Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons

Dumoulin, Andréa; Lévi, Sabine; Riveau, Béatrice; Gasnier, Bruno; Triller, Antoine

doi:10.1046/j.1460-9568.2000.00271.x

Cited by 83 publications

(73 citation statements)

References 57 publications

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“…Under these conditions, the vast majority of the dendritic vege(2) clusters (80.5 Ϯ 3.5%) colocalized with the presynaptic marker FM4-64 (Fig. 2 A), in agreement with previous results on the colocalization of gephyrin with endogenous GlyR clusters or the vesicular transporter protein VIATT at inhibitory synapses (Dumoulin et al, 2000;Bedet et al, 2006;Hanus et al, 2006). We could, therefore, directly image synaptic gephyrin clusters in living neurons, avoiding fixation and immunolabeling of the receptor.…”

Section: Interference With Gephyrin Clustering In Spinal Cord Neuronssupporting

confidence: 90%

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Calamai

Specht²,

Heller³

et al. 2009

Journal of Neuroscience

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149

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High local concentrations of glycine receptors (GlyRs) at inhibitory postsynaptic sites are achieved through their binding to the scaffold protein gephyrin. The N-and C-terminal domains of gephyrin are believed to trimerize and dimerize, respectively, thus contributing to the formation of submembranous gephyrin clusters at synapses. GlyRs are associated with gephyrin also at extrasynaptic locations. We have investigated how gephyrin oligomerization influences GlyR dynamics and clustering in COS-7 cells and in cultured spinal cord neurons. To this aim, we have expressed isolated N-and C-terminal domains of gephyrin that interfere with the oligomerization of the full-length protein. We also studied the effect of an endogenous splice variant, ge(2,4,5), with a decreased propensity to trimerize. A reduction of the size and number of gephyrin-GlyR clusters was found in cells expressing the various interfering gephyrin constructs. Using fluorescence recovery after photobleaching, we studied the exchange kinetics of synaptic gephyrin clusters. Real-time singleparticle tracking was used to analyze the mobility of GlyRs. We found that all the tested constructs displayed faster rates of recovery than wild-type gephyrin and increased the mobility of extrasynaptic receptors, showing that gephyrin-gephyrin interactions modulate the lateral diffusion of GlyRs. Furthermore, we observed an inverse correlation between GlyR diffusion properties and gephyrin cluster size that depended on the number of binding sites blocked by the different constructs. Since alterations in the oligomerization properties of gephyrin are related to the dynamics of GlyRs, the gephyrin splice variant ge(2,4,5) may be implicated in the modulation of synaptic strength.

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Section: Interference With Gephyrin Clustering In Spinal Cord Neuronssupporting

confidence: 90%

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Calamai

Specht²,

Heller³

et al. 2009

Journal of Neuroscience

Self Cite

149

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“…These results support the notion that GlyRs tracked at FM4-64-stained synapses were mainly located at inhibitory synapses. This is consistent with (1) the predominance of inhibitory contacts on spinal cord neurons (Dumoulin et al, 1999) and (2) the low probability of finding GlyRs at excitatory synapses where they are not accumulated (Dumoulin et al, 2000).…”

Section: Distinct Effects Of F-actin and Mictrotubule Disruption On Gsupporting

confidence: 86%

Cytoskeleton Regulation of Glycine Receptor Number at Synapses and Diffusion in the Plasma Membrane

et al. 2006

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Lateral diffusion of neurotransmitter receptors in and out of synapses has been postulated as a core mechanism for rapid changes in receptor number at synapses during plastic processes. In this study, we have used single particle tracking to investigate how changes in glycine receptor (GlyR) lateral diffusion properties might account for changes in receptor number at synapses after disruption of the cytoskeleton in dissociated spinal cord neurons. We found that pharmacological disruption of F-actin and microtubules decreased the amount of GlyR and gephyrin, the backbone of the inhibitory postsynaptic scaffold, at synapses. F-actin and microtubule disruption increased GlyR exchanges between the synaptic and extrasynaptic membranes and decreased receptor dwell time at synapses. GlyR lateral diffusion was predominantly controlled by microtubules in the extrasynaptic membrane and by actin at synapses. Both diffusion coefficients and confinement at synapses were affected after F-actin disruption. Our results indicate that receptor exchanges between the synaptic and extrasynaptic compartments depend on the properties of both the postsynaptic differentiation and the extrasynaptic membrane. Consequently, GlyR number at synapses may be rapidly modulated by the cytoskeleton through the regulation of lateral diffusion in the plasma membrane and of receptor stabilization at synapses.

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“…We were unable to determine whether GABARs or gephyrin localize to synapses first in developing wild-type neurons, because we could consistently detect only both or neither. However, Dumoulin et al (2000) reported in spinal cord neurons that GABA A R localizes to synapses before gephyrin.…”

Section: Role Of Gephyrin In Synaptic Clustering Of Gaba a Rsmentioning

confidence: 99%

Gephyrin Is Critical for Glycine Receptor Clustering But Not for the Formation of Functional GABAergic Synapses in Hippocampal Neurons

Lévi¹,

Logan²,

Tovar³

et al. 2004

J. Neurosci.

193

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The role of the scaffolding protein gephyrin at hippocampal inhibitory synapses is not well understood. A previous study (Kneussel et al., 1999) reported a complete loss of synaptic clusters of the major GABA A R subunits ␣2 and ␥2 in hippocampal neurons lacking gephyrin. In contrast, we show here that GABA A R ␣2 and ␥2 subunits do cluster at pyramidal synapses in hippocampal cultures from gephyrinϪ/Ϫ mice, albeit at reduced levels compared with control neurons. Synaptic aggregation of GABA A R ␣1 on interneurons was identical between the culture types. Furthermore, we recorded miniature IPSCs (mIPSCs) from gephyrinϪ/Ϫ neurons. Although the mean mIPSC amplitude was reduced (by 23%) compared with control, the frequency of these events was unchanged. Cell surface labeling experiments indicated that gephyrin contributes, in part, to aggregation but not to insertion or stabilization of GABA A R ␣2 and ␥2 in the plasma membrane. Thus, a major gephyrin-independent component of hippocampal inhibitory synapse development must exist. We also report that glycine receptors cluster at GABAergic synapses in a subset of hippocampal interneurons and pyramidal neurons. Unlike GABA A Rs, synaptic clustering of glycine receptors was completely abolished in gephyrinϪ/Ϫ neurons. Finally, artificial extrasynaptic aggregation of GABA A R was able to redistribute and cocluster gephyrin by a mechanism requiring a neuron-specific modification or intermediary protein. We propose a model of hippocampal inhibitory synapse development in which some GABA A Rs cluster at synapses by a gephyrinindependent mechanism and recruit gephyrin. This clustered gephyrin may then recruit glycine receptors, additional GABA A Rs, and other signal-transducing components.

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Formation of mixed glycine and GABAergic synapses in cultured spinal cord neurons

Cited by 83 publications

References 57 publications

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Gephyrin Oligomerization Controls GlyR Mobility and Synaptic Clustering

Cytoskeleton Regulation of Glycine Receptor Number at Synapses and Diffusion in the Plasma Membrane

Gephyrin Is Critical for Glycine Receptor Clustering But Not for the Formation of Functional GABAergic Synapses in Hippocampal Neurons

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