Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Neurochemical correlates of cross-sensitization between repeated d=amphetamine administration and male… Fiorino, Dennis Frank 1999

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1999-388859.pdf [ 6.36MB ]
Metadata
JSON: 831-1.0089228.json
JSON-LD: 831-1.0089228-ld.json
RDF/XML (Pretty): 831-1.0089228-rdf.xml
RDF/JSON: 831-1.0089228-rdf.json
Turtle: 831-1.0089228-turtle.txt
N-Triples: 831-1.0089228-rdf-ntriples.txt
Original Record: 831-1.0089228-source.json
Full Text
831-1.0089228-fulltext.txt
Citation
831-1.0089228.ris

Full Text

N E U R O C H E M I C A L CORRELATES OF CROSS-SENSITIZATION B E T W E E N REPEATED ^ - A M P H E T A M I N E ADMINISTRATION A N D M A L E R A T S E X U A L BEHAVIOR by DENNIS F R A N K FIORINO B. Sc., University of Lethbridge, 1990 M . S c , University of British Columbia, 1993 A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Neuroscience We accept this thesis as conforming to the required standards THE UNIVERSITY OF BRITISH C O L U M B I A October 1998 © Dennis Frank Fiorino, 1998 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of , , M B O ( ^ Q < 5 C \ \2NCfr The University of British Columbia Vancouver, Canada DE-6 (2/88) 11 Abstract Behavioral sensitization due to repeated and intermittent administration of psychostimulants, such as cocaine and d-amphetamine, is accompanied by enhanced function in limbic-motor circuitry that is involved in the generation of motivated behavior. The present experiments investigated the effect of sensitization, induced by repeated injections of d-amphetamine, on sexual behavior in the naive male rat tested in a drug-free state. It was hypothesized that prior d-amphetamine treatment would facilitate male rat sexual behavior. Injections of either d-amphetamine (1.5 mg/kg, IP) or saline were given every other day for a total of 10 injections, and this regimen induced behavioral sensitization of locomotor activity in drug-treated male rats. After a three-week post-drug period, d-amphetamine-treated rats exhibited facilitated sexual behavior as indicated by shorter latencies to mount and intromit, as well as a general increase in the amount of copulation. Furthermore, sensitized rats displayed a facilitated acquisition of sexual behavior (i.e., mount and intromission latency < 300s for three consecutive days). After repeated sexual experience, rats pre-treated with d-amphetamine also showed an augmented increase in appetitive locomotor activity (i.e., level changes) made in anticipation of the presentation of a receptive female. In a separate experiment, enhanced sexual behavior was observed to be independent of the environment in which repeated administration of d-amphetamine occurred, indicating that facilitation was not a consequence of conditioned associations between drug and test environment. In a final microdialysis experiment, there was an augmented efflux of dopamine (DA) in the nucleus accumbens (NAC) of d-amphetamine-sensitized rats compared to non-sensitized control rats when a receptive female was present behind a screen (35% vs 17%)). Although there was a significant increase in N A C D A concentrations from baseline in both sensitized and non-sensitized rats during copulation, there was a greater increase in D A efflux in the N A C of sensitized rats during the first 10-min copulatory sample (60% vs 37%). These results demonstrate that behavioral sensitization due to repeated psychostimulant administration can "cross-sensitize" to a natural behavior, such as sex, and that increased N A C D A release may contribute to the facilitation of appetitive and consummatory aspects of this behavior. Furthermore, the subsequent facilitation of anticipatory sexual behavior (i.e., level changes) after repeated experience in rats previously treated with rf-amphetamine, suggests that behavioral sensitization can influence incentive learning. Table of Contents Abstract i i List of Tables vii List of Figures viii List of Abbreviations x Acknowledgements xi INTRODUCTION 1 The Neurobiology of Dopamine 1 Monitoring Dopamine Transmission In Vivo 8 The Neurobiology of Behavioral Sensitization to Psychostimulants 11 Dopamine and Incentive Motivation 21 Mechanisms of Male Sexual Behavior 24 The Role of Mesolimbic Dopamine in Male Sexual Behavior 26 Cross-sensitization Between Drugs and Sexual Behavior 32 Overview of the Present Experiments 34 GENERAL METHODS 36 EXPERIMENT I: THE EFFECT OF REPEATED AND INTERMITTENT (/-AMPHETAMINE ADMINISTRATION ON MALE RAT SEXUAL BEHAVIOR 47 Method Results Discussion 49 49 61 EXPERIMENT II: THE ROLE OF CONDITIONING FACTORS IN THE EFFECT OF </-AMPHETAMINE-INDUCED BEHAVIORAL SENSITIZATION ON MALE RAT SEXUAL BEHAVIOR 63 Method 64 Result 65 Discussion 69 EXPERIMENT in: THE EFFECT OF REPEATED AND INTERMITTENT (/-AMPHETAMINE ADMINISTRATION ON MALE RAT SEXUAL BEHAVIOR IN UNILEVEL CHAMBERS 74 Method 75 Results 76 Discussion 82 EXPERIMENT IV: IN VIVO MEASUREMENT OF NAC DA EFFLUX DURING SEXUAL BEHAVIOR IN (/-AMPHETAMINE-SENSITIZED MALE RATS 83 Method 85 Results 85 vi Discussion 105 GENERAL DISCUSSION 109 Behavioral Sensitization by Non-Pharmacological Stimuli 114 Presynaptic Mechanisms Subserving the Expression of Behavior Facilitated by Psychostimulant-Induced Sensitization 119 The Role of Mesolimbic Dopamine in Motivated Behavior 122 Clinical Implications 127 REFERENCES 130 APPENDIX A 151 vii List of Tables Table 1. Measures of sexual behavior from Experiment I 55 Table 2. Measures of sexual behavior from Experiment II 68 Table 3. Measures of sexual behavior from Experiment III 79 Table 4. Measures of sexual behavior from Experiment IV 89 Table 5. Mean basal concentrations of microdialysis analytes 92 vm List of Figures Figure 1. Diagram of major D A projections of the rat central nervous system 4 Figure 2. Diagram of bilevel testing chamber 40 Figure 3. Diagram of unilevel testing chamber 42 Figure 4. Effect of repeated ^-amphetamine or saline administration on activity in Experiment I 51 Figure 5. Anticipatory level changes made by rats sensitized to rf-amphetamine or non-sensitized control rats in Experiment 1 54 Figure 6. Effect of (/-amphetamine or saline pre-treatment and sexual experience on latencies to mount and intromit in Experiment I 58 Figure 7. Acquisition of sexual behavior in sensitized and non-sensitized rats in Experiment 1 60 Figure 8. Effect of repeated J-amphetamine or saline administration on activity in Experiment II 67 Figure 9. Effect of J-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment II 71 Figure 10. Effect of repeated (/-amphetamine or saline administration on activity in Experiment III 78 Figure 11. Effect of (/-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment III 81 Figure 12. Effect of repeated (/-amphetamine or saline administration on activity in Experiment IV 87 IX Figure 13. Effect of d-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment IV 91 Figure 14. Changes in nucleus accumbens dopamine efflux associated with sexual behavior 94 Figure 15. Changes in DOPAC and H V A concentrations in the nucleus accumbens associated with sexual behavior 97 Figure 16. Changes in locomotor behavior and nucleus accumbens dopamine efflux in response to a d-amphetamine challenge 100 Figure 17. Changes in nucleus accumbens DOPAC and H V A efflux in response to a d-amphetamine challenge 102 Figure 18. Location of microdialysis probes within the N A C of rats used in Experiment IV 104 List of Abbreviations 6-OHDA 6-hydroxydopamine AMPH d-amphetamine-treated [experimental group] ANOVA analysis of variance BST bed nucleus of the stria terminalis CaM-KII calcium-calmodulin kinase II CONT saline-treated [control group] CREB cAMP response binding element DA dopamine DAT dopamine uptake transporter DOPAC dihydroxyphenylacetic acid EC electrochemical EF ejaculation frequency EL ejaculation latency GLU glutamate HPLC high pressure liquid chromatography HVA homovanillic acid IE, number of intromissions to the first ejaculation IEM intromission and ejaculatory mechanism IF intromission frequency III inter-intromission interval IL intromission latency IR intromission ratio LC level change MF mount frequency MLB mesolimbic ML mount latency mPOA medial preoptic area NA noradrenaline NAC nucleus accumbens PEI post-ejaculatory interval PKA protein kinase A SAM sexual arousal mechanism VTA ventral tegmental area Acknowledgements Sincere thanks to my supervisor and mentor, Tony Phillips, for his guidance, encouragement, and generous financial support over the years. He delicately balanced discipline and patience, giving me enough latitude to bring out my best while making sure I was "on track". Thank you for the occasional good pun, too, Tony. [That last sentence is not meant to be read as though he offers a good pun on every occasion, although, God help us, he tries.] A very special thanks to Ariane Coury, a valued friend and teacher. I have always appreciated her honesty, sense of humor, and the great deal of support she has given me during difficult times. Her confidence in my abilities encouraged me to excel. I will miss her very much. I am indebted to my supervisory committee, Boris Gorzalka, Chris Fibiger, and Peter Soja, who helped me focus my ideas and designs for the present experiments. A special thanks to Boris for allowing me to use his lab and testing chambers. Many thanks to Jim Pfaus, whose contagious enthusiasm for sexual behavior - er, the study of sexual behavior - led to many valuable discussions and stimulated my research. Thanks to Fred LePiane, upon whom I relied heavily to get my experiments running well. He gave me a lot of good advice on topics ranging from computer programming to how to varnish furniture. The shelves look great. Thank you to Keith Waldron, a very talented technician, who built many superb tools for our laboratory by improving on the designs that were presented to him. To Lucille Hoover, the "glue" of the Biopsychology area, whose knowledge, compassion, and conscientiousness is uncommon: I am grateful for all your help. Thank you to the animal care technicians in the Psychology department, Anne Cheng and Tara Nash, who raised the level of care in our facilities and greatly contributed to the success of my experiments. Thanks to Chuck Blaha, Natalia Gorelova, and Charles Yang, for the rewarding experience I gained through our discussions and all too brief collaborations. To the graduate students that I have worked with in Tony's lab - Soyon Ahn, Alisdair Barr, Pat DiCiano,Chris Duva, Stan Floresco, David Mutch, Jeremy Seamans, Pornarrin Taepavarapruk - and those who just passed through to use our microwave oven - Jason Carr, Jennifer Galloway, Tom Kornekook, and Steve Wicks; I learned something from each of you. Xll Thanks to John Rogers, Leslie Mitchell, and David Mutch, who helped me conduct many experiments, and Ingrid Price, for teaching me how to score male rat sexual behavior. To the biopsychology professors of the fourth floor, including Liisa Galea, John Pinel, Cathy Rankin, Richard Tees, Don Wilkie: thank you for all the things I borrowed over the years. Thanks to the departments of Psychology and Neuroscience at U B C for their financial and administrative support, especially Kimmy Chiu, Liz McCririck, Lee Smith, Rose Tam, and Liz Wong, who made my life run more smoothly. Thank you to the Jinnouchi family, who warmly accepted me into their great family and made my last years at U B C a little easier. Above all, thanks to the extended Fiorino family, who have always encouraged me in my studies, and given me an incredible amount of love and support. This thesis is dedicated to Colette Jinnouchi and Anna Fiorino, who have made me a better man. I love you. 1 NEUROCHEMICAL CORRELATES OF CROSS-SENSITIZATION BETWEEN REPEATED PSYCHOSTIMULANT ADMINISTRATION AND MALE RAT SEXUAL BEHAVIOR The overall objective of this thesis is to describe changes to male sexual behavior resulting from long-term, intermittent psychostimulant administration and, on a separate and more basic level, to obtain a more comprehensive knowledge of the role of the mesolimbic (MLB) dopamine system in the expression of motivated behavior. The repeated and intermittent administration of psychostimulants, such as cocaine and J-amphetamine, can lead to an enhanced behavioral response upon subsequent administration of the same dose of drug. This phenomenon is known as behavioral sensitization and its study may provide important insights into drug addiction, which can be considered an extreme form of motivated behavior (Jaffe, 1990; Robinson and Berridge, 1993), as well as the pathophysiology of psychostimulant-induced psychosis and epilepsy (Robinson and Becker, 198.6; Post and Weiss, 1988; Pierce and Kalivas, 1997a). Research into the phenomena of incentive motivation and behavioral sensitization to psychostimulants has overlapped frequently. This thesis represents one of the first formal investigations into the confluence of these two fields with the overall aim of elucidating the normal function of M L B DA. The Neurobiology of Dopamine Until the mid-1950s, DA, a catecholamine, was regarded only as a precursor to 2 noradrenaline (NA) (Blaschko, 1939). The marked differences in regional distribution of D A and N A , both in the peripheral and central nervous systems, led to the proposal that D A may be an independent neurotransmitter (Von Euler and Lishajko, 1957; Bertler and Rosengren, 1959). The application of histochemical fluorescence methods that permitted the visualization of catecholaminergic cells (Dahlstrom and Fuxe, 1964) and immunohistochemical techniques directed at enzymes involved in the synthesis of specific catecholamines (Hokfelt et a l , 1976) led to a precise mapping of the cell bodies, projections fibers, and terminal areas of DA-containing neurons in the rat central nervous system. Dopaminergic cells of the central nervous system can be classified into three major categories: ultrashort fiber systems, diencephalic, and mesotelencephalic D A systems (Figure 1; Moore and Lookingland, 1995; Bloom, 1996). The ultrashort D A systems include the interplexiform amacrine-like cells of the retina (area A17) and the periglomerular cells of the olfactory bulb (A 16). The diencephalic systems include the hypothalamic D A cells groups: the periventricular neurons (A 14) that project to the medial preoptic area (rnPOA) and anterior hypothalamus; the incertohypothalamic neurons (A 13) which link dorsal and posterior hypothalamus with the dorsal anterior hypothalamus and lateral septal nuclei; the mberinfundibular neurons (A12) which project from the arcuate and periventricular nuclei to the intermediate lobe of the pituitary and the median eminence; and the A l 1 area which sends projections from the posterior dorsal hypothalamus to the spinal cord. The mesotelencephalic D A systems include the projections linking the midbrain D A cells of the ventral tegmental area (VTA; 3 Figure 1. Diagram of major D A projections of the rat central nervous system. A . Mesotelencephalic D A systems. The striped regions represent the major terminal areas of the D A cell groups of origin (i.e., retrorubral, A8; substantia nigra, A9; ventral tegmental area, A10). B. Retinal (A 16), olfactory (A 17), and diencephalic ( A l 1, A12, A13, A14, A15) D A systems. Adapted from Moore (1987), Cooper et al. (1991), and Paxinos and Watson (1997). 4 A. anterior cingulate cortex lateral septum bed nucleus of the stria terminalis hippocampus A8 A10 prefrontal cortex perirhinal cortex nucleus accumbens olfactory pyriform tubercle cortex lateral parabrachial nucleus central nucleus amygdala entorhinal cortex 5 A10), substantia nigra (A9), and the retrorubral area (A8) with forebrain targets, such as the neostriatum (caudate-putamen), the limbic cortex (medial prefrontal, cingulate, and entorhinal areas), and other limbic areas (the nucleus accumbens; olfactory tubercle, septum; amygdala, and piriform cortex). There is a considerable degree of overlap in the mesotelencephalic D A neurons that project from the midbrain to the forebrain. The collection of D A neurons of the substantia nigra whose axons extend to the caudate-putamen is termed the nigrostriatal D A system. The majority of D A neurons in the V T A terminate in the N A C , which is also viewed as part of the ventral striatum, but this nucleus also receives some dopaminergic input from the substantia nigra and the retrorubral area. Collectively, this pathway is known as the M L B D A system. Furthermore, the N A C can be divided into shell and core regions based on anatomical connectivity and neurochemistry (Zahm and Brog, 1992). The core region is related to the caudate-putamen and receives input from D A cells of the substantia nigra, whereas the shell region receives D A projections from the V T A (Heimer et al., 1991; Zahm and Brog, 1992). Based on anatomical connections, it has been proposed that the N A C shell primarily modulates limbic input relaying motivational information, whereas the core is preferentially involved with motor aspects of motivated behavior (Zahm and Brog, 1992; Deutch et al., 1993). In addition, the neurons of the N A C shell are more spontaneously active (Boeijinga et al., 1990) but also appear to be less excitable (O'Donnell and Grace, 1993) than cells of the N A C core. The post-synaptic effects of D A depend on the subtype of D A receptor as well as 6 the action of other neurotransmitters that converge on the same neuron. There are at least five distinct genes that encode D A receptors and they can be classified into two categories: the Di-like and D2-like receptors (Schwartz et a l , 1992; Civelli, 1995). This classification is based on pharmacological and biochemical differences between the Di and D2 subtypes, the first two D A receptors that were identified (Kebabian and Caine, 1979). The Di-like receptors, which include the Di and D 5 receptors, whose genes do not contain introns, bind the benzazepine antagonist SCH 23390 with high affinity and are coupled to G s proteins that activate adenylyl cyclase. In contrast, the D2-like receptors, which include D2, D 3 , and D 4 subtypes, bind the butyrophenones spiperone and haloperidol with high affinity and their genes contain introns. The D2 and D4 receptors are coupled to Gi proteins that inhibit cAMP production. Studies employing in situ hybridization techniques have shown that the mRNA of the D A receptor subtypes is differentially distributed in brain. For example, although D i , D2, and D3 receptor mRNA expression is high throughout the striatum, the cellular expression of Di and D2 mRNA is highest in the lateral caudate-putamen, whereas D3 mRNA expression is found predominantly in the N A C (Mansour and Watson, 1995). A heterogeneous distribution also exists across subcompartments of the N A C . There are higher levels of expression and more cells expressing D3 mRNA in the accumbens shell, while higher levels of expression of Di and D2 mRNA are found in the core (Mansour and Watson, 1995). Psychostimulants, such as cocaine and J-amphetamine, function as indirect D A agonists by increasing synaptic concentrations of D A via the interaction with the D A uptake transporter (DAT). The DAT is a substrate-specific, Na+-dependent membrane transporter which is believed to be the primary mechanism for limiting the extent, duration, and area of D A neurotransmission (Parsons and Justice, 1994; Bannon et al., 1995; Giros et al., 1996). Cocaine acts to block the reuptake of monoamines by the transporter, but release is still dependent on terminal depolarization (Galloway, 1988). Amphetamine appears to mobilize D A from vesicles into the cytoplasm and release D A into the extracellular space in a calcium- and impulse-independent manner via the D A T by reverse transport (Kuczenski and Segal, 1994; Sulzer et al., 1995; Jones et al., 1998). Amphetamine enters the neuron by its specific uptake by the D A T (Liang and Rutledge, 1982a,b; Zaczek et al., 1991a,b; Seiden et al., 1993) but also can cross the plasma membranes by lipophilic diffusion (Mack and Bonisch, 1979; Liang and Rutledge, 1982a; Zaczek et al., 1991a,b). Inside the cell, amphetamine, a weak base, redistributes D A from vesicles to the cytosol by reducing the vesicular proton gradient that provides the free energy necessary for neurotransmitter accumulation (Sulzer et al., 1993). This appears to be the rate-limiting step in amphetamine's action (Jones et al., 1998). Although there is debate on whether or not amphetamine can promote reverse transport of cytosolic D A to the synapse (Sulzer et al., 1995; Jones et a l , 1998), it is clear that the D A T is required for its DA-releasing action (Jones et al., 1998). The D A axons of the N A C shell contain a lower density of plasmalemmal D A T (Nirenberg et al., 1997). Given that D A uptake sites are located predominantly, i f not exclusively, outside the synapse (Pickel et al., 1996), a mechanism exists by which the D A T can regulate the extent of extrasynaptic D A efflux differentially in the N A C shell 8 and core. Indeed, D A was found to diffuse further in the N A C shell as measured by in vivo voltammetry (Garris et al., 1994) and uptake efficiency was reported to be reduced in this region (Jones et al., 1996b). Furthermore, Pontieri et al. (1995) showed that intravenous cocaine and d-amphetamine preferentially enhanced D A efflux in the N A C shell, relative to core, although a contrary finding was reported also (Jones et al., 1996b). This difference in D A T density in the subcompartment of the N A C may also explain why cell of the shell are less vulnerable to the catecholamine-specific neurotoxin, 6-hydroxydopamine (6-OHDA), which must be tranported inside the cell by the D A T to produce its toxic effects (Zahm, 1991). Monitoring DA Transmission In Vivo The ability to monitor local changes in neurotransmitter concentrations in the central nervous system of freely behaving animals is an integral part of understanding the function of that compound in a specific brain region. The use of in vivo techniques, such as microdialysis and voltammetry, have provided important insights about the role of M L B D A transmission in motivated behavior. The basic principle of microdialysis has been described as mimicking a capillary blood vessel (Ungerstedt, 1991); a thin, semi-permeable dialysis membrane tube is perfused by a physiological solution, and extracellular compounds outside of the membrane diffuse across their concentration gradient into the tube. These samples, in turn, are pumped through a closed internal cannula system and are collected for analysis. The concentration of the compound(s) of 9 interest in the collected perfusate is then quantified by chemical analysis (e.g., separation of analytes by high pressure liquid chromatography (HPLC) and quantification using electrochemical (EC) or fluorometric detection). In this manner, qualitative and quantitative changes in neurotransmitter concentrations can be monitored. Voltammetric electrodes, which sample the same extracellular pool as microdialysis, apply an electrical potential in order to oxidize or reduce electroactive compounds present at the electrode surface (O'Neill et al., 1998). There are advantages and disadvantages to each technique concerning issues of temporal and spatial resolution, selectivity, and sensitivity (Finlay and Zigmond, 1995; O'Neill et al., 1998). Voltammetric probes are small, relative to microdialysis probes (1-30 pm vs >200 pm in diameter), and, therefore, can be used to monitor neurochemistry in small brain regions. In addition, voltammetric sampling of compounds can occur as often as every 100 ms, in contrast to the long intervals required for microdialysis (e.g., 1-20 min), making it well-suited for observing brief, phasic changes in the content of the extracellular fluid. The major advantages of microdialysis over voltammetry concern the issues of selectivity and sensitivity. The assessment of extracellular compounds using microdialysis is limited only by what can diffuse across the microdialysis probe and the availability of an assay for quantification of a specific analyte. In contrast, voltammetric assessment is restricted to the detection of electroactive species. Furthermore, i f two compounds have similar or identical oxidizing potentials (e.g., N A and DA), they cannot be distinguished electrochemically. Because separation of analytes is accomplished after collection of the microdialysis sample, the 10 issue of selectivity is not a problem. In this thesis, changes in microdialysate analyte concentrations are described in terms of increased or decreased "efflux", rather than "release", for two reasons. First, certain metabolites (e.g., homovanillic acid) are formed extracellularly, and are not "released". Second, the amount of neurotransmitter collected by microdialysis is more than a reflection of neurotransmitter release; it is a function of what escapes from the synaptic cleft. Therefore, the processes of release, reuptake, metabolism, and catabolism, as well as the complexity of the diffusional path through the tissue matrix (i.e., tortuosity), all contribute to how much neurotransmitter reaches the microdialysis probe. In an early attempt to estimate the absolute concentration of compounds in the extracellular space, Ungerstedt et al. (1982) employed a "recovery" ratio of analyte concentration recovered through the microdialysis probe to analyte concentration present outside the probe in vitro. The assumption that in vitro recovery in a non-stirred medium is the same as in vivo recovery, which is implied in this model, is not correct and leads to an underestimation of extracellular concentrations of neurotransmitters and metabolites (Benveniste and Huttemeier, 1990). Subsequently, a number of theoretical approaches were proposed that take into account neuronal processes and the concept of tortuosity, as well as the permeability of the dialysis (reviewed in Kehr, 1993). Unfortunately, many of these approaches incorporate constants that apply under narrow circumstances or are too mechanistic to be practical (Kehr, 1993). Perhaps the best approaches to calculate absolute extracellular concentrations of compounds are the zero flow (based on Jacobsen et al., 1985; Lerma et al., 1986) and "no-net flux" (Lonnroth et al., 1987) methods. The 11 first approach involves sampling the extracellular fluid using different perfusing rates and extrapolating to zero flow by regression analysis. At zero flow, steady-state conditions apply and recovery is 100%. The no-net flux method, on the other hand, is based on the assumption that when the concentration of analyte within the probe equals that outside the probe, there is no net diffusion across the dialysis membrane (i.e., the point of no-net flux). By including varying amounts of analyte in the perfusate at a constant flow rate., a point of no-net flux is reached eventually, and the extracellular analyte concentration is equal to the analyte concentration introduced into the perfusate. These approaches are useful for estimating basal concentrations of analytes, but are not practical during situations of phasic neurotransmitter release, such as after the administration of a drug or in response to a behavioral challenge. Traditionally, under these circumstances, data are transformed into a value based on the percentage of basal concentration of a specific analyte (uncorrected for probe recovery) during the baseline period just prior to the experimental manipulation. The Neurobiology of Behavioral Sensitization to Psychostimulants Behavioral sensitization to psychostimulants appears to be the result of functional changes which involve a number of different transmitters and nuclei within complex limbic-motor circuitry (Kalivas et al., 1993a,b; White et al., 1995; Pierce and Kalivas, 1997a), however, much of the research on the neurochemical correlates of sensitization has focused on alterations involving D A transmission. There are several reasons for this. First, different classes of psychostimulants share the ability to increase D A transmission 12 acutely (Kuczenski and Segal, 1994; Robinson and Berridge, 1993). Second, many alterations to the M L B D A system have been reported to accompany behavioral sensitization (Kalivas et al., 1993b; Vezina, 1996; Pierce and Kalivas, 1997a). Finally, an extensive literature indicates a key role for the M L B D A system in drug addiction and reward (Fibiger and Phillips, 1986; Wise and Bozarth, 1987; Koob, 1992; Robinson and Berridge, 1993). The occurrence of behavioral sensitization has been conceptualized to involve two components, initiation and expression, that can be defined temporally and with respect to anatomical correlates (Kalivas and Stewart, 1991). Initiation refers to the transient molecular and cellular changes that establish behavioral sensitization. They are believed to occur within the V T A . Enduring changes in the N A C , and connected areas, appear to be necessary for its expression. Although cocaine and d-amphetamine have two different mechanisms of action, their common ability to increase somatodendritic and terminal D A release, via the DAT, is essential for the initiation and expression of behavioral sensitization, respectively (Kalivas and Stewart, 1991; Pierce and Kalivas, 1997a). Initiation Repeated bilateral infusions of d-amphetamine into the V T A , which elicits somatodendritic D A release, which do not produce increases in locomotion, can lead to a sensitized behavioral response to systemic injections of d-amphetamine and cocaine (Kalivas and Weber, 1988; Hooks et al., 1992; Vezina, 1996) or an intra-NAC infusion of d-amphetamine (Perugini and Vezina, 1994). This injection procedure is also sufficient to augment N A C D A release after a systemic d-amphetamine challenge (Vezina, 1993). 