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1 Florida State University Libraries Honors Theses The Division of Undergraduate Studies 2013 The Effects of Mating Experience on Motivation and Anxiety-like Behaviors in Adult Male Rats Erika Vitale Follow this and additional works at the FSU Digital Library. For more information, please contact

2 THE FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES THE EFFECTS OF MATING EXPERIENCE ON MOTIVATION AND ANXIETY-LIKE BEHAVIORS IN ADULT MALE RATS By ERIKA VITALE A Thesis submitted to the Department of Psychology in partial fulfillment of the requirements for graduation with Honors in the Major Degree Awarded: Spring

3 The members of the Defense Committee approve the thesis of Erika Vitale defended on April 18 th, Thesis Director Dr. Elaine Hull Committee Member Dr. Lisa Eckel Outside Committee Member Dr. Susan Blessing 2

4 Acknowledgements This research was supported by Florida State University, the Department of Psychology, and the Program in Neuroscience. This project was funded by R01 MH to Elaine M. Hull as well as the Bess Ward Thesis Award to Erika M. Vitale. I would like to acknowledge the guidance and support of Dr. Elaine Hull and Jenna McHenry, without which none of this project would have been possible. I would like to thank graduate student Chris Robison, as well as undergraduates Gabriella Garcia, Amy Norton, and Raleigh Clarke for their assistance throughout the duration of this project. I would also like to thank my committee members Profs. Lisa Eckel and Susan Blessing. 3

5 Abstract Social and physical aspects of sexual intercourse provide protective benefits on mental and physical health in humans. Similarly, in rodents, mating promotes resistance to anxiety- anddepressive-like behaviors. Furthermore, our lab has shown that sexually experienced males are also more resistant to the effects of stress, as evidenced by decreased neuronal stress-reactivity in the paraventricular nucleus of the hypothalamus. However, little is known about the possible effects of recent mating or mating abstinence on motivation for other natural rewards. Here, we tested whether mating history, at different time periods, would affect measures of motivation and/or anxiety-like behaviors. We hypothesized that recent sexual activity would protect against anxiety-like behavior and anhedonia, whereas loss of sexual activity would have the opposite effect, compared to control males. The study found that recent mating increased motivation for a food reward in an anxiety-provoking novel setting, while abstinence from sexual experience decreased sucrose consumption at lower sucrose concentrations. Keywords: anhedonia, anxiety, depression, sexual behavior, motivation 4

6 Introduction Stress can be defined as a threat to the homeostasis or well-being of an organism. In animals and humans, stress results in the activation of the hypothalamic-pituitary-adrenal (HPA) axis. Activation of this system results in elevation of glucocorticoids, such as cortisol in humans or corticosterone in rodents. Excess circulation of cortisol in the body can have negative effects on mental health and can physically alter the structure and function of brain regions involved in controlling HPA responsiveness (Ulrich & Herman, 2009). Additionally, dysregulation of the HPA axis has been implicated in the development of major depressive disorder as well as other mood and anxiety disorders (Medina et al., 2012). Thus, stress reactivity and depression involve interacting neuronal processes that rely heavily on one another. Anxiety disorders and major depression can also lead to changes in other neural circuits, such as those mediating reward processing. Stress increases reward-seeking behavior, such as lever pressing for drugs of abuse, while rewarding experiences, such as highly palatable foods or sexual intercourse, provide protective benefits for mental and physical health (Ulrich & Herman, 2009). A great deal of research has been done to explore the interaction between natural rewards and anxiety- and depressive-like behaviors. For example, rats exposed to sucrose show attenuated physiological responses to stress, such as lower levels of corticosterone release, and also display less anxiety-like behaviors in the elevated plus maze (Herman et al., 2010). Furthermore, lesions to the basolateral amygdala, a brain region that appears to have a complex role in stress regulation, seem to block the stress attenuation of sucrose experience (Herman et al., 2010). Wheel running is another naturally rewarding stimulus that causes decreases in anxiety-like behaviors in rodents (Onksen et al., 2012). Sexual intercourse is also very closely integrated with stress reactivity and depression in both humans and animals. Both anxiety and depression affect mating behavior - with high levels 5

