Akbar M T, Rattray M, Williams R J, Chong N W, Meldrum B S
Department of Clinical Neurosciences, Institute of Psychiatry, London, UK.
Neuroscience. 1998 Aug;85(4):1235-51. doi: 10.1016/s0306-4522(97)00684-2.
The genetically epilepsy-prone rat is an animal model of inherited generalised tonic-clonic epilepsy that shows abnormal susceptibility to audiogenic seizures and a lowered threshold to a variety of seizure-inducing stimuli. Recent studies suggest a crucial role for glutamate and GABA transporters in epileptogenesis and seizure propagation. The present study examines the levels of expression of the messenger RNAs encoding the glial and neuronal glutamate transporters, GLT-1 and EAAC-1, and the neuronal GABA transporter, GAT-1, in paired male genetically epileptic-prone rats and Sprague Dawley control rats using the technique of in situ hybridization. In a parallel study, semiquantitative immunoblotting was used to assess GLT-1 and EAAC-1 protein levels in similarly paired animals. Animals were assessed for susceptibility to audiogenic seizures on six occasions, and killed seven days following the last audiogenic stimulus exposure. Rat brains were processed for in situ hybridization with radioactive 35S-labelled oligonucleotide probes (EAAC-1 and GAT-1), 35S-labelled riboprobes (GLT-1), and Fluorescein-labelled riboprobes (GLT-1 and GAT-1) or processed for immunoblotting using subtype-specific antibodies for GLT-1 and EAAC-1. Semiquantitative analyses were carried out on X-ray film autoradiograms in several brain regions for both in situ hybridization and immunoblotting studies. Reductions in GAT-1 messenger RNA were found in genetically epileptic-prone rats in all brain regions examined (-8 to -24% compared to control). Similar reductions in GLT-1 messenger RNA expression levels were seen in cortex, striatum, and CA1 (-8 to -12%) of genetically epileptic-prone rats; the largest reduction observed was in the inferior colliculus (-20%). There was a tendency for a reduced expression of EAAC-1 messenger RNA in most regions of the genetically epileptic-prone rat brain although this reached statistical significance only in the striatum (-12%). In contrast, no significant differences in GLT-1 and EAAC-1 protein between genetically epileptic-prone rats and control animals were observed in any region examined, although there was a tendency to follow the changes seen with the corresponding messenger RNAs. These results show differences in the messenger RNA expression levels of three crucial amino acid transporters. For the two glutamate transporters, GLT-1 and EAAC-1, differences in messenger RNA levels are not reflected or are only partially reflected in the expression of the corresponding proteins.
遗传性癫痫易感大鼠是遗传性全身性强直阵挛性癫痫的动物模型，对听源性癫痫发作表现出异常易感性，对多种诱发癫痫的刺激阈值降低。最近的研究表明，谷氨酸和γ-氨基丁酸（GABA）转运体在癫痫发生和癫痫传播中起关键作用。本研究采用原位杂交技术，检测了雄性遗传性癫痫易感大鼠和Sprague Dawley对照大鼠配对组中，编码胶质细胞和神经元谷氨酸转运体GLT-1和EAAC-1以及神经元GABA转运体GAT-1的信使核糖核酸（mRNA）的表达水平。在一项平行研究中，使用半定量免疫印迹法评估了相似配对动物中GLT-1和EAAC-1的蛋白质水平。对动物进行六次听源性癫痫发作易感性评估，并在最后一次听源性刺激暴露后七天处死。大鼠脑用于与放射性35S标记的寡核苷酸探针（EAAC-1和GAT-1）、35S标记的核糖探针（GLT-1）以及荧光素标记的核糖探针（GLT-1和GAT-1）进行原位杂交，或使用针对GLT-1和EAAC-1的亚型特异性抗体进行免疫印迹。对原位杂交和免疫印迹研究的几个脑区的X射线胶片放射自显影片进行了半定量分析。在所有检测的脑区中，遗传性癫痫易感大鼠的GAT-1信使核糖核酸均减少（与对照组相比减少8%至24%）。在遗传性癫痫易感大鼠的皮质、纹状体和CA1区，GLT-1信使核糖核酸表达水平也有类似程度的降低（-8%至-12%）；观察到最大降幅的是下丘（-20%）。遗传性癫痫易感大鼠脑的大多数区域中，EAAC-1信使核糖核酸表达有降低趋势，不过仅在纹状体中达到统计学显著性（-12%）。相比之下，在任何检测区域中，遗传性癫痫易感大鼠和对照动物之间的GLT-1和EAAC-1蛋白质均未观察到显著差异，尽管有随相应信使核糖核酸变化的趋势。这些结果显示了三种关键氨基酸转运体信使核糖核酸表达水平的差异。对于两种谷氨酸转运体GLT-1和EAAC-1，信使核糖核酸水平的差异未在相应蛋白质表达中得到反映或仅部分得到反映。
