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.

项目概要 这项研究的目标是优化用于记录单细胞活动的两用钙离子传感器 来自自由活动小鼠的整个背侧皮层或海马体的光学成像已被广泛接受。 使用基因编码荧光钙传感器是目前获得测量值的唯一方法 在过去的几年中，通过密集采样进行了基因鉴定的神经群体的记录。 双光子显微镜的能力已得到改进，可以记录毫米级组织，并且新的 微型显微镜已经被开发出来来记录移动的小鼠，然而，大多数行为都会发生。 来自多个大脑区域的神经元之间的集体相互作用，这些相互作用不能同时进行 因此，显然需要开发一种新的方法来直接监控。 CaMPARI 是一种独特的钙传感器，可以检测分布式神经回路的同步活动。 以两种钙依赖性方式发挥活性：1) 在紫罗兰色照射下发生永久性颜色变化（绿色到红色） 光，一个称为光转换的过程，以及 2）我们的数据显示荧光强度的动态变化。 CaMPARI 可以在不使用显微镜物镜的情况下记录自由行为小鼠的大脑活动 此外，红色 CaMPARI 蛋白的自然降解能够实现多重纵向。 然而，之前使用体外测定改进 CaMPARI 的尝试减少了一些结果。 体内特性，导致动态记录灵敏度低，光转换率低，需要 较长的光照时间才能积累足够量的红色蛋白质因此，该项目的目标是优化。 CaMPARI 允许在自由移动的小鼠中灵敏地记录细胞分辨率、皮层范围的活动快照， 随后使用双光子和三光子显微镜对同一只小鼠进行后续动态记录。 优化 CaMPARI 的性能，我们将结合纯化蛋白、HEK 细胞和神经元的体外测试， 最具预测性的测定：大规模体内筛选比以前的研究多约 30 倍的构建体。 目标 1 将重点提高 CaMPARI 的光转换效率，以促进大规模记录 Aim 2 将专注于提高 CaMPARI 的动态记录特性和灵敏度。 目标 3，我们将结合目标 1-2 中的有益突变，生成经过优化的新 CaMPARI 我们的概念验证实验将展示多种光转换和动态记录功能。 在一系列行为和认知测试中绘制区域皮质图，以检测细胞分辨率的变化 这种优化的 CAMPARI 有望促进新的假设驱动的研究。 通过提供认知和认知过程中基因靶向神经元的体积、多区域大脑活动数据 对自由移动的小鼠进行行为测试，从而能够进行涉及头部固定和自由移动的研究 相同的小鼠，并利用光遗传学刺激和单细胞测序等互补技术 在行为研究期间研究活性（红色标记）细胞特性的方法。