Chapter 13 Synaptic and extrasynaptic GABAA receptor and gephyrin clusters

Christie, Sean B.; Li, Rong-Wen; Miralles, Celia P.; Riquelme, Raquel; Yang, Bih Y; Charych, Erik I.; Yu, Wendou; Daniels, Stephen B.; Cantino, Marie E.; Blas, Angel L. De

doi:10.1016/s0079-6123(02)36015-1

Cited by 43 publications

(66 citation statements)

References 79 publications

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“…The aforementioned data demonstrate that this isoform heterogeneity in individual hippocampal neurons is also observed at the protein level. It is also worth mentioning that with the exception of ␣ 3 , the other studied subunits are highly expressed in the intact hippocampus (Fritschy and Möhler, 1995;Sperk et al, 1997;Miralles et al, 1999;Christie et al, 2002). However, in the intact hippocampus, the ␣ 3 subunit is expressed at very low levels, as shown with various antibodies, including the one used in the present study.…”

Section: Individual Hippocampal Pyramidal Cells and Interneurons In Cmentioning

confidence: 46%

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

2002

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We have studied the effects of GABAergic innervation on the clustering of GABA A receptors (GABA A Rs) in cultured hippocampal neurons. In the absence of GABAergic innervation, pyramidal cells form small (0.36 Ϯ 0.01 m diameter) GABA A R clusters at their surface in the dendrites and soma. When receiving GABAergic innervation from glutamic acid decarboxylase-containing interneurons, pyramidal cells form large (1.62 Ϯ 0.08 m breadth) GABA A R clusters at GABAergic synapses. This is accompanied by a disappearance of the small GABA A R clusters in the local area surrounding each GABAergic synapse. Although the large synaptic GABA A R clusters of any neuron contained all GABA A R subunits and isoforms expressed by that neuron, the small clusters not localized at GABAergic synapses showed significant heterogeneity in subunit and isoform composition. Another difference between large GABAergic and small non-GABAergic GABA A R clusters was that a significant proportion of the latter was juxtaposed to postsynaptic markers of glutamatergic synapses such as PSD-95 and AMPA receptor GluR1 subunit. The densities of both the glutamate receptor-associated and non-associated small GABA A R clusters were decreased in areas surrounding GABAergic synapses. However, no effect on the density or distribution of glutamate receptor clusters was observed. The results suggest that there are local signals generated at GABAergic synapses that induce both assembly of large synaptic GABA A R clusters at the synapse and disappearance of the small GABA A R clusters in the surrounding area. In the absence of GABAergic innervation, weaker GABA A R-clustering signals, generated at glutamatergic synapses, induce the formation of small postsynaptic GABA A R clusters that remain juxtaposed to glutamate receptors at glutamatergic synapses.

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Section: Individual Hippocampal Pyramidal Cells and Interneurons In Cmentioning

confidence: 46%

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

2002

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“…We have previously shown that gephyrin clusters and γ2-GABA A R clusters are already present in these cultures after 3.5 days and that a large number of these clusters are present after 8.5 days in culture (Christie et al 2002a). Fig 3F and G show that although the density of both gephyrin and γ2-GABA A R clusters increased in non-transfected neurons from day 11 to 15, both densities decreased substantially from 1 day to 5 days after gephyrin-EGFP overexpression, suggesting that the formation of new gephyrin and γ2-GABA A R clusters is seriously compromised in addition to the reduction of existing clusters.…”

Section: Overexpression Of Gephyrin-egfp a Fusion Protein That Formsmentioning

confidence: 80%

“…Transfection of pyramidal cells with gephyrin-EGFP, or gephyrin shRNAs was done at day 10 in culture, a time at which many but not all gephyrin and γ2-GABA A Rs clusters are formed in our cultures (Christie et al, 2002a, and legend to Fig 3). We have previously shown that gephyrin clusters and γ2-GABA A R clusters are already present in these cultures after 3.5 days and that a large number of these clusters are present after 8.5 days in culture (Christie et al 2002a).…”

