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Céline Devroye M2 Neurosciences

Activity - dependent tuning of inhibitory neurotransmission based on GABA A R diffusion dynamics. Hiroko Bannai, Sabine Lévi, Claude Schweizer, Takafumi Inoue , Thomas Launey , Victor Racine, Jean-Baptiste Sibarita , Katsuhiko Mikoshiba and Antoine Triller Neuron , 2009.

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Céline Devroye M2 Neurosciences

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  1. Activity-dependenttuning of inhibitory neurotransmission based on GABAAR diffusion dynamics Hiroko Bannai, Sabine Lévi, Claude Schweizer, TakafumiInoue, Thomas Launey, Victor Racine, Jean-Baptiste Sibarita, KatsuhikoMikoshiba and Antoine Triller Neuron, 2009 Céline Devroye M2 Neurosciences

  2. INTRODUCTION SYNAPTIC STRENGTH Modulation of synapticstrength Learning and memory Depends on : • Number of functional release sites • Release probability • Post-synapticresponse => occursatbothexcitatory and inhibitorysynapses Whatmolecular and cellular mecanismsin inhibitory synapses? GABAergic synapse

  3. INTRODUCTION GABAERGIC SYNAPSE Hippocampus : inhibitory transmission by GABA and GABAAreceptors GABAAR GABA Gephyrin Amount of GABAAR (post synapticresponse) Strength of synapse Correlationbetween changes in GABA currents and GABAAR amounts - Lateral diffusion? - AMPA latdiff are activitydependent - Calcium? Membrane insertion, removal and lateral diffusion number of GABAAR at the synapse Neuronal activity surface expression of GABAAR

  4. INTRODUCTION QUESTION Does neuronal activityregulates the diffusion dynamics of GABAAR?

  5. INTRODUCTION EXPERIMENTAL APPROACH • Hippocampalneurons (culture) • Neuronal activity : • Amounts of GABAAR • Amplitude of GABA currents • Diffusion dynamics of GABAAR

  6. RESULTS NEURONAL ACTIVITY - GABAAR AND GEPHYRIN CLUSTER SIZES Gephyrin: scaffoldingprotein of GABAAR Immunofluorescence • GABAAR γ2 • Gephyrin (scaffoldingprotein) • Synapsin (synapse)  Treatment • CNQX + AP5 (blocks AMPAR and NMDAR)  NEURONAL ACTIVITY • 4AP (blocks potassium channels)  NEURONAL ACTIVITY • Strychnine + SR95531 (blocks GlyR and GABAAR)  NEURONAL ACTIVITY

  7. RESULTS NEURONAL ACTIVITY - GABAAR AND GEPHYRIN CLUSTER SIZES  Treatment • CNQX + AP5 (  neuronal activity) • 4AP (  neuronal activity) • St+ SR (  neuronal activity) Gephyrin clusters GABAAR clusters Increasedexcitatoryactivity decreasessynaptic GABAAR and gephyrin cluster sizes

  8. RESULTS NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE numberof synapticGABAAR Amplitude of mIPSC // 4AP inducedhyperactivity  Patch clamp (wholecell) RecordingmIPSC SR 95531 : antagonizes GABAAR TTX + NBQX  Depolarizing stimuli decreaseGABAergicmIPSC amplitudes

  9. RESULTS NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE  GABAergicmIPSC Internalizationof GABAAR? Immunofluorescence : GABAAR γ2 Treatment:4AP Dynasore : membrane-permeabledynamininhibitor  No dynamin-dependentinternalization of GABAAR

  10. RESULTS NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE Total and surface GABAAR Treatment:4AP • Biotynilation (to isolate surface receptors) • Western blot GABAAR β3 No differencebetween total and surface receptorsamounts Quantification Depolarizing stimuli decreaseGABAergicmIPSCamplitudes withoutinternalization

  11. RESULTS NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR Lateral diffusion and local trapping number of synapse receptors GABAAR GABA Gephyrin Could neuronal activityalteratelateral diffusion?

