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Habituation Learning Is a Widely Affected Mechanism in Drosophila Models of Intellectual Disability and Autism Spectrum Disorders

  • Michaela Fenckova
    Affiliations
    Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Laura E.R. Blok
    Affiliations
    Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Lenke Asztalos
    Affiliations
    Aktogen Limited, Department of Genetics, University of Cambridge, Cambridge, United Kingdom

    Aktogen Hungary Limited, Bay Zoltán Nonprofit Limited for Applied Research, Institute for Biotechnology, Szeged, Hungary
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  • David P. Goodman
    Affiliations
    School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania
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  • Pavel Cizek
    Affiliations
    Centre for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Euginia L. Singgih
    Affiliations
    Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands

    Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Jeffrey C. Glennon
    Affiliations
    Department of Cognitive Neuroscience, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Joanna IntHout
    Affiliations
    Department for Health Evidence, Radboud University Medical Center, Nijmegen, The Netherlands
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  • Christiane Zweier
    Affiliations
    Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen, Germany
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  • Evan E. Eichler
    Affiliations
    Department of Genome Sciences, University of Washington School of Medicine, Seattle, Washington

    Howard Hughes Medical Institute, University of Washington, Seattle, Washington
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  • Catherine R. von Reyn
    Affiliations
    School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, Pennsylvania

    Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania
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  • Raphael A. Bernier
    Affiliations
    Department of Psychiatry and Behavioral Sciences, University of Washington, Seattle, Washington
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  • Zoltan Asztalos
    Affiliations
    Aktogen Limited, Department of Genetics, University of Cambridge, Cambridge, United Kingdom

    Aktogen Hungary Limited, Bay Zoltán Nonprofit Limited for Applied Research, Institute for Biotechnology, Szeged, Hungary

    Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary
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  • Annette Schenck
    Correspondence
    Address correspondence to Annette Schenck, Ph.D., Radboud University Medical Center, Department of Human Genetics (route 855), Geert Grooteplein 10, 6525 GA, Nijmegen, The Netherlands.
    Affiliations
    Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands
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Open AccessPublished:May 08, 2019DOI:https://doi.org/10.1016/j.biopsych.2019.04.029

      Abstract

      Background

      Although habituation is one of the most ancient and fundamental forms of learning, its regulators and its relevance for human disease are poorly understood.

      Methods

      We manipulated the orthologs of 286 genes implicated in intellectual disability (ID) with or without comorbid autism spectrum disorder (ASD) specifically in Drosophila neurons, and we tested these models in light-off jump habituation. We dissected neuronal substrates underlying the identified habituation deficits and integrated genotype–phenotype annotations, gene ontologies, and interaction networks to determine the clinical features and molecular processes that are associated with habituation deficits.

      Results

      We identified >100 genes required for habituation learning. For 93 of these genes, a role in habituation learning was previously unknown. These genes characterize ID disorders with macrocephaly and/or overgrowth and comorbid ASD. Moreover, individuals with ASD from the Simons Simplex Collection carrying damaging de novo mutations in these genes exhibit increased aberrant behaviors associated with inappropriate, stereotypic speech. At the molecular level, ID genes required for normal habituation are enriched in synaptic function and converge on Ras/mitogen-activated protein kinase (Ras/MAPK) signaling. Both increased Ras/MAPK signaling in gamma-aminobutyric acidergic (GABAergic) neurons and decreased Ras/MAPK signaling in cholinergic neurons specifically inhibit the adaptive habituation response.

      Conclusions

      Our work supports the relevance of habituation learning to ASD, identifies an unprecedented number of novel habituation players, supports an emerging role for inhibitory neurons in habituation, and reveals an opposing, circuit-level-based mechanism for Ras/MAPK signaling. These findings establish habituation as a possible, widely applicable functional readout and target for pharmacologic intervention in ID/ASD.

