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

项目摘要 在这里，我们试图开发一种“神经元活动定量诊断”平台和试剂盒，以 将用于测量整个小鼠大脑中最新神经活动的可访问产品传播到 神经科学界。此前，我们已经证明了活性依赖的即刻早期基因蛋白 产品NPAS4和CFO可以使用我们的开发版本进行清晰的检测、成像和量化 平台工具。直接早期基因是指对广泛的 各种细胞刺激。在第二阶段项目中要调查的三个重叠目标是： 1)向客户展示我们的化验结果的保真度。以展示健壮性和准确性 使用另一种众所周知的活性测试方法，我们将进行双光子钙化 在重复视觉刺激下，小鼠初级视皮层的荧光成像。我们会清理的 对大脑进行CFO和NPAS4染色，并证明这些标记最近用于检测的保真度 活跃的神经元。 2)开发和测试我们的软件工具的健壮版本，供研究人员使用，无需专门的 编码或统计知识。我们将把Stichy和BrainSnap开发成强大的、用户友好的软件 这些产品为对全脑成像感兴趣的神经科学家消除了目前的分析瓶颈。 Stichy将是我们内部软件的基于浏览器的改进版本，可以简化和加快这一过程 将薄片显微镜图像拼接在一起。BrainSnap软件将集成我们的高性能3TK- 支持机器学习的量化框架和我们的全脑统计框架 解剖学，3TK-统计，在基于浏览器的界面中。我们已招募最终用户，他们将在 与我们的内部测试进行正面比较。 3)研制并测试神经元活性的脑清除和染色试剂盒。以帮助研究人员产生高 质量清除大脑染色神经元活动标记物，我们将产生神经元活动清除和 CFO和NPAS4染色试剂盒。这些将由合作者以面对面的比较进行测试 我们的大脑内部程序是同时准备的。 在第二阶段结束时，半透明将有一个市场就绪的神经元活动定量诊断 平台包括试剂盒、用户友好的软件和服务选项。

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.