Studler Barbara, Sidler Corinne, Fritschy Jean-Marc
Institute of Pharmacology and Toxicology, University of Zurich, CH-8057 Zurich, Switzerland.
J Comp Neurol. 2005 Apr 11;484(3):344-55. doi: 10.1002/cne.20472.
Gephyrin is a postsynaptic scaffolding protein involved in clustering of glycine- and GABA(A) receptors at inhibitory synapses. The role of gephyrin in GABAergic synapses, the nature of its interactions with GABA(A) receptors, and the mechanisms of targeting to GABAergic synapses are largely unknown. To gain further insights into these questions, the formation of GABA(A) receptor and gephyrin clusters and their distribution relative to presynaptic terminals were investigated in immature cultures of embryonic hippocampal neurons using triple immunofluorescence staining. GABA(A) receptor clusters, labeled for the alpha2 subunit, formed independently of gephyrin clusters, and were distributed on neurites at constant densities, either extrasynaptically or, to a lesser extent, postsynaptically, apposed to synapsin-I-positive axon terminals. In contrast, gephyrin clusters were always associated with GABA(A) receptors and were preferentially localized postsynaptically. Their density increased linearly with the extent of innervation, which developed rapidly during the first week in vitro. These results suggested that GABA(A) receptor clustering is mediated by cell-autonomous mechanisms independent of synapse formation. Their association with gephyrin is dynamically regulated and may contribute to stabilization at postsynaptic sites. Labeling for vesicular glutamate transporters revealed that most synapses in these immature cultures were presumably glutamatergic, implying that postsynaptic GABA(A) receptor and gephyrin clusters initially were located in "mismatched" synapses. However, clusters appropriately localized in GABAergic synapses were distinctly larger and more intensely stained. Altogether, these results demonstrate that the targeting of GABA(A) receptor and gephyrin clusters to GABAergic synapses occurs secondarily and is regulated by presynaptic factors that are not essential for clustering.
桥连蛋白是一种突触后支架蛋白，参与抑制性突触处甘氨酸和GABA(A)受体的聚集。桥连蛋白在GABA能突触中的作用、其与GABA(A)受体相互作用的性质以及靶向GABA能突触的机制在很大程度上尚不清楚。为了进一步深入了解这些问题，我们使用三重免疫荧光染色，在胚胎海马神经元的未成熟培养物中研究了GABA(A)受体和桥连蛋白簇的形成及其相对于突触前终末的分布。用α2亚基标记的GABA(A)受体簇独立于桥连蛋白簇形成，并以恒定密度分布在神经突上，要么在突触外，要么在较小程度上在突触后，与突触素-I阳性轴突终末相对。相比之下，桥连蛋白簇总是与GABA(A)受体相关联，并且优先定位在突触后。它们的密度随着神经支配程度呈线性增加，神经支配在体外培养的第一周迅速发展。这些结果表明，GABA(A)受体簇的聚集是由独立于突触形成的细胞自主机制介导的。它们与桥连蛋白的关联是动态调节的，可能有助于在突触后位点的稳定。囊泡谷氨酸转运体的标记显示，这些未成熟培养物中的大多数突触可能是谷氨酸能的，这意味着突触后GABA(A)受体和桥连蛋白簇最初位于“不匹配”的突触中。然而，适当地定位在GABA能突触中的簇明显更大且染色更强烈。总之，这些结果表明，GABA(A)受体和桥连蛋白簇向GABA能突触的靶向是继发发生的，并且受对聚集并非必需的突触前因子调节。
