The Functional Organization of Synaptic Vesicle Pools in a Retinal Bipolar Neuron

..............................................................................................v TABLE OF CONTENTS.......................................................................................................vii LIST OF FIGURES....................................................................................viii LIST OF TABLES...................................................................................................................x CHAPTER I – GENERAL INTRODUCTION........................................................................1 CHAPTER II – GENERAL METHODS...............................................................................18 CHAPTER III – RELATIONSHIP BETWEEN VESICLE POOLS.....................................26 INTRODUCTION......................................................................................................27 RESULTS...................................................................................................................36 DISCUSSION.............................................................................................................53 CHAPTER IV – VESICLE POOLS IN SNARE COMPLEX...............................................59 INTRODUCTION......................................................................................................60 RESULTS...................................................................................................................67 DISCUSSION.............................................................................................................92 CHAPTER V – CONCLUSIONS AND FUTURE DIRECTIONS.....................................104 APPENDIX A.......................................................................................................................117 APPENDIX B.......................................................................................................................118 REFERENCES ....................................................................................................................124 VITA....................................................................................................................................168 viii LIST OF FIGURES Fig 1.1 The classical synaptic vesicle cycle 5 Fig 1.2 Ultra structure of Retinal ribbon synapses 14 Fig 2.1 Isolated Mb1 neuron and terminal 20 Fig 3.1 Location and size of vesicle pools of the goldfish bipolar cell 31 Fig 3.2 Protocol used for the train stimulus 36 Fig 3.3 Sample trace from a terminal stimulated with a pulse train. 37 Fig 3.4 A pulse train stimuli reveals fusion of multiple vesicle pools. 38 Fig 3.5 The ATP sensitive component. 41 Fig 3.6 ATP is required for the refilling of the releasable and rapid pool. 43 Fig 3.7 Depletion of the releasable pool also depletes the rapid pool 46 Fig 3.8 Depletion of the releasable pool also depletes the rapid pool 47 Fig 3.9 The rapid pool is a subset of the releasable pool 50 Fig 3.10 The rapid pool is a subset of and draws from the releasable pool 51 Fig 3.11 Changes in intracellular calcium during experiment 4 53 Fig 4.1 Syntaxin 3B and scrambled peptides 67 Fig 4.2 Syntaxin 3 peptide prevents formation of the SNARE complex 68 Fig 4.3 Dialysis of the FITC tagged peptides were verified by increase in fluorescence 70 Fig 4.4 A SNARE complex inhibiting peptide blocks fusion of the reserve vesicle but not the releasable pool of vesicles 72 Fig 4.5 The Syntaxin peptide does not affect the number of vesicles in the releasable pool 73 Fig 4.6 The Syntaxin peptide does not affect fusion from the rapid pool 75