13 A l l of these effects can be blocked by either the prior systemic or intra-VTA injections of the Di antagonist SCH-23390 , but not other antagonists with greater affinities to D2 or 5-HT2 receptors (Stewart and Vezina, 1987; Vezina, 1996). Di receptors do not appear to be synthesized in V T A D A neurons (Mansour and Watson, 1995) and intra-VTA stimulation of Di receptors can produce dose-dependent increases in glutamate (GLU) and G A B A (Cameron and Williams, 1993; Kalivas and Duffy, 1995). Therefore, Di receptors in the V T A may be located presynaptically on G L U and G A B A afferents to modulate M L B D A activity via release of these transmitters (Kalivas, 1993; Kalivas and Duffy, 1995). In contrast to intra-VTA infusions, injections of d-amphetamine directly into the N A C are not sufficient to induce behavioral sensitization as assessed by either a local or systemic d-amphetamine challenge (Cador et al., 1995). The action of amphetamine in the N A C , however, is necessary for the expression of behavioral sensitization (Cador et al., 1995). Expression A number of postsynaptic changes in the N A C that involve D A transmission have been reported. Briefly, it appears that enhanced Di receptor function in the N A C is necessary for the expression of behavioral sensitization (Pierce and Kalivas, 1997a). Electrophysiological studies confirm the presence of Di receptor supersensitivity, as indicated by a greater inhibition of glutamate-driven activity in N A C neurons of psychostimulant-sensitized rats (Henry et al., 1989; Henry and White, 1991; White et al., 1995). Although there are few data to support a change in the density of postsynaptic D A 14 receptors in the N A C due to psychostimulant-induced sensitization (see Pierce and Kalivas, 1997a), altered second messenger systems, especially those downstream from D i receptor activation, have been reported (Self and Nestler, 1995; Fitzgerald and Nestler, 1995). Stimulation of D i receptors results in the activation of adenylyl cyclase, and subsequently, cAMP and protein kinase A (PKA), via G-proteins (Duman and Nestler, 1995). A persistent decrease in Gi content in the N A C of cocaine-sensitized rats (Striplin and Kalivas, 1992), in contrast to the lack of change in the amount of G s (Nestler et al., 1990; Terwilliger et al., 1991; Striplin and Kalivas, 1992), suggests that summed alterations in N A C G-protein content, due to psychostimulant-induced sensitization, favor the activation of adenylyl cyclase. Indeed, a study by Miserendino and Nestler (1995) found that repeated cocaine administration augumented activity of adenylyl cyclase and P K A in the N A C . Moreover, intra-NAC injections of a P K A activator produced a significant enhancement of sensitization to cocaine treatment (Miserendino and Nestler, 1995). It is unclear how these events may result in the long-term expression of behavioral sensitization to psychostimulants, although phosphorylation of the glutamate-activated A M P A receptor (Fitzgerald et al., 1996; L u et al., 1997) and voltage-dependent N a + channels (Zhang et al., 1998), as well as an elevation of phosphorylated c A M P response binding element (CREB) (Cole et al., 1995; Simpson et al., 1995) within the N A C , may contribute to this phenomenon. The most widely replicated finding relating to a presynaptic mechanism is the presence of augmented N A C D A efflux following repeated administration of a wide variety of psychostimulants, including ^-amphetamine, methamphetamine, 15 methylphenidate, and cocaine (Robinson et al., 1988; Akimoto et al., 1990; Kalivas et al., 1993b; Parsons and Justice, 1993). In vivo assessment of N A C D A efflux in freely moving animals, using microdialysis or voltammetry, at different times following discontinuation of drug treatment led to the suggestion that this may be a time-dependent characteristic of behavioral sensitization (Kalivas and Stewart, 1991; Kalivas et al., 1993b; but see Warburton et al., 1996; Kuczenski et al., 1997). Whereas most early studies demonstrated enhanced N A C D A efflux associated with behavioral sensitization to d-amphetamine or cocaine (Kalivas and Duffy, 1988; Robinson et al., 1988; Paulson et a l , 1991) others did not (Hurd et al., 1989; Segal and Kuczenski, 1992a,b). The latter studies measured extracellular D A content in response to a psychostimulant injection less than 48 h after discontinuing repeated cocaine or d-amphetamine administration. Given that sensitized behavioral responses are most robust when a longer period of time is allowed to elapse after discontinuing drug treatment (Antelman, 1988; Kalivas and Duffy, 1993; Paulson and Robinson, 1995; Heidbreder et al., 1996), subsequent studies examined N A C D A responses at different times after the last sensitizing injection. Kalivas and Duffy (1993) examined the ability of an acute cocaine challenge to increase N A C D A efflux at different withdrawal times following daily cocaine administration and confirmed the "time-dependence" phenomenon. By day 1 of withdrawal, behavioral sensitization was present, but there was no augmentation in the capacity of an acute cocaine injection to elevate N A C DA. However, following 2-3 weeks of withdrawal, both behavioral and neurochemical sensitization were present. Paulson and Robinson (1995) also reported a time-dependency for the expression of behavioral and neurochemical sensitization to d-amphetamine. Rats injected on an escalating-dose d-16 amphetamine regimen (1 to 10 mg/kg twice daily over a 6-week period) were challenged with a low dose of d-amphetamine (0.5 mg/kg) at 3, 7, or 28 days after the last injection. Rats tested after only 3 or 7 days of withdrawal showed neither behavioral sensitization or augmented N A C D A release. However, rats tested after 28 days of withdrawal were sensitized to the behavioral effects of d-amphetamine and also showed an enhanced d-amphetamine-induced release of N A C D A relative to saline controls. Heidbreder et al. (1996) confirmed the presence of enhanced N A C D A efflux in response to a cocaine challenge after extended (i.e., 22 days), rather than shorter (i.e., 2 days), withdrawal times following the cessation of cocaine pre-treatment (20 mg/kg/day x 5 days) in rats. In contrast to the above studies, Warburton et al. (1996) demonstrated that just a single injection of d-amphetamine (1 mg/kg) was sufficient to induce behavioral sensitization of rats to the same dose of drug 24 h later and augment d-amphetamine-induced N A C D A efflux by greater than 150% of baseline levels relative to saline controls, as assessed by microdialysis. It is important to note, however, that intermittent repeated doses, as opposed to a single or continuous infusion of psychostimulant, is necessary to produce optimal behavioral sensitization (Post, 1980; Robinson and Becker, 1986). Examination of the neurochemical correlates underlying behavioral sensitization to psychostimulants has revealed a dissociation between different regions of the N A C . Behavioral sensitization to cocaine was associated with more robust increases in N A C D A efflux in the shell, rather than the core, in response to local administration of d-amphetamine (Pierce and Kalivas, 1995). Whereas behavioral sensitization to a local injection of d-amphetamine was observed when administered to the shell at late 17 withdrawal periods (20-22 days), no behavioral enhancement was seen with intra-core injections at either late or early (1-3 days) withdrawal times. Moreover, D A efflux was greater in the N A C shell, relative to saline controls, in response to (/-amphetamine administration via the microdialysis probe after long withdrawal periods (Pierce and Kalivas, 1995). Recently, Kuczenski et al. (1997) found that, despite extended withdrawal periods (14 days) following the discontinuation of repeated (/-amphetamine administration and a similar dosage regimen to experiments cited above (i.e., 2.5 mg/kg/day x 7 days), behavioral sensitization to a (/-amphetamine challenge (2.5 mg/kg) was not accompanied by enhanced caudate-putamen or N A C D A efflux as assessed by microdialysis. These authors concluded that augmented striatal D A release cannot be a necessary correlate of behavioral sensitization. The observation that different types of (/-amphetamine-induced behaviors (i.e., locomotion or stereotypy) become more intense after repeated drug administration, without an alteration in their duration, suggested that the altered behavioral profile is not a simple consequence of increased sensitivity of D A systems (Segal and Kuczenski, 1994). Although most data suggest N A C D A is an important transmitter in the expression of behavioral sensitization to psychostimulants, it is clear that other transmitters systems are involved. In this regard, there are data that implicate augmented N A C glutamate transmission in the expression of behavioral sensitization to cocaine, although the role of glutamate in this context is not clear (reviewed in Pierce and Kalivas, 1997a). There is little evidence to suggest that D A autoreceptors play a significant role in 18 the expression of behavioral sensitization to psychostimulants. Most midbrain D A neurons possess D A autoreceptors that modulate D A release, as well as cell excitability and D A synthesis (Chiodo et al., 1995). It has been proposed that decrements in autoreceptor sensitivity may contribute to the enhanced release of D A associated with behavioral sensitization (Antelman and Chiodo, 1981). The results of in vitro studies examining D A autoreceptor function in the N A C or caudate-putamen of psychostimulant-sensitized rats have been equivocal, with increases (Dwoskin et al., 1988), decreases (Yi and Johnson, 1990), or no change (Fitzgerald and Reid, 1991; Gifford and Johnson, 1992; King et al., 1994) in sensitivity being reported. The sensitivity of D A autoreceptors was assessed recently by the ability of local perfusion of the D2/D3 agonist, quinpirole, to lower extracellular D A efflux in the N A C in vivo at different stages of drug withdrawal. Although desensitization of the D A autoreceptor was present during the first week of drug withdrawal (Pierce et al., 1995; Jones et al., 1996a) there was no change 20-21 days following cessation of daily cocaine administration (Pierce et al., 1995). Therefore, it is unlikely that changes in D A autoreceptor function contribute to the augmentation of N A C D A release associated with long-term expression of behavioral sensitization. Even though increased D A neuron activity may contribute to increased D A release in sensitized rats (White and Wang, 1984), augmented D A efflux has been demonstrated in N A C and striatal tissue slices (Robinson and Becker, 1982; Castaneda et a l , 1988; Peris et al., 1990) or in response to local application of psychostimulants (Pierce and Kalivas, 1995; Pierce and Kalivas, 1997b). These data suggest that 19 independent mechanisms exist at the level of the terminal that are responsible for augmenting striatal D A release, regardless of any change in D A cell activity. Increased calcium transduction may underlie the augmentation of D A release in the striatum of sensitized rats. The expression of behavioral sensitization to psychostimulants can be blocked by systemic administration of L-type calcium channel antagonists (Karler et al., 1991; Martin-Iverson and Reimer, 1994) despite the fact that the acute DA-release action of rf-amphetamine is calcium-independent. Furthermore, augmented D A efflux in the N A C of cocaine-sensitized rats, in response to local administration of J-amphetamine, was blocked by local co-administration of L-type or N-type calcium channel antagonists (Pierce and Kalivas, 1997b) or by replacing calcium in the microdialysis perfusion fluid with magnesium in rats pre-treated with d-amphetamine (Warburton et al., 1996). Repeated d-amphetamine administration was reported to increase the activity of stratial calmodulin (Gnegy et al., 1991), a calcium-dependent phosphoprotein which plays a key regulatory role in neurotransmitter release. In addition, prior exposure to J-amphetamine was found to increase calcium-calmodulin kinase II (CaM-KII) phosphorylation of synapsin I in the striatum (Iwata et al., 1996) and the intra-NAC application of KN-93, a CaM-KII antagonist, prevented enhanced efflux of D A by d-amphetamine in cocaine-sensitized rats (Pierce and Kalivas, 1997b). As mentioned above, reuptake by the DAT is the primary mechanism by which D A neurons limit the duration and extent of D A transmission and, therefore, may be an important mechanism by which behavior and neurochemistry are altered by long-term psychostimulant treatment. Changes in striatal DAT density or kinetics due to repeated 20 cocaine administration appear to occur in a manner which is similar to the time-dependent emergence of striatal D A release. Generally, no change was detected in studies that examined D A T number or affinity in the striatum of rats during early withdrawal (i.e., less than 10 days) after repeated cocaine administration, (Allard et al., 1990; Izenwasser et al., 1990; Y i and Johnson,1990; Peris et a l , 1990; Kula et al., 1991; Benmansour et al., 1992; Reith and Selmeci, 1992; Cass et al., 1993). If D A T binding was assessed following longer periods (i.e., greater than 10 days) after discontinuing cocaine treatment, the number of DAT binding sites was found to be reduced in the N A C shell (Pilotte et al., 1994; Kuhar and Pilotte, 1996), as well as other part of the striatum and prefrontal cortex (Sharpe et al., 1991; Farfel et al., 1992; Wilson et al., 1994). These findings may help to explain why it has been difficult to observe enhanced N A C D A release during early withdrawal times in psychostimulant-sensitized rats. Changes in the V T A that are present at longer withdrawal periods may also contribute to the long-term expression of behavioral sensitization to psychostimulants. Increases in tyrosine hydroxylase activity (Masserano et al., 1996) and tyrosine hydroxylase mRNA content (Zhang and Angulo, 1996) have been observed in the V T A 15 days following the discontinuation of repeated cocaine or methamphetamine, thereby raising the possibility that a persisting increase in D A synthesis may occur as a consequence of drug treatment. Persistent changes to DAT expression in the V T A may also accompany behavioral sensitization to psychostimulants. Pre-treatment with d-amphetamine (2.5 mg/kg/day x 5 days) was reported to induce an upregulation of D A T mRNA in the V T A which was present 7 days following discontinuation of drug treatment 21 (Shilling et al., 1997). Lu and Wolf (1997) also reported increases in D A T mRNA in the V T A during early (3-day) and late (14-day) withdrawal following repeated d-amphetamine treatment (5 mg/kg/day x 5 days) in rats. In contrast, repeated cocaine adrninistration reduced the amount of DAT mRNA in ventral midbrain neurons assessed up to 10 days following drug withdrawal (Xia et al., 1992; Cerruti et al., 1994; Letchworth et al., 1997). In summary, in order to observe robust behavioral sensitization to d-amphetamine, it is important that the administration of the drug be intermittent (i.e., repeated and non-continuous) and that a sufficiently long period of time should elapse after the last drug injection (e.g., 3 weeks) before a pharmacological (or behavioral) challenge. In this respect, long-term expression of behavioral sensitization to psychostimulants appears to be associated with enhanced M L B D A transmission; this is reflected most commonly as an augmentation of N A C D A efflux in response to a challenge, as measured by microdialysis, and may show preferential enhancement in the N A C shell. Dopamine and Incentive Motivation Mesolimbic D A neurotransmission appears to act as a primary modulator in complex integrative processes that involve the evaluation of environmental stimuli and the organization of goal-directed behaviors (Phillips et al., 1991; Blackburn et al., 1992; Kalivas et al., 1993a; Kiyatkin, 1995; LeMoal, 1995). Goal-directed, or motivated behaviors are performed for their consequences which includes gaining access to 22 valuable rewards (e.g., food, water, mates) or to avoid threatening events (e.g., predation, injury), and, collectively, they ensure the well-being of the individual or the species. Traditionally, these behaviors have been partitioned, somewhat artifically, into appetitive and consummatory components (Blackburn et al., 1992). Appetitive behaviors, which include all preparatory and anticipatory behaviors, are made in reponse to incentive stimuli that precede to the consummation of the motivated behavior. Consummatory behaviors include eating, drinking, escape from predation, and copulation. Motivated behaviors are influenced by the assignment of an incentive value to the experienced outcome of those actions in a process known as incentive learning (Dickinson and Balleine, 1995). There is a large body of evidence indicating that the M L B D A system plays an important role in the incentive motivational effects of natural rewards (e.g., food, sex), as well as addictive drugs (Fibiger and Phillips, 1986; Phillips et al., 1991; Robinson and Berridge, 1993; Kiyatkin, 1995). It has been suggested that a common characteristic of addictive drugs is their acute ability to increase M L B D A activity (Wise and Bozarth, 1987; Robinson and Berridge, 1993), a property shared by natural rewards (Pfaus et al., 1990a; Schultz et al., 1993; Wilson et al., 1995). In their "incentive-sensitization" theory of drug addiction, Robinson and Berridge (1993) brought together research from the areas of incentive motivation, neurobiology of mesotelencephlic D A systems, and behavioral sensitization, to propose a biopsychological basis for drug addiction. Specifically, they propose that during incentive learning processes, neutral stimuli associated with natural rewards are attributed with incentive salience via reward-activated D A systems. Under normal 23 circumstances, this ensures that these stimuli become attractive and wanted. Addictive drugs also increase mesotelencephalic D A activity, but in this case, the progressive enhancement in function, that occurs in D A systems as a consequence of repeated drug administration (i.e., behavioral sensitization), leads to the attribution of excessive salience to the act of drug taking and stimuli associated with the drug. This sensitization of incentive salience leads to the excessive drug wanting or "craving" which is characteristic of drug addiction (Jaffe, 1990). In addition, the theory proposes a distinction between neural systems that underly the attribution of incentive salience (i.e., mesotelencephalic D A systems) and those that mediate the hedonic qualities of reward (i.e., non-dopaminergic); further emphasizing the distinction between "wanting" and the hedonic processes related to "liking". Moreover, the neural systems underlying "wanting" and "liking" can be altered independently to the extent that excessive drug craving can occur in the absence of any pleasurable effects from the drug. Because incentive salience is attributed to drug-associated stimuli, the theory implies that the behavioral expression of activity in sensitized neural systems should come under strong associative control (Robinson and Berridge, 1993). Consistent with this prediction are studies that have demonstrated the context-dependent expression of behavioral sensitization to J-amphetamine. For example, an injection regimen that induced robust behavioral sensitization to d-amphetamine, was shown to be completely specific to the environment in which rats received repeated drug treatment (Anagnostaras and Robinson, 1996). Furthermore, Crombag et al. (1996) reported the absence of the 24 expression of behavioral sensitization when other cues associated with repeated administration of (/-amphetamine, such as handling by the experimenter in preparation for drug administration or an IP injection, were not present. A logical corollary of this "incentive-sensitization" theory is that the neuroadaptations in midbrain D A systems as a consequence of repeated and intermittent psychostimulant administration also should be manifest as enhanced motivation toward natural rewards, such as food or a receptive mate. A long-standing challenge to behavioral neuroscientists has been to construct experimental paradigms that allow meaningful inferences about incentive motivation to be made. Furthermore, such animal models should provide analagous and adequate indices of various subcomponents of the human condition. The study of the sexual behavior of the male rat has been particular^ successful in this regard (Pfaus and Everitt, 1995; Pfaus, 1996). Mechanisms of Male Sexual Behavior A l l male sexual behavior can be divided into appetitive and consummatory phases. While this separation may divide a unified behavior artificially, it has heuristic value for understanding the neural bases of male sexual behavior (Sachs and Meisel, 1988). Beach (1956) first proposed the existence of two relatively independent and consecutive mechanisms underlying the appetitive and consummatory phases of male sexual behavior. The first, a sexual arousal mechanism (SAM), mediates appetitive behavioral responses leading to the first copulatory acts of mounting and intromission. Once this copulatory threshold is attained, a second process, the intromission and 25 ejaculatory mechanism (IEM), is engaged which serves, through successive mounts and intromissions, to bring the male to ejaculation. Using factor analyses, Sachs (1978) concluded that four factors underlie male copulation: initiation, a precopulatory factor which includes latencies to mount and intromit, and three measures of copulatory rate and efficiency named copulatory rate, hit rate, and intromission rate. Whereas copulatory rate, hit rate, and intromission rate can be viewed as interactive processes related to the ejaculatory mechanism, the initiation factor closely resembles Beach's S A M (Everitt, 1990). The use of a bilevel testing chamber, introduced by Mendelson and Gorzalka (1987), yielded a new measure associated with sexual behavior in rats, known as anticipatory level changes (Mendelson and Pfaus, 1989). This represents the number of level changes made by an experienced male in the period just prior to the presentation of a receptive female. Statistical analyses of male sexual behavior in bilevel chambers proved the existence of Sach's four factors, as well as an additional factor named anticipation, which is reflected by these level changes, as well as latency to change levels, (Pfaus et al., 1990b) and these can be considered as meaures of incentive motivation (Pfaus and Everitt, 1995; Van Furth and Van Ree, 1996a,b). Manipulating sexual motivation by allowing male rats to ejaculate prior to tests of anticipatory level changes in bilevel chambers lends support to the use of this measure as an index of appetitive sexual behavior (Van Furth and Van Ree, 1996b). In this study, ejaculation prior to testing, which has been shown to decrease appetitive sexual behavior as assessed by instrumental responding under a second-order schedule of sexual reinforcement (Everitt et al., 1987), reduced the number of appetitive level changes made 26 in anticipation of a receptive female. Furthermore, the attenuation of behavior was greater when rats ejaculated twice compared to level changes made after a single ejaculation. The Role of Mesolimbic Dopamine in Male Sexual Behavior Data from lesion and pharmacological studies, as well as experiments that have monitored D A neurotransmission in vivo, support a general facilitatory role for D A in male rat sexual behavior. Lesion Studies Strong evidence for the neuroanatomical basis of the appetitive/consummatory distinction came from lesion experiments by Everitt and Stacey (1989). Excitotoxic lesions of the mPOA in male rats, while having no effect on the rates of bar-pressing for an estrous female, prevented copulation in the presence of females. Depletion of ventral striatal D A by injection of 6-OHDA increased latencies to mount and intromit, but had no effect on copulatory behavior, such as the number of mounts and intromissions (Everitt, 1990). 