ORIENTING IN 3D SPACE: BEHAVIORAL AND NEUROPHYSIOLOGICAL STUDIES IN BIG BROWN BATS By Ninad B. Kothari A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland December 2017 © 2017 Ninad B. Kothari All Rights Reserved Abstract In their natural environment, animals engage in a wide range of behavioral tasks that require them to orient to stimuli in three-dimensional space, such as navigating around obstacles, reaching for objects and escaping from predators. Echolocating bats, for example, have evolved a high-resolution 3D acoustic orienting system that allows them to localize and track small moving targets in azimuth, elevation and range. The bat’s active control over the features of its echolocation signals contributes directly to the information represented in its sonar receiver, and its adaptive adjustments in sonar signal design provide a window into the acoustic features that are important for different behavioral tasks. When bats inspect sonar objects and require accurate 3D localization of targets, they produce sonar sound groups (SSGs), which are clusters of sonar calls produced at short intervals and flanked by long interval calls. SSGs are hypothesized to enhance the bat’s range resolution, but this hypothesis has not been directly tested. We first, in Chapter 2, provide a comprehensive comparison of SSG production of bats flying in the field and in the lab under different environmental conditions. Further, in Chapter 3, we devise an experiment to specifically compare SSG production under conditions when target motion is predictable and unpredictable, with the latter mimicking natural conditions where bats chase erratically moving prey. Data from both of these studies are consistent with the hypothesis that SSGs improve the bat’s spatio-temporal resolution of target range, and provide a behavioral foundation for the analysis and interpretation of neural recording data in chapters 4 and 6. The complex orienting behaviors exhibited by animals can be understood as a feedback loop between sensing and action. A primary brain structure involved in sensorimotor integration is the midbrain superior colliculus (SC). The SC is a widely studied brain region and has been ii implicated in species-specific orienting behaviors. However, most studies of the SC have investigated its functional organization using synthetic 2D (azimuth and elevation) stimuli in restrained animals, leaving gaps in our knowledge of how 3D space (azimuth, elevation and distance) is represented in the CNS. In contrast, the representation of stimulus distance in the auditory systems of bats has been widely studied. Almost all of these studies have been conducted in passively listening bats, thus severing the loop between sensing and action and leaving gaps in our knowledge regarding how target distance is represented in the auditory system of actively echolocating bats. In chapters 4, 5 and 6, we attempt to fill gaps in our knowledge by recording from the SC of free flying echolocating bats engaged in a naturalistic navigation task where bats produce SSGs. In chapter 4, we provide a framework to compute time- of-arrival and direction of the instantaneous echo stimuli received at the bats ears. In chapters 5 and 6, we provide an algorithm to classify neural activity in the SC as sensory, sensorimotor and premotor and then compute spatial receptive fields of SC neurons. Our results show that neurons in the SC of the free-flying echolocating bat respond selectively to stimulus azimuth, elevation and range. Importantly, we find that SC neuron response profiles are modulated by the bat’s behavioral state, indicated by the production of SSG. Broadly, we use both behavior and electrophysiology to understand the action- perception loop that supports spatial orientation by echolocation. We believe that the results and methodological advances presented here will open doors to further studies of sensorimotor integration in freely behaving animals. iii Advisor: Dr. Cynthia Moss Internal Committee: Drs. Peter Holland, Shreesh Mysore and Susan Courtney. External Committee: Drs. Veit Stuphorn, Mounya Elhilali and Noah Cowan iv Dedication To my family In loving memory of Dr. Chintan Oza vi Acknowledgements MENTORS I am thankful to Cindy (Dr. Moss) for her support, guidance, encouragement and the extraordinary amount of freedom and trust she conferred on me. I have learnt many things from Cindy and a few of them (of the many) I hope to always keep with me are, hard work and dedication to ones work, always appreciating the good qualities in people, always and immediately responding to emails (as she says – it only takes a few seconds), sharing data and information with others and, the