Synaptic Energetics: Consumption of ATP in the Presynaptic and Postsynaptic Terminal and the Interaction of ATP with the Presynaptic Protein Synapsin Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY” by Alexandra Stavsky Submitted to the Senate of Ben-Gurion University of the Negev 05.10.2019 Beer-Sheva Synaptic Energetics: Consumption of ATP in the Presynaptic and Postsynaptic Terminal and the Interaction of ATP with the Presynaptic Protein Synapsin Thesis submitted in partial fulfillment of the requirements for the degree of “DOCTOR OF PHILOSOPHY” by Alexandra Stavsky Submitted to the Senate of Ben-Gurion University of the Negev Approved by the advisor ______________ Approved by the Dean of the Kreitman School of Advanced Graduate Studies __________________________________ 05.10.2019 Beer-Sheva This work was carried out under the supervision of Dr. Daniel Gitler and Prof. Yael Amitai In the Department Physiology and Cell biology Faculty of Health Science Research-Student's Affidavit when Submitting the Doctoral Thesis for Judgment I, Alexandra Stavsky, whose signature appears below, hereby declare that (Please mark the appropriate statements): X I have written this Thesis by myself, except for the help and guidance offered by my Thesis Advisors. X The scientific materials included in this Thesis are products of my own research, culled from the period during which I was a research student. X This Thesis incorporates research materials produced in cooperation with others, excluding the technical help commonly received during experimental work. Therefore, I am attaching another affidavit stating the contributions made by myself and the other participants in this research, which has been approved by them and submitted with their approval. Date: 05.11.2019 Student's name: Alexandra Stavsky Signature: Affidavit stating the contributions of the other participants in this research Dr. Yoav Shulman, a former doctoral student in our lab, and Prof. Inna Slutsky, our collaborator from Tel-Aviv University, contributed to this research. I state their contribution in the body of the thesis (in the Methods and Results sections) where their data presented with their agreement: Dr. Yoav Shulman carried out all the intracellular electrophysiological recordings in autaptic cultures and measured synaptic puncta intensity or CI of Syb2. Prof. Inna Slutsky and her lab members (mentioned in the paper) measured the FR of SypI-AT1.03 in their confocal system during electrical stimulation. Alexandra Stavsky Signature: Date: 08.11.2019 I hereby declare that the author’s above statement about my contribution to this research is true and accurate, and I agree with the presentation of these results in her thesis. Prof. Inna Slutsky Signature: Date: 11.11.2019 Dr. Yoav Shulman Signature: Date: 13.11.2019 Acknowledgments First I would like to thank Dr. Daniel Gitler. Thank you for teaching me everything! How to think, reconsider, question and solve problems. Thank you for your endless patience during my work in the lab. Thank you for what you have thought me. You showed me what passion for science and knowledge is. Prof. Yael Amitai, thank you for your elegant and incredibly important inputs. Huge thanks to Prof. Israel Sekler. Thank you for the small project you gave me the opportunity to grow into this huge topic and took me into the mitochondria world. Thank you for always pushing me forward and believing in me. Thank you for teaching me what pure optimism in science looks like and the importance of it. Prof. Ilya Fleidervish, thank you for sharing with me your electrophysiology knowledge. You gave me permission to use your set-up, without any experience, and play with it. Dr. Joy Kahn, thank you for teaching me, for your inputs, for your care and help. Thank you for guiding me, in research and in life. Thank you for the enlightening and fascinating talks we had. You were my voice of reason in the darkest times… I couldn't be where I am and who I am without you. To all my department members, who became my very good friends a long time ago: Arik, Ivana, Tomer, Moshe, Oron, Leenor, Milos, Marcel, Marko, Zaga – thank you for being there for me, for the laughs and the fun we had. Thanks to Ohad Stoler – for teaching me, counseling and helping with everything. For the late dinners and the passion for sweets and food in general. Special thanks to Maya Rozenfeld and Shirel Arguetti – you're amazing, and I am very lucky to have you both in my life… Thanks to Shamchal whom I met just recently at the beginning of my writing… You added color to my life and made writing of this thesis into a much easier task. What will I do without you?? To my lab members: Eden – Thank you for your creativity and unique way of thinking. Thank you for the thoughts and inputs. Thank you for teaching all of us on the importance of good food and drinks… Yoav – Thank you for everything you thought me, for the talks and the laughs. 