7 of stress impairing sexual performance and motivation. Specifically, a loss of sexual interest is a core symptom seen in individuals with major depressive disorder, with 72% of patients with unipolar and 77% of patients with bipolar depression reporting loss of sexual interest (Casper et al., 1985).One study showed that sexual experience causes acute HPA activation in rats similar to the activation seen in social-defeat (Buwalda et al, 2012); however, repeated mating seems to have protective benefits against stress reactivity. According to recent studies in our lab, males who received acute plus chronic sexual behavior exhibited fewer c-fos activated cells in the paraventricular nucleus of the hypothalamus (PVN) in response to acute restraint stress compared to unmated males (McHenry, Vitale, and Hull, 2012). This stress buffering effect may be mediated in part by activation of androgens in the medial preoptic area (MPOA) (McHenry, Vitale, and Hull 2012). Repeated mating seems to have implications both in modulating anti-depressant behaviors and attenuating stress responsiveness; however, little is known about whether abstinence from sexual activity will have opposite effects, such as an increase in anxiety-like and depressive-like behavior (including anhedonia). Similarly, little research has been done to examine both the effects of recent sexual experience and abstinence from sexual experience on motivation-seeking and anxiety-like behaviors. The present study examined the effects of recent mating and mating abstinence on preference for natural rewards including sex and sucrose. We hypothesize that previous mating experience will increase motivation for sucrose shortly following ejaculation, but that prolonged abstinence from mating will result in less sucrose consumption and anhedonia-like behavior. Furthermore, we predict that abstinence from mating will result in decreased sexual motivation and female preference in the X-maze test. 6

8 Additional behavioral paradigms in the study include elevated plus maze testing following a two week abstinence from mating, and the novelty-induced hypophagia test immediately following sexual behavior to determine whether sexual experience and mating abstinence affect anxiety-like behaviors. It is hypothesized that previously mated animals will exhibit fewer anxiety-like behaviors in the novelty-induced hypophagia test and that abstained animals will exhibit greater anxiety in the elevated plus maze compared to control animals. Materials and Methods Subjects Adult male Long-Evans rats (Harlan Laboratories; Indianapolis, IN) were used as subjects in these experiments. Rats were habituated to the colony rooms for one week before any experimental testing began. Rats were singly housed in standard polycarbonate cages and were given food and water ad libitum. Rats were kept on a reverse 14 hour light: 10 hour dark cycle, with the 14 hour light cycle commencing at 9:00 PM. All behavioral testing was conducted during the dark phase of the cycle. Behavioral Testing Sexual Behavior Testing Copulatory behavior testing was performed in a test room under dim red light. Male rats received six 30 minute sessions with a receptive stimulus female presented into the male's home cage. Rats then received a final copulation test to one ejaculation. The control group stayed sexually naïve and instead received control handling. The following measures were recorded during copulation testing: mount latency (ML), the time from the introduction of the female to the first mount; intromission latency (IL), the time from the introduction of the female to the first 7