      Keywords

      Habituation is one of the most ancient and fundamental forms of learning, and it is conserved across the animal kingdom (
      • Peeke H.V.S.
      • Herz M.J.
      ). It causes an organism’s initial response to repeated meaningless stimuli to gradually decline. Learning to ignore irrelevant stimuli as a result of habituation is thought to represent a filter mechanism that prevents information overload, allowing for selective attention, thereby focusing cognitive resources on relevant matters. Habituation learning has been proposed to represent an important prerequisite for higher cognitive functions (
      • Colombo J.
      • Mitchell D.W.
      Infant visual habituation.
      ,
      • Rankin C.H.
      • Abrams T.
      • Barry R.J.
      • Bhatnagar S.
      • Clayton D.
      • Colombo J.
      • et al.
      Habituation revisited: An updated and revised description of the behavioral characteristics of habituation.
      ,
      • Barron H.C.
      • Vogels T.P.
      • Behrens T.E.
      • Ramaswami M.
      Inhibitory engrams in perception and memory.
      ). In line with this, habituation in infants correlates better than other measures with later cognitive abilities (
      • Kavšek M.
      Predicting later IQ from infant visual habituation and dishabituation: A meta-analysis.
      ). However, key players and molecular mechanisms underlying habituation are poorly understood (
      • Schmid S.
      • Wilson D.A.
      • Rankin C.H.
      Habituation mechanisms and their importance for cognitive function.
      ).
      In humans, deficits in habituation have been reported in a number of neuropsychiatric and behavioral disorders. In particular, defective cortical filtering of sensory stimuli and information overload, as expected to arise from habituation deficits, are thought to represent mechanisms contributing to autism spectrum disorder (ASD) (
      • Ramaswami M.
      Network plasticity in adaptive filtering and behavioral habituation.
      ,
      • Sinha P.
      • Kjelgaard M.M.
      • Gandhi T.K.
      • Tsourides K.
      • Cardinaux A.L.
      • Pantazis D.
      • et al.
      Autism as a disorder of prediction.
      ). A decreased ability to habituate has been described in a fraction of individuals with ASD (
      • Pellicano E.
      • Rhodes G.
      • Calder A.J.
      Reduced gaze aftereffects are related to difficulties categorising gaze direction in children with autism.
      ,
      • Ewbank M.P.
      • Rhodes G.
      • Von Dem Hagen E.A.H.
      • Powell T.E.
      • Bright N.
      • Stoyanova R.S.
      • et al.
      Repetition suppression in ventral visual cortex is diminished as a function of increasing autistic traits.
      ,
      • Swartz J.R.
      • Wiggins J.L.
      • Carrasco M.
      • Lord C.
      • Monk C.S.
      Amygdala habituation and prefrontal functional connectivity in youth with autism spectrum disorders.
      ) but has not yet been connected to specific genetic defects, with a single exception. Recently, two independent studies demonstrated habituation deficits in patients with fragile X syndrome, the most common monogenic cause of intellectual disability (ID) and ASD (
      • Ethridge L.E.
      • White S.P.
      • Mosconi M.W.
      • Wang J.
      • Byerly M.J.
      • Sweeney J.A.
      Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in fragile X syndrome.
      ,
      • Rigoulot S.
      • Knoth I.S.
      • Lafontaine M.-P.
      • Vannasing P.
      • Major P.
      • Jacquemont S.
      • et al.
      Altered visual repetition suppression in fragile X syndrome: New evidence from ERPs and oscillatory activity.
      ), confirming previously reported habituation deficits in Fmr1 knockout mice (
      • Restivo L.
      • Ferrari F.
      • Passino E.
      • Sgobio C.
      • Bock J.
      • Oostra B.A.
      • et al.
      Enriched environment promotes behavioral and morphological recovery in a mouse model for the fragile X syndrome.
      ,
      • Lovelace J.W.
      • Wen T.H.
      • Reinhard S.
      • Hsu M.S.
      • Sidhu H.
      • Ethell I.M.
      • et al.
      Matrix metalloproteinase-9 deletion rescues auditory evoked potential habituation deficit in a mouse model of fragile X syndrome.
      ). Habituation deficits have also been reported in a limited number of other ID or ASD (ID/ASD) disease models (
      • Wolman M.A.
      • deGroh E.D.
      • McBride S.M.
      • Jongens T.A.
      • Granato M.
      • Epstein J.A.
      Modulation of cAMP and Ras signaling pathways improves distinct behavioral deficits in a zebrafish model of neurofibromatosis type 1.
      ,
      • Kirshenbaum G.S.
      • Clapcote S.J.
      • Duffy S.
      • Burgess C.R.
      • Petersen J.
      • Jarowek K.J.
      • et al.
      Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump.
      ,
      • Cheli V.T.
      • Adrover M.F.
      • Blanco C.
      • Verde E.R.
      • Guyot-Revol V.
      • Vidal R.
      • et al.
      Gene transfer of NMDAR1 subunit sequences to the rat CNS using herpes simplex virus vectors interfered with habituation.
      ,
      • Walsh J.
      • Desbonnet L.
      • Clarke N.
      • Waddington J.L.
      • O’Tuathaigh C.M.P.
      Disruption of exploratory and habituation behavior in mice with mutation of DISC1: An ethologically based analysis.
      ).
      Because assessing human gene function in habituation is challenging, we used a cross-species approach. We apply light-off jump habituation in Drosophila to increase our knowledge on the genetic control of habituation and, at the same time, to address the relevance of decreased habituation in ID and in comorbid ASD disorders. Since ID is present in 70% of individuals with ASD (
      • Schwartz C.E.
      • Neri G.
      Autism and intellectual disability: Two sides of the same coin.
      ), monogenic causes of ID provide unique molecular windows to ASD pathology (
      • Chahrour M.
      • O’Roak B.J.
      • Santini E.
      • Samaco R.C.
      • Kleiman R.J.
      • Manzini M.C.
      Current perspectives in autism spectrum disorder: From genes to therapy.
      ). Drosophila provides a powerful, well-established model for ID (
      • McBride S.M.J.
      • Choi C.H.
      • Wang Y.
      • Liebelt D.
      • Braunstein E.
      • Ferreiro D.
      • et al.
      Pharmacological rescue of synaptic plasticity, courtship behavior, and mushroom body defects in a Drosophila model of fragile X syndrome.
      ,
      • Kramer J.M.
      • Kochinke K.
      • Oortveld M.A.W.
      • Marks H.
      • Kramer D.
      • de Jong E.K.
      • et al.
      Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.
      ,
      • van der Voet M.
      • Nijhof B.
      • Oortveld M.A.W.
      • Schenck A.
      Drosophila models of early onset cognitive disorders and their clinical applications.
      ) and offers genome-wide resources to study gene function in large scale (
      • Dietzl G.
      • Chen D.
      • Schnorrer F.
      • Su K.-C.
      • Barinova Y.
      • Fellner M.
      • et al.
      A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.
      ,
      • Bellen H.J.
      • Tong C.
      • Tsuda H.
      100 years of Drosophila research and its impact on vertebrate neuroscience: A history lesson for the future.
      ). Several forms of habituation have been established in Drosophila (
      • Duerr J.S.
      • Quinn W.G.
      Three Drosophila mutations that block associative learning also affect habituation and sensitization.
      ,
      • Asztalos Z.
      • Arora N.
      • Tully T.
      Olfactory jump reflex habituation in drosophila and effects of classical conditioning mutations.
      ,
      • Larkin A.
      • Karak S.
      • Priya R.
      • Das A.
      • Ayyub C.
      • Ito K.
      • et al.
      Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae.
      ,
      • Acevedo S.F.
      • Froudarakis E.I.
      • Kanellopoulos A.
      • Skoulakis E.M.C.
      Protection from premature habituation requires functional mushroom bodies in Drosophila.
      ,
      • Engel J.E.
      • Wu C.F.
      Altered habituation of an identified escape circuit in Drosophila memory mutants.
      ). Deficits in light-off jump habituation have already been reported in several ID models (
      • Kramer J.M.
      • Kochinke K.
      • Oortveld M.A.W.
      • Marks H.
      • Kramer D.
      • de Jong E.K.
      • et al.
      Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.
      ,
      • van Bon B.W.M.
      • Oortveld M.A.W.
      • Nijtmans L.G.
      • Fenckova M.
      • Nijhof B.
      • Besseling J.
      • et al.
      CEP89 is required for mitochondrial metabolism and neuronal function in man and fly.
      ,
      • Willemsen M.H.
      • Nijhof B.
      • Fenckova M.
      • Nillesen W.M.
      • Bongers E.M.
      • Castells-Nobau A.
      • et al.
      GATAD2B loss-of-function mutations cause a recognisable syndrome with intellectual disability and are associated with learning deficits and synaptic undergrowth in Drosophila.
      ,
      • Lugtenberg D.
      • Reijnders M.R.F.
      • Fenckova M.
      • Bijlsma E.K.
      • Bernier R.
      • van Bon B.W.M.
      • et al.
      De novo loss-of-function mutations in WAC cause a recognizable intellectual disability syndrome and learning deficits in Drosophila.
      ,
      • Stessman H.A.F.
      • Willemsen M.H.
      • Fenckova M.
      • Penn O.
      • Hoischen A.
      • Xiong B.
      • et al.
      Disruption of POGZ is associated with intellectual disability and autism spectrum disorders.
      ,
      • Esmaeeli-Nieh S.
      • Fenckova M.
      • Porter I.M.
      • Motazacker M.M.
      • Nijhof B.
      • Castells-Nobau A.
      • et al.
      BOD1 is required for cognitive function in humans and Drosophila.
      ) and in classical learning and memory mutants (
      • Asztalos Z.
      • Arora N.
      • Tully T.
      Olfactory jump reflex habituation in drosophila and effects of classical conditioning mutations.
      ,
      • Engel J.E.
      • Wu C.F.
      Altered habituation of an identified escape circuit in Drosophila memory mutants.
      ). Moreover, this form of habituation can be assessed in a high-throughput manner. In the light-off jump paradigm, the initial jump response to repeated light-off stimuli gradually wanes; this has been demonstrated to result not from sensory adaptation (a decrease in detecting the stimulus) or motor fatigue (a decrease in the ability to execute the response) but from learned adaptation of the startle circuit (
      • Engel J.E.
      • Wu C.F.
      Altered habituation of an identified escape circuit in Drosophila memory mutants.
      ). This behavior meets all habituation criteria (
      • Rankin C.H.
      • Abrams T.
      • Barry R.J.
      • Bhatnagar S.
      • Clayton D.
      • Colombo J.
      • et al.
      Habituation revisited: An updated and revised description of the behavioral characteristics of habituation.
      ), including spontaneous recovery and dishabituation with a novel stimulus (
      • Engel J.E.
      • Wu C.F.
      Altered habituation of an identified escape circuit in Drosophila memory mutants.
      ,
      • Kelly L.E.
      An altered electroretinogram transient associated with an unusual jump response in a mutant of Drosophila.
      ).
      Here, we use inducible RNA interference (RNAi) in Drosophila to systematically assess the role of Drosophila orthologs of 286 genes that are well-established as causes of ID in humans when mutated (hereinafter referred to as “ID genes”). Of these ID genes, 68 (20%) have also been implicated in ASD (
      • Basu S.N.
      • Kollu R.
      • Banerjee-Basu S.
      AutDB: A gene reference resource for autism research.
      ,
      • Fischbach G.D.
      • Lord C.
      The Simons Simplex Collection: A resource for identification of autism genetic risk factors.
      ) (Supplemental Table S1), hereinafter referred to as “ID plus ASD–associated genes.”

