Simultaneous, Cell-Resolved, Bioluminescent Recording From Microcircuits

总结 在知道神经元接线图的情况下，同时测量许多单个神经元的活动， 关于网络组件如何交互的令人兴奋的信息。接线图的知识迅速 由于连接组学领域的进步，以及同时测量许多 单个神经元随着大规模记录技术呈指数增长。然而， 来联合收割机这样的测量。配准用于追踪神经投射的高分辨率成像 诸如电极阵列的电生理测量是极其困难的。通过光学成像， 追踪是可能的，但神经活动测量通常限于特定的几何形状，最常见的是 z轴上的一个平面虽然体积成像的新成像进展已经在一定程度上缓解了这种限制， 复杂的仪器使得这些技术对于大多数实验室来说遥不可及。该提案针对这一点 通过使用水母发光蛋白-荧光蛋白（Aeq-FP）作为荧光结构示踪剂和 记录钙活性的功能指标。Aeq-FP是钙的生物发光指示剂 在一些实施方案中，细胞可以具有从整个细胞（包括树突和轴突乔木）发光的浓度。拟议 方案中，每个神经元将表达Aeq-FP颜色的唯一组合，使得其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。 在不解析光的起源的空间位置的情况下，生物发光。这使得同时 记录任意空间排列的许多细胞的活动，包括来自细胞中不同层的细胞的活动。 皮层通过将Aeq-FP的表达限制在作为一个突触的神经元来识别连接的网络 远离“起始”细胞，使用跨突触病毒载体（用于逆行转运和腺病毒的修饰狂犬病， 相关病毒（AAV）用于顺行运输）。每个单元格中表达的独特颜色组合还 便于结构追踪。通过这些技术的结合， 与单个“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 在一个单一的审判。在Aim 1中，起始细胞是来自网络的突触后，因此这些数据将显示启动细胞是如何从网络中分离出来的。 突触前网络涉及跨皮层的多种不同类型的细胞，影响起始细胞 活动在目标2中，起始细胞与标记的网络突触前，并将表达通道视紫红质。 光刺激起始细胞将显示网络活动如何受到单个细胞的调制的影响。 cell.这种测量能力将使新型的实验，有关结构和活动， 可以被很多实验室采用。

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.