University of St Andrews Full metadata for this thesis is available in St Andrews Research Repository at: http://research-repository.st-andrews.ac.uk/ This thesis is protected by original copyright "Electrophysiological Studies On The Caudal Photoreceptor Of The Crayfish, Pacifasticus leniusculus". RHODA ELLIS Thesis submitted for the degree of Master of Philosophy of the University of St. Andrews July 2004 School of Biology Bute Building University of St. Andrews St. Andrews Fife KY16 9TS ABSTRACT ■ The caudal photoreceptors (CPRs) are bilaterally-paired neurons located in the terminal ganglion of the crayfish ventral nerve cord. Each CPR has an axon which runs the length of the animal on the contralateral side of the nerve cord. When activated, they induce behaviors such as backwards walking. The CPRs are also mechanosensory integrating intemeurons. ■ The photic response was recorded intracellularly in the presence of the voltage-gated Na+ channel blocker, tetrodotoxin (TTX), to reveal the shape of the underlying generator potential. ■ The generator potential has an initial transient peak, followed by a sustained plateau. There is a small "off response. This waveform reflects the shape of the spike frequency response in the intact preparation. ■ Application of the neuromodulator serotonin changes the spike frequency response of the CPR. It induces spontaneous firing by altering the properties of synaptic inputs to the CPR but does not significantly alter the generator potential of the photic response. ■ Lithium reduced the photic response and caffeine increased it, confirming the involvement of IP3 and Ca2+ as second messengers in the transduction cascade. Staurosporine had no effect, suggesting the absence of a phosphorylation step. ■ The generator potential was reduced in amplitude by bathing the preparation in 20% sodium saline in the presence of TTX, indicating that it is sodium-dependent. ■ No evidence was found for a membrane conductance change during the photic response. ■ Application of tetraethyl ammonium ions (TEA) enhanced the magnitude of the generator potential depolarization, suggesting that voltage-gated K+ channels normally limit and sculpt the shape of the potential. TEA also induced "flickering" of the generator potential which means that it might directly affect the transduction mechanism. 278 Words DECLARATIONS (i) I, Rhoda Ellis, hereby certify that this thesis, which is approximately 26,510 words in length, has been written by me, that it is the record of work carried out by me and that it has not been submitted in any previous application for a higher degree. (ii) I was admitted as a research student in October, 1999 and as a candidate for the degree of MPhil. in March, 2004; the higher study for which this is a record was carried out in the University of St Andrews between 1999 and 2004. (iii) I hereby certify that the candidate has fulfilled the conditions of the Resolution and Regulations appropriate for the degree of MPhil. in the University of St Andrews and that the candidate is qualified to submit this thesis in application for that degree. COPYRIGHT DECLARATION A Unrestricted In submitting this thesis to the University of St Andrews I understand that I am giving permission for it to be made available for use in accordance with the regulations of the University Library for the time being in force, subject to any copyright vested in the work not being affected thereby. I also understand that the title and abstract will be published, and that a copy of the work may be made and supplied to any bona fide library or research worker. date- Signature of candidate ACKNOWLEDGEMENTS I would like to thank the Maitland Ramsey Bequest for funding this research and Dr. William Heitler for his supervision and encouragement. I would also like to thank my parents, Duncan, and all my friends who have been constant their support and understanding. ABBREVIATIONS CAMP 3'5'-cyclic monophosphate cGMP Cylic guanosine mono-phosphate CPR Caudal photoreceptor DAG diacylglycerol EPSP Excitatory Post-Synaptic Potential EOP Extra-ocular photoreceptors GDP guanosine di-phosphate 5'-GMP guanosine mono-phosphate GTP guanosine tri-phosphate IP3 Inositol triphosphate IPSP Inhibitory Post-Synaptic Potential KDa kilo Dalton LG Lateral Giant Fibre mV Millivolt PDE Phosphodiesterase PI phosphatidylinositide PIP2 phosphatidylinositol bisphosphate PI-PLC phosphatidylinositol phospholipase C PLC phospholipase-C R Rhodopsin R* Activared rhodopsin SRC Subrhabdomeric cisternae TEA Tetraethyl ammonium TTX Tetrodotoxin CONTENTS CHAPTER 1: INTRODUCTION 1 1.1 General Introduction 1 1.1.1 The Vertebrate Visual Systems 1 1.1.2 The Transduction Cascade in the Mammalian Rod Photoreceptor 2 1.1.3 The Invertebrate Compound Eye 5 1.1.4 Transducti on in the Compound Eye 6 1.1.5 Extra-ocular Photoreception in Vertebrate and Invertebrate Animals 7 1.2 The Crayfish Caudal Photoreceptor 12 1.2.1 Function of the Crayfish Caudal Photoreceptor 13 1.2.2 Does The Caudal Photoreceptor Play A Role In The Entrainment Of Circadian Rhythms? 