NEURAL AND ENDOCRINE MECHANISMS UNDERLYING ADOLESCENT MATURATION OF SOCIAL REWARD By Margaret R Bell A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Neuroscience 2012 Abstract NEURAL AND ENDOCRINE MECHANISMS UNDERLYING ADOLESCENT MATURATION OF SOCIAL REWARD By Margaret R. Bell The adolescent transition from childhood to adulthood requires a qualitative shift in the focus on social interactions and reward. The male Syrian hamster provides a unique model for understanding how social information processing matures throughout adolescence, as juvenile males are not attracted to female vaginal secretions (VS) that are essential for sexual behavior in adulthood. I have shown that VS are rewarding in gonad-intact adult but not juvenile hamsters using conditioned place preference (CPP), and that testosterone treatment facilitates a VS-induced CPP in juveniles that is dopamine dependent. Moreover, VS induces Fos expression throughout the mesocorticolimbic system in adult but not juvenile hamsters. This body of work demonstrates that 1) the pubertal rise in testosterone changes the perception of a social stimulus, allowing it to serve as an unconditioned reward; 2) adolescent maturation of social reward is mediated by dopaminergic mechanisms and involves engagement of the mesocorticolimbic reward system. Acknowledgements I would like to express my great appreciation to my committee members, Drs. Jim Galligan, Joe Lonstein, Laura Smale, and Cheryl Sisk. Thank you for your time, energy, thoughts, and guidance for the last six years. Cheryl, thank you for your constant support and encouragement. Your enthusiasm and creative thinking are so valued and make your lab a wonderful place to grow as a scientist. I would also like to express my gratitude to the Neuroscience Program as a whole, including their past and current director, support staff, faculty and students. I am so lucky to have been a part of such an inviting and intelligent community. In particular, Jim and Shari Stockmeyer; thank you for dealing with all of my issues so cheerfully. I cannot thank the past and present members of the Sisk lab enough for all their help. This dissertation was only possible because of their contributions. Past members include Rayson Figueira, Heather Molinda-Figueira, Sarah Meerts, Kaliris SalasRamirez, Kalynn Schulz, Eman Ahmed, and all contributed their thoughts and hands. Undergraduate help was also essential to completing the many hours of tissue analysis and conditioned place preference behavioral work. Many thanks to Dana Gradl, Allison Melkonian, Joshua Olszewicz, Bradley Lawrence, Elise Craig, Shannon O’Conner, Susie Sonnenshine, Nicole Griep, Ashley Pratt, Robyn Weston, Genevieve Trombly, Jennifer La, Christine Azizkhan, and Steven Townsend. Andrew Kneynsberg has also been a terrific help with all things science (and food) related. Jane Venier deserves special thanks for her many (late and weekend) hours in the basement and constant thoughtful help. Thank you, Jane, for keeping the lab going and solving all of our iii problems! Kayla De Lorme and Maggie Mohr, you have made the office such a happy, productive, and thoughtful place, and I cannot imagine better lab mates. Thank you for letting me bother you with my questions, helping with experiments, reading my work, and being my friends. Finally, my utmost thanks to my family and, in particular, my husband, Lance Brinkman, for their unending support and love. My parents have been tireless moralboosters; thank you for your interest and enthusiasm when I need it most. Lance has shown inexhaustible patience with my unusual work schedule and often long hours. I cannot express how much your support and understanding means to me. Thank you for making this possible. iv TABLE OF CONTENTS List of Tables vi List of Figures vii Key to Abbreviations ix Chapter 1 Introduction 1 Chapter 2 Male Syrian hamsters demonstrate a conditioned place preference for sexual behavior and female chemosensory stimuli 41 Chapter 3 Adolescent gain in positive valence of a socially relevant stimulus: engagement of the mesocorticolimbic reward circuitry 55 Chapter 4 Adolescent maturation of mesocorticolimbic responses to a rewarding social cue is gonadal hormone independent 88 Chapter 5 Dopamine mediates testosterone-induced social reward in juvenile male hamsters 114 Chapter 6 Discussion 146 Appendix Select populations of dopaminergic cells do not show an immediate early gene response to VS 162 Literature Cited 173 v List of Tables Table 3.1 Immunohistochemical reagents 64 Table 4.1 Complete list of age, hormone, stimulus and region effects with interactions 99 Table 4.2 Peripheral measures of hamsters at time of sacrifice 105 Table 5.1 Group size, body weight, and plasma testosterone concentration. 137 vi List of Figures Figure 1.1 Diagram of brain regions involved in social behavior and motivation 34 Figure 2.1 CPP to sexual interaction in adult hamsters 49 Figure 2.2 CPP to VS in adult hamsters 50 Figure 3.1 Figures and images of brain regions of interest 67 Figure 3.2 Preference and difference scores for control and experimental groups when tested for a CPP for VS, palatable food, and cocaine 72 Figure 3.3 Fos-ir cell density in mesocorticolimbic and hypothalamic clusters 73 Figure 3.4 Images of Fos/TH immunohistochemistry 74 Figure 3.5 Single-labeled Fos-ir cell density in mesocorticolimbic cluster regions, PFC, Acb, MeP, and VTA 76 Figure 3.6 Fos-ir cell density in hypothalamic cluster regions, DM/PeF and LH and VMH 77 Figure 3.7. TH-ir measures in IF, PN, PBP, and Tail VTA 78 Figure 3.8 Orexin measures in the DM/PeF and LH 79 Figure 3.9 Plasma testosterone concentrations from animals in Exp 1a and Exp 2 80 Figure 3.10 Schematic diagram of adolescent changes in VS-induced Fos responses 81 Figure 4.1 Representative subregion contours placed over atlas diagrams 95 Figure 4.2 Cluster analysis of Fos responses to VS 100 Figure 4.3 Fos-ir cell density in all brain regions analyzed, in blank- or testosterone- treated juveniles and adults exposed to clean or VS-oil. 102 vii Figure 4.4 TH- and Fos/TH-ir cell density in the VTA, in blank- or testosterone- treated juveniles and adults exposed to clean or VS-oil 104 Figure 5.1 CPP to VS in hormone manipulated adult hamsters 127 Figure 5.2 CPP to VS in hormone manipulated juvenile and adult hamsters 129 CPP to VS in hormone and dopamine manipulated juvenile hamsters 131 Figure 5.4 CPA to dopamine antagonist in juvenile hamsters 133 Figure 5.5 Locomotor activity in dopamine manipulated juvenile hamsters 134 Figure 5.6 Fecal boli in dopamine manipulated juvenile hamsters 135 Figure 5.7 Food detection in dopamine manipulated juvenile hamsters 136 Figure A.1 Total number of TH-ir and Fos/TH-ir cells in the posterior medial amygdala 166 Figure A.2 Fos-ir, TH-ir, and Fos/TH-ir cell density in the full extent or magnocellular region of the paraventricular nucleus 168 Figure A.3 Fos-ir, TH-ir, and Fos/TH-ir cell density in the posterior hypothalamus and supramammilary nucleus 170 Figure 5.3 viii Key to Abbreviations Brain Regions of Interest Acb nucleus accumbens AcbC nucleus accumbens core AcbSh nucleus accumbens shell AMY amygdala AOB accessory olfactory bulb BNST bed nucleus of the stria terminalis Cg1 anterior cingulate medial prefrontal cortex DM/PeF dorsomedial hypothalamus / perifornical area IF interfascicular nucleus of the ventral tegmental area IL infralimbic medial prefrontal cortex LH lateral hypothalamus Me medial amygdala MeP posterior medial amygdala MePD posterdorsal medial amygdala MePV posteroventral medial amygdala MOB medial olfactory bulb mPFC medial prefrontal cortex MPOA medial preoptic area PBP parabrachial pigmented nucleus of the ventral tegmental area PeVN periventricular nucleus of the hypothalamus PH posterior hypothalamus PN paranigral nucleus of the ventral tegmental area ix PrL prelimbic medial prefrontal cortex PVN paraventricular nucleus of the hypothalamus SuM supramammillary nucleus Tail tail nucleus of the ventral tegmental area VMH ventromedial hypothalamus VMHL lateral ventromedial hypothalamus VMHM medial ventromedial hypothalamus VTA ventral tegmental area Other Acronyms and Abbreviations ANOVA analysis of variance CPP conditioned place preference CPA conditioned place aversion DOPAC 3,4-dihydroxyphenylacetic acid GABA gamma-aminobutyric acid GnRH gonadotropin-releasing hormone ir immunoreactive Orx orexin SB sex behavior TBS Triton Buffered Saline TH tyrosine hydroxylase VS vaginal secretions x Chapter 1: Introduction Adolescent maturation of social information processing: relevance to human mental health Adolescence, the behavioral transition from child to adult, is a time of dramatic developments in abstract reasoning, cognitive control, and social skills necessary to understand others’ emotions and mental state (reviewed in (Spear, 2000, Casey et al., 2005, Blakemore, 2008). Perhaps enabled by this gain in social cognition, adolescents switch their focus from family to peers (Nelson et al., 2005) and are more sensitive to peer influences than are adults (Gardner and Steinberg, 2005). This transition has been described as a time of social reorientation, and, amongst other things, involves changes in the interpretation of social stimuli (Herba and Phillips, 2004). For example, while adults consistently describe a stimulus face as scared, adolescents interpret it as sad, shocked, or confused (Baird et al., 1999). Adolescents also have difficulties shifting their attention from emotional to non-emotional aspects of a face (Monk et al., 2003). These changes in social information processing may affect the performance of normal social behaviors, but also may be associated with mental illness. Anxiety and mood disorders all increase in prevalence during adolescence (Hayward and Sanborn, 2002), and involve distorted interpretation of social stimuli: patients with anxiety disorders show hyper-vigilance or exaggerated attention toward threat (Mogg and Bradley, 1998), and depression is characterized as a bias to interpret information negatively (Gotlib et al., 2004). Thus, it is possible that these disorders may be the result of unsuccessful social reorientation. 1 Coincident with the general social reorientation is a shift in social and motivational tendencies (Forbes and Dahl, 2010). Adolescence is characterized by increased reward seeking behavior (Galvan, 2010), with frequency of behavior following an inverted U-shaped curve, peaking between10-15 years of age (Steinberg, 2008). This increase in reward seeking may result from reduced responsiveness to potential rewards, as evidenced by reduced activation of reward related brain regions when waiting for an expected reward in adolescents compared to adults, in fMRI studies (Bjork et al., 2004). On the other hand, greater neural activation in response to rewards in adolescence has also been widely documented by fMRI studies (Geier and Luna, 2009). Whether adolescence is a time of partial anhedonia or enhanced hedonic sensitivity is an ongoing debate, but both theories posit immature interpretations of potentially rewarding stimuli in adolescents (Galvan, 2010). The alterations in reward and sensation seeking may affect a range of mental health disorders that increase in prevalence during adolescence. One obvious potential effect of reward dysregulation is that of substance abuse and addiction. Indeed, many suggest that adolescents may be particularly vulnerable to the effects of psychotropic drugs (Chambers et al., 2003). Pubertal changes in reproductive function Concurrent with adolescence, puberty specifically is the time of reproductive maturity, and is typically achieved before adolescence is completed (Nussey and Whitehead, 2001). Pubertal onset is determined by a developmentally timed pulsatile release of gonadotropin-releasing hormone from neurons throughout the hypothalamus (as reviewed in (Sisk and Foster, 2004)). This peptide reaches the anterior pituitary gland 2 via the portal vasculature, and induces the release of luteinizing hormone and follicle stimulating hormone. These gonadotrophins then increase steroidogenesis by acting at Leydig or theca cells in the testes or ovaries, respectively (Nussey and Whitehead, 2001). In the male, Leydig cells produce androgens, including testosterone, androstenedione, and dihydrotestosterone which act at androgen receptors. In addition testosterone can be enzymatically converted by aromatase in the brain to an estrogen, and act at estrogen receptors. Therefore, the effects of testosterone manipulations in many studies (and in this dissertation) may occur through binding either androgen or estrogen receptors. Steroid hormone receptors classically act in the nucleus as transcription factors to regulate gene expression (Spelsberg et al., 1989). While relatively recent findings have revealed non-genomic actions of estrogen and androgen receptors (Boonyaratanakornkit and Edwards, 2007), classical nuclear receptors are likely most relevant to the studies contained within this dissertation. Human adolescent brain development The processing of social information has been theorized to include three major processes: 1) detection of the social stimulus, 2) affective and motivational processes and 3) and cognitive and regulatory control (Adolphs, 2001, 2009). In models based on human and non-human primates, the detection node is centered in temporal-parietal cortices important for vision and face detection. Adaptation to rodents would also include olfactory regions in this node. Affective processes are guided by the amygdala (AMY) and accumbens (Acb), while cognitive-regulatory processes are largely controlled by the medial prefrontal cortex (mPFC). In humans, the mPFC and AMY are 3 involved in the recognition of socially relevant stimuli, judging attractiveness, and social and non-social reward (Hoffman and Haxby, 2000, Aharon et al., 2001, Guyer et al., 2008, Steinberg, 2008). Importantly, these regions all receive extensive dopaminergic and non-dopaminergic projections from the ventral tegmental area (VTA) as part of the complexly and reciprocally connected circuits of the mesocorticolimbic system (Swanson, 1982a, Oades and Halliday, 1987, Thompson and Swanson, 2010). The “social information processing network” has been adapted to describe adolescent changes in behavior (Nelson et al., 2005). This model suggests that while the detective node is largely functional in childhood, major developmental changes occur within the affective and cognitive nodes that alter emotional attributions to social stimuli and enable complex regulation of social behavior (Nelson et al., 2005). This model of social information processing is strikingly similar to that of motivated behavior, called the “Triadic Model” (Ernst et al., 2006). This “Triadic Model” also proposes a regulatory node that mirrors the above cognitive/regulatory node, and is centered in the mPFC. However, the Triadic Model breaks up the “affective” social information processing node into two nodes to distinguish between “approach” and “avoidance” functions controlled by the Acb and AMY, respectively. The triadic model also describes the adolescent shift in the balance between nodes to control motivated behavior. The increased reward seeking in the face of potentially negative consequences may be due to heightened activity in the approach/reward function of the Acb, a weak harm-avoidant system in the AMY, and/or an inefficient regulatory system in the mPFC (Ernst and Fudge, 2009). Indeed, adolescence is characterized by immature prefrontal and enhanced striatal and amygdalar reactivity to rewards in 4 comparison to adults (Forbes et al., 2010), as demonstrated with monetary gambling rewards (Ernst et al., 2005, Galvan et al., 2007, Van Leijenhorst et al., 2010a, Van Leijenhorst et al., 2010b), and self-appraisals from peers (Guyer et al., 2009). Thus, a coordinated shift in neural activity may promote social and motivated behaviors during adolescence. Many of these functional developments may be explained by structural changes in the brain during adolescence. Gross morphological changes have been observed across adolescence: the volume of PFC grey matter and then declines curvilinearly, while white matter increases linearly across adolescence (Giedd et al., 1999, Sowell et al., 2002, Gogtay et al., 2004, Giedd et al., 2006, Lenroot et al., 2007). Also, massive spine and synapse overproduction and pruning occur in the PFC during adolescence (Huttenlocher, 1984, Mrzljak et al., 1990, Casey et al., 2005). Recent work suggests that there are also developmental changes in white matter tracts between the PFC and the AMY and Acb (Eluvathingal et al., 2007), and a general trend throughout the brain to transition from diffuse and short projections to more focused, longer projections across adolescence (Peper et al., 2011b). In the striatum (including the Acb), dopamine receptors are overproduced and then pruned to adult levels (Seeman et al., 1987) and there is an increase in presynaptic dopaminergic markers throughout adolescence (Haycock et al., 2003). Therefore, behavioral changes during adolescence are correlated with neuroanatomical changes in relevant brain regions (Spear, 2000). It is possible that many of the changes observed during adolescence relate to circulating gonadal hormones (Peper et al., 2011a). In adolescents matched for age, circulating testosterone measures correlate with whole brain white matter in boys but 5 not in girls; interestingly, the effect is increased in males with a short androgen receptor polymorphism associated with higher basal testosterone (Perrin et al., 2008). This androgen receptor polymorphism also modifies the association between age and white matter volume in a similar fashion (Paus et al., 2010). Acute effects of testosterone have also been observed; a single dose of testosterone in women can cause a decrease in basal activity coupling between the amygdala and orbitofrontal cortex and an increase between the amygdala and thalamus (van Wingen et al., 2010). These effects may also be relevant to socioemotional abilities, as testosterone can cause a decrease in prefrontal-amygdalo connectivity in males performing a task whereby participants were asked to override a natural tendency to approach a happy face and avoid an angry face (Volman et al., 2011). The intersection of age-dependent maturational change with the increase in circulating testosterone likely dictates the effects of both on social behavior and reward, and are a focus of this dissertation. While great gains are being made in understanding the neurobiology of adolescence, we know very little about the specific neural mechanisms behind adolescent changes in the perception of social stimuli (Kipke, 1999). Moreover, as both social and motivated behaviors change across adolescence, using a social stimulus as the experimental reward might provide a relevant perspective on normative changes in behavior during this developmental period (Galvan, 2010). The overall goal of this proposal is to determine the neural mechanisms important for mature behavioral and physiological responses to female pheromones, a rodent version of social reward, and how these neural systems change across adolescence and hormone treatment. 6 The Syrian hamster model of social information processing Animal models offer us a chance to investigate the hormonal and neural mechanisms behind the assessment and interpretation of social stimuli, which is indexed in this dissertation by physiological and behavioral responses to a stimulus in a specific social context. The male Syrian hamster is an excellent model for studying a reproductivelyrelevant social stimulus as their performance of sociosexual behavior is highly dependent on chemosensory cues, especially female hamster vaginal secretions (VS) (Murphy and Schneider, 1970, Johnston, 1986, Coppola and O'Connell, 1988). Normal adult male copulatory behavior consists of repeated sequences of anogenital investigation, mounts, intromissions and ejaculations (Hull and Dominguez, 2007). In sexually naïve adult males, mating is dependent on the perception of VS, as removal of the olfactory bulb completely blocks odor preferences and copulatory behavior in males (Murphy and Schneider, 1970, Petrulis and Johnston, 1995, Petrulis). In fact, VS are such powerful stimuli that, when applied to a castrated or anesthetized male hamster, they can induce males to mount those same-sex partners (Murphy, 1973, Johnston, 1986). Sexually-naïve adult males show a preference for VS over male odors (Maras and Petrulis, 2006), and an unconditioned attraction to VS, as they spend more time licking VS-containing bottles over clean bottles (Johnston, 1974, Landauer et al., 1977). This salient and socially-relevant cue will be used in this dissertation as a model of a social reward. VS serve to communicate upcoming sexual receptivity of the female to the male, as females actively apply VS around their burrow entrance by vaginal marking (Petrulis, 2009). While secretions from both non-estrous and ovariectomized females are 7 attractive to male hamsters, males can detect differences between secretions from different females and prefer secretions from estrous females over non-estrous (Johnston, 1974, Macrides et al., 1984a, Huck et al., 1989, delBarco-Trillo et al., 2009). VS contain a mixture of volatile and nonvolatile compounds, and the relative importance of these fractions in sexual behavior have been well-researched (Clancy et al., 1984, Singer et al., 1984b, Steel and Keverne, 1985). One volatile compound, dimethyl disulfide, peaks in concentration on the day of ovulation, and promotes male investigatory behavior but does not promote sexual behavior (Singer et al., 1984a). However, the importance of this fraction has been questioned recently, as males, females, and castrated males are equally attracted to dimethyl disulfide (Petrulis and Johnston, 1995). Another major component of VS with sexually attractive properties has been identified as “aphrodisin” (Singer et al., 1986). Specifically, it is a high molecular weight, soluble, glycosylated protein in the lipocalin superfamily; therefore, it is possible that aphrodisin is either a carrier protein for some other unidentified molecule, a pheromone in its own right, or both (Henzel et al., 1988, Briand et al., 2004). Regardless of the chemical composition, VS are impressively potent and concentrated; a water dilution experiment showed that one female equivalent (i.e., the amount typically extruded by one estrous female in response to tactile genital stimulation) can be diluted one hundred-fold without significantly reducing male interest (Macrides et al., 1984a). Experiments contained in this dissertation use combined volatile and non-volatile components of VS well-above levels of detection. Attraction to VS and the performance of sexual behavior are regulated by circulating gonadal steroid hormones in male hamsters (Whalen and Debold, 1974, 8 Gregory and Bishop, 1975). These effects require activation of both androgen and estrogen receptors, as long-term treatment of neither dihydrotestosterone nor estradiol alone is sufficient to match testosterone effects, but combined treatment brings behavioral levels close to that observed in testosterone-treated animals (DeBold and Clemens, 1978, Steel, 1982, Powers and Bergondy, 1983, Powers et al., 1985, Steel and Hutchison, 1986, 1988). The reduction in attraction to VS after castration is not a result of olfactory deficits, as long-term castrates can still locate diluted VS just as well as gonad-intact males (Peters et al., 2004), and VS activates equivalent numbers of neurons in the olfactory bulb of intact and gonadectomized hamster (Fiber et al., 1993). In addition to long-term modulation of these behaviors, testosterone may also serve to provide short-term feedback in response to chemosensory and sexual cues. In gonadintact animals, exposure to VS causes a surge in circulating testosterone 30 minutes after exposure (Pfeiffer and Johnston, 1992). Therefore, testosterone may serve as a cue in reinforcement and learning to promote sexual behavior, an effect that will be investigated in this dissertation. Adolescent developmental of hamster social behavior and evaluation of VS The process of social reorientation, in its many forms, is observed across species and likely evolved as an adaptive mechanism to leave the nest, achieve independence, try new foods, and gain new territory and mates. Male hamster puberty and adolescence is typically defined as the period between postnatal day (P) 28 and P56, according to when they leave the nest, begin to show adult-like reproductive behaviors, and produce mature concentrations of gonadal hormones (Miller et al., 1977, Vomachka and 9 Greenwald, 1979, Schoenfeld and Leonard, 1985, Schulz and Sisk, 2006, delBarcoTrillo et al., 2011). The attraction to VS over control odors reaches significance only after P40, suggesting that the pheromones gain hedonic value through adolescent maturation (Johnston and Coplin, 1979). Gonad-intact adults show higher levels of anogenital investigation than gonad-intact juvenile animals, although the differences are not statistically significant (Meek et al., 1997). Gonad-intact juvenile male Syrian hamsters also display very low levels of mounts and intromissions, and absolutely no ejaculations (Meek et al., 1997). In fact, mounting behavior reaches only 50% of adult levels by P42, halfway through adolescence (Miller et al., 1977). Both the latency to perform anogenital investigations and mounts are longer in juvenile animals as compared to adults, suggesting that juvenile animals are less motivated to perform the behaviors (Schulz, 2007). Juvenile hamsters also fail to show an elevation in testosterone in response to VS (Romeo et al., 1998, Romeo et al., 2002). Therefore, male hamsters display developmental changes in behavioral and endocrine response to a social cue. Given the importance of testosterone in regulating male sexual behaviors, and the dramatic changes in gonadal hormones during adolescence, it follows that the adolescent gain in sociosexual behavior is mediated by gonadal hormones. Indeed, testosterone treatment can induce attractive behaviors to VS in P25 juvenile animals (Johnston and Coplin, 1979), and adult-like levels of anogenital investigation in P28 juvenile males (Meek et al., 1997). However, testosterone treatment cannot induce adult-like levels of mounts, intromissions and ejactulatory behaviors in juvenile males (Meek et al., 1997), even after 17 days of treatment that approximate the normal 10 adolescent period (Schulz et al., 2009). The immature sex behavior responses to testosterone cannot be explained by immature actions of androgen receptors, estrogen receptors or aromatase, as juveniles and adults have similar expression patterns and levels of activity (Meek et al., 1997, Romeo et al., 1998, Romeo et al., 1999). Together, these data suggest the juvenile brain shows some immature responses to female social stimuli, and that these behaviors can only partially be activated by testosterone prior to adolescence. As vaginal secretions may act as a reinforcer or promoter of sexual behavior in adulthood, experiments contained in this dissertation will investigate the rewarding properties of VS to gain more insight into the perception of this stimulus across adolescence. Chemosensory detection and neural evaluation As mentioned earlier, chemosensory information is essential to allow mating behavior in Syrian hamsters, as olfactory bulbectomy completely blocks copulatory behavior (Powers et al., 1979). Olfactory cues stimulate two anatomically and functionally distinct systems. The main olfactory system includes the olfactory epithelium, where olfactory receptors quickly detect volatile molecules and project to the main olfactory bulb (MOB). In contrast, the accessory olfactory system generally detects heavier nonvolatile cues transported up to the vomeronasal organ, which then projects to the accessory olfactory bulb (Meredith, 1991, Keverne, 2004). Both these systems are likely involved in sexual behavior and attraction to VS (Powers et al., 1979, O'Connell and Meredith, 1984, Petrulis, 2009), however the accessory olfactory system is important in processing the nonvolatile aphrodisin components of VS (Clancy et al., 11 1984, Singer et al., 1987) and is required for sexual behavior in sexually naïve animals (Meredith, 1986, Pfeiffer and Johnston, 1994). Both the main and accessory olfactory bulbs project centrally, but by different pathways. The MOB projects to the amygdala mainly via the olfactory cortex, but also with some direct projections to the anterior medial amygdala (Me), whereas the AOB projects directly to the anterior and posterior Me (Davis et al., 1978, Kevetter and Winans, 1981c, b). Therefore, the Me is positioned to integrate social olfactory information for the promotion of social behaviors. Indeed, the Me is essential for mating and chemoinvestigatory behavior (Lehman et al., 1980). The posterodorsal medial amygdala (MePd) is especially important for chemosensory attraction, as lesions there reduce preferences for female over male odors and chemoinvestigatory behavior (Lehman et al., 1980, Maras and Petrulis, 2006, Petrulis). The Me is part of a well-characterized and tight network of interconnected regions, including the bed nucleus of the stria terminalis (BNST) and the medial preoptic area (MPOA). Lesions in these regions also reduce copulatory and motivated behavior; (Lehman and Winans, 1983, Kondo, 1992, Paredes et al., 1993, Kondo and Arai, 1995, Liu et al., 1997, Sato et al., 1999, Simmons and Yahr, 2002, Melis et al., 2003). Specifically, lesions that disrupt connectivity between the Me and the BNST eliminate preferences for volatile female odors, but not non-volatile compounds or sexual behavior in male hamsters (Been and Petrulis, 2012). On the other hand, functionally disconnecting the Me and the MPOA eliminates copulatory behavior but not olfactory preferences (Been and Petrulis, 2011). Neural activation in response to VS in these brain regions has also been characterized by immunohistochemical identification of the 12 expression of immediate early genes like Fos. Sexual behavior and VS exposure both induce Fos in Me, BNST, and magnocellular subdivision of the medial preoptic nucleus (MPNmag) above that of handled controls, with sex inducing more Fos than VS exposure alone (Kollack and Newman, 1992, Fiber et al., 1993, Fernandez-Fewell and Meredith, 1994, Kollack-Walker and Newman, 1995, Fiber and Swann, 1996, KollackWalker and Newman, 1997). Studying Fos expression is particularly useful when interested in multiple brain regions, it will be used in these dissertation studies. Just as gonadal hormones regulate the performance of sexual and chemosensory behavior, they also modulate responses of the chemosensory brain to sexual stimuli. The Me, BNST, and MPOA all express large numbers of androgen and estrogen receptors (Simerly et al., 1990, Wood and Newman, 1999) and mediate some effects of testosterone on sexual behavior. Implanting testosterone into the MPOA or MeP of castrated male hamsters restores anogenital investigation and mounts (Wood and Newman, 1995b), while selective blockade of androgen receptors in the MPOA inhibits mating (McGinnis et al., 1996, Harding and McGinnis, 2004). Integration of chemosensory and gonadal steroid information is also essential for the expression of chemoinvestigatory and copulatory behaviors (Wood and Newman, 1995a, Wood and Coolen, 1997, Hull and Dominguez, 2007). In a series of elegant studies in long-term castrated male hamsters, Wood and colleagues gave replacement testosterone unilaterally in the MPOA to enhance sexual behaviors, as described above. This stimulatory effect of testosterone was blocked by unilateral olfactory bulbectomy, but only when ipsilateral to the testosterone treatment (Wood and Newman, 1995a). That is, the MPOA needed to integrate both chemosensory and hormonal information within 13 the same hemisphere to promote sexual behavior. The MeP serves a similar function, but is more sensitive to disruptions in olfactory information, as removing contralateral olfactory information also affects sexual behavior (Wood and Coolen, 1997). Interestingly, long-term gonadectomy reduces Fos activation to VS in the MPNmag but not MePd or BNST (Fiber and Swann, 1996), demonstrating some site-specific effects of testosterone in the regulation of Fos responses to VS. The Me, BNST, and MPOA are also connected to several hypothalamic regions that have been implicated in sexual behavior and chemosensory responses (Orsini et al., 1985, Gréco et al., 1996, McGinnis et al., 1996, Kollack-Walker and Newman, 1997, Coolen and Wood, 1998, Choi et al., 2005, Harding and McGinnis, 2005, Wood and Swann, 2005). Namely, the Me projects both directly and indirectly, via the BNST and MPOA, to the paraventricular nucleus (PVN), periventricular nucleus (PeVN), lateral hypothalamus (LH) and the ventromedial hypothalamus (VMH) (Simerly and Swanson, 1986, 1988, Fahrbach et al., 1989, Gomez and Newman, 1992, Coolen and Wood, 1998, Wood and Swann, 2005, Yoshida et al., 2006). Me and MPOA are also connected to the mesocorticolimbic system, previously described as a complex circuitry important for a range of social and motivated behaviors. The Me sends some sparse projections to the Acb and VTA (Coolen and Wood, 1998, Geisler and Zahm, 2005), while basolateral and central amygdala send more robust projections to the Acb, mPFC, and VTA (Phillips et al., 2003). The MPOA, having received chemosensory information via the MePd, is also a major source of input to the VTA and Acb (Simerly and Swanson, 1988, Groenewegen et al., 1999, 14 Cunningham et al., 2002, Phillips et al., 2003, Usunoff et al., 2009). The neuroanatomy and function of these different neural circuits are discussed below. Mesocorticolimbic circuitry and dopaminergic function The literature describing the mesocorticolimbic system, the VTA, Acb, and mPFC, is extensive, and cannot be reviewed exhaustively here. Instead, I