  12. RESULTS NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR Single particletracking (SPT) / of GABAAR => QD GABAAR GABAAR GABA Gephyrin  FM4-64: active synapses Excitatoryactivity modifies GABAAR trappingat synapses Most have diffusive behavior Streptavidin Quantum dot Extra synaptic Synaptic Extra synaptic BiotynilatedFab Anti GABAAR γ2  Treatment • CNQX + AP5 ( neuronal activity) • 4AP ( neuronal activity) • St+ SR ( neuronal activity) GABAAR

  13. RESULTS NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR Surface exploration and trajectories of GABAAR Excitatoryactivity modifies GABAAR trappingat synapses GABAAR-QD FM4-64 Most have diffusive behavior Synaptic Extrasynaptic

  14. RESULTS NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR Synapticdwell time (time a receptorremains in a synapse) and synaptic in-out transitions Dwell time Synaptic in-out transitions Synapticstabilityof GABAARisreducedwhen neuronal activityisincreased

  15. RESULTS NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR Neuronal activity diffusion coefficients and confinement of GABAAR? GABAAR-QD  FM4-64 (synaptic area) Diffusion coefficients  Increased neuronal activityincreases diffusion coefficients of GABAAR

  16. RESULTS NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR Inhibitory or excitatory synapses?  Venus-taggedgephyrinconstruct Confinement size (surface of lateral diffusion)  Increased neuronal activityincreases confinement size of GABAAR

  17. RESULTS NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR Neuronal activity changes in Ca2+ Extracellular or intracellular source of Ca2+? EGTA : Ca2+chelator Ryanodine blocks ryanodinereceptors 2APB blocks IP3 receptors  Ca2+ entry from the extracellularspaceisrequired for 4-AP dependentregulation of GABAAR dynamics

  18. RESULTS NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR CA2+ /NMDA receptorreducesGABAergic transmission CA2+ entry / NMDAR Diffusion dynamics of GABAAR Increased diffusion coefficients of GABAAR-QD Increased confinement size of GABAAR-QD Intracellular Ca2+ Activation of NMDA receptorsfavors the escape of GABAAR from synapses by lateral diffusion

  19. RESULTS NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR Ca2+ entry / NMDAR Long termdepressionatGABAergic synapses by activation of phosphatase calcineurin FT Calcineurin : Ca2+ sensitive phosphatase FT Phosphatase calcineurin GABAAR diffusion dynamics

  20. RESULTS NEURONAL ACTIVITY / DIFFUSION PROPERTIES OF GABAAR Phosphatase calcineurin GABAAR diffusion dynamics Cyclosporin-A (CysA) : inhibitor of calcineurin NMDAR-Calcineurinsignalingpathwayisinvolved in the activity-dependentregulation of GABAAR diffusion dynamics

  21. CONCLUSIONS Synaptic accumulation and diffusion dynamicsof GABAAR are controlled by excitatoryactivity Increased neuronal activity - ↘ GABAAR and gephyrin cluster sizes - ↘ mIPSC/ GABAAR - ↗ diffusion coefficients(synaptic and extrasynaptic) and confinement size, mediated by Ca2+ entry / NMDAR and calcineurin REGULATION OF TRAPPING AT SYNAPSE

  22. CONCLUSIONS Functional implications : Duringonset of LTP, depression of IPSC → enhance the ability of EPSC to elicit action potentials? Activity-dependentregulation of diffusion dynamics of GABAAR → LTP?

  23. Thankyou for your attention

  24. RESULTS NEURONAL ACTIVITY / GABAERGIC mIPSC AMPLITUDE  Patch clamp (wholecell) ↘ 78 % Recovering control levels : 10 min // recoveringlevels of synapticgephyrin (figure S3) No change in decay time = no channelkinetic changes / treatment  Depolarizing stimuli ↘ GABAergicmIPSC amplitudes

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