smith et al., 1996). Therefore, a major motivation for the studies in Chapter 4 was to determine the source of activation of this non-dopaminergic population. While the overall goal of Chapter 4 was to determine the relationship of sex-activated neurons throughout the brain with other components of the limbic system, a specific emphasis was placed on the mPFC-VTA projection, for the reasons outlined above. To determine the relative significance of inputs from different injection sites, a novel method of quantification was employed. This technique combined immunostaining Fos, the anterograde tracer BDA, and the neuronal cytoplasmic marker NeuN, which allowed comparisons of the numbers of neurons receiving putative synaptic contacts from a particular injection site relative to the number of sex-activated neurons in a given brain region. These studies demonstrate that the ILA and PL regions of the mPFC provide the

144 most robust input to the VTA. In particular, the majority of sex-activated cells in the

VTA received putative contacts from either the ILA or PL, and the majority of neurons that received contacts from the ILA or PL were activated during sexual behavior. Thus the PFC is in an anatomical position to provide activating input to the VTA during sexual behavior. In addition, the present study confirms that the mPFC-VTA projection is glutamatergic, and moreover, the majority of sex-activated cells in the VTA contain the

NMDA receptor. Taken together, these data suggest that the mPFC may contribute to the activation of the VTA during sexual behavior via glutamate release. However, this data is highly correlative, and pharmacological and lesion studies are needed to determine definitively if glutamatergic input from the mPFC is responsible for the activation of these neurons.

The studies described in Chapters 2 and 3 provide insight into the mechanism by which sexual behavior activates the neural circuitry governing motivation and reward. A hypothetical circuitry model is presented here (Figure 1), which attempts to incorporate the new data presented in this thesis with the current literature.

The first set of studies demonstrated that sex-associated cues cause opioid release and DA neuron activation in the VTA. Activation of VTA DA neurons results in DA release in the NAc, and the subsequent motivated motor response of approach behavior.

At the same time, sensory information is processed and sent to the mPFC, which in turn stimulates the VTA as well as other areas important for the consummatory aspects of sexual behavior (MPN, BST, etc). Previous studies have shown that the PFC-VTA input

145 obeys a strict target specificity, such that PFC afferents project to DA neurons which project back to the PFC, but not those that project to the NAc. Conversely. PFC afferents project to GABA cells that project to the NAc, but not those that project back to the PFC

(Carr and Sesack, 2000a). Both the GABAergic projection to the NAc and the dopaminergic projection to the mPFC are inhibitory. Thus. PFC activation may result in a feed-forward inhibition of the system, in essence providing a “brake” on the system once motivated motor behavior has begun.

In humans, the PFC is activated during episodes of cue-conditioned cocaine craving (Childress et al., 1988), and may be involved in the activation of reward systems by conditioned stimuli. Everitt has compared drug addiction to a “PFC syndrome” in which long-term drug use renders the PFC incapable of inhibiting inappropriate conditioned responses (Cardinal et al., 2002; Everitt, 2002). In support of this hypothesis, in rodent models, PFC-lesioned animals are unable to stop performing an operant task, even when the conditioned stimulus has been paired with an aversive injection of lithium chloride. This is analogous to drug addicts who compulsively seek drugs despite the adverse consequences. The role of the PFC in natural reward and conditioning remains unclear. In the studies described in Chapter 2, robust conditioned

Fos expression was observed in the VTA in response to sex-associated cues. Because the mPFC provides such a strong input to the sex-activated neurons in the VTA, future studies will investigate the effects of lesions and pharmacological manipulations on this conditioned Fos response.

146 The studies presented in Chapters 2 and 3 strongly suggest that mesolimbic circuits are activated during sexual behavior. Therefore, the goal of the final set of studies was to determine if repeated endogenous activation of these circuits results in similar functional changes as are observed with repeated drug administration. Indeed, sexually experienced animals displayed an increased locomotor response to amphetamine compared to naïve animals. Moreover, this effect persisted for up to one month, suggesting that endogenous activation of reward systems results in a long-lasting change in the sensitivity of the DA system. The precise mechanism of this sex experience induced sensitization is unclear at this time. However, the PFC-VTA projection appears to be a critical component of drug-induced behavioral sensitization. Both PFC lesions and intra-VTA administration of glutamate receptor antagonists block the development of amphetamine or cocaine induced sensitization (Wolf et al., 1994; Kim and Vezina, 1998;

Li et al., 1999; Vezina and Queen, 2000). Similar manipulations are needed to determine if sexual behavior exerts its effects via a similar pathway.

Interestingly, environment appears to play less of a role in sex-induced sensitization than in drug-induced sensitization. Robinson and colleagues have reported that the expression of amphetamine sensitization is most robust when the challenge is given in a drug-paired environment that is distinct from the home environment, and is not expressed when the animals are challenged in an environment other than that in which they received the sensitizing drug regimen (Badiani et al., 1995; Anagnostaras and

Robinson, 1996; Badiani et al., 1997; Robinson et al., 1998). In contrast, sex-induced sensitization is expressed regardless of the environmental context. Thus, conditioned

147 environmental stimuli appear to be less important in the expression of sex-induced sensitization. The significance of this is currently not known, but this suggests that subtle differences exist in the mechanisms by which natural reward and drugs of abuse affect the mesolimbic circuitry.