will provide a general description of the circuitry and synaptic effects of dopamine as a foundation for understanding adolescent changes in mesocorticolimbic structure and function. In addition, I will describe structural and functional heterogeneity within components of the mesocorticolimbic circuit that forms the basis for evaluating particular subregions of these components in certain experiments in this dissertation. Overall the VTA, mPFC, AMY, and Acb form a network of looping circuits (Thompson and Swanson, 2010), functioning in synchrony to regulate motivated behaviors. The mesocorticolimbic circuitry is known for its robust projections of dopaminergic cells from the A10 population in the VTA to corticolimbic structures (Oades and Halliday, 1987, Koob and Nestler, 1997, Tzschentke, 2000). The released dopamine can act on two families of dopamine receptors, D1 and D2 with different functional properties, imparting to dopamine a wide range of actions in the CNS (Missale et al., 1998). D1-like receptors (D1 and D5) exist primarily in a low-affinity state for dopamine and require high levels of dopamine to be activated, while D2-like receptors have a higher dopamine affinity and are more likely to be activated at lower concentrations of dopamine (Richfield et al., 1989, Goto and Grace, 2005). Additionally, D1 receptors are linked to stimulatory G proteins which increase cyclic adenosine 15 monophosphate (cAMP) activity, while the D2-like receptors (D2, D3, and D4) are linked to inhibitory G proteins and reduce cAMP (Kebabian and Calne, 1979, Surmeier et al., 2007, Carlezon and Thomas, 2009). Both receptors can also modulate a range of ion channels, including AMPA and NMDA glutamate receptors. D2 receptors can also at as autoreceptors on presynaptic dopaminergic terminals and on dopamine cell bodies in the VTA, regulating dopamine release and uptake (Missale et al., 1998). The ventral tegmental area (VTA). The VTA is known for its population of dopaminergic cells. These dopamine neurons typically fire at some spontaneous tonic rate until glutamatergic stimulation induces bursts of firing (Grace and Bunney, 1984b, a, Goto et al., 2007). This burst firing raises extracellular DA from tonic baseline concentrations to phasic transients (Goto et al 2007). Thus, tonic DA cell activity may induce basal levels of the high-affinity D2 receptor activation, while phasic release can activate D1 receptors (Creese et al., 1983, Richfield et al., 1989, Grace, 1991). Only 60% of the cells in the VTA are actually dopaminergic; 30-35% are gammaaminobutyric acid (GABA)-ergic, and 2-3% glutamatergic (Swanson, 1982b, NairRoberts et al., 2008, Dobi et al., 2010). While distinct populations of cells have been identified based on either their location, co-expression of peptides such as cholecystokinin and neurotensin, whether they are autoinhibited, and their firing patterns, recent evidence suggests that a combination of techniques are required for accurate phenotyping (Swanson, 1982a, Hokfelt et al., 1984, Seroogy et al., 1987, Rayport et al., 1992, Lammel et al., 2008, Margolis et al., 2008). VTA gets input from a range of brain regions characterized as the “isodendritic core”, similar to that observed in the reticular formation (Geisler and Zahm, 2005). In 16 addition, mesopontine tegmental area neurons provide cholinergic modulation (Maskos, 2008). However, the major excitatory and inhibitory inputs to the VTA are from the mPFC and striatum, respectively, and are discussed below. These connections are bidirectional, as the VTA sends distinct projections to the striatum (including the Acb), PFC, amygdala, and hypothalamus (Margolis et al., 2008). Both dopaminergic and GABAergic cells can either synapse locally or project to corticolimbic sites (Groenewegen et al., 1999, Tzschentke, 2000, Brenhouse et al., 2008, Margolis et al., 2008, Carlezon and Thomas, 2009, Ernst et al., 2009). Projections from the VTA to the striatum follow a roughly topographic continuum, with medial regions, including the interfascicular (IF) and paranigral (PN) subregions, projecting to medial accumbens (including the medial shell), and more lateral subdivisions, including the parabrachial pigmented nucleus (PBP), projecting to lateral accumbens (including the core) (Fallon and Moore, 1978, Ikemoto, 2007). There are also many interconnections between these subdivisions of the midbrain (Ikemoto, 2007, Ferreira et al., 2008). In addition, distinct responses to local drug manipulations and genetic manipulations have also been reported along the rostro-caudal extent of the VTA (Ikemoto and Kohl, 1997, Carlezon et al., 2000, Olson and Nestler, 2007) and the posterior hypothalamus (PH) / supramammilary nucleus (SuM) and “Tail” have been suggested as rostral and caudal extensions of the VTA, respectively (Ikemoto, 2010). Medial prefrontal cortex (mPFC). The mPFC contributes both affective and regulatory processes for the performance of motivated behavior (Seitz et al., 2006). Within the mPFC, both GABAergic interneurons and glutamatergic pyramidal neurons receive dopaminergic input from the VTA (Tzschentke, 2000). D1 receptors are 17 expressed in a greater proportion of GABAergic interneurons than pyramidal cells, however, both D1 and D2 receptors are expressed in both cell types. Also, D2 receptors are expressed mainly in cortical layer V (known for its subcortical projections), while D1 receptors are expressed relatively evenly in all layers (Santana et al., 2009). In addition to afferents from the VTA, the mPFC also receives major inputs from the mediodorsal thalamus (a source of striatal and olfactory information), the insula (a source of emotional information), and the SuM nucleus (an extension of the VTA, described above) (Heimer, 1972, Hoover and Vertes, 2007, Ikemoto, 2010, Damasio et al., 2012). The mPFC sends glutamatergic projections to the accumbens, where it modulates accumbens activity by binding AMPA and NMDA postsynaptically, but also on presynaptic dopaminergic terminals in complex three-way synapses (David et al., 2005). The mPFC also sends glutamatergic projections to the VTA; these glutamatergic cells preferentially target GABAergic VTA cells that project to the Acb, and dopaminergic VTA cells that project back to the mPFC (Carr and Sesack, 2000b). Like the rest of the mesocorticolimbic circuitry, the mPFC can be segregated into subregions with distinct functional and anatomical characteristics (Phillipson, 1979, Heidbreder and Groenewegen, 2003, Geisler and Zahm, 2005). The most dorsal subregion is the anterior cingulate (Cg), which is involved in motor control and is less interconnected with limbic and VTA regions (Vertes, 2006). The ventral mPFC includes the prelimbic (PrL) and infralimbic (IL) subregions, which, on the basis of connectivity and behavioral relevance, have been associated with the executive functions of the dorsolateral PFC and affective functions of the orbitomedial PFC in primates, respectively (Uylings et al., 2003, Vertes, 2006). While both the PrL and IL are tightly 18 connected with the Acb, amygdala, and VTA, the IL projects more to the BNST and hypothalamus than PrL (Vertes, 2004). Amygdala (AMY). In addition to the chemosensory role the Me plays in rodent social behavior, the basolateral and central amygdala is also important in fear and avoidance conditioning (LeDoux, 2007). The VTA sends dopaminergic projections to the central and basolateral amygdala (Inglis and Moghaddam, 1999), where it releases dopamine in response to stressors to enhance amygdalar dependent fear-conditioning and aversive responses. This likely occurs via dopamine dependent inhibition of a specific group of GABAergic neurons (Marowsky et al., 2005), which then disinhibit amygdalar glutamatergic cells that project to the Acb (Charara and Grace, 2003) and mPFC (Hoover and Vertes, 2007). Accumbens (Acb). The Acb is generally thought of as the link between limbic processes and motor output, and receives projections from the VTA, mPFC, and AMY (Sesack and Grace, 2009). The Acb primarily consists of GABAergic medium spiny neurons that express D1 and D2 receptors, cholinergic interneurons that express D2 but not D1 receptors, and GABAergic interneurons that express several peptides but not dopamine receptors (Meredith, 1999, Bertran-Gonzalez et al., 2008). The Acb is interconnected with the dorsal striatum, and may display similar cellular organization. The medium spiny neurons in the dorsal striatum have been organized into two populations that exert balanced but antagonistic influences on their midbrain target and motor output (Albin et al., 1989, Surmeier et al., 2007). The “direct” population of cells typically expresses D1-type dopamine receptors on their cell bodies and dendrites and co-expresses dynorphin. This cell group feed backs to the substantia nigra to inhibit 19 non-dopaminergic cells via GABA receptor activation, and is generally thought to facilitate motor output. The “indirect” population expresses D2 family receptors and coexpress enkephalin. It projects mainly to the ventral pallidum and subthalamic nuclei to transynaptically reduce motor output. A parallel organization of direct and indirect cell populations is present in the Acb, but with considerably less exclusive projection patterns (Sesack and Grace, 2009, Lobo et al., 2010). Indeed, recent evidence suggests that a small percentage (6-17%) of Acb neurons even express both D1 and D2 receptors (Bertran-Gonzalez et al., 2008). Generally, though, the direct and indirect pathway disinhibits and inhibits, respectively, motor pathways for reward acquisition and motivated behavior (Sesack and Grace, 2009). The Acb is divided anatomically into two subdivisions, called the core and shell, which vary in their neurochemistry and projection patterns (Groenewegen et al., 1999). The core projects more to substantia nigra while shell projects more to VTA (Heimer et al., 1991). The shell projections synapse on GABAergic, and not dopaminergic, cells in the VTA, including local interneurons and GABAergic projections back to the Acb (Xia et al., 2011). The shell is often associated with functions in drug reward, while core has been implicated more in cue-conditioning; however these are likely oversimplifications of their complex functions (Sesack and Grace, 2009). While D1 and D2 receptors have typically been described as excitatory or inhibitory of cellular membrane potential and activity, more recent data demonstrate that this is an oversimplification (reviewed in (Seamans and Yang, 2004). Instead, the effect of activating either receptor can depend on whether or not the post-synaptic neuron has been recently activated (Arbuthnott and Wickens, 2007). The membrane potentials of 20 medium spiny neurons in the accumbens and pyramidal cells in the cortex fluctuate from periods of hyperpolarization to plateaus of relative depolarization, creating baseline states of “down” or “up” (O'Donnell and Grace, 1995). In “up” states, lower amplitude excitatory post-synaptic potentials are required to induce an action potential. Thus, multiple coordinated synaptic inputs are needed to most readily induce cell firing; in the Acb, this is often means glutamatergic input from the amygdala and mPFC (Seamans and Yang, 2004). Importantly, dopamine modulates these up states to amplify the signal:noise ratio: D1 receptors tend to holding the membrane potential at the “up” state longer, while D2 receptors serve to reducing the number of spontaneously evoked action potentials (O'Donnell, 2003). Generally, dopaminergic action is thought to be more modulatory than other typical neurotransmitters. Thus, the Acb integrates glutamate release from the mPFC and amygdala with dopamine modulation from the VTA and, often in complicated three-way synapses, encodes motivational salience and potentiates goal-directed behavior. This circuitry is widely studied for its ability to sustain site-directed selfadministration of drugs and electrical stimulation (as reviewed in (Ikemoto, 2007). For example, animals self-stimulate in the mPFC, supporting its role in reward circuits, and this activity induces Fos in the Acb, MPOA and VTA (Avanitogiannis et al., 2000). However, this circuitry evolved in response to natural, not artificial, rewards. As such, the VTA, Acb, and mPFC are activated by chemosensory sexual stimuli and copulatory behaviors (Greco et al., 1998, Balfour et al., 2004, Balfour et al., 2006), and are important in sexual motivation and chemosensory attraction. Specifically, lesions of the mPFC and Acb inhibit anogenital investigation, sexual behavior and motivation 21 (Hendricks and Scheetz, 1973, Pfaus and Phillips, 1991, Fernandez-Gausti et al., 1994, Agmo and Villalpando, 1995, Liu et al., 1998, Kippin et al., 2004, Afonso et al., 2007). Behavioral paradigms of reward and motivation Before a more nuanced discussion of the mesocorticolimbic function in sociosexual behaviors, concepts of reward must be discussed. Reward has three different components: hedonic value, motivational salience, and learning (Berridge and Robinson, 2003). Hedonic value, or how pleasurable or “liked” a stimulus is, can be determined without previous learning and is primarily expressed as an affective reaction (e.g. lateral tongue protrusions upon sucrose administration). In contrast, motivational salience is the process whereby a stimulus is deemed attention-grabbing and worthy of goal directed behavior, or “wanted”. While stimuli that are “wanted” are often initially “liked”, these qualities can be dissociated from one another, e.g., drug addicts who no longer enjoy the drug but still have cravings. This theory suggests that mesocorticolimbic dopamine is important for imbuing rewarding stimuli with motivational salience, or wanting (Berridge and Robinson, 1998, Ikemoto and Panksepp, 1999, Salamone et al., 2005, Berridge, 2007, Ikemoto, 2007). While I acknowledge these different facets of reward, almost all behavioral tasks fail to discriminate between the components; therefore I will use “reward” throughout the dissertation to refer to all three. A rewarding stimulus may be used to condition an operant behavior. In operant behavior tasks, such as bar pressing or nose pokes, an animal’s behavior is modified by receiving a reinforcement or punishment. This form of leaning requires action on the part of the subject, and is contrasted with Pavlovian conditioning. In Pavlovian 22 conditioning, an initially rewarding (unconditioned) stimulus is repeatedly paired with a neutral stimulus; the animal then learns that the neutral stimulus is associated with the unconditioned reward, such that it is also “wanted.” Both forms of conditioning require leaning about a stimulus and can indicate how motivated an animal is to receive the reward. One behavioral paradigm of Pavlovian conditioning useful for the rewarding value of a stimulus is conditioned place preference (CPP, (Perks and Clifton, 1997). In this paradigm, an unconditioned reward (that is liked) is repeatedly paired with a neutral stimulus (a conditioning compartment), which then acquires rewarding properties of its own. These rewarding properties are determined in a test whereby the subject “wants” that conditioned environment, and spends more time in the stimulus-paired compartment over an unconditioned compartment. CPP has long been used in determining the rewarding aspects of drugs, copulatory and feeding behaviors (Schechter and Calcagnetti, 1993), as well as which neurotransmitters, hormones, and brain regions are important for reward (Schechter and Calcagnetti, 1993, Tzschentke, 1998). Male and female rodents show a CPP to sexual interactions (Miller and Baum, 1987, Agmo and Berenfeld, 1990, Hughes et al., 1990, Mehrara and Baum, 1990, Oldenburger et al., 1992, Agmo and Gomez, 1993, Meisel and Joppa, 1994, Meisel et al., 1996, Paredes and Alonso, 1997, Pankevich et al., 2006, Agustin-Pavon et al., 2007, Martínez-Ricós et al., 2007). Dopamine has been widely implicated in CPP, as both systemic and intra-Acb delivered D1 and D2 agonists and antagonists induce a CPP or conditioned place aversion, respectively (Hoffman and Beninger, 1988, Papp, 1988, Hoffman and Beninger, 1989, Shippenberg et al., 1991, White et al., 1991, Papp et al., 1993, Acquas and Di Chiara, 1994, Tzschentke, 1998). 23 More recently, optogenetic techniques have demonstrated that activation of phasic firing of VTA dopaminergic neurons is sufficient to induce a CPP (Tsai et al., 2009). Qualities of sexual reward and motivation have been assessed with a range of behavioral tasks and parameters. The classic appetitive / consummatory dichotomy (Everitt, 1990) has been used to parse out motivational aspects of sexual interactions. Simply stated, stereotyped acts of sexual behavior including mounts, intromissions, and ejaculations, are deemed consummatory. Behaviors prior to the first direct copulatory act are described as appetitive. For example, attraction or preferences for stimuli, anticipatory behavior or locomotor excitement in advance of access to a receptive mate and investigatory behavior have all be used as indicators of sexual arousal and motivation (Everitt, 1990). However, many of these behavioral measures are dependent on locomotor ability, which may be affected by age and dopaminergic manipulations. Indeed, Everitt proposed using instrumental conditioning and CPP to better assess sexual reward and motivation. Drug and age confounds are avoided in CPP, as animals are tested for their place preference in a drug-free state, and are compared with age-matched controls that did not received conditioning (Schechter and Calcagnetti, 1993). In addition, a conditioned place aversion (CPA, (Acquas et al., 1989) can be used to determine the aversive qualities of stimuli. Multiple indicators of reward provide the most complete understanding of the motivational properties of a stimulus; CPP will be used in this dissertation to complement previous work on attractive responses to VS, in our attempt to measure rewarding properties of the important social cue. 24 Dopamine and sociosexual reward In general, dopamine appears important for male sexual behavior, as systemic apomorphine (a D1/D2 agonist) increases performance of copulatory behavior in male rats and hamsters (Malmnas, 1976, Agmo and Berenfeld, 1990, Scaletta and Hull, 1990, Mas et al., 1995, Rodríguez-Manzo, 1999, Lopez and Ettenberg, 2000, 2001, Arteaga et al., 2002, Lopez and Ettenberg, 2002, Niikura et al., 2002, Hull and Dominguez, 2007). Several of the behavioral paradigms described above have been used to parse sexual motivation and reward from the consummatory aspects of sexual behavior. Systemic flupenthixol (a D1/D2 antagonist) decreases the number of operant conditioning responses for access to a receptive female at a dose that did not affect copulatory behavior (Everitt, 1990). Similarly, systemic haloperidol (a D2 antagonist) reduces anticipatory level changes for a female in a two-level chamber, while only affecting sexual behavior at high doses that likely affect general motor behavior (Pfaus and Phillips, 1989, Pfaus and Phillips, 1991). D2 receptors also appear important in motivation for female cues, as systemic haloperidol reduced the unconditioned incentive value of estrous female cues in sexually-naïve male rats and sex-associated cues after conditioning (Lopez and Ettenberg, 2001, 2002). The effects of systemic dopamine manipulations in CPP have been mixed. Pimozide, a D2 antagonist, fails to block ejaculation-induced CPP in male rats (Agmo and Berenfeld, 1990), and systemic D1 D2 antagonists fail to block a CPP for male bedding in female mice (Martínez-Hernández et al., 2006, Agustin-Pavon et al., 2007). However, in hamsters, sulpiride, but not raclopride, both D2 antagonists, blocked the sexual behavior-induced CPP (Meisel et 25 al., 1996). Thus, systemic manipulations, often with D2 receptors, can selectively affect some species-specific, appetitive, motivational aspects of sociosexual behavior. Dopamine in the mesocorticolimbic system has also been implicated in sexual performance and motivation (Asmus, 1994, Hull et al., 1995, van Furth et al., 1995, Berridge and Robinson, 1998, Balfour et al., 2004, Balfour et al., 2006). Dopamine activity increases in the male rat Acb in response to female chemosensory and auditory cues and during copulation in male and female rats and hamsters (Mas et al., 1990, Damsma et al., 1992, Mitchell and Gratton, 1992, Wenkstern et al., 1993). Intra-Acb delivery of D1 and D2 antagonists decreases anticipatory behavior for access to a receptive female and increases latencies to mount the female, while agonists increase sex behavior (Pfaus et al., 1990, Pfaus and Phillips, 1991, Meisel et al., 1993). However, one study of unconditioned chemosensory reward with site-specific manipulations found that dopamine depletion in the shell did not affect female mouse attraction to male bedding (Martínez-Hernández et al., 2012). Thus, dopamine release in the accumbens may be important for appetitive preparatory sexual activity and copulation, however its role in sexual reward requires further study. Effects of dopamine in the mPFC on the performance of sexual behavior or reward have not been well-characterized. In the MPOA, dopamine appears important for mediating male sexual behavior, as MPOA dopamine is released in response to female cues and during copulation (Hull et al., 1995, Sato et al., 1995). These dopamine responses to females are dependent on chemosensory stimuli in male hamsters (Wood, 2004, Triemstra et al., 2005). Moreover, microinjections of nonspecific dopamine agonists (apomorphine) or 26 antagonist (flupenthixol) into the MPOA promotes or attenuates sexual behavior in male rats, respectively (Hull et al., 1986, Warner et al., 1991, Arteaga et al., 2002). MPOA dopamine is also important in penile responses: D1 receptors appear important for erections (which, when spontaneous, may indicate motivation), while D2 receptors appear important for seminal emission associated with ejaculation (Hull et al., 1992). While some have proposed that MPOA dopamine effects are mainly consummatory, as opposed to appetitive (Everitt, 1990), there is some evidence to the contrary. Namely, the D2 antagonist raclopride into the MPOA reduces the number of animals choosing a female box in a 4 choice X-maze, and also reduces the number of animals copulating once they reached her, without affecting movement (Moses et al., 1995). Thus, MPOA dopamine appears important in both motivational and consummatory aspects of copulatory behavior. Lesion and knife cut studies suggest that almost all of the dopamine input to the MPOA is from dopaminergic incerto-hypothalamic projections from the A14 cell group in the PeVN (Bjorklund et al., 1975, Palkovits et al., 1977, Day et al., 1980, Horvath et al., 1993). However a recent anatomical tracer study suggests that the MPOA may receive dopamine innervation from a wide range of brain regions, including the MPOA itself, PVN, SuM, PH, and VTA (Oades and Halliday, 1987, Miller and Lonstein, 2009). Through an extensive series of studies, Hull and colleagues have demonstrated that basal and female-induced release of dopamine from MPOA terminals is the result of local glutamate to release nitric oxide (Lorrain and Hull, 1993, Lorrain et al., 1996, Sato et al., 1998, Dominguez et al., 2004). Moreover, increasing and decreasing both glutamate and nitric oxide in the MPOA increase and decrease sexual behavior, ex 27 copula erections, and anticipatory ultrasonic vocalizations (Giuliano et al., 1997, Moses and Hull, 1999, Brudzynski and Pniak, 2002, Lagoda et al., 2004, Dominguez et al., 2006). An important source of glutamate to the MPOA relevant to sexual behavior is the MeP. Lesioning the MeP reduces both sexual behavior and the release of dopamine in the MPOA in response to a stimulus female, and supplementing this MPOA dopamine release with a D1/D2 agonist (apomorphine) restores sexual behavior to prelesion levels (Dominguez et al., 2001, Dominguez and Hull, 2001). These studies emphasize the importance of MeP input to the MPOA to relay chemosensory information important for sociosexual behaviors. Dopamine is also released in response to female cues and during copulation in the nearby PVN (Melis et al., 2003), are shares many similarities with MPOA dopamine, described above. The PVN is also innervated by dopaminergic neurons from the A14 cell group in the PeVN and A13 cell group in the medial zona incerta (Bjorklund et al., 1975, Swanson and Sawchenko, 1980, Buijs et al., 1984, Lindvall et al., 1984, Cheung et al., 1998), and is the site of oxytocin neurons that project to the spinal cord to promote erections (Melis and Argiolas, 2003). Dopamine release in the PVN acts on D2 receptors to increase nitric oxide, stimulate these oxytocin neurons, and increase noncontact erections in response to female cues (Melis et al., 1987, Eaton et al., 1991, Melis et al., 1997, Melis et al., 1998, Melis et al., 2003). Non-contact erections in response to female cues may be an indicator of sexual motivation (Melis and Argiolas, 2003). Interestingly, the dopamine stimulated PVN oxytocin neurons also project to the VTA, where they increase nitric oxide to stimulate VTA dopaminergic neurons and increase the release dopamine in the Acb (Melis et al., 2007, Succu et al., 2007, Succu 28 et al., 2008). The role of PVN DA in these erections, and its connectivity with mesocorticolimbic dopamine, suggests that the PVN is also involved in both consummatory and motivational aspects of sexual behavior Orexin and sociosexual reward Another neural mediator of reward is orexin (also known as hypocretin), a neuropeptide expressed exclusively in the lateral hypothalamic area (LHA) and the perifornical– dorsomedial hypothalamus (DM-PeF) in two forms, orexin-A and orexin-B (de Lecea et al., 1998, Sakurai et al., 1998). While initially implicated in regulating food intake and sleep and arousal, recent studies have demonstrated its involvement in food, drug, and sexual reward. Specifically, orexin cells express Fos in response to copulation (Muschamp et al., 2007), food, cocaine, and morphine (Harris et al., 2005), and orexin agonists and antagonists increase and decrease male rat sexual behavior, respectively (Gulia et al., 2003, Muschamp et al., 2007, Di Sebastiano et al., 2010). Moreover, orexin neurons appear essential for sex and morphine- induced CPP (Harris et al., 2007, Di Sebastiano et al., 2011). Although drug reward effects seem specific to cells lateral of the fornix, copulation increases Fos throughout the cell group. There are dense orexinergic projections to, and Orx 1 and 2 receptor expression in, the accumbens, BNST, MPOA, PVN, Me, VMH, SuM and VTA (Peyron et al., 1998, Trivedi et al., 1998, Marcus et al., 2001, Fadel and Deutch, 2002, Schmitt et al., 2012), suggesting that some of these reward-related functions may be via interactions with the mesocorticolimbic system. Indeed, orexin excites VTA dopaminergic and GABAergic 29 neurons, and causes dopamine release in the Acb and PFC (Korotkova et al., 2003, Narita et al., 2006, Muschamp et al., 2007, Vittoz et al., 2008). Testosterone and sociosexual reward As noted above, many chemosensory and sociosexual behaviors are dependent on testosterone. Therefore, it follows that testosterone may also play a role in the rewarding and motivational properties of these stimuli. Accordingly, gonadectomy blocks male rats’ ability to form a CPP to an estrous female and to emit ultrasonic vocalizations in response to female bedding, another indicator of sexual motivation (Harding and McGinnis, 2004). Testosterone-induced restoration of this CPP and vocalization is blocked by either infusion of the antiandrogen flutamide into or lesion of the VMH (Harding and McGinnis, 2004). While these studies suggest some local action of testosterone within VMH, VMH could also relay hormonal information via projections to the BNST, Me, MPOA, and PVN. However, the VMH is not well-connected with mesocorticolimbic structures (Saper et al., 1976, Canteras et al., 1994). One study also reports rapid effects of testosterone, after aromatization to estradiol, on cellular firing in the male rat lateral hypothalamus (Orsini, 1982). Effects of testosterone on the orexin system have not been well-studied, except for one report of gonadecomy reducing orexin expression in the anterior hypothalamus, but not prefrontal cortex (Silveyra et al., 2009). The dopaminergic dynamics in the MPOA are also highly sensitive to testosterone. Several studies have demonstrated that long-term (2-8 weeks) gonadectomy results in an increase in dopamine in MPOA tissue punches (Engel et al., 30 1979, Simpkins et al., 1983, Mitchell and Stewart, 1989, Du et al., 1998). Further study demonstrated that this increase in intracellular dopamine was accompanied by a decrease in basal extracellular dopamine (and associated low dopamine turnover), and an increase in amphetamine-induced extracellular dopamine (Gunnet et al., 1986, Du et al., 1998), without a change in dopaminergic cell number in the MPOA or PeVN (Du and Hull, 1999). This suggests that testosterone is necessary for MPOA dopamine release, but not synthesis. The MPOA dopaminergic release in response to sexual stimuli in the MPOA is also dependent on testosterone in male rats (Hull et al., 1995, Putnam et al., 2003). As discussed above, nitric oxide is important in the release of dopamine, both at basal levels and in response to a sexual stimulus. Importantly, nitric oxide is also sensitive to testosterone, as long-term gonadectomy reduces expression of nitric oxide synthase in the MPOA both hamsters and rats (Hadeishi and Wood, 1996, Du and Hull, 1999)). This effect of testosterone is due to its metabolism to estradiol, which, after gonadectomy, maintains nitric oxide synthase and intracellular dopamine levels at that of testosterone treated male rats (Putnam et al., 2005). Thus, dopamine release in the MPOA, and presumably the PVN, is sensitive to gonadal hormones. In addition to hypothalamic sites of action, testosterone may exert effects on sociosexual reward via modulation of dopaminergic action in the mesocorticolimbic circuit. Long-term castration (30, but not 14, days) causes an increase in the number of TH-ir cells in the VTA of adult male rats (McArthur et al., 2007, Johnson et al., 2010). This parallels effects observed in the mPFC, where 28 day gonadectormy causes an increase in TH innervation and extracellular dopamine (Aubele and Kritzer, 2011b). Moreover, long-term gonadectomy blocks or reverses the effects of AMPA and NMDA 31 glutamate receptor administration on dopaminergic tone in the mPFC, respectively (Aubele and Kritzer, 2011a). Testosterone could affect dopamine in the mPFC via androgen receptors in dopaminergic cells in the VTA (Kritzer, 1997, Kritzer and Creutz, 2008), or alternatively, via androgen sensitive MPOA projections to the VTA (Sato et al., 2008). The functional relevance of these studies is unclear: gonadectomy reduces performance on PFC dependent operant tasks (Kritzer et al., 2007). However, these tasks are also known to be dependent on dopamine innervation, making the gonadectomy induced increase in PFC dopamine perplexing. In addition, short-term (4 day) gonadectomy causes a decrease in TH innervation and extracellular dopamine, effects that are opposite those observed with 28 day gonadectomy (Aubele and Kritzer, 2011a). Thus, the typical effect of testosterone on prefrontal dopamine is still undetermined. Effects of short-term (>14days) castration in the ventral striatum have not been observed (Engel et al., 1979, Mitchell and Stewart, 1989) however 28 day gonadectomy generally reduces dopamine and DOPAC concentrations in Acb tissue (Alderson and Baum, 1981, Mitchell and Stewart, 1989) but see (Baum et al., 1986). While further study is needed to determine the effects of testosterone treatment, as opposed to withdrawal, these studies demonstrate that testosterone may be needed to maintain dopamine function in the Acb. Testosterone itself also has intrinsically reinforcing properties (Wood, 2004). Animals form a CPP to peripheral injections of testosterone (De Beun et al., 1992, Alexander et al., 1994, Wood, 2004); the CPP can be disrupted by flupenthixol (D1/D2 antagonist) infusion into the Acb on the test day (Packard et al., 1998). Moreover, CPP 32 is also formed to intra-Acb (Frye et al., 2002) and intra-MPOA (King et al., 1999) testosterone injections. Therefore, the endogenous testosterone surge in response to copulatory behavior or chemosensory stimuli may imbue them with rewarding properties. However, gonadectomized adult hamsters with replacement testosterone are still attracted to VS (Gregory and Bishop, 1975), suggesting that tonic levels of circulating testosterone, without elevations in testosterone specifically in response to pheromones, are sufficient for attraction to female pheromones. This dissertation will test whether exogenous testosterone is also sufficient for a conditioned place preference for VS. Overall, it is clear that there are many interactions between mesocorticolimbic and hypothalamic circuitry, including the actions of dopamine, orexin, and testosterone, that may mediate sociosexual reward. A simplified presentation of these brain regions and neurochemicals is shown below in Figure 1.1. Importantly, many of these regions and neurochemicals mature in structure and function across adolescence, and these developmental changes may underlie the behavioral shifts in sociosexual responses at that same time. 33 Figure 1.1: Diagram of brain regions involved in social behavior and motivation. Olfactory information is integrated with hormonal cues in the amygdala, sent to the preoptic area and hypothalamus, and then relayed to the mesocorticolimbic system for motivated behavioral output. Developmental changes in motivational neural circuitry As sexual behavior and attractive responses to VS mature across adolescence, neural responses to VS (as indexed by Fos expression) in brain regions important in the initial olfactory processing