      Methods and Materials

      Investigated ID Genes

      A systematic source of ID genes and their Drosophila orthologs is available online [SysID database, sysid.cmbi.umcn.nl (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      )]. We investigated the Drosophila orthologs of 286 human ID genes from the SysID category primary ID genes (Supplemental Table S1) containing mutations with robust published evidence for causality (see Supplemental Methods). SysID inclusion criteria and inclusion and exclusion criteria of experimentally investigated genes are indicated in the Supplemental Methods. In brief, the vast majority of genes are from the first data freeze of the SysID database (status of mid 2010). Genes have been included based on conservation in Drosophila, available tools (RNAi) from large-scale resources, and viability as a prerequisite for behavioral testing. No selection was performed.

      Light-off Jump Habituation Assay

      Flies of 3 to 7 days of age were subjected to the light-off jump habituation paradigm in two independent 16-unit light-off jump systems (manufactured and distributed by Aktogen Ltd., Cambridge, United Kingdom). After a 5-minute adaptation period, flies were simultaneously exposed to a series of 100 light-off pulses (15 ms) with a 1-second interval. The noise amplitude of wing vibration during jump responses was recorded. An appropriate threshold (0.8 V) was applied to filter out background noise. Data were collected by a custom-made LabVIEW software (National Instruments, Austin, TX). Flies were considered habituated when they were not jumping in five consecutive light-off trials (no-jump criterion). Habituation was quantified as the number of trials required to reach the no-jump criterion (trials to criterion).
      Information about the identification of Drosophila orthologs, proposed disease mechanism, Drosophila stocks, phenotype reproducibility, validation of the automated jump scoring and of jump specificity, fatigue assay, quality criteria for RNAi lines, annotation of ID plus ASD–associated genes, enrichment analysis, comparison of behavior and cognition in individuals with ASD from the Simons Simplex Collection (SSC), molecular interaction network, clustering, physical interaction enrichment, data visualization, and statistics are described in the Supplemental Methods.

      Results

      Systematic Identification of Habituation Deficits in Drosophila Models of ID

      To identify novel genes implicated in habituation, we systematically investigated the role of 278 Drosophila orthologs representing 286 human ID genes in the light-off jump habituation paradigm. We induced neuron-specific knockdowns of each ID gene ortholog by RNAi (
      • Dietzl G.
      • Chen D.
      • Schnorrer F.
      • Su K.-C.
      • Barinova Y.
      • Fellner M.
      • et al.
      A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.
      ) using 513 RNAi lines that fulfilled previously established quality criteria (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      ,
      • Oortveld M.A.W.
      • Keerthikumar S.
      • Oti M.
      • Nijhof B.
      • Fernandes A.C.
      • Kochinke K.
      • et al.
      Human intellectual disability genes form conserved functional modules in Drosophila.
      ), with two independent constructs per gene whenever available. These were crossed to the panneuronal elav-Gal4 driver line (see Supplemental Methods). Knockdown is a suitable approach for modeling of the here-investigated human disease conditions since full or partial loss of function is considered to be the underlying mechanism in the vast majority of these disorders (
      • Oortveld M.A.W.
      • Keerthikumar S.
      • Oti M.
      • Nijhof B.
      • Fernandes A.C.
      • Kochinke K.
      • et al.
      Human intellectual disability genes form conserved functional modules in Drosophila.
      ) (Supplemental Table S1). Restricting gene knockdown to neurons eliminates potential effects on viability or behavioral performance originating from an essential role of genes in other tissues and establishes neuron-autonomous mechanisms.
      Knockdown and control flies of identical genetic background were subjected to a series of 100 light-off stimuli, hereinafter referred to as trials, in the light-off jump habituation paradigm. The screening procedure and paradigm allowed us to distinguish the following parameters: viability, initial jump response (percentage of flies that jumped in at least one of the first five trials), and premature and reduced habituation, with the latter representing the learning-defective phenotype category of main interest. Genotypes with an initial jump response ≥50% but premature habituation were subjected to a secondary assay to exclude fatigue as a confounder of premature habituation (see Supplemental Methods, Supplemental Table S2, and Supplemental Figure S4). Based on these parameters, genes were assigned to at least one of four phenotype categories (Figure 1A): 1) “not affected”: (both) tested RNAi lines targeting such genes were viable, showed good initial jump response, and had no significant effect on habituation (based on the false discovery rate–corrected p value) (see Supplemental Methods); 2) “non-performers”: at least one RNAi line led to lethality, poor jump response (<50% initial jumpers), or premature habituation because of increased fatigue; 3) “habituation deficient”: at least one RNAi line showed good initial jump response but failed to suppress the response with the increasing number of light-off trials (based on the false discovery rate–corrected p value); and 4) “premature habituation”: at least one RNAi line showed good initial jump response followed by faster decline (based on the false discovery rate–corrected p value), without fatigue being detectable in the secondary assay. Still, this latter phenotype category can result from defects other than improved habituation, and it will be investigated elsewhere. In this study, we focus on habituation deficits (phenotype category 3) corresponding to the phenotype that has been shown in ID and ASD (
      • Pellicano E.
      • Rhodes G.
      • Calder A.J.
      Reduced gaze aftereffects are related to difficulties categorising gaze direction in children with autism.
      ,
      • Ewbank M.P.
      • Rhodes G.
      • Von Dem Hagen E.A.H.
      • Powell T.E.
      • Bright N.
      • Stoyanova R.S.
      • et al.
      Repetition suppression in ventral visual cortex is diminished as a function of increasing autistic traits.
      ,
      • Swartz J.R.
      • Wiggins J.L.
      • Carrasco M.
      • Lord C.
      • Monk C.S.
      Amygdala habituation and prefrontal functional connectivity in youth with autism spectrum disorders.
      ,
      • Ethridge L.E.
      • White S.P.
      • Mosconi M.W.
      • Wang J.
      • Byerly M.J.
      • Sweeney J.A.
      Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in fragile X syndrome.
      ,
      • Rigoulot S.
      • Knoth I.S.
      • Lafontaine M.-P.
      • Vannasing P.
      • Major P.
      • Jacquemont S.
      • et al.
      Altered visual repetition suppression in fragile X syndrome: New evidence from ERPs and oscillatory activity.
      ).
      Figure thumbnail gr1
      Figure 1Habituation screen of intellectual disability (ID) genes, phenotype distribution, and proof of principle. (A) Procedure, phenotype categories, and phenotype distribution of the light-off jump habituation screen. Knockdowns that resulted in lethality, no jumper phenotype (defined as <50% of flies jumping in at least one of the first five light-off trials), or premature habituation plus increased fatigue were assigned to the category “non-performers” and their habituation was not further analyzed. Other phenotype categories are “habituation deficient,” “not affected,” and “premature habituation” (the latter if no fatigue was detected in a secondary assay; see example in ). Drosophila orthologs of 34% of the investigated human ID genes were associated with defects in habituation learning. See also . (B–D) Defective habituation upon neuron-specific RNAi-mediated knockdown of G9a, Synapsin (syn), and dunce (dnc) (2xGMR-wIR/+; UAS-RNAi/elav-Gal4, UAS-Dicer-2, in red) compared with that of their respective genetic background controls (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+, in gray). Jump response curves show the average jump response (% of jumping flies) over 100 light-off trials at 1-second intertrial interval. Mean trials to criterion (TTC): the mean number of trials that flies needed to reach the no-jump criterion (see Methods and Materials) presented as mean TTC ± SEM. ***padj < .001, **padj < .01, based on false discovery rate–corrected general linear model analysis. A complete list of ID genes with previously identified habituation defects is provided as , adding further proof of principle. dsRNA, double-strand RNA; hab., habituation; prem., premature; RNAi, RNA interference.
      We validated the experimental approach to identify genes that, if manipulated, cause habituation deficits (hereinafter referred to as habituation deficient genes) by recapitulating published habituation deficits of Drosophila ID null mutant models G9a (
      • Kramer J.M.
      • Kochinke K.
      • Oortveld M.A.W.
      • Marks H.
      • Kramer D.
      • de Jong E.K.
      • et al.
      Epigenetic regulation of learning and memory by Drosophila EHMT/G9a.
      ) and Synapsin (
      • Sadanandappa M.K.
      • Blanco Redondo B.
      • Michels B.
      • Rodrigues V.
      • Gerber B.
      • VijayRaghavan K.
      • et al.
      Synapsin function in GABA-ergic interneurons is required for short-term olfactory habituation.
      ), and of the classical learning and memory mutant dunce (
      • Asztalos Z.
      • Arora N.
      • Tully T.
      Olfactory jump reflex habituation in drosophila and effects of classical conditioning mutations.
      ,
      • Dudai Y.
      • Jan Y.N.
      • Byers D.
      • Quinn W.G.
      • Benzer S.
      dunce, a mutant of Drosophila deficient in learning.
      ,
      • Tempel B.L.
      • Bonini N.
      • Dawson D.R.
      • Quinn W.G.
      Reward learning in normal and mutant Drosophila.
      ) (Figure 1B–D). This demonstrated that light-off jump habituation upon RNAi can efficiently identify genetic regulators of habituation learning. We also validated the technical accuracy of the automated jump scoring methodology by comparing automated and manually assessed jumping of controls and a number of ID models (Supplemental Methods and Supplemental Figure S1).
      In our screen, we found that the Drosophila orthologs of 98 human ID genes (35% of all investigated orthologs) are required, in neurons, for habituation learning. This phenotype represents a highly specific defect in behavioral adaptation to the stimulus; flies keep on jumping in response to the repetitive light-off stimulus, illustrating that they do not suffer from broad neuronal transmission deficits (which would disable jumping), fatigue, or sensory or other deficiencies. No excessive locomotion was observed when handling the flies, and no stimulus hypersensitivity or random jumping was found (see Supplemental Methods and Supplemental Figures S2 and S3 for validation of light-off jump habituation assay specificity). Of all ID gene orthologs, 27% had no effect on habituation, 41% fell into the category of “non-performers”, and 8% showed “premature habituation” without detectable fatigue. The complete list of habituation screen results and distribution of human ID genes in phenotype categories can be found in Supplemental Tables S2 and S3. The screen thus identified nearly a hundred orthologs of disease genes controlling habituation learning.