The Fos studies performed in Chapter 4 further demonstrate that repeated exposure to a natural rewarding stimulus causes changes in neural activity in the mesolimbic system. In particular, sexually experienced animals challenged with saline had increased numbers of Fos-IR cells compared to sexually naïve animals in the rostral portion of the NAc Core, suggesting a basal change in neural activity. The NAc Core has been implicated in instrumental learning behaviors and may be involved in the development of drug sensitization (Di Chiara, 2002). In particular, drug-induced sensitization is associated with increased dopamine transmission in the NAc Core but not the Shell (Cadoni et al., 2000), suggesting that these compartments play a distinct roles in this behavior. However, when challenged with amphetamine, sexually experienced animals did not exhibit increased Fos expression in the NAc compared to naïve animals, whereas drug sensitized animals challenged with amphetamine do show an increase in

Fos expression in the NAc compared to drug-naïve animals (Hedou et al., 2002). This provides further evidence that the mechanisms governing sex-induced sensitization and drug-induced sensitization are not entirely the same.

Overall, this study demonstrates that a sexual behavior activates the mesolimbic reward circuitry that has been implicated in drug addiction. Furthermore, endogenous

148 activation of these systems leads to similar long-term changes in their function.

However, while many similarities exist between natural reward and drugs of abuse, subtle difference must exist to explain why repeated drug exposure use to a pathological state of addiction while repeated sexual behavior does not. Sensitization has been proposed as a model of drug craving, in which hyperresponsiveness of the dopamine system results in increased “wanting” or craving of the drug (Robinson and Berridge, 1993). In the present experiments the sensitization paradigm was used simply as a marker for increased sensitivity of the DA system. There is no evidence that sexual behavior leads to craving or addiction in rodents. To the contrary, while both stress and drug priming trigger relapse in rodent self-administration models, sexual behavior fails to do so (Shaham et al., 1997). Sexual addiction in humans is a more controversial topic, and there is disagreement as to whether it is rightly classified as an addictive, obsessive-compulsive, or impulse-control disorder (Gold and Heffner, 1998). If sex addiction is a true addictive disorder, it is appears to be less addictive than drugs of abuse. Repeated self- administration of drugs can readily lead to drug dependence and addiction. In contrast, of all the individuals who regularly engage in sexual behavior, only a small portion develops a “sex-addiction.”

The techniques utilized in this study may not be sensitive enough to detect more subtle differences between endogenous and exogenous reward pathways. Therefore, this work has prompted a detailed comparison between sex-induced and drug-induced activation of the mesolimbic reward system. These ongoing studies utilize microarray technology in order to screen for changes in the expression of thousands of genes

149 simultaneously. Preliminary data suggests that many genes that have been implicated in drug addiction are also involved in sexual reward. The challenge will be to determine how these genes contribute to the functional changes and neuroadaptations that occur during both normal behavior and drug addiction. Comparative analyses such as this may one day reveal targets for the treatment of addiction.

150 REFERENCES

Agmo A (2002) Copulation-contingent aversive conditioning and sexual incentive motivation in male rats: evidence for a two-stage process of sexual behavior. Physiol Behav 77:425-435. Agmo A, Berenfeld R (1990) Reinforcing properties of ejaculation in the male rat: role of opioids and dopamine. Behav Neurosci 104:177-182. Agmo A, Gomez M (1993) Sexual reinforcement is blocked by infusion of naloxone into the medial preoptic area. Behav Neurosci 107:812-818. Agmo A, Villalpando A, Picker Z, Fernandez H (1995) Lesions of the medial prefrontal cortex and sexual behavior in the male rat. Brain Res 696:177-186. Alderson LM, Baum MJ (1981) Differential effects of gonadal steroids on dopamine metabolism in mesolimbic and nigro-striatal pathways of male rat brain. Brain Res 218:189-206. Anagnostaras SG, Robinson TE (1996) Sensitization to the psychomotor stimulant effects of amphetamine: modulation by associative learning. Behav Neurosci 110:1397- 1414. APA (1994) Diagnostic and Statistical Manual of Mental Disorders, 4th Edition. Washington, DC: APA Press. Avena NM, Hoebel BG (2003) Amphetamine-sensitized rats show sugar-induced hyperactivity (cross-sensitization) and sugar hyperphagia. Pharmacol Biochem Behav 74:635-639. Badiani A, Anagnostaras SG, Robinson TE (1995) The development of sensitization to the psychomotor stimulant effects of amphetamine is enhanced in a novel environment. Psychopharmacology (Berl) 117:443-452. Badiani A, Camp DM, Robinson TE (1997) Enduring enhancement of amphetamine sensitization by drug-associated environmental stimuli. J Pharmacol Exp Ther 282:787-794. Baker DA, Khroyan TV, O'Dell LE, Fuchs RA, Neisewander JL (1996) Differential effects of intra-accumbens sulpiride on cocaine-induced locomotion and conditioned place preference. J Pharmacol Exp Ther 279:392-401. Balfour ME, Yu L, Coolen LM (2003) Sexual behavior and a sex-associated environment cause activation of the mesolimbic system. Submitted. Band LC, Hull EM (1990) Morphine and dynorphin(1-13) microinjected into the medial preoptic area and nucleus accumbens: effects on sexual behavior in male rats. Brain Res 524:77-84. Bassareo V, Di Chiara G (1999) Differential responsiveness of dopamine transmission to food-stimuli in nucleus accumbens shell/core compartments. Neuroscience 89:637-641. Baum MJ, Melamed E, Globus M (1986) Dissociation of the effects of castration and testosterone replacement on sexual behavior and neural metabolism of dopamine in the male rat. Brain Res Bull 16:145-148. Becker JB, Molenda H, Hummer DL (2001) Gender differences in the behavioral responses to cocaine and amphetamine. Implications for mechanisms mediating gender differences in drug abuse. Ann N Y Acad Sci 937:172-187.

