activity-dependent tuning of inhibitory neurotransmission based on gaba a r diffusion dynamics...

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