SPATIOTEMPORALLY PRECISE OPTICAL STIMULATION SYSTEM FOR CONTROLLING NEURAL ACTIVITY IN-VITRO By PERRY THOMAS TWYFORD A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011 1 © 2011 Perry Twyford 2 To my family, Loren, Stefani, Evan, Julie, and Jack 3 ACKNOWLEDGMENTS I would like to thank Dr. Tom DeMarse for his support and inspiration throughout my time at the University of Florida. I thank the other members of my committee, Dr. William Ogle, Dr. Bruce Wheeler, and Dr. Arunava Banerjee for helpful feedback. I also thank Phil Barish for virus synthesis and general help with matters of genetic engineering. I thank the members of the Wheeler lab, Eric Franca for his long hours conducting cell culture preps, Sankar Alagapan, Kucku Varghese and Liangbin Pan for insightful and spirited experimental discussions. I would also like to extend my thanks to the J. Crayton Pruitt Family and the University of Florida Alumni Foundation for their financial support of this research. Finally, I thank my family and friends, without whose support this work would not have been possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATIONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 INTRODUCTION AND BACKGROUND ................................................................. 12 Micro-Electrode Arrays ........................................................................................... 12 Photostimulation ..................................................................................................... 13 Uncaging Methods ........................................................................................... 13 Chemically Modified Ion Channels ................................................................... 14 Photosensitive Ion Channels ............................................................................ 15 History ........................................................................................................ 16 Basic characterization ................................................................................ 18 Opsin targeting ........................................................................................... 21 Light delivery methods ............................................................................... 24 Other in-vivo work ...................................................................................... 26 Advantages over electrical stimulation ....................................................... 27 Disadvantages ........................................................................................... 30 Future of Optogenetics ..................................................................................... 31 Previous Work ........................................................................................................ 33 Optogenetics on MEAs ..................................................................................... 33 Structured Light Application .............................................................................. 35 Digital Light Processing (DLP) ................................................................................ 35 Objective ................................................................................................................. 37 2 EXPERIMENTAL METHODS ................................................................................. 44 Neural Cultures ....................................................................................................... 44 Cell Culture ....................................................................................................... 44 Viral Infection .................................................................................................... 44 Natural Activity ................................................................................................. 45 Experimental Setup ................................................................................................ 46 Recording Suite ................................................................................................ 46 Bath Light Application ....................................................................................... 46 DLP Light Application ....................................................................................... 47 Data Analysis .......................................................................................................... 47 5 Spike rate ......................................................................................................... 47 Bursts ............................................................................................................... 48 Input-Output Separability .................................................................................. 48 Cross-Trial Similarity ........................................................................................ 49 Plots ................................................................................................................. 49 3 BATH LIGHT APPLICATION .................................................................................. 55 Background ............................................................................................................. 55 Experiments ............................................................................................................ 57 Results and Discussion........................................................................................... 58 Duration Modulation ......................................................................................... 58 Frequency Modulation ...................................................................................... 63 Conclusions ............................................................................................................ 68 4 DLP SYSTEM ......................................................................................................... 85 DLP Operation ........................................................................................................ 85 DLP Device – First Generation ............................................................................... 88 Construction ..................................................................................................... 88 Issues ............................................................................................................... 89 DLP Device – Second Generation .......................................................................... 89 Hardware .......................................................................................................... 89 Software ........................................................................................................... 91 Assembly .......................................................................................................... 92 Power ............................................................................................................... 93 5 DLP EXPERIMENTS ............................................................................................ 102 Experiment 5-1: Quadrant Analysis ...................................................................... 102 Results ........................................................................................................... 103 Discussion ...................................................................................................... 105 Experiment 5-2: Parametric Spot Size Analysis .................................................... 106 Results ........................................................................................................... 107 Discussion ...................................................................................................... 109 Experiment 5-3: High-frequency Photostimulation ................................................ 110 Results ........................................................................................................... 110 Discussion ...................................................................................................... 112 General Discussion ............................................................................................... 112 6 CONCLUSIONS ................................................................................................. 126 Discussion ............................................................................................................ 126 Future Work .......................................................................................................... 127 LIST OF REFERENCES ............................................................................................. 130 BIOGRAPHICAL SKETCH .........................................................................................