6-OHDA lesions of the N A C also decrease the number of anticipatory level changes during testing in bilevel chambers (Van Furth et al., 1995). Thus, lesion studies suggest that N A C D A mediates anticipatory and appetitive male sexual behavior, while the mPOA subserves consummatory aspects (Everitt, 1990; Van Furth et al., 1995). A l l lesion studies come with the inherent problem of compensatory mechanisms, within the affected area or by supporting systems, that may call the results of these experiments into question. Pharmacology 27 Pharmacological experiments conducted over the last 30 years support a facilitatory role of dopaminergic systems in male rat sexual behavior (Everitt, 1990; Melis and Argiolas, 1995; Pfaus and Everitt, 1995). Systemic injections of d-amphetamine have been found to decrease latencies to mount and intromit in intact male rats but differences relative to controls are greater in castrated rats given low-dose testosterone replacement or inexperienced rats (Bignami, 1966; Agmo and Fernandez, 1989; Agmo and Picker, 1990). Distinct effects on precopulatory behavior have been demonstrated using specific Di and D2 D A antagonists. Systemic injections of low doses of the selective Di antagonist SCH-23390 and both typical (haloperidol and pimozide) and atypical neuroleptics (clozapine and sulpiride) disrupted both anticipatory and appetitive male sexual behavior, indicated by a reduced number of level changes and increased mount and intromission latencies, respectively (Pfaus and Phillips, 1991). Haloperidol, pimozide, and SCH-23390 also affected copulation by decreasing the number of intromissions required for ejaculation. In contrast, low doses of the atypical neuroleptic clozapine and the D2-selective atypical neuroleptic sulpiride, drugs believed to affect N A C D A activity preferentially (White and Wang, 1983), did not alter copulatory measures. However, at high doses, all of these drugs abolished both level changing and copulatory behavior in most rats (Pfaus and Phillips, 1991). Local injections of D A agonists and antagonists into various brain regions provide data that complement the precopulatory/consummatory distinction male sexual behavior. Bilateral infusions of apomorphine, SCH-23390, haloperidol, or the mixed D1/D2 antagonist cis-flupenthixol into the mPOA alter both the rate and initiation of copulation, as well as genital reflexes (Hull et al., 1989; Pfaus and Phillips, 1991; Warner 28 et al., 1991). Manipulation of N A C D A appears to affect anticipatory and appetitive behaviors preferentially. Injecting apomorphine directly into the N A C was found to reduce intromission latencies (Hull et al., 1986) while intra-NAC d-amphetamine was found to increase responding for an estrous female in a dose-dependent manner, reduce mount and intromission latencies, but not alter copulation, per se (Everitt, 1990). Pfaus and Phillips (1991) reported that bilateral injections of the D2 antagonists haloperidol or raclopride into the N A C reduced level changes, while sparing consummatory measures of copulation. Control groups, including sexually naive rats tested without the presentation of receptive females and sexually experienced rats presented with non-receptive females, showed increased number of level changes across weekly tests (Van Furth and Van Ree 1996a). However, the authors recognize that no effort was made to control for residual [sexual] odors remaining from prior test sessions. It has been reported that repeated exposure to sex-related olfactory stimuli is associated with augmented M L B D A efflux over repeated testing as assessed by in vivo chronamperometry (Mitchell and Gratton, 1991). Given the role of M L B D A in anticipatory level changing behavior and the induction of behavior sensitization, this finding may explain why control groups in the study by Van Furth and Van Ree (1996a) displayed increased level changing behavior. It must be noted, however, that Mendelson and Pfaus (1989) did not report increases in level changes made by male rats presented with non-estrous female rats over repeated test sessions. The discrepencies between these two studies may have been the result of using two different strains of rats; the earlier study used Long-Evans rats, while Van Furth and Van Ree used Wistar albinos (Van Furth and Van Ree 1996a). 29 Anticipatory level change activity by male rats is dependent on olfactory stimulation. Sexually experienced rats that received an intranasal application of zinc sulphate solution to induce an olfactory deficit displayed low numbers of level changes during their first test in the bilevel chamber as well as failing to show an increase over four weekly tests (Van Furth and Van Ree 1996a). In Vivo Neurochemistry The most direct evidence for increased dopaminergic transmission during male sexual behavior comes from the use of in vivo voltammetry and microdialysis. Although reports have demonstrated increased D A efflux in the mPOA during exposure to receptive females and copulation (Hull et al, 1995; Mas, 1995), the majority of studies have examined changes in striatal D A efflux. Using differential pulse voltammetry, Mas et al. (1990) found a significant increase in electrochemical signals corresponding to D A (+60%) and the D A metabolite, dihydroxyphenylacetic acid (DOPAC), (+40%) in the N A C of copulating male rats. The D A signal remained elevated 60 min after removal of the female. Neurochemical signals were attenuated greatly when males were exposed to either non-receptive females or other males. Pleim et al. (1990) reported increases in N A C D A concentrations during sexual behavior in male rats using microdialysis. After a baseline period, receptive female rats were introduced and male rats were allowed to copulate for three consecutive 30-min periods. Copulation increased N A C D A concentrations reaching a maximal increase of 58% of baseline during the second 30-min sample. The first microdialysis experiment to address directly the role of striatal D A in appetitive and consummatory components of male sexual behavior was conducted by Pfaus et al. (1990a). There were four phases of this experiment: 1) a baseline period in 30 which the male was present on one side of a testing chamber which was divided by a wire-mesh screen; 2) a 10-min appetitive phase in which a receptive female was placed behind the screen; 3) a 30-min consummatory period which began after the screen was removed and copulation ensued; 4) an 80-rnin post-copulatory period following the removal of the female. A 30% increase in N A C D A efflux was observed during the appetitive phase which was followed by a further increase to greater than 95% above baseline during copulation. In contrast, increases in caudate-putamen D A concentrations were smaller and observed only during copulation (40% above baseline) implying a more dominant role for N A C D A during appetitive behaviors. In a carefully controlled study, Damsma et al. (1992) showed that pronounced increases in striatal D A concentrations observed could not be explained by increased motoric activity during copulation by demonstrating that forced locomotion on a rotating drum only resulted in slight increases in D A in these regions (approximately 10%). Neither prior exposure to estrous females nor copulatory experience is a necessary prerequisite for increased D A efflux in males during exposure to sex-related olfactory stimuli or copulation. A significant increase in the voltammetric signal corresponding to N A C D A was observed when sexually naive males were placed in cages which previously contained receptive females (Louilot et al, 1991). This increase was much larger than when the cages had previously contained non-receptive females, intact males, or no animals. Concentrations of D A in microdialysis samples obtained from the N A C of sexually naive male rats increased significantly (to 135% of baseline) during copulation with receptive females (Wenkstern et al., 1993). Male rats exposed to non-receptive females and sexually sluggish rats exposed to receptive females also 31 showed increases in N A C D A (to 126% and 127% of baseline, respectively), but they were not significant. As indicated above, there is evidence for increased N A C D A efflux during both appetitive and consummatory aspects of male sexual behavior. In fact, copulation-associated increases in N A C D A efflux are greater than those occurring in appetitive phases. These findings have led to the suggestion that M L B D A is involved in both appetitive and consummatory motivated behavior (Fibiger, 1993; Wilson et al., 1995). Further, the preferential disruption of appetitive behaviors by neuroleptics may be the result of sub-effective concentrations of competitive D A antagonist required to overcome D A efflux during the consummatory phase of behavior (Fibiger, 1993). Recently, support for M L B D A in the reinitiation of sexual behavior was provided by a microdialysis study which monitored N A C D A during the Coolidge effect in the male rat (Fiorino et al., 1997a). The Coolidge effect describes the reinitiation of sexual behavior in a "sexually satiated" animal in response to a novel receptive mate. In agreement with earlier reports, both the presentation of an estrous female behind a screen and copulation were associated with significant increases in N A C D A efflux (44% and 95%, respectively). The return of N A C D A concentrations to baseline values coincided with a period of sexual satiety, although concentrations of the D A metabolites, D O P A C and homovanillic acid (HVA), remained elevated. The presentation of a novel receptive female behind a screen resulted in a slight increase in N A C D A (12%) which was augmented significantly during renewed copulation with the novel female (34%). The results of this experiment suggested that the incentive stimulus properties of a novel receptive female may serve to increase N A C D A transmission in a "sexually satiated" 32 male rat and this, in turn, may be related to the reinitiation of sexual behavior. Cross-sensitization Between Drugs and Sexual Behavior Given the extensive literature indicating that long-term administration of psychostimulants, sufficient to induce behavioral sensitization, can augment M L B D A transmission (Kalivas and Stewart, 1991; Kalivas et al., 1993a; Pierce and Kalivas, 1997a), sensitization of this system may also enhance its function to modulate the motivational value of natural incentive stimuli (Kalivas et al., 1993a; Robinson and Berridge, 1993) resulting in a facilitation of motivated behavior. The study of sexual behavior provides a unique opportunity to examine this hypothesis as it permits experimental control over the subjects' motivational history. The use of naive rats would remove the influence of conditioned stimuli, thus providing a much greater degree of control than could be achieved with alternative rewards, such as food or water, which are consumed on a daily basis. Male sexual behavior can be analyzed in terms of appetitive/motivational (i.e., leading to copulation) and consummatory (i.e., copulation) components and indices of appetitive motivation include latencies to mount and intromit, the percentage of animals that copulate, and activity associated with anticipation of the receptive female in bilevel testing chambers (Mendelson and Pfaus, 1989; Pfaus et al., 1990b). Progressive changes in these measures occur and rates of acquisition can be assessed as naive animals gain sexual experience (Everitt, 1990; Pfaus and Phillips, 1991). Sexual-inexperience also may increase the probability of observing facilitated sexual behavior after a treatment such as repeated exposure to psychostimulants because many measures (e.g., latency to mount) reflect a 33 maximal level of performance in experienced copulators. Sexual behavior differs from other natural motivated behaviors (e.g., feeding, drinking) in some fundamental ways: it does not serve to maintain essential physiological/metabolic balances nor is it influenced by a state of deprivation, and it does not serve to improve the well-being of the individual (Toates, 1986). External incentives (e.g., the attractiveness of a prospective mate) and the arousability/sensitivity of the nervous system are the two main factors which influence sexual motivation. Therefore, sexually naive male rats may be more sensitive to the effect of psychostimulant-induced sensitization on incentive-motivation because proceptive cues from the receptive female play a more prominent role in the initiation of sexual behavior in these animals (Beach, 1941; Madlafousek and Hlinak, 1983). Mitchell and Stewart (1990) demonstrated that sexually experienced castrated rats, given a low replacement dose of testosterone, exhibited facilitated sexual behavior as indicated by reduced mount and intromission latencies when tested in the same environment in which they received systemic morphine injections. While they proposed that the conditioning state induced by stimuli associated with opiate reward were responsible for facilitated sexual behavior, it is interesting to note that the injection regimen used in this experiment (4 injections of 10 mg/kg morphine sulfate, one every other day) may induce behavioral sensitization to the locomotor effects of opiates (Kalivas and Stewart, 1991). Moreover, there is strong evidence that the expression of behavioral sensitization is context-dependent (Kalivas and Stewart, 1991; Stewart and Badiani, 1993; Anagnostaras and Robinson, 1996; Robinson et al., 1998) which may explain why rats tested for sexual behavior in an environment different from where they 34 received morphine injections did not display facilitated sexual behavior. There are also some clinical data that suggest drug-sex cross-sensitization. Over 70% of patients admitted into a prominent cocaine addiction treatment program were found to also suffer from compulsive sexuality (Washton and Stone-Washton, 1993) and a number of studies have reported the occurrence of high sexual drives in (/-amphetamine addicts, although it was suggested that drug use augmented an already "disturbed" sexual pattern (Bell and Trethowan, 1961; Ellinwood, 1967; Greaves, 1972). Overview of the Present Experiments Experiments in this thesis utilized a "cross-sensitization" procedure to examine how a drug treatment, known to sensitize the M L B D A system, affected a natural motivated behavior. Specifically, the effect of (/-amphetamine-induced behavioral sensitization on male sexual behavior was investigated. Each experiments consisted of two main parts: 1) induction of behavioral sensitization; and 2) tests of sexual behavior. Special consideration was given to appetitive and consummatory components of male sexual behavior as well as the role of environmental conditioning. The use of in vivo microdialysis to monitor dynamic changes in M L B D A transmission in freely behaving animals is well established (Pfaus et al., 1990a; Fiorino et al., 1993; Finlay and Zigmond, 1995; Fiorino et al., 1997a) and this technique was applied to measure changes to increases in neurotransmission associated with sexual behavior as a consequence of prior drug treatment. Experiment I examined the effect of repeated and intermittent (/-amphetamine on the sexual behavior of naive male rats tested in bilevel chambers. Repeated testing 35 afforded the opportunity to examine the effect of prior d-amphetamine exposure on various components of male sexual behavior during the acquisition of sexual behavior. A l l injections and behavioral testing were conducted in bilevel chambers to keep environmental cues constant and to gain another measure of incentive motivation prior to sexual behavior: namely, anticipatory level changes (Pfaus and Phillips, 1991; Pfaus and Everitt, 1995). Experiment II investigated the role of conditional factors in the effect of d-amphetamine-induced behavioral sensitization on male rat sexual behavior. As mentioned above, conditional factors have been shown to play an important role in the development and expression of behavioral sensitization (Stewart and Badiani, 1993; Anagnostaras and Robinson, 1996; Robinson et al., 1998). Experiment II examined whether d-amphetamine-induced behavioral sensitization could facilitate male sexual behavior irrespective of conditioned associations between the testing environment and drug administration. This was accomplished by administering d-amphetamine injections in the unilevel chamber, an apparatus which was different from home cages and the bilevel chamber used, subsequently, to test sexual behavior. In Experiment III, the effect of repeated and intermittent d-amphetamine on male rat sexual behavior was assessed in unilevel chambers. Although bilevel chambers provide valuable information about sexual motivation, the layout of the apparatus makes it impossible to conduct a microdialysis experiment. Experiment III sought to determine whether earlier findings about the effect of repeated and intermittent administration of d-amphetamine on sexual behavior could be reproduced in unilevel chambers prior to conducting a microdialysis experiment. 36 Once it was established that facilitation of male sexual behavior due to repeated (/-amphetamine administration could be replicated in unilevel chambers, Experiment IV was conducted to monitor changes in N A C D A efflux in sexually inexperienced male rats previously sensitized to (/-amphetamine. Increased D A efflux in the N A C of male rats after the presentation of a receptive female or during copulation is robust and has been replicated in this laboratory and that of others (Damsma et al.,1992; Melis and Argiolas, 1995; Fiorino et al., 1997a). This response also has been demonstrated in sexually inexperienced male rats (Wenkstern et al., 1993). Experiment IV also examined whether any changes in appetitive or consummatory sexual behavior due to (/-amphetamine-sensitization were associated with changes in N A C D A efflux. G E N E R A L M E T H O D Subjects Male Sprague-Dawley rats (225-250g) were obtained from the U B C Animal Care Centre and housed individually in plastic cages with paper bedding (Carefresh, Absorption Corp., Bellingham, WA), except in Experiment I when wire-mesh cages were used. The colony room was maintained on a reverse light-dark cycle (lights off 7a.m.-7p.m.). Ambient temperature was approximately 20° C and rats had unlimited access to food and water. Female Long-Evans rats (Charles River Canada, Inc., St. Constant, Quebec) were housed in a separate colony room on the same reverse light-dark cycle. Rats were bilaterally ovariectomized under halothane gas anesthesia (Fluothane, Ayerst 37 Laboratories) at least four weeks prior to testing. Sexual receptivity in the stimulus females was induced by subcutaneous injections of estradiol benzoate (10 pg) and progesterone (500 pg), 48 h and 4 h, respectively, before each test session. A l l females were sexually experienced before tests of sexual behavior were initiated. Testing occurred during the middle third of the dark cycle. These studies were conducted according to national guidelines on the care and use of experimental animals (Canadian Council on Animal Care, 1993). 38 Apparati Two testing apparati were used in the present experiments: bilevel and unilevel chambers. Bilevel chambers (Figure 2) have been described previously (Mendelson & Gorzalka, 1987). Briefly, these chambers (51 x 60.5 x 15 cm) consisted of two platforms (length, 30.5 cm), one approximately 28 cm above the other. On both ends of the platforms, a set of ramps and narrow landings allowed rats to move from one level to the other. Crosses over the midline of either of the two main levels of the chamber, or vertical movements between either platform and the narrow landings were each scored as an activity count. Unilevel chambers (48 x 24 x 35 cm) were bisected by a removable transparent Plexiglas partition (32 cm) creating two 12-cm wide alleys along the length (Figure 3). These chambers were approximately the same size as the bilevel chambers in terms of floor area, but had only one floor surface. Rats could move freely between each side on both ends of the chamber. An antechamber (24 x 8 x 16 cm), which was used to hold an estrous female in Experiments III and IV, was located at one end of the apparatus and separated from the main body of the chamber by a wire mesh screen. Infrared photobeam emitters/detectors allowed the number of activity counts (movements from one side of the partition to the other) to be monitored automatically by a computer (2 Hz scan rate). In order to minimize the effect of residual rat odors on subsequent tests of activity or sexual behavior and to make the odor of each type of environment distinct, testing apparati were cleaned with dilute solutions of Windex (bilevel chambers) or detergent (unilevel chambers). Figure 2. Diagram of bilevel testing chamber. Front view. 40 60.5 cm 41 Figure 3. Diagram of unilevel testing chamber. 42 Top 48 cm 43 Surgery (Experiment III and IV) Male rats were anesthetized with ketamine hydrochloride (100 mg/kg, IP) and xylazine (10 mg/kg, IP) prior to stereotaxic surgery. Microdialysis probe guide cannulae (19 ga.) were implanted over the N A C (coordinates from Bregma: anterior +1.7 mm; medial -1.1 mm; ventral -1.0 mm; flat skull), and secured to the skull with dental acrylic and jeweler's screws. A 19 ga. wire "training post" was cemented on top of the skull behind the guide cannulae. Induction of Behavioral Sensitization Male rats were assigned randomly to either the repeated d-amphetamine (AMPH) or saline control (CONT) groups. Rats were placed into testing chambers and, after a 30-min habituation period, given IP injections of either d-amphetamine sulfate (1.5 mg/kg; Smith-Kline Beecham, Oakville, ON) or saline vehicle (1 mL/kg; Baxter Corp., Toronto, ON). Two hours after the injection, rats were returned to their home cages. Injections were given once every two days for a total of 10 injections. Behavior associated with the first and tenth injections was videotaped for subsequent scoring of activity, i f necessary, by an observer blind to treatment condition. Activity counts were collected in 10-min bins and a significant increase in total activity counts over 2h post-injection from the first to the tenth test was taken as evidence for behavioral sensitization. Previous reports indicate that a single administration of d-amphetamine may be sufficient to induce behavioral sensitization (Robinson, 1984; Warburton, 1996); therefore, to ensure that the CONT group would remain unsensitized, an amphetamine challenge was not administered prior to the assessment of sexual behavior. 44 Sexual Behavior A number of studies have reported that behavioral sensitization is more robust when conducted 21 days or more after the discontinuation of drug treatment (Antelman, 1988; Paulson and Robinson, 1995; Pierce and Kalivas, 1997a). Therefore, male rats in the present study were tested for sexual behavior 21 days after the tenth injection. Each test of copulation was 30 min in duration. A l l sexual behavior was videotaped. Subsequently, a computer and appropriate software (courtesy of Sonoko Ogawa, Rockefeller University) were used to record standard measures of consummatory sexual behavior: 1) mount frequency (MF), 2) intromission frequency (IF), 3) ejaculation frequency (EF), 4) mount and 5) intromission latencies (ML and IL; the time (s) from the presentation of the female to the first mount or intromission), 6) ejaculation latency (EL; time (s) from the first intromission to the first ejaculation), 7) number of intromissions to the first ejaculation (IEi), 8) post-ejaculatory interval (PEI; the time (s) from the first ejaculation to the next intromission), 9) inter-intromission interval (III; EL/ IEi), and 10) the intromission ratio (IR; IF/( M F + IF)). Criteria for mounts and intromissions are described in Sachs and Barfield (1976). Microdialysis and HPLC-EC detection (Experiment IV) Rats were implanted with microdialysis probes 12-18 h prior to the experiment and placed in the testing chamber with free access to food and water. On the morning of the experiment, microdialysis samples were collected every 10 min. The experiment consisted of four phases: 1) baseline (at least 60 min); 2) estrous female behind the screen (10 min); 3) copulation with estrous female (30 min); and 4) a post-copulation 45 interval (60 min). Microdialysis probes were concentric in design with a semi-permeable hollow fiber membrane (2 mm membrane exposed; 340 pm o.d., 65 000 M.W. cut-off, Filtral 12, Hospal) at the distal end. Probes were perfused at 1.0 pL/min with a modified Ringer's solution (0.01 M sodium phosphate buffer, pH 7.4, 1.3 m M CaCl 2 , 3.0 m M KC1, 1.0 m M MgCh, 147 m M NaCl) using a gastight syringe (Hamilton) and a syringe pump (Model 22, Harvard). A microdialysis probe guide collar was used to secure the microdialysis probe inside the guide cannula. A steel coil, attached to a liquid swivel (Instech 375 s) that was mounted on top of the testing chamber, was used to protect the probe tubing (see Fiorino et al., 1993 for details). Microdialysate analytes, which included D A and its metabolites DOPAC and H V A , were separated by reverse-phase chromatography (Beckman ultrasphere column, ODS 5 pm, 15 cm, 4.6 mm i.d.) using a 0.083 M sodium acetate buffer (pH 3.5, 5% methanol). Analyte concentrations were quantified by EC detection. The apparatus consisted of a BioRad pump, a Valco Instruments 2-Position injector (EC10W), an ESA Coulochem II EC detector, and a dual channel chart recorder (Kipp and Zonen). Electrochemical detector parameters were: electrode 1, +450 mV; electrode 2, -300 mV; and guard cell, -450 mV. Unfortunately, at the time experiments were conducted, the detection apparatus was not sensitive enough to quantify D A concentrations in the mPOA reliably. Thus, only microdialysis samples from Experiment IV were analyzed. Typical probe recoveries, conducted in vitro and at room temperature, were: for D A , 20.0 ± 0.9%; DOPAC, 15.2 ± 0.9%; and H V A , 14.2 ± 0.6%. 46 Following the microdialysis experiment, animals were given an overdose of chloral hydrate and perfused intracardially with saline and formalin (4%). Frozen brains were sliced and coronal sections were stained with cresyl violet to determine placement of microdialysis probes. Rats with probe placements within the N A C in Experiment IV were used for behavioral and neurochemical analyses. Statistics Activity counts and measures of sexual behavior were assessed using analyses of variance (ANOVAs). When a significant main effect was obtained, between-groups comparisons were made using simple main effects analyses. Within-groups post-hoc comparisons of total activity counts were made using the Newman-Keuls test. Neurochemistry was analyzed in the same manner except that Dunnett's test was used to make within-group comparisons from control means. A separate statistical analysis of sexual behavior data employed Kaplan-Meier plots of both M L and IL which were assessed by survival analyses (Bloch et al., 1993; Liu et al., 1997). The latencies to mount and intromit and the percentage of subjects showing these behaviors are measures of motivational components of sexual behavior. Both of these measures are utilized in Kaplan-Meier plots, generated by plotting latencies against the percentage of rats that showed the behavior within the test. This procedure has the added advantage that data from subjects who fail to mount or intromit are not omitted or given an arbitrary value. Analyses were performed using SPSS and Statistica statistical software packages. 