Optimization of Calcium and RNA multiplexed activity imaging for highly parallelized evaluation of cell type functions in deep-brain structures

钙和 RNA 多重活性成像的优化，用于高度并行评估深部脑结构中的细胞类型功能

• 批准号: 10401603
• 负责人: Meng Cui
• 金额: $ 84.28万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10401603
• 关键词: Adoption; Animal Behavior; Anterior; Area; Axon; BRAIN initiative; Back; Bar Codes; Behavior; Behavioral; Brain; Brain Diseases; Calcium; Cell physiology; Cells; Censuses; Characteristics; Classification; Code; Collaborations; Communities; Computing Methodologies; Data; Engineering; Evaluation; Fluorescent in Situ Hybridization; Fright; Funding; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetic Transcription; Goals; Hunger; Hypothalamic structure; Image; In Situ Hybridization; Individual; Investigation; Lateral; Lead; Measurement; Methodology; Methods; Microscopy; Molecular; Mus; NIH Program Announcements; Nervous system structure; Neurons; Neurosciences; Noise; Obesity; Pattern; Physiological; Procedures; Property; Pupil; RNA; Research; Role; Serotyping; Signal Transduction; Speed; Structure; System; Technology; Thick; Thirst; Tissues; Transcript; Viral; Viral Vector; Work; adaptive optics; cell type; computerized tools; emotional functioning; ex vivo imaging; experimental study; high dimensionality; hindbrain; image registration; improved; in vivo; in vivo calcium imaging; in vivo imaging; indexing; information processing; lens; motivated behavior; multimodal data; neural patterning; neuronal cell body; response; success; tool; transcriptomics; two-photon

PROJECT SUMMARY A central goal of neuroscience is to understand animal behavior in the context of the information processing properties of neuron ensembles. Neurons are circuit nodes in the brain information processing network, but they are also cells that express hundreds of genes that may make important contributions to determining their activity patterns. The relationship between gene expression, neuron projections, neuron activity, and behavior have been, individually, major avenues for investigation in both molecular and systems neuroscience. Integrating these levels of investigation would greatly advance understanding the influence of molecular properties on neuron information processing characteristics in healthy brains and disease states. In this project, which we refer to as CaRMA 2.0, we propose to develop and democratize methodologies to integrate measurements of transcriptomics, projectomics, calcium dynamics, and behavior in deep-brain structures. This proposal builds on our past success to develop and implement Calcium and RNA Multiplexed Activity (CaRMA) imaging, which combines these levels of investigation into a single large-scale experiment. This is achieved by deep-brain two-photon calcium imaging in a behaving mouse, followed by removing and sectioning the brain, aligning the ex vivo image volume with the in vivo-imaged neurons, and then performing multiple rounds of RNA-fluorescence in situ hybridization (FISH) on the same tissue. To reliably produce these high dimensional, multi-modal datasets requires significant advancements for “Optimization of Transformative Technologies for Large Scale Recording and Modulation in the Nervous System” as indicated in this RFA-NS-18-019 program announcement. We will develop improved technologies for deep-brain calcium imaging as well as highly multiplexed gene expression analysis on the same cells and apply this pipeline to investigate the mPFC, hindbrain, and the role of the lateral hypothalamus in hunger, thirst, and fear.

项目概要 神经科学的一个中心目标是在神经元集合的信息处理特性的背景下理解动物的行为。神经元是大脑信息处理网络中的电路节点，但它们也是表达数百个基因的细胞，这些基因可能对确定其活动模式做出重要贡献。基因表达、神经元投射、神经元活动和行为之间的关系一直是分子和系统神经科学研究的主要途径。整合这些水平的研究将极大地促进对健康大脑和疾病状态中分子特性对神经元信息处理特征的影响的理解。在这个我们称为 CaRMA 2.0 的项目中，我们建议开发和民主化方法，以整合转录组学、投影组学、钙动力学和深部大脑结构行为的测量。该提案建立在我们过去成功开发和实施钙和 RNA 多重活性 (CaRMA) 成像的基础上，该成像将这些水平的研究结合到一个大规模实验中。这是通过对行为小鼠进行深脑双光子钙成像来实现的，然后取出大脑并切片，将离体图像体积与体内成像神经元对齐，然后在同一组织上进行多轮RNA荧光原位杂交（FISH）。为了可靠地生成这些高维、多模态数据集，需要在“神经系统大规模记录和调制的变革技术优化”方面取得重大进展，如 RFA-NS-18-019 计划公告中所述。我们将开发改进的深脑钙成像技术以及对相同细胞进行高度多重基因表达分析，并应用该流程来研究 mPFC、后脑以及外侧下丘脑在饥饿、口渴和恐惧中的作用。