Augood S J, Waldvogel H J, Münkle M C, Faull R L, Emson P C
Department of Neurobiology, Babraham Institute, Cambridge, UK.
Neuroscience. 1999 Jan;88(2):521-34. doi: 10.1016/s0306-4522(98)00226-7.
The distribution of messenger RNA encoding the human GAT-1 (a high-affinity GABA transporter) was investigated in the subthalamic nucleus of 10 neurologically normal human post mortem cases. Further, the distribution of messenger RNA and protein encoding the three neuronally expressed calcium-binding proteins (calbindin D28k, parvalbumin and calretinin) was similarly investigated using in situ hybridization and immunohistochemical techniques. Cellular sites of calbindin D28k, parvalbumin, calretinin and GAT-1 messenger RNA expression were localized using human-specific oligonucleotide probes radiolabelled with [35S]dATP. Sites of protein localization were visualized using specific anti-calbindin D28k, anti-parvalbumin and anti-calretinin antisera. Examination of emulsion-coated tissue sections processed for in situ hybridization revealed an intense signal for GAT-1 messenger RNA within the human subthalamic nucleus, indeed the majority of Methylene Blue-counterstained cells were enriched in this transcript. Further, a marked heterogeneity was noted with regard to the expression of the messenger RNA's encoding the three calcium-binding proteins; this elliptical nucleus was highly enriched in parvalbumin messenger RNA-positive neurons and calretinin mRNA-positive cells but not calbindin messenger RNA-positive cells. Indeed, only an occasional calbindin messenger RNA-positive cell was detected within the mediolateral extent of the nucleus. In marked contrast, numerous parvalbumin messenger RNA-positive cells and calretinin messenger RNA-positive cells were detected and they were topographically distributed; parvalbumin messenger RNA-positive cells were highly enriched in the dorsal subthalamic nucleus extending mediolaterally; calretinin messenger RNA-positive cells were more enriched ventrally although some degree of overlap was apparent. Computer-assisted analysis of the average cross-sectional somatic area of parvalbumin, calretinin and GAT-1 messenger RNA-positive neurons revealed them all to be in the range of 300 microm2. The unique patterns of calcium-binding protein gene expression were similarly reflected at the protein level; an abundance of parvalbumin- and calretinin-immunopositive neurons were observed whereas only occasional intensely-labelled calbindin-immunopositive fibres were seen, no calbindin-immunopositive cells were detected. Single and double labelling studies show that parvalbumin-immunopositive neurons were mainly localized in the dorsal region of the nucleus, and calretinin-immunopositive neurons were mainly localized in the ventral region although there was overlap with double-labelled neurons located in the middle and dorsal regions. The significance of these findings, in particular the expression of GAT-1, a high-affinity GABA uptake protein, for basal ganglia signalling is discussed.
在10例神经功能正常的人类尸检病例的丘脑底核中，研究了编码人类GAT-1（一种高亲和力γ-氨基丁酸转运体）的信使核糖核酸的分布。此外，使用原位杂交和免疫组织化学技术，同样研究了编码三种神经元表达的钙结合蛋白（钙结合蛋白D28k、小白蛋白和钙视网膜蛋白）的信使核糖核酸和蛋白质的分布。使用用[35S]dATP放射性标记的人类特异性寡核苷酸探针，对钙结合蛋白D28k、小白蛋白、钙视网膜蛋白和GAT-1信使核糖核酸表达的细胞位点进行定位。使用特异性抗钙结合蛋白D28k、抗小白蛋白和抗钙视网膜蛋白抗血清，使蛋白质定位位点可视化。对经原位杂交处理的乳胶包被组织切片的检查显示，在人类丘脑底核内GAT-1信使核糖核酸有强烈信号，实际上，大多数亚甲蓝复染细胞富含这种转录本。此外，在编码三种钙结合蛋白的信使核糖核酸的表达方面观察到明显的异质性；这个椭圆形核富含小白蛋白信使核糖核酸阳性神经元和钙视网膜蛋白信使核糖核酸阳性细胞，但不含钙结合蛋白信使核糖核酸阳性细胞。实际上，在核的中外侧范围内仅偶尔检测到钙结合蛋白信使核糖核酸阳性细胞。与之形成鲜明对比的是，检测到大量小白蛋白信使核糖核酸阳性细胞和钙视网膜蛋白信使核糖核酸阳性细胞，并且它们呈拓扑分布；小白蛋白信使核糖核酸阳性细胞在丘脑底核背侧高度富集，向中外侧延伸；钙视网膜蛋白信使核糖核酸阳性细胞在腹侧更富集，尽管有一定程度的重叠。对小白蛋白、钙视网膜蛋白和GAT-1信使核糖核酸阳性神经元的平均横截面积进行计算机辅助分析，结果显示它们均在300平方微米范围内。钙结合蛋白基因表达的独特模式在蛋白质水平上也有类似反映；观察到大量小白蛋白和钙视网膜蛋白免疫阳性神经元，而仅偶尔见到强烈标记的钙结合蛋白免疫阳性纤维，未检测到钙结合蛋白免疫阳性细胞。单标记和双标记研究表明，小白蛋白免疫阳性神经元主要位于核的背侧区域，钙视网膜蛋白免疫阳性神经元主要位于腹侧区域，尽管在中侧和背侧区域有双标记神经元存在重叠。讨论了这些发现的意义，特别是高亲和力γ-氨基丁酸摄取蛋白GAT-1的表达对基底神经节信号传导的意义。
