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
基本信息
- 批准号:10472700
- 负责人:
- 金额:$ 64.81万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AffinityAreaBehaviorBehavior assessmentBenchmarkingBindingBiological AssayBrainBrain regionCalciumCalcium ionCellsCharacteristicsColorDataDetectionDevelopmentDevicesDorsalEnsureEnvironmentEquipmentFluorescenceFutureGenesGenetic IdentityGoalsHeadHippocampusImmunohistochemistryImplantIn VitroLabelLibrariesLightLightingLinkMapsMeasurementMethodsMicroscopeMicroscopyMonitorMovementMusMutateMutationNeuronsNoiseOpticsPathologyPatternPerformancePopulationPositioning AttributeProcessPropertyProtein EngineeringProteinsResearchResolutionSamplingSignal TransductionStep TestsSurfaceSystemTechniquesTechnologyTestingTissuesVariantViolaViral VectorVisual Cortexadvanced systemage relatedbehavior testbehavioral studycell typecognitive testingcortex mappingdetection sensitivityexperienceexperimental studyflexibilityimaging systemimplantable deviceimprovedin vitro Assayin vitro testingin vivoin vivo evaluationinterestmillimeterminiaturizeneural circuitnovel strategiesoptical imagingoptogeneticspredictive testprotein expressionprotein purificationresponsesample fixationscreeningsensorsingle cell sequencingthree photon microscopytranslational neurosciencetwo-photon
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Robert E. Campbell其他文献
Chemi-genetic sensors for metal ions using green fluorescent protein
使用绿色荧光蛋白的金属离子化学遗传传感器
- DOI:
- 发表时间:
2022 - 期刊:
- 影响因子:0
- 作者:
Takuya Terai;Robert E. Campbell - 通讯作者:
Robert E. Campbell
細胞内イメージングを目指したケミジェネティックNa+センサーの開発と評価
用于细胞内成像的化学基因Na+传感器的开发和评估
- DOI:
- 发表时间:
2022 - 期刊:
- 影响因子:0
- 作者:
竹内志織;朱文超;寺井琢也;Robert E. Campbell - 通讯作者:
Robert E. Campbell
Pushing the wavelength frontier for genetically encoded biosensors of neural activity
推动神经活动基因编码生物传感器的波长前沿
- DOI:
- 发表时间:
2022 - 期刊:
- 影响因子:0
- 作者:
Issei Yamaguchi;Yusuke Nasu;Takuya Terai;Robert E Campbell;Robert E. Campbell - 通讯作者:
Robert E. Campbell
High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate
用于柠檬酸盐的高性能强度直接和反向响应基因编码生物传感器
- DOI:
10.1021/acscentsci.0c00518 - 发表时间:
2020-07 - 期刊:
- 影响因子:18.2
- 作者:
Yufeng Zhao;Yi Shen;Yurong Wen;Robert E. Campbell - 通讯作者:
Robert E. Campbell
An Experimental Infection Model in Sheep and Goats to Evaluate Salmonella Colonization in Deep Tissue Lymph Nodes and after Carcass Vascular Rinsing with Bacteriophages in Goats.
绵羊和山羊的实验感染模型,用于评估山羊深部组织淋巴结和胴体血管用噬菌体冲洗后沙门氏菌的定植。
- DOI:
10.1016/j.jfp.2024.100312 - 发表时间:
2024 - 期刊:
- 影响因子:2
- 作者:
Koeun Hwang;Serhat Al;Robert E. Campbell;Kathleen Glass;Kurt D. Vogel;J. R. Claus - 通讯作者:
J. R. Claus
Robert E. Campbell的其他文献
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{{ truncateString('Robert E. Campbell', 18)}}的其他基金
High-Resolution Bidirectional Optical-Acoustic Mesoscopic Neural Interface for Image-Guided Neuromodulation in Behaving Animals - RF1 Admin Supplement
用于行为动物图像引导神经调节的高分辨率双向光声介观神经接口 - RF1 管理补充
- 批准号:
10712937 - 财政年份:2023
- 资助金额:
$ 64.81万 - 项目类别:
High-resolution bidirectional optical-acoustic mesoscopic neural interface for image-guided neuromodulation in behaving animals
用于行为动物图像引导神经调节的高分辨率双向光声介观神经接口
- 批准号:
10407380 - 财政年份:2022
- 资助金额:
$ 64.81万 - 项目类别:
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:
10293936 - 财政年份:2021
- 资助金额:
$ 64.81万 - 项目类别:
Northern Lights collaboration for better 2-photon probes
北极光合作打造更好的 2 光子探测器
- 批准号:
9336980 - 财政年份:2015
- 资助金额:
$ 64.81万 - 项目类别:
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