151 Berendse HW, Groenewegen HJ, Lohman AH (1992a) Compartmental distribution of ventral striatal neurons projecting to the mesencephalon in the rat. J Neurosci 12:2079-2103. Berendse HW, Galis-de Graaf Y, Groenewegen HJ (1992b) Topographical organization and relationship with ventral striatal compartments of prefrontal corticostriatal projections in the rat. J Comp Neurol 316:314-347. Berke JD, Hyman SE (2000) Addiction, dopamine, and the molecular mechanisms of memory. Neuron 25:515-532. Berridge KC, Robinson TE (1998) What is the role of dopamine in reward: hedonic impact, reward learning, or incentive salience? Brain Res Brain Res Rev 28:309- 369. Bocher M, Chisin R, Parag Y, Freedman N, Meir Weil Y, Lester H, Mishani E, Bonne O (2001) Cerebral activation associated with sexual arousal in response to a pornographic clip: A 15O-H2O PET study in heterosexual men. Neuroimage 14:105-117. Bontempi B, Sharp FR (1997) Systemic morphine-induced Fos protein in the rat striatum and nucleus accumbens is regulated by mu opioid receptors in the substantia nigra and ventral tegmental area. J Neurosci 17:8596-8612. Bouyer JJ, Park DH, Joh TH, Pickel VM (1984) Chemical and structural analysis of the relation between cortical inputs and tyrosine hydroxylase-containing terminals in rat neostriatum. Brain Res 302:267-275. Bradley KC, Meisel RL (2001) Sexual behavior induction of c-Fos in the nucleus accumbens and amphetamine-stimulated locomotor activity are sensitized by previous sexual experience in female Syrian hamsters. J Neurosci 21:2123-2130. Brandt HM, Apkarian AV (1992) Biotin-dextran: a sensitive anterograde tracer for neuroanatomic studies in rat and monkey. J Neurosci Methods 45:35-40. Broekkamp CL, Phillips AG, Cools AR (1979) Stimulant effects of enkephalin microinjection into the dopaminergic A10 area. Nature 278:560-562. Cadoni C, Solinas M, Di Chiara G (2000) Psychostimulant sensitization: differential changes in accumbal shell and core dopamine. Eur J Pharmacol 388:69-76. Cardinal RN, Parkinson JA, Hall J, Everitt BJ (2002) Emotion and motivation: the role of the amygdala, ventral striatum, and prefrontal cortex. Neurosci Biobehav Rev 26:321-352. Carr DB, Sesack SR (2000a) Projections from the rat prefrontal cortex to the ventral tegmental area: target specificity in the synaptic associations with mesoaccumbens and mesocortical neurons. J Neurosci 20:3864-3873. Carr DB, Sesack SR (2000b) GABA-containing neurons in the rat ventral tegmental area project to the prefrontal cortex. Synapse 38:114-123. Castner SA, Xiao L, Becker JB (1993) Sex differences in striatal dopamine: in vivo microdialysis and behavioral studies. Brain Res 610:127-134. Childress A, Ehrman R, McLellan AT, O'Brien C (1988) Conditioned craving and arousal in cocaine addiction: a preliminary report. NIDA Res Monogr 81:74-80. Coolen LM, Peters HJ, Veening JG (1996) Fos immunoreactivity in the rat brain following consummatory elements of sexual behavior: a sex comparison. Brain Res 738:67-82.

