Characterization of voltage-dependent Ca2+ currents in identified Drosophila motoneurons in situ.

Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmalpha1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.

[1]  Y. Jan,et al.  Properties of the larval neuromuscular junction in Drosophila melanogaster. , 1976, The Journal of physiology.

[2]  C. Heckman,et al.  Synaptic integration in bistable motoneurons. , 1999, Progress in brain research.

[3]  C. Heckman.  Active Conductances in Motoneuron Dendrites Enhance Movement Capabilities , 2003, Exercise and sport sciences reviews.

[4]  C. Heckman,et al.  Adjustable Amplification of Synaptic Input in the Dendrites of Spinal Motoneurons In Vivo , 2000, The Journal of Neuroscience.

[5]  F. Kawasaki,et al.  Synaptic Calcium-Channel Function in Drosophila: Analysis and Transformation Rescue of Temperature-Sensitive Paralytic and Lethal Mutations of Cacophony , 2002, The Journal of Neuroscience.

[6]  W. Catterall.  Structure and regulation of voltage-gated Ca2+ channels. , 2000, Annual review of cell and developmental biology.

[7]  C. Heckman,et al.  Movement‐related receptive fields of spinal motoneurones with active dendrites , 2008, The Journal of physiology.

[8]  R. Cooper,et al.  Developmental consequences of neuromuscular junctions with reduced presynaptic calcium channel function , 2005, Synapse.

[9]  J. C. Hall,et al.  Courtship and visual defects of cacophony mutants reveal functional complexity of a calcium-channel alpha1 subunit in Drosophila. , 1998, Genetics.

[10]  Michael Bate,et al.  Electrophysiological Development of Central Neurons in theDrosophila Embryo , 1998, The Journal of Neuroscience.

[11]  L. Byerly,et al.  Ionic currents of Drosophila neurons in embryonic cultures , 1988, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[12]  C. Heckman,et al.  Persistent Inward Currents in Spinal Motoneurons and Their Influence on Human Motoneuron Firing Patterns , 2008, The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry.

[13]  D. Eberl,et al.  A Mutation Affecting Dihydropyridine-Sensitive Current Levels and Activation Kinetics in Drosophila Muscle and Mammalian Heart Calcium Channels , 1998, The Journal of Neuroscience.

[14]  C Duch,et al.  Remodeling of Membrane Properties and Dendritic Architecture Accompanies the Postembryonic Conversion of a Slow into a Fast Motoneuron , 2000, The Journal of Neuroscience.

[15]  Hilmar Bading,et al.  Distinct functions of nuclear and cytoplasmic calcium in the control of gene expression , 1997, Nature.

[16]  N. Patel,et al.  Even-skipped, acting as a repressor, regulates axonal projections in Drosophila , 2003, Development.

[17]  D. Johnston,et al.  Different Ca2+ channels in soma and dendrites of hippocampal pyramidal neurons mediate spike-induced Ca2+ influx. , 1995, Journal of neurophysiology.

[18]  J. Littleton,et al.  Presynaptic N-type Calcium Channels Regulate Synaptic Growth* , 2003, Journal of Biological Chemistry.

[19]  William A. Catterall,et al.  Clustering of L-type Ca2+ channels at the base of major dendrites in hippocampal pyramidal neurons , 1990, Nature.

[20]  R. Levine,et al.  Changes in calcium signaling during postembryonic dendritic growth in Manduca sexta. , 2002, Journal of neurophysiology.

[21]  Kendal Broadie,et al.  Electrophysiological analysis of synaptic transmission in central neurons of Drosophila larvae. , 2002, Journal of neurophysiology.

[22]  F. Kawasaki,et al.  Active Zone Localization of Presynaptic Calcium Channels Encoded by the cacophony Locus of Drosophila , 2004, The Journal of Neuroscience.

[23]  A. Chiba,et al.  Single-cell analysis of Drosophila larval neuromuscular synapses. , 2001, Developmental biology.

[24]  G. King.  Modulation of insect Ca(v) channels by peptidic spider toxins. , 2007, Toxicon : official journal of the International Society on Toxinology.