5 Shoham – Thank you for your humor and unique sarcasm, thank you for all the fun time we had while teaching together – I'll miss that a lot! Yaara – Thank you for your questions and curiosity. For listening and helping me. Thank you for pushing me forward and keeping my feet on the ground. And Merav! Thank you for being there with me for all those years. For the times we spent talking about science and life, for the laughs and cries, for your words and your deeds. You've been my mentor, my guide and my friend. I couldn't have reached this stage without you! Last but not least, I'm forever grateful to my parents, Ella Stavsky and Oleg Vilkov. You are my solid cornerstone, my voice of reason and my love. Thanks to my sister Vita and brother Miron, my grandmother Marra and my nieces, Eva and Olivia for making my day much better… 6 Table of content ACKNOWLEDGMENTS ............................................................................................ 5 TABLE OF CONTENT ............................................................................................... 7 LIST OF TABLES ..................................................................................................... 11 LIST OF FIGURES ................................................................................................... 12 ABBREVIATIONS ................................................................................................... 14 SUMMARY ........................................................................................................... 16 INTRODUCTION .................................................................................................... 18 1 SYNAPTIC TRANSMISSION................................................................................. 18 2 SYNAPTIC VESICLE POOLS ................................................................................. 19 2.1 THE READILY RELEASABLE POOL ............................................................................. 19 2.2 THE RECYCLING POOL ......................................................................................... 20 2.3 THE RESTING POOL ............................................................................................ 20 2.4 THE SUPERPOOL AND VESICLE MOBILITY .................................................................. 20 3 THE SYNAPSIN FAMILY ..................................................................................... 21 3.1 FUNCTIONAL IMPLICATIONS OF THE DELETION OF ALL SYNAPSINS .................................... 22 3.2 THE ROLE OF ATP BINDING IN SYNAPSIN ACTIVITY ...................................................... 22 4 SYNAPTIC CALCIUM HOMEOSTASIS ................................................................... 23 5 SYNAPTIC PLASTICITY ....................................................................................... 25 5.1 SHORT-TERM SYNAPTIC PLASTICITY ........................................................................ 25 5.2 LONG-TERM SYNAPTIC PLASTICITY .......................................................................... 25 5.3 THE ROLE OF SYNAPSINS IN SYNAPTIC PLASTICITY ........................................................ 27 6 SYNAPTIC MITOCHONDRIA ............................................................................... 29 6.1 MITOCHONDRIAL PROVISION OF ATP ..................................................................... 29 6.2 MITOCHONDRIAL CALCIUM SIGNALING .................................................................... 31 6.3 MITOCHONDRIAL CALCIUM INFLUX MECHANISM ........................................................ 32 6.4 MITOCHONDRIAL CALCIUM EFFLUX MECHANISM: THE MITOCHONDRIAL SODIUM/CALCIUM EXCHANGER ……………………………………………………………………………………………………………33 6.5 THE ROLE OF MITOCHONDRIA IN SYNAPTIC PLASTICITY ................................................. 34 RESEARCH OBJECTIVES ......................................................................................... 36 RESEARCH SIGNIFICANCE ...................................................................................... 38 MATERIALS AND METHODS .................................................................................. 39 7 7 SOLUTIONS, MATERIALS AND ANTIBODIES ........................................................ 39 8 MICE …………………………………………………………………………………………………………….. 42 9 NEURONAL CULTURES ...................................................................................... 43 9.1 PRIMARY DENSE HIPPOCAMPAL CULTURES................................................................ 43 9.2 AUTAPTIC