Rapid Evaluation of Neuronal Activity in the Intact Whole Brain at Single Cell Resolution

项目摘要 在这里，我们寻求开发“神经元活动定量诊断”平台和试剂盒， 传播可访问的产品，用于测量整个小鼠大脑的最近神经活动， 神经科学社区以前我们已经表明，活性依赖的立即早期基因蛋白， 产品Npas 4和cFos可以清楚地检测，成像，并使用我们的开发版本的量化 平台工具。立即早期基因是响应于广泛的免疫应答而瞬时且快速激活的基因。 各种细胞刺激。在第二阶段项目中将调查的三个重叠目标是： 1)向客户证明我们的检测试剂的准确性。为了证明耐用性和准确性， 我们的神经元活性测定与另一个众所周知的活性测定，我们将进行双光子钙离子 荧光成像在小鼠的初级视觉皮层呈现重复的视觉刺激。我们将清除 并对大脑进行cFos和Npas 4染色，并证明这些标记物用于检测最近 活跃的神经元 2)开发和beta测试我们的软件工具的强大版本，可供研究人员使用， 编码或统计知识。我们将把Stitchy和BrainSnap开发成强大的、用户友好的软件 为对全脑成像感兴趣的神经科学家消除当前分析瓶颈的产品。 Stitchy将是我们内部软件的一个改进的基于浏览器的版本，它简化并加快了这一过程 将光片显微镜图像拼接在一起。BrainSnap软件将整合我们的高性能3 TK- 用于机器学习量化的Quant框架和我们的全脑统计框架 解剖学，3 TK统计，在一个基于浏览器的界面。我们已经招募了最终用户，他们将在 与我们的内部测试进行对比 3)开发并测试一个用于神经元活动的大脑清除和染色试剂盒。帮助研究人员生产高 质量清除大脑染色的神经元活动标记，我们将产生神经元活动清除， cFos和Npas 4染色试剂盒。这些将由合作者在头对头比较中进行测试 与我们的大脑内部程序并行。 在第二阶段结束时，半透明将有一个市场准备的神经元活动定量诊断 该平台包括试剂盒、用户友好型软件和服务选项。

Project Summary Here we seek to develop a “Neuronal Activity Quantitative Diagnostic” platform and reagent kits to disseminate accessible products for measuring recent neural activity across the entire mouse brain to the neuroscience community. Previously we have shown that the activity-dependent immediate-early gene protein products Npas4 and cFos can be clearly detected, imaged, and quantified using developmental versions of our platform tools. Immediate early genes are genes that are activated transiently and rapidly in response to a wide variety of cellular stimuli. The three overlapping aims to be investigated in this Phase II project are: 1) Demonstration of the fidelity of our assay for our customers. To demonstrate the robustness and accuracy of our neuronal activity assay with another well-known assay for activity, we will perform two photon calcium fluorescence imaging in the primary visual cortex of mice presented with repeated visual stimuli. We will clear and stain the brains for cFos and Npas4 and demonstrate the fidelity of these markers for detecting recently active neurons. 2) Develop and beta test robust versions of our software tools useable by researchers with no specialized coding or statistics knowledge. We will develop Stitchy and BrainSnap into robust, user-friendly software products that remove the current analysis bottleneck for neuroscientists interested in whole-brain imaging. Stitchy will be an improved browser-based version of our internal software that simplifies and speeds the process of stitching together light sheet microscope images. BrainSnap software will integrate our high-performance 3TK- Quant framework for machine learning-enabled quantification and our statistical framework for whole-brain anatomics, 3TK-Stats, in a browser-based interface. We have recruited end users who will test this software in a head-to-head comparison with our in-house testing. 3) Develop and test a brain clearing and staining kit for neuronal activity. To help researchers produce high quality cleared brains stained with neuronal activity markers, we will generate Neuronal Activity clearing and staining reagents kits for cFos and Npas4. These will be tested by collaborators in head-to-head comparisons with our in-house procedures on brains prepared in parallel. At the end of Phase II, Translucence will have a market-ready Neuronal Activity Quantitative Diagnostic platform including reagent kits, user-friendly software, and service options.