47 EXPERIMENT I: THE EFFECT OF REPEATED A N D INTERMITTENT d -AMPHETAMINE ADMINISTRATION ON M A L E RAT S E X U A L B E H A V I O R Behavioral sensitization to psychostimulants is the product of changes which involve a number of different neurotransmitters and nuclei within complex limbic-motor circuitry resulting in a persistent augmentation of function within this system (Kalivas et al., 1993b; Robinson and Berridge, 1993; Self andNestler, 1995; Pierce and Kalivas, 1997a). The limbic-motor circuitry involved in the initiation and expression of behavioral sensitization is known to play an important role in natural motivated behaviors, such as exploration, feeding, drinking, and sexual behavior (Phillips et al., 1991; Kalivas et al., 1993a; Robinson and Berridge, 1993; Pennartz et al., 1994; Robbins and Everitt, 1996). A logical corollary of this relationship is that sensitization induced by repeated psychostimulant administration should be manifest as enhanced motivation toward natural rewards. Experiment I examined this hypothesis by determining if d-amphetamine-induced behavioral sensitization can facilitate sexual behavior in sexually inexperienced male rats tested in a drug-free state. Anticipatory measures of sexual motivation were assessed during repeated testing in bilevel chambers which also facilitated the scoring of copulatory behavior (Mendelson and Gorzalka, 1987; Mendelson and Pfaus, 1989; Pfaus etal., 1990b). As mentioned in the introduction, the study of sexual behavior provides a good 48 opportunity to examine this hypothesis because it permits experimental control over the subjects' motivational history. In addition, there are a number of good measures of appetitive/motivational, as well as consummatory, components of male rat sexual behavior. Indices of appetitive motivation include latencies to mount and intromit, the percentage of animals that copulate, and activity associated with anticipation of the receptive female in bilevel testing chambers (Mendelson and Pfaus, 1989; Pfaus et al., 1990b). Sexually naive male rats may be more sensitive to the effect of psychostimulant-induced sensitization on incentive-motivation because proceptive cues from the receptive female play a more prominent role in the initiation of sexual behavior in these animals (Beach, 1941; Madlafousek and Hlinak, 1983). In this respect, latencies to mount and intromit are the most likely to be altered by prior (/-amphetamine treatment. As male rats gain sexual experience, however, there is a progressive decrease in latencies to mount and intromit, to the point that these measures reach minimal values that reflect a maximal level of performance. Thus, sexual experience may cloud facilitatory effects due to drug-induced sensitization. Monitoring of anticipatory level changes can provide another index of appetitive sexual behavior which may be enhanced, and observed, in psychostimulant-treated, sexually experienced rats. In this manner, progressive changes that occur in appetitive measures as naive animals gain sexual experience (Everitt, 1990; Pfaus and Phillips, 1991) can be monitored, and rates of acquisition as a consequence of prior (/-amphetamine treatment can be assessed. The main hypothesis addressed in the present experiment is that (/-amphetamine administration, a treatment that results in a long-term augmentation of function in limbic-motor circuitry, would facilitate male sexual behavior, a natural motivated behavior 49 dependent upon the integrity of this circuitry. The question of whether d-amphetamine-induced behavioral sensitization alters the appetitive or consummatory aspects of sexual behavior differentially was also addressed. Method Subjects were obtained and housed as outlined in the General Methods section. A l l tests of activity and sexual behavior were conducted in bilevel chambers. Male rats in the A M P H group (n=20) and CONT groups («=20) received injections according to the Induction of Behavioral Sensitization paragraph in the General Methods section. Following the 21-day abstinence period, rats were given 10 tests of sexual behavior, conducted once every 4 days. Each test session was 35 min in duration. Male rats were placed into the testing chamber for a 5-min test of activity after which a sexually receptive female was presented for the remaining 30 min. Behavior during the 5-min pre-copulatory period was scored for the number of level changes (LC), defined as a complete movement from one of the main levels of the test chamber to the other. Copulatory behavior was scored as indicated in the Sexual Behavior paragraph of the General Methods section. Results Activity Over the course of the 10-injection regimen, A M P H rats displayed a progressively sensitized activity response to d-amphetamine administration (Figure 4). A repeated measures A N O V A on the total number of activity counts over a 2 h post-injection 50 Figure 4. Effect of repeated d-amphetamine (AMPH) or saline (CONT) injections on activity in Experiment I. The data are represented as mean (± SEM) activity counts. A . The line graphs represent the activity profile after injection 1 (upper panel) and injection 10 (lower panel). B. The histogram represents cumulated activity counts after injections 1 and 10 for each group. */?<0.05, ***/><0.001, using simple main effects analysis. J/K0.001, using Newman-Keuls post-hoc test. A. u 1 o 1—1 > PQ 15 o U • i—i > •1—I -•-> o 120 -100 -80 60 40 " T T 20 " o -Injection 1 + - A -T - A - ~ A -120 100 80 60 40 20 0 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection 10 1 i i S A — - A A-~ A A 51 - A - CONT - • - AMPH 0 10 20 30 40 50 60 70 80 90 100 110 120 Injection Number 52 interval revealed a significant interaction between group and injection number [F(l,37)=13.87;p<0.0l]. Simple main effects analyses yielded significant differences between groups for injection number 1 [F(l,37)=6.06;p<0.05] and number 10 [F(l,37)=22.81; /K0.001]. Subsequent Newman-Keuls tests revealed a significant increase in the total number of activity counts across injections in the A M P H group [p<0.001], confirming that sensitization had occurred. One rat in the A M P H group was sacrificed due to i l l health; activity and sexual behavior data from this animal was excluded. Sexual behavior Rats in both A M P H and CONT groups increased the number of anticipatory level changes with sexual experience (Figure 5). A significant interaction of group x test number [F(l,37)=8.22; p<0.0\] was obtained and Newman-Keuls post-hoc tests revealed significant increase in level change scores between Test 1 and 10 within both groups. More importantly, the A M P H group made significantly more level changes than the CONT group on Test 10 as indicated by a simple main effects analysis [F(l,37)=7.791; /?<0.001]; this implies that d-amphetamine treatment enhanced the attribution of incentive value to cues associated with the expectation of a receptive female. During Test 1, the CONT and A M P H groups displayed similar level change scores in the 5 min prior to the presentation of a receptive female, indicating that general activity did not increase in the A M P H group in anticipation of an amphetamine injection. Table 1 summarizes measures of sexual behavior for both groups during Tests 1 and 10 in Experiment I. A M P H rats displayed facilitated sexual behavior during Test 1 as indicated by significantly shorter latencies to mount [F(l,27)=5.12;p<0.05]. By Test 53 Figure 5. Mean number of level changes (± SEM) in the five minute period before presentation of the receptive female during test 1 and 10 of sexual behavior by rats sensitized to (/-amphetamine (AMPH) or non-sensitized control rats (CONT) in Experiment I. */K0.05, ***/K0.001, using simple main effects analysis. {pO.OOl, using Newman-Keulspost-hoc test. 54 i Test Number 55 Table 1 Measures of sexual behavior from Experiment I. Data are expressed as the mean ± S E M . TEST 1 TEST 10 CONT A M P H CONT A M P H L C 7.5 ± 1.0 7.0 ± 0.7 9.4 ± 1.3f 12.5 ± 1.0***$ M L (s) 332.7 ± 87.3 162.3 ±24.5* 219.1 ± 125.7 150.1 ±93.6 IL(s) 493.6 ± 137.1 315.8 ±78.1 281.4 ± 141.2| 161.1 ±98.8 EL (s) 636.4 ± 145.1 761.3 ±95.0 446.3 ±91.3 543.1 ± 132.5+ PEI (s) 283.3 ± 12.8 308.3 ± 16.9 387.7 ± 68.0 333.8 ±20.5 MF 3.8 ± 1.1 11.0 ± 1.8** 14.7 ±2.3f 15.9 ±2 .2 IE, 9.3 ± 1.9 11.4 ± 1.1 9.7 ± 0.9 10.3 ±0 .9 IF 5.8 ± 1.8 12.6 ± 1.9* 13.9 ± 2.3f 16 .8±2 .4 t EF 0.7 ± 0.2 2.0 ± 0.9* 1.6 ±0 .3 2.1 ±0 .3 III 71.4 ±9.5 69.5 ± 8.4 49.9 ± 12.0| 54.1 ± 13.8| IR 0.49 ±0.10 0.53 ± 0.06 0.44 ± 0.06 0.46 ± 0.05 Abbreviations: L C , level changes; M L , mount latency; IL, intromission latency; E L , ejaculation latency; PEI, post-ejaculatory interval; M F , mount frequency; IE,, intromissions before the first ejaculation; IF, intromission frequency; EF, ejaculation frequency; III, inter-intromission interval; IR, intromission ratio. Different than CONT group for a given measure: */?<0.05, **/?<0.01, ***/?<0.001 by simple main effects analysis. Within-group comparisons across tests in Experiment 1: t/><0.05, $p<0.001 using Newman-Keulspost-hoc test. 56 10, there was no significant difference in M L between groups. Survival analysis of latencies to mount (Figure 6) yielded a similar result showing a facilitation of sexual behavior in the A M P H group for Test 1 [Log Rank statistic = 7.42, p=0.0064], but not Test 10. A simple main effects A N O V A did not reveal a significant group effect in IL during either Test 1 or 10. However, survival analyses did reveal a significant enhancement of the IL measure of sexual behavior on Test 1 (Figure 6) [Log Rank statistic = 9.93, p=0.0016]. A decrease in IEi is interpreted as a facilitation of rat sexual behavior, but, in this study, there was no difference in IEi measures between groups. The A M P H group displayed a significantly greater number of mounts [F(l,37)=12.74; p<0.0l] and intromissions [F(l,37)=7.07;p<0.05] than CONT subjects in Test 1 and these data reflect an increase in the number of bouts of copulation, as indicated by significantly more ejaculations by the A M P H group [F(l,37)=5.02; p<0.05]. In previous experiments, we have used the criteria of M L and IL < 300 s and E L < 900 s, for three consecutive days, as indices of efficient copulatory performance in a sexually experienced male rat (Fiorino et al., 1995). Independent A N O V A s conducted on measures of the number of test days required to achieve these criteria revealed that rats in the A M P H group displayed a facilitated acquisition of sexual behavior because they reached this level of performance more quickly than CONT rats (Figure 7) for M L [F(l,32)=6.56;/K0.05] and IL [F(l,31)=8.10; p<0.0\]. Furthermore, a greater percentage of rats in the A M P H group reached two of these criteria during Test 1 relative to CONT rats (ML, 74% vs 30%; IL, 63% vs 20%). Rats from both groups improved their sexual performance over the ten test 57 Figure 6. Effect of d-amphetamine or saline pre-treatment and sexual experience on latencies to mount and intromit in Experiment I. A . Kaplan-Meier curve for mount latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 7.42, p=0.0064). B. Kaplan-Meier curve for intromission latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 9.93,^=0.0016). A. --A-- CONT-Test 1 - • - AMPH-Test 1 -O- CONT-Test 10 - • - AMPH-Test 10 100 f 8 0 | o fl o 3 60 • • P 4 « 4 0 1 O £ 20 0 - o -- o --A-,-A' • • / A ' ' A A ,-A TA' 4> fA ' 1 r 0 300 600 900 1200 1500 1800 Mount Latency (s) B. 100 too | 80 • o 60 W « 4 0 O P 20 0 , - o -•O' P / - f A' O • .A (i "A' A-_ - - A - A " ,-A' 0 300 600 900 1200 1500 1800 Intromission Latency (s) 59 Figure 7. Acquisition of sexual behavior in sensitized (AMPH) and non-sensitized (CONT) rats in Experiment I. Data are expressed as the mean number of days (± SEM) to reach a criterion of three consecutive days where M L < 300s, IL < 300s, or E L < 900s. */?<0.05, **/?<0.01 using simple main effects analysis. 60 CONT A M P H M L < 300 s IL < 300 s E L < 900 s Behavioral Criteria 61 sessions. Specifically, there was a significant reduction in latencies to mount, intromit, and ejaculate. In addition, there were increases in the amount of sexual activity as indicated by a greater number of mounts and intromissions during Test 10 as compared to Test 1. Finally, there was a significant decrease in the III over tests in both groups. Graphed data for all tests of sexual behavior are found in Appendix A . Discussion Repeated and intermittent (/-amphetamine administration, sufficient to induce behavioral sensitization, facilitated sexual behavior in the naive male rat. This is the first demonstration that psychostimulant-induced behavioral sensitization, linked to functional changes in limbic-motor circuitry, can influence natural motivated behaviors displayed when rats are in a drug-free state. In the present study, although both appetitive and consummatory measures of sexual behavior were facilitated by prior exposure to (/-amphetamine, the data suggest that appetitive/motivational components were affected to a greater degree (e.g., decreased M L and IL in naive rats, increased LCs after sexual experience). Similar results were reported by Mitchell and Stewart (1990) in a study that examined the effect of repeated pairings of systemic administration of morphine with the test chamber in which male rats were tested subsequently for sexual behavior. It is also clear that the overall amount of copulation as indicated by M F , IF, and EF was increased in A M P H rats. However, sensitization had no effect on consummatory measures within bouts of copulation. For instance, IEj, III, IR, EL , and PEI did not differ between groups. This suggests that increased copulation over the test session in the 62 A M P H group was secondary and possibly a consequence of a preferentially enhanced appetitive mechanism. If a sensitized M L B D A system is contributing to these behavioral changes, it supports a preferential role for this pathway in appetitive behavior and the attribution of incentive salience to a reward (Robinson and Berridge, 1993). Pre-treatment with d-amphetamine also facilitated the acquisition of sexual behavior as indicated by criteria that reflect efficient appetitive sexual behavior in experienced rats (Figure 7). This index is based on how long it took A M P H and CONT groups to meet mounting and intromitting criteria for three consecutive days. Therefore, it is clear that the A M P H group met the criteria more quickly than the CONT group, and in slightly over the minimum number of days required (i.e., 3 days). Nevertheless, d-amphetamine-sensitization did not guarantee that all rats would copulate during their first test of sexual behavior. After repeated testing, however, 100% of rats in the A M P H group did copulate. Our results are consistent with the incentive-sensitization theory of drug addiction (Robinson and Berridge, 1993). This theory proposes that repeated activation of mesotelencephalic D A transmission by psychostimulants, and other addictive drugs, sensitizes this neural system and thereby enhances its main function, which is the attribution of "incentive salience" to stimuli associated with activation of the system. In the present study, there was evidence for attribution of incentive salience and incentive learning after psychostimulant-induced sensitization, as indicated by a greater increase in the number of anticipatory level changes made by the A M P H group relative to CONT rats as they gained copulatory experience. This implies that changes in limbic-motor circuits due to repeated psychostimulant administration, and their behavioral 63 consequences, can be modified further by associative stimuli subsequent to the development of sensitization. Sensitization was not by itself sufficient to produce maximal sexual responding; experience and learning also played a major role. EXPERIMENT II: THE R O L E OF CONDITIONING FACTORS IN THE EFFECT OF d-AMPHETAMINE-INDUCED B E H A V I O R A L SENSITIZATION O N M A L E RAT S E X U A L BEHAVIOR In Experiment I, prior intermittent exposure to J-amphetamine facilitated behavior in the sexually naive rat tested in a drug-free state. One important question that must be addressed is the role of conditioning factors in the expression of this phenomenon. A conditioned association between the drug and cues in the testing environment may have led to enhanced sexual behavior observed in Experiment I which may be independent of a sensitization-like processes. Mitchell and Stewart (1990) reported that sexual behavior in male rats was enhanced when tests were conducted in the same environment in which the animals received systemic morphine injections. As in Experiment I, facilitation was restricted to appetitive measures of sexual behavior including increases in anogenital exploration, pursuit activity, and shorter latencies to mount the female, rather than changes in copulation proper. They proposed that a conditioned state induced by stimuli associated with opiate reward was responsible for facilitated sexual behavior (Mitchell and Stewart, 1990) 64 Many authors (Vezina and Stewart, 1984; Robinson et al., 1998) have demonstrated that environmental cues can exert a powerful effect over behavioral sensitization, to the extent that the absence of such cues can prevent its expression. It has been suggested, however, that this context-dependence may play a lesser role when the circumstances surrounding the development of behavioral sensitization are highly arousing, such as in novel environments (Badiani et al., 1995a,b). The present experiment investigated whether prior exposure to d-amphetamine could facilitate sexual behavior in sexually inexperienced male rats, irrespective of the context in which the drug was administered. Male rats were injected according to the same protocol with respect to dosage and intermittency that was used in Experiment I. In this experiment, however, rats received all injections in a unilevel test chamber that was distinct from both bilevel chambers and home cages, with respect to tactile, visual, and odor cues. A positive finding would rule out a significant role for environmental cues in the effect of d-amphetamine-induced sensitization on subsequent sexual behavior. In contrast, a negative finding would leave open the relative contributions of conditioning effects due to drug-environment association and the control of the expression of sensitization by conditioned cues. Method Rats were obtained and housed as outlined in the General Methods section. In order to control for the effects of conditioning due to environment-drug pairing, both A M P H («=10) and CONT («=10) rats received injections in unilevel chambers according to the regimen outlined in the General Methods section. The change in environment was 65 achieved by testing rats in the A M P H and CONT groups for sexual behavior in bilevel chambers; a single test, identical in design to those of Experiment I, was conducted 21 days after the tenth injection. Copulatory behavior was scored as indicated in the Sexual Behavior paragraph of the General Methods section. Results Activity Behavioral sensitization to (/-amphetamine was observed when rats in the A M P H group were tested in unilevel chambers (Figure 8). A significant interaction between group and injection number [F(l,18)=12.44;p<0.01] was observed on the total activity counts post-injection. Subsequent simple main effects analyses indicated that (/-amphetamine produced significant increases in total activity counts relative to vehicle treatment after the first [F(l,18)=41.93;/?<0.001] and tenth injection[F(l,18)=51.10; /K0.001]. Most importantly, rats in the A M P H group displayed a sensitized behavioral response to (/-amphetamine as indicated by a significant increase in the total number of activity counts from injection 1 to injection 10 [Newman-Keuls; /K0.001]. Sexual behavior The facilitation of sexual behavior following repeated injections of (/-amphetamine was unaffected by a change in test environment to the bilevel chamber from the unilevel chamber employed for drug injections. Table 2 summarizes the measures of sexual behavior in A M P H and CONT groups in Experiment II. Again, the A M P H group displayed enhanced sexual behavior as indicated by a significantly shorter M L : A N O V A [F(l,15)=4.58;/K0.05]; survival analysis [Log Rank statistic = 7.91, 66 Figure 8. Effect of repeated d-amphetamine (AMPH) or saline (CONT) injections on activity in Experiment II. The data are represented as mean (± SEM) activity counts. A . The line graphs represent the activity profile after injection 1 (upper panel) and injection 10 (lower panel). B. The histogram represents cumulated activity counts after injections 1 and 10 for each group. *p<0.05, ***p<0.001, using simple main effects analysis. Jp<0.001, using Newman-Keuls post-hoc test. A. B. 1 U 13 > D • i—i S3 O U • i—i > • i-^ o < 50 40 30 20 10 0 50 40 30 20 10 0 I n j e c t i o n 1 1 A -£A " " — A i i 1 1 1 1 r 1 — 0 10 20 30 40 50 60 70 80 90 100 110120 I n j e c t i o n 10 U U I 1 N U i i =4= =4= 0 10 20 30 40 50 60 70 80 90 100 110 120 T i m e ( m i n ) 67 CONT AMPH •<-> fl O U • i—i • o < Is CONT AMPH I n j e c t i o n N u m b e r 68 Table 2 Measures of sexual behavior from Experiment II. Data are expressed as the mean ± S E M . CONT A M P H L C 2.5 ± 1.1 3.2 ± 0.9 M L (s) 146.9 ±41.8 73.5 ± 15.0* IL(s) 246.8 ± 106.7 95.3 ± 22.7 EL (s) 517.8 ±78.2 448.2 ± 122.6 PEI (s) 758.8 ± 160.6 543.5 ± 124.0 M F 5.3 ± 1.7 8.1 ±2 .3 IE, 6.4 ± 1.3 7.0 ±0 .8 IF 7.6 ±2 .4 13.7 ±0 .9* EF 1.1 ±0 .4 2.5 ±0.2** III 96.2 ± 28.0 62.3 ±11.2 IR 0.65 ±0.14 0.65 ± 0.07 Abbreviations: L C , level changes; M L , mount latency; IL, intromission latency; E L , ejaculation latency; PEI, post-ejaculatory interval; M F , mount frequency; IE,, intromissions before the first ejaculation; IF, intromission frequency; EF, ejaculation frequency; III, inter-intromission interval; IR, intromission ratio. Different than CONT group for a given measure: */?<0.05, **/?<0.01 by simple main effects analysis. 69 ^=0.0049] of Kaplan-Meier curves (Figure 9). Survival analysis of Kaplan-Meier curves for IL (Figure 9) also revealed a significant facilitation in the A M P H group [Log Rank statistic = 10.50, /?=0.0012]. Further evidence for enhanced sexual behavior in the A M P H group was provided by a significantly greater number of intromissions [F(l,18)=6.31;p<0.05] and ejaculations [F(l,18)=9.09;p<0.0l] than was achieved by the CONT group. Discussion Repeated and intermittent d-amphetamine administration, sufficient to induce behavioral sensitization, facilitated sexual behavior in the naive male rat, independent of the environmental context in which the drug was administered. Once again, although both appetitive and consummatory (e.g., M F , IF, EF) measures of sexual behavior were facilitated by prior exposure to d-amphetamine, the data suggest that appetitive/motivational components were affected to a greater degree (e.g., decreased M L and IL). The finding that consummatory measures within the first bout of copulation (IEi, III, IR, EL , and PEI) were not affected by d-amphetamine-sensitization was also consistent with the results from Experiment I. The present results differ from the findings of Mitchell and Stewart (1990), who demonstrated a facilitation of sexual behavior by cues associated with systemic morphine adrninistration. This experiment differed from their study in several important respects. First, d-amphetamine was administered, not morphine. Second, the administration regimen was tailored to optimize behavioral sensitization by: 1) increasing the number of intermittent injections (10 instead of 4) and; 2) allowing a three-week period to elapse 70 Figure 9. Effect of d-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment II. A . Kaplan-Meier curve for mount latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 7.91,/J=0.0049). B . Kaplan-Meier curve for intromission latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 10.50, p=0.0012). A — A— CONT - Test 1 - • - AMPH - Test 1 100 g> 80 o ^ 60 E 40 CD o I 20 0 • 2 A 0 B. 100 | 80 g 60 J-H t= 4 0 CD O 20 0 A' 7^  0 A-300 600 900 1200 1500 1800 Mount Latency (s) A " .A - - A -300 600 900 Intromission Latency (s) 1200 1500 1800 72 after the last injection before testing for sexual behavior. Third, sexually inexperienced male rats were used instead of experienced rats that were castrated and given low level testosterone replacement. Finally, control rats did not receive drug injections in their home cage, but in a novel environment which also differed from the bilevel chamber. The novelty of the injection environment may have enhanced the development and subsequent expression of behavioral sensitization (Badiani et al., 1995a,b). The present study does not provide evidence for associative gating of the expression of sensitization: d-amphetamine-induced sensitization facilitated behavior elicited by natural incentive stimuli, irrespective of whether environmental stimuli associated with drug administration were present. This is in contrast to a number of studies demonstrating associative control over the expression of behavioral sensitization to psychostimulants (reviewed in Robinson et al., 1998). For example, in a carefully controlled study, Crombag et al. (1996) reported the absence of the expression of behavioral sensitization i f cues associated with repeated administration amphetamine were absent. Although the facilitation of sexual behavior by pre-treatment with d-amphetamine was not environment specific, it cannot be said that conditioned stimuli did not play a role in this effect. In fact, sources of conditioned stimuli were present in the experiment. For example, the manner in which rats were handled and transported to the testing room before each administration of drug or saline and the test for sexual behavior were similar. The influence of contextual cues on the expression of behavioral sensitization may be diminished when rats self-administer psychostimulants or are challenged with a motivational task. Indeed, Mendrek et al. (1998) found an increased motivation to self-73 administer drug even though rats had received passive injections of (/-amphetamine in a different environment. In the present study, the multimodal primary incentive qualities of the receptive female, including the presence of pheromones, ultrasonic vocalizations, earwiggling, and darting, played a dominant role in the enhanced sexual behavior observed in sensitized rats. These results emphasize the presence of two distinct components of behavioral sensitization: non-associative adaptations that are independent of conditioned environmental cues and associative neural adaptations that exercise conditioned control over its expression (Pierce and Kalivas, 1997a). Control rats in Experiment 2 displayed shorter latencies to mount and intromit than control rats in Experiment 1 (Test 1) that were tested in the same chamber in which they received saline injections. It is unlikely that this was due to the novelty of the testing chamber since sexually naive male rats exhibit longer latencies to mount and intromit in a novel chamber compared to sexually inexperienced male rats pre-exposed to the test chamber (Pfaus and Wilkins, 1995). Alternatively, this effect may be the result of housing conditions. Due to a change in animal care protocol between experiments, rats were housed in large plastic cages in Experiment 2. Perhaps the combined effect of separate housing in a restricted environment of small wire cages depressed sexual behavior in rats of Experiment 1. The simplest explanation for these differences may lie in the normal variance in behavior observed across different litters of rats or be due to testing at different times of the year. Nevertheless, these differences in the behavior of control groups emphasizes the importance of restricting conclusions to comparisons between the experimental and control groups within a single experiment. The results from Experiment II confirm that prior (/-amphetamine exposure 74 enhances sexual behavior in naive male rats. Latencies to mount and intromit appear to be preferentially facilitated during the first test of sexual behavior using inexperienced male rats. EXPERIMENT III: THE EFFECT OF REPEATED A N D INTERMITTENT d-AMPHETAMINE ADMINISTRATION O N M A L E R A T S E X U A L B E H A V I O R IN U N I L E V E L C H A M B E R S Several factors that can influence the quality and quantity of sexual behavior displayed by male rats include the strain and experience of subjects, the incentive attributes of the estrous female, and, in particular, the design of the testing apparatus (Pfaus, 1996). The use of bilevel chambers can provide valuable indices of male rat sexual behavior, such as anticipatory level changes, but these chambers also function to slow down the pace of sexual behavior resulting in an overall reduction in the amount of copulation observed. This is important when an experimental manipulation, such as repeated d-amphetamine administration, is predicted to facilitate sexual behavior. Facilitation of some measures of sexual motivation, such as decreases in latencies to mount or intromit, which would otherwise be difficult to observe in chambers that did not allow pacing by the female or obstacles between the male and receptive female because of floor effects, can be revealed in bilevel chambers. Unfortunately, by their design, bilevel chambers would obstruct movement during microdialysis experiments because of 75 the tubing required to connect the implanted microdialysis probe to the infusion pump. Unilevel chambers were constructed to be approximately the same size as bilevel chambers, in terms of floor area. The use of a removable transparent Plexiglas partition located in the center of the floor surface created two alleys, each of which can be considered analogous to the two platforms of the bilevel chamber. Rats can move freely between each side on both ends of the chamber. This arrangement offers a perimeter "track" area around which the female can pace sexual behavior. The aim of the present experiment was to determine if -^amphetamine-induced behavioral sensitization can facilitate sexual behavior in sexually naive male rats tested in unilevel chambers during a "mock" microdialysis experiment. This experiment also differed from Experiments I and II by the inclusion of a 10-min appetitive phase during which a sexually receptive female was present behind a screen. Method Subjects were obtained and housed as outlined in the General Methods section. Microdialysis probe cannulae were implanted over the NAC according to procedures in the Surgery paragraph of the General Methods section. Both AMPH («=10) and CONT («=10) rats received injections in unilevel chambers following the regimen outlined in the General Methods section. At the beginning of each activity test, a steel coil attached to the liquid swivel was secured to the rats' training post using a microdialysis probe guide collar. In this manner, rats became accustomed to the apparatus necessary for microdialysis. Twenty days after the tenth injection, the microdialysis apparatus was attached and rats were left overnight in unilevel chambers. The next day, a mock 76 microdialysis experiment was conducted as described in the Microdialysis paragraph of the General Methods section. Copulatory behavior was scored as indicated in the Sexual Behavior paragraph of the General Methods section. Results Activity Rats in the A M P H group showed a progressive enhancement in d-amphetamine-induced activity over the course of 10 injections (Figure 10). There was a significant interaction between group and injection number revealed by a repeated measures A N O V A on the total number of activity counts in the 2-h post-injection period [F(i?i6) = 5.60; pO.05]. Subsequent simple main effects analyses yielded significant differences in total activity counts between groups for injection number 1 [F(i,i6) = 29.74; pO.OOl] and number 10 [F(i ;i6) = 33.19;pO.OOl]. Most importantly, there was a significant increase in the total number of activity counts across injections in the A M P H group [Newman-Keuls: pO.01] indicating behavioral sensitization to (/-amphetamine. Sexual behavior Table 3 summarizes measures of sexual behavior in Experiment III. There was facilitation of sexual behavior in the A M P H group relative to the CONT group as indicated by significantly shorter latencies to mount [F(i,i4) = 4.80; pO.05] and intromit [F(i,i4) = 5.92; pO.05]. Survival analyses of Kaplin-Meier curves for mount latency (Figure 11) and intromission latency (Figure 11) confirmed a facilitation of behavior in AMPH-treated rats [Log Rank statistic = 9.43,p=0.0021 and Log Rank statistic = 10.48, p=0.0012, respectively]. 