152 Coolen LM, Peters HJ, Veening JG (1997a) Distribution of Fos immunoreactivity following mating versus anogenital investigation in the male rat brain. Neuroscience 77:1151-1161. Coolen LM, Olivier B, Peters HJ, Veening JG (1997b) Demonstration of ejaculation- induced neural activity in the male rat brain using 5-HT1A agonist 8-OH-DPAT. Physiol Behav 62:881-891. Coolen LM, Fitzgerald ME, Wells AB, Yu L, Lehman MN (2003) Activation of mu opioid receptors in the medial preoptic area following copulation in male rats. Submitted. Cunningham ST, Kelley AE (1992) Evidence for opiate-dopamine cross-sensitization in nucleus accumbens: studies of conditioned reward. Brain Res Bull 29:675-680. Cunningham ST, Finn M, Kelley AE (1997) Sensitization of the locomotor response to psychostimulants after repeated opiate exposure: role of the nucleus accumbens. Neuropsychopharmacology 16:147-155. Curran T, Morgan JI (1995) Fos: an immediate-early transcription factor in neurons. J Neurobiol 26:403-412. Damsma G, Pfaus JG, Wenkstern D, Phillips AG, Fibiger HC (1992) Sexual behavior increases dopamine transmission in the nucleus accumbens and striatum of male rats: comparison with novelty and locomotion. Behav Neurosci 106:181-191. Dauge V, Kalivas PW, Duffy T, Roques BP (1992) Effect of inhibiting enkephalin catabolism in the VTA on motor activity and extracellular dopamine. Brain Res 599:209-214. Davis JF, Loos M, Coolen LM (2003) Lesions of the medial prefrontal cortex do not disrupt sexual behavior in male rats. In: Society for Behavioral Neurendocrinology, p 45. Cincinanti, OH: Hormones and Behavior. Devine DP, Leone P, Pocock D, Wise RA (1993) Differential involvement of ventral tegmental mu, delta and kappa opioid receptors in modulation of basal mesolimbic dopamine release: in vivo microdialysis studies. J Pharmacol Exp Ther 266:1236-1246. Di Chiara G (2002) Nucleus accumbens shell and core dopamine: differential role in behavior and addiction. Behav Brain Res 137:75-114. Di Chiara G, Imperato A (1988) Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proc Natl Acad Sci U S A 85:5274-5278. Dilts RP, Kalivas PW (1990) Autoradiographic localization of delta opioid receptors within the mesocorticolimbic dopamine system using radioiodinated [2-D- penicillamine, 5-D-penicillamine]enkephalin (125I-DPDPE). Synapse 6:121-132. Eckersell CB, Popper P, Micevych PE (1998) Estrogen-induced alteration of mu-opioid receptor immunoreactivity in the medial preoptic nucleus and medial amygdala. J Neurosci 18:3967-3976. Everitt BJ (2002) Neural and Psychological Mechanisms Underlying Drug Addiction: The Impact of Learning and Prospects for Treatment. In: Society for Neuroscience 32ns Annual Meeting. Orlando, FL. Everitt BJ, Stacey P (1987) Studies of instrumental behavior with sexual reinforcement in male rats (Rattus norvegicus): II. Effects of preoptic area lesions, castration, and testosterone. J Comp Psychol 101:407-419.

153 Everitt BJ, Cador M, Robbins TW (1989) Interactions between the amygdala and ventral striatum in stimulus-reward associations: studies using a second-order schedule of sexual reinforcement. Neuroscience 30:63-75. Everitt BJ, Fray P, Kostarczyk E, Taylor S, Stacey P (1987) Studies of instrumental behavior with sexual reinforcement in male rats (Rattus norvegicus): I. Control by brief visual stimuli paired with a receptive female. J Comp Psychol 101:395-406. Fallon JH, Leslie FM (1986) Distribution of dynorphin and enkephalin peptides in the rat brain. J Comp Neurol 249:293-336. Feldman RS, Meyer JS, Quenzer LF (1997) Principles of Neuropsychopharmacology. Sunderland, Massachusetts: Sinauer Associates. Fernandez-Guasti A, Omana-Zapata I, Lujan M, Condes-Lara M (1994) Actions of sciatic nerve ligature on sexual behavior of sexually experienced and inexperienced male rats: effects of frontal pole decortication. Physiol Behav 55:577-581. Fiorino DF, Phillips AG (1999) Facilitation of sexual behavior and enhanced dopamine efflux in the nucleus accumbens of male rats after D-amphetamine-induced behavioral sensitization. J Neurosci 19:456-463. Freund TF, Powell JF, Smith AD (1984) Tyrosine hydroxylase-immunoreactive boutons in synaptic contact with identified striatonigral neurons, with particular reference to dendritic spines. Neuroscience 13:1189-1215. Garzon M, Pickel VM (2001) Plasmalemmal mu-opioid receptor distribution mainly in nondopaminergic neurons in the rat ventral tegmental area. Synapse 41:311-328. Glickman SE, Schiff BB (1967) A biological theory of reinforcement. Psychol Rev 74:81-109. Gold SN, Heffner CL (1998) Sexual addiction: many conceptions, minimal data. Clin Psychol Rev 18:367-381. Greenwell TN, Zangen A, Martin-Schild S, Wise RA, Zadina JE (2002) Endomorphin-1 and -2 immunoreactive cells in the hypothalamus are labeled by fluoro-gold injections to the ventral tegmental area. In: International Narcotics Research Conference, p 26. Pacific Grove, CA. Hajnal A, Norgren R (2001) Accumbens dopamine mechanisms in sucrose intake. Brain Res 904:76-84. Hedou G, Jongen-Relo AL, Murphy CA, Heidbreder CA, Feldon J (2002) Sensitized Fos expression in subterritories of the rat medial prefrontal cortex and nucleus accumbens following amphetamine sensitization as revealed by stereology. Brain Res 950:165-179. Heimer L, Zahm DS, Churchill L, Kalivas PW, Wohltmann C (1991) Specificity in the projection patterns of accumbal core and shell in the rat. Neuroscience 41:89-125. Heimer L, Alheid GF, de Olmos JS, Groenewegen HJ, Haber SN, Harlan RE, Zahm DS (1997) The accumbens: beyond the core-shell dichotomy. J Neuropsychiatry Clin Neurosci 9:354-381. Hendricks SE, Scheetz HA (1973) Interaction of hypothalamic structures in the mediation of male sexual behavior. Physiol Behav 10:711-716. Hernandez-Gonzalez M, Guevara MA, Cervantes M, Morali G, Corsi-Cabrera M (1998) Characteristic frequency bands of the cortico-frontal EEG during the sexual

