Simultaneous, Cell-Resolved, Bioluminescent Recording From Microcircuits

摘要 同时测量许多单个神经元的活动，同时知道它们的接线图将提供 有关网络组件如何交互的令人兴奋的信息。对接线图的了解迅速 由于连接学领域的进步以及同时测量多个 随着大规模记录技术的发展，单个神经元的数量呈指数级增长。然而，这仍然是困难的 把这样的测量结合起来。将用于追踪神经投射的高分辨率成像与 电生理测量，如电极阵列，是极其困难的。通过光学成像，例如 追踪是可能的，但神经活动的测量通常局限于特定的几何形状，最常见的是 Z中的单个平面。尽管体积成像的新成像进展在一定程度上缓解了这一限制， 复杂的仪器设备使这些技术对大多数实验室来说遥不可及。这项提议解决了这个问题 使用多色水通道蛋白荧光蛋白(AEQ-FP)作为荧光结构示踪剂和 用于记录钙活性的功能指示器。AEQ-FP是钙的生物发光指示剂 从包括树突和轴突在内的整个细胞发出光的浓度。在建议的 方案中，每个神经元将表达AEQ-FP颜色的唯一组合，以便它被颜色编码以具有其 自己的光谱特征。单个神经元的活动可以从发射的光谱中区分出来。 生物发光而不解析光原点的空间位置。这使得能够同时 记录任意空间排列中的许多细胞的活动，包括来自 大脑皮层。通过将AEQ-FP的表达限制在作为一个突触的神经元上来识别连接的网络 使用跨突触病毒载体(用于逆行运输和腺病毒的改良狂犬病--译者注)远离“起始”细胞 顺行运输的相关病毒(AAV)。每个单元格中表达的独特颜色组合也 促进结构跟踪。有了这些组合技术，微电路网络由 到单个“起始”细胞的连通性将在三个维度上被追踪，并与活动的测量相关联 在一次审判中。在目标1中，起始细胞是来自网络的突触后细胞，所以这个数据将显示 涉及多个不同类型细胞的跨皮层突触前网络影响起始细胞 活动。在目标2中，起始细胞是标记网络的突触前细胞，并将表达通道视紫红质。 光刺激起始器细胞将显示网络活动如何受单个 手机。这种测量能力将使新类型的实验能够将结构和活动联系起来，并 很容易被许多实验室采用。

Summary Measuring the activity of many individual neurons at once while knowing their wiring diagrams would provide exciting information on how the components of a network interact. Knowledge of wiring diagrams has rapidly improved due to advances in the field of connectomics, and capabilities for simultaneous measurement of many individual neurons has increased exponentially with large-scale recording techniques. However, it is still difficult to combine such measurements. Registering high-resolution imaging for tracing neural projections with electrophysiological measurements, such as electrode arrays, is extremely difficult. With optical imaging, such tracing is possible, but neural activity measurements are often limited to particular geometries, most commonly a single plane in z. Although new imaging advances for volumetric imaging have eased this limitation somewhat, complicated instrumentation puts such technologies out of reach for most labs. This proposal addresses this challenge by using multicolor aequorin-fluorescent proteins (Aeq-FPs) as both fluorescent structural tracers and functional indicators for recording calcium activity. Aeq-FPs are bioluminescent indicators of calcium concentration that emit light from the entire cell including the dendritic and axonal arbors. In the proposed scheme, each neuron will express a unique combination of Aeq-FP colors so that it is color-coded to have its own spectral signature. The activity of individual neurons can be distinguished from the spectrum of the emitted bioluminescence without resolving the spatial position of the origin of the light. This enables simultaneous recording of the activity of many cells in arbitrary spatial arrangements including from different layers in the cortex. Connected networks are identified by limiting expression of the Aeq-FPs to neurons that are one synapse away from “starter” cells using transsynaptic viral vectors (modified rabies for retrograde transport and adeno- associated viruses (AAVs) for anterograde transport). The unique color combinations expressed in each cell also facilitate structural tracing. With these combined technologies, the network of microcircuits defined by connectivity to a single “starter” cell will be traced in three dimensions and correlated to measurements of activity in a single trial. In Aim 1, the starter cell is postsynaptic from the network, so this data will show how the presynaptic network involving multiple different types of cells from across cortical layers affects starter cell activity. In Aim 2, the starter cell is presynaptic to the labeled network and will express channelrhodopsin. Optically stimulating the starter cell will show how the network activity is affected by the modulation of the single cell. Such measurement capabilities will enable new types of experiments relating structure and activity and could be readily adopted by many labs.