were investigated in juvenile and adult male hamsters. Gonadintact juvenile and adult hamsters expressed increases in Fos expression in response to VS in the MeP, BNST, MPN, and MPNmag (Romeo et al., 1998, Parfitt et al., 1999). Therefore, although this study suggests juveniles can detect VS so as to induce neural activation in olfactory-related brain regions, it failed to identify the neural mechanism behind the behavioral changes in response to VS. While Fos expression was similar between juvenile and adult hamsters, juvenile did hamsters fail to show the adult-like MPOA dopamine release (indexed via dihydroxyphenylacetic acid concentrations in 34 tissue punches) in response to VS (Schulz et al., 2003), highlighting the MPOA as one possible site of maturation. However, further research is needed to identify the neural maturation behind changes in chemoinvestigatory and sexual behaviors during adolescence. As dopamine has been strongly implicated in reward, motivational salience and sexual motivation pathways (Berridge and Robinson, 1998, Andersen et al., 2002, Bjork et al., 2004, Hull and Dominguez, 2007), changes in dopamine circuitry that occur during adolescence may be important for the maturation of social stimulus interpretation and assessment (Bjork et al., 2004). Indeed, there is good evidence for mesocorticolimbic and dopaminergic maturation over adolescence (Spear, 2000). However, it should be mentioned that the great majority of these studies were performed in rats and mice. While the overarching processes are likely similar to that in hamsters, exact timing could differ between the species. These studies are given to demonstrate possibilities, and future studies are needed in hamsters. Multiple aspects of the mesocorticolimbic dopaminergic circuit mature during adolescence. In the midbrain, the firing rates of dopaminergic neurons peak midadolescence (McCutcheon and Marinelli, 2009). In male rats, striatal tissue content of dopamine reaches adult levels by P60 (Giorgi et al., 1987, Broaddus and Bennett, 1990), and microdialysis studies show extracellular dopamine peaks at mid adolescence in the Acb (Philpot and Kirstein, 2004, Badanich et al., 2006, Philpot et al., 2009). Dopamine transporter density, another marker of innervation, also peaks in late adolescence in the accumbens (Coulter et al., 1996). In the mPFC, there is an relatively linear adolescent increase in dopamine innervation and therefore dopamine 35 tissue concentrations in the mPFC in male monkeys (Rosenberg and Lewis, 1995) and rats (Kalsbeek et al., 1988), and an increase in amygdalar input in male rats (Cunningham et al., 2002). At the same time, the number of neurons in the PrL and IL decrease (Markham et al., 2007), and glutamatergic projections from the PFC to the accumbens increase (Brenhouse et al., 2008) during adolescence in male rats. Together, these data suggest gains in dopaminergic and glutamatergic activity and projections throughout adolescence. In addition to changes in production and innervation, dopamine receptor expression matures during adolescence in both humans (Seeman et al., 1987) and rats. Specifically, there is an overproduction and then pruning of D1 and D2 dopamine receptors in the dorsal striatum and Acb in males (and less so in females), as evidenced by binding assays (Teicher et al., 1995, Andersen et al., 1997b). In the mPFC, D1 and D2 density also peak mid-adolescence, particularly on pyramidal cells that project to the AcbC (Andersen et al., 2000). Studies also suggest that autoreceptor function in the mPFC is only present prior to adolescence (Teicher et al., 1991, Andersen et al., 1997a). Thus, even if dopamine is released in a similar fashion between adults and juveniles, its postsynaptic consequences could be very different. Lastly, not only does density of receptor expression shift, but the functional outcome of receptor activation changes during adolescence. In both the mPFC and Acb, D2 receptor activation excites GABAergic interneurons through some unknown mechanism, but only after P50 (Tseng and O'Donnell, 2007, Benoit-Marand and O'Donnell, 2008). These GABAergic interneurons then attenuate pyramidal and medium spiny neuron responses to excitatory input, potentially enhancing the 36 signal:noise ratio in adulthood (Tseng and O'Donnell, 2007, Benoit-Marand and O'Donnell, 2008). In the mPFC, interactions between dopamine and glutamate receptors also mature during adolescence (O'Donnell, 2003). As discussed previously, the membrane potential of mPFC pyramidal neurons can be brought into a more excitable “up” state by coordinated activation of glutamatergic NMDA and dopaminergic D1 receptor activation. Importantly, the amplitude of these up states is lower in juvenile animals compared to adults (O’Donnell et al., 2002), and the D1 facilitation is only present after P48 (Tseng and O'Donnell, 2005). In the striatum, excitatory post synaptic potentials in response to cortical input reach adult levels between P30 and P40, while spontaneous burst firing patterns do not mature until after P40 (Tepper et al., 1998). Together, all these changes suggest that any dopamine dependent behavioral responses to a social cue may change dramatically during adolescence, due to the extensive developmental changes in the relevant circuitry and electrophysiological properties. There have been many attempts to relate these developmental changes in dopamine circuits to a range of reward-related behaviors. For example, the midadolescent peak in D1 expression on mPFC pyramidal cells that project to the Acb have been associated with a peak in cocaine reward at P40 in male rats (Brenhouse et al., 2008). Similarly, Acb dopamine transients to a conspecific fail to show adult-like habituation to the social stimulus in a subsequent social interaction in juveniles, and have been linked to enhanced levels of peer-directed social interactions at this same age (Varlinskaya and Spear, 2008, Robinson et al., 2011). Enhanced reward sensitivity to palatable food, cocaine, and amphetamine in juveniles compared to adults are 37 detected both behaviorally and with Fos expression in the Acb and mPFC (Andersen et al., 2001, Cao et al., 2007, Friemel et al., 2010). As Fos expression is a useful way to monitor cellular activity in many brain regions at once, it will be used in this dissertation to provide a systems-level perspective for adolescent changes in responses to VS. There are also reports of orexinergic maturation during adolescence in male rats. Dramatic increases in prepro-orexin mRNA (the uncleaved precursor to orexin A and B) are seen from P1 – P25, and reached adult levels at P40 (Yamamoto et al., 2000). However, orexin-A and orexin-B immunoreacitve cells peak at P70, and then decline to adult levels over the next 30 days (Sawai et al., 2010). Given the known interactions between orexinergic and dopaminergic systems in motivated behaviors, development in both systems may interact to affect reward-seeking behavior. Summary of Dissertation Experiments The differences between gonad-intact juvenile and adult male hamsters in their unconditioned behavioral and physiological responses to social stimuli suggest that female cues are being interpreted differently at the two ages. This dissertation tested the hypothesis that adult but not juvenile animals find VS rewarding and that this shift in stimulus interpretation is mediated by changes in dopaminergic mesocorticolimbic circuitry due to pubertal increases in testosterone. To do this, behavioral, immunohistochemical and pharmacological methods were used to test predictions of this hypothesis. Chapter 2 tested the hypothesis that adult male hamsters find VS rewarding. Conditioned place preference was used by training males to associate one 38 of two chambers with female pheromones and then determining in which chamber the males choose to spend more time. Additionally, CPP was used to compare the reinforcing properties of VS to that of sexual behavior. Chapter 3 tested the hypothesis that both rewarding and mesocorticolimbic responses to VS are gained during adolescence. Conditioned place preference (CPP) paradigm was used to compare the rewarding properties of VS between juvenile and adult hamsters. In parallel, immunohistochemistry for Fos and tyrosine hydroxylase was used to compare mesocorticolimbic activation patterns in response to VS between juveniles and adults. Hamsters were gonad intact in order to determine normal developmental changes in the evaluation of female pheromones. Chapter 4 tested the hypothesis that testosterone treatment is sufficient in juveniles, and necessary in adults, for mesocorticolimbic responses to VS. Immunohistochemistry for Fos and tyrosine hydroxylase was used to compare mesocorticolimbic activation patterns in response to VS between gonadectomized juvenile and adult males with or without testosterone replacement. Chapter 5 tested the hypothesis that dopamine receptor activation mediates the testosterone-dependent CPP formation for VS. Adult hamsters were gonadectomized and juvenile hamsters were given testosterone prior to testing for a CPP to VS. Additionally, testosterone-treated juveniles were given a dopamine antagonist to block a CPP to VS. Although other brain regions, including the bed nucleus of the stria terminalis and dorsal striatum, and other neurotransmitters, including oxytocin and opioids, are involved in social information processing, social behaviors, and social reward, the 39 dramatic adolescent changes in mesocorticolimbic dopamine circuitry make it a good candidate for focused study in this dissertation. Overall, this work furthers our understanding of the neurobiological mechanisms underlying the adolescent maturation of social information processing and social reward. 40 Chapter 2: Male Syrian hamsters demonstrate a conditioned place preference for sexual behavior and female chemosensory stimuli Introduction Sexual activity is an effective natural reward in rodents; male rodents approach and prefer opposite sex conspecifics and perform operant behavior tasks for sexual stimuli (Murphy, 1973, Everitt, 1990, Crawford et al., 1993, Agmo, 2003, Paredes, 2009). Another paradigm used extensively to assess the rewarding properties of a range of stimuli is conditioned place preference (CPP) (Schechter and Calcagnetti, 1993, Tzschentke, 1998, Tzschentke, 2007). CPP is a form of classical conditioning in which animals develop a preference for a distinctive environment associated with a rewarding stimulus such as an addictive drug, food, water, copulation, and other social behaviors (Pfaus and Phillips, 1991, Robbins and Everitt, 1996, Kelley and Berridge, 2002). CPP procedures have demonstrated the rewarding value of sexual behavior in male and female rats and mice, and in female hamsters (Miller and Baum, 1987, Agmo and Berenfeld, 1990, Agmo and Picker, 1990, Hughes et al., 1990, Mehrara and Baum, 1990, Kippin and van der Kooy, 2003, Popik et al., 2003, Harding and McGinnis, 2004). Specific components of male sexual behavior, including somatosensory stimuli resulting from intromission and ejaculation, are rewarding in and of themselves (Agmo and Gomez, 1993, Kudwa et al., 2005, Tenk et al., 2009). Chemosensory stimuli encountered during sexual behavior may also be rewarding, but this component of sexual interactions has not been as thoroughly investigated. Of the several rodents species for which sexual behavior induces a CPP, only mice are known to show a CPP for volatile (but not non-volatile) chemosensory stimuli derived from the opposite sex 41 (Pankevich et al., 2006, Pierman et al., 2006, Agustin-Pavon et al., 2007, MartínezRicós et al., 2007). Male Syrian hamsters are ideal for investigating the rewarding properties of chemosensory stimuli, because hamsters are unique from rats and mice in their utter reliance on female chemosensory stimuli for the initiation of sexual behavior (Murphy and Schneider, 1970, Johnston, 1986, Coppola and O'Connell, 1988). For example, olfactory bulb removal eliminates odor preferences and copulatory behavior in sexually experienced male hamsters (Murphy and Schneider, 1970, Powers and Winans, 1975, Petrulis and Johnston, 1995, Petrulis, 2009). Vaginal secretions (VS) are the primary source of female chemosensory stimuli (Murphy and Schneider, 1970). Sexually-naïve male hamsters show an unconditioned attraction to VS (Johnston, 1974, Landauer et al., 1977), and a preference for VS over male odors (Maras and Petrulis, 2006). In fact, VS are such powerful stimuli that male hamsters will mount another castrated or anesthetized male scented with VS (Murphy, 1973, Johnston, 1986). The data showing that female chemosensory stimuli are strongly attractive to male hamsters suggest that neural processing of female chemosensory stimuli encountered during anogenital investigation may be a specific component of sexual behavior that is rewarding for male hamsters (Wood, 2004). Surprisingly, however, male hamsters fail to bar press for female hamster VS, but operant conditioning may not have been well-suited to detect responses for VS (Coppola and O'Connell, 1988). Whether sexual behavior or its components can induce a CPP in male Syrian hamsters is unknown. Syrian hamsters are an animal model that has yielded important insights into the mechanisms underlying the integration of sensory stimuli and hormones that are crucial 42 to the expression of appropriate behaviors during sexual and social interactions (Wood, 2004, Sato et al., 2008, Petrulis, 2009). As we continue to investigate the neurobiology underlying sexual behavior using this model, we must determine whether sexual behavior and related chemosensory stimuli are rewarding to better direct research on brain regions and neurotransmitters involved in these behaviors. To assess the rewarding components of sexual behavior in the male Syrian hamster model, Experiment 1 tested the prediction that male hamsters demonstrate a CPP for sexual behavior, and Experiment 2 tested the prediction that male hamsters demonstrate a CPP for VS. The present study is the first to report that both sexual behavior and VS alone induce a CPP in male hamsters. Methods Animals Forty sexually naïve adult male Syrian hamsters (Mesocricetus auratus) obtained from Harlan Sprague-Dawley laboratories (Madison, WI) were experimental subjects. All testing began when hamsters were 61-70 days old. Twenty-eight ovariectomized adult female hamsters, approximately 12 months old, were primed with estradiol benzoate and progesterone, 52 h and 4 h respectively, prior to use as stimulus females. Before use in Experiment 1, the females were tested for receptivity by placing a nonexperimental, sexually experienced male from our colony in the females’ home cage until the female showed lordosis but before the male achieved an intromission. All hamsters were singly housed in clear polycarbonate cages (30.5 x 10.2 x 20.3 cm) with ad libitum access to food (Teklad Rodent diet no. 8640, Harlan, Madison, WI) and 43 water. Male and female hamsters were housed in separate temperature- and humiditycontrolled vivaria with a reversed light:dark cycle (14:10; lights off at 1400 h). Hamsters were treated in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. Experimental Design Place preference conditioning occurred in an apparatus with three distinct compartments (Med Associates, St. Albans, VT). The middle compartment (12 x 21 x 21 cm) was gray with a smooth Plexiglas floor and was connected to the two outer compartments (28 x 21 x 21 cm) by manually controlled sliding guillotine doors. One outer compartment was white, with metal grid flooring. Fresh pine pellets were placed in the waste pan beneath the floor before each conditioning session. The other outer compartment was black, with black scalloped solid Plexiglas flooring, and scented with a 2% acetic acid solution swabbed along the top of the walls and ceiling before each conditioning session. These contextual cues were used in both Experiment 1 and 2, as we felt that that an olfactory contextual cue was important for this olfactory-oriented species and would not interfere with conditioning for VS. Pilot experiments determined that these conditions reduced initial preferences for one or the other compartment. Time spent in each compartment was recorded using MED-PC software connected to infrared photobeams spaced at five cm intervals along the bottom of the apparatus. All conditioning and tests were conducted under dim red light at least one hour into the dark phase. 44 An initial place preference test, here called the pretest, was used to determine each hamster’s initial compartment preference. Following a 5-min habituation period in the middle gray compartment, the doors were raised and the hamster could move freely throughout the apparatus for 15 min. The outer compartment in which the hamster spent the most time was defined as the initially preferred compartment. Experimental and control groups, n = 10/group, were matched such that average initial preferences were similar across the groups. Following the pretest, the hamsters received a series of 30 min conditioning sessions, one session per day on consecutive days. Stimuluspaired (sex behavior, SB or vaginal secretions, VS) or no-stimulus conditioning sessions occurred on alternating days, beginning with the no-stimulus conditioning session. Although three stimulus-paired sessions were sufficient to induce a CPP for SB (Experiment 1), in a pilot study using similar procedures, three stimulus-paired sessions were insufficient to induce a CPP for VS. Therefore, in Experiment 2 we increased the number of stimulus-paired sessions to five with the idea that more exposure to VS would lead to a CPP. During the no-stimulus conditioning sessions, hamsters in both the experimental and control groups were taken directly from their homecages and placed into their initially preferred compartments, where they remained alone for 30 min. During stimulus-paired conditioning sessions, hamsters in the experimental group were taken from their homecages, placed in the initially non-preferred compartments and given access to SB or VS. The hamsters in the control group were also placed in their initially non-preferred compartments, but were not given the stimulus so that their experiences in the two compartments were comparable. The CPP apparatus was cleaned thoroughly with 25% ethanol following each conditioning session and test. 45 Twenty-four hours after the last conditioning session, hamsters were tested for their place preference following the same procedure used for the pretest. Experiment 1: CPP for Sexual Behavior. In Experiment 1, the stimulus tested for its rewarding value was sexual behavior with a receptive female hamster. In the SBpaired conditioning sessions, a female was placed into the compartment immediately before the male. Hamsters were observed for the duration of these sessions to verify that each male performed sexual behavior. They engaged in bouts of sexual behavior, including ejaculations, throughout the 30-min session, although behavior was not quantified. Hamsters in the control group were alone in compartments for both conditioning sessions, while hamsters in the SB group were alone in the compartment during the no-stimulus sessions. Six total conditioning sessions occurred, including three no-stimulus and three stimulus-paired sessions. The experiment took place over eight consecutive days including the pretest and test days. Experiment 2: CPP for Vaginal Secretions. Experiment 2 was similar to Experiment 1, except the experimental stimuli tested for rewarding value were VS. VS were collected by vaginally palpating 14 hormone-primed female hamsters approximately two hours before conditioning sessions began. Because both volatile and non-volatile components of VS have important, and potentially different, roles in male hamster sexual behavior, the paradigm was designed to ensure that both were present throughout the conditioning session. Several pilot studies were performed to determine the optimal method for VS delivery, including cotton swabs and Eppendorf tubes. The latter proved to be the most effective at keeping VS moist and allowing for continued exposure to volatile cues throughout the conditioning session. Approximately 46 20 µl of VS were applied to water-moistened cotton gauze packed into a 2 ml Eppendorf tube. The tube was out of reach of the male, taped to the top of the back wall of the initially non-preferred compartment in VS-paired conditioning sessions for the VS group. Empty Eppendorf tubes were used for the control group in all conditioning sessions and for the VS group in the no-stimulus conditioning sessions. To ensure exposure to nonvolatile components of VS, the remaining VS were mixed with 1.5 ml of mineral oil (see (Woodley and Baum, 2004)), and approximately 50 µl of this mixture was applied with a metal spatula directly onto the nose of the hamsters in the VS group immediately before being placed in the VS-paired compartment. The concentration of VS used in this experiment far exceeds that which is attractive to male hamsters, but mimics the amount available to a male hamster upon anogenital investigation of an estrous female (Macrides et al., 1984b). Clean oil was applied to the nose of hamsters in the control group for all conditioning sessions and in the VS group for no-stimulus conditioning sessions. Ten total conditioning sessions occurred, including five no-stimulus and five stimulus-paired sessions. The experiment took place over 12 consecutive days including the pretest and test days. Data Analysis To assess whether the stimuli (Experiment 1 SB; Experiment 2 VS) induced a CPP, data from the pretests and final tests were used to calculate a preference score, defined as time in the stimulus-paired compartment / (time in stimulus-paired compartment + time in no-stimulus compartment), and a difference score, defined as the time in the no-stimulus compartment – time in the stimulus-paired compartment 47 (Meisel and Joppa, 1994, Paredes and Alonso, 1997, Martínez and Paredes, 2001, Meerts and Clark, 2007, 2009c, b, a, Tenk et al., 2009, Parada et al., 2010). Paired ttests were used to evaluate the change in preference score and difference score preand post-conditioning, and the alpha level was set at p < 0.05 (Meisel and Joppa, 1994, Paredes and Alonso, 1997, Martínez and Paredes, 2001, Meerts and Clark, 2007, 2009c, b, a, Tenk et al., 2009, Parada et al., 2010). One methodological concern when using CPP is that the change in time spent in the compartments may be due to habituation across the conditioning sessions. This concern was addressed in these experiments by the use of the control group that received the same experience in the testing apparatus without any stimulus pairings (Meisel and Joppa, 1994). Results Experiment 1: CPP for Sexual Behavior. Male hamsters in the sexual behavior group showed a CPP for the SB-paired compartment, whereas control hamsters did not (Figure 2.1). Paired t-tests showed that the preference score increased significantly for the SB group, t(9)=-4.26, p<0.01, but not the controls, t(9)=.49, p=0.63. Likewise, the difference score decreased significantly for the SB group, t(9)=4.11, p<0.01, but not the controls, t(9)=-.54, p=0.60. 48 Figure 2.1. CPP to sexual interaction in adult hamsters. Mean±SEM preference score (top) and difference score (bottom) on pretest (gray bars) and test (black bars) for control and SB groups, n=10 per group. Asterisks indicate that the SB group showed a CPP for the compartment associated with sexual behavior; there was a significant change in preference score and difference score for the SB group but not control group, p<0.05. Experiment 2: CPP for Vaginal Secretions. Male hamsters that were given VS showed a CPP for the VS-paired compartment, whereas control hamsters did not (Figure 2.2). Paired t-tests showed that the preference score increased significantly for 49 the VS group, t(9)=-2.772, p<0.05, but not the controls, t(9)=-0.89, p=0.40. Likewise, the difference score decreased significantly for the VS group, t(9)=0.35, p<0.01, but not the controls, t(9)=2.07, p=0.07. Figure 2.2. CPP to VS in adult hamsters. Mean±SEM preference score (top) and difference score (bottom) on pretest (gray bars) and test (black bars) for control and VS groups, n=10 per group. Asterisks indicate that the VS group showed a CPP for the compartment associated with VS; there was a significant change in preference score and difference score for the VS group but not control group, p<0.05. 50 Discussion The present study is the first to demonstrate that male Syrian hamsters show a CPP for both sexual behavior with a receptive female hamster and VS, reinforcing the concept that sexual activity is a strong natural reward for male rodents (Pfaus and Phillips, 1991). Moreover, the CPP for VS indicates that female chemosensory stimuli are an unconditioned reward to male hamsters because the males were sexually naïve and VS had not been previously associated with sexual activity. Fewer conditioning sessions may be needed to induce a CPP for sexual behavior than for VS because the additional somatosensory, auditory and visual aspects likely contribute to the rewarding properties of sexual behavior. However, differences in VS stimulus intensity when presented by an experimenter versus by a female hamster during a sexual encounter may also be a factor. The present data agree with those from mice showing that chemosensory stimuli are rewarding (Pankevich et al., 2006, Agustin-Pavon et al., 2007, Martínez-Ricós et al., 2007). Mice are also sensitive to chemosensory stimuli in certain reproductive contexts (e.g., pregnancy block and puberty acceleration (Bruce, 1959, Vandenbergh, 1973). Together, the findings from hamsters and mice suggest that the behavioral salience and rewarding value of a chemosensory stimulus may be positively correlated. Rewarding Components of VS The specific compounds in VS that are rewarding remain to be identified. The chemosensory stimulus used in the present study contained both volatile and nonvolatile components. The volatile component of VS, dimethyl disulfide, has been proposed to promote anogenital investigation of the female (Singer et al., 1976, 51 Macrides et al., 1977, Pfeiffer and Johnston, 1994), whereas the non-volatile component, aphrodisin, is thought to stimulate sexual arousal and copulatory behavior (Macrides et al., 1977; (Singer et al., 1984a, Singer et al., 1987). Generally speaking, the different components bind different receptors; volatile olfactants bind receptors located in the main olfactory epithelium, whereas non-volatile molecules bind those in the vomeronasal organ (Rowe and Edwards, 1972, Meredith, 1991, Keverne, 2004, Keller et al., 2009). In male hamsters, the vomeronasal organ is important for the display of sexual behavior and plasma testosterone responses to VS (Meredith, 1986). Main olfactory epithelium lesions have little effect on sexual behavior whereas vomeronasal organ lesions alone reduce sexual behavior in sexually naïve male hamsters; combined lesions completely eliminate male courtship and sexual activity (Powers and Winans, 1975). The accessory olfactory system also appears to be critical for chemosensory reward in mice (Pankevich et al., 2006, Martínez-Ricós et al., 2007). Although additional studies are needed to pinpoint the specific components of VS and their site of action that lead to a CPP in male hamsters, the literature suggests that the accessory olfactory system is important to motivate sexual behavior in male hamsters. Neural Mechanisms of Sexual Reward Dopamine and opioids have both been implicated in the rewarding aspects of rodent sexual behavior. Dopamine is released in the medial preoptic area (MPOA) and Acb following copulation and exposure to chemosensory stimuli in male and female rodents (Mas et al., 1990, Pfaus et al., 1990, Damsma et al., 1992, Mitchell and Gratton, 1992, Meisel et al., 1993, Wenkstern et al., 1993, Asmus, 1994, Sato et al., 1995, Schulz et 52 al., 2003). In fact, the MPOA dopamine release in hamsters requires exposure to female chemosensory stimuli (Triemstra et al., 2005), further reinforcing the obligatory nature of chemosensory stimuli for sexual responses in this species. Dopamine is implicated in CPP for sexual behavior in hamsters, as CPP induced by sexual behavior in female hamsters is blocked by administration of a D2 receptor antagonist (Meisel et al., 1996) but see (Agmo and Berenfeld, 1990, Agustin-Pavon et al., 2007) for different findings in other rodent species. Opioid receptor activation has also been observed after male rat copulation (Balfour et al., 2004). Studies on the role of opioids in sexual reward have consistently shown that opiate receptor antagonists block CPP for sexual behavior in male rats (Miller and Baum, 1987, Agmo and Berenfeld, 1990, Mehrara and Baum, 1990, Agmo and Gomez, 1993). Although opioids may modulate dopamine activity in reward-related responses in male rats (Balfour et al., 2004), more research is needed to determine the role of dopamine and opioids in mediating the rewarding value of VS in male hamsters. Testosterone is another potential mediator of chemosensory reward because sexual stimuli elicit an acute increase in plasma testosterone (Pfeiffer and Johnston, 1994, Romeo et al., 1998), and testosterone is intrinsically rewarding. For example, hamsters self-administer intracerebroventricularly-delivered testosterone (Wood, 2004) and rats and mice show a CPP for systemic testosterone injections (De Beun et al., 1992, Alexander et al., 1994). In addition, castration prevents formation of CPP for an estrous female rat (Harding and McGinnis, 2004). Thus, the sex- and VS-induced increase in testosterone in hamsters (Pfeiffer and Johnston, 1994, Romeo et al., 1998) 53 may be a neuroendocrine response that contributes to the rewarding aspects of these stimuli. In summary, adult male hamsters show a CPP for sexual behavior and VS, indicating that the chemosensory stimuli encountered during anogenital investigation is one specific component of sexual behavior in male hamsters that is rewarding. It is noteworthy that the chemosensory stimuli are rewarding in both hamsters and mice, as the animals had never before associated the chemosensory stimuli with sexual behavior. Both these species are heavily influenced by chemosensory stimuli in different aspects of social behavior, suggesting that there may be an evolutionary connection between the dependence on these stimuli for social behaviors and their intrinsically rewarding properties. 54 Chapter 3: Adolescent gain in positive valence of a socially relevant stimulus: engagement of the mesocorticolimbic reward circuitry Introduction A universal feature of mammalian adolescence is the restructuring of social spheres as interactions with peers become more salient than those with family (Nelson et al., 2005). This reallocation of interest involves maturation of social information processing, i.e., the perception of and responses to social stimuli. Social stimuli evoke different neural responses in human adolescents as compared with adults, particularly in the prefrontal cortex (Blakemore, 2008), but the neuronal mechanisms underlying this developmental change remain unclear. Given the importance of appropriately interpreting social stimuli in successful adult social interactions, the overall goal of this study is to determine which brain regions and neurotransmitter systems are associated with adolescent changes in the perception of social stimuli. The male Syrian hamster is an ideal animal model for investigating the neural substrates of adolescent maturation of social information processing. Information about female reproductive status is conveyed via pheromone-containing vaginal secretions (VS). Appropriate neural processing of VS is required for the performance of male sexual behavior and is sufficient to induce a conditioned place preference (CPP) in sexually naïve adult male hamsters, indicating that VS are inherently rewarding to adults (Murphy and Schneider, 1970, Petrulis, 2009, Bell et al., 2010). In contrast, juvenile hamsters are not attracted to VS (Johnston and Coplin, 1979), nor do they mate with a receptive female when primed with exogenous testosterone (Meek et al., 1997, Schulz et al., 2009). Studies using the immediate early gene Fos as a proxy for neural 55 activation reveal that juvenile hamsters do detect VS, as it elicits an increase in Fos expression in brain regions typically associated with processing of chemosensory social stimuli, e.g. the medial amygdala (Romeo et al., 1998). Thus, the social salience of VS changes across adolescent development, and this naturally occurring maturation of social information processing is critical for successful reproduction. The neural underpinnings of these age-related changes in responses to VS are unknown. The ability of adults to form a CPP for VS suggests involvement of rewardrelated neural systems in the processing of this social stimulus. In particular, the rodent mesocorticolimbic dopamine and hypothalamic orexin systems are implicated in sexual, food and psychotropic drug reward (Meisel et al., 1996, Becker et al., 2001b, Harris et al., 2005, Muschamp et al., 2007, Ikemoto, 2010, Lajtha and Sershen, 2010, Di Sebastiano et al., 2011), and these systems often operate in concert (Fadel and Deutch, 2002, Korotkova et al., 2003, Narita et al., 2006). Both dopaminergic and orexinergic circuitries undergo functional and structural changes during adolescence (Kuhn et al., 2010, Sawai et al., 2010), however developmental changes in response to social stimuli, including VS, have not been examined within them. The present study seeks to determine if juveniles differ from adults in their proclivity to 1) show CPP for VS, and 2) express Fos in response to VS in mesocorticolimbic and hypothalamic reward circuits. 