      Habituation Deficits Characterize ID Genes With Synaptic Function

      We first asked whether genes characterized by habituation deficits in Drosophila converge on specific biological process. ID genes are known to be enriched in a number of biological processes, but which are important for habituation? Performing an enrichment analysis of ID-enriched gene ontology (GO)–based categories (see Supplemental Methods) against the background of the investigated ID genes, we found that “habituation deficient” genes are significantly enriched in a sole GO-based category: processes related to the synapse (22/44 ID genes, Enrichment = 1.59, p = .024) (Figure 2 and Supplemental Table S4). No enriched GO terms were found in the “not affected” category. Together, our results support the idea that synaptic processes are crucial for habituation, as previously shown for other forms of this behavior (
      • Castellucci V.
      • Pinsker H.
      • Kupfermann I.
      • Kandel E.R.
      Neuronal mechanisms of habituation and dishabituation of the gill withdrawal reflex in Aplysia.
      ,
      • Poon C.-S.
      • Young D.L.
      Nonassociative learning as gated neural integrator and differentiator in stimulus-response pathways.
      ).
      Figure thumbnail gr2
      Figure 2Habituation deficits in Drosophila characterize intellectual disability genes with synapse-related functions. Of 25 gene ontology–based processes, “habituation deficient” genes are specifically and significantly enriched in processes related to synapse (Enrichment = 1.59, p = .024). Genes with no effect on habituation do not show significant enrichment in any gene ontology–based process. *p < .05, based on Fisher’s exact test. All enrichment scores, p values, and enriched genes are listed in . Glut., glutamate; GTPase, guanosine triphosphatase; MAPK, mitogen-activated protein kinase; TOR, target of rapamycin.

      Drosophila Habituation Deficits Characterize ID Genes Associated With Macrocephaly

      To understand whether habituation deficits in Drosophila represent a proxy of specific phenotypes in human individuals, we performed enrichment analysis among ID-associated clinical features (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      ). We found that orthologs of ID genes characterized by habituation deficits in Drosophila are specifically enriched among ID genes associated with macrocephaly and/or overgrowth (Enrichment = 2.19, p = .018) (Figure 3 and Supplemental Table S4). In contrast, ID genes characterized as “non-performers” show enrichment in different, severe ID-associated features such as endocrine, limb and eye anomalies, brain malformations and obesity (Supplemental Figure S5 and Supplemental Table S4). Moreover, ID genes not giving rise to habituation deficits (“not affected” category) did not show any enrichment among ID-associated clinical features (Figure 3 and Supplemental Table S4).
      Figure thumbnail gr3
      Figure 3Habituation deficits in Drosophila characterize intellectual disability genes associated with macrocephaly in humans. Enrichment of Drosophila phenotype categories across 27 intellectual disability–accompanying clinical features
      (
      • Oortveld M.A.W.
      • Keerthikumar S.
      • Oti M.
      • Nijhof B.
      • Fernandes A.C.
      • Kochinke K.
      • et al.
      Human intellectual disability genes form conserved functional modules in Drosophila.
      )
      . “Habituation deficient” genes show specificity for macrocephaly and/or overgrowth (Enrichment = 2.19, p = .018). *p < .05, based on Fisher’s exact test. For enrichment among the “non-performers” category, see . Enrichment scores, p values, and enriched genes are listed in . MRI, magnetic resonance imaging.

      Habituation Deficits Characterize ID Genes Associated With ASD and Deficits in Specific ASD-Relevant Behavioral Domains

