Glycine Receptor Clusters Research Articles

Quantal size is correlated with receptor cluster area at glycinergic synapses in the rat brainstem.

1. Whole-cell patch electrode recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were obtained in neurons of the rat anteroventral cochlear nucleus (AVCN). Mean mIPSC peak amplitude was found to vary considerably between AVCN neurons (range, -19.1 to -317.9 pA; mean +/- s.d., -159.1 +/- 100.7 pA; 14 cells). 2. Immunolabelling of glycinergic receptor clusters in AVCN neurons was performed using antibodies against the glycine receptor clustering protein gephyrin. Measurements of the area of gephyrin immunoreactive clusters were obtained using confocal fluorescence microscopy. These measurements showed a large variability in cluster area, not only in the same cell (mean coefficient of variation, c.v., 0.66 +/- 0.18; 16 cells), but also in mean cluster area between cells (range, 0.21-0.84 microm2; 16 cells). 3. A possible relationship between mIPSC amplitude and receptor cluster area was investigated in a further series of experiments, in which mIPSCs recordings and immunolabelling of glycine receptor clusters were obtained for the same cells. In these experiments, AVCN neurons were identified using intracellular labelling with neurobiotin. Successful results using a combination of whole-cell recordings, neurobiotin identification and immunolabelling were obtained for a total of 10 AVCN neurons. Analysis of the results revealed a positive, statistically significant correlation between mean receptor cluster size and mean mIPSC amplitude (P < 0.05, 10 cells, Spearman's correlation test). 4. These results provide direct experimental evidence supporting a hypothesis of central glycinergic transmission in which synaptic strength may be regulated by changes in the size of the postsynaptic receptor region.

Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity.

Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin. Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes. Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues. The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.

Effects of botulinum neurotoxin type A on the expression of gephyrin in cat abducens motoneurons

In this study, we investigated the effects of long-term synaptic blockade on postsynaptic receptor clustering at central inhibitory glycinergic synapses. High doses of botulinum neurotoxin type A injected in the lateral rectus muscle completely abolishes inhibitory postsynaptic potentials onto abducens motoneurons within 2 days postinjection, and transmission remains blocked for at least 2 months. Using this model, we analyzed the expression of gephyrin, a glycine receptor clustering protein, on the membrane of motoneuron somata after botulinum neurotoxin type A injection in their target muscle. Immunofluorescence or electron microscopy immunohistochemistry revealed gephyrin-immunoreactive clusters (most < 0.5 microm in diameter) densely covering the surface of control abducens motoneurons. Ultrastructurally, presynaptic terminals containing flattened synaptic vesicles (F terminals) were found associated with multiple gephyrin-immunoreactive postsynaptic densities (average 1.24 gephyrin clusters/F+ profile). No significant changes in gephyrin-immunoreactive clusters were observed at 5 days postinjection, but we found significant reductions (25-40%) in the density of gephyrin clusters 19 and 35 days postinjection. Hence, the physiological alterations reported in this model precede structural changes on postsynaptic receptor cluster density. The decrease in gephyrin-immunoreactive clusters was paralleled by reductions in synaptic covering (F+ terminals per 100 microm of membrane). Presumed inactive F+ terminals that remained attached to the motoneuron surface displayed normal gephyrin-immunoreactive clusters; however, the pre- and postsynaptic membranes in between synaptic active zones frequently appeared separated by enlarged extracellular spaces. We concluded that postsynaptic receptor cluster dissolution seemed more directly related to terminal retraction than to inactivity alone.

Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters.

The cellular and molecular mechanisms underlying the postsynaptic aggregation of ionotropic receptors in the central nervous system are not understood. The glycine receptor (GlyR) and its cytoplasmic domain-associated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions. The glycine receptor is inhibited by strychnine. The effects of chronic strychnine treatment on the expression and cellular distribution of gephyrin and glycine receptor were therefore tested using primary cultures of spinal cord neurons. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the glycine receptor alpha1, alpha2, beta subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactive cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The proportion of gephyrin and glycine receptor-immunoreactive (IR) cells was unaffected by strychnine treatment. Confocal microscopy revealed that the glycine receptor was mainly localized intracellularly near the nucleus. This cytoplasmic glycine receptor was not associated with the Golgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins. The number of GlyR clusters on the somato-dendritic membrane was dramatically reduced on neurons displaying intracellular staining. In contrast, the subcellular distribution and the number of gephyrin clusters was not modified by the treatment. The fact that gephyrin postsynaptic localization was not modified by strychnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms. The distribution of other cell surface molecules such as NCAM or GABAA receptor beta2/3 subunits was not modified by strychnine treatment. Chronic exposure of the cultures to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation. Taken together, these results indicate that functional glycine receptor, but not electrical synaptic activity, is required for the formation of glycine receptor clusters.

