Minelli A, DeBiasi S, Brecha N C, Zuccarello L V, Conti F
Institute of Human Physiology, University of Ancona, Italy.
J Neurosci. 1996 Oct 1;16(19):6255-64. doi: 10.1523/JNEUROSCI.16-19-06255.1996.
The termination of GABA synaptic action by high-affinity, Na(+)-dependent, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathological conditions. We have investigated the cellular localization and distribution in the cerebral cortex of adult rats of one GABA transporter (GAT), GAT-3, by immunocytochemistry with affinity-purified polyclonal antibodies directed to its predicted C terminus that react monospecifically with a protein of approximately 70 kDa. Light microscopic studies revealed specific GAT-3 immunoreactivity (ir) in small punctate structures, and it was never observed in fibers or cell bodies. No changes in immunostaining were observed in sections incubated with GAT-3 antibodies preadsorbed with the related rat GAT-1 or mouse GAT-2/ BGT-1 C-terminal peptides, whereas in sections incubated with GAT-3 antibodies preadsorbed with rat GAT-3 C-terminal peptide, ir was not present. The highest number of GAT-3-positive puncta was in layer IV and in a narrow band corresponding to layer Vb, followed by layers II and III. Many GAT-3-positive puncta were in close association with pyramidal and nonpyramidal neuron cell bodies. Ultrastructural studies showed that GAT-3 ir was localized exclusively to astrocytic processes, which were found in the neuropil and adjacent to axon terminals having either symmetric or asymmetric specializations. In sections processed by both preembedding labeling for GAT-3 and postembedding immunogold labeling for GABA, only some of the GAT-3-positive astrocytic processes were found close to GABAergic profiles. These findings on the localization of GAT-3 in the cerebral cortex indicate that this transporter mediates GABA uptake into glial cells, and suggest that glial GABA uptake may function to limit the spread of GABA from the synapse, as well as to regulate overall GABA levels in the neuropil.
高亲和力、依赖钠离子、存在于神经元和神经胶质细胞膜的转运体对γ-氨基丁酸（GABA）突触作用的终止，在生理和病理条件下调节神经元活动中发挥着重要作用。我们通过免疫细胞化学方法，使用针对预测的C末端的亲和纯化多克隆抗体，对成年大鼠大脑皮质中一种GABA转运体（GAT）即GAT-3进行了细胞定位和分布研究，该抗体与大约70 kDa的蛋白质发生单特异性反应。光学显微镜研究显示，在小的点状结构中有特异性的GAT-3免疫反应性（ir），在纤维或细胞体中从未观察到。用相关大鼠GAT-1或小鼠GAT-2/BGT-1 C末端肽预吸附的GAT-3抗体孵育的切片中，未观察到免疫染色变化，而用大鼠GAT-3 C末端肽预吸附的GAT-3抗体孵育的切片中，不存在ir。GAT-3阳性点状结构数量最多的是IV层和对应于Vb层的窄带，其次是II层和III层。许多GAT-3阳性点状结构与锥体和非锥体神经元细胞体紧密相连。超微结构研究表明，GAT-3 ir仅定位于星形胶质细胞突起，这些突起存在于神经毡中，且与具有对称或不对称特化的轴突终末相邻。在通过GAT-3预包埋标记和GABA包埋后免疫金标记处理的切片中，仅发现一些GAT-3阳性星形胶质细胞突起靠近GABA能结构。这些关于GAT-3在大脑皮质中定位的发现表明，该转运体介导GABA摄取到神经胶质细胞中，并提示神经胶质细胞对GABA的摄取可能起到限制GABA从突触扩散以及调节神经毡中总体GABA水平的作用。
