Thalamocortical network dysfunction in a novel genetic model of GRIN2D developmental and epileptic encephalopathy

项目摘要 发育性和癫痫性脑病（DEE）是一组严重的儿童癫痫发作障碍， 一个重要而多样的遗传成分。DEE患者通常会经历显著的癫痫发作负担， 患有认知和发育障碍，以及睡眠和运动障碍。的50 更多携带DEE突变的基因包括“GRIN”基因，它编码 膜蛋白复合物是重要的电化学信号之间的神经元。突变 GRIN 2D亚基引起特别严重和难治性DEE。我们最近开发了一种小鼠模型， GRIN 2D携带突变，在GRIN 2D DEE患者中复发多次。这些老鼠有非常 强烈的癫痫特征，包括已知的惊厥性发作和非惊厥性发作 由大脑皮层和丘脑之间的信号传导调节（即丘脑皮层网络）。这 丘脑皮层网络也与睡眠，意识和许多其他活动的调节有关。 大脑区域。在初步的研究中，我们还注意到Grin 2D的蛋白产物表达于 皮质的兴奋性和抑制性细胞，而丘脑的抑制性细胞中富集。 此外，神经元信号传导的主要部位-突触-在神经元中具有不寻常的结构特征。 皮质，这表明它们没有正常发育。总而言之，这些初步数据激发了 研究Grin 2D突变如何改变皮质和丘脑的功能。在这个试点项目中，我们 将应用电生理学方法来测量GRIN 2D DEE模型中突触的功能， 确定结构变化是否对应于功能障碍。我们还将识别神经元 GRIN 2D DEE中丘脑皮质网络内受影响最严重的连接。aim 1将使用 切片电生理学方法来检查皮质和丘脑内的群体水平反应， 因为这些区域的敏感性产生了类似神经元的活动。我们还将采用创新方法， 解剖皮质中反应的组成部分，这将使我们能够有效地查明 大脑皮层中的许多连接，这些连接很可能被Grin 2D突变所改变。这些网络级 在Aim 2中，将使用突触的细胞内全细胞膜片钳记录来进一步探索这种作用。 水流Aim 2将评估GRIN 2D信号传导的两种不同模式，突触和紧张，这两种模式与GRIN 2D信号传导的神经元功能相关。 具有不同的细胞后果，并且可能参与GRIN 2D DEE致病机制。 这些实验将一起确定GRIN 2D DEE突变对以下活性的影响： 神经回路，这是牵连在这些动物的癫痫发作的产生，并将决定，在 细胞水平，癫痫发生的潜在机制。这项研究是确定 GRIN 2D如何塑造丘脑皮层回路的发育和维持（在正常条件下 以及在DEE模型中），这些信息将指导未来开发靶向矫正疗法的努力。

PROJECT SUMMARY Developmental and epileptic encephalopathy (DEE) is a collection of severe childhood seizure disorders, with a significant and diverse genetic component. DEE patients usually experience a significant seizure burden and suffer from cognitive and developmental impairments, as well as sleep and motor disturbances. The 50 or more genes harboring DEE-causing mutations include the “GRIN” genes, which encode components of membrane protein complexes that are important for electrochemical signaling between neurons. Mutations in the GRIN2D subunit cause particularly severe and intractable DEE. We recently developed a mouse model of GRIN2D carrying a mutation that has recurred several times in GRIN2D DEE patients. These mice have very robust epileptic features, including both convulsive seizures and non-convulsive seizures known to be regulated by signaling between the cerebral cortex and the thalamus (i.e. thalamocortical network). This thalamocortical network is also associated with regulation of sleep, awareness, and the activity of many other brain regions. In preliminary findings, we also noticed that the protein product of Grin2D is expressed in excitatory and inhibitory cells of the cortex, while it is enriched in the inhibitory cells of the thalamus. Additionally, the primary sites of neuronal signaling – the synapse – have unusual structural features in the cortex, suggesting that they do not develop normally. Altogether, these preliminary data motivate an examination of how mutation in Grin2D alters the function of the cortex and thalamus. In this pilot program, we will apply electrophysiological approaches to measure the function of synapses in the GRIN2D DEE model to determine if structural changes correspond to functional impairments. We will also identify the neuronal connections that are most severely affected within the thalamocortical network in GRIN2D DEE. Aim1 will use slice electrophysiology methods to examine population-level responses within the cortex and thalamus to look for susceptibility of these regions to generate seizure-like activity. We will also adapt innovative approaches to dissect the component parts of the response in the cortex, which will allow us to efficiently pinpoint amongst the many connections in the cortex, which are most likely altered by the Grin2D mutation. These network-level effects will be further explored in Aim2 using intracellular whole-cell patch clamp recordings of synaptic currents. Aim2 will assess two distinct modes of GRIN2D signaling, synaptic and tonic, which are associated with different cellular consequences and are likely involved in the GRIN2D DEE pathogenic mechanism. Together these experiments will determine the consequence of the GRIN2D DEE mutation on the activity of neural circuits, which are implicated in the generation of seizures in these animals, and will determine, at the cellular level, the underlying mechanism of epileptogenesis. This study is an important first step for determining how GRIN2D shapes the development and maintenance of thalamocortical circuits (under normal conditions and in a DEE model), information which will guide future efforts to develop targeted corrective therapeutics.