Section: Overexpression Of Gephyrin-egfp a Fusion Protein That Formsmentioning

confidence: 99%

Gephyrin clustering is required for the stability of GABAergic synapses

Jiang

Miralles

et al. 2007

Molecular and Cellular Neuroscience

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Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. We report here that knocking down gephyrin expression with small hairpin RNAs (shRNAs) in cultured hippocampal pyramidal cells decreased both the number of gephyrin and GABA(A) receptor clusters. Similar results were obtained by disrupting the clustering of endogenous gephyrin by overexpressing a gephyrin-EGFP fusion protein that formed aggregates with the endogenous gephyrin. Disrupting postsynaptic gephyrin clusters also had transynaptic effects leading to a significant reduction of GABAergic presynaptic boutons contacting the transfected pyramidal cells. Consistent with the morphological decrease of GABAergic synapses, electrophysiological analysis revealed a significant reduction in both the amplitude and frequency of the spontaneous inhibitory postsynaptic currents (sIPSCs). However, no change in the whole-cell GABA currents was detected, suggesting a selective effect of gephyrin on GABA(A) receptor clustering at postsynaptic sites. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses.

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“…The guinea pig anti-rat ␥2 (to amino acids [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] antibody was raised in our laboratory and affinity-purified on immobilized peptide. The specificity of the guinea pig anti-rat ␥2 antibody has been determined previously (21)(22)(23)(24)(25)(26)(27)(28). The Rb anti-gephyrin antiserum used in immunoblotting was a gift from Dr. Ben Bahr (University of Connecticut) (29), and the mouse monoclonal antibody to gephyrin (clone 7a) used in rat brain immunocytochemistry was from Synaptic Systems (Gottingen, Germany).…”

Section: Methodsmentioning

confidence: 99%

“…Identification Goslin et al (34), as described elsewhere (22,23,35). HEK 293 cells or rat neuroblastoma B103 cells were cultured on poly-L-lysinecoated 18-mm coverslips, as described elsewhere (25,36).…”

Section: Subcellular Fractionation Of Rat Brain and Preparation And Fmentioning

confidence: 99%

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Serwanski

Miralles

et al. 2009

Journal of Biological Chemistry

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Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 ؎ 4% of the GABAergic synapses and 25 ؎ 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.We have recently developed a method for the preparation of a brain fraction enriched in GABAergic postsynaptic complex (1). This fraction, insoluble in Triton X-100, was enriched in Gray's type-II postsynaptic densities (type-II PSDs) 2 and in the postsynaptic GABAergic markers GABA A receptors (GABA A Rs) and gephyrin. Here we report that septin 11 is a major component of the type-II PSD fraction. Septins are a family of proteins with GTPase activity that form heterooligomeric filaments and ringlike structures that act as diffusion barriers and scaffolds. Septins are involved in cytokinesis, positioning of the mitotic spindle, cellular morphology, vesicle trafficking, apoptosis, neurodegeneration, and neoplasia (2-5). In mammals, 14 septin genes have been identified. Each septin gene is expressed in several spliced forms. Although most septins are highly expressed in the brain (6), only recently is their role in neuronal function (7-9) and in neuropathology (10 -14) is beginning to be addressed for some septins.Septin 11 is expressed in various tissues, including the brain (15), but little is known about the role of septin 11 in the brain. Septins 3, 5, 6, and 7 are localized in the presynaptic terminals, frequently associated with synaptic vesicles (6,16,17). In neurons, septin 11 forms heterooligomeric complexes with septin 7 and septin 5 (9, 18). Nevertheless, the regional and developmental distribution of septin 11 in the brain and in hippocampal cultures is not identical to that of septin 7 or septin 5 (8). These result...

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Chapter 13 Synaptic and extrasynaptic GABAA receptor and gephyrin clusters

Cited by 43 publications

References 79 publications

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

Gephyrin clustering is required for the stability of GABAergic synapses

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

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Chapter 13 Synaptic and extrasynaptic GABAA receptor and gephyrin clusters

Cited by 43 publications

References 79 publications

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABAAReceptor Clustering in Cultured Hippocampal Neurons

Gephyrin clustering is required for the stability of GABAergic synapses

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

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GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons

GABAergic Innervation Organizes Synaptic and Extrasynaptic GABA_AReceptor Clustering in Cultured Hippocampal Neurons