77 Figure 10. Effect of repeated d-amphetamine (AMPH) or saline (CONT) injections on activity in Experiment III. The data are represented as mean (± SEM) activity counts. A . The line graphs represent the activity profile after injection 1 (upper panel) and injection 10 (lower panel). B. The histogram represents cumulated activity counts (± SEM) after injections 1 and 10 for each group. ***/><0.001, using simple main effects analysis. $/?<0.001, using Newman-Keuls post-hoc test. A. S-H c<3 u > • I - H 1 o . > %-> o 60 50 40 30 20 10 0 I n j e c t i o n 1 I I > L J 1 I I*-- A A - - A A - = 4 -0 10 20 30 40 50 60 70 80 90 100 110 120 60 50 40 30 20 1 10 I n j e c t i o n 10 f t * -4=4= A- -& A - A — A 0 10 20 30 40 50 60 70 80 90 100 110 120 T i m e ( m i n ) 78 CONT AMPH B. w GO I O U > •1— I o < E2 CONT AMPH I n j e c t i o n N u m b e r 79 Table 3 Measures of sexual behavior from Experiment III. Data are expressed as the mean ± S E M . CONT A M P H M L (s) 219.1 ± 111.7 31.3 ± 12.1* IL(s) 271.0 ± 106.2 54.4 ±21.9* EL (s) 621.5 ± 181.3 583.0 ± 101.3 PEI (s) 428.6 ± 124.9 389.2 ±40.1 MF 9.9 ±2.9 15.7 ±2 .3 IEi 8.3 ± 1.4 10.3 ± 1.3 IF 10.4 ±2 .8 17.9 ± 1.7* EF 1.1 ±0.4 2.3 ± 0 . 3 * III 76.6 ± 16.1 57.1 ±8 .5 IR 0.58 ± 0.09 0.58 ± 0.04 Abbreviations: L C , level changes; M L , mount latency; IL, intromission latency; E L , ejaculation latency; PEI, post-ejaculatory interval; M F , mount frequency; IEi , intromissions before the first ejaculation; IF, intromission frequency; EF, ejaculation frequency; III, inter-intromission interval; IR, intromission ratio. Different than CONT group for a given measure: */?<0.05 by simple main effects analysis. 80 Figure 11. Effect of d-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment III. A . Kaplan-Meier curve for mount latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 9.43, />=0.0021). B. Kaplan-Meier curve for intromission latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 10.48,/?=0.0012). -A - CONT - • - AMPH A. • ^ O O PH B. o H O PH 100 80 60 40 20 0 i „-A-A A A IA 0 300 600 900 1200 1500 1800 Mount Latency (s) 100 80 60 40 20 0 • / A-A" A ,k A' A 0 300 600 900 1200 1500 1800 Intromission Latency (s) 82 There was an overall increase in the amount of sexual behavior due to prior d-amphetamine exposure as indicated by a significantly greater number of intromissions [^( i , i6) = 5.89; /K0.05] and ejaculations [F^^) = 6.37; /><0.05] over the 30-rnin copulation period as compared to the CONT group. However, other measures from the first ejaculatory series once copulation was initiated, such as EL , PEI, IEi, III, and IR, were not different between groups. Discussion Repeated and intermittent administration of d-amphetamine facilitated sexual behavior in male rats when tested in unilevel chambers. Results from the present experiment were consistent with those from Experiments I and II; prior exposure to d-amphetamine again preferentially enhanced appetitive measures of sexual behavior, although there was also an increase in the number of copulatory bouts (i.e., ejaculations) in the A M P H group. Unilevel chambers offer a number of advantages in the study of male rat sexual behavior. Similar to bilevel chambers, they encourage the exposure of the rats' flanks to the experimenter during copulation, thereby facilitating scoring of sexual behavior (Mendelson and Gorzalka, 1987). Other practical attributes of the unilevel chamber include the ease of introducing and removing rats from the chamber, and ease of cleaning relative to the bilevel chamber. Recently, our laboratory has observed that the number of side changes (i.e., complete movements from one side of the Plexiglas partition to the other) made by male rats in anticipation of a sexually receptive female in unilevel 109chambers increase upon repeated testing for sexual behavior (unpublished 83 observations). Furthermore, control rats that were not presented with a female after the 5-min "anticipatory" period did not show a progressive increase in side changes after repeated testing. Thus, side changes in unilevel chambers can be considered a measure of appetitive male rat sexual behavior, which are analogous to anticipatory level changes observed in bilevel chambers. The unilevel chambers also had the added convenience of infrared photobeam emitters/detectors that allowed side changes to be monitored automatically by a computer. Experiment III established that the phenomenon of d-amphetamine-induced sensitization of sexual behavior could be replicated in unilevel chambers. Another advantage to the simple design of these chambers is the ability to conduct a microdialysis experiment - indeed, it was the main impetus for their construction. In Experiment IV, changes in N A C D A efflux in sexually inexperienced male rats previously sensitized to d-amphetamine were monitored by in vivo microdialysis in unilevel chambers. EXPERIMENT IV: IN VIVO M E A S U R E M E N T OF N A C D A E F F L U X DURING S E X U A L B E H A V I O R IN d-AMPHETAMINE-SENSITIZED M A L E RATS It is well established that the incentive properties of a sexually receptive female are critical for sexual behavior in inexperienced male rats (Beach, 1941; Madlafousek and Hlinak, 1983). Mesolimbic dopamine is known to play an important facilitatory role in copulation (Everitt, 1990; Pfaus and Phillips, 1991; Mas 1995; Melis and Argiolas, 84 1995) and a number of microdialysis studies have reported increased N A C D A efflux associated with both appetitive/motivational and consummatory components of sexual behavior in male rats (Damsma et al., 1992; Wenkstern et al., 1993; Mas, 1995; Fiorino et al., 1997a). In Experiments I-III, prior exposure to d-amphetamine facilitated sexual behavior in sexually inexperienced male rats. Given the hypothesis that this facilitation of sexual behavior is mediated by enhanced release of N A C DA, the present experiment employed microdialysis to determine whether the increase in extracellular N A C D A concentration is augmented during sexual behavior in sexually inexperienced male rats sensitized to d-amphetamine. Subsequently, a d-amphetamine challenge was given to provide independent confirmation that augmented N A C DA efflux is evident in rf-amphetamine-sensitized rats. A number of issues will be addressed in Experiment IV. The presence of augmented D A efflux in (/-amphetamine-sensitized rats during an unambiguously appetitive phase of the experiment, where a receptive female is present behind a wire-mesh screen, would provide evidence for dopaminergic mechanisms underlying facilitated sexual behavior . Furthermore, augmented D A efflux during copulation in the (/-amphetamine-treated group would suggest that sensitization to J-amphetamine leads to increased N A C D A release during both appetitive and consummatory components of male sexual behavior. Following completion of the tests for sexual behavior, a ^-amphetamine challenge was administered to confirm behavioral sensitization and to determine if there is augmented N A C D A efflux in sensitized animals as indicated in earlier reports, as reviewed in the introduction. 85 Method Subjects were obtained and housed as outlined in the General Methods section. Microdialysis probe cannulae were implanted over the N A C according to procedures in the Surgery paragraph of the General Methods section. Both A M P H («=10) and CONT («=10) rats received injections in unilevel chambers following the identical protocol used in Experiment III. Microdialysis experiments were conducted 21 days after the tenth injection as outlined in the Microdialysis paragraph of the General Methods section. Copulatory behavior was scored as indicated in the Sexual Behavior paragraph of the General Methods section. Following the fourth phase (i.e., post-copulation baseline) of the microdialysis experiment, rats were injected with ^-amphetamine (1.5 mg/kg, IP). Microdialysis samples were collected and activity was monitored for an additional 120 min post-injection. Results Activity Repeated J-amphetamine administration again induced behavioral sensitization in A M P H rats (Figure 12). A repeated measures A N O V A on the total activity counts post-injection revealed a significant interaction between group and injection number [F(i,i8) = 12.44; pO.01]. Subsequent simple main effects analyses showed that ^-amphetamine produced significant increases in total activity counts relative to vehicle treatment after the first [F(i,i8) = 41.93;pO.OOl] and tenth injection [F ( i , i 8 ) = 51.10; pO.OOl]. Once again, rats in the A M P H group displayed a sensitized behavioral response to ^-amphetamine as indicated by a significant increase in the total number of activity 86 Figure 12. Effect of repeated d-amphetamine (AMPH) or saline (CONT) injections on activity in Experiment IV. The data are represented as mean (± SEM) activity counts. A . The line graphs represent the activity profile after injection 1 (upper panel) and injection 10 (lower panel). B. The histogram represents cumulated activity counts (± SEM) after injections 1 and 10 for each group. ***p<0.001, u s i n g simple main effects analysis. tp<0.01, using Newman-Keuls post-hoc test. A. B. 1 u 13 > • ? ^ o U > +-> o < 60 50 40 30 20 10 0 60 50 40 30 20 10 0 I n j e c t i o n 1 1 A -r i - f ^ i -A A A A A A -T-• I -A ' A ==A-0 10 20 30 40 50 60 70 80 90 100 110 120 I n j e c t i o n 10 T A^ ± •4= -A A -T - A v 87 CONT - • - AMPH 0 10 20 30 40 50 60 70 80 90 100 110 120 T i m e ( m i n ) g O U > •l-H o CONT AMPH I n j e c t i o n N u m b e r 88 counts from injection 1 to injection 10 [Newman-Keuls; /?<0.05]. Sexual behavior Measures of sexual behavior in Experiment 4 are shown in Table 4. Although the A M P H group displayed shorter latencies to mount and intromit, these measures did not differ significantly from those of the CONT group. However, survival analyses of Kaplan-Meier curves for M L and IL (Figure 13) did reveal a significant enhancement of sexual behavior in A M P H rats [Log Rank statistic = 6.12,^=0.0134, Log Rank statistic = 4.38,/>=0.0364, respectively]. The A M P H group also achieved more ejaculations within the 30-min copulatory period than the CONT group [F(i,i4) = 5.56; p<0.03]. Neurochemistry of sexual behavior Prior exposure to d-amphetamine did not alter basal concentrations of D A , DOPAC, or H V A (Table 5). There was an overall change in N A C D A efflux associated with sexual behavior (Figure 14). Whereas concentrations of D A in A M P H rats were significantly elevated relative to baseline throughout all phases of the test session, including the period when the female was present behind a screen, during copulation, and for the 20 min period following her removal. In contrast, significant increases in D A concentrations in CONT rats were restricted to samples associated with copulation. Presentation of a receptive female behind the screen (sample 2) resulted in a significant increase in N A C D A concentrations from baseline values in the A M P H group (+35%,/?<0.01) but not in the CONT group (+17%). There was a further increase in D A efflux during copulation in both groups. In A M P H rats, D A reached maximum concentrations during the first copulatory sample (+60%), and N A C D A remained elevated for 20 min after removal of the female. Maximum D A concentrations in the Table 4 Measures of sexual behavior from Experiment IV. Data are expressed as the mean ± S E M . CONT A M P H M L (s) 37.3 ± 18.6 18.4 ±4.5 IL(s) 53.8 ± 36.2 24.3 ±3.5 EL(s) 611.2 ±76.9 493.6 ± 64.8 PEI (s) 357.3 ±25.0 330.9 ±21.9 MF 3.5 ± 1.1 6.3 ± 1.4 IE, 11.5 ± 1.4 10.6 ±2 .1 IF 16.0 ±4 .3 20.9 ± 2.8 EF 1.9 ±0.5 3.1 ± 0 . 3 * III 59.7 ±15.3 52.6 ± 8.3 IR 0.82 ± 0.06 0.72 ± 0.05 Abbreviations: L C , level changes; M L , mount latency; IL, intromission latency; E L , ejaculation latency; PEI, post-ejaculatory interval; M F , mount frequency; IEi, intromissions before the first ejaculation; IF, intromission frequency; EF, ejaculation frequency; III, inter-intromission interval; IR, intromission ratio. Different than CONT group for a given measure: */K0.05 by simple main effects analysis. 90 Figure 13. Effect of J-amphetamine or saline pre-treatment on latencies to mount and intromit in Experiment IV. A . Kaplan-Meier curve for mount latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 6.12,/?=0.0134). B. Kaplan-Meier curve for intromission latency. Independent survival analyses showed a significant difference between sensitized (AMPH) and non-sensitized (CONT) rats on test 1 (Log Rank statistic = 4.38,^=0.0364). A. A - CONT AMPH too c -I—> o a O PH 100 80 60 40 20 0 A' A Ii ir A 0 B. too B o H o S-H PH 100 80 60 40 20 0 .A--/h 100 200 1600 1700 Mount Latency (s) 1800 , - - A -I A " r I o 100 200 — i — i — < — i — i — ' — i 1600 1700 1800 Intromission Latency (s) 92 Table 5 Mean basal concentrations of microdialysis analytes. These values correspond to 100% baseline. Data are expressed as the mean ± S E M (uncorrected for probe recovery). CONT A M P H D A (nM) 3.9 ± 0.7 3.3 ±0 .5 DOPAC (nM) 820.8 ± 139.4 618.8 ±94.4 H V A (nM) 283.1 ±39.1 243.9 ±29.1 Abbreviations: DA, dopamine; DOPAC, dihydroxyphenylacetic acid; H V A , homovanillic acid. 93 Figure! 4. Changes in nucleus accumbens dopamine efflux (line graph) during baseline (Bas), while a receptive female was present behind the screen (Scr), copulation, and post-copulation for CONT and A M P H rats. Bar graphs show the number of mounts plus intromissions (upper bar graph) and ejaculations (lower bar graph) displayed for each group during three 10-min samples. *p<0.05, **p<0.01 using simple main effects analysis. tp<0.05, |p<0.01, using Newman-Keuls post-hoc test. 94 fl o • 1—1 fl ^ <D .S fl 22 CD 8 fl r OH o Q - A - CONT - • - A M P H Mounts + Intromissions Ejaculations 12 8 4 0 0 Scr Scr Copulation Bas Female Present i 1 2 i i i 3 4 5 • i i i i 6 7 8 9 10 n o fl I—* • o fl i-t CD •§ CD fl O Sample Number 95 CONT group occurred in the second 10-min copulatory sample (+47%). Furthermore, the increase in N A C D A in the A M P H group was significantly greater than the increase obtained from the CONT group, both when the female was behind the screen (sample 2) [F^^i) = 4.76; jt?<0.05] and during the first 10 min of copulation (sample 3) [F(isig2) = 6.32; p<0.05]. The significant augmentation of N A C D A efflux in the A M P H group (sample 3) coincided with an increased number of mounts + intromissions [F^^s) = 18.56; p<0.0\] and ejaculations[F(i;i8> = 5.09; p<0.05] relative to the CONT group. Extracellular concentrations of DOPAC and H V A also increased following copulation (Figure 15). A significant increase in DOPAC concentrations relative to baseline (sample 1) occurred during the first copulatory period (sample 3) in both groups and remained significantly elevated until sample 10 in CONT rats (maximum, sample 4: +40%>) and sample 9 in A M P H rats (maximum, sample 4: +55%). Increases in the concentration of H V A from baseline achieved statistical significance in the first 10-min period of copulation (sample 3) for the A M P H group (maximum, sample 6: +50%) and remained significantly elevated for 30-min post-copulation (sample 8). However, H V A concentrations in A M P H rats were still high just prior to the c/-amphetamine challenge (+28%»). Concentrations of H V A in CONT rats were significantly elevated from the last 10-min copulatory period (sample 5) until the ^-amphetamine injection (sample 11), and reached a maximum increase of +49% (sample 7). There was no statistical difference in metabolite concentrations between groups at any point in the experiment. Behavior and neurochemistry after an amphetamine challenge Systemic administration of J-amphetamine (1.5 mg/kg) resulted in a sensitized 96 Figure 15. Changes in DOPAC (top panel) and H V A (bottom panel) concentrations in the nucleus accumbens during baseline (Bas), while a receptive female was present behind the screen (Scr), copulation, and post-copulation. f/?<0.05, %p<0.0\, using Newman-Keuls post-hoc test. 97 - A - CONT Scr Scr Copulation Bas Female Present 1 2 3 4 5 6 7 8 9 1 0 Sample Number 98 behavioral and neurochemical response by the A M P H group relative to the CONT group (Figure 16). There was a significant overall group by time interaction [F< 14,196) = 28.50; p<0.0\] following a repeated measures A N O V A . The increase in activity counts after d-amphetamine administration persisted for 2 h after the injection in both groups relative to baseline, as assessed by Newman-Keuls post-hoc tests. The A M P H group displayed a significantly greater number of activity counts than the CONT group in the first three 10-min periods after the drug injection (samples 12-14) and with respect to total activity counts post-injection (413.5 ± 37.7 vs 303.6 ± 37.8) [ F ( U 4 ) = 4.84; p<0.05]. There was a concomitant increase in extracellular N A C D A post-injection in the CONT and A M P H groups [F(i4,i96) = 39.16;p<0.0\], and the increase observed in the A M P H group was significantly greater than the CONT group in the first four samples after injection (Figure 16, top panel). Both metabolites of D A decreased post-injection in the CONT and A M P H groups (Figure 17). Systemic administration of J-amphetamine resulted in a significant decrease in DOPAC efflux after 10 min post-injection, reaching a minimum concentration of 48% in the CONT group (sample 15) and 44% in the A M P H group (sample 15). The maximum decrease in H V A concentrations was delayed further, reaching significantly depressed values after 30 min (sample 15) and 40 min (sample 16) in CONT (minimum, sample 18: 82%) and A M P H (minimum, sample 20: 83%) rats, respectively. Histology Microdialysis probes tracts were found in both the shell and core regions of the N A C in a range extending +1.60 to +2.20 mm from Bregma (Figure 18). 99 Figure 16. Changes in locomotor activity and nucleus accumbens dopamine efflux in response to a d-amphetamine challenge (1.5 mg/kg, IP). */?<0.05, **p<0.01 using simple main effects analysis. Dopamine concentrations and activity counts remained elevated throughout the 2-h post-injection period relative to baseline (Bas) in both groups. 100 fl 2 <L> .B fl 3 O CQ fl fc r o - A - CONT : - • - A M P H I B a s P o s t - I n j e c t i o n i 1 1 1 1 1 1 1—-I—-11 12 13 14 15 16 17 18 19 20 21 22 23 Sample Number 101 Figure 17. Changes in locomotor activity and nucleus accumbens DOPAC (top panel) and H V A (bottom panel) efflux in response to a J-amphetamine challenge (1.5 mg/kg, IP). *p<0.05, **p<0.01 using simple main effects analysis. f/?<0.05, JpO.Ol , using Newman-Keulspost-hoc test. 102 0 o • i—I o ^ fl O ^ -s CD 120 100 80 60 40 150 130 110 90 70 - A - CONT - • - AMPH Bas[ Post-Injection 11 12 13 14 15 16 17 18 19 20 21 22 23 Sample Number 103 Figure 18. Location of microdialysis probes within the N A C of rats used in Experiment IV. Vertical black lines correspond to the location of the active fiber area of the microdialysis probes. Coronal brain sections modified from Paxinos and Watson (1997). 104 105 Discussion Repeated and intermittent exposure to J-amphetamine, sufficient to induce behavioral sensitization, facilitated sexual behavior in sexually inexperienced male rats, thus confirming previous observations in Experiments I-III. The present microdialysis experiment showed clearly that enhanced sexual behavior was correlated with augmented N A C D A efflux. A n earlier study found that sexual behavior was facilitated in male rats when tested in an environment which had been paired repeatedly with systemic morphine injections (Mitchell and Stewart, 1990). Specifically, there was a preferential enhancement in motivational measures, such as the amount of anogenital exploration, the percentage of animals copulating, and latency to mount, rather than indices of copulation itself. Although the aim of that study was to investigate whether sexual behavior could be enhanced by a conditioned association between environmental cues and opiate reward, the injection regimen employed has been reported to induce behavioral sensitization to the locomotor activating effects of morphine (Kalivas and Stewart, 1991). The present experiment directly examined the influence of behavioral sensitization on sexual behavior in male rats and found a similar facilitation of motivational components of sexual behavior, including mount and intromission latencies. Although the overall amount of copulation was increased in sensitized rats, as indicated by a greater number of intromissions plus mounts and ejaculations during the first 10 min of copulation, consummatory measures within bouts of copulation, such as IEi, III, IR, and E L were not significantly altered. The preferential effect of sensitization on appetitive aspects of sexual behavior confirms previous observations in bilevel chambers (Experiments I and 106 II) and unilevel chambers (Experiment III). It should be noted that, despite the fact that all rats in the A M P H group copulated in experiments conducted in unilevel chambers, d-amphetamine sensitization, induced by this injection regimen, does not guarantee that sexually naive rats will copulate. Nevertheless, a very high percentage of rats pre-exposed to J-amphetamine copulated during their first test of sexual behavior (i.e., >85% in Experiments I and II). Our observation that increased M L B D A transmission was associated with copulation in naive rats is in agreement with the results of an earlier microdialysis experiment (Wenkstern et al., 1993). The inclusion of a purely appetitive condition in the present study, in which a receptive female was presented behind a screen, addresses the issue of whether unconditioned sexual incentives, not confounded by previous copulatory activity, can induce N A C D A release. There was no significant appetitive increase in N A C D A efflux in the CONT group. However, two CONT rats (25%) did not copulate and the corresponding change in mean N A C D A concentrations in response to the receptive female in the CONT group was attenuated. Previous reports have noted that increases in mPOA or N A C D A efflux in response to a receptive female behind a screen were only observed in male rats that also copulated once the screen was removed (Hull et al., 1995; Wang et al., 1995; Fiorino et al., 1997a). The same relationship was observed in the present study. Therefore, increases in N A C D A concentrations in response to sexual incentives probably occur unconditionally, but only in rats that copulate subsequently. There was a significant augmentation of N A C D A efflux in the A M P H group, relative to the CONT group, during sexual behavior and this was evident during both the 107 appetitive phase (i.e., female behind the screen) and the first copulatory sample. As mentioned above, non-copulating CONT rats did not contribute to the appetitive or copulatory rise in NAC DA concentrations. Therefore, the increased amount of copulation observed during the first copulatory sample in AMPH rats relative to CONT rats, and in particular the greater number of ejaculations, may explain the augmented efflux of NAC DA in the AMPH group. Chronoamperometry experiments conducted in our laboratory have shown that peak oxidation currents associated with DA are correlated to ejaculations (Phillips et al., 1991; Fiorino et al., 1997b), although it is difficult to determine whether ejaculation or the vigorous pursuit activity leading to ejaculation is correlated with maximal NAC DA efflux. In this regard, it is important to note that the "consummatory" phase of male rat sexual behavior (i.e., copulation) contains many appetitive components (Fiorino et al., 1997a) and it is impossible, in the present study, to correlate NAC DA efflux preferentially with one component or the other. Nevertheless, the intense behavior of sensitized rats during the first copulatory sample, relative to CONT rats, may account for differences in neurochemical profiles between groups. Augmented dopaminergic transmission in the NAC induced by repeated d-amphetamine administration lends support to the observation that enhanced dopaminergic activity can facilitate the initiation of sexual behavior in sexually naive male rats (Agmo and Picker, 1990). A systemic d-amphetamine challenge also resulted in augmented locomotor behavior and NAC DA efflux in the AMPH group relative to the CONT group. This finding contributes to the growing literature demonstrating that increased striatal DA transmission accompanies behavioral sensitization to psychostimulants, when assessed 108 following extended periods (>14 days) after discontinuation of drug treatment (reviewed in Pierce and Kalivas, 1997a; although see Kuczenski et al., 1997). Caution must be exercised, however, in comparing these results with previous reports because the present drug challenge was administered after a period of sexual activity and residual sex-related odors may have contributed, perhaps differentially, to neurochemical responses in both groups. Bedding from the cages of estrous females was reported to increase extracellular N A C D A in male rats (Mitchell and Gratton, 1992). These data also have implications about using in vivo metabolite efflux as an index of D A neurotransmission. Disparate changes in extracellular N A C D A metabolite concentrations occurred in response to sexual incentives and