Gastaldi M, Bartolomei F, Massacrier A, Planells R, Robaglia-Schlupp A, Cau P
Laboratoire de Biologie Cellulaire, Faculté de Médécine, Marseille, France.
Brain Res Mol Brain Res. 1997 Mar;44(2):179-90. doi: 10.1016/s0169-328x(96)00199-4.
Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.
在成年大鼠脑区，对谷氨酸诱导癫痫发作后的钠通道α亚基mRNA的I、II和III亚型进行了分析。从枕叶新皮质、海马CA1和CA3区以及齿状回进行组织样本显微切割。采用三种逆转录聚合酶链反应（RT-PCR）方案扩增这些mRNA。扩增产物经限制性酶消化、电泳分离和凝胶图谱的光密度分析后进行区分。PCR 1证实了mRNA I、II和III以及新生II和III亚型异构体的相对百分比，这些异构体是由选择性剪接产生的。分别进行PCR 2和3以关注II和III亚型中新生型与成年型的比例。结果显示癫痫发作可诱导两种新生亚型增加，这表明在剪接水平发生了改变。这些变化呈现出特殊的脑区分布，在齿状回和海马CA1区观察到最大效应。使用地高辛标记的针对新生II和III mRNA的寡核苷酸探针进行原位杂交实验，证实了新生mRNA亚型的这种增加。这些变化是短暂的，在药物注射后6小时达到最大值，然后在12至48小时之间消失。通过用NMDA受体的非竞争性拮抗剂MK-801对动物进行预处理可防止这些变化。因此，这项研究表明，谷氨酸诱导的癫痫发作可能伴随着钠通道α亚基mRNA剪接模式的短暂改变，这导致成年大鼠脑局部区域内其新生异构体的表达增加。
