Brain region-specific epileptogenesis in a conditional mouse model

尽管越来越多的癫痫基因被鉴定出来，并阐明了异源表达系统中致病突变的分子机制，但癫痫是如何在遗传缺陷的基础上发展起来的确切机制仍然知之甚少。神经元和动物模型的功能研究为理解癫痫发生的机制提供了有趣的机会，并可能为新的治疗方案铺平道路。根据这个研究单位(RU)的主要假设，癫痫的发生可能是三个相互作用的过程的结果：基因突变本身的神经生理效应，由突变触发的致痫过程，以及大脑内网络特性的发育调节生理变化。因此，我们打算了解由分子遗传功能障碍引起的级联反应，这种功能障碍触发神经元兴奋性的细胞变化，并随着时间的推移连续影响网络功能，最终导致癫痫发作。我们将使用条件性小鼠模型，在该模型中，我们可以以区域和时间特定的方式打开遗传缺陷，以追求以下目标：(I)使用条件区域特异性突变基因的表达来识别与某些癫痫类型的产生有关的脑区和关键的发育时间窗；我们将从条件Scn1aA1783V小鼠模型开始，在该模型中，我们通过局部注射转导Cre重组酶的病毒来激活突变；(Ii)在相同的模型中，利用电生理学和体内电生理学和钙成像技术，探索具有激活突变的脑区域中抑制和兴奋神经元和回路的贡献和活动模式；(3)利用特定区域和特定时间的突变激活前后的特定细胞RNA-SEQ分析来了解癫痫发生过程中的转录变化，作为(I)和(Ii)的神经生理学研究的补充。此外，我们将(Iv)通过使用膜片钳和细胞外记录来研究在项目P1-3中新检测到的有趣的突变，无论是在转基因/转导的神经细胞培养中还是在Ki小鼠模型的脑片中。

Despite the identification of a steadily increasing number of epilepsy genes and elucidation of molecular mechanisms of disease-causing mutations in heterologous expression systems, the exact mechanisms as to how epilepsy develops on the basis of a genetic defect is poorly understood. Functional studies in neurons and animal models offer interesting opportunities to understand the mechanisms underlying epileptogenesis and may pave the way for novel treatment options. Following the main hypothesis of this Research Unit (RU), epilepsy likely develops as a consequence of three interacting processes: the neurophysiological effect of the genetic mutation itself, an epileptogenic process triggered by the mutation, and developmentally regulated physiological changes of network properties within the brain. Therefore, we intend to understand the cascade caused by a molecular genetic dysfunction that triggers cellular changes of neuronal excitability and consecutively affects network function over time ending in epileptic seizures. We will use conditional mouse models in which we can switch on the genetic defect in a region- and time-specific manner to pursue the following aims: (i) to identify relevant brain regions and critical developmental time windows involved in the generation of certain seizure types using conditional region-specific expression of mutant genes; we will start with a conditional Scn1aA1783V mouse model in which we activate the mutation by local injection of viruses transducing Cre-recombinase; (ii) to explore in the same models the contribution and activity patterns of inhibitory and excitatory neurons and circuits in brain regions with an activated mutation using electrophysiology and Ca2+ imaging in vitro and in vivo; (iii) to understand transcriptional changes during epileptogenesis using cell-specific RNA-seq analyses before and after area- and time-specific activation of the mutation as a complementation of the neurophysiological studies of (i) and (ii). In addition, we will (iv) study interesting mutations that have been newly detected in projects P1-3 by studying their neurophysiological consequences using patch clamping and extracellular recordings either in transfected /transduced neuronal cell cultures or in brain slices from KI mouse models.