      There is a long-known relationship between macrocephaly and ASD (
      • Lainhart J.E.
      • Piven J.
      • Wzorek M.
      • Landa R.
      • Santangelo S.L.
      • Coon H.
      • Folstein S.E.
      Macrocephaly in children and adults with autism.
      ). For this reason and because of the potential relevance of habituation deficits to ASD (
      • Pellicano E.
      • Rhodes G.
      • Calder A.J.
      Reduced gaze aftereffects are related to difficulties categorising gaze direction in children with autism.
      ,
      • Ewbank M.P.
      • Rhodes G.
      • Von Dem Hagen E.A.H.
      • Powell T.E.
      • Bright N.
      • Stoyanova R.S.
      • et al.
      Repetition suppression in ventral visual cortex is diminished as a function of increasing autistic traits.
      ,
      • Swartz J.R.
      • Wiggins J.L.
      • Carrasco M.
      • Lord C.
      • Monk C.S.
      Amygdala habituation and prefrontal functional connectivity in youth with autism spectrum disorders.
      ), we decided to further investigate the relationship of Drosophila habituation and human ASD. We used the Simons Simplex Collection (SSC) (
      • Fischbach G.D.
      • Lord C.
      The Simons Simplex Collection: A resource for identification of autism genetic risk factors.
      ), a genetically and phenotypically well-characterized cohort of individuals with sporadic ASD. We matched genes with likely gene-disrupting and likely damaging de novo mutations (
      • Iossifov I.
      • O’Roak B.J.
      • Sanders S.J.
      • Ronemus M.
      • Krumm N.
      • Levy D.
      • et al.
      The contribution of de novo coding mutations to autism spectrum disorder.
      ,
      • Sanders S.J.
      • He X.
      • Willsey A.J.
      • Ercan-Sencicek A.G.
      • Samocha K.E.
      • Cicek A.E.
      • et al.
      Insights into Autism Spectrum Disorder Genomic Architecture and Biology from 71 Risk Loci.
      ) in this ASD cohort to those included in our experimental Drosophila habituation approach. In all, 47 individuals with ASD carried mutations in 33 of the investigated genes (Supplemental Table S5). We first asked whether these ID plus ASD–associated genes preferentially fall into a specific Drosophila phenotype category. They are significantly enriched among the genes that in Drosophila caused habituation deficits (Enrichment = 1.64, p = .029, ASD SSC) (Figure 4A and Supplemental Table S4). Independently, significant enrichment was obtained for high-confidence ID plus ASD–associated genes identified from the Simons Foundation Autism Research Initiative database (
      • Fischbach G.D.
      • Lord C.
      The Simons Simplex Collection: A resource for identification of autism genetic risk factors.
      ) (38 investigated genes, Enrichment = 1.65, p = .016) (Figure 4B and Supplemental Table S4), suggesting a relationship between Drosophila habituation deficits and human ASD.
      Figure thumbnail gr4
      Figure 4Habituation deficits in Drosophila characterize intellectual disability genes associated with autism spectrum disorder (ASD) and deficits in specific behavioral domains. Enrichment of Drosophila phenotype categories “habituation deficient” and “not affected” in intellectual disability plus ASD–associated genes identified in (A) the Simons Simplex Collection cohort (ASD SSC, Enrichment = 1.64, p = .029) and (B) the Simons Foundation Autism Research Initiative database (ASD SFARI, Enrichment = 1.65, p = .016). Circles represent the total number of tested intellectual disability plus ASD–associated genes. (C) Genes associated with “habituation deficient” vs. “not affected” phenotype categories in Drosophila show a tendency for more aberrant behaviors on the Aberrant Behavior Checklist (ABC) (p = .04) in the ASD SSC cohort. Data are presented as mean score ± SEM. *p < .05, based on multivariate analysis of variance. See also (list of ASD SSC and ASD SFARI genes) and (complete multivariate analysis of variance results). CBCL-Ext, Child Behavior Checklist–Externalizing Disorders; CBCL-Int, Child Behavior Checklist–Internalizing Disorders; SRS, Social Responsiveness Scale.
      To further characterize the relationship between Drosophila habituation and human phenotypes, we divided the SSC individuals into two distinct clusters based on their habituation phenotype in the corresponding fly models: habituation deficits (n = 22 individuals, 17 genes) and no habituation deficits (n = 12 individuals, nine genes) (Supplemental Table S5) (another n = 13 individuals, seven genes fall into the noninformative phenotype groups “non-performers” or “premature habituation”). We compared both groups across five broad quantitative measures of behavior and cognition: cognitive ability (full-scale IQ); Social Responsiveness Scale score; depression and anxiety (Child Behavior Checklist–Internalizing Disorders score); impulsivity, attention, and conduct (Child Behavior Checklist–Externalizing Disorders score); and atypical behavior (Aberrant Behavior Checklist score). There was no significant difference for IQ (p = .61), Social Responsiveness Scale score (p = .62), Child Behavior Checklist–Internalizing Disorders score (p = .59) or Child Behavior Checklist–Externalizing Disorders score (p = .37), but a significant trend for Aberrant Behavior Checklist score was found (p = .04) (Figure 4C and Supplemental Table S6). This effect is mainly driven by the Aberrant Behavior Checklist subdomain of inappropriate, stereotypic speech (p = .0003), not by the subdomains of irritability (p = .1), hyperactivity (p = .86), lethargy (p = .54), or stereotypy (p = .91) (Supplemental Table S6). In summary, these data indicate that habituation deficits in Drosophila are relevant to ASD-implicated genes. They also suggest that SSC individuals carrying de novo mutations in genes associated with habituation deficits in Drosophila show a higher rate and/or severity of atypical behaviors associated with inappropriate and stereotypic speech.

      Molecular Networks and Modules Underlying Habituation

      With the rich repertoire of nearly a hundred genes required for habituation that moreover show specificity for ASD and synapse function, we set out to determine the molecular pathways in which these genes operate. ID gene products are significantly interconnected via protein–protein interactions (
      • Cristino A.S.
      • Williams S.M.
      • Hawi Z.
      • An J.-Y.
      • Bellgrove M.A.
      • Schwartz C.E.
      • et al.
      Neurodevelopmental and neuropsychiatric disorders represent an interconnected molecular system.
      ,
      • Honti F.
      • Meader S.
      • Webber C.
      Unbiased functional clustering of gene variants with a phenotypic-linkage network.
      ). Consistent with previously published findings (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      ), ID genes investigated in our screen are 1.69 times enriched in interactions compared with 1000 randomly chosen protein sets of the same size and number of known interactions [physical interaction enrichment score (
      • Sama I.E.
      • Huynen M.A.
      Measuring the physical cohesiveness of proteins using physical interaction enrichment (PIE).
      ) = 1.69; p < .001]. To identify biologically relevant modules, we resolved this network into communities with even tighter interconnectivity, using unsupervised community clustering (
      • Kalinka A.T.
      • Tomancak P.
      linkcomm: An R package for the generation, visualization, and analysis of link communities in networks of arbitrary size and type.
      ). This analysis resulted in 26 communities containing 109 proteins (Figure 5A and Supplemental Table S7). Their proximity and specificity for ID-enriched, GO-based processes are depicted in Supplemental Figure S6. Mapping “habituation deficient” genes onto the communities highlighted modules with high incidence of habituation deficits (Figure 5A).
      Figure thumbnail gr5
      Figure 5A central role for Ras/mitogen-activated protein kinase (Ras/MAPK) signaling in habituation learning. (A) Highly connected communities are identified by unbiased community clustering, colored by their functional proximity (). Red circles and gene names highlight nodes representing “habituation deficient” genes. For a complete list of communities and genes, see . (B) Nodes connecting four communities from the central module represent the core components of Ras/MAPK signaling. (C) Schematic representation of Ras/MAPK signaling and associated mechanisms in intellectual disability (ID) disorders called Rasopathies. (D) Increasing Ras signaling either by inducing loss of function (LOF) of negative Ras regulators (left side of pathway scheme) or by constitutively activating Ras (GOF, right side) disrupts habituation learning. (Left panel) Defective habituation upon neuron-specific knockdown of negative Ras regulators, Nf1 (2xGMR-wIR/+; Nf1-RNAivdrc35877/elav-Gal4, UAS-Dicer-2, n = 72, in red) and Spred (2xGMR-wIR/+; Spred-RNAivdrc18024/elav-Gal4, UAS-Dicer-2, n = 73, in red) compared with that of their corresponding genetic background controls (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+, n = 55 and n = 20, respectively, in gray). ***padj < .001, based on general linear model analysis and false discovery rate correction in the screen (see Methods and Materials). (Right panel) Defects in habituation learning in a heterozygous, constitutively active Ras mutant (Ras1R68Q/+, n = 55, in green) compared with that of its genetic background control (n = 43, in gray), and upon neuron-specific expression of Ras1R68Q (elav>Ras1R68Q: UAS-Ras1R68Q/2xGMR-wIR; elav-Gal4, UAS-Dicer-2/+, n = 52, in green) compared with that of its genetic background control (2xGMR-wIR/+; elav-Gal4, UAS-Dicer-2/+, n = 34, in gray). ***p < .001, based on general linear model analysis. Data are presented as mean trials to criterion (TTC) ± SEM. GAP, GTPase activating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GOF, gain of function; GTP, guanosine triphosphate; MEK, MAPK/ERK kinase; RNAi, RNA interference.

