Koulen P, Brandstätter J H, Enz R, Bormann J, Wässle H
Max-Planck-Institut für Hirnforschung, Frankfurt, Germany.
Eur J Neurosci. 1998 Jan;10(1):115-27. doi: 10.1046/j.1460-9568.1998.00005.x.
Polyclonal antibodies which recognize the rho-subunits of the GABA(C) receptor were applied to sections of the rat retina. Strong punctate immunoreactivity was found in the inner plexiform layer (IPL), which was shown by electron microscopy to represent a clustering of the GABA(C) receptors at synaptic sites. During postnatal development diffuse rho-immunoreactivity was first observed at postnatal day P3. Distinct labelling of bipolar cells appeared at P7 and punctate, synaptic labelling was observed at P10. In order to show that the rho-immunoreactive puncta coincide with the axons of bipolar cells, double immunostainings of retinal sections with an antiserum against syntaxin 3 and with the rho-antiserum were performed. The experiments showed that rho-immunoreactive puncta are preferentially located on the axon terminals of rod and cone bipolar cells. In order to determine whether GABA(C) receptor rho-subunits coassemble with GABA(A) receptor subunits, double-labelling experiments were performed with subunit specific antisera. Punctate, putative synaptic clustering was observed with all antisera applied, however, GABA(C) receptor expressing puncta did not coincide with GABA(A) receptor containing puncta. This suggests that there are no synaptic GABA receptors in the retina in which GABA(A) and GABA(C) receptor subunits are coassembled. Similar double-labelling experiments were also performed to find out whether GABA(C) receptors and glycine receptors are colocalized. They were clustered at different synapses. This suggests that synaptic GABA(C) receptors consist of rho-subunits and are not coassembled with GABA(A)- or glycine-receptor subunits.
将识别GABA(C)受体rho亚基的多克隆抗体应用于大鼠视网膜切片。在内网状层（IPL）发现了强烈的点状免疫反应性，电子显微镜显示这代表了GABA(C)受体在突触部位的聚集。在出生后发育过程中，弥漫性的rho免疫反应性在出生后第3天首次被观察到。双极细胞的明显标记在出生后第7天出现，点状的突触标记在出生后第10天被观察到。为了表明rho免疫反应性斑点与双极细胞的轴突重合，用抗 syntaxin 3抗血清和rho抗血清对视网膜切片进行了双重免疫染色。实验表明，rho免疫反应性斑点优先位于视杆和视锥双极细胞的轴突末端。为了确定GABA(C)受体rho亚基是否与GABA(A)受体亚基共同组装，用亚基特异性抗血清进行了双重标记实验。使用的所有抗血清都观察到了点状的、假定的突触聚集，然而，表达GABA(C)受体的斑点与含有GABA(A)受体的斑点并不重合。这表明视网膜中不存在GABA(A)和GABA(C)受体亚基共同组装的突触GABA受体。还进行了类似的双重标记实验，以确定GABA(C)受体和甘氨酸受体是否共定位。它们聚集在不同的突触处。这表明突触GABA(C)受体由rho亚基组成，并且不与GABA(A)或甘氨酸受体亚基共同组装。