154 interaction of the male rat as a result of factorial analysis. J Physiol Paris 92:43- 50. Hiroi N, White NM (1991) The amphetamine conditioned place preference: differential involvement of dopamine receptor subtypes and two dopaminergic terminal areas. Brain Res 552:141-152. Hu M, Becker JB (2003) Effects of sex and estrogen on behavioral sensitization to cocaine in rats. J Neurosci 23:693-699. Hull EM, Meisel RL, Sachs BD (2002) Male Sexual Behavior. In: Hormones, Brain and Behavior (Pfaff DW, Arnold AP, Etgen AM, Fahrbach SE, Rubin RT, eds), pp 1- 138. San Diego, CA: Elsevier Science (USA). Hull EM, Bazzett TJ, Warner RK, Eaton RC, Thompson JT (1990) Dopamine receptors in the ventral tegmental area modulate male sexual behavior in rats. Brain Res 512:1-6. Hyman SE, Malenka RC (2001) Addiction and the brain: the neurobiology of compulsion and its persistence. Nat Rev Neurosci 2:695-703. Ikemoto S, Kohl RR, McBride WJ (1997) GABA(A) receptor blockade in the anterior ventral tegmental area increases extracellular levels of dopamine in the nucleus accumbens of rats. J Neurochem 69:137-143. Jackson ME, Frost AS, Moghaddam B (2001) Stimulation of prefrontal cortex at physiologically relevant frequencies inhibits dopamine release in the nucleus accumbens. J Neurochem 78:920-923. Johnson RP, Sar M, Stumpf WE (1980) A topographic localization of enkephalin on the dopamine neurons of the rat substantia nigra and ventral tegmental area demonstrated by combined histofluorescence-immunocytochemistry. Brain Res 194:566-571. Johnson SW, North RA (1992) Opioids excite dopamine neurons by hyperpolarization of local interneurons. J Neurosci 12:483-488. Kalivas PW, Stewart J (1991) Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res Brain Res Rev 16:223-244. Karler R, Calder LD, Thai DK, Bedingfield JB (1998a) The role of dopamine in the mouse frontal cortex: a new hypothesis of behavioral sensitization to amphetamine and cocaine. Pharmacol Biochem Behav 61:435-443. Karler R, Calder LD, Thai DK, Bedingfield JB (1998b) The role of dopamine and GABA in the frontal cortex of mice in modulating a motor-stimulant effect of amphetamine and cocaine. Pharmacol Biochem Behav 60:237-244. Keith DE, Anton B, Murray SR, Zaki PA, Chu PC, Lissin DV, Monteillet-Agius G, Stewart PL, Evans CJ, von Zastrow M (1998) mu-Opioid receptor internalization: opiate drugs have differential effects on a conserved endocytic mechanism in vitro and in the mammalian brain. Mol Pharmacol 53:377-384. Kelley AE (1999) Functional specificity of ventral striatal compartments in appetitive behaviors. Ann N Y Acad Sci 877:71-90. Kim JH, Vezina P (1998) Metabotropic glutamate receptors are necessary for sensitization by amphetamine. Neuroreport 9:403-406. Kiyatkin EA, Rebec GV (1997) Activity of presumed dopamine neurons in the ventral tegmental area during heroin self-administration. Neuroreport 8:2581-2585.

