Simultaneous, Cell-Resolved, Bioluminescent Recording From Microcircuits

微电路同步、细胞解析、生物发光记录

• 批准号: 10294095
• 负责人: Nozomi Nishimura
• 金额: $ 24.6万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10294095
• 关键词: 3-Dimensional; Action Potentials; Address; Adopted; Aequorin; Affect; Axon; Behavioral Paradigm; Bioluminescence; Calcium; Cells; Code; Collection; Color; Complex; Coupled; Data; Dendrites; Dependovirus; Electrodes; Electrophysiology (science); Equilibrium; Fluorescence; Functional Imaging; Genetic; Geometry; Goals; Grant; Head; Image; Individual; Infection; Interneurons; Ion Channel; Knowledge; Label; Learning; Light; LoxP-flanked allele; Maps; Measurement; Measures; Methodology; Methods; Microinjections; Microscope; Microscopy; Modernization; Neurites; Neurons; Neurosciences; Optics; Pattern; Plasmids; Positioning Attribute; Proteins; Rabies; Rabies virus; Scanning; Scheme; Signal Transduction; Structure; Synapses; Techniques; Technology; Tetanus Helper Peptide; Tracer; Viral; Viral Vector; Virus; Work; adeno-associated viral vector; anterograde transport; base; brain volume; calcium indicator; cell type; experimental study; genetic approach; high resolution imaging; improved; instrumentation; light emission; optical fiber; optical imaging; optogenetics; postsynaptic; presynaptic; presynaptic neurons; recombinase; relating to nervous system; retrograde transport; technology development; tool; vector

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.

概括 同时测量许多单个神经元的活动，同时知道其接线图将提供 有关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的能力，改善了许多 通过大规模记录技术，单个神经元指数增加。但是，仍然很困难 结合此类测量。注册高分辨率成像，以追踪神经项目 电生理测量（例如电极阵列）非常困难。使用光学成像，这样 追踪是可能的，但是神经活动测量通常仅限于特定的几何形状，最常见 Z中的一架平面。尽管体积成像的新成像进步具有容易的限制，但 对于大多数实验室来说，复杂的仪器使此类技术无法触及。该提议解决了这个问题 通过使用多色Aequorin荧光蛋白（AEQ-FPS）作为荧光结构示踪剂和 记录钙活性的功能指标。 AEQ-FPS是钙的生物发光指标 从整个细胞中发出光的浓度，包括树突状和轴突轴。在提议中 方案，每个神经元将表达AEQ-FP颜色的独特组合，以使其具有颜色编码以具有其颜色 自己的光谱签名。可以将单个神经元的活性与发射的光谱区分开 生物发光，而无需解决光的起源的空间位置。这可以同时进行 记录许多细胞在任意空间排列中的活性，包括来自不同层的不同层 皮质。通过将AEQ-FPS的表达限制为一个是突触的神经元，可以识别连接的网络 使用经突触病毒载体远离“起动器”细胞（改良的狂犬病逆行和腺 相关病毒（AAVS）用于顺行传输）。每个单元格也表达的独特颜色组合 促进结构追踪。通过这些组合技术，由微电路网络定义 与单个“启动器”单元的连接性将以三个维度追踪，并与活动的测量相关 在一次试验中。在AIM 1中，起动器细胞是网络后的突触，因此此数据将显示如何 涉及多种不同类型的细胞跨皮层层的突触前网络会影响开胃细胞 活动。在AIM 2中，起动器细胞是标记网络的突触前，并将表达通道旋转。 光刺激起动器单元将显示网络活动如何受单个调制的影响 细胞。这种测量功能将使与结构和活动有关的新型实验以及 许多实验室可以很容易地采用。