9 intromission; ejaculation latency (EL), the time from the first intromission to the first ejaculation; post-ejaculatory interval (PEI), the time from an ejaculation to the next intromission; total mount frequency (MF); total intromission frequency (IF); total ejaculation frequency (EF); intromission ratio (IR) [IR = I/(M+I)]; and inter-intromission interval (III = EL 1 /IF 1 ). Sucrose Preference The sucrose preference test measures a rat s motivation for reward and is most often used as a measure of depression by modeling anhedonia, which is defined as a lack of pleasure in activities usually found enjoyable (Willner et al, 1992). In the first experiment, rats were first habituated to drink from two small glass bottles for one day. They were then habituated to a 1% sucrose solution for two days. Rats were allowed food and water ad libitum during all sucrose preference habituation and testing. Following one day of no sucrose exposure, rats received a baseline sucrose preference test where they were given a choice between a bottle of water and a bottle of 1% sucrose solution. Bottles were weighed before placement on the cages, 2 hours after bottle placement (short-term test) and 24 hours after bottle placement (long-term test). Rats in the sexually experienced group then received 2 weeks of copulation testing. Following the final copulation test to one ejaculation, rats in the experienced group were given access to one bottle of water and one bottle of sucrose one hour after ejaculation. Meanwhile, rats in the naïve group were also given access to one bottle of water and one bottle of sucrose. All bottles were measured two hours after placement and 24 hours after placement. Rats in the experienced group were then given a two week abstinence period from sexual behavior. Sucrose preference tests were given after 7 days of abstinence and 14 days of abstinence. In experiment 2, rats in the experienced group received sexual experience to one ejaculation every other day throughout sucrose preference testing. Immediately following the end 8

10 of their post-ejaculatory interval (or control handling for the naïve group), one bottle of water and one bottle of sucrose was placed on the cage and measured 2 hours, 24 hours, and 48 hours after bottle placement. The concentration of sucrose was changed after each sexual experience. The concentrations of sucrose are as follows: 0.075%, 0.125%, and 0.25%. In experiment 3, rats in the experienced group received one sexual experience to one ejaculation. Immediately following ejaculation (or control handling for the naïve group) one bottle of water and one bottle of 0.25% sucrose was placed on the cage and measured in 24 hour increments for 5 days after sexual experience. Elevated Plus Maze The Elevated Plus Maze (EPM) is a task in which animals may engage in both exploratory and fearful behaviors (Montgomery, 1955). The test is frequently used as a measure of anxiety-like behavior in rodents. This test was used to determine anxiety level of the naive rats compared to the experienced group after two weeks (14 days) of sexual abstinence. The EPM (Med Associates, Georgia VT) is a plus-shaped maze with two open (without walls) arms and two closed (with walls; H = 39.5 cm). Rats began the EPM test in the center hub and were oriented either toward the left or the right open arm (counterbalanced between animals). The camera above the maze and the motion sensors on each arm recorded the behavior of each animal (time spent in each arm, arm entrances, and time spent in the center hub). X-Maze The X-maze was used to assess sexual motivation of the sexually experienced group before and after abstinence by recording running speed to goal boxes of an X-shaped maze, and percentage of trials on which the male chose the goal box containing a receptive female. All X- maze testing was conducted during the dark phase of the light cycle under dim red light. The X- 9

11 maze protocol was adapted from Warner and Hull et al The X-maze is constructed of plywood and painted gray, with a goal box at the end of each of the 4 arms. Each arm, excluding the goal box, is cm wide and extends cm from a cm central hub. The goal box ( cm) is recessed to one side and is separated from the alley by a Plexiglas door, which can be raised to admit the male into the goal box. One of the goal boxes contained a receptive female, the goal box on the opposite side contained a non-cycling female, and the other two boxes remained empty. Rats were first placed in the empty X-maze and allowed 10 minutes to explore the maze. Rats then received two days of conditioning in the X-maze until they chose the female s chamber on at least 50% of the trials. At the beginning of each conditioning day the male was placed in the female's compartment until he achieved an initial intromission. He was then placed into the center of the maze, and subsequent arm choices were recorded. A line of tape was placed at the end of each arm and if the male crossed this tape marker with both front paws, the Plexiglas door was raised and he was allowed inside the chamber. If the goal box contained the non-cycling female or was empty, he remained in the box for 30s before being placed in the center to start a new trial. If the male chose the receptive female s chamber, he was allowed one intromission or 5 minutes in the box if he failed to intromit. The male was then placed in the center of the maze again to start a new trial. The direction in which the male faced when placed into the center of the maze was alternated for each trial. All tests were terminated after the male ejaculated, after 25 trials if the male failed to ejaculate, or after 3 trials where the male chose the receptive female s chamber but failed to copulate within 5 minutes. The day after conditioning trials ended, rats received a final test that was timed and video recorded to determine their sexual motivation. During the test, rats were not given an initial intromission, but placed directly in the 10

