Organization of Circadian Behavior Relies on Glycinergic Transmission.

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.

[1]  M. F. Ceriani,et al.  Communication between circadian clusters: The key to a plastic network , 2015, FEBS letters.

[2]  M. Rosbash,et al.  Circadian Neuron Feedback Controls the Drosophila Sleep-Activity Profile , 2016, Nature.

[3]  C. Kyriacou,et al.  The Logic of Circadian Organization in Drosophila , 2014, Current Biology.

[4]  J. Lynch,et al.  Investigating ion channel conformational changes using voltage clamp fluorometry , 2015, Neuropharmacology.

[5]  C. Helfrich-Förster,et al.  Neuropeptide F immunoreactive clock neurons modify evening locomotor activity and free‐running period in Drosophila melanogaster , 2012, The Journal of comparative neurology.

[6]  Thomas E. Ferrin,et al.  Using Sequence Similarity Networks for Visualization of Relationships Across Diverse Protein Superfamilies , 2009, PloS one.

[7]  M. F. Ceriani,et al.  Experimental assessment of the network properties of the Drosophila circadian clock , 2015, The Journal of comparative neurology.

[8]  A. Sehgal,et al.  Identification of a Circadian Output Circuit for Rest:Activity Rhythms in Drosophila , 2014, Cell.

[9]  Eric Gouaux,et al.  Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters , 2005, Nature.

[10]  P. Schofield,et al.  Distinct agonist- and antagonist-binding sites on the glycine receptor , 1992, Neuron.

[11]  M. F. Ceriani,et al.  Adult-Specific Electrical Silencing of Pacemaker Neurons Uncouples Molecular Clock from Circadian Outputs , 2011, Current Biology.

[12]  R. Vandenberg,et al.  Extracellular Loops 2 and 4 of GLYT2 Are Required for N-Arachidonylglycine Inhibition of Glycine Transport* , 2009, The Journal of Biological Chemistry.

[13]  V. Kilman,et al.  DN1p Circadian Neurons Coordinate Acute Light and PDF Inputs to Produce Robust Daily Behavior in Drosophila , 2010, Current Biology.

[14]  M. F. Ceriani,et al.  Circadian Pacemaker Neurons Change Synaptic Contacts across the Day , 2014, Current Biology.

[15]  C. Helfrich-Förster,et al.  The Neuropeptide Pigment-Dispersing Factor Adjusts Period and Phase of Drosophila's Clock , 2009, The Journal of Neuroscience.

[16]  R. Vandenberg,et al.  Molecular Basis for Substrate Discrimination by Glycine Transporters* , 2007, Journal of Biological Chemistry.

[17]  J. Lynch,et al.  Molecular structure and function of the glycine receptor chloride channel. , 2004, Physiological reviews.

[18]  P. Pévet,et al.  Activation of glycine receptor phase‐shifts the circadian rhythm in neuronal activity in the mouse suprachiasmatic nucleus , 2011, The Journal of physiology.

[19]  C. Helfrich-Förster,et al.  Reevaluation of Drosophila melanogaster's neuronal circadian pacemakers reveals new neuronal classes , 2006, The Journal of comparative neurology.

[20]  G. Stormo,et al.  The Neuropeptide Pigment-Dispersing Factor Coordinates Pacemaker Interactions in the Drosophila Circadian System , 2004, The Journal of Neuroscience.

[21]  Kirsten Harvey,et al.  A Critical Role for Glycine Transporters in Hyperexcitability Disorders , 2008, Frontiers in molecular neuroscience.

[22]  O. Shafer,et al.  Pigment-Dispersing Factor Signaling and Circadian Rhythms in Insect Locomotor Activity. , 2014, Current opinion in insect science.

[23]  C. Helfrich-Förster,et al.  Functional Analysis of Circadian Pacemaker Neurons in Drosophila melanogaster , 2006, The Journal of Neuroscience.

[24]  V. Kilman,et al.  Perturbing Dynamin Reveals Potent Effects on the Drosophila Circadian Clock , 2009, PloS one.

[25]  Gonzalo Yevenes,et al.  Fast synaptic inhibition in spinal sensory processing and pain control. , 2012, Physiological reviews.

[26]  Blocking synaptic transmission with tetanus toxin light chain reveals modes of neurotransmission in the PDF-positive circadian clock neurons of Drosophila melanogaster. , 2011, Journal of insect physiology.

[27]  B. Swinderen,et al.  Sleep Restores Behavioral Plasticity to Drosophila Mutants , 2015, Current Biology.

[28]  Dan Stoleru,et al.  A resetting signal between Drosophila pacemakers synchronizes morning and evening activity , 2005, Nature.

[29]  Ian A Meinertzhagen,et al.  Synaptic connections of PDF‐immunoreactive lateral neurons projecting to the dorsal protocerebrum of Drosophila melanogaster , 2010, The Journal of comparative neurology.

[30]  H. Xue,et al.  The evolution of GABAA receptor-like genes. , 2007, Molecular biology and evolution.

[31]  Cori Bargmann,et al.  GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems , 2008, Neuron.

[32]  V. Kilman,et al.  Dual PDF Signaling Pathways Reset Clocks Via TIMELESS and Acutely Excite Target Neurons to Control Circadian Behavior , 2014, PLoS biology.

[33]  C. Helfrich-Förster,et al.  Peptidergic clock neurons in Drosophila: Ion transport peptide and short neuropeptide F in subsets of dorsal and ventral lateral neurons , 2009, The Journal of comparative neurology.

[34]  J. Berg,et al.  Comparative sequence analysis and tissue localization of members of the SLC6 family of transporters in adult Drosophila melanogaster , 2006, Journal of Experimental Biology.

