Sonnenberg J L, Frantz G D, Lee S, Heick A, Chu C, Tobin A J, Christakos S
Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark 07103.
Brain Res Mol Brain Res. 1991 Feb;9(3):179-90. doi: 10.1016/0169-328x(91)90001-e.
In order to determine whether calcium binding protein (calbindin-D28k or CaBP) and glutamate decarboxylase (GAD) may be involved in the process underlying the generation of seizure activity, changes in CaBP protein and mRNA and in GAD mRNA were examined in the kindling model of epilepsy. Following amygdaloid (AK) and commissure (CK) kindling significant decreases in the concentration of CaBP of 20% and 30%, respectively, were specifically observed in the hippocampal formation. However, using a cDNA specific to mammalian CaBP, Northern analysis of poly(A+) RNA and slot blot analysis of total RNA revealed no changes in the levels of CaBP mRNA in hippocampus, subcortical area (including amygdala, substantia nigra and striatum) or cerebellum of rats sacrificed 30 min, 1 h, 6 h or 24 h after the last kindled seizure. Similarly when these blots were reprobed with a cDNA specific to mammalian GAD, no changes in GAD gene expression were observed. However, fos gene expression was markedly enhanced at 1 h after seizure. We also tested whether changes in CaBP or GAD mRNA could be detected at any of the various stages of the kindling process. Slot blot analysis of cortex, subcortical structures and hippocampus revealed no changes in CaBP or GAD mRNA during the course of commissure kindling. In situ hybridization studies with GAD and CaBP 35S-labeled antisense probes also indicated no obvious changes upon visual analysis of autoradiographs. However, when silver grains were counted, significant changes in GAD mRNA in individual cells in hippocampus and substantia nigra were noted after kindling induced epilepsy. Our results indicate that, unlike fos gene expression, prominent alterations in GAD and CaBP mRNA in gross brain regions (as measured by slot blot and Northern blot analyses) are not observed in the kindling process. However, our in situ hybridization studies suggest that changes in GAD mRNA in individual cells may be involved in the process underlying kindling induced seizure activity.
为了确定钙结合蛋白（钙结合蛋白-D28k或CaBP）和谷氨酸脱羧酶（GAD）是否可能参与癫痫发作活动产生的潜在过程，我们在癫痫点燃模型中检测了CaBP蛋白和mRNA以及GAD mRNA的变化。在杏仁核（AK）和联合（CK）点燃后，在海马结构中分别特异性观察到CaBP浓度显著降低20%和30%。然而，使用哺乳动物CaBP特异性cDNA，对聚腺苷酸（A+）RNA进行Northern分析以及对总RNA进行狭缝印迹分析发现，在最后一次点燃性癫痫发作后30分钟、1小时、6小时或24小时处死的大鼠海马、皮层下区域（包括杏仁核、黑质和纹状体）或小脑中，CaBP mRNA水平没有变化。同样，当用哺乳动物GAD特异性cDNA重新检测这些印迹时，未观察到GAD基因表达的变化。然而，癫痫发作后1小时fos基因表达明显增强。我们还测试了在点燃过程的任何不同阶段是否能检测到CaBP或GAD mRNA的变化。对皮层、皮层下结构和海马的狭缝印迹分析显示，在联合点燃过程中CaBP或GAD mRNA没有变化。用GAD和CaBP 35S标记的反义探针进行原位杂交研究，在对放射自显影片进行视觉分析时也未显示明显变化。然而，当对银颗粒进行计数时，发现点燃诱导癫痫后海马和黑质单个细胞中的GAD mRNA有显著变化。我们的结果表明，与fos基因表达不同，在点燃过程中未观察到大脑总体区域中GAD和CaBP mRNA的显著改变（通过狭缝印迹和Northern印迹分析测量）。然而，我们的原位杂交研究表明，单个细胞中GAD mRNA的变化可能参与了点燃诱导癫痫发作活动的潜在过程。
