Connexins in Neuronal and Glial Gap Junctions in the Central Nervous System

描述(申请人提供)：缝隙连接是微小的细胞间连接，在大脑、视网膜和脊髓的神经细胞之间提供直接的细胞间离子、电和代谢耦合。以前，神经元之间的缝隙连接被认为由数百或数千个连接通道(连接蛋白)组成，但也被认为是罕见的，只出现在几种类型的神经元之间，并且只出现在中枢神经系统的有限的非认知区域。“微型”缝隙连接(&lt；100个连接蛋白)的发现及其在大脑中大量存在的初步证据表明，“微型”缝隙连接，特别是那些位于“混合”(化学+电子)突触的缝隙连接，可能为检测分布在大脑各处的主神经元中微小的亚阈值电“小穗”或“部分棘波”提供结构基础。缝隙连接/部分棘波被认为是调节和优化高频神经元振荡同步的关键，高频神经元振荡同步被认为是意识、睡眠唤醒、认知、学习和记忆的关联绑定以及精细运动控制的基础，并在癫痫、精神分裂症、帕金森病和自闭症谱系障碍中发生病理改变。我们将结合激光扫描共聚焦免疫荧光显微镜和新开发的冷冻骨折复制免疫金标记术(FRIL)电子显微镜来检测、量化、确定小鼠大脑复杂回路中缝隙连接的蛋白质组成和测量其大小，对小鼠和人脑的大脑皮层和海马区的“迷你”缝隙连接进行详细测量，并确定由“迷你”缝隙连接连接的神经元亚型。我们将重点分析那些主要负责思考和意识(大脑皮层)和学习和记忆(海马体)的区域，这些区域代表癫痫放电的主要起源部位。这些互补的方法将允许从大规模(整个小鼠大脑)到超微结构和分子水平的数据直接关联。将获得的数据将是理解意识是如何产生的，睡眠状态和清醒状态的区别，意识在全身麻醉期间是如何改变的，以及记忆是如何创造的，也将是识别亚细胞部位的关键，这些亚细胞部位可能成为设计治疗大脑电突触通讯障碍的新药的靶点。公共卫生相关性一种新发现的“微型”缝隙连接，在许多类别的神经元之间大量存在，可能为神经元之间的微小电子“小穗”的传播提供结构基础，从而调节与人类意识相关的大脑皮层和海马体的振荡同步，并为学习和记忆提供关联绑定。我们将检测、定位、量化和确定小鼠大脑中“迷你”缝隙连接的连接蛋白组成，包括电突触和“混合”(化学+电)突触，并比较小鼠与正常人类大脑皮层和海马区缝隙连接及其组成连接蛋白的分布。这些数据将作为测量癫痫、精神分裂症、帕金森病和自闭症谱系障碍等人类疾病这些结构变化的前奏。

DESCRIPTION (provided by applicant): Gap junctions are microscopic intercellular junctions that provide for direct intercellular ionic, electrical, and metabolic coupling between nerve cells in the brain, retina, and spinal cord. Previously, gap junctions between neurons were thought to consist of hundreds or thousands of connecting channels ("connexons"), but they were also thought to be rare, to occur only between a few types of neurons, and to occur only in limited, non- cognitive areas of the central nervous system. The discovery of "miniature" gap junctions (<100 connexons) and preliminary evidence for their abundance throughout the brain suggests that "mini" gap junctions, particularly those at "mixed" (chemical plus electrical) synapses, may provide the structural basis for the detection of tiny sub-threshold electrical "spikelets" or "partial spikes" in principal neurons that are distributed throughout the brain. Gap junctions / partial spikes are thought to be essential for regulating and optimizing the high-frequency neuronal oscillatory synchronizations that are thought to underlie consciousness, arousal from sleep, cognition, associative binding for learning and memory, and fine motor control, and which become pathologically altered in epilepsy, schizophrenia, Parkinson's disease, and autism spectral disorders. We will combine laser scanning confocal immunofluorescence microscopy with newly-developed freeze-fracture replica immunogold labeling (FRIL) electron microscopy to detect, quantify, determine the protein composition of, and measure the sizes of gap junctions throughout the complex circuitry of the mouse brain, to make detailed measurements of "mini" gap junctions in cerebral cortex and hippocampus of both mouse and human brain, and to identify the neuronal subtypes linked by "mini" gap junctions. We will emphasize analysis of those regions that are primarily responsible for thinking and consciousness (cerebral cortex) and for learning and memory (hippocampus) and that represent the primary sites of origin of epileptic discharges. These complementary approaches will allow direct correlation of data from large-scale (whole mouse brain) to ultrastructural and molecular levels. The data to be obtained will be essential for understanding how consciousness is created, what distinguishes sleeping vs. awake states, how consciousness is altered during general anesthesia, and how memories are created, and will also be essential for identifying subcellular sites that may become targets for designing new drugs to treat disorders of electrical synaptic communication in the brain. PUBLIC HEALTH RELEVANCE A newly-discovered class of "miniature" gap junctions, found to be abundant between many classes of neurons, may provide the structural basis for propagation of small electrical "spikelets" between neurons, and thereby, to regulate the oscillatory synchronizations of cerebral cortex and hippocampus that are associated with human consciousness and for associative binding for learning and memory. We will detect, map, quantify, and determine the connexin protein composition of "mini" gap junctions throughout mouse brain, both at electrical and at "mixed" (chemical plus electrical) synapses, and compare the distributions of gap junctions and their constituent connexins proteins in mouse vs. normal human cerebral cortex and hippocampus. These data will serve as a prelude to measuring changes in these structures in human disease such as epilepsy, schizophrenia, Parkinson's disease, and autism spectral disorders.