12 center of the maze and latency to choose any arm was recorded. Aside from this, conditioning and test trials were conducted the same. Rats then received a 7 day abstinence period where they received no sexual experience. A second test was given after the 7 day abstinence period to determine whether sexual motivation was altered as a result of abstinence from sexual reward. This test was video recorded and conducted exactly the same as the previous test. Novelty-induced Hypophagia and Open Field Tests The Novelty-induced Hypophagia (NIH) test measures both anxiety- and depressive-like behaviors in rodents by measuring latency to consume a familiar palatable food in a novel environment (Dulawa & Hen, 2005). The protocol for the novelty-induced hypophagia test was adapted from Onksen et al All rats were given access to a palatable food (Reese s peanut butter chips) in plastic weigh boat in their home cage for 3 consecutive days. On the fourth day, rats were again given access to the palatable food and latency to consume the food in their home cage which was observed. On the fifth day, rats in the experienced group were given sexual experience to one ejaculation and 30 minutes later were placed in an open field box with the familiar palatable food in the center. Each session was video recorded to determine the latency of each rat to begin feeding and lasted 10 minutes so that anxiety-like behaviors (center time and locomotor activity) could be analyzed. Results Upon comparing the mean preference for 1% sucrose for experiment 1 using an independent samples t-test, no significant differences were found between naïve and experienced groups following sexual experience, one week of abstinence, or two weeks of abstinence (all p- values>0.05). In experiment 2, no significant differences were found between naïve rats and 11

13 sexually experienced rats 24 hours or 48 hours after sexual experience with 0.075% sucrose and 0.125% sucrose (all p-values>0.05). However, a trend can be observed when comparing the mean preference for 0.25% sucrose at the first (t(1,16)=2.002, p = 0.063) (mean±sem: naïve = ± 3.09 percent preference; experienced = ± 5.05 percent preference) and second (t(1,16)=2.106, p = 0.051) (mean±sem: naïve = ± 2.25 percent preference; experienced = ± 5.53 percent preference) 24 hour intervals following sexual experience, with the sexually experienced rats having a reduced sucrose preference compared to the naïve rats (Figure 1) No significant differences between groups were seen at any other time points following 48 hours after sexual experience (all p values > 0.05). Upon analyzing the novelty-induced hypophagia data with an independent samples t-test, there was no significant difference in the amount of food consumed between the naïve group and the experienced group in the home cage test. However, significant differences were found when comparing the mean amount of food consumed in the novelty setting for both the naïve group (t(1,8.236)=-2.852, p<0.05) (mean±sem = ± percent consumption) and the sexually experienced group (mean±sem = ± 1.85 percent consumption) (Figure 2A). Similarly, no significance between naïve and experienced groups was found when comparing the mean latency to begin consumption of the palatable food in the home cage test, while significance was found when comparing the mean latency to begin consumption of the food in the anxiety-provoking novelty setting for the naïve group (t(1,16) = 4.031, p = 0.001) (mean±sem = ± s)and the sexually experienced group (mean±sem = ± s) (Figure 2B). Paired sample t-tests were used to analyze the X-maze results. No significant differences were found when comparing the mean latencies to reach the receptive female s chamber, to mount, to intromit, or to ejaculate before and after abstinence from sexual experience (all p 12

