activity-dependent tuning of inhibitory neurotransmission based on gaba a r diffusion dynamics...
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Activity-dependent tuning of inhibitory neurotransmission based on GABAAR diffusion
dynamics
Hiroko Bannai, Sabine Lévi, Claude Schweizer, Takafumi Inoue, Thomas Launey, Victor Racine, Jean-Baptiste Sibarita,
Katsuhiko Mikoshiba and Antoine TrillerNeuron, 2009
Céline DevroyeM2 Neurosciences
=> 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
Neuronal activity surface expression of GABAAR
GABAERGIC SYNAPSE
INTRODUCTION
Hippocampus : inhibitory transmission by GABA and GABAA receptors
Strength of synapse
Amount of GABAAR(post synaptic response)
Correlation between changes in GABA currents
and GABAAR amounts
GABAARGABAGephyrin
Membrane insertion, removal and lateral diffusion number of GABAAR at the synapse
QUESTION
INTRODUCTION
Does neuronal activity regulates the diffusion dynamics of GABAAR?
Hippocampal neurons (culture)
Neuronal activity :
Amounts of GABAAR
Amplitude of GABA currents
Diffusion dynamics of GABAAR
EXPERIMENTAL APPROACH
INTRODUCTION
Immunofluorescence• GABAAR γ2 • Gephyrin (scaffolding protein) • Synapsin (synapse)
NEURONAL ACTIVITY - GABAAR 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 GABAAR) NEURONAL ACTIVITY
Gephyrin: scaffolding protein of GABAAR
GABAAR clusters Gephyrin clusters
Treatment • CNQX + AP5 ( neuronal activity) • 4AP ( neuronal activity) • St+ SR ( neuronal activity)
Increased excitatory activity decreases synaptic GABAAR and gephyrin cluster sizes
NEURONAL ACTIVITY - GABAAR AND GEPHYRIN CLUSTER SIZES
RESULTS
number of synaptic GABAAR Amplitude of mIPSC
NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE
RESULTS
Patch clamp (whole cell)Recording mIPSC SR 95531 :
antagonizes GABAAR
Depolarizing stimuli decrease GABAergic mIPSC amplitudes
Dynasore : membrane-permeable dynamin inhibitor
Immunofluorescence : GABAAR γ2
Treatment : 4AP
GABAergic mIPSC Internalization of GABAAR?
NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE
RESULTS
No dynamin-dependent internalization of GABAAR
Biotynilation (to isolate surface receptors)
Western blot GABAAR β3
Quantification
Depolarizing stimuli decrease GABAergic mIPSC amplitudeswithout internalization
NEURONAL ACTIVITY - GABAERGIC mIPSC AMPLITUDE
RESULTS
Treatment : 4AP
Total and surface GABAAR
Lateral diffusion and local trapping
NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR
RESULTS
GABAARGABAGephyrin
Could neuronal activity alterate lateral diffusion?
number of synapse receptors
Treatment • CNQX + AP5 ( neuronal activity) • 4AP ( neuronal activity) • St+ SR ( neuronal activity)
GABAAR
Anti GABAAR γ2
Quantum dot
Biotynilated Fab
Streptavidin
Single particle tracking (SPT) / of GABAAR=> QD GABAAR
FM4-64: active synapses
GABAARGABAGephyrin
SynapticExtra synaptic Extra synaptic
NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR
RESULTS
NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR
RESULTS
GABAAR-QDFM4-64
SynapticExtrasynaptic
Surface exploration and trajectories of GABAAR
Synaptic dwell time (time a receptor remains in a synapse) and synaptic in-out transitions
Synaptic stability of GABAAR is reduced when neuronal activity is increased
Synaptic in-out transitionsDwell time
NEURONAL ACTIVITY - LATERAL DIFFUSION OF GABAAR
RESULTS
NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR
RESULTS
GABAAR-QD
FM4-64 (synaptic area)
Neuronal activity diffusion coefficients and confinement of GABAAR?
Increased neuronal activity increases diffusion coefficients of GABAAR
Diffusion coefficients
NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR
RESULTS
Inhibitory or excitatory synapses?
Venus-tagged gephyrin construct
Confinement size (surface of lateral diffusion)
Increased neuronal activity increases confinement size of GABAAR
EGTA : Ca2+ chelator
Neuronal activity changes in Ca2+
Ryanodine blocks ryanodine receptors
2APB blocks IP3 receptors
NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR
RESULTS
Ca2+ entry from the extracellular space is required for 4-AP dependent regulation of GABAAR dynamics
Extracellular or intracellular source of Ca2+?
CA2+ entry / NMDAR Diffusion dynamics of GABAAR
CA2+ /NMDA receptor reduces GABAergic transmission
Activation of NMDA receptors favors the escape of GABAAR from synapses by lateral diffusion
Intracellular Ca2+Increased diffusion
coefficients of GABAAR-QD
Increased confinement size of GABAAR-QD
NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR
RESULTS
Calcineurin : Ca2+ sensitive phosphatase
Ca2+ entry / NMDAR
Long term depression at GABAergic synapses by activation of phosphatase calcineurin
FT
FT
Phosphatase calcineurin GABAAR diffusion dynamics
NEURONAL ACTIVITY - DIFFUSION PROPERTIES OF GABAAR
RESULTS
Cyclosporin-A (CysA) : inhibitor of calcineurin
NMDAR-Calcineurin signaling pathway is involved in the activity-dependent regulation of GABAAR diffusion dynamics
Phosphatase calcineurin GABAAR diffusion dynamics
NEURONAL ACTIVITY / DIFFUSION PROPERTIES OF GABAAR
RESULTS
CONCLUSIONS
Synaptic accumulation and diffusion dynamics of GABAAR are controlled by excitatory activity
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
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 GABAAR → LTP?
Thank you for your attention
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