דוקטור לפילוסופיה Doctor of Philosophy

עבודת גמר )תזה( לתואר Thesis for the degree דוקטור לפילוסופיה Doctor of Philosophy

מוגשת למועצה המדעית של Submitted to the Scientific Council of the מכון ויצמן למדע Weizmann Institute of Science רחובות, ישראל Rehovot, Israel

מאת By ימית בני שפר Yamit Beny Shefer

חקר המנגנון הנוירוביולוגי המבקר העדפה מינית המתווכת ע"י פרומונים בעכברים זכרים

Exploring the neurobiology mechanism regulating pheromone-mediated sexual preference in male mice

מנחה: :Advisor פרופ' טלי קמחי Prof. Tali Kimchi

ניסן התשע"ז March 2017

Acknowledgements

This Ph.D. thesis would not have been possible without the help of many people, to whom I wish to express my appreciation. First and foremost, I would like to express my gratitude to my Principal Investigator Prof. Tali Kimchi for the useful comments, and support and for guiding me during my PhD studies. Moreover, I would like to thank my current and former lab members for their support, assistance and the fun we had in the last years: Dr. Molly Dayan, Dr. Yael Lavi-Avnon, Dr. Niv scott, Dr. Einat Elharrar, Yefim Pen, Nadav Bezalel, and Efi Massasa. And especially Dr. Noga Zilkha, Itsik Sofer, and Dana Rubi Levi, who turned into true friends. Special thanks to Dr. Assaf Ramot for his priceless assistance with the optogenetic experiments; Dr. Ofer Yizhar and Prof. Alon Chen for sharing equipment and viruses; and to all the supportive units in the Weizmann Institute of Science: especially to Sharon Ovadia, Omri Meir, Tzofar Hajbi and Beni Siani from the MMTK animal facility, for taking good care of me and my mice; and Alex Jahanfard from the engineering and instrumentation branch for fulfilling all my requests in a flash and always with a smile. Finally, my dear parents, Zahala and Rony, my siblings Nir and Daniella, and my grandmother Lea, thank you for your endless support and love during my years of study, this journey would not have been possible without you! Last but not least, I wish to thank my husband Roy for always being there for me, providing me with strength and lifting my spirit.

Declaration I am declaring that this thesis summarizes my independent research. All experiments in this projected were planned, executed or conducted by me. Exceptional are the in-vivo microdialysis experiments (Fig. 9c,d) and measuring dopamine content in the nucleus accumbens (Fig. 10b) which were planned and conducted by Dr. Yael Lavi-Avnon and Dr. Molly Dayan from the lab. Dr. Tali Kimchi guided and supervised all the experiments in this thesis.

Table of contents

List of abbreviations ______4

Abstract ______5

1. Introduction ______7 1.1. Sexual behavior in rodents ______7 1.2. What are pheromones? ______7 1.3. The reward system and sexual behavior ______9 1.4. Experience-dependent plasticity in innate sexual behaviors ______10 1.5. TrpC2 knock-out mouse model ______11

2. Research goals ______13

3. Methods ______14 3.1. Animals ______14 3.2. Conditioned odor aversion (COA) to conspecific female odors ______14 3.3. Conditioned odor aversion (COA) to male odors ______18 3.4. Sucrose preference test ______18 3.5. RNA purification, cDNA synthesis and qPCR ______18 3.6. D1R-antagonist microinjections ______18 3.7. VTA-NAc dopaminergic neurons activation ______20 3.8. Statistical analysis ______21

4. Results ______22 4.1. Female-specific COA impairs innate precopulatory and copulatory responses in WT males ______22 4.2. Male-specific COA does not alter olfactory preference ______29 4.3. TrpC2-/- males display diminished responses towards sexual rewards ______31 4.4. Female-specific COA impairs precopulatory and copulatory behaviors in TrpC2-/- males (Beny & Kimchi, 2016) _ 33 4.5. Blocking D1R in the NAc disrupts sexual preference ______37 4.6. Optogenetic activation of VTA-NAc dopaminergic neurons in TrpC2-/- males ______39

5. Discussion ______47 5.1. Flexibility in innate sexual behaviors through olfactory learning ______47 5.2. VNO inputs mediate the innate rewarding value of female cues ______49 5.3. NAc dopamine signaling through D1R is required for pheromone-induced place preference ______50 5.4. Targeting VTA to NAc projections rescued sexual preference of TrpC2-/- males ______51

6. References ______54

List of abbreviations

AAV- Adeno associated virus MeA- Medial amygdala

AOB- Accessory olfactory bulb MOE- Main olfactory epithelium

BLA- Basolateral amygdala mPFC- Medial prefrontal cortex

BNST- Bed nucleus of the stria terminalis MSN- Medium spiny neurons

ChR2- Channelrhodopsin 2 MUP- Major urinary protein

COA- Conditioned odor aversion NAc- Nucleus accumbens

COA- Cortical amygdala OCNC1- Olfactory specific cyclic nucleotide- gated channel CPP- Conditioned place preference OR- Olfactory receptors Cre- Cre recombinase OT- Olfactory tubercle D1R-A- Dopamine receptor antagonist PBS- Phosphate buffered saline DA- Dopamine PFA- Paraformaldehyde DAT- Dopamine transporter TrpC2- transient receptor potential channel DIO- Double-floxed inverted open reading frame USV- Ultrasonic vocalization

EPM- Elevated plus maze VNO- Vomeronasal organ

KO- Knockout VR- Vomeronasal receptor

LiCl- Lithium Chloride VTA- Ventral tagmental ares

WT- Wild-type

Abstract

Male sexual acts are highly rewarding action patterns, controlled by hard-wired brain circuits, and activated in response to external sensory stimuli, without the need for previous learning. Female pheromones, which are detected by the vomeronasal organ (VNO) located in the nasal cavity of the mouse, elicit goal-directed approach behavior in naïve male mice (appetitive phase), which leads to the sexual act. Moreover they are strongly attractive to males, and possess intrinsic rewarding value, stimulating the mesolimbic dopamine reward pathway, which connects the ventral tegmental are (VTA) to the nucleus accumbens (NAc). Pheromone signal transduction through the VNO requires the TrpC2 ion channel. TrpC2 mutant (TrpC2-/-) males do not present sexual preference for female over male odors and do not exhibit NAc dopamine increase following exposure to receptive female, typically seen in wild- type males. The main goal of this study was to explore experience-dependent plasticity in neuronal circuits underlying pheromone-mediated sexual responses, and to elucidate the role of dopamine in mediating the appetitive phase of sexual behavior. We found that innate sexual-preference towards female odors can be modified throughout life following a negative experience associated with female pheromones. Moreover, since TrpC2-/- males acquired specific aversion to female odors and did not generalize the aversion to male odors, we propose that VNO-mediated inputs are not necessary for sexual discrimination. Next, by selectively blocking the dopamine D1 receptor in the NAc of wild-type males we prevented the formation of a conditioned place preference to female pheromones and impaired sexual preference for female over male odors. Finally, optogenetic activation of VTA dopaminergic projections to the NAc, restored the lost sexual preference of TrpC2-/- males to female odors and increased sexual behavior. Together, our results suggest that VNO- mediated inputs are necessary for assigning an innate incentive value to female signals. The process appears to be mediated by D1-type dopamine receptor signaling in the NAc, which promotes reward- seeking behavior and sexual preference for female cues.

תקציר

התנהגות מינית האופיינית לזכרים הינה התנהגות סטריאוטיפית ומתגמלת, שנשלטת על ידי מסלולים מוחיים מולדים, ומופעלת בתגובה לגירויים סנסורים חיצוניים כמו פרומונים נקביים. פרומונים המופרשים על ידי נקבות נקלטים באיבר יעקובסון )VNO( הממוקם בחלל האף של העכבר, ומעוררים העדפה מינית לנקבות אצל זכרים נאיביים, המובילות להתנהגות המינית. בנוסף יש לפרומונים נקביים ערך מתגמל גבוה והם מפעילים את המערכת המזולימבית במוח הזכרי הכוללת תאי עצב דופמינרגיים שנמצאים בטגמנטום הגחוני )VTA( ומגיעים לגרעין האקומבנס )NAc(. העברת סיגנלים דרך ה- VNO, תלויה בתעלת ה- TrpC2. לזכרים בעלי מוטציה בגן המקודד לתעלה זו )-/TrpC2-( אין העדפה מינית מולדת לסיגנליים נקביים על פני זכריים ולא נמדדה אצלם עלייה בכמות דופמין ב- NAc לאחר חשיפה לנקבה, שאופיינית לעכברי wild-type. המטרה העיקרית של עבודת גמר זו הינה לחקור האם מתרחשים תהליכים פלסטיים במסלולים מוחיים המתווכחים התנהגות מינית ואת המעורבות של המערכת הדופמינרגית בהוצאה לפועל של העדפה מינית בקרב עכברים זכרים. התוצאות שלנו מראות שהתנהגויות מולדות, כמו העדפה מינית, הינם פלסטיות ויכולות לעבור שינוי בעקבות חוויה אברסיבית המקושרת לפרומונים נקביים. בנוסף, מכיוון שעכברי -/TrpC2- למדו אברסיה ספציפית לנקבות ולא הכלילו אותה על זכרים, אנחנו מסיקים שאותות של פרומונים המגיעים דרך ה- VNO אינם הכרחיים להבדלה בין המינים. כמו כן, כאשר אנחנו חוסמים את הקליטה של דופמין ב- NAc על ידי חסימה של הקולטן שלו D1R, אנחנו מונעים למידה של העדפת מקום המקושר עם סיגנלים נקביים, ופוגעים בהעדפה המינית לנקבה. לבסוף, על ידי הפעלה אופטוגנטית של תאי עצב דופמינרגיים מה-VTA המעצבבים את ה-NAc, הצלחנו להחזיר לעכברי -/TrpC2- את ההעדפה לפרומונים נקביים ולגרום לעלייה בפעילות המינית שלהם. לסיכום, עבודת הגמר שמוצגת לפניכם מראה שפרומונים שנקלטים דרך ה-VNO, הכרחיים על מנת להקנות לפרומונים הנקביים ערך מתגמל חיובי בקרב הזכרים. אנחנו מציעים שתהליך זה מתבצע על ידי שחרור של דופמין ל-NAc, והקשרות לקולטן שלו, D1R, והינו קריטי והכרחי על מנת שנראה התנהגות של העדפה מינית ומשיכה של זכרים לנקבות.

1. Introduction

1.1. Sexual behavior in rodents Why do animals mate? The typical ultimate cause is to produce offspring. However, since the pregnancy rate following mating, both in rodents and humans are low (Agmo, 1999), the answer might be different. The proximal cause might be that mating is simply an instinctive response to an opposite-sex stimulus, which occurs under specific physiological state. This response further activates motivational systems in the brain to drive the behavior (Meisel & Mullins, 2006). Sexual behavior is one of the most complex and intriguing innate behaviors, presented in a sexually dimorphic manner, across many animal species, including humans. In rodents, the sexual act is commonly divided into two distinct and progressive phases: the appetitive (precopulatory) and consummatory phases (Hashikawa et al., 2016). The precopulatory phase is mainly characterized by sexual preference to the opposite sex, manifested in the motivation to approach and engage in extensive olfactory investigation, and omission of ultrasonic vocalizations (USV) to attract the opposite sex. These actions lead to the consummatory phase, which in males includes mounting, intromissions, and ejaculation (Veening & Coolen, 2014), and in females the lordosis posture (Neunuebel et al., 2015, Veening et al., 2014). The organization/activation model of sexual differentiation is the main hypothesis underlying the development of innate sexually dimorphic sexual behavior (Alexander et al., 2011). According to this model, hard-wired, sexually dimorphic brain circuits are shaped early in development, when the brain is differentiating in the presence of specific sex steroids, emerging in accordance to the genetic sex (XX versus XY). In adulthood, those sex steroids activate the differentiated brain circuits to produce sexually dimorphic behaviors. The permanent developmental effects are named “organizational”, while the later, transient effects “activational” (Sisk, 2016, Yang & Shah, 2014). Sexual behavior is triggered also by external cues. In mammals, sex-specific pheromonal signals secreted from the animal, attracts the opposite sex, initiating behavioral and endocrinological responses, which increases the likelihood of successful sexual act (Stowers & Liberles, 2016).

