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 1 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. 2 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 3 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 4 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. 5 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