d-amphetamine administration (i.e., increase and decrease, respectively) during a period when D A efflux invariably increased. This emphasizes the inherent problems associated with inferring D A transmission by changes in D A metabolite efflux (Fiorino et al., 1997a; O'Neil l et al., 1998). In the context of the role of M L B D A neurons in incentive motivation, psychostimulant-induced sensitization may further lower the threshold for eliciting behaviors in response to the quality and intensity of environmental stimuli (Blackburn et al., 1992; Salamone, 1996). Our results are consistent with the incentive-sensitization theory of drug addiction (Robinson and Berridge, 1993) which proposes that the incentive value of cues associated with reward is enhanced due to repeated administration of psychostimulants via an augmentation of mesotelencephalic D A function; enhanced mesotelencephalic D A transmission ultimately contributes to compulsive drug seeking and drug taking which are defining features of drug addiction (Jaffe, 1990). Previous 109 experiments with drug reward found that pre-exposure of animals to psychostimulants facilitated various measures of self-administration of drugs of abuse (Woolverton et al., 1984; Lett, 1989; Horger et al., 1990,1992; Piazza et al., 1990; Mendrek et al., 1998; although see L i et al., 1994). In a recent study, Mendrek et al. (1998) demonstrated that d-amphetamine-sensitized rats exhibited increased motivation to self-administer d-amphetamine as indicated by a significantly higher break point under a progressive ratio schedule of reinforcement. The present study extends the enhancement of motivated behaviors due to repeated psychostimulant administration to those elicited by natural incentives. In this case, prior d-amphetamine treatment may have amplified the importance of unconditioned incentive cues of the estrous female, which included pheromones, ultrasonic vocalizations, ear wiggling, and darting, and led to a facilitation of sexual behavior. The presence of augmented N A C D A transmission in response to an estrous female located behind a screen strengthens the argument that the M L B D A system contributes to this effect. The results of Experiment IV not only provide support for the role of M L B D A in motivated behavior, but buttress the hypothesis that changes in limbic-motor circuitry, specifically to M L B dopaminergic pathways, contribute to sensitized behavior in response to both psychostimulant administration and natural incentives. G E N E R A L DISCUSSION The objectives of this thesis were to test the hypothesis that male sexual behavior would be facilitated by psychostimulant-induced behavioral sensitization and, in so 110 doing, to obtain a more comprehensive knowledge of the role of MLB DA in the expression of motivated behavior. The general discussion begins with a review of the key findings of each experiment to illustrate how these objectives were met. Experiment I demonstrated that prior, intermittent exposure to d-amphetamine, sufficient to induce behavioral sensitization, facilitated sexual behavior in male rats tested in bilevel chambers, while in a drug-free state. During the first test of sexual behavior, this was manifest as shorter latencies to mount and intromit by sensitized rats, and also as a general increase in the amount of copulation over the 30-min session. The repeated testing of sexually naive rats also allowed the acquisition of sexual behavior to be monitored. Sensitized rats displayed a facilitated acquisition of sexual behavior (i.e., reached the criteria for ML and IL < 300s for three consecutive days more quickly). By the tenth test of sexual behavior, there were no differences in any of these measures between groups. The use of bilevel chambers, however, provided another index of appetitive sexual behavior: namely, anticipatory level changes. After repeated sexual experience, rats pre-treated with J-amphetamine also showed an augmented increase in level changes made in anticipation of the presentation of a receptive female. These data suggest that appetitive, rather than consummatory, components of male rat sexual behavior were preferentially enhanced by d-amphetamine-induced sensitization (i.e., decreased latencies to mount and intromit in naive rats; augmented anticipatory level changes after sexual experience). Given the evidence for enhanced function in limbic-motor circuitry that accompanies the behavioral expression of psychostimulant sensitization (Pierce and Kalivas, 1997a), the results of Experiment I suggest that this circuitry is involved in the appetitive components of motivated behavior. Ill Furthermore, the subsequent facilitation of anticipatory sexual behavior (i.e., level changes) after repeated experience in rats previously treated with ^-amphetamine, suggests that behavioral sensitization can influence incentive learning. Finally, this experiment provided the procedural template for subsequent experiments. Rats in Experiment I were tested for sexual behavior in the same environment associated with repeated injections of J-amphetamine, therefore, the possibility remained that this facilitation of sexual behavior was the result of conditioned arousal by drug-related cues (Mitchell and Stewart, 1990), independent of psychostimulant-induced sensitization. In Experiment II, rats received repeated injections of d-amphetamine or saline in unilevel chambers. These chambers were distinct from home cages and the bilevel chambers in which rats were tested for sexual behavior. This experiment demonstrated that enhanced sexual behavior was independent of the environment in which repeated administration of d-amphetamine occurred, indicating that facilitation was not a consequence of conditioned associations between drug and test environment. Once again, there was a preferential facilitation of appetitive components of male rat sexual behavior. Drug-associated cues can have a strong influence over behavioral sensitization to psychostimulants, to the extent that the absence of such cues can prevent its initiation or expression (Vezina and Stewart, 1984; Crombag et al., 1996; reviewed in Robinson et al., 1998). Experiment II did not provide evidence for associative gating of the expression of sensitization. It is possible that the power to control the expression of sensitized behavior by conditioned cues is diminished under experimental conditions that provide a motivational challenge, such as a receptive female in Experiment II. These results emphasize that there are two components of behavioral sensitization: non-associative adaptations that are independent of conditioned environmental cues and associative neural adaptations that exercise conditioned control over its expression (Pierce and Kalivas, 1997a). The replication of the behavioral phenomenon in bilevel chambers set the stage for conducting an experiment to monitor N A C D A efflux during sexual behavior to determine if the facilitation of sexual behavior in sensitized rats is accompanied by an augmentation of M L B D A transmission. Because the structure of the bilevel chamber cannot accommodate a microdialysis experiment, an additional behavioral experiment was conducted in a unilevel chamber. This experiment was necessary to ensure that the behavioral phenomena observed in Experiment I and II could be replicated in a different apparatus. It is well-documented that the design of a testing apparatus can exert a strong influence on the quality and quantity of sexual behavior observed (Pfaus, 1996). This experiment had the added benefit of providing a behavioral control for microdialysis procedures employed in Experiment IV, conducted in exactly the same manner. Prior ^-amphetamine exposure facilitated sexual behavior in male rats when tested in unilevel chambers during a mock-microdialysis experiment. As in Experiments I and II, psychostimulant-induced sensitization preferentially enhanced appetitive measures of sexual behavior. Experiment IV employed in vivo microdialysis to monitor changes in N A C D A efflux in male rats previously sensitized to ^-amphetamine. Many laboratories have reported increased D A efflux in the N A C of male rats in response to the presentation of a receptive female and during copulation (Pfaus et al., 1990a; Damsma et al.,1992; 113 Wenkstera et al., 1993; Melis and Argiolas, 1995; Fiorino et al., 1997a). In addition, the enhanced behavioral response to a drug challenge in psychostimulant-sensitized rats has been associated most often with augmented efflux of N A C D A (reviewed in Pierce and Kalivas, 1997a). Once again, repeated intermittent exposure to d-amphetamine facilitated appetitive/motivational and consummatory components of sexual behavior and the results of the microdialysis experiment showed clearly that enhanced sexual behavior was correlated with augmented N A C D A efflux. The inclusion of a purely appetitive condition, in which a receptive female was presented behind a screen, also demonstrated that increases in N A C D A concentrations in response to sexual incentives occur unconditionally in those rats that copulated. A subsequent systemic d-amphetamine injection resulted in augmented locomotor behavior and N A C D A efflux in d-amphetamine-treated rats. This finding adds to the growing literature demonstrating that increased striatal D A transmission accompanies behavioral sensitization to psychostimulants, when assessed after extended periods following drug withdrawal (cf., Pierce and Kalivas, 1997a). Taken together, these experiments not only provide support for the role of M L B D A in motivated behavior, but further strengthen the hypothesis an enhancement of function in limbic-motor circuitry, specifically within the M L B dopaminergic pathway, contributes to sensitized behavior in response to both psychostimulant administration and natural incentives. 114 Behavioral Sensitization by Non-Pharmacological Stimuli Experiments in the present thesis demonstrated that behavioral sensitization due to repeated psychostimulant administration could "cross-sensitize" to a natural motivated behavior, such as sex. Based on the conclusion that activation of midbrain D A cell bodies is necessary and sufficient for the initiation of behavioral sensitization to psychostimulants (Kalivas and Stewart, 1991; Cador et al., 1995), it follows that repeated presentation/administration of any stimulus that can activate these D A neurons will result in sensitization of this system and, subsequently, enhance motivated behavior. This is also predicted by the incentive-sensitization theory of drug addiction, although the expression of behavior may come under strong associative control (Robinson and Berridge, 1993). Several examples of sensitized behavior due to repeated administration of non-pharmacological stimuli support this contention. Although there is evidence for DA-independent reward circuits, rewarding electrical stimulation of many brain sites activate the mesotelencephalic D A system (Fibiger and Phillips, 1987; Fiorino et al., 1993). Stimulation of these DA-mediated reward sites induce locomotor activity, exploratory movement, and approach behavior. Moreover, repeated rewarding electrical brain-stimulation can induce behavioral sensitization. Repeated stimulation of the V T A resulted in a lowering of the threshold current necessary for eliciting a locomotor response relative to sham-stimulated rats, and this enhancement was still evident seven months after testing, indicating a persistence which is characteristic of sensitization (Glenthqj et al., 1993). In a cross-sensitization experiment, self-stimulation of the V T A was shown to enhance the behavioral effects of 115 a subsequent J-amphetamine challenge (Ben-Shahar and Ettenberg, 1994). Repeated electrical stimulation in terminal areas of mesotelencephalic D A neurons can also produce behavioral sensitization to psychostimulants. Rats that received repeated electrical stimulation of the N A C or caudate-putamen displayed an enhanced locomotor response to af-amphetamine (Kokkinidis et al., 1989). Repeated electrical self-stimulation of the N A C , substantia nigra, and the medial prefrontal cortex sensitized rats to the anorectic and stereotyped behavior-inducing effects of d-amphetamine (Eichler and Antelman, 1979). As mentioned above, activation of V T A D A neurons is necessary for the initiation of behavioral sensitization (Kalivas and Stewart, 1991; Cador et al., 1995). Presumably, antidromic activation of midbrain D A cell bodies and somatodendritic D A release occur when terminal areas are electrically stimulated, thus providing a mechanism by which sensitization of this system could occur. Cross-sensitization between self-stimulation and ^-amphetamine is reciprocal. Repeated J-amphetamine administration enhanced self-stimulation of the substantia nigra (Kokkinidis and Zacharko, 1980), and N A C (Predy and Kokkinidis, 1984). These results, and those from the present thesis, emphasize the dual capacity of certain stimuli to activate and sensitize midbrain D A neurons, as well as elicit an enhanced behavioral response as a result of sensitization. Rewarding electrical brain-stimulation and most doses of psychostimulants used to induce behavioral sensitization probably stimulate midbrain D A neurons to a greater extent than incentive cues from natural rewards and, therefore, are more likely to result in robust and persistent sensitization of this system. Nevertheless, indirect evidence 116 suggests that naturally occurring sensitization-like processes, that involves changes in the M L B D A neurons, may underlie some aspects of the acquisition of motivated behaviors, such as male sexual behavior. Sexual behavior in sexually naive male rats is dependent to a critical degree on proceptive cues from the female (Madlafousek and Hlinak, 1983) and is very sensitive to disruption by the destruction of sensory modalities (Beach, 1941). Intact, experienced rats require only limited sensory stimulation in order to initiate sexual behavior, illustrated by short mount latencies and the lack of correlation between female proceptive behavior and the intensity of male sexual behavior (Agmo and Picker, 1990). Further, the injection of the D2 antagonist raclopride directly into the N A C of experienced male rats was effective in disrupting the initiation of male sexual behavior only i f estrous females were pretreated with a-flupenthixol, a treatment which abolished all proceptive behavior (Everitt, 1990). These data suggest that as a consequence of sexual experience, neural changes have rendered these animals more sensitive to the stimulus properties of receptive females. Given its role in the evaluation of salient incentive stimuli, the M L B D A system is a likely candidate for the site of these changes. Acquisition of sexual behavior in the male rat is characterized by a progressive decrease in mount and intromission latencies, as well as progressive increases in measures of anticipation, such as level changes, and responding for primary and secondary reinforcers (Everitt, 1990; Pfaus and Phillips, 1991). These measures, that are dependent on N A C D A transmission, are facilitated as male rats become more experienced. Repeated activation of the M L B D A system by sexual behavior may render this system hypersensitive in a manner analogous to the initiation of behavioral sensitization to psychostimulants. This hypothesis receives further support from the 117 results of Experiment I in which psychostimulant-induced behavioral sensitization facilitated acquisition of male sexual behavior. Furthermore, preliminary cross-sensitization experiments in our laboratory demonstrate that sexually experienced male rats show an enhanced locomotor response to a d-amphetamine challenge, relative to male rats that were repeatedly presented with a non-estrous female. Although the presence of increased M L B D A transmission accompanying the acquisition of male rat sexual behavior has not been tested directly (i.e., comparison of N A C D A efflux in naive versus experienced male rats during sexual behavior -preferably, using a within-subjects design), there are other data that support this possibility. In Experiment IV of the present thesis, augmented N A C D A efflux associated with enhanced sexual behavior was observed in d-amphetamine-sensitized male rats. Finally, repeated exposure of estrous female bedding in male rats progressively increased N A C D A efflux in male rats as assessed by high speed chronoamperometry (Mitchell and Gratton, 1992). Given evidence for the ability of a wide variety of stimuli to induce behavioral sensitization independently via activation of midbrain D A neurons, it is possible that the concurrent administration of different DA-activating stimuli could enhance the development and expression of behavioral sensitization. An interesting experiment, in this regard, examined the effect of repeated tail pinch on male rat sexual behavior (Leyton and Stewart, 1996). Stress and psychostimulants share an ability to induce sensitization of M L B D A transmission. N A C D A efflux is enhanced by stressors, such as tail pinch (Abercrombie et al., 1989), foot shock (Sorg and Kalivas, 1991), restraint (Imperato et al, 1992). Upon 118 repeated adrrunistration, footshock stress can cross-sensitize to the behavioral and neurochemical effects of an acute ^-amphetamine challenge (Deroche et al., 1995). Repeated tail pinch also can sensitize a rat to the behavioral effects of <f-amphetamine, and vice versa (Antelman et al., 1980). Furthermore, prior stress can pre-dispose rats to the acquisition of intravenous self-administration of d-amphetamine (Piazza et al., 1989), which reflects an enhanced motivation toward psychostimulant reward. It is well-established that arousing stimulation, such as tail pinch or electric foot shock, can induce copulation in sexually naive (Cagguila and Eibergen, 1969), non-copulating (Crowley et al., 1973), and gonadectomized male rats (Barfield and Sachs, 1970). In fact, facilitation of sexual behavior due to tail pinch or shock was present during subsequent tests of sexual behavior, even when such stimulation was not administered (Crowley et al., 1973). Leyton and Stewart (1996) replicated these findings and demonstrated that repeated tail pinch "sensitized" sexual behavior in castrated male rats. That is, the percentage of male rats that showed copulatory behavior (e.g., mount, intromission, or ejaculation) increased progressively during three successive tests during which tail pinch was administered. It was proposed that the progressive facilitation of sexual behavior induced by repeated tail pinch may be mediated by sensitization of the M L B D A system (Leyton and Stewart, 1996). The expression of enhanced sexual behavior by repeated tail pinch was prevented by systemic administration of the D2 antagonist, pimozide, or the opioid antagonist, naloxone, at doses that preferentially block p-opioid receptors (Leyton and Stewart, 1996). More importantly, the administration of naloxone prevented the progressive enhancement of sexual behavior by tail pinch. Antagonism of V T A opioid receptors 119 (including p-type receptors) by local naltrexone injections has been shown to block the initiation of behavioral cross-sensitization to enkephalin (Kalivas and Abhold, 1987) and repeated activation of V T A p-opioid receptors sensitized behavior and N A C D A efflux (Kalivas and Stewart, 1991). Taken together, these data suggest that the progressive facilitation of sexual behavior induced by repeated tail pinch may be mediated by sensitization of the M L B D A system via stimulation of the V T A by endogenous opioids released by tail pinch. It appears that any stimulus capable of activating midbrain D A neurons has the potential to sensitize this system and enhance motivated behavior. Moreover, the concurrent presence of different stimuli, that share the ability to enhance mesotelencephalic D A transmission, may promote the development of behavioral sensitization, leading to an augmented enhancement of motivated behavior that may be, in some cases, dysfunctional (see Clinical Implications below). Presynaptic Mechanisms Subserving the Expression of Behavior Facilitated by Psychostimulant-induced Sensitization The present experiments contribute to the growing literature demonstrating that enhanced N A C D A transmission is associated with the expression of behavioral sensitization to psychostimulants. It is important to emphasize that enhancement of D A transmission in other forebrain areas also may have contributed to the facilitation of sexual behavior by d-amphetamine-induced behavioral sensitization. Dopamine in the mPOA is involved in both appetitive and consummatory aspects of male rat sexual behavior (Pfaus and Phillips, 1991; Hull et al., 1993, 1995; Shimura et al., 1994; Mas et 120 al., 1995; Sato et al., 1995) and enhanced mPOA D A transmission may have contributed to facilitation of sexual behavior observed in the present experiments. It is interesting that the A14 periventricular hypothalamic D A neurons, which terminate in the mPOA, share characteristics with A10 D A neurons. For example, A14 and A10 cell bodies are excited by morphine via p-opioid receptors, and possess D A autoreceptors that negatively regulate their activity (Moore, 1987). Furthermore, neurotensin and cholecystokinin are co-localized with D A in neurons of both cell groups (Moore and Lookingland, 1995). A finding that militates against the possibility that similar mesotelencephalic-type of sensitizing mechanisms occur in hypothalamic D A systems is the surprising absence of an increase in mPOA D A efflux after acute administration of J-amphetamine (1 mg/kg, IP; Du et al., 1998). The fact, however, that their laboratory begins microdialysis sampling shortly after probe implantation (i.e., 4 h) may have contributed to these results. There is experimental evidence that dialyzing before a 10-16 h period after probe implantation may reflect damage-induced, rather than physiological/pharmacological precipitated, release of neurotransmitter (Finlay and Zigmond, 1995). The bed nucleus of the stria terminalis (BST) is another structure which deserves consideration in the context of the present results. Lesions studies implicate this structure in male copulatory behavior, causing such deficits as increased inter-intromission intervals and ejaculatory latencies (Emery and Sachs, 1976; Claro et al., 1995; Liu et al., 1997). Recently, it was demonstrated that radiofrequency lesions of the BST result in a reduction of non-contact erections in response to cues from a receptive 121 female (Liu et al., 1997). The expression of non-contact erections can be considered a measure of sexual arousal in the male rat (Sachs, 1995). The BST receives dopaminergic input from the mesencephalon (see Figure 1), including the V T A . There is a possibility that augmented D A release in the BST contributed to enhanced sexual behavior observed in d-amphetamine-sensitized male rats. Increased D A efflux may contribute to the expression of behavioral sensitization, but what mechanisms mediate this enhanced release? Increased calcium transduction may underlie the augmentation of D A release in the striatum of sensitized rats, despite the fact that d-amphetamine increases extracellular D A in a calcium- and impulse-independent manner via the D A transporter (Kuczenski and Segal, 1994; Sulzer et al., 1995; Jones et al., 1998). The expression of behavioral sensitization to psychostimulants can be blocked by systemic administration of L-type calcium channel antagonists (Karler et al., 1991; Martin-Iverson and Reimer, 1994). Augmented D A efflux in the N A C of cocaine-sensitized rats, in response to local administration of d-amphetamine, was blocked by local co-administration of L-type or N-type calcium channel antagonists (Pierce and Kalivas, 1997b) or by replacing calcium in the microdialysis perfusion fluid with magnesium in rats pretreated with d-amphetamine (Warburton et al., 1996). In addition, local electrical stimulation was found to induce a greater efflux of D A in striatal tissue from d-amphetamine-sensitized rats (Castaneda et al., 1988). The efflux of N A C DA, as assessed by microdialysis under normal physiological conditions (e.g., during behavior), is dramatically attenuated by local application of the sodium channel blocker, tetrodotoxin, or abolished by reducing calcium ion concentration in the microdialysis perfusate to zero (Benveniste and Huttemeier, 1990). 