155 Klitenick MA, DeWitte P, Kalivas PW (1992) Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study. J Neurosci 12:2623-2632. Lende DH, Smith EO (2002) Evolution meets biopsychosociality: an analysis of addictive behavior. Addiction 97:447-458. Leone P, Pocock D, Wise RA (1991) Morphine-dopamine interaction: ventral tegmental morphine increases nucleus accumbens dopamine release. Pharmacol Biochem Behav 39:469-472. Li Y, Kolb B, Robinson TE (2003) The Location of Persistent Amphetamine-Induced Changes in the Density of Dendritic Spines on Medium Spiny Neurons in the Nucleus Accumbens and Caudate-Putamen. Neuropsychopharmacology. Li Y, Hu XT, Berney TG, Vartanian AJ, Stine CD, Wolf ME, White FJ (1999) Both glutamate receptor antagonists and prefrontal cortex lesions prevent induction of cocaine sensitization and associated neuroadaptations. Synapse 34:169-180. Lopez HH, Olster DH, Ettenberg A (1999) Sexual motivation in the male rat: the role of primary incentives and copulatory experience. Horm Behav 36:176-185. Lyness WH, Friedle NM, Moore KE (1979) Destruction of dopaminergic nerve terminals in nucleus accumbens: effect on d-amphetamine self-administration. Pharmacol Biochem Behav 11:553-556. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1987) Autoradiographic differentiation of mu, delta, and kappa opioid receptors in the rat forebrain and midbrain. J Neurosci 7:2445-2464. Mansour A, Khachaturian H, Lewis ME, Akil H, Watson SJ (1988) Anatomy of CNS opioid receptors. Trends Neurosci 11:308-314. Martinez I, Paredes RG (2001) Only self-paced mating is rewarding in rats of both sexes. Horm Behav 40:510-517. Matthews RT, German DC (1984) Electrophysiological evidence for excitation of rat ventral tegmental area dopamine neurons by morphine. Neuroscience 11:617-625. Meisel RL, Joppa MA, Rowe RK (1996) Dopamine receptor antagonists attenuate conditioned place preference following sexual behavior in female Syrian hamsters. Eur J Pharmacol 309:21-24. Michal EK (1973) Effects of limbic lesions on behavior sequences and courtship behavior of male rats (Rattus norvegicus). Behaviour 44:264-285. Mitchell JB, Stewart J (1990) Facilitation of sexual behaviors in the male rat associated with intra-VTA injections of opiates. Pharmacol Biochem Behav 35:643-650. Mitchell JB, Gratton A (1992) Partial dopamine depletion of the prefrontal cortex leads to enhanced mesolimbic dopamine release elicited by repeated exposure to naturally reinforcing stimuli. J Neurosci 12:3609-3618. Mogenson GJ, Jones DL, Yim CY (1980) From motivation to action: functional interface between the limbic system and the motor system. Prog Neurobiol 14:69-97. Moses J, Loucks JA, Watson HL, Matuszewich L, Hull EM (1995) Dopaminergic drugs in the medial preoptic area and nucleus accumbens: effects on motor activity, sexual motivation, and sexual performance. Pharmacol Biochem Behav 51:681- 686. Nestler EJ (2001) Molecular basis of long-term plasticity underlying addiction. Nat Rev Neurosci 2:119-128.

156 Paredes RG, Alonso A (1997) Sexual behavior regulated (paced) by the female induces conditioned place preference. Behav Neurosci 111:123-128. Paulson PE, Camp DM, Robinson TE (1991) Time course of transient behavioral depression and persistent behavioral sensitization in relation to regional brain monoamine concentrations during amphetamine withdrawal in rats. Psychopharmacology (Berl) 103:480-492. Peters RH (1983) Learned aversions to copulatory behaviors in male rats. Behav Neurosci 97:140-145. Pettit HO, Ettenberg A, Bloom FE, Koob GF (1984) Destruction of dopamine in the nucleus accumbens selectively attenuates cocaine but not heroin self- administration in rats. Psychopharmacology (Berl) 84:167-173. Pfaus JG, Phillips AG (1989) Differential effects of dopamine receptor antagonists on the sexual behavior of male rats. Psychopharmacology (Berl) 98:363-368. Pfaus JG, Phillips AG (1991) Role of dopamine in anticipatory and consummatory aspects of sexual behavior in the male rat. Behav Neurosci 105:727-743. Pfaus JG, Damsma G, Nomikos GG, Wenkstern DG, Blaha CD, Phillips AG, Fibiger HC (1990) Sexual behavior enhances central dopamine transmission in the male rat. Brain Res 530:345-348. Roberts DC, Koob GF (1982) Disruption of cocaine self-administration following 6- hydroxydopamine lesions of the ventral tegmental area in rats. Pharmacol Biochem Behav 17:901-904. Roberts DC, Corcoran ME, Fibiger HC (1977) On the role of ascending catecholaminergic systems in intravenous self-administration of cocaine. Pharmacol Biochem Behav 6:615-620. Roberts DC, Koob GF, Klonoff P, Fibiger HC (1980) Extinction and recovery of cocaine self-administration following 6-hydroxydopamine lesions of the nucleus accumbens. Pharmacol Biochem Behav 12:781-787. Robertson GS, Pfaus JG, Atkinson LJ, Matsumura H, Phillips AG, Fibiger HC (1991) Sexual behavior increases c-fos expression in the forebrain of the male rat. Brain Res 564:352-357. Robinson DL, Phillips PE, Budygin EA, Trafton BJ, Garris PA, Wightman RM (2001a) Sub-second changes in accumbal dopamine during sexual behavior in male rats. Neuroreport 12:2549-2552. Robinson TE, Becker JB (1986) Enduring changes in brain and behavior produced by chronic amphetamine administration: a review and evaluation of animal models of amphetamine psychosis. Brain Res 396:157-198. Robinson TE, Berridge KC (1993) The neural basis of drug craving: an incentive- sensitization theory of addiction. Brain Res Brain Res Rev 18:247-291. Robinson TE, Kolb B (1997) Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine. J Neurosci 17:8491-8497. Robinson TE, Kolb B (1999a) Alterations in the morphology of dendrites and dendritic spines in the nucleus accumbens and prefrontal cortex following repeated treatment with amphetamine or cocaine. Eur J Neurosci 11:1598-1604. Robinson TE, Kolb B (1999b) Morphine alters the structure of neurons in the nucleus accumbens and neocortex of rats. Synapse 33:160-162.