56 Methods Animals Syrian hamsters (Mesocricetus auratus) were obtained from Harlan Laboratories (Madison, WI) and were housed in temperature- and humidity-controlled vivaria with a shifted light:dark cycle (14:10, dark phase began at 1:00pm) and ad libitum access to food (Teklad Rodent diet 8640, Harlan, Madison, WI) and water. Juveniles in Exp 1 arrived at postnatal day 13 (P13) and were housed with their littermates and biological mother until weaning at P18. Adults in Exp 1 arrived at ages ranging from P56-62, juveniles in Exp 2 at P20, and adults in Exp 2 at P54. Weanlings and adult males were singly housed in clear polycarbonate cages (30.5 x 10.2 x 20.3 cm) and were all sexually naïve. Sixty adult female hamsters, approximately 12 months old, were housed under similar conditions in separate vivaria and used as the source of VS. Female hamsters were ovariectomized several weeks before hormone administration and collection of VS. They were injected subcutaneously with 10 µg estradiol benzoate and 500 µg progesterone in sesame oil, 52 and 4 hours respectively, prior to collection of VS by gentle vaginal palpation. All experiments were conducted under <4 lux red light 1-5 hours into the dark phase. Hamsters were treated in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. 57 Experiment 1: Conditioned Place Preference for potential rewards Experimental Design: Place preference conditioning occurred as described previously (Bell et al., 2010) in an apparatus with one middle compartment and two outer compartments distinct in their visual, tactile and olfactory cues (Med Associates, St. Albans, VT). To acclimate subjects to handling and novel chambers, male hamsters were placed in glass aquaria in the behavioral testing room for 10 minutes every day, for three days prior to the start of the CPP regimen. A 17-minute pretest (2 minutes in the middle compartment followed by 15 minutes access to all compartments) was used to determine each hamster's initial compartment preference and to create groups with similar initial preferences, when possible. The outer compartment in which the hamster spent more time was defined as the initially preferred compartment. Hamsters that did not enter each compartment at least 5 times were excluded from further training. Following the pretest, the hamsters received a series of 30-minute conditioning sessions in the side compartments, one session per day on consecutive days, alternating no-stimulus or stimulus-paired sessions. During the no-stimulus conditioning sessions, hamsters in both the experimental and the control groups were placed in their initially preferred compartments, where they remained alone. During stimulus-paired conditioning sessions, hamsters in the experimental group were placed in the initially nonpreferred compartments with the stimulus. The hamsters in the control groups were also placed in their initially nonpreferred compartments but were not given the stimulus. This group served to demonstrate that any change in preference or difference score across tests seen in the experimental group was not due to chance or habituation during conditioning. Twenty-four hours after the last conditioning session, hamsters 58 were tested for their place preference following the same procedure used for the pretest. Pretest, conditioning sessions, and test all occurred at the same time of day (+/- an hour) for each hamster. VS were used as a stimulus in the first experiment, and palatable food and cocaine were used as stimuli in two other experiments. Experiment 1a: CPP for VS. To test for a CPP for VS, 22 sexually naïve adult and 18 juvenile hamsters were assigned to control and experimental groups, n=9-11. In order to reduce the number of cohorts required and prevent exposing control animals to the smell of the stimuli, control animals were housed in a separate but similar room in which the dark phase began at 8:00am, and testing at 9:00am. Ten total conditioning sessions occurred, including 5 no-stimulus and 5 stimulus-paired sessions. Including the pretest and test, the experiment took place over 12 consecutive days, from P20 to 31 for juvenile animals and P63-69 to 74-80 for adult animals. An hour before use, VS were collected from 30 females and mixed together to total approximately 500µl. Approximately 15 µl of VS were applied to water-moistened cotton gauze packed into a 2-ml Eppendorf tube, one tube for each male. Immediately before testing, the tube was placed out of reach from the male at the top of the back wall in the initially nonpreferred compartment in VS-paired conditioning sessions for the VS group. Empty Eppendorf tubes were used for the control group in all conditioning sessions and for the VS group in the no-stimulus conditioning sessions. To ensure exposure to nonvolatile components of VS, the remaining ~200 µl of VS were mixed with 1.5 ml of mineral oil, and approximately 20 µl of this mixture was applied with a metal spatula directly onto the nose of hamsters in the VS group immediately before being placed in the VS-paired compartment. Clean oil was applied to the nose of hamsters in the control group for all 59 conditioning sessions and in the VS group for no-stimulus conditioning sessions. One hour after completion of the CPP test, hamsters were euthanized with an overdose of sodium pentobarbital (150 mg/kg, ip) and a terminal blood sample was collected via cardiac puncture for radioimmunoassay of circulating plasma testosterone. Experiment 1b: CPP for palatable food. To test for a CPP for palatable food, 22 juvenile hamsters were assigned to control and experimental groups, n=11. Ten total conditioning sessions occurred, including 5 no-stimulus and 5 stimulus-paired sessions, and the experiment lasted 12 days from P20-31. Approximately 10 pieces of Hoola Fruits cereal (~2.5 g, Our Family Foods, Minneapolis, MN) were scattered on the floor of the initially non-preferred compartment, while none were present in the initially preferred compartment for the experimental group. There was no palatable food placed in either compartment for the control group. Quantity of normal chow and palatable food consumed and body weights were recorded each day. Experiment 1c: CPP for cocaine. To test for a CPP for cocaine, 16 juvenile hamsters were assigned to control and experimental groups, n=8. Six total conditioning sessions occurred, including 3 no-stimulus and 3 stimulus-paired sessions, and the experiment lasted 8 days from P23-30. Hamsters in the experimental group were injected intraperitoneally with cocaine (20mg/kg, Lannett Company, Inc. Philadelphia, PA) immediately before being placed in the initially nonpreferred compartment for stimulus-paired sessions, whereas they received a 0.9% saline vehicle injection before being placed in the initially preferred compartment for no-stimulus sessions. The control group received saline injections before being placed in either compartment in conditioning sessions. 60 Statistical analysis. To assess whether the stimuli (VS, palatable food, or cocaine) induced a CPP, data from the pretests and final tests were used to calculate a preference score, defined as [time in the stimulus-paired compartment/(time in stimuluspaired compartment+time in no-stimulus compartment)], and a difference score, defined as [time in the no-stimulus compartment−time in the stimulus-paired compartment] (Bell et al., 2010). Paired t-tests within each group were used to evaluate the change in preference score and difference score pre- and post-conditioning (Bell et al., 2010). Here and with all other reported analyses, p < 0.05 was considered significant, and all statistical analyses were done with SPSS software (PASW Statistics 18; SPSS: An IBM Company, Chicago, IL). Experiment 2: Neural Responses to VS Experimental Design. 16 juvenile (P28) and 16 adult (P64) hamsters were weighed and randomly assigned to either the VS or control group, n = 8. VS were collected less than two hours before use and delivered via cotton applicator swabs to minimize effects of handling the animals on Fos expression. VS-containing or clean blank swabs were dropped into VS or control hamsters’ home cages, respectively, and behavior was monitored while the hamsters interacted with the swab for one hour. Hamsters were often observed to pick up the swab, chew on it, and place it in their cheek pouches for several minutes at a time. To prevent control hamsters from smelling volatile components of VS, they were given access to blank swabs and then removed from the room for tissue collection prior to giving the VS group the VScontaining swabs. Blank and VS-containing swabs were delivered 1-2 and 3-4 hours 61 after lights off, respectively. One hour after introduction of swab into the cage, hamsters were euthanized with an overdose of sodium pentobarbital (150 mg/kg, ip) and a terminal blood sample was collected via cardiac puncture for radioimmunoassay of circulating plasma testosterone. Hamsters were perfused transcardially with heparinized buffered saline rinse followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 24 hours and stored in 20% sucrose/phosphate buffered saline solution until sectioning. Histological procedures. Brains were sectioned with a cryostat into 4 series of 40 µm thick sections and stored in cryoprotectant at -20°C until histological processing. The first series of sections was mounted onto glass slides, dehydrated with a series of ethanols, and stained with cresyl violet before coverslipping for identification of regions of interest. A second and third series of sections were used to double-label cFos with tyrosine hydroxylase (TH) and Orexin-A (Orx) immunoreactivity, respectively, with freefloating immunohistochemistry. cFos is an immediate early gene used to indicate transcriptional activation (Sheng and Greenberg, 1990, Hughes and Dragunow, 1995), in many studies on sex and VS-induced neural activity. Thus, the use of Fos allows for more facile inter-study comparisons and provides an excellent way to survey general activity patters in many brain regions at once. TH is the rate-limiting enzyme for catecholamine production. Dopamine-β-hydroxylase, the enzyme that converts dopamine to norepinephrine, is absent in the ventral tegmental area in hamsters (Vincent, 1988), thus TH immunoreactivity in the ventral tegmental area was used here to identify dopaminergic cells. Orexin-A is one of two active orexinergic peptides (de Lecea et al., 1998, Sakurai et al., 1998), and, in particular, has been implicated in 62 sexual reward (Muschamp et al., 2007, Di Sebastiano et al., 2011). While both TH and Orx have AP-1 binding sites in their promoters (Hughes and Dragunow, 1995, Chen and Randeva, 2010), conclusions about DA or orexinergic activity upon observations of double-labeling must be made cautiously (Hoffman and Lyo, 2002). Immunohistochemistry work occurred at room temperature unless otherwise noted. Rinses with Trizma buffered saline (TBS, 0.05M, pH = 7.6) occurred initially and between steps, and all antibodies were diluted in 2% of the appropriate serum and 0.3% Triton-X TBS (see Table 3.1). To visualize Fos and TH, residual aldehydes were removed with 0.1% sodium borohydride after the first series of TBS rinses, and endogenous peroxidase activity was quenched with 1% hydrogen peroxide. Tissue was blocked and made permeable with 20% goat serum and 0.3% Triton-X TBS, followed by incubation in the cFos primary antibody for 48 hours at 4°C. Tissue was then incubated consecutively in the Fos secondary antibody and avidin-biotin complex for one hour each. Lastly, sections were reacted for ~2 minutes with 10 mg 3,3’Diaminobenzideine tetrahydrochloride in 50 ml TBS and 45 µl of 30% hydrogen peroxide to produce a dark brown reaction product in the nucleus of Fosimmunoreactive (ir) cells. After rinsing, tissue was again blocked and made permeable and then incubated overnight in TH primary antibody. TH secondary antibody and avidin-biotin complex were then each applied consecutively for 1 hour. Finally, sections were reacted for ~2 minutes with 1 drop of Vector SG enzyme substrate in 7 ml TBS and 50 µl 30% hydrogen peroxide to produce a cytoplasmic blue reaction product in THir cells. To visualize Fos and Orx, a similar immunohistochemistry protocol was used, but with the appropriate reagents (see Table 3.1). 63 Table 3.1. Immunohistochemical reagents Fos/TH Immunohistochemistry Fos Normal goat serum. Pel-Freez Biologicals, Rogers, AR Primary c-Fos (4): rabbit, sc-52. 1:10,000, 0.02µg IgG/ml solution, Santa Cruz Biotech, Santa Cruz, CA Secondary Biotinylated goat anti-rabbit IgG (H+L). 1:500, 3µg IgG/ml solution, Vector Laboratories, Burlingame, CA Serum Normal goat serum. Pel-Freez Biologicals, Rogers, AR Primary Mouse anti-TH monoclonal antibody. 1:2,000, MilliporeChemicon, Billerica, MA Secondary TH Serum Biotinylated goat anti-mouse IgG (H+L). 1:500, 3µg IgG/ml solution, Vector Laboratories, Burlingame, CA Fos/Orx Immunohistochemistry Normal donkey serum. Jackson ImmunoResearch Laboratories, West Grove, PA c-Fos (4): rabbit, sc-52. 1:10,000, 0.02µg IgG/ml solution, Santa Cruz Biotech, Santa Cruz, CA Secondary Biotinylated donkey anti-rabbit IgG (H+L). 1:500, 2.4µg IgG/ml solution, Jackson ImmunoResearch, West Grove, PA Serum Normal horse serum, S-2000. Vector Laboratories, Burlingame, CA Primary Orexin-A (C-19): goat, sc-8070, 1:5,000, 0.04µg IgG/ml solution, Santa Cruz Biotech, Santa Cruz, CA Secondary Orx Serum Primary Fos Biotinylated horse anti-goat IgG (H+L), 1:500, 3µg IgG/ml solution, Vector Laboratories, Burlingame, CA Other reagents Avid :Biotin Complex Peroxidase- Vectastain ABC Kit PK-6100, Vector Laboratories, Burlingame, CA Enzyme Substrate Peroxidase- Vector SG Substrate Kit SK-4700, Vector Laboratories, Burlingame, CA Enzyme Substrate Peroxidase- 3,3’ Diaminobenzidine tetrahydrochloride, SigmaAldrich, St. Louis, MO Primary and secondary antibody deletion control studies were run on separate sections. Non-specific background staining was low or absent in these sections. 64 Tissue sections were mounted onto glass slides and dehydrated with a series of ethanols before coverslipping. Microscopic analysis. Regions of interest included the nucleus accumbens (Acb), medial prefrontal cortex (mPFC), and ventral tegmental area (VTA) because they are primary components of the mesocorticolimbic dopamine circuitry (Fibiger and Phillips, 1988); the lateral hypothalamus (LH) because of its orexinergic cell population (AstonJones et al., 2009); the ventromedial hypothalamus (VMH) because of its role in gating reproductive and defensive behaviors (Choi et al., 2005); and the posterior medial amygdala (MeP) as a positive control region known to express Fos in response to VS in both juvenile and adult male hamsters (Romeo et al., 1998). Regions were subdivided according to the hamster brain atlas (Morin and Wood, 2001), as indicated by previous research demonstrating distinct functional and anatomical characteristics of the subregions (Groenewegen et al., 1999, Bradley and Meisel, 2001, Heidbreder and Groenewegen, 2003, Balfour et al., 2006, Ikemoto, 2007). The mPFC included the anterior cingulate (Cg1), prelimbic (PrL), and infralimbic (IL) subregions; the Acb included the core (AcbC) and medial portion of the shell (AcbSh); the MeP included the dorsal (MePD) and ventral (MePV) subregions; the VMH included medial (VMHM) and lateral (VMHL) portions; and the VTA included interfasicular (IF), paranigral (PN), parabrachial pigmented (PBP), and Tail nuclei (Figure 3.1). The hypothalamic area containing orexinergic cells was subdivided into the dorsomedial hypothalamus and perifornical area (DM/PeF) and lateral hypothalamus (LH), relative to the lateral edge of the fornix because of functional specificity of these two populations of orexinergic neurons (Harris et al., 2005), Figure 3.1). 65 The VTA was further subdivided along its rostrocaudal extent because of previous reports of functional specificity in rats and mice (Olson et al., 2005, Ikemoto, 2007) and a relative lack of region-specific analysis in the hamster. Rostral sections were defined as having TH cells adjacent to the fasciculus retroflexus prior to the onset of the interpeduncular nucleus; caudal sections were defined as having interpeduncular nucleus present prior to the medial lemniscus merging with the cerebral peduncle; tail sections were defined as having a rounded interpeduncular nucleus prior to the oral part of the pontine nuclei (Figure 3.1). Upon completion of microscopic inspection and analysis, similar effects of age and swab exposure were found in the rostral and caudal portions of each subregion; therefore, data from rostral and caudal IF, PN, and PBP sections were combined within subregion for statistical analysis and presentation here. Anatomically matched tissue sections throughout the extent of each ROI (2-5 sections per subregion, depending on size) were selected at 4x objective. In the Acb, Me, and VMH, subregion contours were manually traced bilaterally according to the atlas and cytoarchitecture in Nissl-stained sections and then overlaid onto corresponding immunohistochemistry-treated tissue sections for cell counting. In the mPFC, 600 x 600 µm boxes were placed in the mPFC relative to brain contour and corpus callosum landmarks. In the hypothalamus, boxes were drawn to surround all Orx-ir cells medial or lateral to the lateral edge of the fornix in immunohistochemistry tissue sections. In the VTA, contours were drawn unilaterally in immunohistochemistry tissue sections. 66 A. Prefrontal Cortex (PFC) 3.2 – 2.4 mm from bregma E. Hypothalamus -1.8 – -2.3 mm from bregma Cg1 LH PrL DM/PeF IL B. Nucleus Accumbens (Acb) 2.4 – 1.8 mm from bregma F. Rostral Ventral Tegmental Area (VTA) -3.7 mm from bregma AcbC AcbSh IF PBP PN G. Caudal Ventral Tegmental Area (VTA) -4.0 – -4.3mm from bregma C. Medial Amgydala (Me) -1.5 – -2.3 mm from bregma PN MePV D. Ventromedial hypothalamus (VMH) -1.8 – -2.3 mm from bregma PBP IF MePD H. Tail Ventral Tegmental Area (VTA) -4.6 – -4.9 mm from bregma VMHM Tail VMHL Figure 3.1. Figures and images of brain regions of interest. Representative subregion contours drawn over atlas diagrams (Morin and Wood, 2001) and low-magnification photomicrographs of immunohistochemically-treated tissue sections from VS-exposed 67 adult animals; scale bar is 500µm. For color images, the reader is referred to the electronic version of this dissertation. Cells counts were made within a contour by a single experimenter blind to hamster treatment with an UPlanSApo 40x (0.9NA) objective on double-labeled immunohistochemistry tissue. Cells were considered Fos-ir if they had a distinct nucleus with visible puncta stained dark red-brown and TH- or Orx-ir if the cytoplasm was stained gray-blue. In all regions, single-labeled Fos-ir cells were counted, and measures describing Fos-ir cells do not include double-labeled cells. The number of Fos-ir cells within each subregion contour was divided by the area of that contour to create a measure of cell density within a section. These density data control for any change in subregion area with age, and generally detect similar effects of treatment as do cell count data. In the VTA, single-labeled TH-ir cells and cells double-labeled for both TH and Fos, here called TH/Fos-ir, were counted and used in the same density calculations as the Fos-ir cells. In the LH, single-labeled Orx-ir cells and cells doublelabeled for both Orx and Fos, here called Orx/Fos-ir, were counted within an area defined by the presence of the Orx-ir cells. Therefore number, not density, of Orx-ir and Orx/Fos-ir cells per section are reported here. Measurements from each tissue section were averaged across sections to create one measurement per subregion per hamster. All analyses were performed on an Olympus BX51 microscope under brightfield illumination using Neurolucida (version 7; Microbrightfield, Williston, VT). Statistical Analysis. With data from so many subregions within each hamster, one goal of our statistical approach was to simplify the data and present it at a circuit level by identifying clusters of regions that showed similar patterns of Fos expression 68 across animals. To do so, we used a combination of factor analysis and descriptive correlational analyses to inform our expectations given previous functional and anatomical findings. Factor analysis, with principle axis factoring and a promax rotation, identified two clusters of subregions. Cluster 1 included Cg1, PrL, IL, AcbC, AcbSh, MePD, MePV, IF, PN, PBP, and Tail, and we refer to regions in this cluster as mesocorticolimbic. Cluster 2 included DM/PeF, LH, VMHM, and VMHL, and we refer to regions in this cluster as hypothalamic. Given the potential problems of using factor analysis with a small sample, we computed the correlations among the regions within each cluster as well as between the two clusters. The average within-cluster correlation was 0.34 in the MCL cluster and 0.42 in the hypothalamic cluster, based on 55 and 6 correlations, respectively. These indicate that Fos expression in subregions within the same cluster were consistently correlated with one another. We also examined correlations between regions falling into the two different clusters, and here the average of the 44 between-cluster correlations was 0.05 supporting the idea that Fos responses in these two clusters are relatively independent. Fos-ir cell density was next analyzed with multilevel modeling treating animal as the upper-level sampling unit and brain region as the lower-level sampling unit. In this analysis, the cluster the region belonged to (mesocorticolimbic vs. hypothalamic) was treated as a within-subject variable, and age (juvenile vs. adult) and swab (blank vs. VS) were treated as between-subject independent variables. Multilevel modeling provides a more powerful analysis than a traditional repeated measures analysis of variance (ANOVA) because it allows for analysis even if data from all subregions were 69 unavailable for each hamster (as was the case in two juvenile and one adult hamsters due to poor quality tissue sections). The error structure was modeled to impose the traditional homoscedasticity assumption used in ANOVA. Our hypotheses predicted that swab (blank vs. VS) will differentially affect Fos expression in adults and juvenile animals in some subregions. Therefore we also tested a set of planned comparisons within each subregion, comparing swab vs. blank groups within juvenile and adult animals separately. A different statistical approach was required when analyzing densities of TH-ir and TH/Fos-ir cells and numbers of Orx-ir and Orx/Fos-ir cells.because 1) they were only quantified in one region per animal and 2) we were interested in both age and swab effects. Therefore, two way ANOVAs were used to analyze the effects of age (juvenile vs. adult) and swab (blank vs. VS) on these variables. Plasma testosterone measures. Duplicate 50 µl samples of plasma testosterone were analyzed within a single assay using the Coat-A-Count Total T Kit (Diagnostic Products, Los Angeles, CA). The minimum detectable concentration was 0.1ng/ml. The intra-assay coefficient of variation was 6.4% and 6.7% for Experiments 1 and 2, respectively. Two way ANOVA (age x swab) was used to analyze plasma testosterone concentrations between groups. 70 Results Experiment 1: CPP for potential rewards Expt 1a. CPP for VS. Adult hamsters showed a CPP for VS (Figure 3.2a). Paired t-tests showed that the preference score increased significantly for the adult VS group (t(10) = 2.74, p < 0.05) but not the control group, which did not receive pairings of VS during conditioning trials (t(9) = .78, NS). Likewise, the difference score decreased significantly for the adult VS group (t(10) = 3.74, p < 0.01), but not controls (t(9) = .46, NS). On the other hand, juvenile hamsters did not show a CPP for VS (Figure 3.2b). In juveniles, paired t-tests showed that neither the preference scores for the VS group (t(8) = -1.08, NS) and control group (t(8) = 0.19, NS), nor the differences score for the VS group (t(8) = 1.82, NS) and control group (t(8) = -0.53, NS) changed between pretest and test. Expt 1b. CPP for palatable food. Juvenile hamsters showed a CPP for palatable food (Figure 3.2c). Paired t-tests showed that the preference score increased significantly for the palatable food group (t(10) = -2.275, p < 0.05) but not the controls (t(9) = -1.39, NS), and the difference score decreased significantly for the palatable food group (t(10) = 3.145, p < 0.01) but not the controls (t(9) = 1.99, NS). Exposure to palatable food did not affect chow intake or body weight (data not shown). Expt. 1c. CPP for cocaine. Juvenile hamsters showed a CPP for cocaine (Figure 3.2d). Paired t-tests showed that the preference score increased significantly for the cocaine group (t(7) = -3.38, p < 0.01) but not the control group (t(7) = -1.67, NS), just as the difference score decreased significantly for the cocaine group (t(7) = 3.48, p < 0.01) but not the control group (t(7) = 1.60, NS). 71 Preference Score Difference Score A. Adult Vaginal Secretions * 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 200 100 0 -100 200 100 0 -100 * Preference Score C. Juvenile Palatable Food 0.6 B. Juvenile Vaginal Secretions D. Juvenile Cocaine * Pretest Test * 0.6 0.4 0.2 0.2 0.0 0.0 200 100 0 -100 200 100 0 -100 Difference Score 0.4 * Control Exper. * Control Exper. Figure 3.2. Preference and difference scores for control and experimental groups when tested for a CPP for VS (A and B), palatable food (C), and cocaine (D). Adult males showed a CPP for VS, whereas juvenile males did not; however, juvenile males did show a CPP for palatable food and cocaine. * indicates a significant difference between pretest and test within a group, p < 0.05 with paired t-test. 72 Experiment 2: Neural Responses to VS Fos-ir. Multilevel modeling revealed a main effect of cluster (F(1,429) = 13.86, p < 0.01), but no main effect of age or swab on Fos-ir cell density (Figure 3.3). This main effect of cluster was qualified by an interaction between cluster and swab (F(1,429) = 10.53, p < 0.01), such that the effect of swab varied depending on the cluster (Figure 3.3). Follow-up multilevel modeling, analyzing Cluster 1 and 2 separately, indicated an increase in Fos-ir cell density in response to VS in the mesocorticolimbic cluster (F(1,30) = 20.366, p < 0.01), but no effect of swab in the hypothalamic cluster (F(1,28) = 2.41, Number of single Fos-ir cells / mm2 NS). A. Cluster 1 500 400 300 200 100 0 # B. Cluster 2 Blank VS 400 300 200 100 Juvenile Adult 0 Juvenile Adult Figure 3.3. Fos-ir cell density in mesocorticolimbic (A) and hypothalamic (B) clusters. There was a greater density of Fos-ir cell in VS exposed hamsters compared to blank hamsters in the mesocorticolimbic but not hypothalamic clusters. # indicates a main effect of swab, p < 0.01 with multilevel modeling analysis. 73 Figure 3.4. Images of Fos/TH immunohistochemistry. High magnification photomicrographs of representative Fos- (brown nuclei) and TH- (blue cytoplasm) immunoreactive tissue sections from juvenile and adult animals exposed either blank or VS swabs in the AcbC, MePD, and IF. Scale bar is 50µm. 74 Planned contrasts were performed to analyze differences in Fos-ir cell density between blank and VS-exposed animals within an age for each ROI, because the a priori hypotheses predicted that adult and juvenile hamsters would show different responses to VS. Within the mesocorticolimbic cluster, in both juvenile and adult hamsters, VS elicited an increase in Fos-ir cell density in the MePD (t(26) =5.33, p < 0.01 and t(26) = 6.61, p < 0.01, respectively; Figure 3.4 and 3.5C), MePV (t(26) =5.49, p < 0.01 and t(26) = 5.06, p < 0.01, respectively; Figure 3.5C), and PN (t(28) =2.16, p < 0.05 and t(28) = 2.490, p < 0.05, respectively; Figure 3.5D). In contrast, the Fos response to VS in other mesocorticolimbic cluster subregions between adult and juvenile responses diverged. Adult, but not juvenile, hamsters showed greater Fos-ir cell density when exposed to VS compared to blank swabs in the IL (t(26) = 2.26, p = 0.03 and t(26) = 1.35, NS, respectively, Figure 3.5A), AcbC (t(26) = 2.33, p = 0.03 and t(26) = 0.78, NS, respectively, Figure 3.4 and 3.5B), IF (t(28) = 4.61, p < 0.01 and t(28) = 1.746, NS, respectively, Figure 3.4 and 3.5D), and PBP (t(28) = 3.56, p < 0.01 and t(28) = 1.53, NS, respectively, Figure 3.5D). VS did not elicit a Fos response in either juvenile or adult hamsters in the remaining mesocorticolimbic cluster subregions, which included Cg1, PrL, AcbSh, and VTA Tail (Figure 3.5). VS did not evoke a Fos response in any hypothalamic cluster subregions in either age group, as indicated by similar Fos-ir cell densities in the blank- and VS-exposed groups in both ages (Figure 3.6). 75 A. Prefrontal Cortex Anterior Cingulate Prelimbic Infralimbic Number of single Fos-ir cells / mm 600 600 400 2 600 400 400 200 200 200 0 0 Juvenile Adult B. Nucleus Accumbens Core 600 600 * 400 200 0 Shell 400 0 Juvenile Adult D. Ventral Tegmental Area Interfascicular Paranigral 1500 250 * * * 200 1000 500 0 Juvenile Adult 150 100 50 0 Blank VS Juvenile Adult C. Medial Amygdala Posterodorsal 200 Juvenile Adult 0 Juvenile Adult * Juvenile Adult 800 600 400 200 0 * * Juvenile Adult 800 600 400 200 0 * Juvenile Adult Juvenile Adult Tail Parabrachial 250 200 150 100 50 0 Posteroventral * * 250 200 150 100 50 0 Juvenile Adult Figure 3.5. Single-labeled Fos-ir cell density in mesocorticolimbic cluster regions, PFC (A), Acb (B), MeP(C), and VTA (D). There was a greater density of Fos-ir cells in animals exposed to VS swabs compared to those exposed to blank swabs in the MeP and PN in both juveniles and adults, and in the AcbC, IL, and IF in only adults. * indicates difference between Blank and VS swab with planned contrasts within an age group, p < 0.05. Note differences in y-axis in D. 76 2 Number of single Fos-ir cells / mm A. Hypothalamus Dorsomedial/ Lateral Perifornical 400 400 300 300 200 200 100 100 0 0 B. Ventromedial Hypothalamus Medial Lateral 400 400 300 300 200 200 100 100 0 0 Juvenile Juvenile Adult Blank VS Adult Figure 3.6. Fos-ir cell density in hypothalamic cluster regions, DM/PeF and LH (A) and VMH (B). No significant differences between blank- and VS-exposed groups were observed in either age with planned contrasts within an age. TH-ir and TH/Fos-ir cells. The densities of TH-ir and TH/Fos-ir cells were calculated for VTA and analyzed by two-way ANOVA. In IF, a main effect of age was observed on the density of TH/Fos-ir cells (F(1,28) =88.246, p < 0.01, Figure 3.7a). Specifically, adults showed greater density of double-labeled cells independent of swab exposure. No effect of age was observed on density of TH-ir cells, and no significant effects of swab or age x swab interaction was observed on TH-related measures in IF. In PN and PBP, a main effect of swab was observed on the density of TH/Fos-ir cells (F(1,28) = 12.51, p < 0.01, Figure 3.7b and F(1,28) = 23.63, p < 0.01, Figure 3.7c, respectively), such that hamsters exposed to VS expressed a greater density of double77 labeled cells compared to those exposed to a blank swab. No effect of swab was observed on density of TH-ir cells, and no effect of age or age x swab interaction was observed on any TH-related measure in PN or PBP. In Tail, a main effect of age was observed on TH-ir cell density (F(1,28) = 4.524, p < 0.05), such that juvenile hamsters expressed a greater TH-ir cell density than adults, regardless of swab condition (Figure 3.7d). No effect of age was observed on density of TH/Fos-ir cells, and no effect of swab or age x swab interaction was observed on any Number of double Number of single TH/Fos-ir cells/mm2 TH-ir cells/mm2 TH-related measure in Tail. A. Interfascicular B. Paranigral C. Parabrachial D. Tail 2000 1500 1000 500 0 1500 1500 1500 1000 1000 1000 500 500 500 0 0 Blank VS 0 500 400 300 200 100 0 + Juvenile Adult 100 80 60 40 20 0 # Juvenile Adult # 10 8 6 4 2 0 Juvenile Adult 10 8 6 4 2 0 + Juvenile Adult Figure 3.7. TH-ir measures in IF (A), PN (B), PBP (C) and Tail (D) VTA. First row shows number of single-labeled TH-ir cells and second row shows number of double-labeled TH/Fos-ir cells. In IF and Tail, adults and juveniles differ in immunoreactivity, independent of swab exposure; in PN and PBP, both adults and juveniles show greater immunoreactivity when exposed to a VS swab compared to a blank swab. + indicates main effect of age, # indicates main effect of swab, p < 0.05 with a two-way ANOVA. Note different y axis scales in throughout. 78 Orx-ir and Orx/Fos-ir cells. The number of Orx-ir cells and Orx/Fos-ir cells were determined in the LH and analyzed by two-way ANOVA (Figure 3.8). A main effect of age was observed on number of Orx-ir cells in both LHM (F(1,25) = 35.80, p < 0.01) and LHL (F(1,25)=17.79, p < 0.01), such that juvenile hamsters expressed a greater number of Orx-ir cells than adults, independent of swab condition. There was also a main effect of age on the number (F(1,25) = 7.12, p = 0.01) of Orx/Fos-ir cells in LHM, such that adults expressed greater levels than juvenile hamsters on both measures. There was no effect of age on number of Orx/Fos-ir cells in the LHL, nor was there an effect of Number of double Orx/Fos-ir cells Number of single Orx-ir cells swab or age x swab interaction on any measure in LHM and LHL. A. Dorsomedial Perifornical + 100 80 60 40 20 0 + B. Lateral Hypothalamus 100 80 60 40 20 0 60 40 20 + 60 40 Blank VS 20 0 Juvenile Adult 0 Juvenile Adult Figure 3.8. Orexin measures in the DM/PeF (A) and LH (B). First row shows number of single-labeled orexin cells and second row shows number of double-labeled Orx/Fos-ir cells. Adults and juveniles show differences in immunoreactivity, independent of swab exposure. + indicates an effect of age, p < 0.01 with a two-way ANOVA. 