FEAR AND ASSESSMENT OF SAFETY IN RATS SELECTIVELY BRED FOR DIFFERNTIAL EMISSION OF 50 KHZ ULTRASONIC VOCALIZATIONS Emily S. Webber A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment for the degree of Masters of Arts August 2009 Committee: Howard Casey Cromwell, Ph.D, Advisor Verner Bingman, Ph.D. Laura Dilley, Ph.D. i Abstract: H. Casey Cromwell, Advisor The goal of this study was to explore fear in rats that were selectively bred for variations in 50 kHz ultrasonic vocalizations (USVs) emission. Animals’ USVs are related to affective states and have been shown to be vital for communication and social interactions. 50 kHz USV emission during a tickle paradigm was used as the selection criterion for choosing breeders. Three animal lines were bred: high, low and random. The high line animals emit significantly more 50 kHz USVs when compared to the random line animals while the low line animals emit significantly fewer 50 kHz USVs than the random line animals. Random line animals were produced by arbitrarily choosing 2 breeders from two different litters. Prior studies have suggested a variation of affective states in these animals. Behavioral strategies were used as measures of whether or not the animals diverge on traits related to fear and the assessment of safety: 1) social recognition, 2) play suppression, and 3) prepulse inhibition (PPI). The social recognition task requires basic discrimination abilities and examined the ability of these animals to assess safety and familiarity in social situations. The play behavior paradigm investigated instinctual fear responses, conditioning and extinction of play suppression by observing play behavior for several days after exposure to the aversive unconditioned stimulus. The PPI test was used to assess reflexive fear and sensorimotor gating by measuring the acoustic startle response and the inhibition of this response. Results showed that low line animals had impaired social recognition abilities compared to random and high line animals. During play high line animals demonstrated exaggerated conditioned fear to the play apparatus. Conversely, low line animals failed to show contextual fear conditioning. ii High and low line animals had deficiencies in PPI. High line animals emitted more 22 kHz USVs than the other lines. Overall these findings suggest that the high line animals may have exaggerated emotional conditioning abilities and the low line animals may have deficits in emotional associative learning. This animal model may serve as a useful tool in examining the basic genetics and neurobiology of emotional learning. iii “As soon as there is life, there is danger.” - Ralph Emerson iv This manuscript is dedicated to Karl and Lillian Englund, their never ending support and love has helped to educate me, providing me with the knowledge and skills to pursue my passion. They are forever in my heart, my mind and my determination to succeed. v ACKNOWLEDGEMENTS I thank my advisor H. Casey Cromwell for all of guidance and support that he has given me throughout the course of this project. In addition you have provided me with essential feedback required to write this manuscript. Thank you for all of your help and efforts. I also would like to thank my committee members Verner Bingman and Laura Dilley for their service on this committee and advice given throughout this process. In addition, I thank Jaak Panksepp for agreeing to be a special guest at my thesis proposal meeting; his insight has guided the interpretation and analysis of my results. I also acknowledge and thank Jeff Burgdorf, for helping me learn the scoring process of USVs on spectrograms. Another individual who has helped me grasp the prior research on this topic is graduate student, Kelley Harmon. Thank you for all your help in the interpretation of both the prior research and the project at hand. Your advice has been invaluable to me. I also thank Travis Beckwith and Samantha Peña for all their help in running the experiments, you did an exemplary job. This project was funded by the Hope for Depression Research Foundation. I also thank the J.P. Scott Center for Neuroscience Mind and Behavior for providing an atmosphere of collaboration and constructive criticism necessary for my development as a graduate student. Employees of University Animal Facilities are also in my debt, as they took care of the animals’ daily needs throughout this project. vi TABLE OF CONTENTS Page CHAPTER I. INTRODUCTION ..……………………………………………….... 1 Animal Models of Emotion ………………………………………… 1 Ultrasonic Vocalizations and Rat Behavior ……………………….. 2 Neurobiology of USVs ……………………………………………. 5 Selective Breeding ………………………………………………… 9 Fear as a Key Social Emotion …………………………………….. 13 Behavioral Paradigms Used in this Study …………………………. 