1.2. What are pheromones? Pheromones are complex chemosignals used for social communication among a large variety of species, from single cell bacteria to mammals (Wyatt, 2009) and humans (Gelstein et al., 2011). In mammals, specific pheromones, secreted by one individual, convey various information regarding its species, age, sex (Cheetham et al., 2007, He et al., 2008, Isogai et al., 2011), reproductive/endocrine state, familiarity, and social status (Ben-Shaul et al., 2010, Bergan et al., 2014, Hurst & Beynon, 2004). For example, the sulfated steroid, 17β-diol disulphate (E1050), has been shown to signal estrus females

7 and promotes mounting behavior in male mice (Haga-Yamanaka et al., 2014, Nodari et al., 2008). Pheromones differ in their chemical properties, as some are small volatile molecules exerting their effect from distance, and others are larger, non-volatile molecules like peptides and proteins (Stowers & Kuo, 2015). Darcin, a nonvolatile, major urinary protein (MUP20), abundantly secreted by male mice, was found to innately attract females, and induce learned preference to the previously unattractive volatile urinary odors (Roberts et al., 2012, Roberts et al., 2010). Moreover, pheromones elicit a variety of endocrinological and innate behavioral reactions depended on the context and the receiver animal (Stowers & Liberles, 2016, Wyatt, 2010). For example, dehydro-exo-brevicomin (brevicomin) and 2-(sec- butyl)-4,5-dihydrothiazole (thiazole) are volatile testosterone-dependent pheromones that are found in male urine and evoke immediate, stereotypical male-male aggressive responses (Novotny, 2003). In contrast, when detected by females, the same pheromones exert delayed responses through activation of the neuroendocrine system, such as acceleration of puberty onset and induction of estrus (Whitten effect) (Jemiolo et al., 1986, Novotny et al., 1999). Pheromones are emitted to the outside by the releaser animal, and detected by olfactory sensory organs specific to the recipient animal. Rodents detect pheromones through olfactory receptors (OR) and vomeronasal receptors (VR) found in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO), respectively (Dulac & Wagner, 2006a). From the periphery, the pheromonal cue is further processed in different brain areas. MOE-derived signals are transmitted to the main olfactory bulb, and further to third-order nuclei such as the piriform cortex and cortical amygdala (Root et al., 2014, Stowers & Liberles, 2016). In contrast, VNO-detected pheromones reach the accessory olfactory bulb and relay further to limbic areas as the medial amygdala (MeA) and posteromedial cortical amygdala (Beny & Kimchi, 2014, Cádiz-Moretti et al., 2013). Finally, the medial and cortical amygdaloid areas project to the medial hypothalamus, which plays an essential role in initiation of different innate behavioral responses (Hashikawa et al., 2016, Sokolowski & Corbin, 2012). Pheromones mediate a range of innate appetitive and aversive responses, such as sexual, aggressive and maternal behaviors and fear and anxiety-like behaviors, in naïve animals without the need of previous learning (Brennan & Zufall, 2006, Stowers & Marton, 2005). More specifically, female pheromones elicit a set of copulatory behavioral and physiological responses in sexually naïve males (Amstislavskaya et al., 2013, Goldey & Van Anders, 2014, Swaney et al., 2012), and may serve as unconditioned natural reinforcers inducing associative conditioned learning [reviewed in (Beny & Kimchi, 2014, Griffiths & Brennan, 2015, Pfaus et al., 2001)]. For example, male mice emit courtship 70-kHz USV as a response to receptive female and her odors, as part of courtship behavior leading to mating (Nyby et al., 1977). When artificial odors were paired with access to receptive female, those odors alone elicited the USV, via acquisition of sex-signaling properties (Nyby et al., 1978). Moreover, following association between a 8 neutral cue and female signals, the cue itself triggered endocrinological changes normally seen following an exposure to a female, such as elevations in luteinizing hormone and testosterone secretion (Graham & Desjardins, 1980).

1.3. The reward system and sexual behavior Sexual behavior is one of few natural rewards necessary for the survival and reproduction of the species (Hu, 2016). As was implied earlier, female pheromones have been shown to be intrinsically rewarding and strongly attractive to males (Bell et al., 2013, Beny & Kimchi, 2014, Trezza et al., 2011), eliciting anticipatory locomotion (Mendelson & Pfaus, 1989, Trezza et al., 2011) and preference to approach and investigate female over male stimuli (Ago et al., 2015, Brown, 1977). The nucleus accumbens (NAc), which is innervated by dopaminergic (DA) neurons of the ventral tegmental area (VTA), have a crucial role in sexual motivation and behavior (Brom et al., 2014, Pfaus & Phillips, 1991). A recent study compared expression patterns of the Fos protein, a molecular marker for neuronal activity, between c-fos-GFP male mice following 90 sec exposure to either male or female stimuli (Kim et al., 2015). This comparison revealed strong and specific female-induced activation in brain areas associated with behavioral motivation such as the olfactory tubercle (OT), NAc shell, and prefrontal cortical areas (Kim et al., 2015). In order to directly assess the role of dopamine in sexual reward studies are using in-vivo microdialysis and fast-scan cyclic voltammetry, which measures extracellular dopamine secretions (Fiorino et al., 1997, Wenkstern et al., 1993, Wightman & Robinson, 2002). Using microdialysis, Malkesman et al., showed that sexually naïve male mice present an increase in DA concentrations in the NAc following exposure to female urine (Malkesman et al., 2010). Moreover, fast-scan cyclic voltammetry studies demonstrated that naïve male rats exhibited transient DA elevations in the NAc when receptive females were presented, approached and investigated, all of which comprise the appetitive aspect of sexual behavior (Robinson et al., 2002, Robinson et al., 2001). Thus, this elevation in DA release appeared most robust during the initial exposure to receptive female and occurred alongside pre-copulatory behaviors such as olfactory investigation of the female. In the NAc there are medium spiny neurons (MSN) that express either the D1-type or D2-type dopamine receptors (Ikemoto et al., 1997). D1 and D2 dopamine receptors are two G-protein-coupled receptors which have distinct signaling cascades and targets and are thought to have different roles in modulating general reward (Gerfen & Surmeier, 2011). For example, administering the antagonist of D1R, but not D2R, into the NAc impairs reward-seeking behavior (Stuber et al., 2011, Young et al., 2014). With regards to sexual reward, intraperitoneal injection (i.p) of the selective D1-like agonist, but not D2-like, facilitated the expression of sexual behavior in male rats (Guadarrama-Bazante et al., 2014). Moreover, a recent study in male mice showed an increased signaling in MSNs expressing the D1R

9 among frequently mated males, while infrequently mated males exhibited an increased activity in D2- expressing MSNs (Goto et al., 2015). The role of NAc DA in pheromone-mediated sexual preference mainly focuses on the sexual preference of female subjects and is largely controversial. One study showed that innate preference for male odors was still present in female mice after blocking dopamine signaling via lesions of the VTA (Martinez-Hernandez et al., 2006) or administrating dopamine antagonists (Agustin-Pavon et al., 2007). However, a more recent work demonstrated that dopamine innervations to the NAc and medial OT are involved in opposite-sex urinary odor preference in female mice (Dibenedictis et al., 2014, Dibenedictis et al., 2015). In recent years, genetically defined, light-based control of neural circuits allowed a specific examination of the role of VTA dopaminergic neurons in reward and aversion (Adamantidis et al., 2011, Lammel et al., 2012, Steinberg et al., 2014, Tsai et al., 2009). Moreover, several studies have shown the involvement of midbrain dopaminergic neurons in social behaviors using optogenetic methods. For example, Yu et al., selectively expressed the light-gated ion channel channelrhodopsin2 (ChR2) in VTA dopaminergic neurons of Dopamine-transporter Cre-driver-line (DAT:Cre) males. Blue-light illumination of these neurons during male-male social encounter increased aggressive behavior duration (Yu et al., 2014). Additionally, optogenetic tools allow specific projection targeting by expressing the ChR2 in the region containing the cell bodies, and implanting the light-delivery device (optic fiber) above the region with the axonal projections. In one study, stimulation of VTA-NAc dopamine neuronal projections, but not VTA-medial prefrontal cortex (mPFC) projections during social interaction increased the duration of social interaction, and this effect was attenuated by injecting antagonist to the dopamine receptor D1, but not D2, in the NAc (Gunaydin et al., 2014). As of today, to our knowledge, viral-based optogenetic methods were not used to manipulate dopaminergic neurons in the VTA to test their role in pheromone- mediated precopulatory and copulatory sexual responses.

1.4. Experience-dependent plasticity in innate sexual behaviors After a successful sexual encounter, plasticity mechanisms occur that improve the sexual performance of the experienced male and contributes to the sexual act by increasing the chances to successfully mate and reproduce. For example, sexual experienced males emit higher USV as a response to female (Roullet et al., 2011), and show higher preference for female signals when other social signals are present (Pitchers et al., 2012). In addition sexual experience changes sensitivity to sexual rewards, as experienced males, but not naïve males, developed conditioned place preference (CPP) to a chamber that was previously associated with sexual intromissions (Tenk et al., 2009). As regards to consummatory behaviors, sexual experienced males have shorter mounting, intromission and ejaculation latency, even 7 days following the last sexual act (Pitchers et al., 2012). In female mice, following first exposure to

10 innately attractive non-volatile male pheromones, like darcin (MUP20), females develop acquired preference for previously unattractive volatile male pheromones (Moncho-Bogani et al., 2002, Roberts et al., 2012, Roberts et al., 2010). The adaptive value of this plasticity change is the increasing chance of detecting a mating partner from distance. Overall, sexual preference is considered innate, preprogrammed behavior, determined prenatally as the brain undergoes sexual differentiation under the control of sex steroids (Alexander et al., 2011). Stress and steroid hormonal exposure during prenatal period may interfere with brain sexual differentiation and may produce inversion in sexual preference of male rats and mice (Arnold & Breedlove, 1985, Dela Cruz & Pereira, 2012, Lupien et al., 2009, Meek et al., 2006, Popova et al., 2011). Thus, the innate rewarding nature of reproductive behaviors, suggests that negative environmental conditions, associated with these behaviors and experienced by the adult animal, will have a limited effect (if any) on their execution. However, there are some early studies conducted in male rodents, which demonstrate that taste or odor aversive conditioning to female stimuli could alter pre-copulatory aspects of sexual behavior. Aversive conditioning is induced by paring either a taste or an odor stimulus with unpleasant noxious feeling caused by lithium chloride (LiCl) injections (Yamamoto, 1993). For example, administering LiCl following copulation with an estrous female reduced sexual motivation in male rats (Agmo, 2002), and pairing ingestion of vaginal secretion with LiCl significantly reduced the attraction to the secretion and its consumption in male hamsters (Johnston & Zahorik, 1975, Johnston et al., 1978, Zahorik & Johnston, 1976). Similar experiments on male mice were performed by Kay and Nyby, which tested the effect of LiCl aversive conditioning on olfactory preference and ultrasonic vocalizations elicited by female urine. They reported that odor aversion to female urine caused only minor and transitory changes in these pre-copulatory behaviors (Kay & Nyby, 1992). Moreover, studies have shown that copulatory behaviors can also be affected by aversive conditioning as following pairings of LiCl and copulation with an estrous females, male rats ceased to copulate and reduced sexual behavior was witnessed (Agmo, 2002, Peters, 1983). Yet, the precise and long-term influence of negative experience on plasticity of innate reproductive behaviors and its limitations are less clear.