1 2 2 This implies that the concentration of neurotransmitter in microdialysis samples during behavior is calcium- and depolarization-dependent. Thus, Experiment IV is the first demonstration that behaviorally induced, and presumably calcium-dependent, neuronal depolarization can augment M L B D A efflux in d-amphetamine sensitized rats and, therefore, is consistent with the view that repeated psychostimulant treatment may modify presynaptic calcium-mediated mechanisms in the N A C (Pierce and Kalivas, 1997b). Although there is a great deal of evidence for enhanced function in mesotelencephalic D A transmission in the expression of behavioral sensitization, other transmitters systems are undoubtedly involved. For example, acute administration of d-amphetamine increases extracellular concentrations of N A , as well as D A (Kuczenski and Segal, 1992), and a general role for N A in psychostimulant sensitization has been proposed (Kokkinidis and Anisman, 1980). Nevertheless, there is a paucity of data concerning N A in this regard. Recently, Camp et al. (1997) demonstrated the presence of augmented efflux of hippocampal N A in response to a d-amphetamine challenge in sensitized rats after a 30-day withdrawal period, but only when data were expressed in absolute values and not in terms of percent baseline. In contrast, a d-amphetamine challenge resulted in enhanced N A efflux in the hypothalamus of sensitized rats only when data were expressed as percent baseline. Regardless, alterations in N A transmission in these areas may have contributed to observed changes in the sexual behavior of psychostimulant-sensitized male rats. The Role of Mesolimbic Dopamine in Motivated Behavior 123 A number of theories have been proposed to explain the role of M L B D A in motivated behavior. Mesolimbic D A has been described as a mediator of pleasure (Wise, 1982), reward learning (Schultz et al., 1997; Beninger and Miller, 1998), incentive motivation (Blackburn et al.. 1992), and the attribution of incentive salience (Robinson and Berridge, 1993). One of the most influential theories of D A function, based on the ability of neuroleptic drugs to attenuate operant behavior, is the anhedonia hypothesis, which proposes that central D A systems mediate the pleasure produced by rewards, such as sex, food, or addictive drugs (Wise, 1982). Observations that ventral striatal D A activity is preferentially increased during appetitive situations, prior to contact with the "reward" and the experience of the pleasure, cast serious doubt on the validity of this theory of D A function because peak D A activity would be predicted to correspond to periods of maximal pleasure. For example, using in vivo voltammetry, Phillips et al. (1993) reported that striatal D A efflux increased in response to conditioned stimuli prior to a meal and remained elevated until after the meal ended. Electrophysiological experiments in behaving monkeys also provide evidence of anticipatory activation of midbrain D A neurons. Although D A neurons were activated by liquid food reward injected directly into the monkey's mouth when the animal was inexperienced, after repeated pairing of a stimulus with presentation of food reward, these neurons stopped responding to the reward and instead fired in response to the conditioned stimulus (Schultz, 1992). As training continued and the stimulus became highly predictive of reward, these neurons were no longer activated by either the stimulus or reward. Microdialysis studies, however, have produced seemingly conflicting results. For example, Wilson et al. (1995) reported that, whereas presentation of a palatable liquid 124 food behind a screen significantly enhanced N A C D A efflux in food-deprived rats, peak increases in striatal D A efflux were associated with "consummatory" behavior (i.e., when rats consumed the food). A similar neurochemical profile was observed in the ventral striatum of male rats engaged in sexual behavior. Although significant increases in striatal D A efflux occur when a receptive female is presented behind a screen, extracellular D A concentrations increase further during copulation in male rats (Pfaus et al., 1990a; Damsma et al., 1992; Fiorino et al., 1997a). Although we can view consummatory behaviors as being associated with enhanced N A C D A release (Wenkstern et al., 1993; Wilson et al., 1995), it is important to examine the terms "appetitive" and "consummatory" in the context of sexual behavior. Whereas the phase in which the female is present behind the screen is exclusively appetitive or preparatory, the behavior during the copulation phase cannot be considered purely consummatory. In as much as "appetitive" can be used to describe all behaviors leading to the consummation of a motivated behavior (copulation), the primary behavior the male exhibits while active in the "consummatory" phase is best described as appetitive; the male spends most of his time and effort pursuing the female in order to copulate. Therefore, it is probably more accurate to conclude that maximal N A C D A transmission is linked to consummatory as well as intense appetitive components of male rat sexual behavior. The relatively poor temporal resolution of microdialysis in monitoring extracellular concentrations of N A C D A does not permit a more accurate description at this time. The incentive-sensitization theory of drug addiction, which stems from earlier theories of incentive motivation, proposes a novel role for mesotelencephalic D A neurons 125 (Robinson and Berridge, 1993). The theory posits that the normal function of mesotelencephalic D A is to attribute incentive salience to cues associated with activity of this system. Robinson and Berridge (1993) describe incentive salience as "a psychological process that transforms the perception of stimuli, imbuing them with salience, making them attractive, 'wanted' incentive stimuli." Neutral stimuli become associated with natural rewards by learning processes and are attributed with incentive salience via reward-activated D A systems. In this manner, these stimuli become attractive and wanted. The repeated use of addictive drugs, such as psychostimulants, also increase mesotelencephalic D A activity, but in this case, induces a progressive enhancement in function that leads to excessive salience being attributed to the act of drug taking and stimuli associated with the drug. The theory further proposes that the mesotelencephalic D A system is independent from the system that mediates "liking" (i.e., pleasure). In as much as M L B D A transmission was enhanced selectively as a consequence of prior exposure to ^-amphetamine, the present results shed light on the normal function of N A C D A and support the incentive-sensitization theory . Evidence that D A is involved in the attribution of incentive salience to incentive cues associated with reward is compelling. Repeated ^-amphetamine administration, which augmented M L B D A function, enhanced the incentive salience of unconditioned incentive cues of the estrous female, resulting in facilitated appetitive sexual behavior in sensitized male rats. The presence of augmented N A C D A transmission in response to an estrous female located behind a screen strengthens the argument that the M L B D A system contributes to this effect, and not to "pleasure" that may result subsequently from copulation. 126 In addition, through mechanisms of incentive learning, the incentive value of environmental cues associated with copulation increased with sexual experience. That is, appetitive level change activity increased across successive copulatory sessions. Furthermore, psychostimulant-induced sensitization enhanced the progressive increase in appetitive activity, suggesting a role for M L B D A in this regard. Although this finding supports the incentive-sensitization theory of incentive learning, it does not exclude mechanisms by which the association between the pleasure of copulating and cues of the chamber could facilitate appetitive measures of sexual behavior. Much of the pharmacological literature on the role of D A in incentive learning is complex and contradictory, especially with regard to experiments that used D A agonists (Beninger, 1993; Beninger and Miller, 1998). Results of learning experiments employing specific D A antagonists are more straightforward; Di-like antagonists are associated with reduced reward, whereas D2-like antagonists tend to impair motor performance (Miller et al., 1990; Beninger, 1993). A recent review of learning experiments across a variety of paradigms and species supports the hypothesis that stimulation of D, receptors and the subsequent cascade of intracellular events, such as cAMP and P K A activation, may be key processes that lead to the enduring neural changes underlying incentive learning (Beninger and Miller, 1998). In this regard, the parallels between behavioral sensitization to psychostimulants and incentive learning are interesting. The long-term expression of psychostimulant-induced behavioral sensitization also is associated with a persistent augmentation of Di receptor function. As mentioned in the introduction, repeated administration of cocaine can lead to a persistent enhancement of c A M P and P K A activity in the N A C and a P K A activator enhanced the expression of behavioral 127 sensitization (Miserendino and Nesfler, 1995). In a similar manner, the injection of the P K A inhibitor RPp-cAMPS into the N A C blocked the establishment of a conditioned place preference to amphetamine (Beninger and Miller, 1998). The transcription modulating protein CREB, which is phosphorylated by P K A , also appears to play an important role in memory formation (DeZazzo and Tully, 1995) and has been shown to be active after long-term administration of psychostimulants (Cole et al., 1995; Simpson et al., 1995). These data support the contention that underlying mechanisms of incentive learning involve sensitization-like processes. While the systematic analysis of factors underlying the initiation and expression of behavioral sensitization may be necessary in order to understand the neurobiology of drug addiction, the examination of "de-sensitizing" processes is equally important and, indeed, critical for its treatment. Some fundamental and practical questions arise from this perspective. Are the effects of repeated administration of addictive drugs on limbic-motor circuitry and motivated behavior permanent? Can they be reversed by normal mechanisms of incentive learning or the pharmacological manipulation of these processes? The use of simple, well-studied animal models of motivated behavior, such as male rat sexual behavior, may prove to be valuable in answering these questions. Clinical implications Important practical implications arise from the finding that behavioral sensitization induced by repeated exposure to psychostimulants, and the possible attendant modifications of limbic-motor circuitry, can generalize to a natural motivated behavior, such as sex. As many as 70% of patients admitted to a New York cocaine 128 addiction treatment program were also reported to suffer from compulsive sexuality (Washton and Stone-Washton 1993). Drug-related compulsive sexual behavior in these patients was treated as a part of a dual-addiction pattern. The authors report that many of these patients are "trapped in a reciprocal relapse pattern in which compulsive sexual behavior precipitates relapse to cocaine and vice versa" (Washton and Stone-Washton 1993). It is clear that the interplay between drug taking and sexual behavior must be an important consideration when treating patients addicted to psychostimulants. Considering the consequences of high-risk sexual activity today, such as the transmission of HIV and hepatitis C, the treatment of comorbid compulsive behavior is imperative to the health of the addict. The present finding that repeated administration of psychomotor stimulants can facilitate sexual behavior in the male rat raises an intriguing question: could a similar drug-protocol be used in the treatment of motivational dysfunction, such as sexual desire disorders? Results from the present thesis provide a theoretical basis for a prospective treatment. In this regard, it is of interest to note that bupropion, an antidepressant drug which can increase D A transmission via blockade of the D A transporter (Cooper et al., 1980; Stamford et al. 1988; Nomikos et al., 1989), enhanced sexual function in male and female patients treated for sexual dysfunction (Crenshaw and Goldberg, 1995) and psychiatric disorders (Modell et al., 1997). The ability of bupropion to block D A uptake is a property of many drugs which induce behavioral sensitization (Robinson and Berridge, 1993; Pierce and Kalivas, 1997a). The clinical effects of bupropion appear only after a few weeks of treatment, a time frame that is similar to that required for psychostimulants to induce robust behavioral sensitization. These parallels raise the 129 possibility that the mechanism responsible for bupropion's efficacy in treating sexual dysfunction may involve sensitization of the M L B D A system and suggest that comparable clinical effects may be obtained with the repeated administration of other dopaminergic agonists. It is interesting that pre-clinical studies have shown that long-term (10 mg/kg b.i.d. x 21 days), but not acute (10 mg/kg b.i.d. x 2 days), administration of bupropion resulted in augmented N A C D A efflux in response to a bupropion challenge (Nomikos et al, 1989, 1992). In contrast, there was no significant augmentation of D A transmission observed in the striatum after chronic bupropion treatment (Nomikos et al., 1992). The development of the prosexual effects of bupropion may be a consequence of neural changes similar to those involved in the initiation and expression of behavioral sensitization to psychostimulants. Therefore, the ability of a compound to induce long-term functional enhancement in the M L B D A system may provide valuable information about its potential to treat sexual dysfunction. 130 R E F E R E N C E S Abercrombie ED, Keefe K A , DiFrischia DS, Zigmond M J (1989) Differential effect of stress on in vivo dopamine release in striatum, nucleus accumbens, and medial frontal cortex. J Neurochem 52:1655-1658. Agmo A , Fernandez H (1989) Dopamine and sexual behavior in the male rat: a reevaluation. J Neural Transm 77:21-37. Agmo A, Picker Z (1990) Catecholamines and the initiation of sexual behavior in male rats without sexual experience. Pharmacol Biochem Behav 35:327-334. Akimoto K , Hamamura T, Kazahaya Y , Akiyama K , Otsuki S (1990) Enhanced extracellular dopamine level may be the fundamental neuropharmacological basis of cross-behavioral sensitization between methamphetamine and cocaine - an in vivo dialysis study in freely moving.Brain Res 507:344-6. Allard P, Eriksson K , Ross SB, Marcusson JO (1990) Unaltered [3H]GBR12935 binding after chronic administration of cocaine. Synapse 14:314-323. Anagnostaras SG, Robinson TE (1996) Sensitization to the psychomotor stimulant effects of amphetamine: modulation by associative learning. Behav Neurosci 110:1397-1414. Antelman S (1988) Stressor-induced sensitization to subsequent stress: implications for the development and treatment of clinical disorders. In: Kalivas PW, Barnes C D (eds) Sensitization in the nervous system. Telford Press: Caldwell, pp 227-254. Antleman S M , Chiodo L A (1981) Dopamine autoreceptor subsensitivity: a mechanism common to the treatment of depression and the induction of amphetamine psychosis. Biol Psychiatry 16:717-727. Antelman SM, Eichler AJ , Black CA, Kocan D (1980) Interchangeability of stress and amphetamine in sensitization. Science 207:329-331. Badiani A , Anagnostaras SG, Robinson TE (1995a) The development of sensitization to the psychomotor stimulant effects of amphetamine is enhanced in a novel environment. Psychopharmacology 117:443-452. Badiani A , Browman K E , Robinson TE (1995b) The influence of novel versus home environments on sensitization to the psychomotor stimulant effects of cocaine and amphetmaine. Brain Res 674:291-298. 1 3 1 Bannon MJ , Granneman JG, Kapatos G (1995) The dopamine transporter: potential involvement in neuropsychiatric disorders. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 179-188. Barfield RJ, Sachs B D (1970) Effect of shock on copulatory behavior in castrate male rats. Horm Behav 1:247-253. Beach F A (1941) Analysis of the stimuli adequate to elicit mating behavior in the sexually-inexperienced male rat. J Comp Psychol 33: 163-207. Beach F A (1956) Characteristics of masculine "sex drive." In: Jones M R (ed) Nebraska symposium on motivation. University of Nebraska Press, Lincoln, 1956:1-31. Bell DS, Trethowan W H (1961) Amphetamine addiction and disturbed sexuality. Arch Gen Psychiatry 4:74-78. Beninger RJ (1993) The role of D i and D2 receptors in learning. In: Waddington (ed) Di:D2 dopamine receptor interactions. Academic Press, London, pp 115-157. Beninger RJ, Miller R (1998) Dopamine Dl-l ike receptors and reward-related incentive learning. Neurosci Biobeh Rev 22:335-345. Benmansour S, Tejani-Butt SM, Hauptmann M , Brunswick DJ (1992) Lack of effect of high-dose cocaine on monoamine uptake sites in rat brain as measured by quantitative autoradiography. Psychopharmacology 106:459-462. Ben-Shahar O, Ettenberg A (1994) Repeated stimulation of the ventral tegmental area sensitizes the hyperlocomotor response to amphetamine. Pharmacology Biochem Behav 48:1005-1009. Benveniste H , Huttemeier PC (1990) Microdialysis: theory and application. Prog Neurobiol 35:195-215. Bertler A , Rosengren E (1959) Occurrence and distribution of dopamine in brain and other tissues. Experentia 15:10. Bignami G (1966) Pharmacological influences on mating behavior in the male rat. Psychopharmacologia 10:44-58. Blackburn JR, Pfaus JG, Phillips A G (1992) Dopamine functions in appetitive and defensive behaviours. Prog Neurobiol 39:247-79. Blaschko H (1939) The specific action of 1-dopa decarboxylase. J. Physiol 96:50. 132 Bloch GJ, Butler PC, Kohlert JG, Bloch D A (1993) Microinjection of galanin into the medial preoptic nucleus facilitates copulatory behavior in the male rat. Physiol Behav 54:615-624. Bloom FE (1996) Neurotransmission and the central nervous system. In: Hardman JG, Limbird L E , Molinoff PB, Ruddon RW, Gilman A G (eds) Goodman & Gilman's The pharmacological basis of therapeutics (9th ed). McGraw-Hill, New York, pp 267-293. Boeijinga PH, Pennartz C M A , Lopes da Silva F H (1990) Paired-pulse facilitation in the nucleus accumbens following stimulation of subicular inputs in the rat. Neurosci 35:310-311. Cador M , Bjijou Y , Stinus L (1995) Evidence of a complete independence of the neurobiological substrates for the induction and expression of behavioral sensitization to amphetamine. Neurosci 65: 385-395. Cagguila AR, Eibergen R (1969) Copulation of virgin male rats evoked by painful peripheral stimulation. J Comp Physiol Psychol 69:414-419 Cameron DL, Williams JT (1993) Dopamine D l receptors facilitate transmitter release. Nature 366:344-347. Camp D M , DeJonghe DK, Robinson TE (1997) Time-dependent effects of repeated amphetamine treatment on norepinephrine in the hypothalamus and hippocampus assessed with in vivo microdialysis. Neuropsychopharmacology 17:130-140. Canadian Council on Animal Care (1993) Guide to the care and use of experimental animals.Volume 1, 2nd ed. Olfert ED, Cross B M , McWilliam A A (eds). Cass W A , Gerhardt G A , Gillespie K , Curella P, Mayfield RD, Zahniser N R (1993) Reduced clearance of exogenous dopamine in rat nucleus accumbens, but not dorsal striatum following cocaine challenge in rats withdrawn from repeated cocaine. J Neurochem 61:273-283. Castaneda E, Becker JB, Robinson TE (1988) The long-term effects of repeated amphetamine treatment in vivo on amphetamine, KC1 and electrical stimulation evoked striatal dopamine release in vitro. Life Sci 42:2447-2456. Cerruti NS, Pilotte NS, Uhl G, Kuhar M J (1994) Reduction in dopamine transporter mRNA after cessation of repeated cocaine treatment. Mol Brain Res 22:132-138. Chiodo L A , Freeman AS, Bunney BS (1995) Dopamine autoreceptor signal transduction and regulation. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 221-226. 133 Civelli O (1995) Molecular biology of the dopamine receptor subtypes. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 155-161. Claro F, Segovia S, Guilamon A , del Abril A (1995) Lesions in the medial posterior region of the BST impair sexual behavior in sexually experienced and inexperienced male rats. Brain Res Bull 36:1-10. Cole RL , Konradi C, Douglass J, Hyman SE (1995) Neuronal adaptation to amphetamine and dopamine: molecular mechanisms of prodynorphin gene regulation in rat striatum. Neuron 14:813-823. Cooper BR, Hester TJ, Maxwell R A (1980) Behavioral and biochemical effects of the antidepressant bupropion (Wellbutrin): evidence for selective blockade of dopamine uptake in vivo. J Pharmacol Exp Ther 215:127-134. Cooper JR, Bloom FE, Roth R H (1991) The biochemical basis of neuropharmacology. Oxford University Press, New York, Oxford. Crenshaw TL, Goldberg JP (1995) Sexual pharmacology: drugs that affect sexual functioning. WW Norton and Company, New York, London. Crombag HS, Badiani A , Robinson TE (1996) Signalled versus unsignalled intravenous amphetamine: large differences in the acute psychomotor response and sensitization. Brain Res 722:227-31. Crowley WR, Popolow HB, Ward OB Jr (1973) From dud to stud: copulatory behavior elicited through conditioned arousal in sexually inactive male rats. Physiol Behav 10:391-394. Dahlstrom A , Fuxe K (1964) Evidence for the existence of monoamine neurons in the central nervous system. I. Demonstration of monoamines in cell bodies of brainstem neurons. Acta Physiol Scand Suppl 64:232-278. Damsma G, Pfaus JG, Wenkstern D, Phillips A G , 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. Deroche V , Marinelli M , Maccari S, LeMoal M , Simon H, Piazza PV (1995) Stress-induced sensitization and glucocorticoids. I. Sensitization of dopamine-dependent of amphetamine and morphine depends on stress-induced corticosterone secretion. J Neurosci 15:7181-7188. 134 Deutch A Y , Bourdelais A J , Zahm DS (1993) The nucleus accumbens core and shell: accumbal compartments and their functional attributes. In: Kalivas PW, Barnes C D (eds) Limbic motor circuits and neuropsychiatry. CRC Press, Boca Raton, pp 45-88. DeZazzo J, Tully T (1995) Dissection of memory formation: from behavioral pharmacology to molecular genetics. TINS 18:212-8. Dickinson A , Balleine B (1995) Motivational control of instrumental action. Curr Dir Psychol Sci 4:162-167. Du J, Lorrain DS, Hull E M (1998) Castration decreases extracellular, but increases intracellular dopamine in medial preoptic area of male rats. Brain Res 782:11-17. Duman RS, Nestler EJ (1995) Signal transduction pathways for catecholamine receptors. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 303-320. Dwoskin LP, Peris J, Yasuda RP, Philpott K , Zahniser N R (1988) Repeated cocaine administration results in supersensitivity of striatal D-2 dopamine autoreceptors to pergolide. Life Sci 42:255-262. Eichler A J , Antelman S M (1979) Sensitization to amphetamine and stress may involve nucleus accumbens and medial frontal cortex. Brain Res 176:412-416. Ellinwood E H (1967) Amphetamine psychosis: I. Description of the individuals and process. J Nerv Ment Disord 144:273-283. Emery DE, Sachs BD (1976) Copulatory behavior in male rats with lesions in the bed nucleus of the stria terminalis. Physiol Behav 17:803-806. Everitt BJ (1990) Sexual motivation: a neural and behavioural analysis of the mechanisms underlying appetitive and copulatory responses of male rats. Neurosci Biobehav Rev 14:217-232. 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. Farfel G M , Kleven MS, Woolverton W L , Seiden LS, Perry B D (1992) Effects of repeated injections of cocaine on catecholamine receptor binding sites, dopamine transporter binding sites and behavior in rhesus monkeys. Brain Res 578:235-243. Fibiger HC (1993) Mesolimbic dopamine: an analysis of its role in motivate behavior. Semin Neurosci 5:321-327. 135 Fibiger HC, Phillips A G (1986) Reward, motivation, cognition: psychobiology of mesotelencephalic dopamine systems. In: Bloom FE, Geiger SD (eds) Handbook of physiology: the nervous system IV. American Physiological Society, Bethesda, pp 647-675. Fibiger HC, Phillips A G (1987) Role of catecholamine transmitters in brain reward systems: implication for the neurobiology of affect. In: Engel J, Oreland L , Ingvar D H , Pernow B, Rossner S, Pellborn L A (eds) Brain reward systems and abuse. Raven Press, New York, pp 647-675. Finlay JM, Zigmond M J (1995) A critical analysis of neurochemical methods for monitoring transmitter dynamics in the brain. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 29-39. Fiorino DF, Coury A , Phillips A G (1997a) Dynamic changes in nucleus accumbens dopamine efflux during the Coolidge effect in male rats. J Neurosci 17(12):4849-4855. Fiorino DF, Coury A , Phillips A G , Fibiger HC (1993) Electrical stimulation of reward sites in the ventral tegmental area increases dopamine transmission in the nucleus accumbens of the rat. Behav Brain Res 55:131-141. Fiorino DF, Phillips A G , Blaha CD (1997b) Fine temporal correlations between nucleus accumbens dopamine efflux and copulatory events in the male rats using in vivo chronoamperometry. Soc Neurosci Abstr 533.1:1358. Fitzgerald JL, Reid JJ (1991) Chronic cocaine treatment does not alter rat striatal D2 autoreceptor sensitisitivy to pergolide. Brain Res 541:327-333. Fitzgerald L W , Nestler EJ (1995) Cocaine regulation of signal transduction pathways. In: Hammer RP Jr (ed) The neurobiology of cocaine. CRC Press, Boca Raton, pp. 225-246. Fitzgerald L W , Ortiz J, Hamedani A G , Nestler EJ (1996) Drugs of abuse and stress increase the expression of GluRl and NMDAR1 glutamate receptor subunits in the rat ventral tegmental area: common adaptations among cross-sensitizing agents. J Neurosci 16:274-282 Galloway M P (1988) Neurochemical interactions of cocaine with dopaminergic systems. Trends Pharmacol Sci 9:451-454. Garris PA, Ciolkowski EL , Pastore P, Wightman R M (1994) Efflux of dopamine from the synaptic cleft in the nucleus accumbens of the rat brain. J Neurosci 14:6084-6093. 136 Gifford A N , Johnson K M (1992) Effect of chronic cocaine treatment on D2 receptors regulating the release of dopamine and acetylcholine in the nucleus accumbens and striatum. Pharmacol Biochem Behav 41:841-846. Giros B , Jaber M , Jones SR, Wightman R M , Caron M G (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379:606-612. Glenthoj B, Mogensen J, Laursen H , Horn S, Hemmingsen R (1993) Electrical sensitization of the meso-limbic dopaminergic system in rats: a pathogenetic model for schizophrenia. Brain Res 619:39-54. Gnegy M E , Hewlett GHK, Yee SL, Welsh M J (1991) Alterations in calmodulin content and localization in areas of rat brain after repeated intermittent amphetamine. Brain Res 562:6-12. Greaves G (1972) Sexual disturbances among chronic amphetamine users. J Nerv Ment Disord 155:363-365. Heidbreder C A , Thompson A C , Shippenberg TS (1996) Role of extracellular dopamine in the initiation and long-term expression of behavioral sensitization to cocaine. J Pharmacol Exp Ther 278:490-502. Heimer L , Zahm DS, Churchill L , Kalivas PW, Wohltmann C (1991) Specificity in the projection patterns of accumbal core and shell. Neurosci 41:89-126. Henry DJ, Greene M A , White FJ (1989) Electrophysiological effects of cocaine in the mesoacumbens dopamine system: repeated administration. J Pharmacol Exp Ther 251:833-839. Henry DJ, White FJ (1991) Repeated cocaine administration causes a persistent enhancement of D l dopamine receptor supersensitivity within the rat nucleus accumbens. J Pharmacol Exp Ther 258:882-890. Hokfelt T, Johansson O, Fuxe K , Goldstein M , Park D (1976) Immunocytochemical studies on the localization and distribution of monoamine neuron systems in the rat brain. Med Biol 55:21-56. Hooks MS, Jones G H , Liem BJ, Justice Jr JB (1992) Sensitization and individual differences to intraperitoneal amphetamine, cocaine or caffeine following repeated intracranial amphetamine infusions. Pharmacol Biochem Behav 43:815-823. Horger B A , Giles M K , Schenk S (1992) Preexposure to amphetamine and nicotine predisposes rats to self-administer a low dose of cocaine. Psychopharmacology 107:271-276. 137 Horger B A , Shelton K , Schenk S (1990) Preexposure sensitizes rats to the rewarding effects of cocaine. Pharmacol Biochem Behav 37:707-711. Hull E M , Bitran D, Pehek E A , Warner RK, Band L C , Holmes G M (1986) Dopaminergic control of male sexual behavior in rats: effects of an intracerebrally infused agonist. Brain Res 370:73-81. Hull E M , Eaton RC, Moses J, Lorrain DS (1993) Copulation increases dopamine activity in the medial preoptic area of male rats. Life Sci 52:935-940. Hull E M , Du J, Lorrain DS, Matuszewich L (1995) Extracellular dopamine in the medial preoptic area: implications for sexual motivation and hormonal control of copulation. J Neurosci 15(11):7465-7471. Hull E M , Warner RK, Bazzett TJ, Eaton RC, Thompson JT, Scaletta L L (1989) D2/D1 ratio in the medial preoptic area affects copulation of male rats. J Pharmacol Exp Ther 251:422-427. Hurd Y L , Weiss F, Koob GF, Ungerstedt N - E U (1989) Cocaine reinforcement and extracellular dopamine overflow in rat nucleus accumbens: an in vivo microdialysis study. Brain Res 498:199-203. Imperato A , Angelucci L , Casolini P, Zocchi A , Puglisi-Allegra S (1992) Repeated stressful experiences differently affect limbic dopamine release during and following stress. Brain Res 577:194-199. Iwata S, Hewlett GHK, Ferrell ST, Czernik AJ , Meiri K , Gnegy M E (1996) Increased in vivo phosphorylation state of neuromodulin and synapsin I in striatum from rats treated with repeated amphetamine. J Pharmacol Exp Ther 278:1428-1434. Izenwasser S, Werling L L , Cox B M (1990) Comparison of the effects of cocaine and other inhibitors of dopamine uptake in rat striatum, nucleus accumbens, olfactory tubercle, and medial prefrontal cortex. Brain Res 520:303-309. Jacobsen I, Sandberg M , Hamberger A (1985) Mass transfer in brain dialysis devices: a. new method for estimation of extracellular amino acids concentration. J Neurosci Methods 15:263-268. Jaffe JH (1990) Drug addiction and drug abuse. In: Gilman A G , Rail TW, Nies AS , Taylor P (eds) Goodman & Gilman's The pharmacological basis of therapeutics (8th ed). McGraw-Hill, New York, pp 532-581. Jones SR, Gainetdinov RR, Wightman R M , Caron M G (1998) Mechanisms of amphetamine action revealed in mice lacking the dopamine transporter. J Neurosci 18:1979-1986. 