14 values > 0.05) (Figure 3B). There were also no significant differences observed when comparing the mean mount or intromission frequencies before and after abstinence (p>0.05). However, a trend exists when comparing the intromission interval (measurement of how long a rat takes between each intermission = ejaculation latency/number of intromissions) before (mean±sem = ± 9.31 s) and after (mean±sem = ± s) (t(1,7)=2.081; p = 0.076) abstinence (Figure 3C). There were also no significant differences found when comparing the percent preference for the female s chamber before (mean±sem = ± 5.33 percent preference) and after (mean±sem = ± 4.48 percent preference) abstinence (Figure 3A). An independent sample t-test revealed that there was no significant difference in the mean time spent in the open arms between naïve (mean±sem = ± 9.94 s) and experienced (mean±sem = ± s) (t(1,16)=-1.053; p>0.05) groups. However, a trend may exist when comparing the mean time spent in the closed arms between naïve (mean±sem = ± s) and experienced (mean± SEM = ± 24.58) (t(1,16)=1.562; p=0.138) animals (Figure 4). Discussion In these experiments we examined whether previous mating experience or mating abstinence would affect motivation for another natural reward (sucrose). We looked at possible anxiety- and depressive-like behaviors as a result of sexual experience or sexual abstinence and found that while neither recent mating nor mating abstinence are sufficient to alter preference of 1% sucrose solution, at lower concentrations (such as 0.25% sucrose), evidence suggests that acute mating abstinence causes a trend for a decrease in sucrose consumption, and therefore an increase in depressive-like behaviors. The experiments also verified the stress-buffering 13

15 capabilities of sexual experience and suggest that these effects may last as long as two weeks after mating. The current study found that previous mating experience and mating abstinence did not cause a significant change in preference for 1% sucrose solution compared to controls; however, when 0.25% sucrose was given to sexually experienced rats and measured at 24 hour intervals post-mating, sexually experienced rats exhibited a decreased preference as abstinence increased, which suggests that abstinence from sexual behavior increases depression-like behaviors. Previous studies have shown that abstinence from sexual experience causes increases in corticotropin releasing hormone (CRH) and that previously mated rats who received 28 days of sexual abstinence exhibit more floating behaviors and less swimming behaviors in the forced swim test (Sebastiano et al. 2011). While these findings suggest that abstinence from mating may cause anxiety- and depressive-like behaviors, no significant differences were found in sucrose preference when comparing rats experiencing 28 days of sexual abstinence with naïve controls (Sebastiano et al. 2012). However, the current study showed that an abstinence period as short as 48 hours was sufficient enough to cause a decrease in sucrose preference compared to naïve controls, suggesting that 28 days may be too long of an abstinence period to see the same effect. These experiments also verified that sexual experience interacts with stress areas of the brain to decrease stress reactivity. In the novelty-induced hypophagia test, rats needed to overcome their fear and anxiety responses to obtain the highly palatable food reward at the center of the novel environment (the open field). Both the sexually experienced and naïve groups exhibited an increased latency to consume the food in the open field compared to their original home cage latencies; however, the sexually experienced rats began consumption more quickly than the naïve rats and consumed more of the food reward. These findings are not only consistent 14

16 with previous findings from our lab (McHenry 2012), but also suggest that sexual behavior increases motivation for reward, even in anxiety-provoking situations. The stress-dampening capabilities of sexual behavior may also be long-lasting. Even though sexually abstinent rats received 14 days without sexual experience, they still had a trend toward exhibiting fewer anxiety-like behaviors in the elevated plus maze, including more time spent in the open arms and less time spent in the closed arms. This is consistent with findings from another study showing that snacking on sucrose dampens stress responsiveness and causes neuroplastic changes in the brain that are still present up to 21 days after last sucrose experience (Christiansen et al 2011). The study showed that deltafosb, a protein implicated in neuronal plasticity in the brain, was present in the basolateral amygdala and the nucleus accumbens 1 day, 6 days, and 21 days after last sucrose experience. DeltaFosB is also activated during sexual behavior (Wallace et al 2008), which implies that sexual experience also induces neuronal plasticity in the brain and thus may cause long-lasting stress attenuation even during abstinence from mating. The current study showed that abstinence from sexual behavior does not significantly alter sexual motivation, though it may decrease copulation efficiency. No changes in female preference or latencies to reach the receptive female, to mount, to intromit, or to ejaculate were observed; however, intromission interval (measure of copulation efficiency calculated by dividing the ejaculation latency by the total number of intromissions) was slightly increased in the rats post-abstinence. Differences in female preference and sexual motivation may not have been observed due to individual differences in copulatory behavior across the group. A recent study showed that rats with either a high-novelty seeking phenotype or a low-novelty seeking phenotype differ significantly in mating behavior (Cummings & Clinton et al 2013). The study 15