1.5. TrpC2 knock-out mouse model In rodents, increasing evidence indicates that chemical signals sensed via the MOE and the VNO function synergistically in the regulation of innate reproductive responses (Keller et al., 2009). Both olfactory systems use different signaling components to encode social chemosignals. In the MOE, the olfactory specific cyclic nucleotide-gated channel (OCNC1) is involved in the signaling cascade, and in the VNO, the transient receptor potential channel (TrpC2) (Dulac & Wagner, 2006b). In the VNO, pheromones are detected through V1 or V2 G-protein-coupled receptors; this binding eventually leads to

11 influx of Ca2+ through the TrpC2 channel and to cell excitation (Yu, 2015). Genetic silencing of VNO or MOE signaling is accomplished via knockout of those genes. TrpC2-/- mutant lab female mice display loss of sexual preference, deficits in sexual approach/olfactory investigations, and male-typical mounting behavior towards both sexes (Chalfin et al., 2014, Kimchi et al., 2007). Additionally, when examining inter-female aggressive behavior, manifested only by wild-backcrossed females, a null mutation in the TrpC2 gene resulted in a decrease in female-directed aggression (Chalfin et al., 2014). In accordance, TrpC2-/- males fail to initiate aggressive behavior towards a strange male mouse, unlike typical wild-type males. Instead, those mutant males display loss of sexual behavioral preference for females and heightened mounting behavior towards alien males (Leypold et al., 2002, Stowers et al., 2002). Furthermore, surgical ablation of the VNO system in sexually naïve males eliminates their preference for female urinary odors (Pankevich et al., 2004). This suggests that sexual olfactory preference and the motivation to approach and investigate potential mating partners are largely mediated by the VNO system. The exact role of the VNO in mediating sexual partner preference is still ambiguous.

2. Research goals

The main goal of this research is to explore the role of mesolimbic dopamine system in regulating VNO- mediated innate and conditioned copulatory behavioral response.

The specific objectives of the studies described in this thesis are:

1. To explore whether we can induce experience-dependent plasticity that will affect innate pheromone-mediated sexual responses of males. We developed a conditioned odor aversion (COA) paradigm, where we associated sex-specific pheromones with an aversive experience, and applied it on wild-type (WT) male mice. 2. To elucidate the role of the VNO in sex discrimination by applying female-specific COA on male mice impaired in VNO-mediated signal transduction (TrpC2-/-). 3. To study the role of NAc DA in encoding the rewarding properties of female pheromones. We applied loss-of-function approach using pharmacological tools. We injected antagonist of DA receptor D1, specifically to the NAc of WT males and examined the effects on acquisition of conditioned place preference to female pheromones. 4. To examine the direct role of VTA-NAc dopaminergic projections in mediating sexual preference by utilizing the TrpC2-/- transgenic mouse model. To that end we used in vivo optogenetic gain-of- function approach to directly excite VTA-NAc DA neurons in TrpC2-/- males and examine whether sexual preference can be rescued.

3. Methods 3.1. Animals For the aversive conditioning and pharmacology experiments, sexually naive adult (10-14 week-old) TrpC2-/- laboratory mice and their wild-type (WT) littermates were used as previously described (Chalfin et al., 2014). The genetic background of the TrpC2 strain is C57BL/6J x 129S1/Sv. For the optogenetic experiments, dopamine transporter (DAT)::IRES-Cre transgenic C57BL/6J mice (Backman et al., 2006) were obtained from Jackson laboratories and mated with TrpC2 mice to generate DATCre+/TrpC2-/-, DATCre+/TrpC2+/+ and DATCre+/TrpC2-/- mice. All mice were bred under standard pathogen-free laboratory conditions with food and water ad libitum. Two weeks before the initiation of behavioral assays, mice were housed in single cages, and were transferred to a reversed 12/12 hours light/dark cycle (lights off at 10 AM). All behavioral procedures were performed during the dark phase under dim red light, except for the motivation assay and anxiety-related assays that were conducted under dim white light. The Institutional Animal Care and Use Committee of the Weizmann Institute of Science approved all experimental procedures.

3.2. Conditioned odor aversion (COA) to conspecific female odors The COA protocol included the following experimental stages (Fig. 1a): Male mice were tested for their baseline olfactory preference on days 1-2 of the experiment. Three conditioning sessions were performed on days 4-6 and a forth was performed on day 13 (see “Conditioning procedure”). On days 8-9 and 11, mice were tested in the olfactory preference and sexual motivation assays, respectively. On day 14, we conducted the social interaction assay with a receptive female intruder and this assay was repeated again on days 17, 20, and 31 with new unfamiliar females as an extinction procedure.

3.2.1. Conditioning procedure Animals were divided randomly into Lithium Chloride (LiCl, 0.2 M, 2% body weight, J.T.Baker) and saline-treated (0.9 % NaCl) groups. One hour before each conditioning session, water and food were removed from the home cage. The saline and LiCl-treated groups were exposed to female-soiled bedding in their home cages for 12 min (Fig. 1c). Intraperitoneal (i.p) injections of LiCl or saline were applied 5 min after the introduction of the bedding, and 7 min following the injections the bedding was removed from the subject’s home cage. This conditioning pairing was conducted three times during three consecutive days (days 4-6). On day 7 mice were exposed in their home cage to clean bedding for 5 min in the same behavioral context as in the conditioning days, except no injections were applied. On day 13, an additional conditioning session was conducted to the LiCl and saline groups, as described above, in order to strengthen the aversive learning. Soiled bedding was taken from cages of unfamiliar sexually

14 mature females housed in groups of 4 (4-6 month old, same strain as the tested mice). Females used as the source for the bedding were weekly exposed to soiled bedding of males to induce estrus. For bedding presentation, 30ml of either female-soiled or clean bedding, were placed in an open polycarbonate cup (5 cm height, 7.5 cm diameter), which allowed physical access to the stimuli (i.e. exposure to both volatile and nonvolatile pheromones).

3.2.2. Olfactory preference assay (OP) The three-chamber apparatus consisted of 2 large side chambers and 1 narrow middle chamber previously described (Karvat & Kimchi, 2012, Zilkha et al., 2016). Procedure: The OP assay was conducted before (days 1-2) and after (days 8-9) the conditioning phase. Prior to the first OP test only, a habituation day was conducted and the mice were placed in the 3 chamber apparatus for 15 min, while they were free to explore the entire apparatus, in order to rule out any side preferences. During the next two days, the mice were tested in the apparatus for their preference of female or male-soiled bedding versus clean bedding (the order of the sex of the bedding stimuli was counter-balanced between the mice). Following a 10 min habituation period, 30 ml of clean bedding were placed in a polycarbonate cup (described above) and a similar amount of soiled bedding was placed in another, and each cup was stationed in a corner of one of the two main chambers (Fig. 1b). Mice were allowed to freely move between the chambers for 5 min. Experiments were recorded and analyzed for the time spent interacting with each of the cups using the EthoVision software (Noldus). An olfactory preference index was defined as the difference between the times spent sniffing the stimulus bedding and clean bedding divided by the total time spent sniffing either bedding, multiplied by 100. Then, the result for male bedding was subtracted from the result for female bedding, such that a positive value indicates that the preference is towards female over male bedding: 퐹푒푚푎푙푒 − 퐶푙푒푎푛 푀푎푙푒 − 퐶푙푒푎푛 푥 100 − 푥 100 퐹푒푚푎푙푒 + 퐶푙푒푎푛 푀푎푙푒 + 퐶푙푒푎푛

3.2.3. Sexual motivation assay (MA) Two red Plexiglass boxes, a start-box and a goal-box (12.5 x 8.5 x 8 cm3), were connected through a transparent narrow tunnel (2.5 x 2.5 x 29 cm3) that contained three thin openings on top, through which 3 barriers could be inserted (Fig. 1d). The barriers were Plexiglass rectangles of increasing heights, making passage underneath each successive barrier more difficult. The barriers differed in their distance from the bottom of the tunnel. The first easiest barrier allowed 15 mm of passage space, while the following barriers allowed 13, and then 11 mm. Procedure: A day before the test, mice were placed for 15 min of habituation in the apparatus and were allowed to freely move in the tunnel and between the boxes with no barriers. Then, they were tested for their motivation to overcome barriers to reach the goal-box that was either empty or contained a stimulus mouse, which was either a receptive female or a sexually mature

15 male (8-10 weeks old). A one-sided door (4.5 x 4.5 cm2) separated the goal-box from the tunnel to prevent the stimulus mice from exiting the goal-box. In the first trial, a mouse had 10 min to cross the tunnel and reach the goal-box. If it succeeded, it was placed back in the start-box and the first barrier was placed for an additional 10 min, and so on until the 3rd barrier was added. If the mouse failed to cross the tunnel after 10 min, the assay was stopped and no further trails were performed. The latency of the mouse to cross the tunnel and overcome 1, 2, or 3 barriers was measured using the Observer XT software (Noldus). The motivation score was defined as the difference in latency times between reaching an empty goal-box and reaching a stimulus goal-box, normalized to the total time of a single trial: 퐸푚푝푡푦 − 퐹푒푚푎푙푒

600 The motivation score was calculated for each barrier, such that the motivation for the 2nd barrier, for example, is calculated for the session where barriers 1 & 2 were present.

3.2.4. Social interaction assay (SI) A female intruder was introduced into the home cage of the tested resident male for 15 min of social interaction, on days 14, 17, 20 and 31 of the experiment (Fig. 1e). Behavior was recorded using digital video cameras and was later scored by a single observer who was blind to the experimental conditions, using the Observer software. Behavioral parameters scored included olfactory investigation, sexual behavior parameters together with anxiety-related behaviors of avoidance and freezing, and aggressive behavior. Intruder females were sexually receptive and naïve 8-weeks old C57BL/6 (Harlan laboratories). For control experiments, the resident males were introduced with an intruder male, a sexually naïve 5- weeks old C57BL/6. The fur of the male intruders was swabbed with 140 µl urine collected from sexually mature and experienced male mice.

3.2.5. Anxiety-related assays To examine anxiety-related behavior we performed the open field assay and the elevated plus maze assay on a separate cohort of WT mice that underwent the conditioning phase followed by the motivation assay. As described previously(Scott et al., 2015), the open field assay was performed in a white Plexiglas box (50 × 50 × 40 cm3) under dim white light (120 Lux). Each mouse was placed in the center of the apparatus and its movement patterns were recorded for 10 min, and analyzed using the EthoVision software. Total distance traveled and number of visits to the center of the apparatus were measured. For the elevated plus maze assay we used a polyvinyl chloride maze comprising a central part (5 × 5 cm2), two opposing open arms (30.5 × 5 cm2), and two opposing closed arms (30.5 × 5 × 15 cm3). The apparatus was raised to a height of 50 cm. The test was performed under dim white light. Each mouse

16 was placed in the center of the apparatus and its locomotion was recorded for 5 min. Time spent in the open arms and number of visits to the open arms were measured using EthoVision.

3.2.6. Testosterone and corticosterone quantification Three days following the completion of the anxiety-related assays, male mice were exposed to female- soiled bedding in their home cages for 10 min. Immediately after the olfactory stimulation, the males were deeply anaesthetized and blood samples were collected from the orbital sinus. The blood was centrifuged at 1000g for 15 min and the supernatant containing the plasma was collected and stored at - 80˚C. Plasma testosterone and corticosterone levels were measured using commercial ELISA kits according to the manufacturer’s protocol (Cayman, Ann Arbor, MI). Additionally, plasma levels of naïve male mice were collected for quantification of baseline testosterone levels, using the same protocol.

