Measuring glutamate transients at Schaffer collateral synapses Dissertation with the aim of achieving a doctoral degree (Dr. rer. nat.) at the Faculty of Mathematics, Informatics and Natural Sciences of the University of Hamburg Center for Molecular Neurobiology, Institute for Synaptic Physiology Submitted by Céline Dürst Lausanne July 2018, Hamburg The following evaluators recommend the admission of the dissertation: Supervisor Prof. Dr. Thomas G. Oertner Institut für Synaptische Physiologie Zentrum für Molekulare Neurobiologie (ZMNH) am Universitätsklinikum Hamburg-Eppendorf Co-Supervisor Prof. Dr. Christian Lohr Abteilung Neurophysiologie Zoologisches Institut an der Universität Hamburg Oral defense: December 7th, 2018 2 Table of Contents 1 Introduction ......................................................................................................................... 11 1.1 Connections of the trisynaptic excitatory hippocampal circuitry ................................. 11 1.2 Discovery of synaptic transmission ............................................................................ 12 1.3 Information transmission ............................................................................................ 13 1.4 The CNS presynaptic terminal ................................................................................... 15 1.4.1 Synaptic vesicle exocytosis and fusion mode ........................................................ 16 1.4.1.1 Fusion mode .................................................................................................... 17 1.4.2 Synaptic vesicles endocytosis ................................................................................ 20 1.4.2.1 The ‘presynaptic quantum’ .............................................................................. 22 1.5 Release probability ..................................................................................................... 24 1.5.1 Pves regulation ......................................................................................................... 25 1.5.2 Number of vesicles (n) ........................................................................................... 27 1.5.3 Modulation of pr and short-term plasticity ............................................................... 27 1.6 Monitoring presynaptic function.................................................................................. 29 1.6.1 Electrophysiological approaches for assessing changes in pr ............................... 29 1.6.2 Indicators of synaptic release ................................................................................. 30 1.6.2.1 FM dyes ........................................................................................................... 31 1.6.2.2 pHluorins .......................................................................................................... 31 1.6.2.3 Optical glutamate sensors ............................................................................... 33 1.6.2.4 Genetically encoded glutamate indicators (GEGI) .......................................... 33 1.7 Quantal analysis at CNS synapses ............................................................................ 35 1.7.1.1 The one site-one vesicle hypothesis ............................................................... 37 1.7.1.2 Multivesicular vs. univesicular release at CA3-CA1 synapses ........................ 39 1.8 Hippocampal organotypic slice cultures ..................................................................... 41 1.9 Two-photon laser scanning microscopy ..................................................................... 41 1.10 Aims of the thesis ....................................................................................................... 43 2 Experimental Methods ........................................................................................................ 44 2.1 Organotypic hippocampal slice cultures ..................................................................... 44 2.2 Plasmids and single-cell electroporation .................................................................... 44 2.3 Solution and electrophysiology................................................................................... 45 2.4 Two-photon microscopy ............................................................................................. 46 2.5 Drift Correction ........................................................................................................... 47 2.6 Analysis of fluorescence transients ............................................................................ 47 2.7 Localization of center of release ................................................................................ 49 2.8 Synapse modeling and glutamate release simulation ................................................ 50 2.9 Quantal analysis ......................................................................................................... 52 2.10 Statistical Analysis ...................................................................................................... 53 3 3 Results ................................................................................................................................ 55 3.1 Expression of the glutamate sensor ........................................................................... 55 3.2 Modulating synaptic release probability affects cleft glutamate concentration........... 60 3.3 Mapping the spatial location of individual fusion events ............................................. 62 3.4 Desynchronized release events reveal quantal size .................................................. 67 3.5 Non-linear response of iGluSnFR to glutamate release ............................................. 68 3.6 The dynamic range of postsynaptic responses .......................................................... 71 3.7 Extracting synaptic parameters by histogram analysis .............................................. 74 3.8 Publication in PNAS 2018........................................................................................... 79 3.8.1 ABSTRACT ............................................................................................................. 80 3.8.2 Introduction ............................................................................................................. 82 3.8.3 Results .................................................................................................................... 84 3.8.3.1 Affinity variants of iGluSnFR by binding site mutations ................................... 84 3.8.3.2 Kinetic measurements of iGluSnFR variants in vitro and in situ ...................... 87 3.8.3.3 Synaptic glutamate dynamics during high frequency stimulation .................... 88 3.8.3.4 Paired-pulse facilitation correlates with rapid recovery from depression ........ 91 3.8.3.5 Response kinetics of iGluSnFR and variants iGluf and iGluu are based on the rate of structural change ......................................................................................................... 93 3.8.4 Discussion .............................................................................................................. 96 3.8.5 Methods .................................................................................................................. 99 3.8.6 Supplementary information ................................................................................... 100 4 Discussion ........................................................................................................................ 120 4.1 Presynaptic dynamic range ...................................................................................... 120 4.1.1 Variability in pr and PPR among boutons ............................................................. 121 4.1.2 Ca2+ dependent release ........................................................................................ 122 4.1.3 Comparing AMPAR-mediated currents to iGluSnFR signals ............................... 123 4.2 Function of MVR for synaptic transmission .............................................................. 124 4.2.1 Spillover ................................................................................................................ 126 4.3 Classical quantal analysis......................................................................................... 126 4.3.1 Monitoring [glu] transients has distinct advantages over EPSCaTs measurements 129 4.3.2 Binomial model ..................................................................................................... 131 4.3.3 Inter and intrasynapse variability in [glu] transients .............................................. 132 4.4 MVR controversy ...................................................................................................... 133 4.4.1 Interpretation of glutamate transients amplitude variability under UVR assumption or partial fusion ............................................................................................................................. 135 4.5 Number of release sites ............................................................................................ 136 4.6 Localization of the sensor ......................................................................................... 138 4.6.1 Non-linearity of the sensor ...................................................................................