REGULATION OF PRESYNAPTIC FUNCTION BY SODIUM PERMEABLE ION CHANNELS AT THE CALYX OF HELD SYNAPSE AN ABSTRACT SUBMITTED ON THE NINETEENTHDAY OF APRIL2021 TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE SCHOOL OF SCIENCEAND ENGINEERING OF TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY BY DAINAN LI Approved: --�--4+· _-_z..__?__ - __ Hai Huang, Ph.D., Advisor Jeff�eyG. Tasker, Ph.D. Laura A. Schrader, Ph.D. �OJ-1 Andrea Zsombok, Ph.D. ABSTRACT Synaptic strength, which is described as the amplitude of postsynaptic response upon a presynaptic spike, is essential for reliable synaptic transmission. Previous work has revealed a presynaptic cytosolic Na+-dependent regulation on vesicular glutamate content and miniature excitatory postsynaptic current (mEPSC) amplitude via activating vacuolar Na+/H+ exchangers (NHEs) expressed on the synaptic vesicles, suggesting a presynaptic determinant of quantal size for synaptic strength (Goh et al., 2011; Huang and Trussell, 2014). Manipulation of the presynaptic Na+ at the calyx of Held synapse with up and down regulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channel activity, which induced a small change of cytosolic resting Na+ level, bidirectionally changed the quantal size (Huang and Trussell, 2014). However, it remains unknown how spike activities control intracellular Na+ at the axon terminals and how the fluctuation of presynaptic Na+ during activities modulates quantal content and contributes to synaptic strength. I studied these questions using the calyx of Held, a giant glutamatergic synapse in the auditory brainstem that allows direct pre- and postsynaptic recordings and manipulation of presynaptic cytosolic environment. With two-photon Na+ imaging, I found that presynaptic Na+ substantially accumulated during spike firing in a frequency and duration-dependent manner. This spike-induced elevation of presynaptic Na+ gradually increased EPSC amplitude by solely affecting vesicular glutamate filling, which was further confirmed as increased amplitude of asynchronous released vesicles, but without affecting the size of readily releasable pool or neurotransmitter release probability. This Na+-dependent modulation of EPSC amplitude resulted in a change of the reliability of transferring presynaptic spike to postsynaptic firing. Finally, blockade of NHEs reduced both EPSC amplitude and reliability of synaptic signaling, suggesting that NHEs are required for presynaptic Na+ regulation of synaptic transmission. Recent studies demonstrated that a TTX- and Cs+-resistant, non-inactivation cation channel NALCN (Na+ leak channel, non-selective), characterized as a major Na+ leak channel, is widely expressed in the central nervous system. I asked whether NALCN channel is also expressed in the axon terminal and if so how it controls intracellular Na+ and synaptic transmission. Immunostaining with antibodies against NALCN revealed the expression of this channel at the calyceal terminals. In line with a role of NALCN in controlling the cell excitability, calyces with conditional knockout (cKO) of NALCN exhibited a more hyperpolarized resting membrane potential compared with the wildtype (WT) calyces. Blockade of NALCN with a non-specific NALCN blocker gadolinium (Gd3+) induced a reduction of basal Na+ level and mEPSC amplitude (quantal size) in the WT but not in cKO group, suggesting the involvement of presynaptic NALCN channels in regulating the vesicular glutamate content. More importantly, two-photon Ca2+ imaging showed that NALCN channels were permeable to Ca2+, and Gd3+ decreased the basal Ca2+ level in WT but not cKO calyces. The Ca2+ permeability was further confirmed by reduced sensitivity of mEPSC frequency in response to increased extracellular Ca2+ concentration in cKO and reduced initial release probability in response to application of Gd3+ to block NALCN channels in WT group. Finally, Gd3+ induced a stronger reduction of EPSC amplitude in WT group compared to cKO group. Overall, these data indicate that NALCN channels regulate glutamate transmission through modulation of both quantal size and initial release probability. REGULATIONOF PRESYNAPTIC FUNCTION BY SODIUM PERMEABLE ION CHANNELS AT THE CALYX OF HELD SYNAPSE A DISSERTATION SUBMITTED ON THE NINETEENTHDAY OF APRIL2021 TO THE DEPARTMENT OF CELL AND MOLECULAR BIOLOGY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE SCHOOL OF SCIENCE AND ENGINEERING OF TULANE UNIVERSITY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DAINAN LI Approved: __.....,_{k --'---·�----- Hai Huang, Ph.D., Advisor � ( l'ya,, JeffreyG. Tasker, Ph.D. c::Zt..