Mushroom Body Specific Transcriptome Analysis Reveals Dynamic Regulation of Learning and Memory Genes After Acquisition of Long-Term Courtship Memory in Drosophila

The formation and recall of long-term memory (LTM) requires neuron activity-induced gene expression. Transcriptome analysis has been used to identify genes that have altered expression after memory acquisition, however, we still have an incomplete picture of the transcriptional changes that are required for LTM formation. The complex spatial and temporal dynamics of memory formation creates significant challenges in defining memory-relevant gene expression changes. The Drosophila mushroom body (MB) is a signaling hub in the insect brain that integrates sensory information to form memories across several different experimental memory paradigms. Here, we performed transcriptome analysis in the MB at two time points after the acquisition of LTM: 1 hr and 24 hr. The MB transcriptome was compared to biologically paired whole head (WH) transcriptomes. In both, we identified more transcript level changes at 1 hr after memory acquisition (WH = 322, MB = 302) than at 24 hr (WH = 23, MB = 20). WH samples showed downregulation of developmental genes and upregulation of sensory response genes. In contrast, MB samples showed vastly different changes in transcripts involved in biological processes that are specifically related to LTM. MB-downregulated genes were highly enriched for metabolic function. MB-upregulated genes were highly enriched for known learning and memory processes, including calcium-mediated neurotransmitter release and cAMP signaling. The neuron activity inducible genes Hr38 and sr were also specifically induced in the MB. These results highlight the importance of sampling time and cell type in capturing biologically relevant transcript level changes involved in learning and memory. Our data suggests that MB cells transiently upregulate known memory-related pathways after memory acquisition and provides a critical frame of reference for further investigation into the role of MB-specific gene regulation in memory.

[1]  John Tyler Bonner,et al.  Morphogenesis , 1965, The Physics of Living Matter: Space, Time and Information.

[2]  J. L. de la Pompa,et al.  A novel source of arterial valve cells linked to bicuspid aortic valve without raphe in mice , 2018, eLife.

[3]  S. Sprecher,et al.  Regulators of Long-Term Memory Revealed by Mushroom Body-Specific Gene Expression Profiling in Drosophila melanogaster , 2018, Genetics.

[4]  Thomas Preat,et al.  A GABAergic Feedback Shapes Dopaminergic Input on the Drosophila Mushroom Body to Promote Appetitive Long-Term Memory , 2018, Current Biology.

[5]  Francesc X. Soriano,et al.  Synaptic activity‐induced glycolysis facilitates membrane lipid provision and neurite outgrowth , 2018, The EMBO journal.

[6]  Paola Cognigni,et al.  Do the right thing: neural network mechanisms of memory formation, expression and update in Drosophila , 2018, Current Opinion in Neurobiology.

[7]  Barry J Dickson,et al.  Persistent activity in a recurrent circuit underlies courtship memory in Drosophila , 2018, eLife.

[8]  Juliana Costa-Silva,et al.  RNA-Seq differential expression analysis: An extended review and a software tool , 2017, PloS one.

[9]  F. W. Wolf,et al.  Mef2 induction of the immediate early gene Hr38/Nr4a is terminated by Sirt1 to promote ethanol tolerance , 2017, bioRxiv.

[10]  Zhiping Weng,et al.  A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes , 2017, PLoS genetics.

[11]  K. Keleman,et al.  Drosophila Courtship Conditioning As a Measure of Learning and Memory , 2017, Journal of visualized experiments : JoVE.

[12]  T. Préat,et al.  Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory , 2017, Nature Communications.

[13]  H. Bading,et al.  Synaptic Activity Drives a Genomic Program That Promotes a Neuronal Warburg Effect* , 2017, The Journal of Biological Chemistry.

[14]  Jin Billy Li,et al.  Evolutionary analysis reveals regulatory and functional landscape of coding and non-coding RNA editing , 2017, PLoS genetics.

[15]  The Gene Ontology Consortium,et al.  Expansion of the Gene Ontology knowledgebase and resources , 2016, Nucleic Acids Res..

[16]  Anushya Muruganujan,et al.  PANTHER version 11: expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements , 2016, Nucleic Acids Res..

[17]  The Gene Ontology Consortium.  Expansion of the Gene Ontology knowledgebase and resources , 2016, Nucleic Acids Res..

[18]  Yanhui Hu,et al.  FlyBase at 25: looking to the future , 2016, Nucleic Acids Res..

[19]  M. Rosbash,et al.  Genome-wide identification of neuronal activity-regulated genes in Drosophila , 2016, eLife.

[20]  M. Saitoe,et al.  Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies , 2016, Nature Communications.