157 Robinson TE, Browman KE, Crombag HS, Badiani A (1998) Modulation of the induction or expression of psychostimulant sensitization by the circumstances surrounding drug administration. Neurosci Biobehav Rev 22:347-354. Robinson TE, Gorny G, Mitton E, Kolb B (2001b) Cocaine self-administration alters the morphology of dendrites and dendritic spines in the nucleus accumbens and neocortex. Synapse 39:257-266. Rossetti ZL, Marcangione C, Wise RA (1998) Increase of extracellular glutamate and expression of Fos-like immunoreactivity in the ventral tegmental area in response to electrical stimulation of the prefrontal cortex. J Neurochem 70:1503-1512. Saal D, Dong Y, Bonci A, Malenka RC (2003) Drugs of abuse and stress trigger a common synaptic adaptation in dopamine neurons. Neuron 37:577-582. Schenk S, Horger BA, Peltier R, Shelton K (1991) Supersensitivity to the reinforcing effects of cocaine following 6-hydroxydopamine lesions to the medial prefrontal cortex in rats. Brain Res 543:227-235. Schultz W (2001) Reward signaling by dopamine neurons. Neuroscientist 7:293-302. Segal DS, Kuczenski R (1987) Individual differences in responsiveness to single and repeated amphetamine administration: behavioral characteristics and neurochemical correlates. J Pharmacol Exp Ther 242:917-926. Sesack SR, Pickel VM (1992) Prefrontal cortical efferents in the rat synapse on unlabeled neuronal targets of catecholamine terminals in the nucleus accumbens septi and on dopamine neurons in the ventral tegmental area. J Comp Neurol 320:145-160. Sesack SR, Deutch AY, Roth RH, Bunney BS (1989) Topographical organization of the efferent projections of the medial prefrontal cortex in the rat: an anterograde tract- tracing study with Phaseolus vulgaris leucoagglutinin. J Comp Neurol 290:213- 242. Shaham Y, Puddicombe J, Stewart J (1997) Sexually arousing events and relapse to heroin-seeking in sexually experienced male rats. Physiol Behav 61:337-341. Sinchak K, Micevych PE (2001) Progesterone blockade of estrogen activation of mu- opioid receptors regulates reproductive behavior. J Neurosci 21:5723-5729. Smith Y, Charara A, Parent A (1996) Synaptic innervation of midbrain dopaminergic neurons by glutamate-enriched terminals in the squirrel monkey. J Comp Neurol 364:231-253. Stinus L, Koob GF, Ling N, Bloom FE, Le Moal M (1980) Locomotor activation induced by infusion of endorphins into the ventral tegmental area: evidence for opiate- dopamine interactions. Proc Natl Acad Sci U S A 77:2323-2327. Swanson LW (1982) The projections of the ventral tegmental area and adjacent regions: a combined fluorescent retrograde tracer and immunofluorescence study in the rat. Brain Res Bull 9:321-353. Takahata R, Moghaddam B (2000) Target-specific glutamatergic regulation of dopamine neurons in the ventral tegmental area. J Neurochem 75:1775-1778. Thierry AM, Tassin JP, Blanc G, Glowinski J (1976) Selective activation of mesocortical DA system by stress. Nature 263:242-244. Thomas MJ, Malenka RC (2003) Synaptic plasticity in the mesolimbic dopamine system. Philos Trans R Soc Lond B Biol Sci 358:815-819. Tong ZY, Overton PG, Martinez-Cue C, Clark D (1998) Do non-dopaminergic neurons in the ventral tegmental area play a role in the responses elicited in A10