� dLaura A. Schrader, Ph.D. �a/2 Andrea Zsombok, Ph.D. © Copyrights by Dainan Li, 2021 All Rights Reserved ACKNOWLEDGMENT It has been seven years since the Department of Cell and Molecular Biology at Tulane University provided the stage for me to pursue my Ph.D. degree. I deeply appreciate this chance and this whole Ph.D. training has become one of the most valuable experience in my life. This is a long journey and the only reason I can survive is that I received tremendous supports from my mentors, colleagues, family, and friends. First and foremost, I would like to express my special thanks of gratitude to my Ph.D. advisor, Dr. Hai Huang. As an excellent and rigorous electrophysiologist, he taught me in many aspects of doing research including basic knowledge, experimental skills, scientific thinking, and troubleshooting since the first day I joined the lab. I have greatly benefited from his suggestions, such as how to understand and learn more information from publications, how to design appropriate experiments to address scientific questions, and how to fully understand and analyze experimental results without missing new discoveries. Without Dr. Huang’s constant guidance and support, I will never finish my dissertation and reach my goal. I also would like to gratefully thank my committee members, Dr. Jeffrey Tasker, Dr. Laura Schrader, and Dr. Andrea Zsombok for their invaluable feedbacks and suggestions, which kept my research work in pace. I not only took their classes to build my basic background of neuroscience, but more importantly received advice from them to help me better understand my work and be confident to explore more scientific questions. I especially want to thank Dr. Tasker for letting me use the equipment and materials in his lab and being a source of motivation. ii Next, I would like to thank all the current and previous members in the Huang lab for creating a lovely environment that I always feel relax and joyful during my stay in the lab. I want to express my thanks to Dr. Yihui Zhang for all the experimental discussion, technical assistance, and encouragement. I would like to thank Yun Zhu, who helped me to finish the capacitance measurement experiment and shared a lot of useful experimental tricks to me. Many thanks go to Youad Darwish for staying with me in the lab in a lot of days, to Dr. Inga Kristaponyte for helping with my dissertation and sharing tasty chocolate cake flavor beer, and to Tianhao Wu for taking care of our animals. Then, I would like to thank all the faculty and Ph.D. students in the CMB for their encouragement and useful advice I received after my annual student presentation. I also would like to thank the nice people in our CMB office, Marnie Elsky, John Drwiega, and Jonathan Flack for their sweet help in our daily work. Last, but certainly not least, I would like to thank my family and friends for their constant support. Special thanks to my husband Xin Fu, who always tried to make me happy during my tough time. iii Table of Contents ACKNOWLEDGMENT ..................................................................................................... ii LIST OF FIGURES ......................................................................................................... vii CHAPTER 1: INTRODUCTION ...................................................................................... 1 1.1 Synaptic Transmission Between Neurons ............................................................. 1 1.2 Strength of Synaptic Transmission ........................................................................ 3 1.2.1 Readily releasable vesicles (n) ....................................................................................................... 5 1.2.2 Release probability (pr) .................................................................................................................. 6 1.2.3 Quantal size (q) .............................................................................................................................. 8 1.2.4 The role of Ca2+ for synaptic strength .......................................................................................... 10 1.3 Research Model: The Calyx of Held Synapse ..................................................... 11 1.4 Formation of Quanta at Glutamatergic Synapse ............................................... 14 1.5 Presynaptic Determinants of Quantal Size in Glutamatergic Synapse ............ 16 1.5.1 Expression level of VGLUTs ...................................................................................................... 16 1.5.2 Cl- gradient .................................................................................................................................. 17 1.5.3 Cation/H+ exchanger ...................................................................................................................