[21]  B. S. Baker,et al.  Memory Elicited by Courtship Conditioning Requires Mushroom Body Neuronal Subsets Similar to Those Utilized in Appetitive Memory , 2016, PloS one.

[22]  Andreas S. Thum,et al.  Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae , 2016, PLoS genetics.

[23]  P. Mermelstein,et al.  Opposite Effects of mGluR1a and mGluR5 Activation on Nucleus Accumbens Medium Spiny Neuron Dendritic Spine Density , 2016, PloS one.

[24]  Sarah C. Ayling,et al.  The Ensembl gene annotation system , 2016, Database J. Biol. Databases Curation.

[25]  M. Janitz,et al.  Transcriptional regulation of long-term potentiation , 2016, neurogenetics.

[26]  Oliver Barnstedt,et al.  Aversive Learning and Appetitive Motivation Toggle Feed-Forward Inhibition in the Drosophila Mushroom Body , 2016, Neuron.

[27]  Mala Murthy,et al.  Cell-Type-Specific Transcriptome Analysis in the Drosophila Mushroom Body Reveals Memory-Related Changes in Gene Expression. , 2016, Cell reports.

[28]  R. Kittel,et al.  Synaptic Vesicle Proteins and Active Zone Plasticity , 2016, Front. Synaptic Neurosci..

[29]  Johannes Felsenberg,et al.  Memory-Relevant Mushroom Body Output Synapses Are Cholinergic , 2016, Neuron.

[30]  Michael A. Cousin,et al.  The iTRAPs: Guardians of Synaptic Vesicle Cargo Retrieval During Endocytosis , 2016, Front. Synaptic Neurosci..

[31]  P. Dayan,et al.  A mathematical model explains saturating axon guidance responses to molecular gradients , 2016, eLife.

[32]  M. Mayford,et al.  Exploring Memory Representations with Activity-Based Genetics. , 2016, Cold Spring Harbor Perspectives in Biology.

[33]  Brian D. Slaughter,et al.  Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator , 2015, Cell.

[34]  J. Veenstra,et al.  SIFamide acts on fruitless neurons to modulate sexual behavior in Drosophila melanogaster , 2015, Peptides.

[35]  P. Magistretti,et al.  Learning-Induced Gene Expression in the Hippocampus Reveals a Role of Neuron -Astrocyte Metabolic Coupling in Long Term Memory , 2015, PloS one.

[36]  Daewoo Lee.  Global and local missions of cAMP signaling in neural plasticity, learning, and memory , 2015, Front. Pharmacol..

[37]  A. Conesa,et al.  Data quality aware analysis of differential expression in RNA-seq with NOISeq R/Bioc package , 2015, Nucleic acids research.

[38]  Nancy R. Zhang,et al.  Memory acquisition and retrieval impact different epigenetic processes that regulate gene expression , 2015, BMC Genomics.

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

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

[41]  R. O. Godinho,et al.  New perspectives in signaling mediated by receptors coupled to stimulatory G protein: the emerging significance of cAMP efflux and extracellular cAMP-adenosine pathway , 2015, Front. Pharmacol..

[42]  Jean-René Martin,et al.  PKA and cAMP/CNG Channels Independently Regulate the Cholinergic Ca2+-Response of Drosophila Mushroom Body Neurons1,2,3 , 2015, eNeuro.

[43]  Shiyong Wu,et al.  The Warburg effect: evolving interpretations of an established concept. , 2015, Free radical biology & medicine.

[44]  B. Hanlon,et al.  Spatio-temporal in vivo recording of dCREB2 dynamics in Drosophila long-term memory processing , 2015, Neurobiology of Learning and Memory.

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

[46]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[47]  C. Mizutani,et al.  The KRÜPPEL-Like Transcription Factor DATILÓGRAFO Is Required in Specific Cholinergic Neurons for Sexual Receptivity in Drosophila Females , 2014, PLoS biology.

[48]  J. David Sweatt,et al.  Histone H2A.Z subunit exchange controls consolidation of recent and remote memory , 2014, Nature.

[49]  Gary D Bader,et al.  Biological Network Exploration with Cytoscape 3 , 2014, Current protocols in bioinformatics.

[50]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[51]  Allan Kuchinsky,et al.  enhancedGraphics: a Cytoscape app for enhanced node graphics , 2014, F1000Research.

[52]  Gary D Bader,et al.  GeneMANIA: Fast gene network construction and function prediction for Cytoscape , 2014, F1000Research.

[53]  Michael Hawrylycz,et al.  Aerobic glycolysis in the human brain is associated with development and neotenous gene expression. , 2014, Cell metabolism.

[54]  R Core Team,et al.  R: A language and environment for statistical computing. , 2014 .