158 dopaminergic neurons by electrical stimulation of the prefrontal cortex? Exp Brain Res 118:466-476. Topple AN, Hunt GE, McGregor IS (1998) Possible neural substrates of beer-craving in rats. Neurosci Lett 252:99-102. Trafton JA, Abbadie C, Marek K, Basbaum AI (2000) Postsynaptic signaling via the [mu]-opioid receptor: responses of dorsal horn neurons to exogenous opioids and noxious stimulation. J Neurosci 20:8578-8584. Tzschentke TM (2000) The medial prefrontal cortex as a part of the brain reward system. Amino Acids 19:211-219. Tzschentke TM (2001) Pharmacology and behavioral pharmacology of the mesocortical dopamine system. Prog Neurobiol 63:241-320. Uslaner J, Badiani A, Day HE, Watson SJ, Akil H, Robinson TE (2001) Environmental context modulates the ability of cocaine and amphetamine to induce c-fos mRNA expression in the neocortex, caudate nucleus, and nucleus accumbens. Brain Res 920:106-116. Valjent E, Corvol JC, Pages C, Besson MJ, Maldonado R, Caboche J (2000) Involvement of the extracellular signal-regulated kinase cascade for cocaine-rewarding properties. J Neurosci 20:8701-8709. Van Bockstaele EJ, Pickel VM (1995) GABA-containing neurons in the ventral tegmental area project to the nucleus accumbens in rat brain. Brain Res 682:215- 221. van Erp AM, Miczek KA (2000) Aggressive behavior, increased accumbal dopamine, and decreased cortical serotonin in rats. J Neurosci 20:9320-9325. van Furth WR, van Ree JM (1996) Sexual motivation: involvement of endogenous opioids in the ventral tegmental area. Brain Res 729:20-28. van Furth WR, Wolterink G, van Ree JM (1995) Regulation of masculine sexual behavior: involvement of brain opioids and dopamine. Brain Res Brain Res Rev 21:162-184. Vanderschuren LJ, Tjon GH, Nestby P, Mulder AH, Schoffelmeer AN, De Vries TJ (1997) Morphine-induced long-term sensitization to the locomotor effects of morphine and amphetamine depends on the temporal pattern of the pretreatment regimen. Psychopharmacology (Berl) 131:115-122. Veening JG, Coolen LM (1998) Neural activation following sexual behavior in the male and female rat brain. Behav Brain Res 92:181-193. Veenman CL, Reiner A, Honig MG (1992) Biotinylated dextran amine as an anterograde tracer for single- and double-labeling studies. J Neurosci Methods 41:239-254. Vezina P, Queen AL (2000) Induction of locomotor sensitization by amphetamine requires the activation of NMDA receptors in the rat ventral tegmental area. Psychopharmacology (Berl) 151:184-191. Vezina P, Giovino AA, Wise RA, Stewart J (1989) Environment-specific cross- sensitization between the locomotor activating effects of morphine and amphetamine. Pharmacol Biochem Behav 32:581-584. Wallace BC (1989) Psychological and environmental determinants of relapse in crack cocaine smokers. J Subst Abuse Treat 6:95-106. Wang T, French ED (1995) NMDA, kainate, and AMPA depolarize nondopamine neurons in the rat ventral tegmentum. Brain Res Bull 36:39-43.

159 Weissenborn R, Robbins TW, Everitt BJ (1997) Effects of medial prefrontal or anterior cingulate cortex lesions on responding for cocaine under fixed-ratio and second- order schedules of reinforcement in rats. Psychopharmacology (Berl) 134:242- 257. Wenkstern D, Pfaus JG, Fibiger HC (1993) Dopamine transmission increases in the nucleus accumbens of male rats during their first exposure to sexually receptive female rats. Brain Res 618:41-46. Wise RA (1996) Neurobiology of addiction. Curr Opin Neurobiol 6:243-251. Wise RA, Bozarth MA (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469-492. Wise RA, Rompre PP (1989) Brain dopamine and reward. Annu Rev Psychol 40:191- 225. Wolf ME, White FJ, Hu XT (1994) MK-801 prevents alterations in the mesoaccumbens dopamine system associated with behavioral sensitization to amphetamine. J Neurosci 14:1735-1745. Wyvell CL, Berridge KC (2001) Incentive sensitization by previous amphetamine exposure: increased cue-triggered "wanting" for sucrose reward. J Neurosci 21:7831-7840. Xia Y, Haddad GG (1991) Ontogeny and distribution of opioid receptors in the rat brainstem. Brain Res 549:181-193. Zahm DS (1999) Functional-anatomical implications of the nucleus accumbens core and shell subterritories. Ann N Y Acad Sci 877:113-128. Zahm DS, Brog JS (1992) On the significance of subterritories in the "accumbens" part of the rat ventral striatum. Neuroscience 50:751-767. Zhang K, Tarazi FI, Baldessarini RJ (2000) Dopamine D(4) receptors in rat forebrain: unchanged with amphetamine-induced behavioral sensitization. Neuroscience 97:211-213.

160 Figure 1

Figure 1. Proposed circuitry model. In this hypothetical model, sex-related visual and olfactory cues stimulate the release of opioids into the VTA, resulting in disinhibition of mesoaccumbens DA neurons and DA release into the NAc, which is then translated to a motivated motor response (blue pathway). Simultaneously, the sex-related cues are processed and sent to the mPFC (red pathway), which then activates brain areas important for sexual performance. In addition, the PFC activates GABA neurons, which project to the NAc to resulting in feed-forward inhibition of the system. The PFC-VTA projection may also cause feedback inhibition on itself, by projecting to DA neurons which then project back to the mPFC and inhibit cortical pyramidal cells via D2 receptors. Black nuclei represent cells in which Fos is expressed during sexual behavior.

161