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.

项目摘要/摘要 由于神经元通过调节膜电位进行了整合和处理信息，因此监测电压的能力对于了解单个神经元的计算方式至关重要。遗传编码的电压指标（GEVI） - 将电压动力学作为亮度变化的荧光蛋白作为首选记录方法出现，因为它们可以跟踪具有高时空分辨率和细胞类型特异性的电压瞬变。在两光子（2p）显微镜下表现良好的GEVI尤其受追捧的GEV，这是在高度散射组织（如啮齿动物脑）中成像神经元活性的选择方法。我们最近证明，2P光学记录方法ULOVE和指示灯JEDI-2P实现了持续的（> 30分钟），快速（> 1 kHz），深部组织（<400 µm）监测醒着的小鼠的单个神经元中电压动力学的电压动力学。但是，Ulove从根本上无法从位于不同焦距的细胞和结构中记录。这是一个关键的局限性，因为神经元计算通常涉及位于不同深度的细胞或神经元。该提案的目的是解决这一技术差距，并在醒着的小鼠中实现三维光电电压记录。我们建议优化3D现期，这是一种新方法，可以使钙动力学的三维记录，但其信噪比较低，阻止了可靠的电压记录。我们提出了几种完整的互补但独立的方法来改善电压记录的信噪比，包括基于硬件的策略有效地令人兴奋的细胞/结构，同时最大程度地减少了运动伪像和Neuropil背景荧光（AIM 1）。我们还提出了一种新一代的GEVI，以改善子阈值和尖峰的检测（AIM 2）和优化GEVI的亚细胞定位方法，以增加感兴趣的特定结构（例如树突状或躯体），同时减少背景污染的信号（AIM 3）。我们预计该项目将为神经科学应用提供改进的通用效用，并采用新的光学方法，以实现体内三维电压记录。这些新技术将使神经科学社区能够提出目前在技术上不可行的问题，从而为对树突状动物的树突一体化和神经网络计算的更详细的了解贴上了道路。