Synaptogenesis in the rat retina: subcellular localization of glycine receptors, GABAA receptors, and the anchoring protein gephyrin

The mechanisms by which neurotransmitter receptors are clustered at postsynaptic sites of neurons are largely unknown. The 93-kDa peripheral membrane protein gephyrin has been shown to be essential for the formation of postsynaptic glycine receptor clusters, and there is now evidence that gephyrin can also be found at gamma-aminobutyric acid (GABA)ergic synapses. In this study, we have analyzed the synaptic localization of glycine receptors, GABA(A) receptors, and the anchoring protein gephyrin in the inner plexiform layer of the developing rat retina, by using immunofluorescence with subunit specific antibodies. At early postnatal stages, the antibodies produced a diffuse staining, suggesting that early retinal neurons can express glycine and GABA(A) receptors. A clustered distribution of the subunits in "hot spots" was also observed. The number of "hot spots" increased during development and reached adult levels in about 2 weeks. Electron microscopy showed that synapses of the conventional type are present in the inner plexiform layer of the postnatal retina and that the hot spots correspond to an aggregation of receptors at postsynaptic sites. Gephyrin was also localized to "hot spots," and double immunofluorescence revealed a colocalization of gephyrin with the alpha2 subunit of the GABA(A) receptor. These results indicate that clustering of receptor subunits occurs in parallel with the formation of morphologically identifiable synaptic specializations and suggest that gephyrin may be involved in clustering of GABA(A) receptors at postsynaptic sites.

Cell-type specific organization of glycine receptor clusters in the mammalian spinal cord

Glycinergic synapses play a major role in shaping the activity of spinal cord neurons. The spatial organization of postsynaptic receptors is likely to determine many functional parameters at these synapses and is probably related to the integrative capabilities of different neurons. In the present study, we have investigated the organization of gephyrin expression along the dendritic membranes of alpha- and gamma-motoneurons, Ia inhibitory interneurons, and Renshaw cells. Gephyrin is a protein responsible for the postsynaptic clustering of glycine receptors, and the features of gephyrin and glycine receptor alpha(1)-subunit immunofluorescent clusters displayed similar characteristics on ventral horn spinal neurons. However, the density of clusters and their topographical organization and architecture varied widely in different neurons and in different dendritic regions. For motoneurons and Ia inhibitory interneurons, cluster size and complexity increased with distance from the soma, perhaps as a mechanism to enhance the influence of distal synapses. Renshaw cells were special in that they displayed an abundant complement of large and morphologically complex clusters concentrated in their somas and proximal dendrites. Serial electron microscopy confirmed that the various immunoreactivity patterns observed with immunofluorescence accurately parallel the variable organization of pre- and postsynaptic active zones of glycinergic synapses. Finally, synaptic boutons from single-labeled axons of glycinergic neurons (Ia inhibitory interneurons) were also associated with postsynaptic receptor clusters of variable shapes and configurations. Our results indicate that mechanisms regulating receptor clustering do so primarily in the context of the postsynaptic neuron identity and localization in the dendritic arbor.

Cell‐type specific organization of glycine receptor clusters in the mammalian spinal cord

Cell‐type specific organization of glycine receptor clusters in the mammalian spinal cord

Selective clustering of GABA(A) and glycine receptors in the mammalian retina

Molecular cloning has revealed a multiplicity of neurotransmitter receptor isoforms with different subunit compositions. Additionally, there is growing evidence that such receptors are clustered at postsynaptic sites of neurons. Thus, the questions arise whether individual neurons express different receptor isoforms and, if so, whether different isoforms are present within the same cluster or are aggregated at distinct postsynaptic sites. We have studied with immunofluorescence methods and antibodies that recognize specific subunits the distribution of glycine and GABA(A) receptors in mammalian retinae. Alpha ganglion cells were injected in rat or rabbit retinae with a fluorescent marker and then immunostained for receptor localization. Clusters of glycine receptors and clusters of the alpha1, and alpha2, alpha3, and gamma2 subunits of the GABA(A) receptor were found on the somatodendritic membranes of Alpha ganglion cells. Double-immunofluorescence experiments with different combinations of the subunit-specific antibodies showed that the alpha1, alpha2, and alpha3 subunits of the GABA(A) receptor are not colocalized within the same clusters. These results indicate that an individual neuron can express several isoforms of the GABA(A) receptor and that these different isoforms are aggregated at distinct postsynaptic sites. This suggests individual sorting mechanisms of GABAa receptors at GABAergic synapses.

