Dumoulin A, Lévi S, Riveau B, Gasnier B, Triller A
Laboratoire de Biologie Cellulaire de la Synapse--Inserm U 497, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France.
Eur J Neurosci. 2000 Nov;12(11):3883-92. doi: 10.1046/j.1460-9568.2000.00271.x.
In the spinal cord, GABA and glycine mediate inhibition at separate or mixed synapses containing glycine and/or GABA(A) receptors (GlyR and GABA(A)R, respectively). We have analysed here the sequence of events leading to inhibitory synapse formation during synaptogenesis of embryonic spinal cord neurons between 1 and 11 days in vitro (DIV). We used immunocytochemical methods to detect simultaneously an antigen specific to inhibitory terminals, the vesicular inhibitory amino acid transporter (VIAAT), and one of the following postsynaptic elements: GlyR, GABA(A)R or gephyrin, the anchoring protein of GlyR, which is also associated with GABA(A)R. Quantitative analysis revealed that until 5 DIV most gephyrin clusters were not adjacent to VIAAT-positive profiles, but became associated with them at later stages. In contrast, GlyR and GABAAR clustered predominantly in front of VIAAT-containing terminals at all stages. However, about 10% of receptor aggregates were detected at nonsynaptic loci. The two receptors colocalized in 66.2+/-2.5% of the inhibitory postsynaptic domains after 11 DIV, while 30.3+/-2.6% and 3.4+/-0.8% of them contained only GlyR and GABA(A)R, respectively. Interestingly, at 3 DIV GABA(A)R clustered at a postsynaptic location prior to gephyrin and GlyR; GABA(A)R could thus be the initiating element in the construction of mixed glycine and GABAergic synapses. The late colocalization of gephyrin with GABA(A)R, and the demonstration by other groups that, in the absence of gephyrin, postsynaptic GABA(A)R is not detected, suggest that gephyrin is involved in the stabilization of GABA(A)R rather than in its initial accumulation at synaptic sites.
在脊髓中，γ-氨基丁酸（GABA）和甘氨酸在分别含有甘氨酸和/或GABA A受体（分别为甘氨酸受体和GABA A受体）的单独或混合突触中介导抑制作用。我们在此分析了体外培养1至11天（DIV）的胚胎脊髓神经元突触形成过程中导致抑制性突触形成的一系列事件。我们使用免疫细胞化学方法同时检测抑制性终末特有的一种抗原——囊泡抑制性氨基酸转运体（VIAAT），以及以下突触后元件之一：甘氨酸受体、GABA A受体或桥连蛋白，即甘氨酸受体的锚定蛋白，它也与GABA A受体相关。定量分析显示，直到5 DIV，大多数桥连蛋白簇并不与VIAAT阳性结构相邻，但在后期阶段与它们相关联。相比之下，甘氨酸受体和GABA A受体在所有阶段主要聚集在含有VIAAT的终末前方。然而，在非突触位点检测到约10%的受体聚集体。11 DIV后，两种受体在66.2±2.5%的抑制性突触后结构域中共定位，而其中30.3±2.6%和3.4±0.8%的结构域分别仅含有甘氨酸受体和GABA A受体。有趣的是，在3 DIV时，GABA A受体在桥连蛋白和甘氨酸受体之前聚集在突触后位置；因此，GABA A受体可能是混合甘氨酸能和GABA能突触构建中的起始元件。桥连蛋白与GABA A受体的后期共定位，以及其他研究小组的证明，即在没有桥连蛋白的情况下，未检测到突触后GABA A受体，表明桥连蛋白参与GABA A受体的稳定，而不是其在突触位点的初始聚集。