      A Key Role for ID and ASD–Associated Ras Signaling in Habituation

      Five communities form a large, interconnected module with high incidences of habituation deficits. However, the tightly interconnected hub at the module's center is characterized by the absence of habituation deficits (Figure 5A). This hub represents the key proteins of Ras/mitogen-activated protein kinase (Ras/MAPK) signaling (Figure 5B). This pathway, best known for its role in cancer, underlies a group of disorders collectively referred to as Rasopathies. Importantly, while 92% of the modeled ID disorders are thought to result from loss of function of the underlying genes, Rasopathies are caused by gain-of-function mutations in the core pathway (Figure 5C and Supplemental Table S1). Our RNAi approach, despite addressing gene function, did thus not recapitulate the molecular pathology of these specific cognitive disorders. However, Rasopathies can also result from loss of function in negative regulators of the pathway. We therefore asked whether the same genetic mechanisms that cause Rasopathies in humans also hold true for habituation deficits in Drosophila. In our screen, we tested habituation of two negative regulators of Ras: neurofibromin 1 (Drosophila Nf1) (
      • Martin G.A.
      • Viskoohil D.
      • Bollag G.
      • McCabe P.C.
      • Crosier W.J.
      • Haubruck H.
      • et al.
      The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21.
      ) and Sprouty-related, EVH1 domain–containing protein 1 (Drosophila Spred) (
      • Dunzendorfer-Matt T.
      • Mercado E.L.
      • Maly K.
      • McCormick F.
      • Scheffzek K.
      The neurofibromin recruitment factor Spred1 binds to the GAP related domain without affecting Ras inactivation.
      ,
      • Hirata Y.
      • Brems H.
      • Suzuki M.
      • Kanamori M.
      • Okada M.
      • Morita R.
      • et al.
      Interaction between a domain of the negative regulator of the ras-ERK pathway, SPRED1 protein, and the GTPase-activating protein-related domain of neurofibromin is implicated in legius syndrome and neurofibromatosis type 1.
      ). Panneuronal knockdown of either regulator caused strong habituation deficits (Figure 5D). We therefore tested a constitutively active Ras mutant, Ras1R68Q (
      • Gafuik C.
      • Steller H.
      A gain-of-function germline mutation in drosophila ras1 affects apoptosis and cell fate during development.
      ). Heterozygous Ras1R68Q flies showed strong habituation deficits compared with the control flies with the same genetic background (p = 3.56 × 10−9) (Figure 5D). The same was true when we overexpressed, specifically in neurons, the Ras1R68Q allele from an inducible transgene (p = 1.96 × 10−6) (Figure 5D). We conclude that increased activity of Ras, which causes Rasopathies and associated cognitive deficits in humans, causes habituation deficits in Drosophila.

      Habituation-Inhibiting Function of Increased Ras/MAPK Signaling Maps to Inhibitory/Gamma-aminobuteryic Acidergic Neurons

      We next aimed to identify in which type of neurons the habituation-inhibiting function of Ras/MAPK signaling resides. Because the well-characterized neurons of the giant fiber circuit controlling the light-off jump response are cholinergic (
      • Allen M.J.
      • Murphey R.K.
      The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.
      ), just as the majority of excitatory neurons in Drosophila, we first tested whether increased Ras/MAPK signaling activity would induce habituation deficits when directed to cholinergic neurons. For this, we adopted the knockdown of negative Ras regulators (Nf1, Spred), expressed constitutively active Ras1 (Ras1R68Q), and tested expression of a gain-of-function allele of Raf (RafGOF), a downstream mediator of Ras signaling. None of these, when driven by the cholinergic Cha-Gal4 driver, recapitulated the panneuronally evoked habituation deficits (Figure 6A).
      Figure thumbnail gr6
      Figure 6Dual, opposing roles of Ras/mitogen-activated protein kinase (Ras/MAPK) signaling in gamma-aminobutyric acidergic (GABAergic) and cholinergic neurons in the regulation of habituation learning. (A) No effect on habituation of Ras1R68Q (n = 51, in green), Nf1-RNAi (n = 38, in red), and Spred-RNAi (n = 55, in red) upon expression in cholinergic neurons compared with that of their respective genetic background controls (Cha-Gal4/+; 2xGMR-wIR/+, n = 54, 45, 54, in gray). Expression of RafGOF in cholinergic neurons resulted in lethality. (B) Defective habituation of Ras1R68Q (n = 52, in green), RafGOF (n = 57, in green), Nf1-RNAi (n = 55, in red), and Spred-RNAi (n = 37, in red) on habituation upon expression in GABAergic neurons compared with that of their respective genetic background controls (Gad1-Gal4/+; 2xGMR-wIR/+, n = 50, 50, 39, 58, in gray). (C) Defective habituation of Csw-RNAi (UAS-Csw-RNAivdrc21756/Y; Cha-Gal4/+; 2xGMR-wIR/+, n = 58), Sos1-RNAi (UAS-Sos1-RNAivdrc42848/Cha-Gal4; 2xGMR-wIR/+, n = 56), Ras1-RNAi (UAS-Ras1-RNAivdrc106642/Cha-Gal4; 2xGMR-wIR/+, n = 55), Raf-RNAi (UAS-Raf-RNAivdrc20909/Cha-Gal4; 2xGMR-wIR/+, n = 59), and Mek-RNAi (Cha-Gal4/+; UAS-Mek-RNAivdrc40026/2xGMR-wIR, n = 58) in cholinergic neurons (in green) compared with that of their respective genetic background controls (Cha-Gal4/+; 2xGMR-wIR/+, n = 62, 54, 34, 46, 46, in gray). (D) No effect on habituation of Csw-RNAi (n = 58), Sos1-RNAi (n = 51), Ras1-RNAi (n = 53), Raf-RNAi (n = 52) and Mek-RNAi (n = 54) in GABAergic neurons (in green) compared with that of their respective genetic background controls (Gad1-Gal4/+; 2xGMR-wIR/+, n = 60, 46, 54, 39, 39, in gray). Data presented as mean trials to criterion (TTC) ± SEM. ***p < .001, **p < .01, *p < .05, based on general linear model analysis. RNAi, RNA interference.
      Because of the recently established role of gamma-aminobutyric acidergic (GABAergic) neurons in Drosophila olfactory and proboscis extension reflex habituation (
      • Larkin A.
      • Karak S.
      • Priya R.
      • Das A.
      • Ayyub C.
      • Ito K.
      • et al.
      Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae.
      ,
      • Das S.
      • Sadanandappa M.K.
      • Dervan A.
      • Larkin A.
      • Lee J.A.
      • Sudhakaran I.P.
      • et al.
      Plasticity of local GABAergic interneurons drives olfactory habituation.
      ,
      • Paranjpe P.
      • Rodrigues V.
      • VijayRaghavan K.
      • Ramaswami M.
      Gustatory habituation in Drosophila relies on rutabaga (adenylate cyclase)-dependent plasticity of GABAergic inhibitory neurons.
      ) and the emerging importance of GABA inhibition in ASD (
      • Robertson C.E.
      • Ratai E.M.
      • Kanwisher N.
      Reduced GABAergic action in the autistic brain.
      ), we next targeted GABA neurons using the Gad1-Gal4 driver and the same toolbox. This consistently induced habituation deficits in all tested conditions (Figure 6B). We conclude that the habituation-inhibiting function of increased Ras/MAPK signaling maps to GABAergic neurons.