[35]  C. Becker,et al.  The inhibitory glycine receptor: prospects for a therapeutic orphan? , 1998, Current pharmaceutical design.

[36]  P. Hardin,et al.  Light and Temperature Control the Contribution of Specific DN1 Neurons to Drosophila Circadian Behavior , 2010, Current Biology.

[37]  Xiangzhong Zheng,et al.  Serotonin Modulates Circadian Entrainment in Drosophila , 2005, Neuron.

[38]  E. Gundelfinger,et al.  Sequence of a Drosophila Ligand‐Gated Ion‐Channel Polypeptide with an Unusual Amino‐Terminal Extracellular Domain , 1994, Journal of neurochemistry.

[39]  Y. Hamasaka,et al.  Mapping of serotonin, dopamine, and histamine in relation to different clock neurons in the brain of Drosophila , 2006, The Journal of comparative neurology.

[40]  J. Lynch,et al.  Native glycine receptor subtypes and their physiological roles , 2009, Neuropharmacology.

[41]  M. F. Ceriani,et al.  Acetylcholine from Visual Circuits Modulates the Activity of Arousal Neurons in Drosophila , 2015, The Journal of Neuroscience.

[42]  C. Helfrich-Förster,et al.  BLOCKING ENDOCYTOSIS IN DROSOPHILA'S CIRCADIAN PACEMAKER NEURONS INTERFERES WITH THE ENDOGENOUS CLOCK IN A PDF-DEPENDENT WAY , 2009, Chronobiology international.

[43]  O. Shafer,et al.  The Drosophila Circadian Clock Is a Variably Coupled Network of Multiple Peptidergic Units , 2014, Science.

[44]  A. Kriegstein,et al.  Nonsynaptic Glycine Receptor Activation during Early Neocortical Development , 1998, Neuron.

[45]  J. Hirsh,et al.  Roles of Dopamine in Circadian Rhythmicity and Extreme Light Sensitivity of Circadian Entrainment , 2010, Current Biology.

[46]  C. Helfrich-Förster,et al.  Moonlight shifts the endogenous clock of Drosophila melanogaster , 2007, Proceedings of the National Academy of Sciences.

[47]  Yan Zhang,et al.  Functional reconstitution of glycinergic synapses incorporating defined glycine receptor subunit combinations , 2015, Neuropharmacology.

[48]  Aaron DiAntonio,et al.  Visualizing glutamatergic cell bodies and synapses in Drosophila larval and adult CNS , 2008, The Journal of comparative neurology.

[49]  Yves Grau,et al.  Glutamate and its metabotropic receptor in Drosophila clock neuron circuits , 2007, The Journal of comparative neurology.

[50]  Alan L. Hutchison,et al.  A Conserved Bicycle Model for Circadian Clock Control of Membrane Excitability , 2015, Cell.

[51]  M. Owen,et al.  Mutations in the gene encoding GlyT2 (SLC6A5) define a presynaptic component of human startle disease , 2006, Nature Genetics.

[52]  José Agosto,et al.  Coupled oscillators control morning and evening locomotor behaviour of Drosophila , 2004, Nature.

[53]  N. Davidson,et al.  Cloning, expression, and localization of a rat brain high-affinity glycine transporter. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[54]  B. Sakmann,et al.  Activation of multiple-conductance state chloride channels in spinal neurones by glycine and GABA , 1983, Nature.

[55]  J. Dupuis,et al.  Homomeric RDL and heteromeric RDL/LCCH3 GABA receptors in the honeybee antennal lobes: two candidates for inhibitory transmission in olfactory processing. , 2010, Journal of neurophysiology.

[56]  T. Lynagh,et al.  Principles of agonist recognition in Cys-loop receptors , 2014, Front. Physiol..

[57]  F. Jackson,et al.  Dispensable, Redundant, Complementary, and Cooperative Roles of Dopamine, Octopamine, and Serotonin in Drosophila melanogaster , 2013, Genetics.

[58]  J. Coyle,et al.  The NMDA receptor 'glycine modulatory site' in schizophrenia: D-serine, glycine, and beyond. , 2015, Current opinion in pharmacology.

[59]  K. Giacomini,et al.  SLC transporters as therapeutic targets: emerging opportunities , 2015, Nature Reviews Drug Discovery.

[60]  C. Pennartz,et al.  Circadian modulation of GABA function in the rat suprachiasmatic nucleus: excitatory effects during the night phase. , 2002, Journal of neurophysiology.

[61]  B. Sakmann,et al.  Patch-clamp measurements of elementary chloride currents activated by the putative inhibitory transmitter GABA and glycine in mammalian spinal neurons. , 1983, Journal of neural transmission. Supplementum.

[62]  Ozge Ozkaya,et al.  The circadian clock of the fly: a neurogenetics journey through time. , 2012, Advances in genetics.

[63]  M. Rosbash,et al.  Autoreceptor control of peptide/neurotransmitter corelease from PDF neurons determines allocation of circadian activity in drosophila. , 2012, Cell reports.

[64]  M. Vansteensel,et al.  A GABAergic Mechanism Is Necessary for Coupling Dissociable Ventral and Dorsal Regional Oscillators within the Circadian Clock , 2005, Current Biology.

[65]  G. Gisselmann,et al.  Drosophila melanogaster GRD and LCCH3 subunits form heteromultimeric GABA‐gated cation channels , 2004, British journal of pharmacology.

[66]  C. Helfrich-Förster,et al.  Period Gene Expression in Four Neurons Is Sufficient for Rhythmic Activity of Drosophila melanogaster under Dim Light Conditions , 2009, Journal of biological rhythms.

[67]  T. Sapsis,et al.  Circadian Rhythms in Rho1 Activity Regulate Neuronal Plasticity and Network Hierarchy , 2015, Cell.