17 showed that bred-high responders (bhrs; rats selectively bred for high-novelty exploration) exhibited more efficient copulatory behavior than the bred-low responders (blr; rats characterized by increased anxiety-like behaviors). Since there was variability in the sexual performance of the rats in this study during X-maze testing, there may be individual effects of anxiety phenotype on overall copulation behavior. Data from this experiment is currently being analyzed to look for correlations between copulation behavior, sucrose consumption, and anxiety-like behaviors. A number of specific strengths are evident in the current study. First, naïve and sexually experienced males were separately housed to assure that naïve rats were not exposed to female odors from the sexually experienced rats. Also, well-validated behavioral measures to test for anxiety- and depressive-like behaviors in rodents were used in these experiments, such as the elevated plus maze (Montgomery, 1955), the sucrose preference test (Willner et al, 1992), and the novelty induced hypophagia test (Dulawa & Hen, 2005). Another strength observed in this study is the use of increasing concentrations of sucrose across various time points post-mating in order to determine whether changes in sucrose preference are dose-dependent. An additional strength is the modification of the sexual behavior paradigm to one ejaculation on days where mating would be followed by an additional behavioral measure. This ensured that any effects that resulted from mating would be standardized across the group and that no extra effects were seen due to additional ejaculations. This study was not without limitations, a key limit being that anxiety- and depressive-like behaviors as a result of mating and abstinence were tested only in male rats. Sex differences in stress responsiveness, as well as prevalence of affective disorders and related behaviors do exist between males and females in both rats and in humans (Goel & Bale, 2010). One study showed 16

18 that acute restraint stress induced greater c-fos expression in the PVN in male rats than in female rats (Sterrenburg et al, 2012). Sex differences are also seen in sexual behavior and sexual motivation between male and female rats (Cummings & Becker, 2012), demonstrating another reason to look at females in addition to males in this experiment. A final limitation is the absence of protein or cell expression analysis in the brains of these rats to determine whether specific proteins are expressed or unexpressed as a result of sexual experience or sucrose experience. Analysis of brain tissue may be able to tell us whether we are seeing an effect even when no significant differences were seen in behavioral tests. The results of the current study suggest several implications for further research. Brain tissue collected from sexually experienced rats versus sexually abstained rats may be analyzed to look at specific expression of proteins involved in reward and anhedonia. For example, dopamine and serotonin are the primary neurotransmitters released during rewarding experiences, while orexin is known to be decreased in rats exhibiting anhedonic symptoms (Harris & Ashton-Jones, 2006). Orexin is of particular interest because it interacts with dopamine neurons from the ventral tegmental area. As a result, orexin may play a major role in the decrease of motivation for rewarding experiences seen in depression in both rats and in humans (Harris & Ashton-Jones, 2006). In addition, recent studies have shown a decrease in orexin peptide levels in the mesolimbic dopamine system, which is important for motivation and reward, in rats displaying both decreased preference for sucrose and decreased sexual interests (Nocjar et al, 2012). Stress also activates orexin neurons: CRF excites orexin neurons and is thought to play a role in maintenance of arousal during stressful situations or events (Harris & Ashton-Jones, 2006). Orexin neurons are also activated with mating behavior (Muschamp et al 17

19 2007), thus orexin may play an important role in the integration of anxiety/depression circuits with areas of reward. In conclusion, mating, anxiety, and depression are highly integrated in the brain circuitry of humans and animals. This study found that mating history effects both HPA activation and motivation for other natural rewards, as evidenced by the decreased latency of sexually experienced rats to consume a food reward in an anxiety-provoking novel setting. There also may be preliminary evidence to suggest that abstention from mating results in depressive-like behaviors including a decreased motivation for sucrose, while none of the experiments were able to conclude that abstinence from sexual experience increases anxiety-like behaviors. Future studies may focus on learning more about the proteins implicated in these behaviors in order to better understand the relationship between the neural circuits implicated in depression, anxiety, and reward. 18

