Memory-Relevant Mushroom Body Output Synapses Are Cholinergic

Summary Memories are stored in the fan-out fan-in neural architectures of the mammalian cerebellum and hippocampus and the insect mushroom bodies. However, whereas key plasticity occurs at glutamatergic synapses in mammals, the neurochemistry of the memory-storing mushroom body Kenyon cell output synapses is unknown. Here we demonstrate a role for acetylcholine (ACh) in Drosophila. Kenyon cells express the ACh-processing proteins ChAT and VAChT, and reducing their expression impairs learned olfactory-driven behavior. Local ACh application, or direct Kenyon cell activation, evokes activity in mushroom body output neurons (MBONs). MBON activation depends on VAChT expression in Kenyon cells and is blocked by ACh receptor antagonism. Furthermore, reducing nicotinic ACh receptor subunit expression in MBONs compromises odor-evoked activation and redirects odor-driven behavior. Lastly, peptidergic corelease enhances ACh-evoked responses in MBONs, suggesting an interaction between the fast- and slow-acting transmitters. Therefore, olfactory memories in Drosophila are likely stored as plasticity of cholinergic synapses.

[1]  E. Gundelfinger,et al.  Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co‐assemble 
into the same receptor complex , 2002, Journal of neurochemistry.

[2]  Scott Waddell,et al.  Shocking Revelations and Saccharin Sweetness in the Study of Drosophila Olfactory Memory , 2013, Current Biology.

[3]  G. Laurent,et al.  Conditional modulation of spike-timing-dependent plasticity for olfactory learning , 2012, Nature.

[4]  B. Dickson,et al.  A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila , 2007, Nature.

[5]  Gilles Laurent,et al.  Testing Odor Response Stereotypy in the Drosophila Mushroom Body , 2008, Neuron.

[6]  A. Wong,et al.  Two-Photon Calcium Imaging Reveals an Odor-Evoked Map of Activity in the Fly Brain , 2003, Cell.

[7]  S. Farris.  Evolution of Complex Higher Brain Centers and Behaviors: Behavioral Correlates of Mushroom Body Elaboration in Insects , 2013, Brain, Behavior and Evolution.

[8]  Pierre Trifilieff,et al.  Intrinsic neurons of Drosophila mushroom bodies express short neuropeptide F: Relations to extrinsic neurons expressing different neurotransmitters , 2008, The Journal of comparative neurology.

[9]  Scott Waddell,et al.  Sweet Taste and Nutrient Value Subdivide Rewarding Dopaminergic Neurons in Drosophila , 2015, Current Biology.

[10]  W. Quinn,et al.  Classical conditioning and retention in normal and mutantDrosophila melanogaster , 1985, Journal of Comparative Physiology A.

[11]  I. Meinertzhagen,et al.  Synaptic organization of the mushroom body calyx in Drosophila melanogaster , 2002, The Journal of comparative neurology.

[12]  G. Rubin,et al.  Mushroom body efferent neurons responsible for aversive olfactory memory retrieval in Drosophila , 2011, Nature Neuroscience.

[13]  Stefan R. Pulver,et al.  Ultra-sensitive fluorescent proteins for imaging neuronal activity , 2013, Nature.

[14]  G. Miesenböck,et al.  Excitatory Local Circuits and Their Implications for Olfactory Processing in the Fly Antennal Lobe , 2007, Cell.

[15]  C. Tabone,et al.  A Putative Vesicular Transporter Expressed in Drosophila Mushroom Bodies that Mediates Sexual Behavior May Define a Neurotransmitter System , 2011, Neuron.

[16]  Paul Antoine Salin,et al.  Cyclic AMP Mediates a Presynaptic Form of LTP at Cerebellar Parallel Fiber Synapses , 1996, Neuron.

[17]  Andrew C. Lin,et al.  Different Kenyon Cell Populations Drive Learned Approach and Avoidance in Drosophila , 2013, Neuron.

