Mechanisms of reduced chloride homeostasis in the human epileptogenic neocortex

GABA是中枢神经系统中重要的抑制性神经递质。GABA介导的快速抑制的效果取决于GABA的释放量、受体亚基的密度和亚型，关键是取决于跨膜氯梯度。在过去的几年中，氯调节的干扰在几种神经系统疾病（颞叶癫痫、精神分裂症、神经性疼痛）中得到证实。我们之前对癫痫手术组织切片的研究表明，单个神经元的氯离子稳态明显受损。最突出的转运体是钾偶联氯离子向外转运机制（KCC2），在健康的啮齿动物皮层中占主导地位（占总转运能力的46%）。在单细胞记录中，我们发现这种转运蛋白在人类致癫痫的新皮层中减少；平均约80%。跨膜氯离子运动的其他途径也大大减少（平均NKCC1约为60%，氯离子通道约为80%）。此外，我们的数据表明，功能损伤在不同的组织，甚至在一个组织内表现出显著的神经元间变异性。然而，新皮层中这些下调的精确分子机制尚不清楚。在提交的拨款申请中，我们将使用互补方法进一步研究人体组织中氯稳态的病理生理改变。除了现场和细胞内记录外，我们将使用2光子荧光寿命成像来研究细胞内氯化物的分布。生理特征切片随后将使用RTqPCR， Western blot和免疫组织化学染色进行处理。这些方法的结合能够定位转录，翻译和功能蛋白整合到膜中的干扰。从我们的研究中，我们期望在人类致癫痫新皮层的病理生理改变方面的知识有显著的收获。癫痫发作后的初始紊乱是特别有趣的，因为它们可能是各种癫痫的共同特征。对神经元氯离子调控和功能缺陷分子机制的深入了解，为设计合理的因果治疗策略以减轻癫痫患者的负担开辟了新的途径。

GABA is the key inhibitory neurotransmitter in the central nervous system. The efficacy of the fast GABA-mediated inhibition depends upon the amount of GABA being released, density and subtypes of receptor subunits and crucially upon the transmembranal chloride gradient. During the last few years disturbances of chloride regulation were demonstrated in several neurological disorders (temporal lobe epilepsy, schizophrenia, neuropathic pain).Our previous studies on slices from epilepsy surgery tissues revealed a marked impairment of chloride homeostasis of individual neurones. The most prominent transporter, a potassium coupled chloride outward transport mechanism (KCC2), dominates in the healthy rodent cortex (46 % of the total transport capacity). In single cell recordings we found this transporter being reduced in the human epileptogenic neocortex; on average by about 80 %. Further routes of transmembrane chloride movement are considerably reduced as well (on average NKCC1 about 60 %, chloride channels about 80 %). In addition, our data indicate that the functional impairments in different tissues and even within a given tissue exhibit a marked interneuronal variability. The precise molecular mechanisms of these downregulations in the neocortex are unsolved, though. In the submitted grant proposal we will further investigate the pathophysiological alterations of chloride homeostasis is human tissues using complementary methods. In addition to field and intracellular recordings, we will investigate the distribution of intracellular chloride using 2 photon fluorescence lifetime imaging. The physiologically characterized slices will subsequently be processed using RTqPCR, Western blot und immunohistochemical staining. The combination of methods enables a localization of the disturbance between transcription, translation and integration of functional proteins into the membrane. From our studies we expect a marked gain in knowledge regarding pathophysiological alterations in the human epileptogenic neocortex. The initial disturbances after seizures are of particular interest as they may present the common denominator of various epilepsies. The indepth understanding of neuronal chloride regulation and of the molecular mechanisms underlying functional deficits open new venues for designing rational causal therapeutic strategies to alleviate the burden of epilepsy patients.