      Ras/MAPK Signaling in Cholinergic Neurons Is Essential for Habituation Learning

      Impaired jump response and/or increased fatigue associated with Ras, Raf, and Mek knockdown in the screen could potentially mask an essential role for Ras signaling in habituation, in addition to the habituation-inhibiting function of increased Ras/MAPK signaling. In fact, our screen also identified habituation deficits upon RNAi of the positive Ras/MAPK regulators Sos and Csw. We therefore downregulated Ras/MAPK activity by crossing the upstream activation sequence (UAS)–based RNAi lines targeting Sos and Csw, but also RNAi lines targeting Ras, Raf, and Mek, to the GABAergic driver Gad1-Gal4. We did not observe any detrimental effect on habituation (Figure 6D). In contrast, downregulating Ras/MAPK signaling in cholinergic neurons consistently prevented normal habituation learning (Figure 6C). We conclude that Ras/MAPK signaling is essential in cholinergic but not in GABAergic neurons. Thus, Ras/MAPK signaling plays a dual, opposing role in inhibitory versus excitatory neurons in habituation learning.

      Discussion

      A Drosophila Screen Demonstrates That Genes Implicated in ASD Are Important for Habituation Learning

      To systematically address the genetic basis of habituation deficits associated with neurodevelopmental disorders, we investigated 286 ID genes with a clear Drosophila ortholog in light-off jump habituation. Panneuronal knockdown of the orthologs of 98 ID genes specifically suppressed the adaptive habituation response to repeated stimulation without affecting organismal health or jump ability. Follow-up work on the Ras/MAPK pathway raised this number to 104. Of these, 93 are novel regulators of habituation, substantially exceeding the sum of previously known regulators of habituation across species and paradigms. Stringent criteria for RNAi specificity and correction for multiple testing (see Supplemental Methods) in our experiments ensured a minimal level of potential false positive discoveries. Of 13 previously identified ID genes with habituation deficits, our screen confirmed ten (Supplemental Table S8). Our approach and data, although based on experiments in another species, suggest that deficits in habituation learning are a widely affected mechanism in ID. Habituation deficits might be a hallmark of even more ID genes than determined here. In particular, the phenotype category of “non-performers” is likely to contain genes with promiscuous functions masking a specific role in habituation learning.
      Enrichment analysis of ID-associated clinical features revealed that “habituation deficient” ID genes are preferentially characterized by macrocephaly/overgrowth, associated for long with ASD (
      • Lainhart J.E.
      • Piven J.
      • Wzorek M.
      • Landa R.
      • Santangelo S.L.
      • Coon H.
      • Folstein S.E.
      Macrocephaly in children and adults with autism.
      ). Strikingly, we found that mutations in genes associated with Drosophila habituation deficits are significantly overrepresented among ID genes that are also implicated in ASD (52% [SSC cohort]; 53% [Simons Foundation Autism Research Initiative database]). In comparison the frequency of habituation deficits among ID genes not associated with ASD is 24%. SSC individuals carrying mutations in these genes show a high rate and/or severity of aberrant behaviors associated with stereotypic speech. Habituation deficits thus represent a common phenotypic signature of ASD in Drosophila and highlight specific behavioral subdomains affected in ASD. Future work has to establish whether habituation deficits are a direct basis for these clinical features, or are one of many factors involved.

      Synapse-Related Processes and Ras/MAPK Signaling Play a Key Role in Habituation

      Synapse biology has been proposed to play a central role in ASD (
      • Bourgeron T.
      From the genetic architecture to synaptic plasticity in autism spectrum disorder.
      ). Our data show that among the investigated disease genes, “habituation deficient” genes are specifically enriched in genes with synaptic function. This is in line with habituation representing a measurable form of synaptic plasticity (
      • Ramaswami M.
      Network plasticity in adaptive filtering and behavioral habituation.
      ,
      • Poon C.-S.
      • Young D.L.
      Nonassociative learning as gated neural integrator and differentiator in stimulus-response pathways.
      ,
      • Glanzman D.L.
      Common mechanisms of synaptic plasticity in vertebrates and invertebrates.
      ).
      Analyzing the distribution of “habituation deficient” genes in ID-specific molecular interaction networks, we discovered that they accumulate in a multiple-community module and connect to the Ras/MAPK pathway core proteins Ras, Raf, and Mek (Figure 5A, B). We observed habituation deficits upon panneuronal knockdown of Ras negative regulators and panneuronal expression of the constitutively active Ras allele Ras1R68Q (Figure 5C), demonstrating that increased Ras-mediated signaling causes habituation deficits. Moreover, proteins encoded by “habituation deficient” genes form a significantly interconnected module (Figure 7). The coherence of this module further supports the validity of the chosen RNAi approach to identify genes and molecular processes regulating habituation learning. The module contains a number of synaptic proteins (Figure 7) with not-yet-investigated roles in Ras signaling. It would be interesting to determine whether some of these enlarge the spectrum of diseases caused by deregulated Ras signaling.
      Figure thumbnail gr7
      Figure 7Connections between “habituation deficient” genes, including Ras, identified in the reference network used for community clustering (see ) with significantly increased connectivity (physical interaction enrichment [PIE] score = 1.89, p < .001). Nodes are colored based on the community to which they belong. Nodes that represent “habituation deficient” genes but are not members of a community are labeled in black.

      Ras/MAPK Signaling Exerts a Dual but Opposing Role in Inhibitory Versus Excitatory Neurons, a Novel Systems-Level Mechanism

      Identification of neuronal substrates in which specific ID genes are required to warrant habituation learning is an important fundamental problem. Restoring the function of affected neurons might also represent a suitable treatment strategy. The light-off jump startle circuit of Drosophila is relatively simple, and its cholinergic nature is well described (
      • Allen M.J.
      • Murphey R.K.
      The chemical component of the mixed GF-TTMn synapse in Drosophila melanogaster uses acetylcholine as its neurotransmitter.
      ). However, it is not known how habituation of this circuit is regulated. The commonly accepted view regards synaptic depression in excitatory neurons, induced by repetitive stimulation, as the underlying mechanism (
      • Castellucci V.
      • Pinsker H.
      • Kupfermann I.
      • Kandel E.R.
      Neuronal mechanisms of habituation and dishabituation of the gill withdrawal reflex in Aplysia.
      ,
      • Weber M.
      • Schnitzler H.U.
      • Schmid S.
      Synaptic plasticity in the acoustic startle pathway: The neuronal basis for short-term habituation?.
      ). This view has recently been challenged by investigators who showed that plasticity of inhibitory, GABAergic neurons drives two nonstartle types of habituation (
      • Das S.
      • Sadanandappa M.K.
      • Dervan A.
      • Larkin A.
      • Lee J.A.
      • Sudhakaran I.P.
      • et al.
      Plasticity of local GABAergic interneurons drives olfactory habituation.
      ,
      • Paranjpe P.
      • Rodrigues V.
      • VijayRaghavan K.
      • Ramaswami M.
      Gustatory habituation in Drosophila relies on rutabaga (adenylate cyclase)-dependent plasticity of GABAergic inhibitory neurons.
      ). We found that increased activity of our identified key pathway, Ras/MAPK, in GABAergic but not in cholinergic neurons causes deficits in light-off jump habituation. Our results thus support inhibitory circuits as crucial components of habituation learning across different paradigms and sensory modalities. Further experiments are needed to establish the direct involvement of GABAergic signaling. At the same time, we identified that decreased Ras/MAPK signaling activity can also lead to habituation deficits. Yet, the neuronal substrates of these deficits are different and map to excitatory, cholinergic neurons. Although our experiments do not distinguish between developmental effects and acute circuit plasticity, the opposing role for Ras/MAPK signaling on habituation may provide new insights into mechanisms of neural plasticity in health and disease. It may also have crucial implications for treatment of Rasopathies. Future clinical trials, as opposed to those that broadly decreased Ras activity and failed (
      • van der Vaart T.
      • Plasschaert E.
      • Rietman A.B.
      • Renard M.
      • Oostenbrink R.
      • Vogels A.
      • et al.
      Simvastatin for cognitive deficits and behavioural problems in patients with neurofibromatosis type 1 (NF1-SIMCODA): A randomised, placebo-controlled trial.
      ), may need more attention toward restoring circuit function and balance.