[18]  J. Changeux,et al.  The diversity of subunit composition in nAChRs: evolutionary origins, physiologic and pharmacologic consequences. , 2002, Journal of neurobiology.

[19]  M. Heisenberg.  Mushroom body memoir: from maps to models , 2003, Nature Reviews Neuroscience.

[20]  Irina Sinakevitch,et al.  Ground plan of the insect mushroom body: Functional and evolutionary implications , 2009, The Journal of comparative neurology.

[21]  Wanhe Li,et al.  Imaging a Population Code for Odor Identity in the Drosophila Mushroom Body , 2013, The Journal of Neuroscience.

[22]  Paul Szyszka,et al.  Converging Circuits Mediate Temperature and Shock Aversive Olfactory Conditioning in Drosophila , 2014, Current Biology.

[23]  Yonatan Loewenstein,et al.  Alternative Sites of Synaptic Plasticity in Two Homologous “Fan-out Fan-in” Learning and Memory Networks , 2011, Current Biology.

[24]  Ann-Shyn Chiang,et al.  Drosophila ORB protein in two mushroom body output neurons is necessary for long-term memory formation , 2013, Proceedings of the National Academy of Sciences.

[25]  E. Buchner,et al.  Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster , 1986, Cell and Tissue Research.

[26]  G. Rubin,et al.  A subset of dopamine neurons signals reward for odour memory in Drosophila , 2012, Nature.

[27]  Robert A. A. Campbell,et al.  Cellular-Resolution Population Imaging Reveals Robust Sparse Coding in the Drosophila Mushroom Body , 2011, The Journal of Neuroscience.

[28]  R. Menzel.  The insect mushroom body, an experience-dependent recoding device , 2014, Journal of Physiology-Paris.

[29]  G. Rubin,et al.  Mushroom body output neurons encode valence and guide memory-based action selection in Drosophila , 2014, eLife.

[30]  J. Storm-Mathisen,et al.  Taurine‐like immunoreactivity in the brain of the honeybee , 1988, The Journal of comparative neurology.

[31]  L. Abbott,et al.  Random Convergence of Olfactory Inputs in the Drosophila Mushroom Body , 2013, Nature.

[32]  Stephan J. Sigrist,et al.  Bruchpilot, a Protein with Homology to ELKS/CAST, Is Required for Structural Integrity and Function of Synaptic Active Zones in Drosophila , 2006, Neuron.

[33]  Scott Waddell,et al.  Drosophila Learn Opposing Components of a Compound Food Stimulus , 2014, Current Biology.

[34]  Kei Ito,et al.  Neuronal assemblies of the Drosophila mushroom body , 2008, The Journal of comparative neurology.

[35]  G. Rubin,et al.  The neuronal architecture of the mushroom body provides a logic for associative learning , 2014, eLife.

[36]  Scott Waddell,et al.  Olfactory learning skews mushroom body output pathways to steer behavioral choice in Drosophila , 2015, Current Opinion in Neurobiology.

[37]  D. Feldman.  The Spike-Timing Dependence of Plasticity , 2012, Neuron.

[38]  R. Greenspan.  Mutations of choline acetyltransferase and associated neural defects , 1980, Journal of comparative physiology.

[39]  Norbert Perrimon,et al.  A Drosophila Resource of Transgenic RNAi Lines for Neurogenetics , 2009, Genetics.

[40]  Johannes Felsenberg,et al.  Activity of Defined Mushroom Body Output Neurons Underlies Learned Olfactory Behavior in Drosophila , 2015, Neuron.

[41]  Gerald M. Rubin,et al.  A Higher Brain Circuit for Immediate Integration of Conflicting Sensory Information in Drosophila , 2015, Current Biology.

[42]  Stefan R. Pulver,et al.  Independent Optical Excitation of Distinct Neural Populations , 2014, Nature Methods.