[25]  D. Dickman,et al.  Mutations in a Drosophila α2δ Voltage-Gated Calcium Channel Subunit Reveal a Crucial Synaptic Function , 2008, The Journal of Neuroscience.

[26]  Jeffrey C. Hall,et al.  A Drosophila Calcium Channel α1 Subunit Gene Maps to a Genetic Locus Associated with Behavioral and Visual Defects , 1996, The Journal of Neuroscience.

[27]  F. Hannan,et al.  Cloning and characterization of a calcium channel alpha 1 subunit from Drosophila melanogaster with similarity to the rat brain type D isoform , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[28]  D. Van Vactor,et al.  Genetic and developmental characterization of Dmca1D, a calcium channel alpha1 subunit gene in Drosophila melanogaster. , 1998, Genetics.

[29]  P. Schwindt,et al.  Role of a persistent inward current in motoneuron bursting during spinal seizures. , 1980, Journal of neurophysiology.

[30]  J. Seamans,et al.  Contributions of Voltage-Gated Ca2+ Channels in the Proximal versus Distal Dendrites to Synaptic Integration in Prefrontal Cortical Neurons , 1997, The Journal of Neuroscience.

[31]  L. Griffith,et al.  Electrophysiological and morphological characterization of identified motor neurons in the Drosophila third instar larva central nervous system. , 2004, Journal of neurophysiology.

[32]  Bruce R. Johnson,et al.  Dopamine modulation of calcium currents in pyloric neurons of the lobster stomatogastric ganglion. , 2003, Journal of neurophysiology.

[33]  D. Lipscombe,et al.  Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Cavα1 , 2002, Molecular Neurobiology.

[34]  W. Branton,et al.  Neurotoxins from Plectreurys spider venom are potent presynaptic blockers in Drosophila , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[35]  Ronald L. Davis,et al.  Spatial and Temporal Control of Gene Expression in Drosophila Using the Inducible GeneSwitch GAL4 System. I. Screen for Larval Nervous System Drivers , 2008, Genetics.

[36]  F. Kawasaki,et al.  A Temperature-Sensitive Paralytic Mutant Defines a Primary Synaptic Calcium Channel in Drosophila , 2000, The Journal of Neuroscience.

[37]  O. McManus.  Calcium-activated potassium channels: Regulation by calcium , 1991, Journal of bioenergetics and biomembranes.

[38]  Jørn Hounsgaard,et al.  Subcellular distribution of L‐type Ca2+ channels responsible for plateau potentials in motoneurons from the lumbar spinal cord of the turtle , 2003, The European journal of neuroscience.

[39]  Neil V Marrion,et al.  Calcium-activated potassium channels , 1998, Current Opinion in Neurobiology.

[40]  J. Littleton,et al.  Ion Channels and Synaptic Organization Analysis of the Drosophila Genome , 2000, Neuron.

[41]  S Singh,et al.  Resolution and pharmacological analysis of the voltage-dependent calcium channels of Drosophila larval muscles , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[42]  A. Dolphin.  Faculty Opinions recommendation of Mutations in a Drosophila alpha2delta voltage-gated calcium channel subunit reveal a crucial synaptic function. , 2008 .

[43]  Chun-Fang Wu,et al.  Drosophila cacophony Channels: A Major Mediator of Neuronal Ca2+ Currents and a Trigger for K+ Channel Homeostatic Regulation , 2007, The Journal of Neuroscience.

[44]  M. Burrows,et al.  ELECTRICAL CHARACTERISTICS OF THE MEMBRANE OF AN IDENTIFIED INSECT MOTOR NEURONE , 1980 .

[45]  D. Johnston,et al.  Characterization of single voltage‐gated Na+ and Ca2+ channels in apical dendrites of rat CA1 pyramidal neurons. , 1995, The Journal of physiology.

[46]  L. Byerly,et al.  Characterization of single calcium channels in Drosophila embryonic nerve and muscle cells , 1991, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[47]  Hans Hultborn,et al.  Key mechanisms for setting the input-output gain across the motoneuron pool. , 2004, Progress in brain research.