16 Social Recognition ……………………………………….... 16 Play Suppression …………………………………………... 19 Prepulse Inhibition …………………………………………. 20 CHAPTER II AIMS OF THIS STUDY …..…….…………………………………22 Aim I: Social Recognition ………………………………………… 22 Aim II. Play Suppression …………………………………………. 23 Aim III: Prepulse Inhibition ………………………………………. 23 CHAPTER III. METHODS ……………………………………………………….. 25 Breeding …………..………………………………………………… 25 Social Recognition .………………………………………………… 25 Play Suppression ….……………………………………………….. 26 Prepulse Inhibition ….……………………………………………… 28 CHAPTER IV. STATISTICAL ANALYSIS ....…………………………………… 30 Social Recognition …………….…………………………………… 30 vii Play Suppression …………………………………………………... 31 Prepulse Inhibition ………………………………………………… 32 CHAPTER V. RESULTS …………………………………………………………. 32 Social Recognition ………………………………………………… 32 Play Suppression …………………………………………………... 45 Prepulse Inhibition ………………………………………………… 68 CHAPTER VI. DISCUSSION ...………………………………………………….. 79 Summary ……………………………………..……………………. 79 Social Recognition ………………………………………………… 79 Play Suppression …………………………………………………... 82 Prepulse Inhibition ………………………………………………… 84 Previous Research: A Closer Look ………………………………... 85 Alternative Hypothesis: Variations in Associative Learning ..…….. 87 Future Research ……………………………………………………. 91 Clinical Implications ………………………………………………. 92 REFERENCES ……………………………………………………………………. 95 viii LIST OF FIGURES Figure Page 1 Percentage of Social Investigation ………………………………………….. 36 2 Percentage of Social Motivation ……………………………………………. 40 3 50 kHz USVs During Social Recognition ………..………………………….. 44 4 Pin Number During Play Suppression …......................................................... 46 5 Pin Duration in seconds During Play Suppression ………………..………… 48 6 Doral Contacts During Play Suppression …………………………………… 50 7 50 kHz USVs on the Worn Cat Collar Day …………………………………. 52 8 ASR for Different Trial Types During PPI ...................................................... 71 9 PPI (%) for the Different Selectively Bred Lines of Animals ………………. 72 10 22 kHz USV emission over different trial types During PPI ……….………. 73 11 22 kHz USV Examination of Trial and Sex Interaction During PPI ….……. 74 12 22 kHz USV Emission of Different Lines of Animals During PPI ……....… 75 13 22 kHz USV Emission Sex Differences During PPI ……………….…….… 76 14 22 kHz USV Emission Over Blocks of Trials During PPI …………….…… 77 15 22 kHz USV Emission Investigating Trial Block and Animal Line Interaction ……………………………………………………………… 78 ix LIST OF TABLES Table Page 1 Independent Samples t-test for Social Investigation Percentage ……………….. 33 2 Means ± S.E.M Social Investigation Percentage ……………………………….. 34 3 Independent Samples t-test for Social Motivation Percentage …......................... 38 4 Means ± S.E.M Social Motivation Percentage ..……………………………….. 39 5 Independent Samples t-test for 50 kHz USVs During Social Recognition ………...……………………………………………………. 42 6 Means ± S.E.M. for 50 kHz USVS during Social Recognition ………………… 43 7 Independent Samples t-tests Pin Number During Play Suppression …...……….……………………………………….................. 53 8 Paired Samples t-tests of Pin Numbers for Random Line Animals During Play Suppression …..…………………................................................... 54 9 Paired Samples t-tests of Pin Numbers for High Line Animals During Play Suppression ……..………………………………………………... 55 10 Paired Samples t-tests of Pin Numbers for Low Line Animals During Play Suppression ……..………………………………………………... 56 11 Means ± S.E.M. of Pin Numbers During Play Suppression ………..…………. 57 12 Independent samples t-tests on Pin Duration During Play Suppression ……..……………………………………………………….. 58 13 Paired Samples t-tests of Pin Duration for Random Line Animals During Play Suppression …………….………………………………………... 59 x 14 Paired Samples t-tests of Pin Duration for High Line Animals During Play Suppression …………………………………………………….... 60 15 Paired Samples t-tests of Pin Duration for Low Line Animals During Play Suppression ……………………………………………………… 61 16 Means ± S.E.M. of Pin Durations During Play Suppression ………….………. 62 17 Independent Samples t-tests Dorsal Contact Number During Play Suppression ……………………………………………………… 63 18 Paired Samples t-tests of Dorsal Contact Numbers for Random Line Animals During Play Suppression ……………………………………………. 64 19 Paired Samples t-tests of Dorsal Contact Numbers for High Line Animals During Play Suppression ……………………………………………. 65 20 Paired Samples t-tests of Dorsal Contact Numbers for Low Line Animals During Play Suppression ……………………………………………. 66 18 Means ± S.E.M. of Dorsal Contact Numbers During Play Suppression ……………………………………………………………… 67 1 CHAPTER I: INTRODUCTION Animal Models of Emotion Animal models have been essential in making progress within psychology and