20 References 1. Arendt DH, Ronan PJ, Oliver KD, Callahan LB, Summers TR, Summers CH. (2013) Depressive behavior and activation of the orexin/hypocretin system. Behav Neurosci. 127(1): Buwalda B, Scholte J, de Boer SF, Coppens CM, Koolhaas JM. (2012) The acute glucocorticoid stress response does not differentiate between rewarding and aversive social stimuli in rats. Horm Behav. 61(2): Casper RC, Redmond DE Jr, Katz MM, Schaffer CB, Davis JM, Koslow SH. (1958) Somatic symptoms in primary affective disorder. Presence and relationship to the classification of depression. Arch Gen Psychiatry. (11): Christiansen AM, Dekloet AD, Ulrich-Lai YM, Herman JP. (2011) "Snacking" causes long term attenuation of HPA axis stress responses and enhancement of brain FosB/deltaFosB expression in rats. Physiol Behav. 103(1): Cummings JA, Becker JB. (2012) Quantitative assessment of female sexual motivation in the rat: Hormonal control of motivation. J Neurosci Methods. 204(2): Cummings JA, Clinton SM, Perry AN, Akil H, Becker JB. (2013) Male rats that differ in novelty exploration demonstrate distinct patterns of sexual behavior. Neurosci. 127(1): Di Sebastiano AR, Coolen LM. (2011) Depression-like behavior following abstinence from sexual behavior in male rats is mediated by activation of corticotrophin releasing factor receptors. 41 st annual meeting Society for Neuroscience. Washington DC 19

21 8. Di Sebastiano AR, Coolen LM. (2012) Loss of Sexual Reward Causes Depression-like Behaviors Including Passive Stress Coping and Anhedonia in Male Rats.16 th annual meeting Society for Behavioral Neuroendocrinology. Maddison Wisconsin. 9. Dulawa SC, Hen R. (2005) Recent advances in animal models of chronic antidepressant effects: the novelty-induced hypophagia test. Neurosci Biobehav Rev. 29(4-5): Duncko R, Kiss A, Skultétyová I, Rusnák M, Jezová D. (2001) Corticotropin-releasing hormone mrna levels in response to chronic mild stress rise in male but not in female rats while tyrosine hydroxylase mrna levels decrease in both sexes. Psychoneuroendocrinology. 26(1): Goel N, Bale TL. (2006) Sex differences in the serotonergic influence on the hypothalamic-pituitary-adrenal stress axis. Endocrinology Apr;151(4): Harris GC, Aston-Jones G. Arousal and reward: a dichotomy in orexin function. Trends Neurosci. 29(10): Heinrichs M, Baumgartner T, Kirschbaum C, Ehlert U. (2003) Social support and oxytocin interact to suppress cortisol and subjective responses to psychosocial stress. Biol Psychiatry. 54(12): McHenry JA, Hull EM. (2012) Neural Mechanisms of Mating-Induced Anxiolysis and Stress Buffering: Exploring the Potential Role of MPOA-PVN Interactions in Male Rats. 16 th annual meeting Society for Behavioral Neuroendocrinology. Maddison Wisconsin. 15. McHenry JA, Vitale EM, Hull EM. (2012) Neural mechanisms of mating-induced anxiolysis and stress buffering: Examining neuromodulators in the MPOA and PVN of male rats. 42 nd annual meeting of Society for Neuroscience. New Orleans, Louisiana. 20