[55]  P. Cui,et al.  Dynamic regulation of genome-wide pre-mRNA splicing and stress tolerance by the Sm-like protein LSm5 in Arabidopsis , 2014, Genome Biology.

[56]  Makoto Sato,et al.  Visualization of Neural Activity in Insect Brains Using a Conserved Immediate Early Gene, Hr38 , 2013, Current Biology.

[57]  H. Ishimoto,et al.  A Novel Role for Ecdysone in Drosophila Conditioned Behavior: Linking GPCR-Mediated Non-canonical Steroid Action to cAMP Signaling in the Adult Brain , 2013, PLoS genetics.

[58]  Brian R Johnson,et al.  The importance of tissue specificity for RNA-seq: highlighting the errors of composite structure extractions , 2013, BMC Genomics.

[59]  Tony D. Southall,et al.  Cell-Type-Specific Profiling of Gene Expression and Chromatin Binding without Cell Isolation: Assaying RNA Pol II Occupancy in Neural Stem Cells , 2013, Developmental cell.

[60]  G. Robinson,et al.  Activity-dependent gene expression in honey bee mushroom bodies in response to orientation flight , 2013, Journal of Experimental Biology.

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

[62]  Pierre Baldi,et al.  The Neuron-specific Chromatin Regulatory Subunit BAF53b is Necessary for Synaptic Plasticity and Memory , 2013, Nature Neuroscience.

[63]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[64]  M. Arbeitman,et al.  Identification of Gene Expression Changes Associated With Long-Term Memory of Courtship Rejection in Drosophila Males , 2012, G3: Genes | Genomes | Genetics.

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

[66]  A. Chiang,et al.  Molecular Genetic Analysis of Sexual Rejection: Roles of Octopamine and Its Receptor OAMB in Drosophila Courtship Conditioning , 2012, The Journal of Neuroscience.

[67]  Jai Y. Yu,et al.  Dopamine neurons modulate pheromone responses in Drosophila courtship learning , 2012, Nature.

[68]  S. Eddy,et al.  Cell type–specific genomics of Drosophila neurons , 2012, Nucleic acids research.

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

[70]  Johannes E. Schindelin,et al.  Fiji: an open-source platform for biological-image analysis , 2012, Nature Methods.

[71]  S. Henikoff,et al.  Cell-type-specific nuclei purification from whole animals for genome-wide expression and chromatin profiling. , 2012, Genome research.

[72]  Stephan J. Sigrist,et al.  RIM-Binding Protein, a Central Part of the Active Zone, Is Essential for Neurotransmitter Release , 2011, Science.

[73]  B. Dauwalder,et al.  The hector G-Protein Coupled Receptor Is Required in a Subset of fruitless Neurons for Male Courtship Behavior , 2011, PloS one.

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

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

[76]  Stephan J. Sigrist,et al.  Presynapses in Kenyon Cell Dendrites in the Mushroom Body Calyx of Drosophila , 2011, The Journal of Neuroscience.

[77]  S. Knapek,et al.  Bruchpilot, A Synaptic Active Zone Protein for Anesthesia-Resistant Memory , 2011, The Journal of Neuroscience.

[78]  Robert A. Edwards,et al.  Quality control and preprocessing of metagenomic datasets , 2011, Bioinform..

[79]  G. E. Carney,et al.  Socially-Responsive Gene Expression in Male Drosophila melanogaster Is Influenced by the Sex of the Interacting Partner , 2011, Genetics.

[80]  Annette Schenck,et al.  Epigenetic Regulation of Learning and Memory by Drosophila EHMT/G9a , 2011, PLoS biology.

[81]  G. E. Carney,et al.  Mating alters gene expression patterns in Drosophila melanogaster male heads , 2010, BMC Genomics.

[82]  S. Henikoff,et al.  A simple method for gene expression and chromatin profiling of individual cell types within a tissue. , 2010, Developmental cell.

[83]  E. Boersma,et al.  Prevention of Catheter-Related Bacteremia with a Daily Ethanol Lock in Patients with Tunnelled Catheters: A Randomized, Placebo-Controlled Trial , 2010, PloS one.

[84]  J. Ferveur,et al.  Drosophila Cuticular Hydrocarbons Revisited: Mating Status Alters Cuticular Profiles , 2010, PloS one.

[85]  Ronald L. Davis,et al.  Dynamics of Learning-Related cAMP Signaling and Stimulus Integration in the Drosophila Olfactory Pathway , 2009, Neuron.

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

[87]  H. Ishimoto,et al.  Ecdysone signaling regulates the formation of long-term courtship memory in adult Drosophila melanogaster , 2009, Proceedings of the National Academy of Sciences.

