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

项目摘要 这项拟议的研究的目标是优化一种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质或海马体。人们普遍认为，光学成像 使用基因编码的荧光钙传感器是目前唯一获得 用密集抽样进行遗传鉴定的神经细胞群体。在过去的几年里，录音 双光子显微镜的能力已经得到改进，可以从毫米级的组织中进行记录，并且新的 微型显微镜已经被开发出来，用来记录老鼠移动的过程。然而，大多数行为都会出现 来自多个大脑区域的神经元之间的集体相互作用，这不可能同时 通过这些系统进行监控。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，可以检测到 在两种钙依赖的方式下的活性：1)紫光照射下的永久性颜色变化(由绿色变为红色) 光，这一过程被称为光转化，以及2)荧光强度的动态变化。我们的数据显示 Campari允许记录行为自由的小鼠的大脑活动，而不需要使用显微镜物镜 或植入的装置。此外，红色Campari蛋白的自然降解使其能够多次纵向 测量。然而，之前使用体外试验改善Campari的尝试减少了一些 活体特性，这导致低动态记录灵敏度和低光转化率，需要 光照时间长，可积累足够量的红色蛋白质。因此，项目目标是优化 Campari允许在自由活动的小鼠中敏感地记录细胞分辨率、大脑皮层范围的活动快照， 随后使用双光子和三光子显微镜从同一小鼠进行随后的动态记录。至 为了优化Campari的性能，我们将结合对纯化蛋白、HEK细胞和神经元的体外测试， 最具预测性的试验：大规模的体内筛选，比以前的研究多出约30倍的结构。 目标1将专注于提高Campari的光转换效率，以促进在 自由活动的老鼠。目标2将专注于改善Campari的动态记录特性和灵敏度。在……里面 目标3，我们将结合目标1-2的有益突变来生成具有优化的新Campari 光转换和动态记录能力。我们的概念验证实验将展示多个 在检测细胞分辨率变化的一系列行为和认知测试期间进行区域皮质标测 在大脑皮层的活动模式中。这一优化的Campari有望促进新的假设驱动的研究 通过提供体积的、多区域的大脑活动数据，在认知和 对自由活动的小鼠进行行为测试，使研究能够同时涉及头部固定和自由活动 并利用光遗传刺激和单细胞测序等互补技术 能够在行为学研究中研究活性(红色标记)细胞的特性的方法。

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.