Synaptic Specializations Mediated by Synaptotagmin Isoforms The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Turecek, Josef. 2019. Synaptic Specializations Mediated by Synaptotagmin Isoforms. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029600 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Synaptic specializations mediated by Synaptotagmin isoforms A dissertation presented by Josef Turecek to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Neurobiology Harvard University Cambridge, Massachusetts March 2019 © 2019 – Josef Turecek All rights reserved Dissertation Advisor: Professor Wade Regehr Josef Turecek Synaptic specializations mediated by Synaptotagmin isoforms The timing of neurotransmitter release in response to presynaptic firing varies widely across synapses in the brain. The release of synaptic vesicles can be synchronous and occur within milliseconds of an action potential, or asynchronous and persist for tens of milliseconds. Release can also undergo short-term facilitation in which it is enhanced by two closely timed action potentials. The molecular properties of synapses that determine the relative timing of synaptic vesicle release have remained poorly understood. We have found that Synaptotagmin 7 (Syt7) plays a major role in multiple aspects of synaptic transmission. Syt7 mediates short-term facilitation at almost all synapses where it is present, including seven different synapses tested here. Loss of Syt7 also eliminated facilitation that was activated by trains of stimuli at Purkinje cell and vestibular synapses, revealing an interplay between short-term facilitation and depression that maintains constant synaptic strength. Changes in the initial probability of release were not detectable in Syt7 KOs at three different synapses, and removal of Syt7 did not reduce the rate of recovery from depression in cerebellar Purkinje cell synapses. Facilitation could be rescued in Syt7 KOs by presynaptic expression of Syt7, but not Ca2+-insensitive Syt7. At cerebellar parallel fibers we found that Syt7 can mediate both facilitation and asynchronous release, but there are additional mechanisms of facilitation and asynchronous release in the absence of Syt7. In contrast to facilitation, the presence or absence of Syt7 does not determine whether asynchronous release occurs. Syt7 was most strongly expressed at Purkinje cell synapses in the cerebellar nuclei, even though no asynchronous release could be detected. Inhibitory synapses in the inferior olive are exclusively asynchronous, and we found that release synchrony is instead determined by the absence of Synaptotagmin 1 and 2. Syt7 plays a role in shaping the kinetics of asynchronous release in the inferior olive, but does not control the magnitude of asynchronous release. Our findings suggest that Syt7 could be a Ca sensor for facilitation that is iii prominent at many synapses, and that it may also play a role in asynchronous release in combination with other additional mechanisms. iv Table of Contents Abstract ....................................................................................................................................................... iii Table of Contents ........................................................................................................................................ v Acknowledgements .................................................................................................................................... vi Chapter 1: Introduction ............................................................................................................................. 1 Chapter 2: Synaptic specializations support frequency-independent Purkinje cell output from the cerebellar cortex ........................................................................................................................................ 15 Chapter 3: Synaptotagmin 7 confers frequency invariance onto specialized depressing synapses .................................................................................................................................................................... 65 Chapter 4: Synaptotagmin 7 mediates both facilitation and asynchronous release at granule cell synapses .................................................................................................................................................... 101 Chapter 5: Neuronal regulation of fast Synaptotagmin isoforms controls the relative contribution of synchronous and asynchronous release ................................................................................................ 134 Chapter 6: Conclusion ........................................................................................................................... 173 Appendix A, The calcium sensor Synaptotagmin 7 is required for synaptic facilitation ................. 178 Appendix B, The role of CaV2.1 channel facilitation in synaptic facilitation ................................... 205 References ................................................................................................................................................ 234 v Acknowledgments I would like to thank my advisor Wade Regehr for his rigorous training and guidance. I also thank current and former members of the Regehr lab for supplying the lab with humor: Kimberly McDaniels, Skyler Jackman, Laurence Witter, Stephanie Rudolph, Jay Wang, Chris Chen, Chris Weyrer, Chong Guo, Kyung Han, Tomas Osorno, Mehak Khan, Monica Thanawala, and Diasynou Fioravante. I thank members of my dissertation advisory committee: Pascal Kaeser, Chinfei Chen, and Bruce Bean for taking the time to provide me with feedback and suggestions. I also thank Rachel Wilson for her career and personal advice. I thank Karen Harmin for keeping a watchful eye over my graduate education. I would like to thank my parents, Frank and Olga Turecek for their endless support, and for providing me with the opportunity to obtain a world-class education. Finally, I would like to thank Keiko Weir for her close friendship, support, wisdom, and love. vi Chapter 1 Introduction The presynaptic terminal Neurotransmitter is typically released from presynaptic terminals by highly specialized machinery. Transmitter is stored in synaptic vesicles that undergo fusion in the presence of calcium. Fusion of the vesicle with the presynaptic membrane releases its internal contents, allowing neurotransmitter to enter the synaptic cleft and be detected post-synaptically by neurotransmitter receptors. Fusion usually occurs at a release site where fusion machinery is concentrated and synaptic vesicles are docked and primed to be released. Synchronous and asynchronous release Several forms of release can occur, and they are distinguished by their timing relative to presynaptic action potentials. When an action potential invades the presynaptic bouton, synchronous release is evoked within a millisecond, closely time-locked to presynaptic spiking (Katz and Miledi, 1965, Sabatini and Regehr, 1996, Borst and Sakmann, 1996). In contrast, asynchronous release is delayed and loosely time-locked to the presynaptic action potential, occurring for tens to hundreds of milliseconds after spiking (Atluri and Regehr, 1998, Lu and Trussell, 2000, Hefft and Jonas, 2005, Iremonger and Bains, 2007, Daw et al., 2009, Best and Regehr, 2009, Peters et al., 2010). Release can also occur in the absence of presynaptic activity in the form of spontaneous release. For individual release sites the rate of spontaneous release is very low and may serve to maintain synapses rather than contributing to the rapid processing of information (but see Carter and Regehr, 2002). Different forms of release are evoked more effectively by different patterns of activity. Synchronous release is often maximally evoked by single stimuli or infrequent presynaptic firing. In contrast, asynchronous release is always weakly evoked by single stimuli and instead builds with repeated stimulation, especially if the frequency of stimulation is high (Atluri and Regehr, 1998, Lu and Trussell, 2000, Hefft and Jonas, 2005, Iremonger and Bains, 2007, Daw et al., 2009, Best and Regehr, 2009, Peters et al., 2010). Thus, the contribution of asynchronous release is only prominent when presynaptic firing is elevated, or during brief bursts of presynaptic activity. 2 The contributions of synchronous, asynchronous and spontaneous release at different synapses in the brain can vary widely. Release at almost all synapses is predominantly synchronous, but some synapses have especially fast synchronized release. At the mature Calyx of Held, synchronous release occurs for less than a millisecond after firing (Borst and Sakmann, 1996). Inhibitory (GABAergic) synapses can also be very rapid. For example, the release of GABA at cerebellar Purkinje cell synapses is restricted to two milliseconds following a spike (Person and Raman, 2012b). Release at these synapses is tightly controlled, and there is no evidence of asynchronous release in mature animals, even after prolonged