79 Plasma testosterone concentration. Plasma testosterone measures revealed a main effect of age in both Expt 1a (F(1,35) = 30.164, p < 0.01) and Expt 2 (F(1,26) = 40.52, p < 0.01), such that adult hamsters had greater testosterone concentrations than juvenile hamsters (Figure 3.9). In addition in Exp 2, a main effect of swab was observed (F(1,26) = 5.16, p = 0.03), in which hamsters exposed to VS had greater testosterone concentrations than those exposed to blank swabs. This main effect appears to be driven solely by an increase in testosterone in VS-exposed adults, although no statistically significant age x swab interaction was detected. Plasma testosterone (ng/ml) A Control Experimental 1.5 Blank VS B + 1.5 + 1.0 1.0 0.5 # 0.5 0.0 Juvenile Adult 0.0 Juvenile Adult Figure 3.9. Plasma testosterone concentrations from animals in Exp 1a (CPP for VS, A) and Exp 2 (neural responses to VS, B). Adults have higher concentrations of testosterone than do juveniles in both experiments, and particularly when exposed to VS in Exp 2. + indicates a main effect of age, p < 0.01. # indicates a main effect of swab, p < 0.05; with a two-way ANOVA. 80 Discussion This report is the first demonstration that adolescent maturation of social information processing includes a transformation of a species-specific, socially-relevant sensory signal from a neutral stimulus to an unconditioned reward in the absence of social experience. This perceptual shift is accompanied by a gain in the ability of the social stimulus to activate midbrain, accumbens, and prefrontal components of the mesocorticolimbic reward pathway, indicating that these particular regions are recruited to mediate the adolescent gain in the perception of VS as rewarding (Figure 3.10). No Fos response to VS Cg mPFC Prl Adult-specific Fos response to VS IL Acb Shell Fos response to VS Core VMH M L VTA IF PN DM LH PeF PBP Tail MeP Dorsal Ventral Figure 3.10. Schematic diagram of adolescent changes in VS-induced Fos responses (indicated by shading). VS activated the posterior medial amygdala and paranigral VTA neurons similarly in juvenile and adult male hamsters, but activated non-dopaminergic interfascicular and parabrachial VTA neurons, nucleus accumbens core, and infralimbic medial prefrontal cortex only in adults (bold outline). This pattern of selective neural activation in adulthood correlates with the ability of VS to serve as an unconditioned 81 stimulus for reward in adult, but not juvenile males, indicating recruitment of these specific cell groups in the adolescent development of social reward. Adolescent gain in the positive valence of VS Juvenile male hamsters failed to show a CPP for VS. However, they did show a CPP to both palatable food and cocaine, demonstrating a pre-adolescent ability to show place preferences for both natural and pharmacological rewards, and consistent with previous reports that peri-adolescent rodents form a CPP to these types of stimuli (Tzschentke, 2007). Adult males did form a CPP for VS, leading to the conclusion that adult, but not prepubertal, male hamsters perceive VS as rewarding. These results provide strong evidence that in the absence of sexual experience, a species-specific social stimulus that is a relatively weak reward or neutral in valence to juveniles becomes a potent unconditioned reward as a consequence of adolescent maturation. This report extends earlier studies on the development of hamsters’ attraction to VS, where adults, but not juveniles, spend significantly more time investigating VS than control stimuli. Preferences for VS are present only after males reach 40 days of age, by which time circulating levels of testosterone are elevated as a result of puberty onset (Johnston and Coplin, 1979). Whether or not elevated testosterone levels influence the perception of VS as rewarding is an open and testable question. In either case, the transformation of VS as a rewarding social stimulus during adolescence is likely critical for successful social interactions in adulthood. 82 Adolescent maturation of VS-induced neural activation patterns The adolescent gain of rewarding properties of VS was correlated with different patterns of VS-induced neural activation within the mesocorticolimbic reward circuitry in adults and juveniles. Factor analysis of Fos expression in the fifteen brain areas analyzed in this study identified two functionally related clusters of cell groups. One cluster included the MeP and members of a complex network of limbic, tegmental, and cortical projections that coordinate reward, incentive motivation and adaptive behavior (reviewed in (Berridge and Robinson, 1998, Ikemoto and Panksepp, 1999, Wise, 2004). This cluster was characterized by neural responsiveness to VS. The second cluster included the hypothalamic subregions, and was characterized by an absence of responsiveness to VS. Thus, developmental dynamics within the mesocorticolimbic cluster appear to underlie the developmental gain in positive valence of VS. The mesocorticolimbic reward system includes extensive dopaminergic and nondopaminergic projections from the VTA to the Acb, mPFC, and MeP, all of which are complexly and reciprocally connected via recurrent circuits (Swanson, 1982a, Oades and Halliday, 1987, Thompson and Swanson, 2010). In rodents, the flow of social chemosensory information to this circuit begins with direct projections from the main and accessory olfactory bulbs to the MeP, which integrates sensory information with the internal hormonal milieu for initial evaluation of the social stimulus (Wood and Newman, 1995a). This first pass evaluation can then be relayed either directly or via preoptic and hypothalamic cell groups to the VTA (Phillipson, 1979, Kevetter and Winans, 1981a, Coolen and Wood, 1998, Geisler and Zahm, 2005). Placing our data within the framework of this circuitry, we propose that VS acquires positive valence through 83 experience-independent alterations in reward circuit responses to initial evaluation of a social stimulus by the amygdala. We base this hypothesis first on the observation that early stage evaluation of VS by the MeP appears to be in place in juveniles and similar to that of adults, because VS elicited similar Fos responses in the amygdala and one of the downstream areas, the VTA PN. Subsequently, over the course of adolescence and in the absence of social experience, VS stimulation comes to engage the IF and PBP nuclei in the VTA, IL of the mPFC, and core of the Acb. This observation suggests that the responses of IF, PBB, IL, and AcbC to evaluative transmissions from the amygdala are altered by developmentally programmed maturational changes, thus associating these cell groups with a positive valence of VS in adulthood. The neurobiological mechanisms that produce the adult-typical behavioral and neural responses to VS are unknown, but our results offer some testable hypotheses. First, it is important to note that within VTA, the populations of cells that were activated by VS only in adulthood were not dopaminergic, and therefore are likely GABAergic projection- or inter-neurons (Van Bockstaele and Pickel, 1995, Carr and Sesack, 2000a, Olson and Nestler, 2007, Ferreira et al., 2008). In fact, dopaminergic (TH-ir) cells in PN and PBP were activated by VS in both juveniles and adults, indicating that these cell groups are involved in social information processing of VS, but in and of themselves do not encode whether VS is rewarding or not. However, dopamine release in Acb is associated with sexual behavior and reward (Wenkstern et al., 1993, Meisel et al., 1996, Becker et al., 2001b, Robinson et al., 2001). Thus, if VS-induced Fos expression in THir VTA neurons is a proxy for dopamine release in corticolimbic regions, then our results suggest that non-dopaminergic VTA projection neurons, activated by VS only in 84 adulthood, may modulate Acb and/or mPFC responses to dopamine release differently in juveniles and adults On the other hand, Fos expression in TH-ir neurons may not reflect a change in dopamine synthesis or release, in which case differential modulation of VTA dopamine neurons by VTA interneurons may underlie the ability of adults, but not juveniles, to form a CPP to VS. The above hypotheses do not preclude the possibility that age-related changes at the accumbens and mPFC projection sites of these dopaminergic neurons may also contribute to the observed pattern of Fos expression. Indeed, a number of adolescent changes in dopaminergic circuitry have been documented, including increased dopamine innervation of the PFC, dopamine synaptic dynamics in the Acb, dopamine receptor expression in the Acb and PFC, and dopamine modulation of PFC GABAergic interneurons (reviewed in (Doremus-Fitzwater et al., 2010, Wahlstrom et al., 2010b). It is also important to note that this corticolimbic restructuring occurs concomitantly with hormonal changes during puberty, and further study is needed to determine the role of gonadal hormones in adolescent maturation of social information processing (Kuhn et al., 2010). The apparent lack of involvement of the hypothalamic cluster cell groups in mediating adolescent change in social information processing is surprising, given their roles in expression of social behaviors and reward. The VMH is involved in sexual behavior (Harding and McGinnis, 2005) and shows increased Fos expression in response to estrous odors in adult male rats (Kippin et al., 2003). However, the rats in this study were sexually experienced, whereas hamster in the current study were sexually naïve, suggesting that a VMH response to estrous odors may be conditioned 85 as a result of previous experience. Hypothalamic orexin is involved in expression of sexual behavior and reward (Muschamp et al., 2007, Di Sebastiano et al., 2011) but the finding that orexinergic neurons were not responsive to VS suggests that the rewarding value of VS is somehow distinct from general sexual reward. Adolescent maturation of mesolimbic dopamine and orexin The number of single-labeled Tail VTA TH-ir and Orx-ir neurons was greater in juveniles than in adults. These results are somewhat difficult to interpret because a reduction in cytoplasmic immunoreactivity could be indicative of either reduced neuropeptide expression or reduced cytoplasmic levels of neuropeptide secondary to enhanced neuropeptide release. The current study also found that, compared with juveniles and independently of VS exposure, adults had greater numbers of Fosexpressing TH-ir and Orx-ir cells in Tail VTA and DM/PeF, respectively. These results may be indicative of heightened vigilance or sensitivity to non-specific stimuli in adulthood (e.g., a clean cotton swab in this study), as both dopamine and DM/PeF orexin have been implicated in general arousal as reviewed in (Harris and Aston-Jones, 2006, Ikemoto, 2007, Boutrel et al., 2010). Conclusion Previous studies have documented adolescent changes in the rewarding properties of drugs of abuse in animals, but less attention has been paid to natural or social rewards (Doremus-Fitzwater et al., 2010). The present study demonstrates an experience-independent gain in the unconditioned rewarding value of a social stimulus 86 over the course of adolescent development, and provides a neuroanatomical basis for the hypothesis that maturational changes within the mesocorticolimbic system mediate this shift in behavioral responses to VS. Because maturation of social information processing is a decisive component of mammalian adolescence, and perturbations in this aspect of development are associated with maladaptive behaviors and certain mental illnesses associated with adolescence, further research into the mechanisms by which social stimuli gain rewarding properties during this critical period of development is warranted. 87 Chapter 4: Adolescent maturation of mesocorticolimbic responses to a rewarding social cue is gonadal hormone independent Introduction The perception of and responses to social cues mature during puberty and adolescence. In particular, interactions with peers become more salient and gain sexual connotations (Spear, 2000, Forbes and Dahl, 2010). Thus, the sources of social reward evolve during the juvenile-to-adult transition, but little is known about the underlying neurobiological mechanisms of this vital aspect of adolescence. To address this question, we have studied developmental changes in endocrine, neural, and behavioral responses of sexually naïve male Syrian hamsters to female hamster vaginal secretions (VS) (Chapter 3 (Romeo et al., 1998). VS signal female receptivity, and must be detected and neurally integrated with internal hormonal information by the male for a successful sexual interaction to occur (Wood and Newman, 1995b, Wood and Coolen, 1997). Importantly, adult, but not juvenile, hamsters are attracted to, and form a conditioned place preference (CPP) for, VS (Johnston and Coplin, 1979) and Chapter 3), demonstrating a shift toward rewarding evaluation of female pheromones contained in VS during reproductive maturation. Sexual reward is often associated with dopaminergic action within the mesocorticolimbic reward circuit (Meisel et al., 1996, Becker et al., 2001b), and this circuit is remodeled during puberty and adolescence (Doremus-Fitzwater et al., 2010, Wahlstrom et al., 2010a). Therefore, the present study investigated the effects of adolescent development and gonadal hormone manipulation on neural responses to VS within the reward circuit. 88 Immediate early gene expression, specifically Fos immunoreactivity, has been used as a proxy for neural activity to reveal neural correlates of male hamster behavioral responses to VS. Initial studies focused on hormone-sensitive brain regions previously implicated in chemosensory responses to social cues, including the bed nucleus of the stria terminalis (BNST), magnocellular region of the medial preoptic nucleus (MPNmag), and the posterior medial amygdala (MeP) (Fiber et al., 1993, Kollack-Walker and Newman, 1997, Wood, 1997). Both gonad-intact adult and juvenile hamsters express more Fos in all of these regions after exposure to VS compared with unexposed age-matched controls (Romeo et al., 1998). However, more recently, we have found that juvenile and adult responses to VS diverge in the mesocorticolimbic circuit (Chapter 3). In Chapter 3, VS again elicited a Fos response in the MeP of both juvenile and adult hamsters, and also in ventral tegmental area (VTA) dopamineproducing neurons. However, in non-dopamine VTA cells, nucleus accumbens (Acb) core, and the infralimbic region of the medial prefrontal cortex (mPFC), VS elicited a Fos response only in adults (Chapter 3). These data suggest that during adolescent development, VS acquires positive valence through alterations in reward circuit processing of sensory information relayed from the medial amygdala. The increase in gonadal hormone secretion that occurs during puberty modulates behavioral responses to VS. While testosterone treatment is sufficient for juveniles to become attracted to VS, it is not sufficient for juveniles to perform adult-like levels of sexual behaviors with a receptive female (Johnston and Coplin, 1979, Meek et al., 1997). Testosterone also affects a range of dopamine-modulated behaviors (Hull et al., 1995, Putnam et al., 2001, Kritzer et al., 2007). These behavioral changes may be 89 mediated by gonadal hormone-induced alterations in the mesocorticolimbic system. For example, in adult rats, gonadectomy results in both short- and long-term effects on basal extracellular dopamine and glutamate-induced dopamine in the mPFC (Aubele and Kritzer, 2011b, a). On the other hand, gonadal hormone-independent changes in dopamine receptor expression in the rat accumbens and mPFC have been observed across adolescence (Andersen et al., 2002). Therefore, adult-like Fos responses to VS may develop as the result of pubertal testicular hormonal influences or developmentally programmed hormone-independent changes within the mesocorticolimbic circuit. The goal of the present study is to determine if treating juvenile hamsters with testosterone results in an adult-like pattern of neural activation within the reward circuit in response to VS. Methods Animals Sexually naïve male Syrian hamsters (Mesocricetus auratus) were obtained from Harlan Laboratories (Madison, WI) and singly housed in clear polycarbonate cages (30.5 x 10.2 x 20.3 cm) in temperature- and humidity-controlled vivaria with a shifted light:dark cycle (14:10, dark phase began at 2:00pm). They were provided with cotton nestlet enrichment and ad libitum access to food (Teklad Rodent diet 8640, Harlan, Madison, WI) and water. Juveniles arrived on postnatal day 18 (P18) or P19 and adults on P4956. Ten-month old adult female hamsters were housed under similar conditions in separate vivaria and used as the source of VS. All experiments were conducted under <4 lux red light 1-5 hours into the dark phase. Hamsters were treated in accordance 90 with the National Institute of Health Guide for Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. Experimental design The experiment used a 2 x 2 x 2 x 2 factorial design, with age (juvenile or adult), hormone status (testosterone or blank capsule treated), stimulus exposure (VS or clean oil), and brain region as independent variables. Because of the large number of animals, stimulus exposure and tissue collection were performed in cohorts on two consecutive days, with all groups represented evenly each day. Two or three days after arrival, on P21 (juveniles) and P51-58 (adults), hamsters were gonadectomized and received subcutaneous implants of two blank or testosterone-containing silastic capsules (one 7 mm and one 15 mm of testosterone, sealed on each end with 4mm of silastic adhesive; inner diameter 1.98 mm; outer diameter 3.18 mm). One week after capsule implants, on P28 (juveniles) and P58-66 (adults), half of each age/hormone group was randomly assigned to be exposed to either clean or VS-containing mineral oil. Hamsters were weighed and moved into a behavior testing room 3 hours prior to exposure. Stimulus exposure Thirty female hamsters were ovariectomized on P70 and used as stimulus animals in other experiments for seven months before hormone administration and collection of VS for use in the two exposure days in this study. They were injected subcutaneously with 91 10 µg estradiol benzoate and 500 µg progesterone in sesame oil 52 and 4 hours, respectively, prior to collection of VS. VS were collected by gentle vaginal palpation and mixed together (total of 0.5ml VS) and stored at -20° for two weeks prior to C thawing and mixing with 1.5 ml of mineral oil one hour prior to exposure. Clean mineral oil was used as the control for VS exposure. To ensure equivalent exposure to nonvolatile components of VS across groups, approximately 20 µl of this mixture were applied with a metal spatula directly onto the nose of hamsters. To prevent control hamsters from smelling volatile components of VS, they were exposed to the clean oil and then removed from the room for tissue collection prior to exposing the VS group to the VS-oil. Clean- and VS-oil were delivered 1-2 and 3-4 hours after lights off, respectively. Tissue collection and analysis Hamsters were euthanized with an overdose of sodium pentobarbital (150 mg/kg, intraperitoneal) sixty minutes after stimulus exposure to allow for peak Fos expression (Sheng and Greenberg, 1990, Hughes and Dragunow, 1995). Flank gland lengths and seminal vesicle weights were recorded as indicators of peripheral testosterone effects (Vandenbergh, 1973). A terminal blood sample was collected via cardiac puncture, held on ice in a heparinized tube for less than four hours, and centrifuged to isolate plasma, which was later used for radioimmunoassay of circulating plasma testosterone. Hamsters were perfused transcardially with heparinized and buffered saline rinse followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde for 12 hours and stored in 20% sucrose/phosphate buffered saline solution until sectioning. 92 Histological procedures. Brains were sectioned with a cryostat into 4 series of 40 µm thick sections and stored in cryoprotectant at -20° unt il histological processing. C One series of sections was used to double-label Fos and tyrosine hydroxylase (TH) immunoreactivity by free-floating immunohistochemistry. TH is the rate-limiting enzyme for catecholamine production; dopamine-β-hydroxylase, the enzyme that converts dopamine to norepinephrine, is absent in the VTA in hamsters (Vincent, 1988), thus TH immunoreactivity in the VTA was used here to identify dopaminergic cells. Immunohistochemistry was performed as in Chapter 3. Briefly, all work occurred at room temperature unless otherwise noted, rinses in Trizma buffered saline (TBS, 0.05M, pH = 7.6) occurred initially and between steps, and all antibodies were diluted in 2% normal goat serum (Pel-Freez Biologicals, Rogers, AR) and 0.3% Triton-X TBS. To visualize Fos and TH, residual aldehydes were removed and endogenous peroxidase activity was quenched before tissue was blocked and made permeable with 20% goat serum and 0.3% Triton-X TBS. Tissue was then incubated in the cFos primary antibody (c-Fos (4): rabbit, sc-52. 1:10,000, 0.02µg IgG/ml solution, Santa Cruz Biotech, Santa Cruz, CA) for 48 hours at 4° cFos secondary antibody (Biotinylated goat anti-rabbit C, IgG (H+L), 1:500, 3µg IgG/ml solution, Vector Laboratories, Burlingame, CA) for one hour, and avidin-biotin complex (Peroxidase- Vectastain ABC Kit PK-6100, Vector) for one hour, consecutively. Then, tissue was reacted with 3,3’-Diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St. Louis, MO) to produce a dark brown reaction product in the nucleus of Fos-immunoreactive (ir) cells. After rinsing, tissue was again blocked and made permeable and then incubated overnight in TH primary antibody (Mouse anti-TH monoclonal antibody, 1:2,000, Millipore-Chemicon, Billerica, MA). TH 93 secondary antibody (Biotinylated goat anti-mouse IgG (H+L), 1:500, 3µg IgG/ml solution, Vector) and avidin-biotin complex (same as above) were then each applied consecutively for 1 hour, and sections were reacted with SG enzyme substrate (Kit SK4700, Vector) to produce a cytoplasmic blue reaction product in TH-ir cells. Primary and secondary antibody deletion control studies were run on separate sections; nonspecific background staining was low or absent in these sections. Tissue sections were mounted onto glass slides and dehydrated with a series of ethanols before coverslipping. Microscopic analysis. Acb, mPFC and VTA were subdivided according to the hamster brain atlas (Morin and Wood, 2001), as indicated by previous research demonstrating distinct functional and anatomical characteristics of the subregions (Groenewegen et al., 1999, Bradley and Meisel, 2001, Heidbreder and Groenewegen, 2003, Balfour et al., 2006, Ikemoto, 2007). The mPFC included the anterior cingulate (Cg1), prelimbic (PrL), and infralimbic (IL) subregions; the Acb included the core (AcbC) and medial portion of the shell (AcbSh); and the VTA included interfasicular (IF), paranigral (PN), and parabrachial pigmented (PBP) nuclei (Figure 4.1). The VTA was further subdivided along its rostro-caudal extent by the presence of the interpeduncular nucleus in caudal sections because of previous reports of rostral/caudal functional differences in rats, mice, and hamsters (Olson et al., 2005, Ikemoto, 2007). Upon completion of microscopic inspection and analysis, virtually all main effects and interactions were observed in the caudal portion of each VTA subregion, and therefore only data from caudal sections are presented here. 94 Anatomically matched tissue sections throughout the extent of the Acb (3 sections), mPFC (3 sections), caudal VTA (cVTA; 2 sections), and posterodorsal medial amygdala (MePd, 1 section, as a control region) were selected at 4x objective and 2 indicated in Figure 4.1. In all regions, 0.064mm rectangular contours were placed bilaterally and consistently according to neuroanatomical landmarks visible in immunohistochemistry treated tissue. Because of the large size of Acb and MePd regions, two boxes were placed in each of those subregions and counts from them were added together within a hemisection. Figure 4.1. Representative subregion contours placed over atlas diagrams (Morin and Wood, 2001). 95 Images were taken within the contour with an UPlanSApo 40x (0.9NA) objective. In the MePd, Acb, and mPFC, only single-labeled Fos-ir cells were observed, and ImageJ (NIH Bethesda, MD) was used to quantify Fos-ir cells. Color images were converted to grayscale, and the image threshold was adjusted to be three standard deviations above and below the minimum and maximum gray values for each image, which consistently detected Fos-ir cells. Particle size was defined as above 200 and below 2000 pixels2, excluding ones on the edge of the image. All particles above threshold were visually confirmed by an experimenter familiar with Fos-ir staining but blind to group assignment. In the VTA, both Fos- and TH-ir cells were present, which precluded ImageJ analysis. Therefore, cells were counted manually using Neurolucida (version 9; Microbrightfield, Williston, VT). Cells were considered Fos-ir if they had a distinct nucleus with visible puncta stained dark red-brown and TH-ir if the cytoplasm was stained gray-blue. All analyses were performed on an Olympus BX51 microscope under brightfield illumination, and images captured with an Optronics MicroFire camera. The number of single-labeled Fos-ir cells (excluding double-labeled cells in the VTA) within each subregion contour was divided by the area of that contour to create a measure of cell density within each hemisection. In the VTA, single-labeled TH-ir cells and cells double-labeled for both TH and Fos, here called TH/Fos-ir, were also counted and converted to density measures as with the Fos-ir cells. In all subregions, density measures were averaged across hemispheres and sections to create one measurement per subregion per hamster. 96 Plasma testosterone measures Plasma testosterone was measured in duplicate 50 µl plasma samples in a single radioimmunoassay using the Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angeles, CA). The minimum detectable concentration was 0.1ng/ml and the intraassay coefficient of variation was 4.7%. Statistical analysis To provide an integrated assessment of independent variable effects on Fos expression across all brain regions studied, multilevel modeling (MLM) was used as in Chapter 3. The model treated hamster as the upper-level sampling unit and brain region as the lower-level sampling unit, with age, hormone status, and stimulus exposure, and brain region as independent variables and Fos-ir cell density as the dependent variable. The error structure was modeled to impose the traditional homoscedasticity assumption used in ANOVA. MLM provides a more powerful analysis than a traditional repeated measures analysis of variance (ANOVA) that uses a within-subject factor, because it integrates non-independence between samples from the same subject in the model. Interactions were followed up by separate MLMs; interactions that involved age were followed up by determining effects within juveniles and adults separately. Region interactions were followed-up by separate analyses for effects of age, hormonal status, or stimulus within a region. However in these analyses, there is just one sample per subject, in which case ANOVA provides the exact same modeling and statistical outcome as MLM. ANOVA was therefore used for analyses within region. Three way ANOVAs were also used to examine effects of age, hormone, and stimulus on TH and 97 Fos/TH-ir in the VTA. P < 0.05 was considered significant, and all statistical analyses were done with SPSS software (PASW Statistics 18; SPSS: An IBM Company, Chicago, IL). Results Fos-ir analysis. Multilevel modeling revealed multiple main effects and two-way interactions that are shown in Table 4.1. Notably, neither main effects of hormone status nor any interactions with hormone status were observed. Of the three two-way interactions observed, only the age x stimulus interaction was independent of brain region and could be followed-up without analyzing brain regions separately. Therefore, the age x stimulus interaction was probed to assess how the Fos response to VS was affected by age, taking all brain regions of interest into account. This analysis revealed an effect of stimulus in adults (F(1,30) = 18.61, p < 0.01) but not juveniles (F(1,28) = 1.55, NS), such that adults, but not juveniles, expressed more Fos-ir cells in response to VS than to clean oil (Figure 4.2). A three-way interaction of age x stimulus x region was also observed and followed-up with separate two-way ANOVAs (age x stimulus) for each region. For every region other than MePD, all main effects were qualified by an age x stimulus interaction (shown in Table 4.1); the appropriate interaction follow-up is described for each region below. 98 Region all 9 MePD AcbC AcbSh Cg1 PrL IL Effect age hormone stimulus region age x hormone age x stimulus age x region hormone x stimulus hormone x region stimulus x region age x hormone x stimulus age x hormone x region age x stimulus x region hormone x stimulus x region age x hormone x stimulus x region age stimulus age x stimulus age stimulus age x stimulus age stimulus age x stimulus age stimulus age x stimulus age stimulus age x stimulus age stimulus age x stimulus F value 63.66 0.55 4.32 360.89 0.30 14.829 49.66 1.87 0.48 6.14 0.03 0.34 6.41 1.74 0.11 5.80 166.19 0.40 1.24 0.01 11.56 0.37 0.15 4.54 1.81 4.28 6.78 0.17 0.76 16.62 0.82 0.85 9.13 df 1, 54 1, 54 1, 54 7, 378 1, 54 1, 54 7, 378 1, 54 7, 378 7, 378 1, 54 7, 378 7, 378 7, 378 7, 378 1,49 1,49 1,49 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 Table 4.1. Complete list of age, hormone, stimulus and region effects with interactions. Salient interactions are followed up and described in text. 99 p value < 0.01 ns ns < 0.01 ns < 0.01 < 0.01 ns ns < 0.01 ns ns < 0.01 ns ns 0.02 <0.01 ns ns ns <0.01 ns ns 0.04 ns 0.04 0.01 ns ns <0.01 ns ns <0.01 Table 4.1 (con’t) age stimulus age x stimulus age stimulus age x stimulus age stimulus age x stimulus IF PN PBP * Number Fos-ir 2 cells / mm 150 56.70 6.62 7.99 26.60 0.04 4.62 1.62 0.01 1.11 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 1, 54 <0.01 0.01 <0.01 <0.01 ns 0.04 ns ns ns Clean VS 100 50 0 Juvenile Adult Figure 4.2. Cluster analysis of Fos responses to VS. Fos-ir cell density using data from all brain regions analyzed as sampling units as in MLM, mean +/- SE, * indicates p < 0.01. An age x stimulus interaction was found, such that only in adults was there a significant increase in the number of Fos-ir cells in response to VS. In the IL and IF (Figure 4.3, left column), the age x stimulus follow-ups revealed an effect of stimulus in adults (F(1,28) = 5.88, p = 0.02 and F(1,30) = 15.51, p < 0.01, respectively), such that adults expressed more Fos-ir cells when in response to VS compared to clean oil. The effect of stimulus in juveniles was not significant in IF, and 100 fell just short of statistical significance in IL (F(1,28) = 0.03, NS and F(1,28) = 3.963, p = 0.056, respectively). In the AcbC and PrL (Figure 4.3, middle column), the age by stimulus follow-ups revealed an effect of stimulus in both adults (F(1,28) = 5.57, p = 0.03 and F(1,28) = 11.62, p = 0.02, respectively), and juveniles (F(1,30) = 6.75, p = 0.01 and F(1,30) = 5.01, p = 0.03, respectively). However, the effects of VS were opposite at the two ages: juveniles expressed fewer Fos-ir cells in response to VS compared to clean oil, but adults expressed more Fos-ir cells in response to VS compared to clean oil. In the PN (Figure 4.3, middle column), no significant effects of stimulus were detected in either age group, however the results follow the same general trend as in AcbC and PrL, with juveniles expressing fewer, and adults more, Fos-ir cells in response to VS. In the AcbSh (Figure 4.3, right column), the age x stimulus follow-up revealed no effect of stimulus in either age group. Therefore, the effect of age (juvenile or adult) was determined in clean oil- and VS- exposed hamsters separately. In clean oilexposed hamsters, an effect of age was observed, F(1,28) = 4.55, p = 0.04, such that juveniles expressed more Fos-ir cells than did adults. In VS-exposed hamsters, no effect of age was detected. In the Cg1 (Figure 4.3, right column), the age x stimulus follow-up revealed an effect of stimulus in juveniles, F(1,28) = 9.90, p < 0.01, such that they expressed fewer Fos-ir cells in response to VS compared to clean oil. No effect of stimulus was detected in adult hamsters. 