Simultaneous, Cell-Resolved, Bioluminescent Recording From Microcircuits

微电路同步、细胞解析、生物发光记录

基本信息

批准号：
10463819
负责人：
Nozomi Nishimura
金额：
$ 24.6万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10463819
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

项目摘要

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，同时了解它们的接线图将提供关于网络组件如何交互的令人兴奋的信息对接线图的了解已经迅速发展。由于连接组学领域的进步以及同时测量许多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，但这仍然很困难。结合这些测量来追踪神经投影。诸如电极阵列之类的电生理测量对于光学成像来说是极其困难的。追踪是可能的，但神经活动测量通常仅限于特定的几何形状，最常见的是尽管体积成像的新成像进步在一定程度上缓解了这一限制，复杂的仪器使大多数实验室无法实现此类技术，该提案解决了这个问题。通过使用多色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 是钙的生物发光指标。从整个细胞（包括树突和轴突乔木）发射光的浓度。方案中，每个神经元将表达 Aeq-FP 颜色的独特组合，以便对其进行颜色编码以使其具有单个神经元的活动可以从发射的光谱中区分出来。生物发光无需解析光源的空间位置，这使得同步成为可能。记录任意空间排列中的许多细胞的活动，包括来自不同层的细胞通过限制 Aeq-FP 表达到作为一个突触的神经元来识别连接的网络。使用跨突触病毒载体（用于逆行运输的改良狂犬病病毒和腺病毒载体）远离“起始”细胞用于顺行运输的相关病毒（AAV））也在每个细胞中表达独特的颜色组合。结合这些技术，微电路网络可以由以下定义：与单个“起始”细胞的连接将在三个维度上进行追踪，并与活动测量相关联在单次试验中，起始细胞位于网络的突触后，因此该数据将显示如何涉及皮质层多种不同类型细胞的突触前网络影响起始细胞在目标 2 中，起始细胞位于标记网络的突触前，并将表达视紫红质通道。光学刺激启动细胞将显示网络活动如何受到单个细胞的调制的影响这种测量能力将使与结构和活性相关的新型实验成为可能。可以很容易地被许多实验室采用。