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Activity-dependent tuning of inhibitory neurotransmission based on GABA A R diffusion dynamics Hiroko Bannai, Sabine Lévi, Claude Schweizer, Takafumi Inoue,

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Presentation on theme: "Activity-dependent tuning of inhibitory neurotransmission based on GABA A R diffusion dynamics Hiroko Bannai, Sabine Lévi, Claude Schweizer, Takafumi Inoue,"— Presentation transcript:

1 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 Céline Devroye M2 Neurosciences

2 => occurs at both excitatory and inhibitory synapses What molecular and cellular mecanisms in inhibitory synapses? GABAergic synapse SYNAPTIC STRENGTH INTRODUCTION Modulation of synaptic strength Learning and memory Depends on : Number of functional release sites Release probability Post-synaptic response

3 Neuronal activity surface expression of GABA A R GABAERGIC SYNAPSE INTRODUCTION Hippocampus : inhibitory transmission by GABA and GABA A receptors - Lateral diffusion? - AMPA lat diff are activity dependent - Calcium? Strength of synapse Amount of GABA A R (post synaptic response) Correlation between changes in GABA currents and GABA A R amounts GABA A R GABA Gephyrin Membrane insertion, removal and lateral diffusionnumber of GABA A R at the synapse

4 QUESTION INTRODUCTION Does neuronal activity regulates the diffusion dynamics of GABA A R?

5 Hippocampal neurons (culture) Neuronal activity : Amounts of GABA A R Amplitude of GABA currents Diffusion dynamics of GABA A R EXPERIMENTAL APPROACH INTRODUCTION

6 Immunofluorescence GABA A R γ2 Gephyrin (scaffolding protein) Synapsin (synapse) NEURONAL ACTIVITY - GABA A R AND GEPHYRIN CLUSTER SIZES RESULTS Treatment CNQX + AP5 (blocks AMPAR and NMDAR) NEURONAL ACTIVITY 4AP (blocks potassium channels) NEURONAL ACTIVITY Strychnine + SR95531 (blocks GlyR and GABA A R) NEURONAL ACTIVITY Gephyrin: scaffolding protein of GABA A R

7 GABA A R clustersGephyrin clusters Treatment CNQX + AP5 ( neuronal activity) 4AP ( neuronal activity) St+ SR ( neuronal activity) Increased excitatory activity decreases synaptic GABA A R and gephyrin cluster sizes NEURONAL ACTIVITY - GABA A R AND GEPHYRIN CLUSTER SIZES RESULTS

8 number of synaptic GABA A R Amplitude of mIPSC // 4AP induced hyperactivity TTX + NBQX NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE RESULTS Patch clamp (whole cell) Recording mIPSC SR 95531 : antagonizes GABA A R Depolarizing stimuli decrease GABAergic mIPSC amplitudes

9 Dynasore : membrane-permeable dynamin inhibitor Immunofluorescence : GABA A R γ2 Treatment : 4AP GABAergic mIPSCInternalization of GABA A R? NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE RESULTS No dynamin-dependent internalization of GABA A R

10 No difference between total and surface receptors amounts Biotynilation (to isolate surface receptors) Western blot GABA A R β3 Quantification Depolarizing stimuli decrease GABAergic mIPSC amplitudes without internalization NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE RESULTS Treatment : 4AP Total and surface GABA A R

11 Lateral diffusion and local trapping NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABA A R RESULTS GABA A R GABA Gephyrin Could neuronal activity alterate lateral diffusion? number of synapse receptors

12 Excitatory activity modifies GABA A R trapping at synapses Most have diffusive behavior Treatment CNQX + AP5 ( neuronal activity) 4AP ( neuronal activity) St+ SR ( neuronal activity) GABA A R Anti GABA A R γ2 Quantum dot Biotynilated Fab Streptavidin Single particle tracking (SPT) / of GABA A R => QD GABA A R FM4-64: active synapses GABA A R GABA Gephyrin Synaptic Extra synaptic NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABA A R RESULTS

13 Excitatory activity modifies GABA A R trapping at synapses Most have diffusive behavior NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABA A R RESULTS GABA A R-QD FM4-64 Synaptic Extrasynaptic Surface exploration and trajectories of GABA A R

14 Synaptic dwell time (time a receptor remains in a synapse) and synaptic in-out transitions Synaptic stability of GABA A R is reduced when neuronal activity is increased Synaptic in-out transitions Dwell time NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABA A R RESULTS

15 NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABA A R RESULTS GABA A R-QD FM4-64 (synaptic area) Neuronal activitydiffusion coefficients and confinement of GABA A R? Increased neuronal activity increases diffusion coefficients of GABA A R Diffusion coefficients

16 NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABA A R RESULTS Inhibitory or excitatory synapses? Venus-tagged gephyrin construct Confinement size (surface of lateral diffusion) Increased neuronal activity increases confinement size of GABA A R

17 EGTA : Ca 2+ chelator Neuronal activitychanges in Ca 2+ Ryanodine blocks ryanodine receptors 2APB blocks IP3 receptors NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABA A R RESULTS Ca 2+ entry from the extracellular space is required for 4-AP dependent regulation of GABA A R dynamics Extracellular or intracellular source of Ca 2+ ?

18 CA 2+ entry / NMDARDiffusion dynamics of GABA A R CA 2+ /NMDA receptor reduces GABAergic transmission Activation of NMDA receptors favors the escape of GABA A R from synapses by lateral diffusion Intracellular Ca 2+ Increased diffusion coefficients of GABA A R-QD Increased confinement size of GABA A R-QD NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABA A R RESULTS

19 Calcineurin : Ca2+ sensitive phosphatase Ca 2+ entry / NMDAR Long term depression at GABAergic synapses by activation of phosphatase calcineurin FT Phosphatase calcineurin GABA A R diffusion dynamics NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABA A R RESULTS

20 Cyclosporin-A (CysA) : inhibitor of calcineurin NMDAR-Calcineurin signaling pathway is involved in the activity-dependent regulation of GABA A R diffusion dynamics Phosphatase calcineurin GABA A R diffusion dynamics NEURONAL ACTIVITY / DIFFUSION PROPERTIES OF GABA A R RESULTS

21 CONCLUSIONS Synaptic accumulation and diffusion dynamics of GABA A R are controlled by excitatory activity Increased neuronal activity - GABA A R and gephyrin cluster sizes - mIPSC / GABA A R - diffusion coefficients (synaptic and extrasynaptic) and confinement size, mediated by Ca 2+ entry / NMDAR and calcineurin REGULATION OF TRAPPING AT SYNAPSE

22 CONCLUSIONS Functional implications : During onset of LTP, depression of IPSC enhance the ability of EPSC to elicit action potentials? Activity-dependent regulation of diffusion dynamics of GABA A R LTP?

23 Thank you for your attention

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25 No change in decay time = no channel kinetic changes / treatment Recovering control levels : 10 min // recovering levels of synaptic gephyrin (figure S3) 78 % Patch clamp (whole cell) NEURONAL ACTIVITY / GABAERGIC mIPSC AMPLITUDE RESULTS Depolarizing stimuli GABAergic mIPSC amplitudes

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