[88]  Yoshinori Aso,et al.  The Mushroom Body of Adult Drosophila Characterized by GAL4 Drivers , 2009, Journal of neurogenetics.

[89]  Barry J Dickson,et al.  Function of the Drosophila CPEB protein Orb2 in long-term courtship memory , 2007, Nature Neuroscience.

[90]  J. Littleton,et al.  A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth , 2007, Nature Neuroscience.

[91]  D. O'Dowd,et al.  nAChR‐mediated calcium responses and plasticity in Drosophila Kenyon cells , 2007, Developmental neurobiology.

[92]  Y. Grosjean,et al.  Prospero Mutants Induce Precocious Sexual Behavior in Drosophila Males , 2007, Behavior genetics.

[93]  J. Levine,et al.  Generalization of Courtship Learning in Drosophila Is Mediated by cis-Vaccenyl Acetate , 2007, Current Biology.

[94]  L. Luo,et al.  Comprehensive Maps of Drosophila Higher Olfactory Centers: Spatially Segregated Fruit and Pheromone Representation , 2007, Cell.

[95]  S. Waddell,et al.  Sequential Use of Mushroom Body Neuron Subsets during Drosophila Odor Memory Processing , 2007, Neuron.

[96]  Ronald L. Davis,et al.  Drosophila α/β Mushroom Body Neurons Form a Branch-Specific, Long-Term Cellular Memory Trace after Spaced Olfactory Conditioning , 2006, Neuron.

[97]  E. Kandel,et al.  Molecular Mechanisms of Memory Storage in Aplysia , 2006, The Biological Bulletin.

[98]  D. O'Dowd,et al.  Cholinergic Synaptic Transmission in Adult Drosophila Kenyon Cells In Situ , 2006, The Journal of Neuroscience.

[99]  Devanand S. Manoli,et al.  Male-specific fruitless specifies the neural substrates of Drosophila courtship behaviour , 2005, Nature.

[100]  E. Kandel,et al.  Chromatin Acetylation, Memory, and LTP Are Impaired in CBP+/− Mice A Model for the Cognitive Deficit in Rubinstein-Taybi Syndrome and Its Amelioration , 2004, Neuron.

[101]  R. Bourtchouladze,et al.  Targeting the CREB pathway for memory enhancers , 2003, Nature Reviews Drug Discovery.

[102]  Ann-Shyn Chiang,et al.  The staufen/pumilio Pathway Is Involved in Drosophila Long-Term Memory , 2003, Current Biology.

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

[104]  V. Hartenstein,et al.  Early development of the Drosophila mushroom body: the roles of eyeless and dachshund. , 2000, Development.

[105]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[106]  W. Gehring,et al.  Genetic control of development of the mushroom bodies, the associative learning centers in the Drosophila brain, by the eyeless, twin of eyeless, and Dachshund genes. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[107]  K. Siwicki,et al.  Mushroom Body Ablation Impairs Short-Term Memory and Long-Term Memory of Courtship Conditioning in Drosophila melanogaster , 1999, Neuron.

[108]  Liqun Luo,et al.  Mosaic Analysis with a Repressible Cell Marker for Studies of Gene Function in Neuronal Morphogenesis , 1999, Neuron.

[109]  R. Burgess,et al.  Distinct Requirements for Evoked and Spontaneous Release of Neurotransmitter Are Revealed by Mutations in theDrosophila Gene neuronal-synaptobrevin , 1998, The Journal of Neuroscience.

[110]  J. Lisman.  The CaM kinase II hypothesis for the storage of synaptic memory , 1994, Trends in Neurosciences.

[111]  M Heisenberg,et al.  Associative odor learning in Drosophila abolished by chemical ablation of mushroom bodies. , 1994, Science.

[112]  Ronald L. Davis,et al.  The Drosophila learning and memory gene rutabaga encodes a Ca 2+ calmodulin -responsive , 1992, Cell.

[113]  E R Kandel,et al.  A critical period for macromolecular synthesis in long-term heterosynaptic facilitation in Aplysia. , 1986, Science.

[114]  M. Livingstone,et al.  Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga, a Drosophila learning mutant , 1984, Cell.

[115]  R. W. Siegel,et al.  Conditioned responses in courtship behavior of normal and mutant Drosophila. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[116]  E R Kandel,et al.  Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP. , 1976, Science.

[117]  J. R. Price,et al.  Looking to the future , 1976, Nature.

[118]  Y. Jan,et al.  dunce, a mutant of Drosophila deficient in learning. , 1976, Proceedings of the National Academy of Sciences of the United States of America.

[119]  H. T. Spieth.  Courtship behavior in Drosophila. , 1974, Annual review of entomology.