Mechanisms of reduced chloride homeostasis in the human epileptogenic neocortex

GABA是中枢神经系统中的关键抑制性神经递质。快速的GABA介导的抑制的效果取决于GABA的释放量、受体亚基的密度和亚型，最重要的是取决于跨膜氯的梯度。在过去的几年中，氯离子调节的障碍被证实存在于几种神经系统疾病(颞叶癫痫、精神分裂症、神经病理性疼痛)中。我们先前对癫痫手术组织切片的研究发现，单个神经元的氯离子稳态显著受损。最重要的转运体，钾偶联氯离子向外转运机制(KCC2)，在健康啮齿动物皮质中占主导地位(占总转运量的46%)。在单细胞记录中，我们发现这种转运蛋白在人类致痫的新皮质中减少了；平均减少了大约80%。进一步的跨膜氯迁移途径也大大减少(平均NKCC1约60%，氯通道约80%)。此外，我们的数据表明，不同组织甚至特定组织内的功能损害表现出明显的神经元间变异性。然而，大脑皮层这些下调的确切分子机制仍未解决。在提交的拨款提案中，我们将使用补充方法进一步研究人体组织氯离子动态平衡的病理生理变化。除了现场记录和细胞内记录外，我们还将使用双光子荧光寿命成像来研究细胞内氯的分布。生理特征切片随后将使用RTqPCR、Western印迹和免疫组织化学染色进行处理。这两种方法的结合使得能够定位转录、翻译和功能蛋白整合到膜中之间的干扰。从我们的研究中，我们期望在人类致痫新皮质的病理生理学改变方面的知识有显着的增长。癫痫发作后的最初干扰特别令人感兴趣，因为它们可能是各种癫痫的共同点。对神经元氯化物调节和潜在功能缺陷的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场所。

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.