22 16. Medina A, Seasholtz AF, Sharma V, Burke S, Bunney W Jr, Myers RM, SchatzbergA, Akil H, Watson SJ. (2013) Glucocorticoid and mineralocorticoid receptor expression in the human hippocampus in major depressive disorder. J Psychiatr Res. 47(3): Montgomery KC. (1955) The relation between fear induced by novel stimulation and exploratory behavior. J Comp Physiol Psychol. 48(4): Muschamp JW, Dominguez JM, Sato SM, Shen RY, Hull EM. (2007) A role for hypocretin(orexin) in male sexual behavior. J Neurosci. 27(11): Nocjar C, Zhang J, Feng P, Panksepp J. (2012) The social defeat animal model of depression shows diminished levels of orexin in mesocortical regions of the dopamine system, and of dynorphin and orexin in the hypothalamus. Neuroscience. 218: Onksen JL, Briand LA, Galante RJ, Pack AI, Blendy JA. (2012) Running-induced anxiety is dependent on increases in hippocampal neurogenesis. Genes Brain Behav. 11(5): Sterrenburg L, Gaszner B, Boerrigter J, Santbergen L, Bramini M, Roubos EW,Peeters BW, Kozicz T. (2012) Sex-dependent and differential responses to acute restraint stress of corticotropin-releasing factor-producing neurons in the rat paraventricular nucleus, central amygdala, and bed nucleus of the striaterminalis. J Neurosci Res. 90(1): Ulrich-Lai YM, Christiansen AM, Ostrander MM, Jones AA, Jones KR, Choi DC,Krause EG, Evanson NK, Furay AR, Davis JF, Solomon MB, de Kloet AD, Tamashiro KL, Sakai RR, Seeley RJ, Woods SC, Herman JP. (2010) Pleasurable behaviors reduce stress via brain reward pathways. ProcNatlAcadSci U S A. 107(47): Ulrich-Lai YM, Herman JP. (2009) Neural regulation of endocrine and autonomic stress responses. Nat Rev Neurosci. 10(6):

23 24. Wallace DL, Vialou V, Rios L, Carle-Florence TL, Chakravarty S, Kumar A,Graham DL, Green TA, Kirk A, Iñiguez SD, Perrotti LI, Barrot M, DiLeone RJ, Nestler EJ, Bolaños-Guzmán CA. (2008) The influence of DeltaFosB in the nucleus accumbens on natural reward-related behavior. J Neurosci. 28(41): Warner RK, Thompson JT, Markowski VP, Loucks JA, Bazzett TJ, Eaton RC, Hull 26. EM. (1991) Microinjection of the dopamine antagonist cis-flupenthixol into the MPOA impairs copulation, penile reflexes and sexual motivation in male rats. Brain Res. 540(1-2): Willner P, Muscat R, Papp M. (1992) Chronic mild stress-induced anhedonia: a realistic animal model of depression. NeurosciBiobehav Rev. 16(4):

24 Figure Legend Figure 1: Preference for 0.25% sucrose in subjects (n=9/group). Experienced and Naïve rats differed slightly in preference for 0.25% at the first 24 hours (p = 0.063) and the second 24 hours (p = 0.051) after sexual experience. Figure 2: Results in the Novelty-induced Hypophagia test (n=9/group). (Figure 2B) Experienced rats consumed a greater percentage of the food reward than Naïve rats (Note: Asterisk (*) indicates p<0.05). (Figure 2A) Experienced rats exhibited a decreased latency to approach and consume food reward compared to Naïve rats (Note: Double Asterisk (**) indicates p<0.01). Figure 3: Performance on X-Maze test (n=9/group). Rats did not show a significant difference in preference for a receptive female before and after one week of mating abstinence (p = 0.279) (Figure 3A). Rats did not show a significant difference in latencies to reach the receptive female, to intromit, or to ejaculate (all p values >0.05) (Figure 3B). Rats showed a trend when comparing intromission interval (average time between intromissions) before and after mating abstinence (p = 0.076) (Figure 3C). Figure 4: Results of the Elevated Plus Maze (n=9/group). Rats in the sexually experienceabstinence group did not differ significantly in time spent in the open arms (p>0.05) or time spent in the closed arms (p = 0.138) compared to naïve controls 23

25 Figure 1 Figure 2 A. 24

26 B. Figure 3 A. 25

27 B. C. 26

28 Figure 4 27