3.2.7. c-Fos quantification Ten adult male mice of ICR strain underwent the COA protocol (n = 5 for Saline and LiCl group). Two days following the social interaction assay, a female ICR mouse, was placed in the subjects’ home-cage for 90 seconds. Approaching behavior and olfactory investigation of the stimuli were recorded for later analysis. Fifty minutes after the initial stimulus exposure, the mouse was anesthetized with veterinary Pental and perfused with cold 0.1M PBS followed by 2.5% paraformaldehyde (PFA). Brains were removed and post-fixed overnight in 2.5% PFA. Using a vibrotome (Leica Microsystems Inc.), brains were sliced into 30μm coronal sections. Free-floating sections were washed in 0.1M PBS, blocked in a solution of 2% normal horse serum (60 min) and incubated in rabbit anti-Fos-related antigens (FRAs) primary antibody solution (SC-52, 1:5000, Santa Cruz Bio/technology, Santa Cruz, CA, USA) for 18 hours at RT. The next day, sections were washed three times, for seven minutes, in 0.1M PBS and incubated in biotinylated goat anti-rabbit secondary antibody solution (1:200; Vector Laboratories, Burlingame, CA, USA) for 90 min. Sections were processed in ABC reagent (Vector Laboratories) for 90 min and stained with diamino benzidine (DAB) (Sigma Laboratories). Image analysis software (ImagePro Plus, Media Cybernetics Inc., Bethesda, MD, USA) was used to count all labeled cell nuclei within the borders of the neuroanatomical nucleus of interest. c-Fos expression was assessed blindly on both sides of the brain in four sections per anatomical area which included anterior and posterior medial amygdala (aMeA and pMeA, respectively), basolateral amygdala (BLA) and nucleus accumbens (NAc).

3.3. Conditioned odor aversion (COA) to male odors The COA protocol to male odors included the following stages, similarly to the COA to female odors: Male mice were tested for their baseline olfactory preference on days 1-2 of the experiment. Three conditioning sessions were performed on days 4-6, and an additional conditioning session was conducted on day 13. In the conditioning phase, the subject male was exposed to male bedding, followed by either saline or LiCl i.p injection. Soiled bedding was taken from cages of unfamiliar sexually mature males housed in groups of 2 mice (4-6 month old, same strain as the tested mice). On days 8-9 and 14, mice were tested in the olfactory preference assay, and social interaction assay, respectively. The olfactory preference and social interaction assays were conducted exactly as described above. In the social interaction assay, the male subject was exposed to a male intruder and for control, to a receptive female intruder.

3.4. Sucrose preference test Mice were habituated to cages with two bottles filled with water for 3 days and two bottles filled with water containing 1% sucrose for an additional 3 days. Then, mice were given access to a two-bottle choice of water or 1% sucrose solution. To measure consumption, bottles were weighted at 12p.m. each day for 3 days. Bottles position was each day to control for side preference. Sucrose preference was calculated as follows: sucrose consumed*100/total volume consumed (water + sucrose).

3.5. RNA purification, cDNA synthesis and qPCR Total RNA from the NAc of WT and TrpC2-/- male mice was extracted with and purified using the RNeasy Kit (Qiagen). NAc area was determined using Franklin and Paxinos Brain Atlas (Paxinos & Franklin, 2004). RNA was converted to cDNA using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). The expression levels of mRNAs were assayed using Power SYBR Green PCR Master Mix according to the manufacturer’s guidelines in AB 7500 thermocycler (Applied Biosystems). Quantification reactions were performed in triplicates for each sample. Forward (F) and reverse (R) primers for D1R were: F 5’-CGTGGTCTCCCAGATCGG-3’, R 5’-GCATTTCTCCTTCAAGCCCC-3’. For D2R: F 5’-TGAACAGGCGGAGAATGG-3’, R 5’-CTGGTGCTTGACAGCATCTC-3’. For beta- ACTIN: F 5’-TCTTTGCAGCTCCTTCGTT-3’, R 5’-CGATGGAGGGGAATACAG -3’.

3.6. D1R-antagonist microinjections 3.6.1. Stereotaxic surgery At the age of 3 months, WT mice were anesthetized by 1:1 solution of ketamine (100 mg/kg) and xylazine (23 mg/kg) diluted x2 with double-distilled water. Then, mice were placed the stereotaxic apparatus with a mice ear bar adapter. Two 24GA guide cannulas (plastics one, VA, USA) were

18 implanted 1mm above the left and right NAc (AP: +1.3, ML: ±2, DV: -4.2, and at a 12° angle relative to bregma and skull surface (Fig. 13). The cannulas were secured to the skull using dental cement. Mice were left to recover for at least two weeks before the beginning of the behavioral assays.

3.6.2. Experimental design The CPP procedure was performed in the 3 chamber apparatus (described in the olfactory preference apparatus) with different texture floor tiles placed in each chamber for spatial cues. Before employing the CPP protocol, mice underwent two days of habituation that included habituation to the apparatus and to the microinjection process where all mice received bilateral injections of saline. CPP protocol consisted of three stages conducted consecutively: (a) 10 min pretest phase in which clean bedding in a polycarbonate cup was presented in both sides of the apparatus, to rule out initial side preference (b) three daily 10 min conditioning sessions in which female-soiled bedding was presented in one side of the chamber (conditioned chamber) and clean bedding was presented on the other side (unconditioned chamber), and (c) a 10 min test phase in which clean bedding was placed in both sides of the apparatus. During all the phases behavior of the mice was video recorded and analyzed using the EthoVision software (Noldus). The durations spent in each chamber and the interaction times with bedding cups were analyzed. In addition the velocity and distance traveled were quantified to verify there were no mobility deficits in the test phase. Two weeks after the CPP test phase, we placed the mice in the 3 chamber apparatus and conducted the sexual preference assay (described above in the COA procedure) after confirming there was no side preference.

3.6.3. Drug infusion Mice were randomly assigned to receive bilateral infusions of saline solution or D1 receptor antagonist, SCH23390 (Sigma Aldrich, 150-300 ng/side), into the NAc, 15 min before each conditioning session. SCH23390 and saline were infused bilaterally in a volume of 0.3 µl/side at a rate of 0.15 µl/min via 31 gauge internal cannulas (plastics one). The internal cannulas were left in place for an additional 1 min to allow for drug diffusion, and mice were left in their home cages for an additional 15 min before being placed in the CPP apparatus.

3.7. VTA-NAc dopaminergic neurons activation 3.7.1. Viral constructs pAAV8-Ef1α-DIO-ChR2-mCherry and pAAV5-EF1α-DIO-EYFP were a gift of the lab of Ofer Yizhar in the Weizmann Institute.

3.7.2. Stereotaxic surgery Mice were anaesthetized with isoflurane and mounted on stereotaxic frame (myNeuroLab). Virus was bilaterally injected to the VTA using a World Precision Instruments syringe at an injection rate of 0.1µl/min. VTA injection coordinates were taken from Franklin and Paxinos Brain Atlas; AP: -3.44mm, ML: ±0.38mm, DV: -4.5mm (Fig. 17) . Injection volumes were 1 µl for ChR2 and AAV-EYFP viruses. Three weeks after surgery, the animals were anesthetized with isoflurane and mounted on stereotaxic frame for fiber implantation surgery. Animals were implanted bilaterally with 200μm fiber-optic cannulae (Thorlabs). Fibers were directed above the NAc (AP: +1.3mm, ML: ±1.1mm, DV: -4.6, angle of 7º; Fig. 18). The cannula was secured using dental cement. Mice were left to recover for at least 3 weeks before behavioral assays.

3.7.3. Optical excitation of VTA-NAc dopaminergic neurons. All behavioral tests were performed 3-4 weeks post-surgery during the dark phase and under dim red light. Behaviors were recorded using a digital video recording unit and scored using Observer® XT or EthoVision software (Noldus Information Technology). A day before the beginning of photostimulation, animals were connected to FC/PC-coupled fiber optic with a 200μm silica core (BFL37–200; Thorlabs) for 10 minutes habituation. To enable free movement during the test, we connected the optical fibers to an optical rotary joint (Doric Lenses QC, Canada). Three photostimulation sessions were conducted for 3 consecutive days. First, the male was connected to the FC/PC-coupled fiber optic, than an estrus female in a holed box was introduced to him (different female for each session) and a photostimulation session began for 5 min. Light stimulation at 473nm was obtained using a DPSS laser (CrystaLaser). The stimulation parameters used during the 5 min behavioral assay were: 8 pulses of 15 ms light flashes delivered at 20Hz with a 5 sec periodicity, and 95 mW mm-2 light power density. Laser pulses were driven by a 33220A Function Waveform Generator (Agilent Technologies, Israel). Female intruders were 9 weeks old C57 mice, which were monitored for estrus cycle status throughout the experiment. Each morning a vaginal smear was collected from all females, stained with Dip Quick stain kit (Jorgensen Laboratories, Inc) and analyzed for the estrous under a light microscope.

3.7.4. Behavioral assays Olfactory preference assay was conducted on days 10 and 11 of the experiment, exactly as described above. Social interaction assay was conducted on day 15 with a receptive female intruder and on day 16, with an intruder male, as described above.

3.7.5. Immunohistochemistry Following behavioral assays mice were euthanized, perfused with 4% paraformaldehyde (PFA), and their brains were sectioned with a vibratome (Leica Microsystems) into 50µm coronal slices. Floating brain slices were collected, washed three times in PBS, and immunostained for tyrosine hydroxylase (TH), or m-Cherry using the following conditions: Blocking: 20% normal horse serum (NHS), 0.04% Triton, Carrier: 2% NHS, 0.04% Triton; primary antibodies: sheep anti-TH (1:1300, Millipore), rabbit anti- mCherry (1:500, Novus), for 48 h at 4º C; secondary antibodies: Alexa Flour 488/594 anti-rabbit, Alexa Flour 488/594 anti-sheep (1:200, Molecular Probes). Stained brain slices were imaged by confocal microscopy (Zeiss, LSM 710) for subsequent analysis.

3.8. Statistical analysis Time spent interacting with the bedding in the olfactory preference and conditioned place preference assays were analyzed by repeated measures ANOVA, followed by Fisher LSD post hoc analysis. As well as the preference index. Behaviors in the motivation and social interaction assays as well as testosterone and corticosterone levels were analyzed using the Mann-Whitney U test for comparison between 2 independent groups or Kruskal Wallis ANOVA test for 3 independent groups. When comparing between the percentages of animals preforming a specific behavior we used the Fisher exact test. Significance in the open field and elevated plus maze assays, as well as velocity and distance travelled in the CPP test and during optogenetic activation, were all analyzed using student’s t-test. All statistical analyses were performed using the Statistica software (Statsoft, Tulsa, OK). Significant results were considered for P ≤ 0.05.