      Translational Value and Application of Cross-Species Habituation Measures for Diagnosis and Treatment of ID and ASD

      Based on our findings that habituation is widely affected in Drosophila models of ID and that habituation deficits are particularly enriched among ID genes also implicated in ASD, we propose that disrupted habituation may be one of the mechanisms that contribute to ID/ASD pathology.
      The emerging importance of inhibitory inputs for habituation [Larkin et al. (
      • Larkin A.
      • Karak S.
      • Priya R.
      • Das A.
      • Ayyub C.
      • Ito K.
      • et al.
      Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae.
      ), Das et al. (
      • Das S.
      • Sadanandappa M.K.
      • Dervan A.
      • Larkin A.
      • Lee J.A.
      • Sudhakaran I.P.
      • et al.
      Plasticity of local GABAergic interneurons drives olfactory habituation.
      ), and this study] and sensory information filtering in the cortical centers of the brain (
      • Isaacson J.S.
      • Scanziani M.
      How inhibition shapes cortical activity.
      ,
      • Geramita M.A.
      • Burton S.D.
      • Urban N.N.
      Distinct lateral inhibitory circuits drive parallel processing of sensory information in the mammalian olfactory bulb.
      ) suggests the existence of an overarching circuit-based mechanism responsible for prevention of inappropriate behavioral responses (
      • Ramaswami M.
      Network plasticity in adaptive filtering and behavioral habituation.
      ). Though our findings that habituation deficits in Drosophila correlate with increased rate and/or severity of atypical ASD-related behaviors in humans should be replicated, we speculate that disrupted habituation arising from GABAergic defects may contribute to these ASD features. If future work can establish a substantial contribution of deficits in habituation learning to patient outcomes, cross-species habituation could become an attractive mechanism-specific functional readout—addressing a pressing need for efficient personalized (pharmacological) treatment in the field of neurodevelopmental disorders. Implementing suitable low-burden protocols for habituation measures in clinical research and diagnostics of ID/ASD, such as those developed for investigation of habituation deficits in fragile X syndrome (
      • Ethridge L.E.
      • White S.P.
      • Mosconi M.W.
      • Wang J.
      • Byerly M.J.
      • Sweeney J.A.
      Reduced habituation of auditory evoked potentials indicate cortical hyper-excitability in fragile X syndrome.
      ,
      • Rigoulot S.
      • Knoth I.S.
      • Lafontaine M.-P.
      • Vannasing P.
      • Major P.
      • Jacquemont S.
      • et al.
      Altered visual repetition suppression in fragile X syndrome: New evidence from ERPs and oscillatory activity.
      ), will help to further delineate the affected cognitive domains that may correlate with or arise from deficient habituation. In future clinical trials, these could serve as objective and quantitative readouts for patient stratification in mechanism-based treatment strategies and for monitoring of drug efficacy. Dissection of the underlying defective mechanisms in Drosophila can at the same time identify novel targets for treatment, with high-throughput light-off jump habituation serving as a translational pipeline for drug testing.

      Acknowledgments and Disclosures

      This research was supported in part by the European Union’s FP7 projects TACTICS, OPTIMISTIC, Aggressotype, and MATRICS (Grant Nos. HEALTH-278948, -305697, -602805, and -603016 [to JCG]), the National Science Foundation (Grant No. CBET-1747506 [to CRvR]), the FP7 large-scale integrated network GENCODYS (Grant No. HEALTH-241995 [to ZA, AS]), The Netherlands Organization for Scientific Research (TOP Grant No. 912-12-109 [to AS]), the European Union's Horizon 2020 Marie Skłodowska-Curie European Training Network MiND (Grant No. 643051 [to AS]), the Jérôme Léjeune Foundation (to AS), the Australian National Health & Medical Research Council Centre for Research Excellence Scheme (Grant No. APP1117394 [to AS]), and the U.S. National Institute for Mental Health (Grant No. R01MH101221 [to EEE] and Grant No. R01MH100047 [to RB]). EEE is an investigator of the Howard Hughes Medical Institute.
      We thank Dr. Erika Virágh and Enikő Csapó (Biological Research Centre, Szeged, Hungary) and moreover Dr. Judit Bíró and Márk Péter-Szabó (Voalaz Ltd., Szeged, Hungary) for their contribution to the validation of the Drosophila semiautomated light-off jump reflex habituation paradigm. We acknowledge the Vienna Drosophila Resource Center and Bloomington Drosophila Stock Center (NIH P40OD018537) for providing Drosophila strains. We thank the anonymous expert referees for constructive feedback. We are grateful to all of the families at the participating Simons Simplex Collection (SSC) sites, as well as the principal investigators (A. Beaudet, R. Bernier, J. Constantino, E. Cook, E. Fombonne, D. Geschwind, R. Goin-Kochel, E. Hanson, D. Grice, A. Klin, D. Ledbetter, C. Lord, C. Martin, D. Martin, R. Maxim, J. Miles, O. Ousley, K. Pelphrey, B. Peterson, J. Piggot, C. Saulnier, M. State, W. Stone, J. Sutcliffe, C. Walsh, Z. Warren, and E. Wijsman). We appreciate obtaining access to phenotypic data on Simons Foundation Autism Research Initiative Base. Approved researchers can obtain the SSC population dataset described in this study (http://sfari.org/resources/simons-simplex-collection) by applying at https://base.sfari.org.
      In the past 3 years, JCG has acted as a consultant to Boehringer Ingelheim GmbH but is not an employee, stock- or shareholder of this company. EEE is on the scientific advisory board of DNAnexus, Inc. ZA is a director and shareholder of Aktogen Ltd. LA is a director of Aktogen Ltd. The commercial light-off jump habituation system was purchased from Aktogen Ltd. Aktogen Ltd. provided training of the personnel, and ∼150 experiments from the initial screen were performed at Aktogen Ltd. by MF and LA. All other authors report no biomedical financial interests or potential conflicts of interest.

      Supplementary Material

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      Linked Article

      • Dysfunction of Habituation Learning: A Novel Pathogenic Paradigm of Intellectual Disability and Autism Spectrum Disorder
        Biological PsychiatryVol. 86Issue 4
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          Intellectual disability (ID) and autism spectrum disorder (ASD) are two neurodevelopmental disorders with a prevalence of more than 1.5% in developed countries. ID is characterized by impaired social, cognitive, and adaptive skills and refers to those with an IQ lower than 70; ASD encompasses a spectrum of neurodevelopmental problems, such as functional deficits in communication skills, impaired linguistic and social aptitude, and repetitive or stereotypical behaviors (1). In many cases, a single gene mutation can effectively result in ID.
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