ENABLING SUBMILLISECOND-TIMESCALE TWO-PHOTON RECORDING OF VOLTAGE DYNAMICS IN THREE DIMENSIONS IN VIVO

实现体内三维电压动态的亚毫秒级双光子记录

• 批准号: 10739579
• 负责人: LAURENT BOURDIEU
• 金额: $ 114.27万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10739579
• 关键词: 3-Dimensional; Acceleration; Action Potentials; Address; Animals; Area; Biological; Biosensor; Brain; Calcium; Cells; Central Nervous System; Communities; Computer Systems; Dendrites; Detection; Disease; Dreams; Engineering; Family; Fluorescence; Generations; Genetic Engineering; Goals; Health; Image; Individual; Interneurons; Learning; Lighting; Location; Measures; Membrane Potentials; Memory; Methods; Microscopy; Mission; Monitor; Morphologic artifacts; Motion; Motor; Mus; Names; Nature; Neurites; Neurons; Neuropil; Neurosciences; Noise; Optics; Paper; Pattern; Perception; Performance; Photons; Population; Process; Proteins; Publications; Publishing; Reporting; Research; Research Project Grants; Resolution; Rodent; Sensory; Shapes; Signal Transduction; Specificity; Structure; Synapses; System; Techniques; Technology; Tissues; Work; artificial neural network; awake; cell type; density; design; digital; experience; improved; in vivo; interest; invention; meter; millisecond; neural; neuroimaging; neuronal cell body; new technology; next generation; prevent; screening; sensor; spatiotemporal; subcellular targeting; tool; two-photon; voltage

PROJECT SUMMARY/ABSTRACT Because neurons integrate and process information via modulation of their membrane potential, the ability to monitor voltage is critical to understanding how single and groups of neurons compute. Genetically encoded voltage indicators (GEVIs) —fluorescent proteins that report voltage dynamics as changes in brightness— are emerging as a preferred recording method because they can track voltage transients with high spatiotemporal resolution and cell type specificity. Particularly sought after are GEVIs that perform well under two-photon (2P) microscopy, the method of choice for imaging neural activity in highly scattering tissue such as the rodent brain. We have recently demonstrated that a combination of the 2P optical recording method ULoVE and the indicator JEDI-2P enable sustained (> 30 min), fast (> 1 kHz), deep-tissue (< 400 µm) monitoring of voltage dynamics in individual neuronal somas in awake behaving mice. However, ULoVE is fundamentally unable to record from cells and structures located in different focal planes. This is a critical limitation as neuronal computations typically involve cells or neurites located at different depths. The goal of this proposal is to address this technology gap and enable three-dimensional optical voltage recordings in awake-behaving mice. We propose to optimize 3D-CASH, a new method that enables the three-dimensional recording of calcium dynamics but whose lower signal-to-noise ratio prevents reliable voltage recordings. We propose several complementary but independent approaches to improving the signal-to-noise ratio of voltage recordings, including hardware-based strategies for efficiently exciting cells/structures while minimizing motion artifacts and neuropil background fluorescence (Aim 1). We also propose a new generation of GEVIs that improve the detectability of subthreshold and spikes (Aim 2) and optimized methods for subcellular localization of GEVIs to increase the signal from specific structures of interest such as dendrites or somas while reducing background contamination (Aim 3). We anticipate that this project will produce improved GEVIs of general utility for neuroscience applications and a new optical approach that enables three-dimensional voltage recordings in vivo. These new technologies will allow the neuroscience community to ask questions that are currently technically infeasible, paving the way for a more detailed understanding of dendritic integration and neural network computations in living animals.

项目总结/摘要 由于神经元通过调节其膜电位来整合和处理信息，因此监测电压的能力对于理解单个和成组神经元如何计算至关重要。遗传编码电压指示器（GEVI）-荧光蛋白，报告电压动态变化的亮度-正在成为一个首选的记录方法，因为它们可以跟踪电压瞬变具有高时空分辨率和细胞类型特异性。特别受欢迎的是在双光子（2 P）显微镜下表现良好的GEVI，这是对高度散射组织（如啮齿动物大脑）中的神经活动进行成像的首选方法。我们最近已经证明，2 P光学记录方法ULoVE和指示剂JEDI-2 P的组合能够持续（> 30分钟），快速（> 1 kHz），深层组织（< 400 µm）监测清醒行为小鼠个体神经元胞体的电压动态。然而，ULoVE根本无法记录位于不同焦平面的细胞和结构。这是一个关键的限制，因为神经元计算通常涉及位于不同深度的细胞或神经突。该提案的目标是解决这一技术差距，并使清醒行为小鼠的三维光学电压记录成为可能。我们建议优化3D-CASH，这是一种新的方法，能够三维记录钙动力学，但其较低的信噪比防止可靠的电压记录。我们提出了几种互补但独立的方法来提高电压记录的信噪比，包括基于硬件的策略，用于有效地激发细胞/结构，同时最大限度地减少运动伪影和神经元背景荧光（目标1）。我们还提出了新一代的GEVI，提高了亚阈值和尖峰的检测能力（目标2）和优化的方法，亚细胞定位的GEVI，以增加信号从特定的结构，如树突或胞体，同时减少背景污染（目标3）。我们预计，该项目将产生改进的GEVI的神经科学应用和一种新的光学方法，使三维电压记录在体内的一般效用。这些新技术将使神经科学界能够提出目前技术上不可行的问题，为更详细地了解活体动物的树突整合和神经网络计算铺平道路。