4. Results

4.1. Female-specific COA impairs innate precopulatory and copulatory responses in WT males In the following set of experiments we performed conditioned odor aversion paradigm towards female odors in WT male mice (see Methods and Fig. 1a-e). Briefly, during three consecutive days, mice were exposed in their home-cage to soiled-bedding from adult female cages, which is naturally embedded with female pheromones (conditioned stimulus, CS). Following bedding presentation, mice received an intraperitoneal injection of either saline (control) or lithium chloride (LiCl), in order to evoke gastrointestinal distress while interacting with the female stimulus, as previously described (Ferry, 2014, Kay & Nyby, 1992). We measured the effects of this female-specific COA on olfactory sexual preference, motivation to invest effort to obtain physical contact with a female, social interaction with a female, and changes in testosterone levels following exposure to female odors. The findings in this section were recently published (Beny & Kimchi, 2016). Differences in the time spent investigating female- and male-soiled bedding before and after the conditioning phase were evaluated for the saline- and LiCl-treated males. Repeated measures ANOVA showed a significant interaction between ‘group’ x ’phase’ x ’stimuli’ (F(1,37) = 8.81, P < 0.01). WT males, which underwent female-specific COA (LiCl group), lost their preference for female odors and spent similar amounts of time interacting with female- and male-soiled bedding (Fig. 1f), stemming from a significant decrease in time spent interacting with female bedding (P < 0.001, Fig. 1f). In the motivation assay, nearly 60% of males from the saline group crossed the 3rd barrier in order to reach a goal-box containing an unfamiliar sexually receptive female, whereas none of the males from the LiCl group crossed the 3rd barrier (P < 0.05, Fig. 1g). In accordance, LiCl WT males received a significantly lower motivation score to cross the 3rd barrier and reach the unfamiliar female (Z = 2.5, P < 0.05; Fig. 1h), whereas the motivation score to reach a male was similar in the LiCl and saline groups (Z = 1.16, P = 0.38; Fig. 4a). LiCl WT males also displayed a significantly lower female-induced testosterone surge compared to the saline WT males (Z = 2.3, P < 0.05; Fig. 1i).

Figure 1: Female-specific COA alters innate precopulatory responses in WT males. (a) Timeline (days) of the conditioned odor aversion (COA) protocol. (b-e) Schematic illustrations of the different behavioral procedures. (f) Time (sec) spent interacting with female and male bedding,

subtracting the time (sec) spent with the respective clean bedding, during the olfactory preference st assay for the LiCl (n = 21) and saline (n = 18) groups. (g) Percentage of animals crossing the 1 , nd rd 2 , and 3 barriers in the motivation assay (nLiCl = 9, nSaline = 7). (h) Motivation score to reach a female reinforcer for saline and LiCl-treated groups. (i) Testosterone surge for the saline- and LiCl- treated males (n = 4 per group) following exposure to female odors, relative to baseline levels of naïve animals. Values are displayed as mean ± SEM. ***P < 0.001, *P < 0.05, NS- not significant.

In order to examine whether COA will affect consummatory (copulatory) behaviors, we tested the sexual behavior of the males towards an unfamiliar sexually receptive female in a 15 min social interaction assay. We found that the LiCl group spent significantly less time engaging in sexual behavior (Z = 2.36, P < 0.05; Fig. 2a) and performed significantly less pelvic thrusts (Z = 2.86, P < 0.01; Fig. 2b) compared to the saline treated males. No difference was detected between the groups in the duration of aggressive behavior towards a male intruder (Z = -0.03, P = 0.97; Fig. 4b). Surprisingly, LiCl males kept presenting significantly shorter durations of sexual behavior (repeated measures ANOVA; ‘group’ effect:

F(1,13) = 7.16, P < 0.05, Fig. 2c) and lower number of pelvic thrusts events (‘group’ effect: F(1,13) = 6.32, P < 0.05, Fig. 2d) even almost a month from the first conditioning session.

Figure 2: Female-specific COA induces long lasting deficits in copulatory behaviors of WT males. (a) Sexual behavior duration and (b) number of pelvic thrusts towards a female intruder in the social interaction assay (n = 12 per group). (c) Sexual behavior duration and (d) number of pelvic thrusts towards females in the social interaction assay conducted at days 17, 20 and 31 of the experiment. Values are displayed as mean ± SEM. **P<0.01, *P < 0.05.

In addition, we measured the average distance males kept from the female during the 15 min assay. LiCl WT males stayed in a significantly larger distance from the female compared to the saline group

(t(20) = -2.21, P < 0.05; Fig. 3a). Surprisingly, we detected anxiety-related behaviors towards the female intruder among males that underwent the aversive conditioning to female odors. A 75% of LiCl WT males presented strong fleeing responses from the receptive female compared to only 30% of males from the saline group (P = 0.08; Fig. 3b). Moreover, LiCl males exhibited significantly greater female-directed avoidance events (Z = -1.95, P = 0.05; Fig. 3c) and freezing behavior (Z = -2.04, P < 0.05; Fig. 3d) than the saline group. LiCl and saline groups presented no significant differences in anxiety related behaviors towards a male intruder (Fig. 4c-d).

Figure 3: Female-specific COA induces social anxiety-like behaviors. (a) Distance from the female intruder in the social interaction assay (nLiCl = 12, nSaline = 10). (b) Percentage of animals fleeing from the female intruder. (c) Female-directed avoidance events and (d) freezing duration for saline and LiCl-treated males. Values are displayed as mean ± SEM. *P < 0.05, #P = 0.08.

Figure 4: Female-specific COA does not affect male-directed behaviors in WT males. (a) Motivation scores for LiCl (n = 9) and saline (n = 8) groups to cross three barriers and reach a male reinforcer. (b) Aggressive behavior duration, (c) freezing durations, and (d) avoidance events towards a male intruder in the social interaction assay (nLiCl = 12, nSaline = 9). Values are displayed as mean ± SEM.

Finally, we measured blood levels of corticosterone following exposure to female odors. No difference was found in corticosterone levels between saline and LiCl groups (Z = 0.87, P = 0.37; Fig. 5e). In addition, no difference was found between the groups in anxiety-related behaviors measured by the elevated plus maze and open field assays (Fig. 5a-d).

Figure 5: Female-specific COA does not affect corticosterone levels or anxiety-related behaviors. (a) Time spent in open arms and (b) number of visits to the open arms in the elevated plus maze. (c) Total distance traveled and (d) number of visits to the center zone in the open field

assay (nLiCl = 8, nSaline = 5). (e) Plasma corticosterone levels (ng/ml) of saline and LiCl-treated groups (nLiCl = 12, nSaline = 8) following exposure to female odors. Values are displayed as mean ± SEM. (f) Standard curve including trendline and linear equation for the corticosterone ELISA assay.

Following exposure to female pheromones, an increase in neuronal activation is reported in brain areas related to olfactory processing including the accessory olfactory bulb (AOB), bed nucleus of stria terminalis (BNST), the medial amygdala (MeA) and basolateral amygdala (BLA), in addition to reward brain areas governing behavioral motivation, as the NAc (Kim et al., 2015). Our next goal was to examine whether the induction of COA protocol will cause a different neuronal activation pattern in specific brain regions, which are activated during pheromonal processing. For this purpose the COA paradigm was induced to new group of 10 adult males (ICR mouse strain). Then, we exposed them to a female intruder for 90 sec and analyzed the amount of c-Fos positive cells in the different brain regions. First, no differences between the groups were observed in duration of sniffing the female intruder during the 90 sec exposure (Fig. 6a). Number of activated c-Fos cells per mm2 in the anterior MeA (aMeA) was significantly higher in the saline group than the LiCl group (student’s t-test, t(7) = 2.81, P = 0.02, Fig. 6b). In addition we analyzed correlation coefficient between number of cFos cells in specific brain areas and aversion level. Aversion level was represented by two parameters: the difference between olfactory preference before and after the aversive conditioning (preference delta) and number of avoidance events from the female intruder. Interestingly, a significant correlation was exhibited between number of activated c-Fos cells/mm2 and the level of aversion exhibited by both saline and LiCl males in the NAc (Pearson correlation; r = -0.65, P = 0.05 for avoidance events, and r = 0.76, P < 0.05 for preference delta; Fig. 6c); and aMeA (r = -0.73, P < 0.05 for avoidance events, and r = 0.84, P < 0.01 for preference delta; Fig. 6d); and a trend towards correlation was found in the BLA (r = -0.60, P = 0.08 for avoidance events, and r = 0.63, P = 0.05 for preference delta; Fig. 6e).

Figure 6: Quantification of cFos+ cells in brain regions of saline and LiCl mice following exposure to female conspecific. (a) Sniffing duration during 90 sec exposure to a female

intruder of the saline (n = 4) and LiCl (n = 5) males. (b) c-Fos positive cells in the anterior and posterior medial amygdala (aMeA and pMeA, respectively), basolateral amygdala (BLA), and, nucleus accumbens core (NAcC) and shell (NAcSh) of males from the saline LiCl groups. (c-e) Correlations between number of c-Fos positive cells per mm2 and preference delta (left) and avoidance events (right) in NAcC (c), aMeA (d), and BLA (e). * P<0.05.

4.2. Male-specific COA does not alter olfactory preference Next, we examined whether we can alter innate behaviors towards male conspecifics by inducing a specific aversion to male-soiled bedding. Repeated measures ANOVA analysis of time interacting with female vs. male soiled bedding revealed a significant effect for the stimulus used (F(1,15) = 49.63, P < 0.001; Fig. 7a). Post hoc analysis showed that following aversion to male odors, LiCl-conditioned males showed a marginally significant decrease in the time they spent interacting with male bedding (P = 0.06; Fig 7a). No significant main effects of ‘phase’ or ‘group’ were found when analyzing the olfactory preference index (‘phase’: F(1,15) = 0.95, P = 0.34; ‘group’: F(1,15) = 0.34, P = 0.56, Fig. 7b). In the social interaction assay, among the LiCl males 100% of executed male-male aggressive attacks, compared to 60% from the saline group (P = 0.08; Fig. 7c). However, no difference was found between the groups in either aggressive score (Z = -1.25, P = 0.21; Fig. 7d), or in anxiety-related behaviors such as freezing or avoiding the male intruder (ZFreezing = 0, P = 1; ZAvoidance = -0.48, P = 0.63; Fig. 7e,f). As for the behavior towards receptive females, we found no difference between the groups in any of the sexual behavioral parameters or anxiety-related behaviors examined (Fig. 8a-c).

Figure 7: Male-specific COA does not alter inter-male aggressive behaviors. (a) Time spent interacting with female and male bedding, normalized to clean bedding in the olfactory preference assay among LiCl (n = 9) and saline (n = 8) groups. (b) Sexual preference index. (c) Percentage of animals presenting aggressive behavior towards a male intruder in the social interaction assay. (d)

Aggression score calculated from aggressive duration and latency to attack. (e) Number of avoidance events and (f) duration of freezing among the saline- and LiCl-treated males. Values are displayed as mean ± SEM. **P < 0.01, ***P < 0.001, #P(a) = 0.06, #P(c) = 0.08.

Figure 8: Male-specific COA has no effect on female-directed behaviors among WT males. (a) Sexual behavior duration, (b) mounting latency and (c) number of avoidance events as measured in the social interaction assay with a receptive female intruder for saline- and LiCl- treated males (nLiCl = 9, nSaline = 9). Values are displayed as mean ± SEM.

4.3. TrpC2-/- males display diminished responses towards sexual rewards WT and TrpC2-/- males were exposed to female soiled-bedding, and their plasma testosterone levels were measured. We found that exposing WT males to female soiled-bedding for 10 min is sufficient to induce a significant increase in plasma testosterone levels compared to baseline levels of naïve males (control) (female bedding: 10.26 ± 1.19 ng/ml, n = 4; control: 0.92 ± 0.46 ng/ml, n = 6, P < 0.001). In contrast, exposing TrpC2-/- males in a similar manner produced no change in testosterone levels (female bedding: 0.93 ± 0.37 ng/ml, n = 4; control: 0.62 ± 0.084 ng/ml, n = 5, P = 0.27). The olfactory preference assay showed that WT males spent significantly more time investigating female-soiled bedding than male-soiled bedding, whereas TrpC2-/- males spent similar amounts of time investigating each (Wilcoxon

-/- matched pairs test; ZWT = 4.01, P < 0.001, ZTrpC2 = 0.08, P = 0.92; Fig. 9a). Moreover, an effort-based sexual motivation assay revealed that the latency to reach a goal-box with a conspecific female was

-/- significantly longer in TrpC2 males than in WT males (Mann-Whitney U-test; ZFemale = -2.06, P < 0.05; Fig. 9b). The latency to reach an empty goal box did not differ significantly between the two genotypes

(ZEmpty = -0.47, P = 0.62; Fig. 9b). Moreover, previous experiments conducted in our lab (by Dr. Yael Lavi-Avnon) used in-vivo microdialysis to measure changes in extracellular NAc DA levels in WT and TrpC2-/- males during 48 min of social interaction with an unfamiliar female or male conspecific (4 consecutive microdialysis fractions of 12 min each). In WT males, NAc DA levels markedly increased by about 70% from baseline levels during the first 12 min of social interaction with a female intruder, while no such change was observed during social interaction with a male intruder (nfemale = 6, nmale = 7; Repeated-Measures ANOVA; F(12,132) = 4.38, P <

0.001 as compared to baseline levels; Fig. 9c). In contrast, in TrpC2-/- males, NAc DA levels remained unchanged during social interaction with either female or male conspecific (nfemale = 6, nmale = 7, F(12,132) = 0.39, P = 0.96; Fig. 9d).

