electrophysiological study of neuronal ion channels in drosophila melanogaster

Electrophysiological study of neuronal ion channels in Drosophila melanogaster A Dissertation Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Program in Neuroscience Leslie Griffith, Advisor In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy by James Choong-Kwan Choi February, 2006 UMI Number: 3200729 UMI Microform 3200729 Copyright 2006 by ProQuest Information and Learning Company All rights reserved This microform edition is protected against unauthorized copying under Title 17, United States Code ProQuest Information and Learning Company 300 North Zeeb Road P.O Box 1346 Ann Arbor, MI 48106-1346 This dissertation, directed and approved by James Choong-Kwan Choi’s committee, has been accepted and approved by the Graduate Faculty of Brandeis University in partial fulfillment of the requirements for the degree of: DOCTOR OF PHILOSOPHY Adam B Jaffe, Dean of Arts and Sciences Dissertation Committee: Leslie Griffith, Chair Gina Turrigiano Eve Marder Rachel Wilson c Copyright by James Choong-Kwan Choi 2006 Acknowledgments I express deep gratitude to all those who have influenced me throughout my graduate school experience Special thanks to my dissertation committee for their longsuffering I thank Demian Park, James Hodge, and Jose Agosto for experimental collaborations detailed here, and all past and current members of the Griffith lab Finally, much thanks to Leslie for her patience and support, and Eve for the opportunity to pursue this research iv Abstract Electrophysiological study of neuronal ion channels in Drosophila melanogaster A dissertation presented to the Faculty of the Graduate School of Arts and Sciences of Brandeis University, Waltham, Massachusetts by James Choong-Kwan Choi This dissertation is a summary of work done in the study of Drosophila neurophysiology This endeavor primarily involves direct observation of channel activity using electrophysiological techniques In third instar larvae, a live preparation was devised to characterize motorneurons in situ By recording from ion channels in their natural milieu, a comprehensive, functional account of five individually identified motorneurons (MN6/7-Ib, MN1-Ib, MN14-Ib, MN 30-Ib and MNISN-Is) was obtained using electrophysiology in combination with labeling, pharmacology and biophysical methods A fast-inactivating, voltage-sensitive potassium channel (Shal) was shown to mediate the delayed spiking response to current pulse Next, transgenic overexpression of a leak potassium channel (Shaw) in full-length and truncated form was studied in the MNISN-Is neuron The truncated overexpression altered the whole cell parameters as recorded from the soma, such that the presumed dominant-negative block of leak current depolarized the cell while decreasing input resistance Also, the direct target and plausible mechanism of therapeutic effect of a common anticonvulsant drug (carbamazepine) was explored using the Drosophila as a model system, and it was found that an effect on fly sleep behavior was evoked via the Rdl channel The cumulative result of this dissertation is, in effect, a showcasing of how multiple aspects of ion channel neurophysiology can be studied in Drosophila by incorporating electrophysiological techniques v Contents Abstract v Introduction Characterization of third instar motorneurons 2.1 Introduction 2.2 Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system 4 The Shaw potassium channel 3.1 Introduction 3.2 Shaw potassium channel genes in Drosophila 35 35 36 Resistance to dieldrin (Rdl) physiology 4.1 Introduction to Rdl 4.2 Carbamazepine affects Drosophila sleep by altering GABAA receptor gating 71 71 A Drosophila embryonic neuronal culture system A.1 Embryonic culture physiology 92 92 vi 74 List of Tables 2.1 Morphology of third instar dorsomedial motorneurons 17 3.1 Effect of Expression of Shaw Transgenes on Viability, Eclosion, Adult Weight, and Shaking Whole Cell Parameters of MNISN-Is 60 63 3.2 vii List of Figures 2.1 2.2 2.3 2.4 2.5 2.6 2.7 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 3.9 4.1 4.2 4.3 4.4 A.1 A.2 A.3 A.4 Ventral ganglion projections of dorsomedial motor neurons Identification of dorsomedial motor neuron targets Firing properties of dorsomedial neurons are stereotyped Delay to spike in MNISN-Is is due to a prepulse- and 4-aminopyridine (4-AP)-sensitive non-Shaker current Total outward currents in MNISN-Is and MN6/7-Ib are equivalent Isolated IA potassium currents in MNISN-Is and MN6/7-Ib Voltage dependence of IA inactivation 16 18 21 Predicted Shawl genomic structure Alignment of Shaw protein sequences Expression of Shaw during embryonic development Expression of Shawl during embryonic development Expression and localization of Shaw transgene products in motor neurons 48 49 51 52 54 Expression and localization of Shaw transgene products in the CCAP neuropeptide pattern Expression of Shaw transgenes cause a infantile phenotype Current clamp recording from motor neurons expressing Shaw transgenes Current-voltage relationships in neurons expressing truncated Shaw 57 59 62 65 CBZ inhibits fly sleep and produces a rebound upon drug withdrawal Mutants of the Drosophila GABAA receptor subunit (Rdl) have altered CBZ- sensitivity CBZ specifically decreases the steady state amplitude of GABA mediated current and the TM2 (A302S) mutation abolishes CBZ effects Analogous TM2 mutations on the mammalian GABAA receptor strongly decrease the potentiating effects of CBZ GFP expression in cultured embryonic neurons T287D decreases peak outward current Different effects of ala expression on outward current eags c29 expression causes a drastic decrease in outward currents viii 23 26 28 28 88 89 90 91 99 100 101 102 Chapter Introduction Ion channels and neural function The nervous system of an organism takes on the remarkable task of controlling and coordinating all aspects of behavior This is made possible by the system’s capacity to sense features of its external and internal environment and encode them into into electrical signals, process and integrate these signals in a functionally relevant manner, and output instructions that their efferent targets will execute At the cellular level, the neuron’s specialized ability to integrate and relay the electrically encoded signal boils down to the function of a few classes of ion channels that enable the flow of ions when activated The intrinsic cellular activity of neurons perpetually seeks to maintain ion channel activity within homeostatic parameters while also retaining functional viability by modulating ion channels through cascades that activate in response to activity itself In essence, voltage and ligand sensitive ion channels are the source and the terminus of functional activity in the nervous system Hence, it makes sense to approach the study of the brain from an ion channel perspective 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