Strategy to map electrical synaptic connectivity in neural networks

摘要 电突触，也称为缝隙连接，在包括人类在内的所有神经系统中频繁出现。 大脑。它们由连接蛋白组成，排列成相邻的、耦合的细胞间通道 细胞。连接蛋白36(Cx36)是中枢神经系统中最主要的连接蛋白。在许多大脑和视网膜回路中，缝隙 连接提供了细胞之间的直接和特定的连接。此外，电突触在 网络属性，如信号平均、降噪和同步。然而，由于他们的 小尺寸、间隙连接在大规模连续EM数据集中不可见。出于这些原因，缝隙连接 往往被低估或干脆被忽视。 这项建议的目标是开发一种组合的方法来成像EM中的缝隙连接 此外，还可以评估缝隙连接的大小、强度和可塑性。我们将研究以下区域 视网膜包含大小和形状截然不同的缝隙连接，使我们能够相互联系 结构和功能。AIM 1将使用高分辨率共聚焦显微镜来确定连接子数 大缝隙和小缝隙连接。分析将确定每个缝隙连接的连接外显子的数量。这些 这些方法将提供一种通用的工具来确定所有大脑区域使用的缝隙连接的大小。 AIM 2将使用3D-EM成像来明确识别FIB-SEM图像中的缝隙连接，这 随后将首次对3D-EM结构中的膜结合蛋白进行免疫金定量。这些 研究将允许对已识别神经元中的缝隙连接和蛋白质进行高分辨率量化。目标3将 使用电生理测量来确定耦合电导，然后开发模型来计算 从形态数据乘以最大电势耦合电导 通道/缝隙连接[特定目标1]乘以连接性(耦合的缝隙连接的数量 细胞)[特定目标2]，乘以Cx36的单位电导。使用配对录音，我们将直接获得 耦合细胞间连接电导的生理测量。然后，与 根据形态数据计算的势能极大值，我们可以计算明渠概率和 对工作范围设定现实的限制。这些是理解 缝隙连接在神经元微电路中的作用。 该程序与列出的其中一个领域完全匹配，即识别缝隙连接和特征的工具 电突触“在资助机会公告中，RFA-MH-20-135。

SUMMARY Electrical synapses, also known as gap junctions, occur frequently in all nervous systems, including the human brain. They are composed of connexins, arranged to form intercellular channels between adjacent, coupled cells. Connexin36 (Cx36) is the predominant connexin in the CNS. In many brain and retinal circuits, gap junctions provide direct and specific connections between cells. In addition, electrical synapses mediate network properties such as signal averaging, noise reduction and synchronization. However, because of their small size, gap junctions are not visible in large-scale serial EM data sets. For these reasons, gap junctions tend to be under-reported or simply ignored. The objective of this proposal is to develop a combined approach to image gap junction connectivity in EM datasets and, in addition, to estimate the size, strength, and plasticity of gap junctions. We will study regions of the retina that contain gap junctions of dramatically different sizes and shapes, to allow us to correlate structure and function. Aim 1 will use high-resolution confocal microscopy to determine connexon number at large and small gap junctions. Analyses will determine the number of connexons per gap junction. These methods will provide a general-purpose tool to determine the size of gap junctions for use in all brain regions. Aim 2 will use 3D-EM imaging to allow unambiguous identification of gap junctions in FIB-SEM images, which will follow with first-ever immunogold quantification of a membrane-bound protein in 3D-EM structures. These studies will allow high-resolution quantification of gap junctions and proteins in identified neurons. Aim 3 will use electrophysiological measures to determine coupling conductance and then develop models to calculate the maximal potential coupling conductance from the morphological data by multiplying the number of channels/gap junction [Specific Aim 1] times the connectivity (the number of gap junctions between coupled cells) [Specific Aim 2], times the unitary conductance of Cx36. Using paired recordings, we will obtain direct physiological measures of the junctional conductance between coupled cells. Then, by comparison with the potential maximum calculated from the morphological data, we can calculate the open channel probability and place realistic limits on the operating range. These are the fundamental properties required to understand the function of gap junctions in neuronal microcircuits. This program is an exact match for one of the listed areas, “Tools to identify gap junctions and characterize electrical synapses” in the Funding Opportunity Announcement, RFA-MH-20-135.