[43]  T. Kitamoto,et al.  Immunocytochemical study of choline acetyltransferase in Drosophila melanogaster: An analysis of cis‐regulatory regions controlling expression in the brain of cDNA‐transformed flies , 1995, The Journal of comparative neurology.

[44]  Julie H. Simpson,et al.  A GAL4-driver line resource for Drosophila neurobiology. , 2012, Cell reports.

[45]  F. Schürmann.  Acetylcholine, GABA, glutamate and NO as putative transmitters indicated by immunocytochemistry in the olfactory mushroom body system of the insect brain. , 2000, Acta biologica Hungarica.

[46]  Ronald L. Davis,et al.  Spatiotemporal Rescue of Memory Dysfunction in Drosophila , 2003, Science.

[47]  Hiromu Tanimoto,et al.  Two pairs of mushroom body efferent neurons are required for appetitive long-term memory retrieval in Drosophila. , 2013, Cell reports.

[48]  T. Kitamoto,et al.  Structure and Organization of the DrosophilaCholinergic Locus* , 1998, The Journal of Biological Chemistry.

[49]  L. Luo,et al.  A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining , 2006, Nature Protocols.

[50]  M Heisenberg,et al.  Localization of a short-term memory in Drosophila. , 2000, Science.

[51]  Raju Tomer,et al.  Profiling by Image Registration Reveals Common Origin of Annelid Mushroom Bodies and Vertebrate Pallium , 2010, Cell.

[52]  Wanhe Li,et al.  Short- and Long-Term Memory in Drosophila Require cAMP Signaling in Distinct Neuron Types , 2009, Current Biology.

[53]  E. Kandel,et al.  The Molecular and Systems Biology of Memory , 2014, Cell.

[54]  A. Chiang,et al.  Parallel circuits control temperature preference in Drosophila during ageing , 2015, Nature Communications.

[55]  K. Han,et al.  D1 Dopamine Receptor dDA1 Is Required in the Mushroom Body Neurons for Aversive and Appetitive Learning in Drosophila , 2007, The Journal of Neuroscience.

[56]  Toby Collins,et al.  The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels , 2012, BMC Neuroscience.

[57]  S. Farris.  Are mushroom bodies cerebellum-like structures? , 2011, Arthropod structure & development.

[58]  Jing W. Wang,et al.  Presynaptic Facilitation by Neuropeptide Signaling Mediates Odor-Driven Food Search , 2011, Cell.

[59]  N. Strausfeld,et al.  Evolution, discovery, and interpretations of arthropod mushroom bodies. , 1998, Learning & memory.

[60]  Kristin Scott,et al.  Gustatory Learning and Processing in the Drosophila Mushroom Bodies , 2015, The Journal of Neuroscience.

[61]  D. Wilkin,et al.  Neuron , 2001, Brain Research.

[62]  David L. Glanzman,et al.  Learning in Aplysia: looking at synaptic plasticity from both sides , 2003, Trends in Neurosciences.

[63]  S. Knapek,et al.  Short Neuropeptide F Acts as a Functional Neuromodulator for Olfactory Memory in Kenyon Cells of Drosophila Mushroom Bodies , 2013, The Journal of Neuroscience.

[64]  Ronald L. Davis,et al.  Eight Different Types of Dopaminergic Neurons Innervate the Drosophila Mushroom Body Neuropil: Anatomical and Physiological Heterogeneity , 2009, Front. Neural Circuits.

[65]  Jeffrey C. Hall,et al.  Immunohistochemical localization of choline acetyltransferase during development and in Chats mutants of Drosophila melanogaster , 1987, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[66]  Z. Weng,et al.  Transposition-Driven Genomic Heterogeneity in the Drosophila Brain , 2013, Science.

[67]  Vikram Chandra,et al.  Neural correlates of water reward in thirsty Drosophila , 2014, Nature Neuroscience.

[68]  Shamik Dasgupta,et al.  A Neural Circuit Mechanism Integrating Motivational State with Memory Expression in Drosophila , 2009, Cell.