Figure 9: Differences between WT and TrpC2-/- males in reward-related responses. (a) Time

of interaction with female- and male-soiled bedding in the olfactory preference assay (nWT = 18, -/- nTrpC2 = 11). (b) Latency to reach an empty goal-box and a female reinforcer in the sexual -/- motivation assay (nWT = 11, nTrpC2 = 8). (c-d) Time course of extracellular DA in the NAc of WT and TrpC2-/- males as a percentage of baseline. Grey boxes (extending from 0-48 min) indicate the time of female or male intruder presence (kindly provided by Dr. Yael Lavi-Avnon). Values are displayed as mean ± SEM. ***P < 0.001, *P < 0.05, NS= Non-significant.

To examine whether the deficit in reward processing is specific to social stimuli, WT and TrpC2-/- males were tested in the sucrose preference assay, and no difference in sucrose consumption was revealed -/- between the groups (Student’s t-test; t(16) = 0.74, P = 0.46; Fig. 10a). Thus, TrpC2 males display reduced physiological and behavioral responses to female reward but not to natural reward. Moreover, we did not observe any difference between the two genotypes in either DA content in the NAc (n = 5 per group; t(8) = -1.16, P = 0.27; Fig. 10b), or mRNA expression of DA receptors in the NAc (n = 7 per group; tD1R(12) = -0.68, P = 0.5, tD2R(12) = -0.56, P = 0.58; Fig. 10c).

-/- Figure 10: TrpC2 males present no general deficits in dopaminergic reward system as compared to WT mice. (a) Preference for 1% sucrose over water of WT and TrpC2-/- mice (n = 9 per group) (b) Total content of baseline DA (ng/ml) in NAc of WT and TrpC2-/- mice (n = 5 per group). (c) Relative expression levels of mRNAs of D1 and D2 receptors in the NAc of WT and TrpC2-/- male mice (n = 7 per group). Values are displayed as mean ± SEM

4.4. Female-specific COA impairs precopulatory and copulatory behaviors in TrpC2-/- males (Beny & Kimchi, 2016) In order to assess whether lack of preference to female odors among TrpC2-/- males is a result of the inability to sex discriminate between male and female odors we employed a female-specific COA protocol on adult, sexually naïve, TrpC2-/- male mice, using the same methodologies applied on the WT males (Fig. 1a-e). We found that TrpC2-/- males develop a female-specific aversion, manifested in a significant interaction effect of ‘group’ x ‘phase’ x ‘stimuli’ (F(1,20) = 7.8, P < 0.05, Fig. 11a). In TrpC2- /- males, similarly to WT males, female-specific COA induced a significant decrease in the time LiCl mice spent olfactory investigating female soiled-bedding (P < 0.001; Fig. 11a). Since prior to the conditioning procedure the TrpC2-/- males had no initial preference towards female odors, following the aversive conditioning they in fact presented olfactory preference towards males, spending more time

33 interacting with male- over female-soiled bedding (P < 0.001; Fig. 11a). The saline group showed no significant change after conditioning (Fig. 11a). LiCl TrpC2-/- males also presented significant lower motivation scores to reach a female than the saline group (Z = 1.92, P = 0.05; Fig. 11b). Unlike WT males, the LiCl and saline TrpC2-/- groups showed no significant difference in testosterone levels following exposure to a female (Fig. 11c). Following the aversive conditioning to female odors we tested TrpC2-/- males in the social interaction assay. It was previously reported that TrpC2-/- males mount both females and males indiscriminately (Leypold et al., 2002, Stowers et al., 2002). When presented with a receptive female, LiCl TrpC2-/- males spent significantly less time engaging in sexual behavior than saline-treated TrpC2-/- males (Z = 2.04, P < 0.05; Fig. 11d). However, no difference was detected in the duration of sexual behavior towards male intruders between the saline and LiCl group (Z = 0.15, P = 0.87; Fig. 12b). Moreover, they exhibited elevated female-directed avoidance events (Z = -2.98, P < 0.01; Fig. 11e) and freezing behavior (Z = - 2.45, P < 0.05; Fig. 11f). LiCl and saline groups of TrpC2-/- males showed no difference in their behaviors towards a male conspecific (Fig. 12b-d). These data suggest that TrpC2-/- males were able to distinguish between male and female odors, acquiring a specific aversion to female signals, which caused a robust alteration in their behavior exclusively towards females.

Figure 11: Female-specific COA induces alteration deficits in precopulatory and copulatory behaviors in TrpC2-/- males. (a) Time spent interacting with female and male soiled bedding, normalized to clean bedding in the olfactory preference assay (n = 11 for saline and LiCl groups). (b) Motivation score to cross the 3rd barrier and reach a female reinforcer (n = 5 for saline and

LiCl groups). (c) Testosterone surge of saline- and LiCl- treated males following exposure to female odors, as relative to baseline levels for naïve animals (n = 4 per group). (d) Sexual behavior duration, (e) avoidance events, and (f) freezing duration as measured for saline and LiCl-treated groups in the social interaction assay (n = 10 per group). Values are displayed as mean ± SEM. **P

< 0.01, *P < 0.05, #P = 0.05.

Figure 12: TrpC2-/- males do not generalize the aversion to male stimuli. (a) Motivation scores for LiCl and saline males (n = 5 per group) to cross three barriers and reach a male reinforcer. (b) Sexual behavior duration, (c) freezing duration, and (d) avoidance events towards a male intruder in the social interaction assay (n= 10 per group). Values are displayed as mean ± SEM.

4.5. Blocking D1R in the NAc disrupts sexual preference Our results suggest that TrpC2-/- males can discriminate between sex-specific odors. In addition, findings obtained in the lab implied that TrpC2-/- males may have deficiencies in processing of social rewarding stimuli. We decided to use loss-of function approach to further examine our hypothesis that dopamine transmission in the NAc is necessary for encoding the rewarding value of female odors, which leads to sexual preference. Thus, in the following experiments WT males were bilaterally injected into the NAc with either saline or the specific D1R antagonist (D1R-A group), SCH-23390, while acquiring conditioned place preference (CPP) to female odors (Fig. 13). In the acquisition period, mice were placed in a 3-chamber apparatus, with different floor textures, following microinjections to the NAc. One chamber contained a cup filled with female soiled bedding and the other clean bedding (Fig. 13b). In the test day, conducted 24 hours after the last conditioning day, place preference was assessed by placing the males in the same apparatus, which contained cups full of clean bedding in both chambers. No brain microinjections preceded the test phase.

Figure 13: Bilateral microinjections of D1R antagonist into NAc before induction of CPP. (a) Timeline (days) for the conditioned place preference (CPP) protocol followed by the olfactory preference assay. (b) Schematic illustration of the conditioning phase. (c and d) Coronal section of the mouse brain (bregma = 1.34mm, 100µm width section) which depicts injection site into the NAc using red beads. NAc= nucleus accumbens.

The saline-treated males spent significantly more time in the chamber previously associated with female odors (Repeated measures ANOVA; F(1,16) = 6.4, P < 0.05; Psaline < 0.01; Fig. 14a). In contrast, the D1R-A group spent similar amounts of time in the female-associated chamber and the clean chamber

(PLiCl = 0.89; Fig. 14a). Importantly, during the conditioning phase, the D1R-A and saline treated groups spent similar amounts of time olfactory investigating the female-soiled bedding (F(2,28) = 1.01, P = 0.37; Fig. 15a). Moreover, during the CPP test no differences were observed in the total distance traveled and mean velocity between the two groups (Total distance: t(15) = -0.78, P = 0.44; Mean velocity t(15) = -0.78, P = 0.44; Fig. 15b, c). When we directly assessed the odor preference of these mice more than 10 days later, the D1R-A males spent significantly less time investigating female-soiled bedding than they did investigating male-soiled bedding (F(1,15) = 16.03, P < 0.01; Fisher LSD post hoc P < 0.05); Fig. 14b). Additionally, time interacting with female bedding was significantly shorter in the D1R-A group, compared to the saline group (P < 0.05; Fig. 14b).

Figure 14: D1R mediated signaling in the NAc is essential for conditioned place preference

and sexual preference. (a) Time spent in the female-associated chamber versus clean-associated chamber during CPP test (nsaline = 9, nD1R-A = 8). (b) Time spent interacting with female and male bedding in the olfactory preference assay. Values are displayed as mean ± SEM. **P < 0.01, *P < 0.05. D1R-A= D1R antagonist.

Figure 15: Bilateral microinjections of D1R antagonist into NAc did not affect overall

olfactory investigation and general mobility. (a) Percentage of time saline- and D1R-A- treated males spent interacting with female bedding relative to clean bedding during three conditioning days. (b) Total distance traveled in m, and (c) mean velocity of movement in cm/s

during the test phase. Saline group: n = 9; D1R-A group: n = 8. Values are displayed as mean ± SEM.

4.6. Optogenetic activation of VTA-NAc dopaminergic neurons in TrpC2-/- males Our results demonstrated that signaling through D1 receptors in the NAc is crucial for sexual preference in males. Next we examined whether specific activation of VTA-NAC dopaminergic neurons will restore sexual preference for female odors in TrpC2-/- males. Whole-brain tracing experiments shows that the dopaminergic neurons located in the posteromedial VTA selectively projects to the ventromedial striatum (including NAc) (Beier et al., 2015, Ikemoto, 2007). Thus, We injected Adeno Associated Viral

(AAV) vector carrying Cre-dependent channelrhodopsin-2 (pAAV8-Ef1α-DIO-ChR2-mCherry) in the VTA of double transgenic DATCre+/TrpC2-/- (ChR2-TrpC2-/-, n = 10) mice (Fig. 16 and Fig. 17). To selectively activate VTA-NAc DA neurons, optic fiber was implanted above the NAc and blue light was delivered (Fig. 16b and Fig. 18). For control we used DATCre+/TrpC2+/+ (eYFP-WT, n = 11) and DATCre+/TrpC2-/- (eYFP-TrpC2-/-, n = 11) mice injected with a control eYFP-expressing virus (pAAV5- EF1α-DIO-EYFP ChR2; Fig 16a).

Figure 16: Projection targeting optogenetic activation in DATCre+/TrpC2-/- mice. (a) -/- Creation of TrpC2 mice and their WT littermates expressing Cre under the dopamine transporter (DAT) promoter. (b) Projection targeting is achieved by AAV-DIO-ChR2 injection to the VTA, followed by implantation of fiber optic above the NAc region, which contains neuronal projections of DA neurons from the VTA.