101 In the MePD (Figure 4.3 left column) and PBP (Figure 4.3, right column), no age x stimulus interactions were observed. A main effect of stimulus was observed in the MePD, such that hamsters expressed more Fos-ir cells in response to VS than to clean oil. A main effect of age was also observed in the MePD, such that juveniles expressed more Fos-ir cells than adult hamsters. Blank Testosterone 30 Posterodorsal (MePd) ME of age, stimulus 40 Accumbens Core (AcbC) * * 30 20 60 Accumbens Shell (AcbSh) + 40 20 10 2 Number Fos-ir cells / mm 20 10 0 0 Infralimbic mPFC (IL) 30 * 30 0 Prelimbic mPFC (Prl) * * Anterior Cingulate mPFC 30 (Cg1) * 20 20 20 10 10 10 0 0 0 Interfascicular VTA (IF) 800 * 200 Paranigral VTA (PN) age x stimulus intx 80 600 150 60 400 100 40 200 50 Parabrachial VTA (PBP) 20 0 Clean VS Juvenile Clean VS Adult 0 Clean VS Juvenile Clean VS Adult 0 Clean VS Juvenile Clean VS Adult Figure 4.3. Fos-ir cell density in all brain regions analyzed, in blank- or testosterone (T)treated juveniles and adults exposed to clean or VS-oil. Mean +/- SE, p < 0.05. In the MePD, main effects of age and stimulus were detected. In all other regions but PB, interactions between age and stimulus were detected. To probe these interactions, 102 effects of stimulus on hamsters within age (*), or effects of age within stimulus (+) were assessed. Interactions occurred either when the effect of stimulus was opposite for the two age groups (AcbC, PrL), when an effect of stimulus was present only in one age (IL, IF, Cg), or when an effect of age was present in only one stimulus group (AcbSh). In the PN, an age x stimulus interaction was detected, but no significant effects of stimulus were found upon follow-up within each age. An interaction was likely detected because the effects of VS were in opposite directions in juveniles and adults. TH-ir and Fos/TH-ir analysis TH-ir. Main effects of age were observed on TH-ir cells in the IF and PN, such that juveniles expressed more TH-ir cells than did adults, F(1,54) = 28.08, p < 0.01 and F(1,54) = 8.23, p < 0.01 respectively (Fig 4.4, top row). A main effect of hormone was also observed on TH-ir cells in the IF, such that blank capsule treated hamsters expressed more TH-ir cells than did T-treated hamsters, F(1,54) = 4.27, p = 0.04. No effects of stimulus or other interactions were observed in any of the three VTA subregions. Fos/TH-ir. Main effects of age were observed on Fos/TH-ir cells in the IF, PN, and PBP such that adults expressed more Fos/TH-ir cells than did juveniles, F(1,54) = 45.96, p < 0.01; F(1,54) = 6.31, p = 0.02; and F(1,54) = 10.68, p < 0.01 respectively (Fig 4.4, bottom row). An age x stimulus interaction was also observed on Fos/TH-ir cells in the PN, F(1,54) = 7.72, p < 0.01. Follow-up revealed an effect of age in VS-exposed 103 hamsters, F(1,30) = 16.28, p < 0.01, but not in clean oil exposed hamsters; when exposed to VS, adults expressed more Fos/TH-ir cells than did juveniles. Number TH-ir 2 cells / mm Number Fos/TH-ir 2 cells / mm Interfascicular (IF) 3000 600 + Blank Testosterone Parabrachial (PBP) Paranigral (PN) + 1500 2000 1500 2000 1000 1000 500 500 0 0 0 1000 + + 150 30 400 100 20 200 50 + 10 0 Clean VS Clean VS Juvenile Adult 0 Clean VS Clean VS Juvenile Adult 0 Clean VS Clean VS Juvenile Adult Figure 4.4. TH- and Fos/TH-ir cell density in the VTA, in blank- or testosterone- treated juveniles and adults exposed to clean or VS-oil. Mean +/- SE, p < 0.05. In the IF and PN, main effects of age were detected (+) on TH-ir cells such that juveniles expressed more than did adults. In the IF, a main effect of hormone was detected on TH-ir cells, such that blank-treated hamsters expressed more than did testosterone-treated. In all three regions, main effects of age were detected (+) on Fos/TH-ir cells, such that adults expressed more than did juveniles. Finally, in the PN, an age x stimulus interaction was detected on Fos/TH-ir cells, such that an effect of age was observed in VS exposed hamsters but not clean oil exposed hamsters. 104 Peripheral and testosterone measures Physiological measures are shown in Table 4.2, and confirm efficacy of testosterone capsules in raising circulating testosterone and increasing flank glands in both ages. Gonadectomy resulted in a decrease in seminal vesicle weight only in adults. Groups of the same age did not differ in body weight. Body Weight (g) Age Juv. Juv. Juv. Juv. Adult Adult Adult Adult Capsule Empty Empty T T Empty Empty T T Stimulus N Blank 7 VS 8 Blank 7 VS 8 Blank 8 VS 8 Blank 8 VS 8 Flank gland length (mm) Seminal Vesicle Weight (g) Mean SD Mean SD Mean 51.03 4.04 3.39 0.82 < 0.01 50.60 4.63 3.30 2.03 < 0.01 50.27 3.03 4.99 1.27 0.03 50.56 5.41 4.64 0.71 0.01 99.28 13.87 6.44 2.21 0.03 96.95 10.57 7.04 1.20 0.08 93.05 8.63 8.41 1.85 0.25 89.34 18.41 7.28 0.86 0.21 SD 0.05 0.04 0.05 0.09 0.27 0.20 Plasma T (ng/ml) Mean <0.10 <0.10 7.76 7.99 <0.10 <0.10 6.19 6.19 SD 0.87 1.19 1.34 1.19 Table 4.2. Peripheral measures of hamsters at time of sacrifice. Mean and standard deviations (SD) of body weight, flank gland length, seminal vesicle weight, and plasma testosterone (T). Adults are heavier, have longer flank glands, heavier seminal vesicles, and less circulating testosterone. Testosterone treated hamsters have longer flank glands, heaver seminal vesicles, and more circulating testosterone. Discussion This study demonstrates that elevating circulating testosterone to adult-like levels in juvenile hamsters is not sufficient to induce adult-like mesocorticolimbic responses to VS. That is, VS elicited a Fos response only in adults, and not in juveniles, whether they were treated with testosterone or not (Figure 4.2). In fact, testosterone treatment 105 did not affect the VS-induced Fos response in adults either. These findings indicate that some developmental process other than the pubertal rise in testosterone accounts for the observed age x stimulus interactions, in which either a VS-induced Fos response occurred in only one age group (as in the infralimbic mPFC, interfascicular VTA, and anterior cingulate mPFC), or the direction of the VS-induced Fos response was in opposite directions in juveniles and adults (as in the accumbens core, prelimbic mPFC, and paranigral VTA). In addition, differences between adults and juveniles in the density of Fos/TH-ir cells only emerged when hamsters were exposed to VS, independent of testosterone treatment, demonstrating that dopaminergic cell responses to VS also mature with age. A one-week period of testosterone treatment, similar to that used here, is sufficient to cause juvenile males to be attracted to VS (Johnston and Coplin, 1979), but not to induce adult-typical sexual behavior (Meek et al., 1997, Schulz and Sisk, 2006). Therefore, the adolescent, but hormone-independent changes in VSinduced Fos expression appear to reflect those observed for sexual behavior, instead of reflecting rewarding effects of VS exposure. Given the involvement of this neural circuitry in linking motivation with motor output (Sesack and Grace, 2009), we propose that the differential Fos responses to VS in adult and juvenile males reflect differences in their drive to perform motor components of sexual behavior. The mesocorticolimbic reward system includes extensive dopaminergic and nondopaminergic projections from the VTA to the Acb, mPFC, and MeP, all of which are complexly and reciprocally connected via recurrent circuits (Swanson, 1982a, Oades and Halliday, 1987, Thompson and Swanson, 2010). In rodents, the MeP is responsible for initial evaluation of a chemosensory social stimulus and integration with hormonal 106 information (Wood and Newman, 1995a), and then relays this information to the VTA, mPFC, and Acb (Phillipson, 1979, Kevetter and Winans, 1981a, Coolen and Wood, 1998, Geisler and Zahm, 2005). Results from this experiment again demonstrate that MeP evaluation of VS in juveniles is similar to that of adults. However, over the course of adolescence and independent of social experience, VS exposure comes to engage non-dopamingeric cells in the IF and PN nuclei in the VTA, and downstream targets in the IL and PrL of the mPFC, and core of the Acb. Therefore, we propose that neural responses to VS mature across adolescence in the mesocorticolimbic system, independently of hormone exposure and sexual experience. Neural mechanisms of adolescent shift in VS response Adolescent development of mesocorticolimbic neural circuitry could explain changes in the pattern of Fos responses to VS across adolescence. Although VS exposure was in general associated with an increase in Fos-ir in adult hamsters, it was unexpectedly associated with reduced constitutive Fos expression in juvenile hamsters throughout the corticolimbic regions. A reduction in Fos in response to a sensory stimulus is uncommon (or at least not often reported), but could be explained mechanistically via specific dopamine receptor activation. D1 receptors are associated with excitatory G proteins and their activation generally causes an increase in Fos expression via activation of the AC/PKA/CREB pathway (Missale et al., 1998). In contrast, D2 receptors are coupled with inhibitory G proteins that have the opposite effect on the intracellular cascade, including intracellular calcium, an important regulator of Fos (Missale et al., 1998, Luo et al., 2011). Synergistic activation of D1 and D2 receptors 107 typically increase Fos-ir within a given brain region, but D2 specific agonists have been observed to reduce Fos expression (Wirtshafter and Asin, 1994, Keefe and Gerfen, 1995)). Likewise, D2 antagonists often induce an increase in Fos expression, which suggests tonic inhibitory effects of DA on D2 receptor activation and Fos expression (Bertran-Gonzalez et al., 2008). Therefore, the reduced constitutive Fos expression in response to VS may result from D2 receptor activation in juveniles and D1/D2 receptor activation in adult animals. There are several possible explanations for D2-shifted activation in juveniles. The lack of increased Fos/TH-ir cells in response to VS in the current study may suggest a lack of burst dopamine cell firing in response to VS. However, this observation does not preclude VS-induced dopamine release in corticolimbic sites, as glutamateric release on to dopaminergic axon terminals has been shown to induce phasic dopamine release (Grace, 1991, David et al., 2005). Release of glutamate in response to VS, either from the amygdala or prefrontal cortex, could therefore induce dopamine release in response to VS. Glutamatergic terminal innervation of the prefrontal cortex and accumbens increases dramatically during adolescence (Cunningham et al., 2002, Brenhouse et al., 2008). Therefore, perhaps glutamate in adult, but not juvenile, hamsters causes a local phasic dopamine release. Previous studies have shown that tonic release of DA at basal activity states can activate highaffinity D2 receptors, while phasic release in response to a stimulus activates both D1 and D2 (Creese et al., 1983, Richfield et al., 1989, Grace, 1991). Thus, perhaps a phasic dopamine release in response to VS in the adult hamsters allows for activation of 108 D1 receptors to increase Fos, while a less robust DA release preferentially activates D2 receptors and causes reduced Fos in juveniles. Additionally, developmental profiles of dopamine receptor expression could explain D2-biased activation in juveniles. Although receptor binding studies have yielded somewhat inconsistent results on developmental profiles of D1 and D2 receptor expression in the striatum and cortex, most find an increase in expression that either peaks at mid-adolescence or continues into adulthood (Andersen et al., 1997b, Andersen et al., 2000, Tarazi and Baldessarini, 2000). Importantly, studies that directly compared D1 and D2 developmental profiles show that the D2:D1 binding ratios are much higher in juveniles than adults in the accumbens (Tarazi and Baldessarini, 2000). This heightened expression of D2 receptors in juvenile animals, paired with sub-adult levels of dopamine and glutamate innervation of the accumbens and cortex (Giorgi et al., 1987, Kalsbeek et al., 1988, Cunningham et al., 2002, Brenhouse et al., 2008, Mathews et al., 2009), would favor expression of D2 receptor activation compared to adults. Voluntary exposure affects neural responses to VS In the previous study, increased Fos expression in response to VS was observed in AcbC, IL mPFC, non-dopamingeric cells in the IF VTA, PN VTA and PBP VTA in adult hamsters (Chapter 3). A similar pattern was observed in the current study, with a few exceptions: an adult response in the PrL was a new observation, and adult Fos responses in the PN and PBP were not present. In addition, both juvenile and adult hamsters also showed Fos/TH-ir responses to VS in the VTA in Chapter 3, and these 109 increases in Fos/TH were not observed in the current study. These inter-study differences may result from methodological differences between the studies. In Chapter 3, immunoreactivity was quantified throughout the regions of interest manually, while in Chapter 4, analyzed smaller rectangular portions of the brain regions. However, these smaller contours either detected similar responses to VS in adult animals (as in the Acb and mPFC), or covered almost the entire extent of the subregion as defined in Chapter 3 (as in the IF and PN). Instead, a more important difference between the studies in Chapter 3 and 4 may be the way in which hamsters were exposed to VS. In the current study, in order to more completely control for equivalent exposure of volatile and non-volatile components of VS across the groups, VS or oil was applied directly to hamsters’ nares, instead of presented on a cotton swab dropped into their home cage as in the previous study. It is possible that more robust VTA responses may be observed when VS exposure is gained voluntarily, as in investigating swabs in Chapter 3, as opposed to involuntarily, as in oil application in the current study. In fact, a similar phenomenon is seen when in response to electrical stimulation of the medial forebrain bundle in male rats. Animals voluntarily seeking self-stimulation showed greater Fos-ir in the VTA than yoked animals; no differences were seen between active and yoked rats in the mPFC and Acb (Hunt and McGregor, 1997). Voluntary exposure may also promote burst firing of dopaminergic VTA cells. Thus, voluntary approach to VS swabs may activate psychological or motor responses not seen in the current study. Therefore, these differential results present an interesting line of questions for future work. 110 Hormone dependent and independent effects of adolescent maturation In general, one week of gonadectomy did not inhibit VS-induced Fos-ir in MePd, NA, mPFC, or VTA in adult hamsters. The lack of an effect in the MePd is not surprising in light of a previous study in which 12 weeks of gonadectomy also failed to block VSinduced Fos-ir in MeP (Fiber and Swann, 1996). However, an effect was seen in a medial preoptic nucleus in that study, demonstrating that responses to VS are hormone sensitive in at least some brain regions. Testosterone has also been previously shown to modulate dopamine-dependent PFC function in male rats, but in a temporally specific pattern: GDX causes a decrease in TH innervation and extracellular dopamine when measured after 4 days of hormone absence, but causes an increase in the same measures after 28 days of hormone absence (Aubele and Kritzer, 2011b). Short-term (10-14 days) castration in the ventral striatum does not affect tissue concentrations of dopamine and DOPAC (Engel et al., 1979, Mitchell and Stewart, 1989), however longterm (28-56 days) gonadectomy generally reduces dopamine and DOPAC concentrations in NA tissue (Alderson and Baum, 1981, Mitchell and Stewart, 1989) but see (Baum et al., 1986). Taken together, it is possible that 1) mesocorticolimbic responses to VS are not modulated by circulating testosterone, as is the case in the MePd, or 2) a longer hormonal absence in adult hamsters could have prevented VS responses in the mPFC and NA. One week of testosterone treatment was selected here to parallel the treatment sufficient to induce CPP responses to VS in juvenile hamsters (Chapter 5), however extending the length of testosterone treatment is an interesting question for future work. 111 Several age-related, VS-independent, changes in Fos expression in VTA dopaminergic cells were observed. The number of Fos/TH-ir cells was greater in adults than juveniles, which may be indicative of heightened vigilance or sensitivity to a range of stimuli in adulthood (mineral oil, in this study). The same effect of age on constitutive Fos expression of dopaminergic cells was observed in gonad intact hamsters (Chapter 3); therefore, this study demonstrates that this change is dependent on adolescent maturation and not circulating hormones. Age also affected the number of singlelabeled VTA TH-ir cells, such that juvenile hamsters expressed more than did adults. This differential labeling could indicate either reduced TH expression within existing cells or reduced TH-expressing cell number in adult hamsters. One mechanism of differential TH expression could be increased dopamine release in adulthood, which then feeds back to reduce TH-expression (as observed in (Stork et al., 1994), or adolescent changes in ion channel activity known to regulate TH-expression (Aumann et al., 2011). An effect of hormone was also observed on TH-ir cells in IF, in that testosteroneexposed hamsters expressed fewer TH-ir cells than did blank-treated hamsters, independent of age. There is some evidence for testosterone suppressing TH expression, as 30 days of gonadectomy causes an increase in the number of TH-ir cells in adult male rats (Johnson et al., 2010). This effect was not seen after 14 days of gonadectomy (McArthur et al., 2007), a time-span that more closely matches 7 days of treatment in this study. However, the temporal pace of responses to hormone removal is often slower than that of responses to hormone replacement (Putnam et al., 2003). Greater numbers of TH-ir cells were previously observed in gonad-intact juveniles 112 compared to adults (Chapter 3), and could be explained by either the developmental or hormonal effects seen here. Conclusion The present study demonstrates that testosterone does not activate adult-typical Fos responses to a rewarding social cue in juvenile male hamsters, indicating that adolescent developmental programming underlies activation of mesocorticolimbic brain regions by female chemosensory cues in adults. These Fos responses may be a neural correlate for motivation or enactment of sexual behavior, which also is not activated by testosterone prior to adolescent development. Normal expression of sexual behaviors is dependent on exposure to testosterone during puberty, as it organizes the brain for adult-typical expression of behavior. Therefore, mesocorticolimbic structures may be a target for these organizational effects of testosterone, and ongoing studies will investigate responses of the Acb to sexual experience in hamsters deprived of pubertal testosterone. As perturbations in adolescent maturation are associated with maladaptive behaviors and certain mental illnesses, these studies are important in expanding our knowledge of this sensitive period of development. 113 Chapter 5: Dopamine mediates testosterone-induced social reward in juvenile male hamsters. Introduction Given the importance of appropriately interpreting social stimuli in successful adult social interactions, determining the neural underpinnings of responses to social cues is essential to understanding social behavior. While social stimuli serve to communicate a range of social information, many salient cues relate to sexual behavior (O'Connell and Hofmann, 2011). Male Syrian hamsters provide a useful model with which to study responses to these cues, as their sexual behavior is dependent on both neural processing of female hamster vaginal secretions (VS) and recent exposure to testosterone (Murphy and Schneider, 1970, Whalen and Debold, 1974, Petrulis, 2009). VS are an unconditioned reward, as sexually-naïve adult male hamsters will form a conditioned place preference (CPP) for them (Bell et al., 2010). Attraction to VS, like the performance of sexual behavior, is dependent on activational effects of testosterone (Gregory and Bishop, 1975). However, it is currently unknown whether the reinforcing value of VS is similarly testosterone-dependent. Therefore, this study seeks to determine the necessity of testicular hormones for VS CPP. An important neural response to chemosensory stimuli and copulation in rodents is the release of dopamine in the medial preoptic area (MPOA) and nucleus accumbens (Acb) (Malmnas, 1976, Mas et al., 1990, Pfaus et al., 1990, Louilot et al., 1991, Damsma et al., 1992, Mitchell and Gratton, 1992, Meisel et al., 1993, Wenkstern et al., 1993, Hull et al., 1995, Schulz et al., 2003). In fact, the MPOA dopamine release in response to sexual interactions requires intact olfactory bulbs in hamsters (Triemstra et 114 al., 2005), further emphasizing the importance of this chemosensory stimuli for sexual responses in this species. Dopamine has been implicated in multiple aspects of sexual reward. For example, systemic administration of haloperidol, a D2 dopamine receptor antagonist, decreases unconditioned motivation for primary female visual, auditory, and chemosensory cues in sexually-naïve male rats, as well as conditioned motivation for olfactory cues previously associated with sexual behavior (Lopez and Ettenberg, 2000, 2002). In addition, formation of conditioned place preference for sexual behavior in female hamsters is blocked by administration of a D2 receptor antagonist (Meisel et al., 1996). However, other studies have found that dopamine receptor activation is not required for CPP for sexual rewards in male rats and mice (Agmo and Berenfeld, 1990, Agustin-Pavon et al., 2007, Ismail et al., 2009). Therefore, this study seeks to determine if dopamine is necessary for CPP to VS in male hamsters. Testosterone regulates dopaminergic circuitry and tone, but with spatial and temporal specificity. Castration causes a decrease in sexual behavior after 2-8 weeks, which coincides with decreases in basal dopamine levels and turnover in the Acb and MPOA (Mitchell and Stewart, 1989). These effects of testosterone on dopaminergic circuitry for the performance on sexual behavior have not been well studied in the Acb; however, testosterone has repeatedly been shown to regulate dopamine in the MPOA. The absence of a precopulatory MPOA dopaminergic response to a stimulus female is predictive of the extinction of copulatory behavior after gonadectomy (Hull et al., 1995). Likewise, the presence of a preoptic dopaminergic response to a stimulus female prior to copulation is predictive of the reinstatement of copulatory behavior upon testosterone replacement in long-term castrated male rats (Putnam et al., 2001). Moreover, sexual 115 behavior can be partially restored in male rats castrated for 3-8 months by systemic and intra-MPOA injections of apomorphine, a dopamine agonist (Scaletta and Hull, 1990). Thus, testosterone may allow for dopaminergic responses to sexual stimuli that are essential for the expression of sexual behavior. Puberty is a time of dramatic change in circulating testicular hormones and mesocorticolimbic maturation (Spear, 2000). Not surprisingly, then, puberty in male Syrian hamsters is coincides with changes in endocrine, neural, and behavioral responses to VS (Meek et al., 1997, Romeo et al., 1998). Juvenile male hamsters do not show adult-typical attraction or CPP to VS (Johnston and Coplin, 1979) and Chapter 3). This behavioral differences between gonad-intact juvenile and adult hamsters are mirrored by their dopaminergic responses to VS. Adult, but not juvenile, hamsters show an increase in dopamine release and metabolism in response to VS in the MPOA (Schulz et al., 2003). Similarly, there are adolescent changes in expression of immediate early gene in response to VS in the dopaminergic mesocorticolimbic system. Adult, but not juvenile hamsters express Fos in response to VS in the ventral tegmental area (VTA), medial prefrontal cortex (mPFC) and Acb in adult but not juvenile hamsters (Chapter 3). However, if juvenile male hamsters are treated with testosterone, they become attracted to VS (Johnston and Coplin, 1979). Given the involvement of testosterone and dopamine in rewarding aspects of sexual behavior, and the changes observed in these endogenous signalers during puberty, I propose that responses to VS will be affected by interactions of the two. This next series of studies tested the effects of testosterone and dopamine manipulations in CPP for VS in adult and juvenile male hamsters. Specifically, the experiments first asked whether testosterone treatment of 116 juvenile males enables formation of a CPP to VS. Finding that this was the case, I then tested the hypothesis that dopamine receptor activation mediates the ability of testosterone to induce rewarding properties of VS in juvenile hamsters. Methods Animals Syrian hamsters (Mesocricetus auratus) were obtained from Harlan Laboratories (Madison, WI) and were housed in temperature- and humidity-controlled vivaria with a light:dark cycle of 14 hr light:10 hr dark, and ad libitum access to food (Teklad Rodent diet 8640, Harlan, Madison, WI) and water. Upon arrival, juvenile males were housed with their male littermates and biological mothers until weaning at P18. Weanling and adult males were singly housed in clear polycarbonate cages (30.5 x 10.2 x 20.3 cm) and were all sexually naïve at the time of study. Sixty adult female hamsters, approximately 12 months old, were housed under similar conditions in separate vivaria and used as the source of VS. Female hamsters were ovariectomized several weeks before hormone administration and collection of VS. They were injected subcutaneously with 10 µg estradiol benzoate and 500 µg progesterone in sesame oil, 52 and 4 hours respectively, prior to collection of VS by gentle vaginal palpation. All experiments were conducted under <4 lux red light 1-5 hours into the dark phase. Hamsters were treated in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, and protocols were approved by the Michigan State University Institutional Animal Care and Use Committee. 117 Surgery and hormone implantation In some experimental groups, hamsters were gonadectomized under isoflurane anesthesia. Bilateral longitudinal scrotal incisions were made, and the testes were removed with a cut distal to ligature or cauterization, in adults and juveniles, respectively. Some groups were also subcutaneously implanted with two blank or testosterone-containing silastic capsules (one 5 mm and one 13 mm of testosterone, Sigma-Aldrich, St. Louis, MO, sealed on each end with 4mm of silastic adhesive; inner diameter 1.98 mm; outer diameter 3.18 mm). These capsules have been used previously in both juveniles and adults to produce adult physiological levels of circulating testosterone (~2-7 ng/ml). Subjects received a subcutaneous injection of ketoprofen analgesic at time of surgery and again 24 hours after. Conditioned place preference (CPP) experimental design Place preference conditioning occurred as described previously (Bell et al., 2010) in an apparatus with one middle compartment and two outer compartments (Med Associates, St. Albans, VT). These outer compartments were designed to allow for compartmentspecific associations: one outer compartment had black walls, scalloped Plexiglas floor, and was scented with 4% acetic acid, while the other had white walls, mesh grid floor, and was scented with pine pellets underneath the floor. To acclimate subjects to handling and novel chambers, male hamsters were placed in glass aquaria in the behavioral testing room for 10 minutes each day, starting two days prior to beginning the CPP regimen. The CPP regimen included a pretest, 10 conditioning sessions, and 118 test, which all occurred at the same time of day (+/- an hour) for each hamster. In order to reduce the number of cohorts required and prevent exposing control animals to the smell of the stimuli, control animals were housed in a separate room in which the dark phase began at 8:00am, and testing at 9:00am. Experimental animals were housed in rooms in which the dark phase began at 2:00pm, and testing at 3:00. A pretest (2 minutes in the middle compartment followed by 15 minutes access to all compartments) was used to determine each hamster's initial compartment preference. The outer compartment in which the hamster spent more time was defined as the initially preferred compartment. A preference score, defined as [time in the initially non-preferred compartment/(time in the initially preferred compartment + time in the initially non-preferred compartment)], and a difference score, defined as [time in the initially-preferred compartment − time in the initially non-preferred compartment] were calculated for each animal (Bell et al., 2010). Hamsters that did not enter each compartment at least 5 times were excluded from further training. To the extent possible, animals were assigned to experimental and control groups so as to equate groups for initial chamber preferences and preference scores, and also to have equal representation of litters in the different groups. Following the pretest, the hamsters received a total of ten 30-minute conditioning sessions in the side compartments, one session per day on consecutive days, alternating five no-stimulus and five stimulus-paired sessions. During the no-stimulus conditioning sessions, hamsters in both the experimental and the control groups were placed in their initially preferred compartments, where they remained alone. During stimulus-paired conditioning sessions, hamsters in the experimental group were placed 119 in the initially nonpreferred compartments with the stimulus. The hamsters in the control groups were also placed in their initially nonpreferred compartments but were not given the stimulus. This group served to demonstrate that any change in preference or difference score across tests seen in the experimental group was not due to chance or habituation during conditioning. The CPP apparatus was cleaned thoroughly with 25% ethanol between each animal, and with 75% ethanol at the end of each conditioning day. In Experiments 1-3, VS were used as the conditioning stimulus. An hour before use, VS were collected from 30 females and mixed together to total approximately 500µl. Approximately 15 µl of VS were applied to water-moistened cotton gauze packed into a 2-ml Eppendorf tube, one tube for each male. Immediately before testing, the tube was placed out of reach from the male at the top of the back wall in the initially nonpreferred compartment in VS-paired conditioning sessions for the VS group. Empty Eppendorf tubes were used for the control group in all conditioning sessions and for the VS group in the no-stimulus conditioning sessions. To ensure exposure to nonvolatile components of VS, the remaining ~200 µl of VS were mixed with 1.5 ml of mineral oil, and approximately 20 µl of this mixture was applied with a metal spatula directly onto the nose of hamsters in the VS group immediately before being placed in the VS-paired compartment. Clean oil was applied to the nose of hamsters in the control group for all conditioning sessions and in the VS group for no-stimulus conditioning sessions. Twenty-four hours after the last conditioning session, hamsters were tested for their place preference following the same procedure used for the pretest. As in the pretest, preference and difference scores were calculated for each animal. One hour 120 after completion of the CPP test, hamsters were euthanized with an overdose of sodium pentobarbital (150 mg/kg, ip) and a terminal blood sample was collected via cardiac puncture for radioimmunoassay of circulating plasma testosterone. Experiment 1: Are testicular hormones necessary for formation of a CPP to VS in adult hamsters? This first experiment tested whether circulating testicular hormones are required for the display of a CPP to VS in adult hamsters. Pilot studies in this laboratory indicated that male hamsters formed a CPP to VS when conditioning began one week after gonadectomy (Bell et al., 2011), suggesting that putative activational effects of testicular hormones do not wash out acutely, as indicated by the gradual decline in sexual behavior over many weeks following gonadectomy in male rodents (Hull and Dominguez, 2007). Therefore, in this experiment, we studied hamsters that had been gonadectomized (GDX) 10 weeks prior to the start of conditioning. All adults arrived at postnatal day P56-63, but staggered so that they could be tested within the same CPP cohort. No-stimulus control animals were left gonad-intact and were pretested at P64-71, while the two experimental groups received conditioning with VS. The GDX+0 group was GDX at P57-64, remained unmanipulated for 10 weeks, and were then implanted with blank capsules at P127-134, one week prior to pretest at P134-141. The GDX+T group was GDX and given testosterone capsules at P57-64, one week prior to pretest at P64-71 to serve as positive controls to demonstrate a significant CPP. This arrangement required different ages of animals. However, we have never observed age-related differences in behavioral or neural responses to testosterone in prior experiments that controlled for this variable in young adults (Schulz et al., 2009). 