art of working efficiently. I deeply share Cindy’s constant endeavor to understand the neural correlates of complex behaviors when animals are freely moving (and flying!) and engaged in naturalistic tasks. Everyone deserves a second chance, and for that, I must thank Cindy for giving me a second chance, when she accepted me in her lab after I spent one year in Dr. Butt’s lab at UMCP. For that matter, I also thank, Dan (Dr. Butt), for giving me my first break in the completely new field (for me) of neuroscience. I am not sure how, I can ever thank Shreesh enough. In the completely new environment at JHU, I found in Shreesh both a mentor and a friend. I will always be indebted to him for time he has spent with me discussing spatial receptive fields and life. I have an immense amount of respect for him and I am extremely excited to join his lab as a post doc. I would like to sincerely thank my entire thesis and proposal committee - Drs. Peter Holland, Veit Stuphorn, Shreesh Mysore, Mounya Elhilali, Susan Courtney, Noah Cowan, Jonathan Flombaum and of course, Cindy – for taking out time from their busy schedules to read my infinitely long thesis. vii I have learnt a lot from all the amazing mentors that I have had the good fortune of working and interacting with, and, I believe, the best way to thank them is to carry these principles forward. LAB MEMBERS In my five (5+) years in the lab, I have made some life-long friendships that I will always carry forward with me. My graduate life would have been very dull without these amazing people. My trajectory in the Batlab would have been very different had it not been for Mel (Dr. Wohlgemuth, as he likes to be addressed in public. Rightfully too!). He has been my mentor as well; I learnt all of my wet lab and surgery skills from him. It is surprising at how fun science can be, when you find a collaborator and friend, like Mel (hopefully, he also learnt a thing or two from me). I will always cherish the ‘lunch, coffee and walk times’ that Mel, Ben and I used to squeeze into our daily schedules (but, we can continue, as I am not going far). The more time you spend with Ben, the more you appreciate him. In the short period of time that Mohit and I overlapped in the lab, we became quite close friends. Mohit actually suggested and pushed me to record from flying bats (it actually worked!). In the past two years, I have had the good fortune of collaborating with Jinhong, whom I have come to respect as a very passionate and meticulous scientist (when are we going to make a video of a bat dancing to the Zootopia song!). And, how can I forget Wei Xian, she is the person, who, behind the scenes, keeps the Batlab machinery so well oiled (Thanks a ton, Wei!). I would also like to thank the many Batlab members that I have had the good fortune of working and interacting with - Silvio, Wu-Jung, Aaron, Michaela, Angie, viii Brittney, Iven, Susanne, Chen, Dallas and Anand. I also want to thank my office mate, first for getting down on his knees and scrubbing our office floor and second, for all the great conversations we have had about science and life. I wish them all the best for their future success and look forward to their scientific contributions. FRIENDS I made lifelong friendships during my PhD years, but I cannot forget the evergreen friendships from the past that gave me the support and strength to carry on for all these years. The undying spirit of Ashutosh Tandon (Ashu) would be enough to make me want to continue with my PhD no matter how difficult and impossible it seemed. The wise and always unbiased advice from Saurabh Gupta would always be the checkpoint for my decisions. Sandeep Pandya and his wife Mamta have always been an inspiration for me. Their unflinching dedication towards their child has given me the confidence that no matter what the odds, there is always a way to overcome all difficulties. I would also like to thanks Sudipto Ghosh and Aditya Mutalik. They (and their families) have been part of our support system in the US. FAMILY Education, and even research, begins first at home and only later on in school or university; this is a maxim common in India, and, is very true in my case. Both of my parents have made tremendous sacrifices for empowering, my brother (Nishad) and me, with education and philosophy of life and values of dedication and passion towards ones work. They gave me the freedom to learn and explore, ask questions and also the confidence and ability to search for answers. I remember, as a 4 year old, my mother would take me to the local park (kamati baug) ix and I would spend the entire day, catching insects, searching for frogs and lizards and playing with other kids.