[69]  Karel Svoboda,et al.  ScanImage: Flexible software for operating laser scanning microscopes , 2003, Biomedical engineering online.

[70]  Thomas Preat,et al.  Parallel Processing of Appetitive Short- and Long-Term Memories In Drosophila , 2011, Current Biology.

[71]  D. Sattelle,et al.  Exploring the pharmacological properties of insect nicotinic acetylcholine receptors. , 2007, Trends in pharmacological sciences.

[72]  Thomas Preat,et al.  Two independent mushroom body output circuits retrieve the six discrete components of Drosophila aversive memory. , 2015, Cell reports.

[73]  Alexander Borst,et al.  Optogenetic and Pharmacologic Dissection of Feedforward Inhibition in Drosophila Motion Vision , 2014, The Journal of Neuroscience.

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

[75]  N. Pírez,et al.  The Drosophila neuropeptides PDF and sNPF have opposing electrophysiological and molecular effects on central neurons. , 2014, Journal of neurophysiology.

[76]  Yoshinori Aso,et al.  Distinct dopamine neurons mediate reward signals for short- and long-term memories , 2014, Proceedings of the National Academy of Sciences.

[77]  N. Strausfeld,et al.  Taurine‐, aspartate‐ and glutamate‐like immunoreactivity identifies chemically distinct subdivisions of Kenyon cells in the cockroach mushroom body , 2001, The Journal of comparative neurology.

[78]  Andrew C. Lin,et al.  Sparse, Decorrelated Odor Coding in the Mushroom Body Enhances Learned Odor Discrimination , 2014, Nature Neuroscience.

[79]  P. Greengard,et al.  Writing Memories with Light-Addressable Reinforcement Circuitry , 2009, Cell.

[80]  G. Rubin,et al.  Shared mushroom body circuits underlie visual and olfactory memories in Drosophila , 2014, eLife.

[81]  Charles F Stevens,et al.  What the fly’s nose tells the fly’s brain , 2015, Proceedings of the National Academy of Sciences.

[82]  Gerald M. Rubin,et al.  Propagation of Homeostatic Sleep Signals by Segregated Synaptic Microcircuits of the Drosophila Mushroom Body , 2015, Current Biology.

[83]  Daryl M. Gohl,et al.  Layered reward signaling through octopamine and dopamine in Drosophila , 2012, Nature.

[84]  Wanhe Li,et al.  Gamma Neurons Mediate Dopaminergic Input during Aversive Olfactory Memory Formation in Drosophila , 2012, Current Biology.

[85]  S. Waddell.  Reinforcement signalling in Drosophila; dopamine does it all after all , 2013, Current Opinion in Neurobiology.

[86]  Jasper Akerboom,et al.  Optimization of a GCaMP Calcium Indicator for Neural Activity Imaging , 2012, The Journal of Neuroscience.

[87]  N. Strausfeld,et al.  The mushroom bodies of Drosophila melanogaster: An immunocytological and golgi study of Kenyon cell organization in the calyces and lobes , 2003, Microscopy research and technique.

[88]  Kouji Yasuyama,et al.  Localization of choline acetyltransferase-expressing neurons in the larval visual system of Drosophila melanogaster. , 1995 .

[89]  Michael J. Krashes,et al.  A Pair of Inhibitory Neurons Are Required to Sustain Labile Memory in the Drosophila Mushroom Body , 2011, Current Biology.

[90]  Gerald M. Rubin,et al.  Heterosynaptic Plasticity Underlies Aversive Olfactory Learning in Drosophila , 2015, Neuron.

[91]  Tim Tully,et al.  Associative Learning Disrupted by Impaired Gs Signaling in Drosophila Mushroom Bodies , 1996, Science.

[92]  Yoshinori Aso,et al.  Three Dopamine Pathways Induce Aversive Odor Memories with Different Stability , 2012, PLoS genetics.