Optimization of CaMPARI for large-scale, cellular-resolution activity recording in freely-moving mice

CaMPARI 的优化，用于自由移动小鼠的大规模细胞分辨率活动记录

• 批准号: 10472700
• 负责人: Robert E. Campbell
• 金额: $ 64.81万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 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 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472700
• 关键词: Affinity; Area; Behavior; Behavior assessment; Benchmarking; Binding; Biological Assay; Brain; Brain region; Calcium; Calcium ion; Cells; Characteristics; Color; Data; Detection; Development; Devices; Dorsal; Ensure; Environment; Equipment; Fluorescence; Future; Genes; Genetic Identity; Goals; Head; Hippocampus; Immunohistochemistry; Implant; In Vitro; Label; Libraries; Light; Lighting; Link; Maps; Measurement; Methods; Microscope; Microscopy; Monitor; Movement; Mus; Mutate; Mutation; Neurons; Noise; Optics; Pathology; Pattern; Performance; Population; Positioning Attribute; Process; Property; Protein Engineering; Proteins; Research; Resolution; Sampling; Signal Transduction; Step Tests; Surface; System; Techniques; Technology; Testing; Tissues; Variant; Viola; Viral Vector; Visual Cortex; advanced system; age related; behavior test; behavioral study; cell type; cognitive testing; cortex mapping; detection sensitivity; experience; experimental study; flexibility; imaging system; implantable device; improved; in vitro Assay; in vitro testing; in vivo; in vivo evaluation; interest; millimeter; miniaturize; neural circuit; novel strategies; optical imaging; optogenetics; predictive test; protein expression; protein purification; response; sample fixation; screening; sensor; single cell sequencing; three photon microscopy; translational neuroscience; two-photon

Project Summary The goal of this proposed research is to optimize a dual-use calcium ion sensor for recording single-cell activity from the entire dorsal cortex or hippocampus of freely-moving mice. It is widely accepted that optical imaging with genetically-encoded fluorescent calcium sensors is currently the only method to obtain measurements of genetically-identified neuronal populations with dense sampling. Over the past several years, the recording capabilities of two-photon microscopes have been improved to record from millimeter-scale tissue, and new miniaturized microscopes have been developed to record from moving mice. However, most behaviors arise from collective interactions between neurons from multiple brain areas, which cannot be simultaneously monitored with these systems. Therefore, there is a clear need to develop a new approach to directly monitor the synchronized activity of distributed neural circuits. CaMPARI is a unique calcium sensor that can detect activity in two calcium-dependent ways: 1) permanent color change (green to red) upon illumination with violet light, a process known as photoconversion, and 2) dynamic changes in fluorescence intensity. Our data show that CaMPARI allows recording of brain activity from freely-behaving mice, without using microscope objectives or implanted devices. Moreover, natural degradation of the red CaMPARI protein enables multiple longitudinal measurements. However, previous attempts to improve CaMPARI using in vitro assays reduced some of its in vivo properties, which resulted in low dynamic recording sensitivity and a low photoconversion rate that requires long illumination times to accumulate a sufficient amount of red protein. Therefore, the project goal is to optimize CaMPARI to allow sensitive recording of cellular-resolution, cortex-wide activity snapshots in freely-moving mice, followed by subsequent dynamic recording from the same mouse using two- and three-photon microscopy. To optimize CaMPARI’s performance, we will combine in vitro testing in purified protein, HEK cells, and neurons, and the most predictive assay: large-scale in vivo screening of ~30-fold more constructs than previous studies. Aim 1 will focus on enhancing CaMPARI’s photoconversion efficiency to facilitate large-scale recordings in freely-moving mice. Aim 2 will focus on improving CaMPARI’s dynamic recording properties and sensitivity. In Aim 3, we will combine beneficial mutations from Aims 1-2 to generate a new CaMPARI with optimized photoconversion and dynamic recording capabilities. Our proof-of-concept experiments will demonstrate multi- regional cortical mapping during a battery of behavioral and cognitive tests to detect cellular-resolution changes in cortex-wide activity patterns. This optimized CAMPARI is expected to facilitate new hypothesis-driven studies by providing volumetric, multi-regional brain activity data of genetically-targeted neurons during cognitive and behavioral testing of freely-moving mice, enabling studies that involve both head-fixation and free movement in the same mice, and to utilize complementary techniques like optogenetic stimulation and single-cell sequencing methods to enable studying the properties of active (red-labeled) cells during behavioral studies.

项目摘要 本研究的目的是优化一种用于记录单细胞活性的两用钙离子传感器 从自由活动的老鼠的整个背侧皮层或海马体中提取。人们普遍认为， 与基因编码的荧光钙传感器是目前唯一的方法来获得测量 密集采样的遗传鉴定的神经元群体。在过去的几年里，记录 双光子显微镜的能力已经得到改进，可以记录毫米级的组织， 已经开发出小型化的显微镜来记录移动的小鼠。然而，大多数行为 来自多个大脑区域的神经元之间的集体相互作用， 监控这些系统。因此，显然需要开发一种新的方法来直接监测 分布式神经回路的同步活动。CaMPARI是一种独特的钙传感器， 以两种钙依赖性方式的活性：1）在用紫色照射时的永久颜色变化（绿色至红色 光，一种称为光转换的过程，以及2）荧光强度的动态变化。我们的数据显示 CaMPARI允许记录自由行为小鼠的大脑活动，而无需使用显微镜物镜 或植入装置。此外，红色CaMPARI蛋白的自然降解使得能够实现多个纵向降解。 测量.然而，先前使用体外测定来改善CaMPARI的尝试减少了其在体内的一些表达。 体内性质，这导致低动态记录灵敏度和低光转换率，需要 长的光照时间以积累足够量的红色蛋白。因此，项目目标是优化 CaMPARI允许灵敏地记录自由移动小鼠的细胞分辨率、全皮质活动快照， 随后使用双光子和三光子显微镜对同一小鼠进行动态记录。到 为了优化CaMPARI的性能，我们将联合收割机在纯化蛋白、HEK细胞和神经元中进行体外测试， 最具预测性的试验：大规模体内筛选的构建体比以前的研究多30倍。 Aim 1将专注于提高CaMPARI的光转换效率，以促进大规模记录， 自由移动的老鼠目标2将专注于改善CaMPARI的动态记录特性和灵敏度。在 目的3，我们将联合收割机组合来自目的1-2的有益突变以产生具有优化的 光转换和动态记录能力。我们的概念验证实验将证明多- 在一系列行为和认知测试中进行区域皮层映射，以检测细胞分辨率的变化 大脑皮层的活动模式这种优化的CAMPARI有望促进新的假设驱动的研究 通过在认知和认知过程中提供遗传靶向神经元的体积、多区域脑活动数据， 自由移动小鼠的行为测试，使研究，涉及头部固定和自由运动， 同样的小鼠，并利用互补技术，如光遗传学刺激和单细胞测序 能够在行为研究期间研究活性（红色标记）细胞特性的方法。