Figure 17: Expression of ChR2-mCherry in dopamine neurons in the VTA. (a) Immunostaining for ChR2-mCherry (red) and TH (green) in the VTA (-3.40mm from bregma), 12 weeks after injection of AAV-DIO-ChR2-mCherry in DATCre+/TrpC2-/- mouse. A portion of the image in magnified in (b). Neurons that express both TH and mCherry are marked with arrows. Scale bar are 200 micron (a) and 50 micron (b).

Figure 18: Expression of ChR2-mCherry in projections of dopamine neurons in the striatum. (a) Arrow indicates optic fiber mark in the tissue. NAc region is marked with a square (1.18 mm from bregma). (b) Confocal images of dopaminergic fibers in the NAc around cell bodies stained for DAPI (blue). Merge image shows that most of the fibers are co-labeled with TH and ChR2-mCherry. Scale bar are 1000 micron (a) and 20 micron (b).

Photostimulation of dopamine neuronal projection was conducted bilaterally during 5 min exposure to a receptive female in a holed box, in days 1, 2, and 3 of the experiment (Fig. 19a and Methods). Analysis of sniffing duration during all 3 days of optogenetic activation and number of sniffing events per day showed no significant difference between the groups (One-Way ANOVA; F(2,26) = 2.25, P = 0.12; Repeated Measures ANOVA; ‘group’ effect: F(2,26) = 1.54, P = 0.23; respectively; Fig. 19b,c). In addition, time spent in proximity to the female stimulus was not different between the groups (F(2,26) = 0.75, P = 0.48; Fig. 19d).

Figure 19: Optogenetic activation during exposure to a receptive female. (a) Timeline in days of the optogenetic activation behavioral protocol and demonstration of behavioral apparatuses. (b) Total sniffing duration of the female stimulus during optogenetic activation. (c) Number of sniffing events per day of the female stimulus. (d) Average time spent in proximity to

the female during 3 days of optogenetic activation. nWT = 9; nTrpC2-/- = 9; nChR2-TrpC2-/- = 11. Values are displayed as mean ± SEM.

At days 10 and 11 an olfactory preference for female odors was conducted as previously described (Fig. 19a and Methods). Repeated measures ANOVA showed a significant main effect for ‘stimuli’

(F(1,28) = 23.07, P < 0.001; Fig. 20a). As expected the WT group presented a significant preference for interacting with female bedding as compared to male (P < 0.001) as opposed to the TrpC2-/- males, which did not prefer neither (P = 0.26). Interestingly, ChR2-TrpC2-/- males presented significant preference for female versus male bedding in the olfactory preference assay (P < 0.01; Fig. 20a). In addition, One-Way ANOVA revealed a trend towards differences in sexual preference index parameter between the 3 groups

(F(2,28) = 2.94, P = 0.06; Fig. 20b).

Figure 20: ChR2-TrpC2-/- males present sexual preference for female odors following VTA- NAc optogenetic activation. (a) Time (sec) spent interacting with female and male bedding, subtracting the time (sec) spent with the respective clean bedding, during the olfactory preference assay for the WT (n = 10) , TrpC2-/- (n = 11) and ChR2-TrpC2-/- (n = 10) groups. (b) Olfactory preference index. Values are displayed as mean ± SEM. ***P<0.001, **P<0.01, ~P=0.06, NS=not significant.

Next, we examined how optogenetic activation during first exposures to a receptive female will affect behavior towards female and male intruders in the social interaction assays (days 15 and 16; Fig 19a). No difference was observed between the groups in sniffing duration of the female intruder (Kruskal-Wallis -/- test; H(2.29) = 4.3, P = 0.11; Fig. 21a). Interestingly, TrpC2 males that received optogenetic stimulation exhibited a robust increase in total sexual behavior as indicated by the significant increase in mounting events (H(2.29) = 10.48, P < 0.01, P < 0.05; Fig. 21c) and a trend towards higher sexual behavior events -/- (H(2.29) = 9.81, P < 0.01, P = 0.09; Fig. 21b) in compare to TrpC2 control males. No difference was detected between the groups in latency time to mount the female (H(2.29) = 2.7, P = 0.25; Fig. 21d).

Figure 21: ChR2-TrpC2-/- males present longer durations of sexual behavior compared to TrpC2-/- males. (a-d) Behavioral parameters measured during social interaction with a female

intruder for WT (n = 10), TrpC2-/- (n = 10) and ChR2-TrpC2-/- (n = 9) groups. Values are displayed as mean ± SEM. *P<0.05, #P=0.09.

When a male intruder was introduced to the home cage, 90% of WT males behaved aggressively as compared to only 20% and 11% of TrpC2-/- and ChR2-TrpC2-/- males, respectively. In accordance, a significant difference between the groups was found in sniffing duration (H(2.29) = 12.48, P < 0.01; Fig.

22a) as well as in aggressive duration (H(2.29) = 16.95, P < 0.001; Fig. 22c) and latency to first attack -/- -/- (H(2.29) = 17.77, P < 0.001; Fig. 22d). Both TrpC2 and ChR2-TrpC2 males preformed less amounts of aggressive responses towards the male intruder and presented longer latencies to attack from the WT males (P < 0.01; Fig. 22c,d). In addition, quantification of male-male mounting revealed that the ChR2- -/- TrpC2 males presented an increase in duration of mounting compared to the WT males (H(2.29) = 6.66, P < 0.05, Fig. 22b).

Figure 22: ChR2-TrpC2-/- show no male-male aggressive behavior similar to control TrpC2- /- males. (a-d) Behavioral parameters measured during social interaction with a male intruder for WT (n = 10), TrpC2-/- (n = 10) and ChR2-TrpC2-/- (n = 9) groups. Values are displayed as mean ± SEM. **P<0.01, *P<0.05.

Last we performed several control measurement to rule out any significant influence of the optogenetic stimulation on locomotion among ChR2-TrpC2-/- males. Parameters extracted from the automated tracking software EthoVision demonstrated no difference between the groups in distance moved (One- Way ANOVA; F(2,29) = 0.24, P = 0.78; Fig. 23b), body rotation frequency (F(2,29) = 1.04, P = 0.36; Fig. 23c) and mobility time (F(1,29) = 1.17, P = 0.32; Fig. 23d) during 5 min of delivering blue light to the NAc.

Figure 23: NAc-VTA neuronal activation does not affect locomotion. (a) Screenshot

extracted from the EthoVision software, while tracking the mouse during 5 min optogenetic

activation. (b) Distance moved in cm, (c) body rotation frequency, and (d) mobility time in sec -/- -/- for WT (n = 11), TrpC2 (n = 10) and ChR2-TrpC2 (n = 11) groups. Values are displayed as mean ± SEM.

5. Discussion

In the current work we set out to explore the underlying reward mechanism regulating pheromone- mediated innate and conditioned copulatory behavioral response. Sexual olfactory preference of males towards female odors is considered innate, precopulatory behavior, leading to the initiation of the copulatory phase. Utilizing TrpC2-/- and DAT:Cre transgenic mice we focused on determining the role of VNO-detected female pheromones and NAc DA in regulating sexual preference of male mice to female signals.

5.1. Flexibility in innate sexual behaviors through olfactory learning Our findings demonstrate that COA to female bedding severely attenuates male-typical pre-copulatory behaviors, including abolishing the olfactory preference to female odors and reducing the intrinsic motivation to reach a sexually receptive female. In addition we show that the aversive conditioning was sufficient to induce deficits in copulatory behaviors towards an unfamiliar sexually receptive female (that was not the donor of the odor stimulus used in the conditioning phase), and that this effect was stable for almost a month following the first conditioning day and resistant to extinction. Remarkably, the aversive conditioning also triggered strong phobic and anxiety-related behavioral responses towards conspecific females, including freezing, avoidance, and escape behaviors, which, to our knowledge, are behaviors typically emerging towards predator-related signals (Brechbuhl et al., 2013, Papes et al., 2010). Importantly, no differences were found between the saline and LiCl group in plasma corticosterone levels following exposure to female bedding, or in general anxiety assays. This suggests that the anxiety-like responses directed towards female conspecifics were not the result of the generally higher anxiety levels of LiCl-treated mice and may be classified as anxiety-related responses specifically triggered by social interactions (Condren et al., 2002, Martin et al., 2010). Generally, female chemosensory signals are considered to be intrinsically rewarding and strongly attractive to males, eliciting activation in the mesolimbic dopamine reward system (Ago et al., 2015, Malkesman et al., 2010, Veening & Coolen, 2014). Our behavioral results led us to argue that by associating a highly rewarding stimulus, as female pheromones, with a stimulus possessing an opposite rewarding value, i.e. LiCl-induced malaise, we caused the devaluation of the incentive salience of female signals. Two main results support this argument. The first is the effect of the aversive conditioning on testosterone blood levels quantified following exposure to female pheromones. Testosterone plays an essential role in mediating the rewarding properties of female cues, as was shown in adult male hamsters where development of conditioned place preference to vaginal secretions was prevented following castration (Harding & Mcginnis, 2004) and restored following testosterone treatment (Bell & Sisk, 2013).

Thus, the testosterone surge induced by female pheromones seems to serve as a neuroendocrine response mediating the intrinsic rewarding value of females, which is required for stimulating male precopulatory behaviors (Bell et al., 2013, Petrulis, 2013). In our study, LiCl-conditioned WT males lacked female- induced testosterone surge, indicating a reduction in the intrinsic rewarding value of female chemosignals. The second result, which supports the devaluation hypothesis, is the correlation between patterns of brain activation, as indicated by c-Fos marker, and the extent of the aversion. We demonstrated that the strongest the aversion is there are less activated cells following exposure to female stimulus in the NAc, BLA and MeA, which are key brain regions associated with behavioral motivation and pheromonal processing (Beny & Kimchi, 2014). Overall, these results support our claim that the intrinsic rewarding value of female chemosignals is dramatically reduced following COA. Together, our data demonstrate that female sexual pheromones, thought to possess an intrinsic positive value, can acquire negative rewarding value, profoundly impairing odor-derived pre-copulatory behaviors and, surprisingly, even driving aversive behaviors towards females. In contrast to our unambiguous results obtained following female-specific aversion, the behavioral effects of male-specific aversion were only minor and included marginally significant decrease in time spent investigating conspecific male bedding, and a trend to increased inter-male aggression. As the COA to male-soiled bedding was performed using the same protocol of COA to female-soiled bedding we suggest that the differential response observed is related solely to the nature of the conditioned stimulus (female vs. male bedding). We propose that this difference might emerge from the distinct incentive saliences assigned to female and male pheromones by male mice. According to the prediction error learning theory, an error is generated when an outcome is different from its prediction (either better than expected or worse than predicted). This leads to an updated prediction, which leads to learning and behavioral change. However, when an outcome matches its predication, no error is generated and behavior remains the same (Hollerman & Schultz, 1998, Pignatelli & Bonci, 2015, Schultz, 2015). In our case, an unpleasant physiological response in males to the presentation of female odors is extremely unexpected thereby triggering learning. On the other hand, the association between male odors and an unpleasant outcome might be more expected, thus it is reasonably that only minor (if any) associative learning will occur, as previously demonstrated (Steinberg et al., 2013, Waelti et al., 2001). This may indicate that female and male odors are placed differently on the “saliency spectrum”, which ranges from aversion to reward (Pignatelli & Bonci, 2015). Additionally, previous studies have demonstrated that factors such as odor type might also affect the magnitude of aversive learning as some odorants cannot be conditioned to induce aversion (Holder & Garcia, 1987, Panhuber, 1982). These characteristics might also explain the differential intensities of the COA between female and male odors.

