NEUROMODULATION OF GANGLION CELL PHOTORECEPTORS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Puneet Sodhi, B.S. Graduate Program in Neuroscience The Ohio State University 2015 Dissertation Committee: Dr. Andrew TE Hartwick, Advisor Dr. Karl Obrietan Dr. Stuart Mangel Dr. Heather Chandler Copyright by Puneet Sodhi 2015 ABSTRACT Intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a rare subset of ganglion cells in the mammalian retina that are primarily involved in non-image forming (NIF) visual processes. In the presence of light, ipRGC photoreceptors exhibit sustained depolarization, in contrast to the transient hyperpolarizing responses of rod and cone photoreceptors. The persistence of this response with light offset underlies the reduced temporal resolution exhibited by these ipRGCs. The overall aim of this thesis was to determine whether the unique temporal dynamics of ipRGC photoresponses are subject to modification by endogenous retinal neuromodulators. As post-synaptic photoreceptors, ipRGCs are capable of integrating photic information transmitted from pre-synaptic neurons regulated by rod- and cone-driven signaling. Given that ipRGCs possess dense dendritic nets that span the entire retina, I hypothesized that these ganglion cell photoreceptors were capable of being modulated by extrinsic input from the retinal network. Using multi-electrode array recordings on rat retinas, I demonstrated that the duration of light-evoked ipRGC spiking can be modified through an intracellular cAMP/PKA-mediated signaling pathway. Specifically, stimulation of the cAMP/PKA pathway leads to prolonged ipRGC light responses. Expanding upon these findings, I next identified an endogenous retinal neuromodulator capable of modulating ipRGC photoresponses through this signaling pathway. I demonstrated that the retinal neuromodulator adenosine suppressed light-evoked ipRGC spiking through activation of the Gi-coupled A1 receptor. These receptors were expressed by ipRGCs themselves, as ii confirmed using immunohistochemistry and calcium imaging experiments on dissociated ipRGCs. Notably, I show that endogenous adenosine A1-mediated suppression of ipRGC photoresponses can occur during dark-adapted conditions, consistent with an elevation in retinal adenosine levels after maintenance in the dark. Dark adaptation serves to increase sensitivity and contrast detection in conventional photoreceptors. However, in ipRGCs, dark adaptation leads to a global suppression of light-evoked responses that does not appear to be due to a reduction in sensitivity. I also showed that ipRGCs can be directly stimulated through a melanopsin-independent pathway by cholinergic activation of muscarinic receptors present on ipRGCs. The prolonged action potential firing evoked by cholinergic compounds was relatively specific to ipRGCs, as compared to other RGCs. Activation of these receptors by endogenous acetylcholine could be elicited by exposing the retina to yellow 6 Hz flickering light stimuli, as application of muscarinic antagonists blocked the robust ipRGC responses induced by this stimulus. Moreover, preliminary evidence suggests that these results could be translatable to humans as examination of pupillary constriction in two subjects revealed that 6 Hz flickering red light produces a greater mean pupil constriction than continuous red light exposure. Taken together, these studies indicate that ipRGC photoresponses are much more complex than previously appreciated. The temporal characteristics of these ganglion cell photoreceptors are not a static feature. Instead, ipRGCs are highly dynamic, capable of altering their responses to light in the presence of retinal neuromodulators such as adenosine and acetylcholine. iii DEDICATION This dissertation is dedicated to my family and to my current and former mentors. iv ACKNOWLEDGMENTS First, I would like to acknowledge my past mentors for the tremendous guidance they provided during my undergraduate years. I thank Eva Feldman for taking the time to steer me towards Jack Parent’s lab, despite the fact that she receives hundreds of emails from undergraduate students looking for research experience and despite the lack of availability in her own lab. If it were not for her guidance, I would have never considered a career as a scientist. I would like to thank Dr. Jack Parent for his support and mentorship throughout my undergraduate studies. I would especially like to thank Patricia Reuter-Lorenz for her mentorship throughout the duration of my time in her lab, as I completed my undergraduate thesis. She truly molded me as a researcher and inspired me to embark upon my current endeavor. I am thankful for her support to this day, as she continues to provide valuable insight. I would truly like to thank Andrew Hartwick for his mentorship throughout my graduate studies. I greatly appreciate his zeal for scientific study and his approach to science as a whole, as I know he has shaped my growth as a scientist in a tremendous capacity. I appreciated the freedom I was afforded in the lab to pursue scientific problems that inspired me. I would also like to thank him for truly supporting my goals and career aspirations. He has molded his mentorship to fit my aspirations, which is uncommon for mentors and for that, I am very thankful. As his first graduate student, I am grateful for his support and close interactions throughout my time at Ohio State. I v would also like to thank David Hackos for his support during my time at Genentech and for his continued mentorship and for his valuable insight as a scientist in industry. I would like to thank my labmates, Patrick and Phil, for their support and friendship. I am especially grateful to Phil for his help with the pupil experiments presented in this thesis. Without him, these experiments would not have been possible. I am grateful for the NGSP faculty at Ohio State for their expertise and support. I would specifically like to thank Karl Obrietan, Stuart Mangel, and Heather Chandler for taking the time to be on my committee. I thank them for their support and feedback on my work. I would especially like to thank Heather for her support and insight, both in relation to scientific matters and beyond, throughout my time at Ohio State. I would like to thank my parents and my brother for their tremendous support and encouragement throughout the years I spent in higher education. Last but not least, I would like to thank my husband Kyle for his patience, encouragement, and insight throughout my graduate studies. As a physician scientist in training himself, he has provided invaluable insight and support during my training. I am forever grateful for his unrelenting encouragement both professionally and personally. vi VITA 2005 ............................................................ Troy High School 2009 ............................................................ B.S. Neuroscience, Psychology (Honors), University of Michigan 2009 to present .......................................... Graduate Research Associate, Department of Neuroscience, The Ohio State University PUBLICATIONS 1. Sodhi P and Hartwick TE (2014). Adenosine modulates light responses of rat ganglion cell photoreceptors through a cAMP-mediated pathway. Journal of Physiology, Epub PMID 25038240. 2. Sodhi P and Hartwick TE (2015). Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors. Journal of Neuroscience (in review) FIELDS OF STUDY Major Field: Neuroscience vii TABLE OF CONTENTS Abstract ........................................................................................................................... ii Dedication ...................................................................................................................... iv Acknowledgments ........................................................................................................... v Vita ................................................................................................................................. vi List of Figures ................................................................................................................ vii Chapters: 1. Introduction ................................................................................................................. 1 2. Modulation of ipRGC photoreseponses through a cAMP/PKA signaling pathway ......37 3. Adenosine modulates ipRGC photoresponses through a cAMP/PKA pathway ..........60 4. Muscarinic acetylcholine receptor-mediated stimulation of ipRGCs ...........................91 5. Conclusions ............................................................................................................. 115 List of References ....................................................................................................... 133 viii LIST OF FIGURES Figure Page 1.1 Differences in vertebrate and invertebrate phototransduction…………………..35 1.2 Proposed model for ipRGC photoresponse regulation through adenosingeric and cholinergic signaling pathways ……………………………………………………………..36 2.1 Effect of spontaneous activity blockade on neonatal ipRGC light responses….54 2.2 Effect of forskolin, an adenylate cyclase stimulator, on neonatal ipRGC photoresponses……………………………………………………………………………….55 2.3 Role for cAMP and PKA in modifying neonatal ipRGC light responses………..57 2.4 Effect of synaptic activity blockade on