14 1.3 The Aims Of This Thesis 15 CHAPTER 2: GENERAL MATERIALS AND METHODS 17 2.1 Animals: Maintenance and Dissection 17 2.2 Electrophysiology 20 2.2.1 Experimental Chamber 20 2.2.2 Illumination 23 2.2.3 Extracellular Recording 24 2.2.4 Extracellular Stimulation 24 2.2.5 Intracellular Recording 24 2.2.5.1 Electrodes 24 2.2.6 Preparation Earth 27 2.2.7 Impalement 27 2.3 Neuron Identification 28 2.4 Data Capture 28 CHAPTER 3: NATURAL PROPERTIES OF THE CAUDAL PHOTORECEPTOR IN THE CRAYFISH, Pastifasticus leniusculus 29 3.1 Introduction 29 3.2 Additional Material And Methods 30 3.3 The Caudal Photoreceptor Of Pacifasticus leniusculus 30 3.3.1 Anatomy 30 3.3.2 Physiology 35 3.3.2.1 The light response of the caudal photoreceptor 35 3.4 Analysis of Caudal Photoreceptor Action Potential 35 3.4.1 Extracellular Caudal Photoreceptor Recording and Template Recognition 35 3.4.2 Intracellular Recording and Threshold Recognition 38 3.4.3 Comparison of Spike Recognition Methods 3 8 3.4.4 Percentage Accuracy 46 3.5 The Relationship Between Generator Potential And Action Potential Frequency 46 3.6 Artificial Current Injection And The Photic Response 51 3.7 Repetitive Light Stimulation 51 3.7.1 Extracellular Recording of Repetitive Stimulation Over 3 Hours 51 3.7.2 Intracellular Recording of Repetitive Stimulation Over 3 Hours 54 3.8 Discussion 61 CHAPTER 4: THE EFFECT OF SEROTONIN ON THE PHOTIC AND MECHANO-SENSORY PROPERTIES OF THE CRAYFISH CAUDAL PHOTORECEPTOR 63 4.1 Introduction 63 4.2 Additional Materials And Methods 65 4.3 Results 66 4.3.1 The Effect of Serotonin [lOOpM] on the Photoreceptive Properties of the Caudal Photoreceptor 68 4.3.2 The Effect of Serotonin on the Generator Potential of the Light Response 71 4.3.3 The Effect of Serotonin [lOOpM] on the Role of the Caudal Photoreceptor as a Mechano-Sensory Integrating Interneuron 80 4.3.3.1 Current evoked EPSPs and IPSPs may be modulated by serotonin 80 4.3.4 The Effect of Serotonin on the Caudal Photoreceptor in the Dark 80 4.4 Discussion 80 4.4.1 The Effect of Serotonin on the Caudal Photoreceptor Generator Potential 86 4.4.2 What Would be the Effects of Serotonin in vivol 87 CHAPTER 5: THE PHOTOTRANSDUCTION PATHWAY OF THE CAUDAL PHOTORECEPTOR OF THE CRAYFISH PACIFASTICUS LENIUSCULUS 88 5.1 Introduction 88 5.1.1 The Site of Photoreception in the Caudal Photoreceptor 88 5.1.2 Light-sensitive Pigment 88 5.1.3 Transduction Cascade 89 5.1.4 Reagents Applied to the Caudal Photoreceptor 92 5.1.4.1 Staurosporine 92 5.1.4.2 Lithium 95 5.1.4.3 Caffeine 95 5.2 Additional Materials and Methods 95 5.2.1 Staurosporine 95 5.2.2 Lithium 95 5.2.3 Caffeine 96 5.3 Results 96 5.3.1 The Effect of Staurosporine on the Light Response of the Crayfish Caudal Photoreceptor 96 5.3.2 The Effect of Lithium on the Light Response of the Crayfish Caudal Photoreceptor 96 5.3.3 The Effect of Caffeine on the Light Response of the Crayfish Caudal Photoreceptor 105 5.4 Discussion 123 5.4.1 Staurosporine 123 5.4.2 Lithium 126 5.4.3 Caffeine 126 CHAPTER 6: IONIC DEPENDENCE OF THE LIGHT RESPONSE OF THE CRAYFISH CAUDAL PHOTORECEPTOR 127 6.1 Introduction 127 6.2 Additional Materials and Methods 128 6.2.1 Reduced Sodium Saline 128 6.2.2 Conductance Measurements 129 6.2.2.1 Method one - Voltage deflection 129 6.2.2.2 Method two - Measuring the time constant of current pulses during the light response of the Caudal Photoreceptor 133 6.2.2.2.1 Exponential Curve 134 6.2.2.2.2 Error function curve 135 6.2.3.1 The effects of tetraethyl ammonium on the light response of the caudal photoreceptor 135 6.2.3.2 TEA controls 136 6.3 Results 136 6.3.1 Low Sodium 136 6.3.2 Conductance Measurements 139 6.3.3 TEA 146 6.3.3.1 Effect of TEA on the light response of the Caudal Photoreceptor 146 6.3.3.2 Control: The effect of TEA on the action potential of the lateral giant 147 6.3.3.3 Control: The effects of TEA on the resting potential of non-Caudal Photoreceptor neurons 147 6.4 Discussion 147 6.4.1 The Effect of Low Sodium Saline on the Generator Potential of the Caudal Photoreceptor 147 6.4.2 Evidence for a Change in Conductance During the Photic Response of the Caudal Photoreceptor 153 6.4.3 The Effect of Tetraethyl Ammonium on the Generator Potential of the Caudal Photoreceptor 15 8 CHAPTER 7: OVERALL DISCUSSION 161 REFERENCES 166 LIST OF TABLES AND FIGURES Figure I.i Vertebrate Phototransduction 4 Figure I.ii Invertebrate Phototransduction 8 Table 1.1 The Main Differences Between Vertebrate And Invertebrate Visual Systems 9 Table 1.2