138 Jones SR, Lee TH, Wightman R M , Ellinwood E H (1996) Effects of intermittent and continuous cocaine administration on dopamine release and uptake regulation in the striatum: in vitro voltammetric assessment. Psychopharmacology 126:331-338. Jones SR, O'Dell SJ, Marshall JF, Wightman R M (1996) Functional and anatomical evidence for different dopamine dynamics in the core and shell of the nucleus accumbens in slices of rat brain. Synapse 23:224-231. Kalivas PW (1993) Neurotransmitter regulation of dopamine neurons in the ventral tegmental area. Brain. Res Rev 18:75-113. Kalivas PW, Abhold R (1987) Enkephalin release into the ventral tegmental area in response to stress: modulation of mesocortical dopamine. Brain Res 414:339-348. Kalivas PW, Churchill L , Klitenick M A (1993a) The circuitry mediating the translation of motivational stimuli into adaptive motor responses. In: Kalivas PW, Barnes C D (eds) Limbic motor circuits and neuropsychiatry. CRC Press, Boca Raton, pp 237-287. Kalivas PW, Duffy P (1988) Effects of daily cocaine and morphine treatment on somatodendritic and terminal field dopamine release. J Neurochem 50:1498-1504. Kalivas PW, Duffy P (1993) Time course of extracellular dopamine and behavioral sensitization to cocaine: I. Dopamine axon terminals. J Neurosci 13:266-275. Kalivas PW, Duffy P (1995) D l receptors modulate glutamate release transmission in the ventral tegmental area. J Neurosci 15:5379-5388. Kalivas PW, Sorg B A , Hooks MS (1993b) The pharmacology and neural circuitry of sensitization to psychostimulants. Behav Pharmacol 4:315-334. Kalivas PW, Stewart J (1991) Dopamine transmission in the initiation and expression of drug- and stress-induced sensitization of motor activity. Brain Res Rev 16:223-244. Kalivas PW, Weber B (1988) Amphetamine injection into the A10 dopamine region sensitizes rats to peripheral amphetamine and cocaine. J Pharmacol Exp Ther 245:1095-1102. Karler R, Turkanis SA, Partlow L M , Calder L D (1991) Calcium channel blockers in behavioral sensitization. Life Sci 49:165-170. Kebabian JW, Caine DB (1979) Multiple receptors for dopamine. Nature 277:93-96. Kehr J (1993) A survey on quantitative microdialysis: theoretical models and practical implications. J Neurosci Methods 48:251-261. 139 King GR, Ellinwood E H Jr, Silvia C, Joyner C M , Xue Z, Caron M G (1994) Withdrawal from continuous or intermittent cocaine administration: changes in D2 receptor function. J Pharmacol Exp Ther 269:743-749. Kiyatkin E A (1995) Functional significance of mesolimbic dopamine. Neurosci BiobehavRev 19(4):573-598. Kokkinidis L , Anisman L (1980) Amphetamine models of paranoid schizophrenia: an overview and elaboration of animal experimentation. Psychol Bull 88:351-379.. Kokkinidis L , Kirkby RD, McCarter BD, Borowski TB (1989) Alterations in amphetamine-induced locomotor activity and stereotypy after electrical stimulation of the nucleus accumbens and neostriatum. Life Sci 44:633-641. Kokkinidis L , Zacharko R M (1980) Response sensitization and depression following long-term amphetamine treatment in a self-stimulation paradigm. Psychopharmacology 68:73-76. Koob GF (1992) Dopamine, addiction and reward. Semin Neurosci 4:139-148. Kuczenski R, Segal DS (1992) Regional norepinephrine response to amphetamine using dialysis: comparison with caudate dopamine. Synapse 11:164-169. Kuczenski R, Segal DS (1994) Neurochemistry of amphetamine. In: Cho A K , Segal DS (eds) Amphetamine and its analogs. Academic Press, San Diego, pp 81-113. Kuczenski R, Segal DS, Todd P K (1997) Behavioral sensitization and extracellular dopamine responses to amphetamine after various treatments. Psychopharmacology 134:221-229. Kuhar MJ , Pilotte NS (1996) Neurochemical changes in cocaine withdrawal. Trends Pharmacol Sci 17:260-4. Kula NS, Baldessarini RJ (1991) Lack of increase in dopamine transporter bidning or function in rat brain tissue after treatment with blockers of neuronal uptake of dopamine. Neuropharmacology 30:89-92. LeMoal M (1995) Mesocorticolimbic dopaminergic neurons. Functional and regulatory roles. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 283-294. Lerma J, Herranz AS , Herreras O, Abraira V , Marin Del Rio R (1986) In vivo determination of extracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis. Brain Res 384:145-155. 140 Letchworth SR, Daunais JB, Hedgecock A A , Porrino L J (1997) Effects of chronic cocaine administration on dopamine transporter mRNA and protein in the rat. Brain Res 750:214-222. Lett BT (1989) Repeated exposures intensify rather than diminish the rewarding effects of amphetamine, morphine, and cocaine. Psychopharmacology 107:271-276. Leyton M , Stewart J (1996) Acute and repeated activation of male sexual behavior by tail pinch: opioid and dopaminergic mechanisms. Physiol Behav 60:77-85. L i D-H, Depoortere R Y , Emmett-Oglesby M W (1994) Tolerance to the reinforcing effects of cocaine in a progressive ratio paradigm. Psychopharmacology 116:326-332. Liang N Y , Rutledge CO (1982a) Comparison of the release of [3H]dopamine from isolated corpus striatum by amphetamine, fenfluramine and unlabelled dopamine. Biochem Pharmacol 31:983-92. Liang N Y , Rutledge CO (1982b) Evidence for carrier-mediated efflux of dopamine from corpus striatum. Biochem Pharmacol 31:2479-84. Liu Y - C , Salamone JD, Sachs BD (1997) Lesions in medial preoptic area and bed nucleus of stria terminalis: differential effects on copulatory behavior and noncontact erection in male rats. J Neurosci 17(13):5245-5253. Lonnroth P, Jansson PA, Smith U (1987) A microdialysis method allowing characterization of intercellular water space in humans. A m J Physiol 253:E228-E231. Louilot A , Gonzalez-Mora JL, Guadalupe T, Mas M (1991) Sex-related olfactory stimuli induce a selective increase in dopamine release in the nucleus accumbens of male rats. A voltammetric study. Brain Res 553:313-317. Lu W, Chen H , Xue C-J, Wolf M E (1997) Repeated amphetamine administration alters the expression of mRNA for A M P A receptor subunits in rat nucleus accumbens and prefrontal cortex. Synapse 26:269-280. Lu W, Wolf M E (1997) Expression of dopamine transport and vesicular monoamine transporter 2 mRNAs in rat midbrain after repeated amphetamine administration. Mol Brain Res 49:137-148. Mack F, Bonisch H (1979) Dissociation constants and lipophilicity of catecholamines and related compounds. Naunyn-Schmiedebergs Archives of Pharmacology 310:1-9 Madlafousek J, Hlinak Z (1983) Importance of female's precopulatory behaviour for the primary initiation of male's copulatory behaviour in the laboratory rat. Behav 86:237-249. 141 Mansour A , Watson SJ Jr (1995) Dopamine receptor expression in the central nervous system. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 207-219. Martin-Iverson MT, Reimer A R (1994) Effects of minodipine and/or haloperidol on the expression of conditioned locomotion and sensitization to cocaine in rats. Psychopharmacology 114:315-320. Mas M (1995) Neurobiological correlates of masculine sexual behavior. Neurosci Biobehav Rev 19:261-277. Mas M , Fumero B, Gonzalez-Mora JL (1995) Voltammetric and microdialysis monitoring of brain monoamine neurotransmitter release during sociosexual interactions. Behav Brain Res 71:69-79. Mas M , Gonzalez-Mora JL, Louilot A , Sole C, Guadalupe T (1990) Increased dopamine release in the nucleus accumbens of copulating male rats as evidenced by in vivo voltammetry. Neurosci Lett 110:303-308. Masserano JM, Baker I, Natsukari N , Wyatt RJ (1996) Chronic cocaine administration increases tyrosine hyrdroxylase activity in the ventral tegmental area through glutamatergic- and dopaminergic D2-receptor mechanisms. Neurosci Lett 217:73-76. Melis M R , Argiolas A (1995) Dopamine and sexual behavior. Neurosci Biobehav Rev 19:19-38. Mendelson SG, Gorzalka B B (1987) An improved chamber for the observation and analysis of the sexual behavior of the female rat. Physiol Behav 39:67-71. Mendelson SG, Pfaus JG (1989) Level searching: a new assay of sexual motivation in the rat. Physiol Behav 45:337-341. Mendrek A , Blaha CD, Phillips A G (1998) Pre-exposure of rats to amphetamine sensitizes self-administration of this drug under a progressive ratio schedule. Psychopharmacology 135:416-422. Miller R, Wickens JR, Beninger RJ (1990) Dopamine D - l and D-2 receptors in relation to reward and performance: a case for the D - l receptor as a primary site of therapeutic action of neuroleptic drugs. Prog Neurobiol 34:143-183. Miserendino MJD, Nestler EJ (1995) Behavioral sensitization to cocaine: modulation by the cyclic A M P system in the nucleus accumbens. Brain Res 674:299-306. Mitchell JB, Gratton A (1991) Opioid modulation and sensitization of dopamine release elicited by sexually relevant stimuli: a high speed chronoamperometric study in freely behaving rats. Brain Res 551:20-27. 142 Mitchell JB, Gratton A (1992) Mesolimbic dopamine release elicited by activation of the accessory olfactory system: a high speed chronoamperometry study. Neurosci Lett 140:81-84. Mitchell JB, Stewart J (1990) Facilitation of sexual behavior in the male rat in the presence of stimuli previously paired with systemic injections of morphine. Pharmacol Biochem Behav 35:367-372. Modell JG, Katholi CR, Modell JD, DePalma R L (1997) Comparative sexual side effects of bupropion, fluoxetine, paroxetine, and sertraline. Clin Pharmacol Ther 61:476-487. Moore K E (1987) Hypothalamic dopaminergic neuronal systems. In: Meltzer H Y (ed) Psychopharmacology: the third generation of progress. Raven Press Ltd., New York, pp 127-139. Moore K E , Lookingland K J (1995) Dopaminergic neuronal systems in the hypothalamus. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 245-256. Nestler EJ, Terwilliger RZ, Walker JR, Sevarino K A , Duman RS (1990) Chronic cocaine treatment decreases levels of the G protein subunits G i a and G o a in discrete regions of rat brain. J Neurochem 55:1079-1082. Nirenberg MJ , Chan J, Pohorille A, Vaughan R A , Uhl GR, Kuhar MJ , Pickel V M (1997) The dopamine transporter: comparative ultrastructure of dopaminergic axons in limbic and motor compartments of the nucleus accumbens. J Neurosci 17(18):6899-6907. Nomikos GG, Damsma G, Wenkstern D, Fibiger HC (1989) Acute effects of bupropion on extracellular dopamine concentrations in rat striatum and nucleus accumbens studied by in vivo microdialysis. Neuropsychopharmacology 2:273-279. Nomikos GG, Damsma G, Wenkstern D, Fibiger HC (1992) Effects of chronic bupropion on interstitial concentrations of dopamine in rat nucleus accumbens and striatum. Neuropsychopharmacology 7:7-14. O'Donnell P, Grace A A (1993) Physiological and morphological properties of accumbens core and shell neurons recorded in vitro. Synapse 13:135-160. O'Neill RD, Lowry JP, Mas M (1998) Monitoring brain chemistry in vivo: voltammetric techniques, sensors, and behavioral applications. Crit Rev Neurobiol 12:69-127. Parsons L H and Justice JB Jr (1993) Serotonin and dopamine sensitization in the nucleus accumbens, ventral tegmental area and dorsal raphe nucleus following repeated cocaine administration. J Neurochem 61:1611-1619. 143 Parsons L H and Justice JB Jr (1994) Quantitative approaches to in vivo microdialysis. Crit Rev Neurobiol 8:189-220 Paulson PE, Camp D M , 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 103:480-492. Paulson PE, Robinson TE (1995) Amphetamine-induced time-dependent sensitization of dopamine neurotransmission in the dorsal and ventral striatum: a microdialysis study in behaving rats. Synapse 19:56-65. Paxinos G, Watson C (1997) The rat brain in stereotaxic coordinates on C D - R O M . Academic Press, San Diego. Peris J, Boyson SJ, Cass W A , Curella P, Dwoskin LP, Larson G, Lin L - H , Yasuda RP, Zahniser N R (1990) Persistence of neurochemical changes in dopamine systems after repeated cocaine administration. J Pharmacol Exp Ther 253:38-44. Perugini M , Vezina P (1994) Amphetamine administered to the ventral tegmental area sensitizes rats to the locomotor effects of nucleus accumbens amphetamine. J Pharmacol Exp Ther 270:690-696. Pfaus JG (1996) Frank A . Beach award. Homologies of animal and human sexual behaviors. Horm Behav 30:187-200. Pfaus JG, Everitt BJ (1995) The psychopharmacology of sexual behavior. In: Bloom FE, Kupfer DJ (eds) Psychopharmacology: the fourth generation of progress. Raven Press Ltd., New York, pp 743-758. Pfaus JG, Damsma G, Nomikos GG, Wenkstern D, Blaha CD, Phillips A G , Fibiger H C (1990a) Sexual behavior enhances central dopamine transmission in the male rat. Brain Res 530:345-348. Pfaus JG, Mendelson SD, Phillips A G (1990b) A correlational and factor analysis of anticipatory and consummatory measures of sexual behavior in the male rat. Psychoneuroendocrinology 15:329-340. Pfaus JG, Phillips A G (1991) Role of dopamine in anticipatory and consummatory aspects of sexual behavior in the male rat. Behav Neurosci 105:727-743. Pfaus JG, Wilkins M F (1995) A novel environment disrupts copulation in sexually naive but not experienced male rats: reversal with naloxone. Physiol Behav 57:1045-9. 144 Phillips A G , Atkinson LJ , Blackburn JR., Blaha CD (1993) Increased extracellular dopamine in the nucleus accumbens of the rat elicited by a conditional stimulus for food: an electrochemical study. Can J Physiol Pharmacol 71:387-93. Phillips A G , Pfaus JG, Blaha CD (1991) Dopamine and motivated behavior: insights provided by in vivo analysis. In: Willner P, Scheel-Kruger J. (eds) The mesocorticolimbic dopamine system: from motivation to action. Wiley, Chichester, pp 199-224. Piazza PV, Deminiere JM, LeMoal M , Simon H (1989) Factors that predict individual vulnerability to amphetamine self-administration. Science 245:1511-1513 Piazza PV, Deminiere JM, LeMoal M , Simon H (1990) Stress- and pharmacologically-induced behavioral sensitization increases vulnerability to acquisition of amphetamine self-administration. Brain Res 514:22-6. Pickel V M , Nirenberg MJ , Milner T A (1996) Ultrastructural view of central catecholaminergic transmisison: immunocytochemical localization of synthesizing enzymes, transporters, and receptors. J Neurocytol 25:843-856. Pierce RC, Duffy P, Kalivas PW (1995) Sensitization to cocaine and dopamine autoreceptor subsensitivity in the nucleus accumbens. Synapse 20:33-36. Pierce RC, Kalivas PW (1995) Amphetamine produces sensitized increases in locomotion and extracellular dopamine preferentially in the nucleus accumbens shell of rats administered repeated cocaine. J Pharmacol Exp Ther 275:1019-1029 Pierce RC, Kalivas PW (1997a) A circuitry model of the expression of behavioral sensitization to amphetamine-like psychostimulants. Brain Res Rev 25:192-216. Pierce RC, Kalivas PW (1997b) Repeated cocaine modifies the mechanism by which amphetamine release dopamine. J Neurosci 17:3245-3261. Pilotte NS, Sharpe L G , Kuhar M J (1994) Withdrawal from repeated intravenous infusions of cocaine persistently reduces binding to dopamine transporters in the nucleus accumbens of Lewis rats. J Pharmacol Exp Ther 269:963-969. Pleim ET, Matochik JA, Barfield RJ, Auerbach SB (1990) Correlation of dopamine release in the nucleus accumbens with masculine sexual behavior in rats. Brain Res 524:160-163. Pontieri FE, Tanda G, DiChiara G (1995) Intravenous cocaine, morphine, and amphetamine preferentially increase extracellular dopamine in the "shell" as compared with the "core" of the rat nucleus accumbens. Proc Natl Acad Sci U S A 92:12304-12308. 145 Post R M (1980) Intermittent versus continuous stimulation: effect of time interval on the development of sensitization or tolerance. Life Sci 26:1275-1282. Post R M , Weiss SRB (1988) Sensitization and kindling: implications for the evolution of psychiatric symptomatology. In: Kalivas PW, Barnes CD (eds) Sensitization in the nervous system. Telford Press: Caldwell, pp 257-292. Predy PA, Kokkinidis L (1984) Sensitization to the effects of repeated amphetamine administration on intracranial self-stimulation: evidence for changes in reward processes. Behav Brain Res 13:251-259. Reith M E A , Selmeci (1992) Cocaine binding sites in mouse striatum, dopamine autoreceptors, and cocaine-induced locomotion. Pharmacol Biochem Behav 41:227-230. Robbins TW, Everitt BJ (1996) Neurobehavioural mechanisms of reward and motivation. Curr Opin Neurobiol 6:228-236. Robinson TE (1984) Behavioral sensitisation: characterisation of enduring changes in rotational behavior produced by intermittent injections of amphetamine in male and female rats. Psychopharmacology 84:466-475. Robinson TE, Becker JB (1982) Behavioral sensitization is accompanied by an enhancement in amphetamine-stimulated dopamine release from striatal tissue in vitro. Eur J Pharmacol 85:253-254. 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 Rev 11:157-198. Robinson TE, Berridge K C (1993) The neural basis of drug craving: an incentive-sensitization theory of addiction. Brain Res Rev 18:247-291. Robinson TE, Browman K E , Crombag HS, Badiani A (1998) Modulation of the induction or expression of psychostimulant sensitization by the circumstances surrounding drug administration. Neurosci Biobeh Rev 22:347-354. Robinson TE, Jurson PA, Bennett JA, Bentgen K M (1988) Persistent sensitization of dopamine neurotransmission in ventral striatum (nucleus accumbens) produced by prior experience with (+)-amphetamine: a microdialysis study in freely moving rats. Brain Res, 462:211-222. Sachs BD (1995) Placing erection in context: the reflexogenic-psychogenic dichotomy reconsidered. Neurosci Biobehav Rev 19:211-224. 146 Sachs B D (1978) Conceptual and neural mechanisms of masculine copulatory behaviour. In: McGi l l TE, Dewsbury DA, Sachs BD (eds) Sex and behaviour. Plenum Press, New York, pp 267-295. Sachs BD, Barfield RW (1976) Functional analysis of masculine copulatory behavior in the rat. In: Rosenblatt JS, Hinde RA, Shaw E, Beer C (eds) Advances in the study of behaviour. (Vol 7) Academic Press, Orlando, pp 91-154. Sachs BD, Meisel R L (1988) The physiology of male sexual behavior. In: Knobil E, Neill JD (eds) The physiology of reproduction. (Vol 2) Raven Press, New York, pp 1393-1485. Salamone JD (1996) The behavioral neurochemistry of motivation: methodological and conceptual issues in studies of the dynamic activity of nucleus accumbens dopamine. J Neurosci Methods 64:137-149. Sato Y , Wada H , Horita H , Suzuki N , Shibuya A , Adachi H , Kato R, Tsukamoto T, Kumamoto Y (1995) Dopamine release in the medial preoptic area during male copulatory behavior in rats. Brain Res 692:66-70. Schultz W (1992) Activity of dopamine neurons in the behaving primate. Semin Neurosci 5:129-138. Schultz W, Dayan P, Montague PR (1997) A neural substrate of prediction and reward. Science 275:1593-9. Schultz W, Apicella P, Ljunberg T (1993) Responses of monkey dopamine neurons to reward and conditioned stimuli during successive steps of learning a delayed response task. J Neurosci 13:900-913. Schwartz J-C, Giros B, Martres M-P, Sokoloff P (1992) The dopamine receptor family: molecular biology and pharmacology. Semin Neurosci 4:99-108. Segal DS, Kuczenski R (1992a) In vivo microdialysis reveals a diminished amphetamine-induced response corresponding to behavioral sensitization produced by repeated amphetamine pretreatment. Brain Res 571:330-337. Segal DS, Kuczenski R (1992b) Repeated cocaine administration induces behavioral sensitization corresponding to decreased extracellular dopamine responses in caudate and accumbens. Brain Res 577:351-355. Segal DS, Kuczenski R (1994) Behavioral pharmacology of amphetamine. In: Cho A K , Segal DS (eds) Amphetamine and its analogs. Academic Press, San Diego, pp 115-150. Seiden LS, Sabol K E , Ricaurte G A (1993) Amphetamine: effects on catecholamine systems and behavior. Ann Rev Pharmacol Toxicol 33:639-77. 147 Self DW, Nestler EJ (1995) Molecular mechanisms of drug reinforcement and addiction. In: Cowan W M , Shooter E M , Stevens CF, Thompson RF (eds) Annual Review of Neurosci. Annual Reviews Inc, Palo Alto, pp 463-495. Sharpe L G , Pilotte NS, Mitchell W M , DeSouza EB (1991) Withdrawal of repeated cocaine decreases autoradiographic [3H] mazindol-labelling of dopamine transporter in rat nucleus acccumbens. Eur J Pharmacol 203:141-144. Shilling PD, Kelsoe JR, Segal DS (1997) Dopamine transporter mRNA is up-regulated in the substantia nigra and the ventral tegmental area of amphetamine-sensitized rats. Neurosci Lett 236:131-134. Shimura T, Yamamoto T, Shimokochi M (1994) The medial preoptic area is involved in both sexual arousal and performance in male rats: re-evaluation of neuron activity in freely moving animals. Brain Res 640:215-222. Simpson JN, Wang JQ, McGinty JF (1995) Repeated amphetamine administration induces a prolonged augmentation of phosphorylated cyclase response element-binding protein and Fos-related antigen immunoreactivity in rat striatum. Neurosci 69:441-457. Sorg B A , Kalivas PW (1991) Effects of cocaine and footshock stress on extracellular dopamine levels in the ventral striatum. Brain Res 559:29-36. Stamford JA, Kruk Z L , Millar J (1988) Effects of uptake inhibitors on stimulated dopamine release and uptake in the nucleus accumbens studied by fast cyclic voltammetry. Brit J Pharmacol: Proceed Supp 34: 348P. Stewart J, Badiani A (1993) Tolerance and sensitization to the behavioral effects of drugs. Behav Pharmacol 4:289-312. Stewart J, Vezina P (1987) Microinjections of SCH-23390 into the ventral tegmental area and substantia nigra pars reticulata attenuate the development of sensitization to the locomotor activating effects of systemic amphetamine. Brain Res 495:401-406. Striplin CD, Kalivas PW (1992) Correlation between behavioral sensitization to cocaine and G protein ADP-ribosylation in the ventral tegmental area. Brain Res 579:181-186. Sulzer D, Maidment NT, Rayport S (1993) Amphetamine and other weak bases act to promote reverse transport of dopamine in ventral midbrain neurons. J Neurochem 60:527-35. Sulzer D, Chen T-K, Lau Y Y , Kristensen H, Rayport S, Ewing A (1995) Amphetamine redistributes dopamine from synaptic vesicles to the cytosol and promotes reverse transport. J Neurosci 15:4102-4108. 148 Terwilliger RZ, Beitner-Johnson D, Sevarino SM, Crain S M , Nestler EJ (1991) A general role for adaptations in G-proteins and cyclic A M P system in mediating the chronic actions of morphine and cocaine on neuronal function. Brain Res 548:100-110. Toates F M (1986) Motivational systems. Cambridge University Press, London. Ungerstedt U (1991) Microdialysis - principles and applications for studies in animals and man. J Internal Med 230:365-373. Ungerstedt U , Herrera-Marschitz M , Jungnelius U , Stahle L , Tossman U , Zetterstrom T (1982) Dopamine synaptic mechanisms reflected in studies combining behavioural recordings and brain dialysis. In: Ktisaka M (ed) Advances in Dopamine Research. Pergamon, New York, pp 219-231. Van Furth W, Van Ree J (1996a) Appetitive sexual behavior in male rats: 1. The role of olfaction in level-changing behavior. Physiol Behav 60(3):999-1005. Van Furth W, Van Ree J (1996b) Appetitive sexual behavior in male rats: 2. Sexual reward and level-changing behavior. Physiol Behav 60(3): 1007-1012. Van Furth WR, Wolterink G, Van Ree J M (1995) Regulation of masculine sexual behavior: involvement of brain opioids and dopamine. Brain Res Rev 21:162-184. Vezina P (1993) Amphetamine injected into the ventral tegmental area sensitizes the nucleus accumbens dopaminergic response to systemic amphetamine: an in vivo microdialysis study in the rat. Brain Res 605: 332-337. Vezina P (1996) D l dopamine receptor activation is necessary for the induction of sensitization by amphetamine in the ventral tegmental area. J Neurosci 16:2411-2420. Vezina P, Stewart J (1984) Conditioning and place-specific sensitization of increases in activity induced by morphine in the V T A . Pharmacol Biochem Behav 20:925-934. Von Euler US, Lishajko F (1957) Dopamine in mammalian lung and spleen. Acta Physiol Pharmacol Neerl, 6:295-303. Wang C-T, Huang R-L, Tai M - Y , Tsai Y-F , Peng M-T (1995) Dopamine release in the nucleus accumbens during sexual behavior in prenatally stressed adult male rats. Neurosci Lett 200:29-32. Warburton EC, Mitchell SN, Joseph M H (1996) Calcium dependence of sensitised dopamine release in rat nucleus accumbens following amphetamine challenge: implications for the disruption of latent inhibition. Behav Pharmacol 7:119-129. 149 Warner R K , Thompson JT, Markowski VP, Loucks JA, Bazzett TJ, Eaton RC, Hull E M (1991) Microinjection of the dopamine antagonist cis-flupenthixol into the M P O A impairs copulation, penile reflexes and sexual motivation in male rats. Brain Res 540:177-182. Washton A , Stone-Washton N (1993) Outpatient treatment of cocaine and crack addiction: a clinical perspective. In: Tims F M , Leukefeld C G (eds) Cocaine treatment: research and clinical perspectives. NIDA Research Monograph (Vol 135), pp 15-30. 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. White FJ, Hu X-T, Zhang X-F , Wolf M E (1995) Repeated administration of cocaine or amphetmaine alters neuronal responses to glutamate in the mesoaccumbens dopamine system. J Pharmacol Exp Ther 273:445-454. White FJ, Wang R Y (1983) Differential effects of classical and atypical antipsychotic drugs on A9 and A10 dopamine neurons. Science 221:1054-57. White FJ, Wang R Y (1984) Electrophysiological evidence for A10 dopamine autoreceptor sensitivity following chronic d-amphetamine treatment. Brain Res 309:283-292. Wilson JM, Nobrega JN, Carroll M E , Niznik HB, Shannak K , Lac ST, Pristupa Z B , Dixon L M , Kish SJ (1994) Heterogenous subregional binding patterns of 3 H - W I N 35,428 and 3 H - G B R 12,935 are differentially regulated by hronic cocaine self-administration. J Neurosci 14:2966-2979. Wilson C, Nomikos GG, Collu M , Fibiger HC (1995) Dopaminergic correlates of motivated behavior: importance of drive. J Neurosci 15:5169-5178. Wise R A (1982) Neuroleptics and operant behavior: the anhedonia hypothesis. Behav Brain Res 5:39-87. Wise R A , Bozarth M A (1987) A psychomotor stimulant theory of addiction. Psychol Rev 94:469-492. Woolverton W L , Cervo L, Johanson CE (1984) Effects of repeated methamphetamine administration on methamphetamine self-administration in rhesus monkeys. Pharmacol Biochem Behav 16:293-30. Xia Y , Goebel DJ, Kapatos G, Bannon M J (1992) Quantitation of rat dopamine transporter mRNA: effects of cocaine treatment and withdrawal. J Neurochem 59:1179-1182. 150 Y i S-J, Johnson K M (1990) Effects of acute and chronic administration of cocaine on striatal uptake, compartmentalization and release of [3H]Dopamine. Neuropharmacology 29:475-486. Zaczek R, Culp S, Goldberg H , Mccann DJ, De Souza EB (1991a) Interactions of [3H]amphetamine with rat brain synaptosomes. I. Saturable sequestration. J Pharmacol Exp Ther 257:820-9. Zaczek R, Culp S, De Souza EB (1991b) Interactions of [3H]amphetamine with rat brain synaptosomes. II. Active transport. J Pharmacol Exp Ther 257: 830-5. Zahm DS (1991) Compartments in the dorsal and ventral striatum revealed following injection of 6-hydroxydopamine into the ventral mesencephalon. Brain Res 552:164-169. Zahm DS, Brog JS (1992) On the significance of subterritories in the "accumbens" part of the rat ventral striatum. Neurosci 50:751-767. Zhang Y , Angulo JA (1996) Contrasting effects of repeated treatment vs. withdrawal of methamphetamine on tyrosing hydroxylase messenger R N A levels in the ventral tegmental area and substantia nigra zona compacta of the rat brain. Synapse 24:218-223. Zhang X - F , Hu X-T, White FJ (1998) Whole-cell plasticity in cocaine withdrawal: reduced sodium currents in nucleus accumbens neurons. J Neurosci 18:488-98. 151 APPENDIX A Measures of sexual behavior in Tests 1 through 10 from Experiment I. Graphed data are expressed as the mean ± SEM. - a - CONT - • - AMPH 1 2 3 4 5 6 7 8 9 10 Test Number 500 i 0 ^  . . , , , , • r-1 2 3 4 5 6 7 8 9 10 Test Number 750 o 1 500 i cl o 1 250 'S p 1 — 1 3 4 5 6 7 Test Number 9 10 c3 3 G i "3 o ea tiT -l-» OS o PH 600 500 400 300 200 100 0 4 5 6 7 Test Number 8 9 10 o e e o 3 5? 1000 800 600 400 200 0 4 5 6 7 Test Number 10 IS 31 c o fi fi 100 80 60 40 20 0 1 T—I 1 2 3 4 5 6 7 8 9 10 Test Number 152 - A - CONT - • - AMPH c 0 J - H 1 > 1 • 1 1 1 1 1 — 0 1 ' 1 1 1 1 1 . . r-1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Test Number Test Number c o 1 2 3 4 5 6 7 Test Number 10 o c w g-u u. fl O 25 20 15 10 5 10 Test Number 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Test Number Test Number 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0089228/manifest

Comment

Related Items