The postsynaptic localization of the glycine receptor-associated protein gephyrin is regulated by the cytoskeleton

The mechanisms underlying the postsynaptic localization of neurotransmitter receptors are poorly understood. Recently, the peripheral membrane protein gephyrin has been shown to be essential for the formation of inhibitory glycine receptor clusters in cultured rat spinal cord neurons. In vitro gephyrin binds with high affinity to polymerized tubulin. Here, the interaction of gephyrin with different components of the cytoskeleton was investigated in primary cultures of rat spinal neurons. After treatment with alkaloids affecting the cytoskeleton, the morphology of post-synaptic gephyrin clusters was analyzed by confocal immunofluorescence microscopy. Depolymerization of microtubules by demecolcine reduced both the percentage of cells with postsynaptic gephyrin clusters and the number of clusters/cell. The size of the remaining gephyrin clusters was increased whereas their gephyrin density was significantly lower than under control conditions. Depolymerization of microfilaments by cytochalasin D in contrast generated smaller clusters of increased gephyrin density. Demecolcine also dispersed postsynaptic glycine receptor clusters as revealed by immunostaining with a specific monoclonal antibody. These findings support the view that in vivo gephyrin anchors receptor polypeptides to the cytoskeleton by a complex interaction with microtubules and microfilaments.

The inhibitory glycine receptor: architecture, synaptic localization and molecular pathology of a postsynaptic ion-channel complex

Significant progress has been made towards the identification of functional domains of the inhibitory glycine receptor. Several residues crucial for ligand binding, ion-channel properties and stoichiometric subunit assembly have been identified. A major recent advance has been the finding that the biogenesis of postsynaptic glycine receptor clusters requires the tubulin-binding protein, gephyrin. Another area of exciting research has focused on mutations of glycine receptor α and β subunit genes, which have been found to be causal for different hereditary motor disorders.

Differential distribution of GABA- and serotonin-containing afferents on an identified central neuron

The distributions of γ-aminobutyric acid (GABA)- and serotonin (5-HT)-containing terminals impinging on the surface of the Mauthner (M-) cell were studied at the light microscopic level using double immunofluorescent labeling and were compared with that of the glycine receptor. The latter was visualized indirectly, using a monoclonal mouse antibody which recognizes its 93-kDa associated protein. This neuron has two large principal dendrites: one extending ventrorostrally (ventral dendrite) and the other dorsolaterally (lateral dendrite). There are also two other classes of smaller processes: one that projects ventrally (small ventral dendrites) and one penetrating in the axons cap (cap dendrites), a peculiar neuropil surrounding the initial segment of the M-cell axon. A cellular regionalization of these afferent systems was found: GABA boutons, labeled for glutamic acid decar☐ylase (GAD), were localized preferentially on the lateral dendrite while 5-HT-filled endings predominated on the ventral one. The density of these two classes of inputs was comparable in the other areas of the M-cell: less of their terminals were in contact with the soma outside the axon cap, and more numerous boutons, which presented either GABA or 5-HT immunoreactivities, were apposed to the small ventral dendrites. This preferential pattern of innervation differed with the ubiquitous presence of glycine receptor clusters on the M-cell membrane. Finally no evidence of a colocalization of GABA and 5-HT in afferent endings was detected at any portion of the M-cell. Since the studied networks are involved in the inhibitory control of this neuron, and in its modulation, our results are discussed in relation with possible heterosynaptic interactions between them, and between other similarly segregated excitatory inputs.

INFLUENCE OF DISCHARGE OF MOTONEURONS UPON EXCITATION OF NEIGHBORING MOTONEURONS

INFLUENCE OF DISCHARGE OF MOTONEURONS UPON EXCITATION OF NEIGHBORING MOTONEURONS