121 Experiment 2: Are adult levels of testosterone sufficient to activate a CPP to VS in juvenile hamsters? This experiment determined whether testosterone treatment of juvenile hamsters is sufficient for formation of a CPP to VS. One week of testosterone is sufficient to induce attractive responses to VS, and this duration of testosterone treatment was therefore used here (Johnston and Coplin, 1979). Juveniles arrived at P6 with their mothers, and were either left gonad-intact as no-stimulus controls or were GDX and given a testosterone capsule at P14. All juveniles were pretested at P21. In addition, age-matched gonad-intact adults were included as positive control and nostimulus control groups; they arrived at P56-63 and were pretested at P70-77. Here the chamber characteristics were modified from that described above in an effort to bring the initial preference scores closer to 0.50. Instead of the mesh floor, a solid Plexiglas floor was used in the white side, without pine pellets as a scent. Experiment 3: Does dopamine receptor activation mediate testosteronedependent CPP to VS in juvenile hamsters? This experiment tested the involvement of dopamine in testosterone-facilitated CPP to VS in juvenile male hamsters. The same chamber stimuli were used as in Experiment 1 and previously published studies, as the changes used in Experiment 2 failed to improve the preference scores. All animals arrived at P12, were pretested at P20, and were run in 3 cohorts. Gonad-intact hamsters were used as no-stimulus controls, while other groups were GDX and given blank or testosterone capsules at P13, one week before testing. The GDX+0 group was included to confirm that juveniles with low levels of testosterone (as in gonad-intact animals) do not show a CPP to VS. A GDX+T was included as a positive control to 122 show that testosterone treatment can induce a CPP to VS. The remaining groups were all GDX+T, and were given injections of haloperidol (a D2 antagonist, 0.05, 0.15, 0.45mg/kg, ip,) or propylene glycol vehicle 30 minutes prior to VS and no-stimulus conditioning sessions, respectively. No-stimulus, GDX+0 and GDX+T control groups received propylene glycol vehicle injections 30 minutes prior to both conditioning sessions. A final group was included to test for any aversive properties of haloperidol. These animals were gonad-intact, and received 0.45mg/kg haloperidol or propylene glycol ip 30 minutes prior to conditioning sessions in the initially preferred and nonpreferred chamber, respectively. Thus, any aversive properties of haloperidol would reduce their initial preference. No VS pairings were made in this group. Experiment 4: Does dopamine receptor antagonism alone alter place preference in juvenile hamsters? This experiment was designed to more completely determine if the doses of haloperidol used in Experiment 3 had any intrinsic aversive qualities in testosterone-treated hamsters. If this were shown to be the case, then the results of Experiment 3 would be difficult to interpret, as an observed prevention of CPP for VS might instead just be an overriding avoidance of the haloperidol-conditioned environment. All animals arrived at P12, were GDX+T at P13, pretested at P20, and run in one cohort. A similar conditioning paradigm was used as above, but haloperidol was given in the initially preferred chamber in an attempt to reduce initial preferences, and no VS were used. Preference scores were converted to aversion scores, and indicated time in initially preferred compartment over time in initially preferred and nonpreferred. Locomotor activity (number of infra-red beam breaks) and fecal boli output 123 during conditioning sessions were also quantified as indicators of physiological effects of haloperidol. Experiment 5: Does dopamine receptor antagonism affect food detection in juvenile hamsters? This experiment determined the potential of haloperidol to block olfactory abilities in testosterone-treated juveniles. Animals from Experiment 4 were used, and tested on P32. Two plastic dishes were placed at opposite ends of glass aquaria (51 x 26 x 31.5 cm) that either contained moistened food pellets or were left empty. Both dishes were covered with opaque foil with small holes punched in the top, so that hamsters could smell but not see the food. Thirty minutes before the test, hamsters were injected with their assigned dose of haloperidol or propylene glycol vehicle. Hamsters were placed in the middle of the aquaria and the time spent in nosecontact with the two dishes was recorded in a five minute test. Aquaria were cleaned with 25% ethanol between tests, and the side of the food was alternated between tests so that half the animals in each group were presented food on the right side of the aquaria. Plasma testosterone measures Duplicate 50 µl samples of plasma testosterone were analyzed within a single assay using the Coat-A-Count Total Testosterone Kit (Diagnostic Products, Los Angeles, CA). The minimum detectable concentration was 0.08ng/ml and the intra-assay coefficient of variation was 7.9%. Five hamsters removed their testosterone capsules midexperiment and were excluded from behavioral or testosterone analysis. Final group 124 sizes are expressed in Table 5.1. Testosterone concentrations from animals in Experiment 4 are not available at this time. Statistical analysis To assess whether VS (or haloperidol) induced a change in place preference, paired ttests within each group were used to evaluate the change in preference (or aversion) score and difference score from pretest to test (Bell et al., 2010). A significant change in both preference score and difference score within a group was required to indicate a CPP. To assess effects of haloperidol on locomotor activity in Experiment 4, a one-way ANOVA and Dunnett post-hoc test were used to compare number of infrared beam breaks in the initially preferred compartment between groups. A separate ANOVA was performed with data from the initially non-preferred compartment (propylene glycolpaired) as an un-drugged control. Because locomotor activity is generally greater in the black compartment than that in the white, only animals that received haloperidol in the white compartment were included in the locomotor analysis, n = 5-6/group. To assess haloperidol effects on fecal boli output, paired t-tests were used to compare number of fecal boli while in haloperidol or vehicle conditioning sessions, within each group. To assess whether haloperidol affected preferences for food smells, paired t-tests were used to compare time spent investigating the empty or food containing dish within each group. P < 0.05 was considered significant, and all statistical analyses were done with SPSS software (PASW Statistics 18; SPSS: An IBM Company, Chicago, IL). 125 Results Experiment 1 – Are testicular hormones necessary for formation of a CPP to VS in adult hamsters? Long-term gonadectomy prevented a CPP for VS in adult hamsters (Figure 5.1). No changes in preference or difference score of the GDX+0 group were seen as a result of conditioning (t(9) = 1.14, NS and t(9) = 0.06, NS, respectively). However, paired t-tests did indicate a significant increase in the preference score and decrease in the difference score for the GDX+T group as a result of conditioning (t(9) = -4.88, p < 0.05 and t(9) = 5.64, p < 0.05, respectively). No changes in preference or difference score between pretest and test were seen in age-matched no-stimulus control group (t(11) = -0.76, NS and t(11) = 1.09, NS, respectively). Therefore, exposure to testicular hormones is necessary for VS-induced CPP. 126 Preference Score 0.8 Pretest Test * 0.6 0.4 0.2 0.0 Difference Score 200 100 * 0 -100 -200 Hormone: Stimulus: 12 10 10 Intact noVS GDX+0 VS GDX+T VS Figure 5.1. CPP to VS in hormone manipulated adult hamsters. Preference and difference scores at pretest and test, mean +/- SE, p < 0.05, with group sample sizes above the x axis. Long-term GDX with blank hormone capsules prevented the CPP for VS observed in the GDX + testosterone group. 127 Experiment 2: Are adult levels of testosterone sufficient to activate a CPP to VS in juvenile hamsters? Testosterone treatment was sufficient to permit a CPP to VS in juvenile animals (Figure 5.2). Paired t-tests showed a significant increase in the preference score and decrease in the difference score for the juvenile GDX+T group as a result of conditioning (t(6) = 2.90, p < 0.05 and t(6) = 2.66, p < 0.05, respectively). No changes in preference or difference score were seen in age-matched no-stimulus control group from pretest to test (t(6) = -1.38, NS and t(6) = 2.20, NS, respectively). Gonad-intact adults also showed a CPP to VS, replicating previous results (Figure 5.2). Paired t-tests showed an increase in the preference score and a decrease in the difference score for the adult gonad-intact group as a result of conditioning (t(7) = -3.88, p < 0.05 and t(7) = 3.95, p < 0.05, respectively). No changes in preference or difference score were seen in agematched no-stimulus control group from pretest to test (t(6) = -1.76, NS and t(6) = 1.98, NS, respectively). 128 Preference Score 0.8 Pretest Test * * 0.6 0.4 0.2 0.0 Difference Score 400 300 200 100 * 0 * -100 7 -200 Age: Adult Hormone: Intact Stimulus: noVS 7 8 7 Adult Intact VS Juvenile Intact noVS Juvenile GDX+T VS Figure 5.2. CPP to VS in hormone manipulated juvenile and adult hamsters. Preference and difference scores at pretest and test; mean +/- SE, p < 0.05, with group sample sizes above the x axis. Testosterone treatment in juvenile hamsters facilitated an adult-like CPP for VS. 129 Experiment 3: Does dopamine receptor activation mediate testosteronedependent CPP to VS in juvenile hamsters? Dopamine receptor antagonism blocked the CPP for VS in T-treated juvenile hamsters (Figure 5.3). The GDX+T VS group that received vehicle injection showed a CPP to VS, as both preference and difference scores showed a significant change as a result of conditioning (t(5) = -4.043, p < 0.05 and t(5) = 4.18, p < 0.05, respectively). This effect was blocked by haloperidol at all three doses: neither the 0.05mg/kg, 0.15mg/kg, nor 0.45mg/kg GDX+T VS groups showed a significant change in preference score (t(7) = 1.11, NS; t(6) = -1.52, NS; and t(7) = -0.68, NS, respectively) or difference score (t(7) = 1.64, NS; t(6) = 1.79, NS; and t(7) = 0.92, NS, respectively) as a result of conditioning. The GDX+0 VS group did not show a significant change in either preference or difference score as a result of conditioning (t(6) = -0.82, NS and t(6) = 1.32, NS, respectively), replicating effects seen in gonad-intact juveniles with similar concentrations of circulating hormone (Chapter 3). The no-stimulus control group did not show a significant change in either preference or difference score from pretest to test (t(10) = -1.33, NS and t(10) = 1.97, NS, respectively). However, the 0.45mg/kg noVS group showed a CPA to haloperidol, as both preference and difference scores showed a significant change across conditioning (t(7) = -2.39, p < 0.05 and t(7) = 2.74, p < 0.05, respectively). This suggests that this high dose is aversive, and precludes interpretation of the results from the GDX+T VS group at that dose. 130 Preference Score 0.8 Pretest Test * * 0.6 0.4 0.2 0.0 Difference Score 200 100 * * 0 -100 11 -200 Hormone: intact Halo mg/kg: 0.00 Stimulus: noVS 7 7 6 8 GDX+0 GDX+T GDX+T GDX+T GDX+T 0.00 0.00 0.45 0.05 0.15 VS VS VS VS VS 7 8 intact 0.45 noVS Figure 5.3. CPP to VS in hormone and dopamine manipulated juvenile hamsters. Preference and difference scores at pretest and test; mean +/- SE, p < 0.05, with group sample sizes above the x axis. Testosterone treatment in juvenile hamsters facilitated an adult-like CPP for VS, as in Experiment 2. Dopamine antagonism prevented the CPP for VS at multiple doses. However, the highest haloperidol dose conditioned a place aversion. 131 Experiment 4: Does dopamine receptor antagonism alone alter place preference in juvenile hamsters? Potential aversive affects of haloperidol at different doses were unable to be determined (Figure 5.4). Most groups, including the no-stimulus control group, 0.15mg/kg group, and 0.45mg/kg group, showed significant changes in preference score (t(6) = -2.78, p < 0.05; t(6) = -5.77, p < 0.01; and t(6) = -3.63, p < 0.01, respectively) and difference score (t(6) = 2.96, p < 0.05; t(6) = 5.57, p < 0.01; and t(6) = 3.29, p < 0.05, respectively). The 0.05mg/kg group showed a significant change in difference score (t(6) = 2.74, p < 0.05) but not preference score (t(6) = -2.16, NS). The observed change in no-stimulus control group indicates that changes in the haloperidol groups are likely not due to the haloperidol, and thus this experiment does not provide evidence one way or the other about the potential aversiveness of haloperidol. 132 Pretest Test Aversion Score 0.8 0.6 * 0.4 * * 0.2 0.0 Difference Score 200 * * 0 * * -200 7 7 7 7 0.00 0.05 0.15 0.45 Haloperidol dose, mg/kg Figure 5.4. CPA to dopamine antagonist in juvenile hamsters. Aversion and difference scores at pretest and test; mean +/- SE, p < 0.05, with group sample sizes above the x axis. Groups showed a significant change in preference and/or difference scores, independent of haloperidol exposure, precluding interpretation. 133 Dopamine receptor antagonism affected activity and fecal boli output, but only at the 0.45mg/kg dose. An effect of dose was found on locomotor activity in the initiallypreferred compartment that was paired with haloperidol, F(19) = 6.44, p < 0.01. The post-hoc test revealed that haloperidol significantly reduced locomotor activity compared to controls, but only at the highest dose, p < 0.01 (Figure 5.5). No differences were found between groups in locomotor activity in the vehicle-paired Infrared beam breaks compartment, F(19) = 2.16, NS. Haloperidol dose, mg/kg 5000 4000 0.00 0.05 0.15 0.45 * 3000 2000 1000 0 Vehicle Haloperidol Compartment Figure 5.5. Locomotor activity in dopamine manipulated juvenile hamsters. Infrared beam breaks, indicating locomotor activity, mean +/- SE, p < 0.01. Animals assigned to 0.00 – 0.45mg/kg haloperidol doses did not differ in locomotor activity when assessed in the vehicle-paired compartment. However, locomotor activity was reduced in the highest haloperidol dose group while under the effects of the drug in the haloperidolpaired compartment. 134 No differences were found in within-animal fecal boli output in initially preferred or non-preferred compartments in the 0.00, 0.05, or 0.15mg/kg haloperidol groups (t(6) = 0.53, NS; t(6) = -0.14, NS; and t(6) = -1.74, NS, respectively. However, the 0.45mg/kg haloperidol group did produce more fecal boli in the initially preferred compartment during haloperidol-paired conditioning sessions, as compared to vehicle-paired conditioning sessions, t(6) = -5.10, p < 0.01 (Figure 5.6). Fecal Boli 4 * Compartment Vehicle Haloperidol 3 2 1 0 0.00 0.05 0.15 0.45 Haloperidol dose, mg/kg Figure 5.6. Fecal boli in dopamine manipulated juvenile hamsters. Number of fecal boli produced while in vehicle or haloperidol paired compartments, mean +/- SE, p < 0.01. Animals assigned to 0.00 – 0.15mg/kg haloperidol doses did not differ in fecal boli output when in the vehicle- or haloperidol-paired compartment. However, fecal boli output was increased in the highest haloperidol dose group while under the effects of the drug in the haloperidol-paired compartment. 135 Experiment 5: Does dopamine receptor antagonism affect food detection in juvenile hamsters? Dopamine receptor antagonism did not affect preferences for the food-containing dish over the control dish, indicating olfactory abilities are intact in haloperidol treated animals (Figure 5.7). The 0.00mg/kg, 0.05mg/kg, 0.15mg/kg and 0.45mg/kg all spent more time investigating the food dish over the control (t(6) = 5.50, p < 0.01; t(6) = 5.80, p < 0.01; t(6) = 4.06, p < 0.01; and t(6) = 6.30, p < 0.01, respectively). Dish Contact 40 Empty Food * 30 20 * * * 0.15 0.45 10 0 0.00 0.05 Haloperidol dose, mg/kg Figure 5.7. Food detection in dopamine manipulated juvenile hamsters. Time spent in nose-contact with the empty or food-containing dish, mean +/- SE, p < 0.01. All doses spent more time in contact with the food dish, indicating intact olfactory abilities even with haloperidol treatment. 136 Physiological measures Physiological measures are shown in Table 5.1, and confirm efficacy of testosterone capsules in raising circulating testosterone in both ages. Groups of the same age did not differ in body weight. Age Hormone Stimulus Experiment 1 Adult Intact noVS Adult GDX+0 VS Adult GDX+T VS Experiment 2 Adult Intact noVS Adult Intact VS Juvenile Intact noVS Juvenile GDX+T VS Experiment 3 Juvenile Intact noVS + 0.00 Juvenile GDX+0 VS + 0.00 Juvenile GDX+T VS + 0.00 Juvenile GDX+T VS + 0.05 Juvenile GDX+T VS + 0.15 Juvenile GDX+T VS + 0.45 Juvenile Intact noVS + 0.45 Experiment 4 & 5 Juvenile GDX+T VS + 0.00 Juvenile GDX+T VS + 0.05 Juvenile GDX+T VS + 0.15 Juvenile GDX+T VS + 0.45 N Body Weight (g) Mean SD 12 10 10 132.50 13.67 1.41 0.53 150.72 11.95 < 0.08 116.77 12.09 2.60 0.87 7 8 7 7 Plasma T (ng/ml) n SD 126.51 11.00 2.76 124.81 9.06 2.24 65.70 5.99 0.94 63.39 3.88 4.06 1.21 0.80 0.66 0.68 11 7 6 8 7 8 8 58.76 54.19 56.05 56.01 56.51 52.34 59.15 3.90 0.89 4.48 < 0.08 5.69 3.87 6.77 2.70 6.91 4.09 4.59 4.09 5.70 0.89 0.50 1.21 1.47 0.65 1.15 0.62 7 7 7 7 54.39 53.06 51.24 52.33 7.04 7.05 5.61 5.06 n/a Table 5.1. Group size, body weight, and plasma testosterone concentration. Adults were consistently larger than juveniles, but groups within an age generally did not differ in body weight. Testosterone pellets produced circulating testosterone concentrations within adult-typical physiological range. 137 Discussion These studies demonstrate that testosterone is necessary for male hamsters to evaluate VS as rewarding. Specifically, long-term gonadal-hormone absence does not permit a CPP to VS in adults, while testosterone treatment of juveniles is sufficient to enable them to form a CPP to VS. These effects of testosterone are similar to those seen for activation of sexual behavior in adulthood and general attraction to VS in juveniles, behaviors that normally increase during adolescence (Whalen and Debold, 1974, Morin and Zucker, 1978, Johnston and Coplin, 1979). The testosterone-mediated gain in VS reward may be dependent on dopaminergic action, as a D2 receptor antagonist prevented expression of a CPP to VS. However, conclusions about the role of dopamine receptor activation in CPP formation must be postponed until completion of important experimental control experiments, which are discussed below. Experimental Controls A dopamine receptor antagonist was used to block dopaminergic responses to VS that may facilitate reward or reinforcement to allow for a CPP, and to keep dopaminergic receptor activity at the usual basal state. However, high doses of dopamine antagonists have the potential to cause aversive or unpleasant psychological or physiological effects. Such an aversive response to haloperidol could conceal an existing CPP by inducing the animal to avoid the haloperidol-associated CPP compartment. Experiment 3 gave some indication that the high dose of haloperidol may indeed be aversive in gonad-intact juveniles, and Experiment 4 was designed to more completely test aversive properties of VS in testosterone-treated juveniles. Unfortunately, the control 138 group that never received haloperidol (and instead received vehicle injections in both initially preferred and non-preferred chambers) showed a significant change in aversion and difference scores from pretest to test. It is unclear what caused this shift in preference in the control group, but regardless, it prevents interpretation of similar changes in haloperidol exposed animals, and the experiment will be repeated. Some useful indicators of physiological and perhaps psychological effects of haloperidol were gleaned from the experiment. Haloperidol reduced locomotor behavior and increased fecal boli output, but only at the highest dose. Fecal boli output has classically been used as an indicator of anxiety and aversion (Sanberg, 1989), however D2 receptors also serve to inhibit gut motility in the enteric nervous system (Li et al.), thus reducing the utility of this measure in this particular case. While doses of 0.2mg/kg have been previously shown to cause aversive side effects (Risinger et al., 1992, Manzanedo et al., 2001), the doses between 0.05mg/kg and 1mg/kg have not cause a conditioned place aversion in other studies that specifically checked for them in mice and rats (Martin-Iverson et al., 1985, Spyraki and Fibiger, 1988, Di Scala and Sandner, 1989, Suzuki and Misawa, 1995, Wu and Zhu, 1999, Imaizumi et al., 2000, Adams et al., 2001, Manzanedo et al., 2001, Tzschentke, 2004, Manzanedo et al., 2005, Miyata et al., 2011). While I acknowledge that effects of drugs can differ among rodent species, I am cautiously hopeful that the next experiment will demonstrate no aversive qualities of 0.05mg/kg haloperidol. Therefore, to allow for a more robust integration of this study with the other data contained in my dissertation, I will discuss the results of this study under the assumption that the lowest dose of haloperidol is not aversive. 139 One other possible mechanism by which haloperidol might block a CPP to VS is by simply blocking the ability of the animals to smell VS. This possibility was a realistic one, as dopamine is a prominent neurotransmitter in the olfactory bulb and modulates the transmission of odorant sensory information from the receptor cells to the mitral cell at the level of the glomerulus (Cave and Baker, 2009). However, Experiment 5 demonstrated that hamsters exposed to even the highest dose of haloperidol can still detect food olfactory cues, as they showed a preference for the food-containing dish over the empty dish. Therefore, we can conclude that haloperidol did not prevent a CPP to VS simply by blocking olfactory detection of the cue. An interesting secondary observation from this study was that haloperidol treated hamsters tended to show less a less dramatic preference for the food. As these effects were present at lower doses of haloperidol that do not otherwise reduce locomotor activity, they may support dopaminergic involvement in motivation for food (Richard and Berridge, 2011). Mechanism of haloperidol action Because we have found that multiple dopamine-sensitive brain regions, including the AMY, Acb, PFC, and VTA, are involved in behavioral responses to VS, systemic intervention was used to antagonize dopamine receptors at multiple putative sites of action. Haloperidol is a potent D2 antagonist, but can also bind the adrenergic and sigma receptor less effectively (NIMH Psychoactive Drug Screening Program). D2 binding attenuates cellular excitability in both pyramidal neurons in the mPFC and medium spiny neurons in the Acb (Cepeda et al., 1999, Tseng and O'Donnell, 2004, Benoit-Marand and O'Donnell, 2008). At these sites, D2 receptor activation serves to 140 reduce the impact of irrelevant information in the integration of corticolimbic inputs, thus allowing more specific learned associations (O'Donnell, 2003). It is likely this aspect of D2 receptor function that is disrupted by haloperidol to block the CPP for VS. Additionally, haloperidol can increase dopamine release from terminals in the accumbens by acting 1) post-synaptically in the “indirect” ventral pallidum pathway that provides feedforward input to the VTA (Valenti and Grace, 2010) and 2) pre-synpatically as an autoreceptor to regulate release and reuptake at the site of dopamine release (Wu et al., 2002). These aspects of D2 receptor activation emphasize the complexity of dopaminergic action, and that it is overly simplistic to think that more dopamine equals more reward. Dopamine generally modulates responses to other neurotransmitters, and may serve to facilitate the formation of specific associations. Dopamine and sexual reward As mentioned previously, dopamine has been implicated in multiple aspects of sexual behavior, including anticipatory or appetitive behaviors (Pfaus and Phillips, 1989), copulatory or consummatory behaviors (Arteaga et al., 2002), and the reinforcing responses to sexual interaction (Meisel et al., 1996). In addition, dopamine is likely important for associating sociosexual stimuli with the environment or other cues. Systemic low doses of a non-specific dopamine antagonist block conditioned mate preference in female rats (Coria-Avila et al., 2008), and a D2 agonist during cohabitation with a scented same-sex partner induces a same-sex partner preference for similarly scented males in male rats (Triana-Del Rio et al., 2011). Work in monogamous prairie voles further supports the importance of D2 receptor in associating sexual 141 reward with stimuli or individuals, as systemic injections of D2, but not D1, receptor agonist and antagonist facilitate and disrupt partner preference in male voles, respectively (Wang et al., 1999). The current study supports the role for dopamine in reinforcing responses to unconditioned social cues in sexually-naïve animals, and parallels the effects of haloperidol in reducing motivation for primary female visual, auditory, and chemosensory cues in sexually-naïve male rats (Lopez and Ettenberg, 2001). The possible site(s) of action of dopamine cannot be determined from this study, however there are several likely candidates. Dopamine agonists and antagonists into MPOA facilitate and reduce the performance of sexual behavior, respectively, in male and female rats (Hull et al., 1986, Bitran et al., 1988, Pehek et al., 1988, Graham and Pfaus, 2010). In addition, the MPOA is also implicated in anticipatory sexual behaviors and female preferences (Pfaus and Phillips, 1991, Moses et al., 1995). The mesolimbic system does not seem to be involved in the performance of copulatory behaviors, except for general motor abilities (Hull et al., 1991, Moses et al., 1995). However, dopaminergic action in the Acb may be involved in anticipatory sexual behavior, such as increased locomotor activity and erections in response to female cues, independent of motor effects (Pfaus and Phillips, 1991, Liu et al., 1998). In addition, the Abc is important in pair bonding and mate-cue association, as evidenced by work in the voles (Gingrich et al., 2000, Aragona et al., 2006). Thus, dopamine in the MPOA, Acb, or both regions may be important for conditioned place preferences to VS. 142 Testosterone and sexual reward Like dopamine, testosterone probably has multiple sites and mechanisms of action in contributing to social reward. Several studies have demonstrated that long-term (2-8 weeks) gonadectomy results in an increase in several measures of dopaminergic tone in the MPOA, including tissue content and amphetamine-induced dopamine release, but a decrease in extracellular dopamine (Engel et al., 1979, Simpkins et al., 1983, Gunnet et al., 1986, Mitchell and Stewart, 1989, Du et al., 1998). These changes occur without a corresponding change in dopaminergic cell number in the MPOA or the A14 periventricular cells (Du and Hull, 1999). Importantly, MPOA dopaminergic responses to female stimuli in male rats are also modulated by testosterone (Hull et al., 1995, Putnam et al., 2001). While effects of castration in the ventral striatum are less consistent than those in the MPOA, 28 day gonadectomy generally reduces dopamine and DOPAC concentrations in Acb tissue (Alderson and Baum, 1981, Baum et al., 1986, Mitchell and Stewart, 1989). In the mPFC, GDX causes a decrease in TH innervation and extracellular dopamine when measured 4 days after hormone absence, but causes an increase in the same measures 28 days after hormone absence (Aubele and Kritzer, 2011b). Interestingly, the mPFC is also the most androgen receptor-enriched source of input to the VTA and, as the VTA itself has few androgen receptors, may indirectly impart hormone sensitivity to the VTA dopaminergic cell population (Aubele and Kritzer, 2011a). Finally, 30, but not 14 days of gonadectomy causes an increase in the number of TH-ir cells in adult male rats (McArthur et al., 2007, Johnson et al., 2010). Taken 143 together, the withdrawal of gonadal hormones affects dopamine systems in site specific ways. The above studies are informative as to the hormonal sensitivity of a brain region, and suggest possible brain regions in which long-term testosterone absence could induce functional deficits that prevent adult hamsters from showing a CPP to VS. However, the application of these studies in understanding mechanisms by with testosterone treatment permits the display of a CPP to VS in juvenile hamsters is limited by several factors. Most of the above studies assessed the effects of gonadal-hormone removal, which can occur in different temporal patterns that gonadal-hormone exposure or replacement. In one study, sexual behavior was recovered after 10 days of testosterone treatment in males castrated for 21 days (Putnam et al., 2001). Moreover, the above studies were performed in adult animals; as neural structures are generally more plastic and responsive to the effects of gonadal-steroids earlier in development, the effects of testosterone exposure in juvenile animals may be different from those in adults (Schulz et al., 2009). Some insights into testosterone and dopaminergic action in VS reward can also be gained from comparisons between juvenile and adult hamsters. Upon VS application to the nose in mineral oil, juvenile hamsters showed Fos immunoreactivity indicative of D2 receptor activation in corticolimbic regions (Chapter 4). This Fos response to VS was observed in both blank and testosterone treated gonadectomized juveniles, suggesting that testosterone was not modulating dopaminergic activity there. In a separate study, gonad-intact juveniles failed to show adult-typical dopamine responses to VS in the MPOA (Schulz et al., 2003). While it is not clear whether or not 144 this preoptic area response to VS is dependent on testosterone, the medial preoptic area is known to be hormone responsive, and thus is a good candidate for testosteronefacilitated, dopamine-dependent, reward. Conclusion Taken together, these studies demonstrate the importance of testosterone and dopamine in rewarding responses to an unconditioned social stimulus. Both testosterone and dopamine systems mature during adolescence, when VS reward is typically acquired. It should be noted that the dopaminergic circuit could be functional in juvenile animals to mediate CPP to VS, but that testosterone-dependent maturation of some other neural circuitry is also necessary for VS reward. Opioids have been implicated in sexual reward, but the literature on hormone-sensitively is less clear. However, the most parsimonious explanation, given the supporting evidence, is that testosterone treatment in juvenile animals mimics the normative elevation in pubertal testosterone, which in turn affects the dopaminergic system to permit VS reward. 145 Chapter 6: Discussion Experiments contained in this dissertation identify an adolescent shift in behavioral and neural responses to an unconditioned chemosensory social reward, female hamster vaginal secretions. The present set of experiments demonstrates both hormone dependent and independent aspects of adolescent and pubertal development: we have observed 1) age, but not testosterone, dependent changes in mesocorticolimbic Fos responses to VS; and 2) testosterone, but not age, dependent CPP to VS. The testosterone-dependent CPP for VS is also dependent on dopamine receptor activation. The normal rise in testosterone during adolescence likely permits the rewarding response to VS in adulthood, as recent exposure to testosterone is necessary for VS CPP then, as well. The various responses to VS in testosterone-treated juveniles in these studies, i.e., the absence of a Fos response to VS in VTA TH neurons, and the efficacy of the dopamine receptor antagonist to block a CPP for VS, may seem contradictory at first. However, upon further reflection on the nuances of Fos expression and dopamine receptor activation, the studies cohesively imply that the balance of dopamine receptor activation in response to VS shifts from being D2-biased in juveniles, to D1-biased in adults. The logic underlying the proposition that D2 receptor activation is the predominant response to VS in juvenile hamsters is as follows. Dopamine must be released from terminals in some motivationally-relevant brain region in testosteronetreated juveniles in response to VS in mineral oil, or else haloperidol would not have blocked the CPP to VS. However, there is no obvious evidence of activation of TH 146 neurons by VS, either in the VTA of testosterone-treated juveniles that received VS on their noses (Chapter 4), or in the PVN/PeVN, SuM, or MeP of adult or juvenile hamsters (Chapter 3, appendix). As the regions (to our knowledge) include the populations of dopaminergic cells that project to the mPFC, Acb, and/or MPOA and are relevant to behavioral responses to VS, either it appears that dopaminergic responses to VS in juveniles are not detected as increases in Fos/TH-immunoreactivity. Failure to detect juvenile dopamine responses to VS with Fos/TH double label could occur in (at least) two ways. The first possibility is that the juvenile response to VS occurs in a subpopulation of TH cells that projects to a specific downstream target. In the VTA, such sub-populations tend to be spatially diffuse throughout the region, and in this case a Fos response within this specific sub-population could be lost in a low signal-to-noise ratio within the whole-VTA analysis. The second possibility is simply that Fos expression is not an index of TH cell activation and/or is not a correlate of dopamine release. This second option is logically appealing for several reasons that are detailed below. Fos expression in TH-ir cells may not always correlate with dopamine release, for several reasons. Dopaminergic release in response to VS in juveniles is suggested by the observed reductions in Fos in PFC and Acb in response to VS that may be the result of D2 receptor activation via their association with inhibitory G-proteins, as discussed in Chapter 4. D2 receptor activation in response to VS is also suggested by the effectiveness of haloperidol to block a CPP to VS in Chapter 5. As mentioned previously, D2 receptor activation can occur in response to tonic levels of dopamine release, when DA cells are firing at relatively low (not coordinated, phasic) rates (Richfield et al., 1989, Goto et al., 2007). These tonic dopamine levels may result from 147 graded increase in dopaminergic cell activity, above baseline but not reaching phasic rates, or glutamate evoked release of dopamine from local axon terminals (David et al., 2005), in response to VS in juveniles. These mechanisms of dopamine release may be correlated with an increase in expression of some other immediate early gene, or is insufficient to evoke Fos expression in TH neurons. Our theoretical understanding of Fos expression emphasizes this latter point. Fos expression is the result of cellular metabolic processes and changes in gene expression in response to some input, and does not reflect changes in membrane potential (Herdegen and Leah, 1998). In other words, Fos cannot indicate neural activity, moment to moment. Instead, it reveals the residual effects of whatever the cell experienced 60 minutes ago. Thus, the absence of Fos does not indicate a lack of cellular activity, or in our case, dopaminergic responses to VS in juvenile hamsters. This nuanced appreciation of Fos expression does not limit our ability to interpret all Fos/TH measures. While the absence of a response does not indicate the lack of cellular activity, the presence of Fos/TH response to VS probably does indicate that a cell released dopamine. Many studies have demonstrated an increase in Fos/TH-ir that occurs in contexts that also cause an increase in dopamine release, including sexual behavior, electrical intracranial self-stimulation, and drug exposure (Asmus, 1994, Hunt and McGregor, 1997, Shim et al., 2000, Thiele et al., 2000, Balfour et al., 2004). Phasic dopamine release might require increased TH (or some other gene product) synthesis to keep up with all the energy needed for generation of action potentials, fill depleted stores of dopamine, etc. This more robust