5.2. VNO inputs mediate the innate rewarding value of female cues The nasal cavity of most mammals contains two anatomically separated chemosensory organs: the VNO and the main olfactory epithelium (MOE) (Ihara et al., 2013). An important genetic model for investigating the role of pheromone stimuli detected by the VNO is offered by TrpC2 mutant mice (Oboti et al., 2014, Perez-Gomez et al., 2014, Yu, 2015, Zufall, 2014). Previous studies report that TrpC2-/- males lose the innate mating preference towards females typically presented by sexually naïve WT males (Kimchi et al., 2007, Leypold et al., 2002, Stowers et al., 2002). This behavioral phenotype could be explained by the inability of these mutant males to detect VNO-mediated signals, which typically allow discrimination between males and females (Ibarra-Soria et al., 2014, Stowers et al., 2002). Our results demonstrate for the first time that TrpC2-/- males can acquire female-specific conditioned odor aversion; similar to WT males, suggesting that experience-dependent plasticity in pheromone-mediated behavioral responses occurs independently of TrpC2-mediated inputs. Thus, signaling through the VNO does not have a role in sexual discrimination. Our results agree with previous studies reporting that sexually naïve males with surgically ablated VNOs can discriminate between female and male conspecifics’ urine, yet they fail to present a sexual preference for the opposite sex (Keller et al., 2006, Liu et al., 2010,

Pankevich et al., 2004). Research in our laboratory has provided us with evidences suggesting that TrpC2-/- males display diminished behavioral and physiological responses towards sexual rewards. We showed that TrpC2-/- males do not present olfactory preference and are significantly less motivated to invest effort in order to overcome obstacles and reach a female reinforcer, as compared to WT. We also found TrpC2-/- males fail to display plasma testosterone responses to female pheromones, which are crucial for mediating the rewarding properties of females, triggering the pre-copulatory acts. This agrees with findings demonstrating that surgical ablation of VNO in male mice prevents the elevation of circulating testosterone levels following presentation of an anesthetized female (Wysocki et al., 1983). Last, a microdialysis study led in our lab by Dr. Yael Lavi-Avnon identified a robust transient increase in DA levels in the NAc of WT males when they encounter strange conspecifics females, but not males. In contrast, social encounters of TrpC2-/- males with either female or male conspecifics failed to induce NAc DA elevation, even though these males presented intense olfactory investigation and mounting behavior towards both sexes. In the literature, an increase in extracellular DA in the NAc is thought to encode reward predications, enhance reinforcement learning and signals the motivational salience of the stimulus (Enomoto et al., 2011, Keiflin & Janak, 2015, Matsumoto & Hikosaka, 2009, Schultz, 2013). Therefore, we suggested an alternative explanation to the loss of mating preference in TrpC2-/- males. We hypothesized that pheromonal processing through the VNO is crucial for appropriate neuronal

49 representation of the intrinsic salient features of female-specific pheromones and in the following experiments we focused our efforts to test this assumption.

5.3. NAc dopamine signaling through D1R is required for pheromone-induced place preference To begin with, we investigated whether NAc DA signaling is necessary for assigning rewarding characteristics to female pheromones. We chose to use the conditioned place preference (CPP) assay, which have provided empirical support that sexual behaviors and sexual pheromones are strong intrinsically rewarding stimuli (Bell et al., 2010, Dominguez-Salazar et al., 2014, Korzan et al., 2013, Tenk et al., 2009, Trezza et al., 2011), and that mice can develop CPP for chemosensory signals derived from the opposite sex (Agustin-Pavon et al., 2007, Martinez-Ricos et al., 2007, Pierman et al., 2006, Roberts et al., 2012). In our study, sexually naïve males had direct access to female pheromone stimuli (bedding soiled by unfamiliar females) located in a particular chamber. We found that three consecutive sessions of 10 min exposures to the female stimuli were sufficient to induce CPP of males to the location previously associated with the female stimuli. This supports the assumption that female pheromones are natural salience stimuli, rewarding for males and mediating unconditioned innate behaviors [reviewed in (Beny & Kimchi, 2014, Pfaus et al., 2012)]. We showed that administering a D1R antagonist into the NAc during the conditioning phase did not affect immediate first interactions with the female reward, also characterized as the ‘liking’ aspect of reward, which is assumed to be independent of dopamine signaling (Agustin-Pavon et al., 2007, Berridge et al., 2009, Pitchers et al., 2013). Instead, the antagonist blocked the development of a preference for the chamber previously associated with female pheromones. Moreover, D1R blockage in the NAc during CPP conditioning reduced the olfactory sexual preference of males towards female pheromones more than 10 days later. Thus, signaling through D1 receptors in the NAc is potentially necessary for the development of female-induced CPP and long-term sexual preference. Our findings are consistent with other studies employing the CPP assay, as they showed that D1 receptors mediate the acquisition of place preference induced by sexual rewards as ejaculation and drug rewards in male mice (Dominguez-Salazar et al., 2014, Pina & Cunningham, 2014). In addition, Ago et al., demonstrated that i.p injections of either the D1 or D2 antagonists significantly attenuated the preference to approach a caged female over a male and the equivalent c-fos response in the NAc (Ago et al., 2015). Moreover, an increase in the transcription factor ∆FosB in the NAc was proved to have a critical role in the reinforcement of sexual behavior and motivation (Pitchers et al., 2010). It has been shown that blocking of DA signaling in the NAc during sexual behavior, using D1R antagonist, but not D2R antagonist, prevented ∆FosB upregulation in the NAc and in turn the reinforcing effect of sexual

50 behavior (Pitchers et al., 2013). In relation to our study, attenuation of female-induced ∆FosB upregulation might be a possible mechanism by which injection of D1R antagonist to the NAc during first exposure to female signals, blocked sexual preference days later. Overall, we demonstrated that female pheromones by themselves possess innately rewarding properties for males that depend on proper dopamine signaling in the NAc through D1 receptors, and further that this is a crucial step required for the display of sexual preference.

5.4. Targeting VTA to NAc projections rescued sexual preference of TrpC2-/- males The next required step to examine our hypothesis, suggesting the existence of reward processing deficits among TrpC2-/- males, was to artificially increase DA secretion in the NAc of TrpC2-/- males during exposure to female stimulus and examine whether sexual preference for female odors can be rescued. In order to achieve this, we expressed ChR2 in VTA dopaminergic neurons while optogenetically targeting their NAc projections. Importantly, previous studies demonstrated that in-vivo optical stimulation of VTA DA neurons (Adamantidis et al., 2011) and in-vitro stimulations of dopamine terminals in the NAc (Stuber et al., 2010), both resulted in dopamine release as measured using fast-scan cyclic voltammetry in the ventral striatum. Moreover, the amounts of dopamine detected following the in- vivo optical stimulation matched dopamine transients triggered by natural rewards (Adamantidis et al., 2011). Additionally, a study conducted in rats showed that selective activation of VTA-NAc pathway lead to an increase in c-Fos positive cells in the NAc and the VTA (Steinberg et al., 2014). Our results demonstrate that selective activation of dopaminergic terminals in the NAc, during exposure to a receptive female, triggered sexual preference for female over male bedding among TrpC2-/- males as opposed to the control TrpC2-/- group, which showed no such preference. Moreover, ChR2-TrpC2-/- males displayed improved sexual behavior towards a receptive female as relative to their matched controls. However, manipulating VTA-NAc dopaminergic pathway during first exposures to female did not influence the behavior towards male conspecific as both ChR2-TrpC2-/- and control TrpC2-/- males did not present inter-male aggression. The behavioral effect of optogenetic activation was not evident immediately, during the activation itself, as no significant different was found between the groups in sniffing duration of the female. Instead, the effect on sexual behavior was witnessed only days later, unassociated temporally from the activation period. This resembles the delayed, but opposite, effect of D1R antagonist on CPP and sexual preference which was witnessed few days following its injection to the NAc, and not immediately. Researchers have dissect reward into 3 separate components: ‘liking’, ‘wanting’, and learning; whereas dopamine has been shown to be involved in the ‘wanting’ aspect of reward, assigning incentive salience to a stimulus and driving reward-seeking behavior (Berridge & Kringelbach, 2015, Berridge et al., 2009, Hu, 2016). The

51 results obtained throughout the study strongly support the notion that dopamine is not involved in the ‘liking’ aspect of reward (Berridge & Kringelbach, 2015). First, during olfactory preference measurements, WT and TrpC2-/- males spent longer time interacting with the stimulus bedding (either male or female) over clean bedding. Additionally, manipulating DA levels in the NAc (either by injection of D1R antagonist or by optogenetically activating DA terminals) had no influence on the immediate consumption of female reward. Thus, it looks as if dopamine did not mediate the current pleasurable effect of female stimulus. We agree with studies suggesting a role for DA in the ‘wanting’ aspect of reward (Berridge, 2007) and postulate that activation of dopaminergic neuronal projections led to attribution of incentive salience to the associated female stimuli, which became the object of desire and motivationally ‘wanted’ over other unassociated stimuli. Hence, the males learned that female signals are attractive and desirable incentives, and since no such attribution occurred towards male signals, we witnessed female-biased approach and investigatory behavior at the following encounters. Other neurotransmitters systems that have been shown to control reward-related behavior, such as VTA GABAergic (Eshel et al., 2015) neurons and dorsal raphe serotonergic neurons (Luo et al., 2015), needs to be accounted for when investigating reward processing of pheromonal-mediated behaviors. Moreover, since there is evidence showing corelease of glutamate (Stuber et al., 2010, Tecuapetla et al., 2010) and GABA (Tritsch et al., 2012) from VTA DA neurons, we should not rule out that the behavioral effect we witnessed following activation of VTA dopaminergic neurons may be mediated by other neurotransmitters rather than dopamine. Our results imply that pheromonal signaling through the VNO mediates dopamine elevation in the NAc, which in turn drives sexual preference and motivation. Sex-specific pheromones, detected through the VNO, activate different regions in the accessory olfactory bulb (AOB) and in turn results in different activation pattern in the central brain (Dudley & Moss, 1999, Hashikawa et al., 2016). From the AOB, female signals relays to the posterodorsal MeA (MeApd), posteromedial bed nucleus of the stria terminalis (BNSTpm) and posteromedial cortical amygdala (COApm), which in turn project to the medial hypothalamus (Cádiz-Moretti et al., 2013, Mohedano-Moriano et al., 2007). The hypothalamus communicates further with motor control areas to release approach and mating behaviors (reviewed in (Hashikawa et al., 2016)). There are two possible indirect ways for the VNO to increase DA levels in the NAc. The first is through connections between the hypothalamus (specifically medial preoptic area) and the VTA (Beier et al., 2015, Watabe-Uchida et al., 2012). The second is via stimulation of the amygdala, which induces an increase in NAc DA in a VTA-independent mechanism (Floresco et al., 1998, Novejarque et al., 2011, Stuber et al., 2011). Research exploring the function of specific neuronal circuits in mediating social behaviors is thriving, however full understanding of behavioral circuits is currently not within reach (Yizhar, 2012). By 52 working on both wild-type and knockout male mice, using various techniques and different approaches we were able to construct a simplistic model representing the important findings of this thesis. Our model suggests the following: female-specific signals, which are detected by the VNO, elicit elevation of DA in the NAc, in a mechanism yet to be discovered. By signaling through the D1 dopamine receptor in the NAc, intrinsic positive rewarding properties (incentive salience) are assigned to female odors and this promotes reward-seeking behavior and sexual preference for female cues. Our results further suggest that the incentive salience of female pheromones, driving innate sexual preference, can be modified throughout life following negative experience and this process appears to be independent of VNO- mediated signaling. In summary, our results provide further support to role of mesolimbic DA in social approach behavior, and are of added value, since they relate specifically to males’ preference to female odors, and focuses on the role of VNO inputs in mediating this innate and learned goal-directed copulatory behavior. Future studies should further explore the mechanism of VNO-dependent DA increase in the NAc, as well as the involvement of other neurotransmitter systems such as serotonin in mediating innate sexual behaviors.

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