increase in action potential firing and dopamine release would also activate D1 receptors downstream, and cause an 148 increase in Fos in the Acb and mPFC (Herdegen and Leah, 1998, Goto et al., 2007). Importantly, we observe these both an increase in Fos/TH in VTA cells, and an increase in Fos in corticolimbic areas in adult animals in response to VS when allowed voluntary approach and investigation of VS (Chapter 3). Therefore, while further study is needed to confirm these findings, the current studies support the activation of VTA cells release of corticolimbic dopamine in response to VS Even though we cannot determine exactly how much dopamine is being released in response to VS in juvenile animals, we can still compare Fos responses across ages to determine that juveniles are not showing adult-like responses to VS. Taken together, these studies suggest an adolescent maturation in mesocorticolimbic circuitry to allow for a shift in dopaminergic responses to VS towards D1 receptor activation in adulthood. It is worth noting that a relatively small proportion of TH-IR cells are activated by VS in adulthood. While somewhat unsatisfying, this seems to be a common finding in many species and dopaminergic cell populations in copulatory behavior (Balfour et al., 2004, Bharati and Goodson, 2006, Vittoz et al., 2008, Northcutt and Lonstein, 2009). Nonetheless, activity in even a small population of cells can be behaviorally-relevant, as these cells are known to have extensive dendritic branching that can amplify effects of cellular activity (Lindvall and Bjorklund, 1978, Arbuthnott and Wickens, 2007). Age-dependent maturation of D1:D2 responses to VS. Given the number of known adolescent changes in mesocorticolimbic circuitry, there are many possible mechanisms by which responses to VS could shift to favor corticolimbic D1 activation across adolescence. We can rule out some possibilities by interpreting 149 reward responses that are present in juvenile animals. For example, CPP to cocaine is intact in juvenile male hamsters (Chapter 2), and may actually be enhanced in juvenile rats compared to adults (Brenhouse et al., 2008). In both juvenile and adult rats, CPP to cocaine seems to be more dependent on D1 than D2 receptor activation, as indicated by selective D1 receptor optogenetic activation promoting the CPP (Lobo et al., 2010). Thus, D1-dependent neural circuitry is sufficiently mature and functional in juvenile hamsters, and itself not the reason for differential Fos responses to VS across adolescence. However, continued development of the mesocorticolimbic system might still shift the balance of receptor activation, and dopaminergic responses specific to VS may still only be sufficient to activate D1 responses in adult hamsters. We begin to explore these mechanisms at the source of dopamine in the VTA. Here, greater activation of dopaminergic cells would favor D1 receptor activation downstream because of the more robust release in dopamine. There is little evidence in the literature for hypo-responsive dopaminergic cells during adolescence: most studies suggest that basal firing rates are achieved by 20 days of age, while some purport a peak in firing rates mid adolescence but with similar rates early adolescence and in adulthood (Pitts et al., 1990, Tepper et al., 1998, Marinelli et al., 2006). No studies, to my knowledge, have investigated the possibility of increased MPOA or amygdalar projections to the VTA during adolescence that could relate to increased activation of transmission of VS-relevant input. However, if we can apply our Fos expression data to compare juveniles and adults, we see VTA cells in juveniles and adults actually respond to VS in similar ways. Voluntary investigation of VS induces Fos expression in non-dopaminergic cells in the PN, and dopaminergic cells in the PN 150 and PBN in both ages, while nose application of VS does not. The one cell group that differs between juveniles and adults in its response to VS in the VTA is GABAergic cells in the IF, that responds to VS only in adults regardless of exposure method. However, there are relatively few GABAergic projections to corticolimbic sites, and GABA typically serves to reduce dopaminergic cell activity. Therefore, neither dopaminergic nor nondopaminergic cell populations in the VTA likely serve as the neural correlate for changes in circuitry responses to VS during adolescence. Two primary targets of dopaminergic innervation, the accumbens and prefrontal cortex, also show dramatic changes during adolescence that could account for increased D1 receptor activation. In the accumbens, while both basal and electrically stimulated levels extracellular dopamine peak mid adolescence, adults show greater levels of these measures and TH expression than do P30 juveniles, a comparison similar to ages used in this dissertation (Philpot and Kirstein, 2004, Marinelli et al., 2006, Walker and Kuhn, 2008, Mathews et al., 2009, Philpot et al., 2009). In the mPFC, dopaminergic innervation increases progressively until P50 – 60, as evidenced by TH fiber density (Kalsbeek et al., 1988, Benes et al., 2000). Recently, fast-scan cyclic voltametry has been used to study dopamine transients that provide high temporal resolution as to burst firing of dopaminergic cells (Robinson et al., 2011). In the Acb core, P28 juvenile and P70 adult animals show similar incidents of spontaneous and same-sex social-contact induced bursting. However, adults show greater burst firing in response to unconditioned odor, visual, and auditory cues. Thus, in both the Acb and PFC, more robust dopamine innervation and burst firing could preferentially activate D1 receptors in adulthood. 151 In addition to an increase in dopaminergic innervations, there are adolescent changes in glutmatatergic projections that could affect neural responses to VS. Basolateral amygdalar input to the mPFC increases during adolescence (Cunningham et al., 2002), which could indirectly relay chemosensory information from the MeP to induce glutamate release in response to VS. Also, there is a dramatic increase in glutamatergic projection from the mPFC to the Acb during adolescence (Brenhouse et al., 2008). This increase in glutamate is if interest because of its ability to induce local release of dopamine from synaptic terminals in the Acb and mPFC (David et al., 2005). Thus, greater glutamate release in response to VS in corticolimbic sites could evoke phasic dopamine release, which could activate D1 receptors only in adult hamsters. Lower levels of both glutamate and dopamine innervations in corticolimbic sites could be associated with tonic dopamine that preferentially activates D2 receptors in juvenile hamsters. Coincident with these increases in dopaminergic tone, there is also a shift in dopamine receptor expression. In the Acb, prior to P28 in male rats, D2 receptors are almost twice as prominent as D1 receptors; by P60, D1 receptors are expressed slightly more than D2 receptors (Tarazi and Baldessarini, 2000). In the mPFC, D2-like autoreceptors are present in juvenile but not adult male rats (Andersen et al., 1997b); as autoreceptors typically serve to modulate DA update and release, the lack of this feedback in adult animals could allow for a more prolonged or robust dopamine release, thereby activating D1 receptors in adulthood. In summary, there are multiple mesocorticolimbic changes during adolescence, including an increase in amygdalar 152 input, an increase in dopaminergic innervation, and a shift in dopamine receptor expression, that could serve as mechanisms for adult-specific Fos responses to VS. Testosterone-dependent CPP to VS We have observed that one week of circulating testosterone is sufficient to allow for formation of a CPP to VS in juvenile hamsters. At the same time, pilot studies suggested that gonadectomized adult hamsters can form a CPP to VS up to at least nineteen days following removal of testosterone, and it is only long term gonadectomized hamsters that fail to form a CPP to VS. Testosterone and gonadectomy could be affecting the gain and loss of CPP to VS via the same mechanism at a different rate, or via different mechanisms in juveniles and adult animals. Perhaps the long-term (28-56 day) effects of castration to reduce Acb tissue content (Alderson and Baum, 1981, Mitchell and Stewart, 1989) correlate with the loss of CPP to VS in 10 week gonadectomized adult males in Chapter 5. Similarly, longterm castration-induced increases TH innervation of the mPFC in male rats (Aubele and Kritzer, 2011b) may relate to the loss of CPP to VS in gonadectomized adults. However, the relationship of an increase in mPFC TH and a decline in performance on mPFC and dopamine-dependent cognitive tasks is still unclear (Kritzer et al., 2007). There are many examples of gonadal hormones regulating dopaminergic circuitry so as to allow for rewarding responses to drugs; however many of them concern estradiol in female rats (Festa and Quinones-Jenab, 2004, Becker and Hu, 2008). For example, cocaine sensitivity and amphetamine sensitization are regulated by estradiol in ovariectomized female rats (Forgie and Stewart, 1994). While T could theoretically be 153 aromatized to E in males, E does not sensitize males’ responses to cocaine in the same manner (Jackson et al., 2005). This result, coupled with the findings that gonadectomy does not affect cocaine or amphetamine sensitivity in adult males (Robinson et al., 1982, Becker, 1999, Becker et al., 2001a, Hu and Becker, 2003, Russo et al., 2003), reduces the likelihood of that T is aromatized to E to affect mesolimbic responses to dopaminergic drugs. However, testosterone withdrawal could affect responses to VS differently than it affects responses to dopaminergic drugs. The studies on the effects of gonadectomy on dopaminergic tissue described throughout this dissertation may not be informative as to the mechanism behind the gain in CPP to VS with testosterone treatment in juveniles. As stated previously, the temporal pattern in functional gains after testosterone replacement can be quite different from the loss of function after testosterone removal. However, our results in Chapter 4 can indicate some sites of testosterone action in responses to VS. Specifically, both blank and testosterone treated juveniles showed a reduction in Fos in response to VS, suggesting that testosterone is likely not affecting the overall balance of D1/D2 receptor activation in the mesocorticolimbic system. While our Fos analysis may not be sensitive enough to detect hormonal effects in specific cell types or populations, these current results and a survey of the literature suggest that testosterone is modulating neural function outside the mesocorticolimbic system. Specifically, I propose that testosterone is modulating dopaminergic function in the MPOA for several reasons. The MPOA Fos response to VS is testosterone dependent (Fiber and Swann, 1996), as is dopaminergic activity within the area (Hull et al., 1995). Gonadectomy tends to reduce MPOA extracellular DA while increasing intracellular DA in the 154 terminals, but only after 20-30 days of hormone absence (Engel et al., 1979, Simpkins et al., 1983, Du et al., 1998, Du and Hull, 1999). Importantly, one study that showed gradual and synchronous decline in both precopulatory dopamine responses to female cues and sexual behavior over 21 days, and then partial and full recovery of both dopamine responses and sexual behavior after 5 and 10 days of testosterone treatment (Putnam et al., 2001). The gradual decline and rapid recovery parallel observations in this dissertation of CPP to VS. Therefore, while MPOA has not been previously implicated in CPP for reward, its hormone sensitivity, dopaminergic function, and responsiveness to VS makes the MPOA an excellent candidate for the effects of T in facilitating a CPP to VS. It should be noted that conclusions about the sources of this MPOA dopamine are incomplete. The TH-ir cells in the MeP, PVN, and SuM all fail to show Fos responses to VS in either juveniles or adults. However, as we know from earlier discussions about Fos/TH-ir cells in the VTA, the absence of Fos/TH expression does not preclude the release of dopamine from terminals. Indeed, like the corticolimbic sites, dopamine release can be regulated at the terminals by glutamate in the MPOA. This MPOA effect occurs via a glutamate-induced increase in local nitric oxide which then causes the release of dopamine from terminals (Dominguez and Hull, 2001, Dominguez et al., 2004). Importantly, testosterone regulates nitric oxide synthase, the enzyme used to produce nitric oxide (Du and Hull, 1999). However, the effect of testosterone has only been shown by gonadectomizing animals and observing a decline in the enzyme (Putnam et al., 2005). Therefore, further study is needed to demonstrate the ability of testosterone to upregulate nitric oxide in juvenile animals. 155 Disconnect between VS reward and sexual behavior When we began this series of studies, we had initially hypothesized that as VS became rewarding to the hamster, it would promote sexual behavior, thus explaining the gain in sexual behavior across adolescence. However, these studies demonstrate that VS can be rewarding, without the associated increase in sexual behavior, in juvenile males treated with testosterone. As such, the question remains: why do juveniles hamsters fail to show adult-like sexual behavior when treated with testosterone and with functional gonadal hormone receptors and enzymes (Meek et al., 1997, Romeo et al., 1998)? Below, I describe several possible explanations for minimal sexual behavior in juvenile animals. While no single factor can explain the behavioral transition, they may all contribute in some degree. As juvenile hamsters tend to show more submissive and fear-related behaviors than adults, it is possible that females (even though they maintain a strict lordosis posture for most of the interaction) are perceived as an aversive or anxiogenic stimulus. To test this in a small pilot study, we determined if sexual behaviors in testosterone treated juvenile hamsters could be increased by treatment with diazepam, an anxiolytic previously shown to reduce anxiety like behavior in an elevated plus maze and to reduce submissive behaviors in a conditioned defeat paradigm in male hamsters (Yannielli et al., 1996, Moise et al., 2008, Gannon et al., 2011). Four groups of animals were used: three groups were gonadectomized and given testosterone pellets one week prior to behavior tests on P31, while a fourth group remained gonad intact. Two of the GDX+T groups received injections of diazepam (2mg/kg and 5mg/kg, ip, doses shown to be affective in above studies), while the other two groups received vehicle injections 156 30 minutes prior to test. Sexually experienced and hormonally-primed females were used as stimuli. Unfortunately, due to video recording malfunction, only data from three different live-scorers are available; therefore, only qualitative descriptions of the results are presented here. In general, both testosterone and diazepam treatment reduced defensive-like behaviors, including flank-marks and escape-dashes. However, neither dose of diazepam paired with testosterone increased sexual behaviors like mounts and intromissive-like behaviors. Therefore, while testosterone may serve to reduce aversive responses to females and dis-inhibit the display of sexual behavior, some other factors are necessary to promote the behaviors. These behavior tests did reveal another possible, though less exciting, explanation for minimal sexual behavior in juvenile hamsters. As the male:female body weight ratio increased, number of mounts also increased. Thus, it is possible that juvenile males, who weighed 50-70g in this study, simply have trouble positioning themselves appropriately at the females’ hindquarters (who weighed 130-160g in this study), to allow for an adult-like mount. However, more exhaustive tests are needed to confirm this possibility. The larger males could also be further along in their pubertal development, and would have shown more sexual behavior regardless the size of the female. To best assess this possibility, juvenile males of similar body should be tested with relatively smaller females. Again, body size ratio may be one of several factors allowing the promotion of sexual behavior. However, it certainly cannot explain immature Fos responses to VS in testosterone-treated juveniles. One other interesting possibility for adult-specific sexual behavior is suggested by increases in luteinizing hormone in response to female urine in adult but not juvenile 157 male mice (Maruniak et al., 1978). Luteinizing hormone release is promoted by gonadotrophin releasing hormone (GnRH) cells scattered throughout the hypothalamus that project to the median eminence (Sisk and Foster, 2004). GnRH cell size (but not number) increases during adolescence (Urbanski et al., 1992), perhaps explaining differential LH responses to female cues during adolescence. In addition, prepubertal hamsters are more sensitive to negative feedback actions of testosterone to reduce GnRH release (Sisk and Turek, 1983). Therefore, testosterone treatment reduces GnRH release in juvenile hamsters, instead of increasing GnRH release in adult animals. This may affect sexual behavior in a meaningful way, as GnRH cells also project through the BNST, Me, and MPOA to facilitate sexual behavior in adult male hamsters whose VNO had been removed (Lehman et al., 1987, Meredith and Howard, 1992). Therefore, the lack of GnRH activation in response to testosterone may explain low levels of sexual behavior prior to adolescence and the disinhibition of the GnRH cells. GnRH is known to amplify Fos responses to VS in the MPOA and MeP (Westberry and Meredith, 2003), regions previously observed to show adult-like Fos expression in response to VS in juvenile animals. It is possible that quantification of Fos expression cannot detect differences between juveniles and adults in VS-induced neural activity, or GnRH may have actions outside these brain regions that have not yet been studied, including mesocorticolimbic regions implicated in this dissertation. An additional insight into the disconnect between VS reward and sexual behavior comes from the Fos expression patterns in response to VS that are age, but not testosterone, dependent. This pattern does not perfectly align with CPP responses to VS, which can be induced by testosterone prepubertally. Instead, the pattern matures 158 across adolescent development, independent of circulating testosterone, as does the expression of sexual behavior. Distinct aspects of sexual behavior have also been observed to parallel Fos expression in female hamsters when challenged by food deprivation (Klingerman et al., 2011). In that study, food deprivation reduced appetitive behaviors like vaginal marking and male preferences, while copulatory behavior, mating-induced CPP, and Acb Fos responses to mating were unaffected. Rewarding qualities of VS may in fact, represent an appetitive sexual behavior, as does anogenital investigation. Thus, Fos responses to sexual stimuli may reflect the motivational drive to perform sexual behaviors. The interpretation of Fos as an indicator for sexual drive is in agreement with our understanding of Fos as an overall readout of mental state. VS reward may be necessary, but not sufficient, for sexual behaviors; the adolescent shift in dopamine receptor activation towards D1, discussed above, might also be necessary for motivated behavior (Seamans and Yang, 2004). Indeed, the mesolimbic system parallels and is interconnected with the nigrostriatal circuit, known to be so important for motor output (Sesack and Grace, 2009). Moreover, the core of the accumbens, where we have observed Fos responses to VS, is more connected with the substantia nigra and ventral pallidum than is the shell. Therefore, Fos expression in the mesocorticolimbic system may best represent the mental state of an animal prepared to execute a behavior in response to a cue. Accordingly, only adult animals show mesocorticolimbic Fos in response to VS, and only adult animals show sexual behavior. In contrast, amygdalar Fos responses to VS may represent chemosensory detection and perception, and are similar between juvenile and adult animals. 159 Adolescence and reward Effects of adolescent development on reward-seeking have been observed in practically every form of drug and natural reward, but not always along the same temporal pattern (Doremus-Fitzwater et al., 2010). Sensitivity to cocaine is generally greater in adolescents compared to adults, and the age of peak sensitivity somewhere between P21-P40 (Laviola et al., 1992, Badanich et al., 2006, Brenhouse et al., 2008). On the other hand, sensitivity to amphetamine, also a dopamine-dependent stimulant, is greater in adulthood than in adolescence at low doses, and vice versa at high doses (Tirelli et al., 2003, Mathews et al., 2009). Reward sensitivity to nicotine is consistently greater in adolescence than in adulthood (Vastola et al., 2002, Belluzzi et al., 2004, Torrella et al., 2004), while sensitivity to ethanol has been more difficult to determine because of age-specific responses to aversive components of the drug and dosedependent effects (Philpot et al., 2003, Dickinson et al., 2009). Characterization of adolescent changes in drug reward is complicated by doses, testing paradigms, and aversive components of the stimuli. In contrast to drug reward, sensitivity to natural rewards, sucrose and social interactions with a same-sex conspecific, is consistently heightened during adolescence compared to adulthood (Douglas et al., 2004, Wilmouth and Spear, 2004, Varlinskaya and Spear, 2008, Friemel et al., 2010). However, the sensory modality used to detect the reward (e.g. olfactory vs visual input), may produce reward-specific temporal patterns due to maturation of neural connectivity between the particular sensory systems with brain regions important in motivation. Indeed, one possible neural 160 mechanism of a gain in VS reward during adolescence is MeP input to the mPFC and Acb. Together, this literature demonstrates the importance in studying a range of motivated stimuli. Male hamster response to VS is an excellent model with which to study changes in adolescent reward for several reasons: 1) they are unconditioned, and does not require learning or sensitization; 2) they are hormone sensitive, a feature useful to tap into behavioral changes that occur during a period of hormonal change; and 3) they are socially relevant, without the confound of a stimulus animal whose behavior could depend on that of the experimental subject. As social behaviors show the most dramatic changes during adolescence, and many other risk-taking behaviors are ultra-sensitive to social input during adolescence, this third feature is perhaps the most important. 161 APPENDIX 162 Appendix: Select populations of dopaminergic cells do not show an immediate early gene response to VS Introduction In addition to the ventral tegemental area (VTA) dopaminergic cell population discussed in the preceding pages, tyrosine hydroxylase (TH)-ir cells also reside in the hamster posterior medial amygdala (MeP), bed nucleus of the stria terminalis (BNST) and medial preoptic area (MPOA). Dopamine beta-hydroxylase and phenylethanolamine Nmethyltransferase are not expressed in these regions, indicating that norepinephrine and epinephrine are not the final end-products of TH activity (Vincent, 1988, Asmus et al., 1992, Asmus, 1993). The MeP and BNST are also immunoreactive for dopamine, confirming their identity as dopaminergic cells (Asmus et al., 1992, Asmus, 1993). However, the MPOA TH-ir cell population does not contain L-amino acid decarboxylase and is not dopamine-ir, suggesting that L-3,4-dihydroxyphenylalanine, and not dopamine, is the end product of TH activity (Vincent and Hope, 1990, Asmus, 1993). Like rats, hamsters also express TH (and not dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase) in the periventricular nucleus (PeVN), paraventricular nucleus (PVN), posterior hypothalamus (PH), and supramammilary nucleus (SuM) (Vincent, 1988). Chapter 1 previously emphasized the importance of these regions in sociosexual behavior: the MeP is an important site for chemosensory cue and gonadal hormone integration (Wood and Coolen, 1997); the PVN is important in sexual arousal in response to female cues, and interconnected with the mesolimbic dopamine circuit 163 (Succu et al 2007); and the PH/SuM are perhaps extensions of the VTA (Ikemoto 2010). In addition, these regions may be sources of dopaminergic input to the medial preoptic area (Miller and Lonstein, 2009 and Lookingland and Moore 1984). Some work has characterized responses of the MeP and PVN TH-ir cells to hamster sexual behavior (Asmus et al 1994), however, little is known about responses specific to VS exposure. Therefore, this appendix contains data from the MeP, PVN, PH, SuM from animals used in Chapter 3, but were not submitted for publication with Chapter 3 data. Methods The methodology is exactly the same as that in Chapter 3, except for details specific to the brain regions below. In the MePD and MePV, contours were drawn as in Chapter 3. Fos-ir cells were presented in the preceding chapter, so only single-labeled TH and double-labeled Fos/TH data are given here. Because so few cells were observed (0-3 per animal), the number of single-labeled TH or double-labeled Fos/TH-ir cells in all Pd and PV sections were summed within an animal. The PVN was partitioned in two different ways. In the first, all tissue sections containing the PVN (Bregma -0.3 to -1.5mm, 6-8 sections per animal) were traced in Nissl-stained sections to also include some adjacent PeVN, and were overlaid onto immunohistochemistry sections. Additionally, a smaller subsection of the PVN was quantified to parallel analyses of PVN Fos responses to sexual behavior in a previous study (Kollack-Walker and Newman, 1997). Therefore, a smaller section of the PVN including the magnocellular cell group along the ventrolateral edge of the previously 164 drawn traces, was re-quantified in two sections per animal and 4 animals per group; these data are also shown below. In both portions of the PVN, the number of singlelabeled Fos-, single-labeled TH-, and double-labeled Fos/TH-ir cells was divided by the 2 area (mm ) of the contour in each section to create a density measurement; density was used for analysis and presentation here. 2 For both the PH and SuM, a 0.064mm rectangular contour was placed in the middle of the section just above and below the supramammillary decussation, respectively, in two anatomically matched immunohistochemistry tissue sections (Bregma -3.2 and -3.5mm). As in the PVN, density of single-labeled Fos-, singlelabeled TH-, and double-labeled Fos/TH-ir cells were analyzed and presented. Results In the MeP, a main effect of age was observed on the number of TH-ir cells (F(1,26) = 9.30, p < 0.01), such that adults had a larger number of cell than did juveniles (Figure A.1). Likewise, a main effect of age was observed on the number of Fos/TH-ir cells (F(1,26) = 24.67, p < 0.01), such that adults expressed more than juveniles. No effects of swab or age x swab interactions were observed on TH-ir or Fos/TH-ir cell number. In adults, approximately 50% of the TH-ir cells were double-labeled. 165 TH-ir cell number * 2.5 Fos/TH-ir cell number Posterior Medial Amygdala 5 Blank VS 2.0 1.5 1.0 0.5 0.0 * 4 3 2 1 0 Juvenile Adult Figure A.1. Total number of TH-ir and Fos/TH-ir cells in the posterior medial amygdala, Mean +/- SEM, * p < 0.01. Adults expressed more TH-ir and Fos/TH-ir cells than did juveniles. In the full quantification of the PVN, main effects of age were observed on Fos-ir, TH-ir, and Fos/TH-ir cell density (F(1,27) = 52.07, p < 0.01; F(1,27) = 33.60, p < 0.01; F(1,27) = 17.48, p < 0.01, respectively), such that adults expressed more of each cell type than did juveniles (Figure A.2). In the more limited quantification of the magnocellular PVN cell group, similar main effects of age were observed on Fos-ir, THir, and Fos/TH-ir cell density (F(1,12) = 5.93, p < 0.05; F(1,12) = 15.29, p < 0.01; F(1,12) = 166 5.58, p < 0.05, respectively), such that adults expressed more of each cell type than did juveniles (Figure A.2). In addition, a main effect of swab was observed on Fos-ir cell density (F(1,12) = 10.19, p < 0.01), such that VS-exposed hamsters expressed more Fos than blank-swab exposed hamsters. This finding replicated the Fos response to VS in adult males observed in Kollack-Walker and Newman, 1997, which was the attempt of this secondary partitioning of the PVN. No other effects of swab or age x swab interactions were found. 167 Magnocellular PVN Fos-ir cell density * 800 TH-ir cell density PeVN/PVN 100 600 500 Main effect of age 400 and swab 300 400 200 200 100 0 0 * 80 100 * 80 60 60 40 40 20 20 0 Fos/TH-ir cell density Blank VS 0 60 60 40 * 20 0 * 40 20 Juvenile Adult 0 Juvenile Adult Figure A.2. Fos-ir, TH-ir, and Fos/TH-ir cell density in the full extent (left) or magnocellular region (right) of the paraventricular nucleus, Mean +/- SEM, * p < 0.01. Adults expressed greater Fos-ir, TH-ir, and Fos/TH-ir cell density than did juveniles in both portions of the region. In addition, VS-exposed hamsters expressed more Fos than did blank swab-exposed hamsters in the magnocellular region. 168 In the PH, a main effect of swab was observed on Fos-ir cell density (F(1,26) = 4.88, p < 0.05, such that VS-exposed hamsters expressed more Fos-ir cells than blankswab exposed hamsters (Figure A.3). No other effect of swab, no effect of age and no age x swab interaction was found on Fos, TH, or Fos/TH-ir cell density in PH. In the SuM, a main effect of age was observed on Fos/TH-ir cell density (F(1,26) = 4.576, p < 0.05, such that adult hamsters expressed more Fos/TH-ir cells than juveniles hamsters (Figure A.3). No other effect of age, no effect of swab, and no age x swab interactions was found on Fos, TH, or Fos/TH-ir cell density in SuM. 169 Fos/TH-ir cell density TH-ir cell density Fos-ir cell density Posterior Hypothalamus 800 Main effect of swab Supramammilary Nuc. 800 600 600 400 400 200 200 0 0 200 200 150 150 100 100 50 50 0 0 200 200 150 150 100 100 50 50 0 Blank VS 0 Juvenile Adult * Juvenile Adult Figure A.3. Fos-ir, TH-ir, and Fos/TH-ir cell density in the posterior hypothalamus and supramammilary nucleus, Mean +/- SEM, * p < 0.01. Adults expressed greater Fos/THir cell density than did juveniles in the supramammilary nucleus. In addition, VSexposed hamsters expressed more Fos than did blank swab-exposed hamsters in the posterior hypothalamus. 170 Discussion An increase in Fos/TH-ir cell expression with age was found in all but one of the brain regions analyzed in this study. A similar effect was observed previously in the MeP, such that approximately 50% of TH-ir cells were active in unhandled control male hamsters, leading the authors to suggest that Fos is involved in constitutive expression of TH (Asmus et al 1993; Hughes et al 1992; Herdegen et al 1993). Therefore, our data may indicate an increase in constitutively active dopaminergic cell population with adolescent development in the MeP, PVN, and SuM. In fact, the same effect was observed in the intrafascicular VTA, as demonstrated in Chapter 3. The functions of these dopaminergic cell populations are not completely understood. The present study suggests that they are not involved in responses to VS, and are likely not the source of enhanced dopamine release in the MPOA upon VS exposure (Schulz et al 2003). This finding is consistent with those of previous studies that failed to functionally link MeP and PVN populations of TH cells to gonadal hormone manipulations (Asmus et al 1993) or sexual behavior (Asmus et al 1994). Instead, handling alone causes an increase of Fos/TH-ir in the PVN, which the authors attribute to arousal or stress functions of the region (Asmus et al 1994). Chronic stress from P28-35 does cause an increase in TH expression in MePD and BNST, but not in the PeVN (the PVN was not studied (Wommack et al., 2004)). Dopamine in the PVN has been clearly implicated in erection (Melis and Argiolis, 2003), however, the TH cells analyzed in the present and Asmus et al 1994 study failed to co-express Fos in response to sexual behavior. It is possible that these analyses did not include the relevant A14 dopamine cells in the PeVN, which extends along the rostral and caudal 171 extent of the third ventricle. While the functions of these MeP, PVN, PH, and SuM dopaminergic cell populations are likely to be region-specific, they may play common roles in arousal states that increase during adolescence. Fos expression, as a proxy for neural activation, was increased in response to VS only in the magnocellular portion of the PVN. This site-specific response to VS replicates previous observations (Kollack-Walker and Newman 1997), and this subregion of PVN overlaps partially with sub-regions that contain oxytocin- and vasopressin-expressing neurons known to be important in female hamster sexual behavior (Morin and Blanchard, 1993, Albers and Bamshad, 1998) and penile erections (Argiolis and Melis, 2003). Thus, perhaps oxytocin responses to VS are of interest for future study, as oxytocin expression also increases during puberty in male rats (Chibbar et al., 1990). The present study demonstrated an increase in number of TH-ir cells between P28 to P56 in the MeP. However, this finding was opposite to that in Wommack et al 2004, that observed a decrease in TH expression in the BNST and MePd in unmanipulated control animals from P28 to P70. Perhaps one reason for this discrepancy is simply the small number of TH-ir cells in this region. Animals were not treated with colchicine in the present study, as that has been observed to affect Fos expression (Ceccatelli et al., 1989); thus, the number TH-ir cells in the MeP are dramatically lower than that in other studies (Asmus et al 1992). 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