Optimizing Tracers For Multicolor Neuronal Profiling

优化多色神经元分析示踪剂

• 批准号: 7418498
• 负责人: Brian David Gray
• 金额: $ 37.01万
• 依托单位: MOLECULAR TARGETING TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-06 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418498
• 关键词: Optimizing; Tracers; Multicolor; Neuronal; Profiling

DESCRIPTION (provided by applicant): Understanding connectional deficits in mutant and transgenic mouse models is essential to a mechanistic understanding of how such deficits contribute to nervous and sensory system disorders. To achieve this requires neurotracing reagents that a) can delineate nerve fibers even in partially defunct cells and b) allow simultaneous tracing of multiple nerve tracts in embryos, juveniles and adults, with equivalent resolution for thick vs. thin and myelinated vs. unmyelinated fibers. Because all nerve cells have membranes, even when defects in critical genes cause aberrant development, lipophilic fluorescent dyes can provide information where other techniques may fail. Tracing with such dyes has been limited by time consuming and cumbersome methodology, largely due to poorly matched spectral and diffusional properties. The project?s long term goal is to build a family of lipophilic fluorescent dyes/reporter molecules to maximize the number of neuronal tracts which can be traced at the same time in individual fixed and/or live specimens. In Phase I, a novel set of lipophilic NeuroVue? dyes with well matched spectral and diffusion properties were developed and commercialized in a simple-to-use coated filter format. These new products enable up to 3- color studies on standard confocal systems and up to 5 color studies on enhanced systems with spectral detection/color unmixing capabilities. This SBIR Phase II proposal will add violet, near-infrared and time- resolved NeuroVue dyes to further increase the number of fibers that can be traced simultaneously, and creation of specialized probe sets for i) detection of developing neuronal connections and ii) long-term neurotracing studies in myelinated/adult tissue. Phase II specific aims are to:1) Expand the Phase I NeuroVue Standard dye set to enable simultaneous tracing of up to 5 neuronal tracts in tissues requiring diffusion periods of at least 4 weeks using standard confocal microscopy; 2) Create optimized dye pair enabling detection of neuronal proximity/connection formation in fixed tissue using standard confocal microscopes (NeuroVue Connections); 3) Expand the Phase I NeuroVue Enhanced set to enable simultaneous tracing of at least 8 neuronal tracts over a diffusion period of at least 4 weeks using enhanced confocal systems with spectral detection/color unmixing, 2-photon excitation and time-resolved fluorescence detection capabilities; 4) Create a dye set optimized for long-term (at least 6 week diffusion) studies of myelinated fibers (e.g. ,tissues from adult mice and, potentially, fixed human specimens), 5) Commercialize the best new dyes from Specific Aims 1-4. Completion of the workplan proposed will: a) Provide a family of well-matched neurotracing probes enabling at least 5-color neurotracing on standard confocal systems and 8-color studies on advanced confocal systems; b) Significantly reduce the cost and number of animals required to trace neuronal circuits, and c) Provide novel tools to study how development of cellular connections are affected by mutations, and are remodeled in the presence of disease, damage or aging. This work will provide novel tools for the study of how connections in the nervous system are altered by genetic defects, disease, trauma or aging. The new techniques will allow more information to be gained from fewer animals, leveraging existing NIH investments in the development of thousands of mutant mouse models with defined genetic defects. These tools and models are expected to provide the neuroscience community with critical insights into the developing brain and how to design more effective therapeutic interventions.

描述(由申请人提供)：了解突变和转基因小鼠模型中的连接缺陷对于从机制上理解这些缺陷如何导致神经和感觉系统障碍至关重要。要做到这一点，需要神经追踪试剂，a)即使在部分死亡的细胞中也能描绘神经纤维，b)允许同时追踪胚胎、青少年和成人的多个神经束，粗纤维与细纤维、有髓纤维与无髓纤维的分辨率相同。因为所有神经细胞都有膜，即使关键基因的缺陷导致发育异常，亲脂性荧光染料也可以提供其他技术可能失败的信息。这类染料的追踪一直受到耗时和繁琐的方法的限制，这主要是由于光谱和扩散特性匹配不佳。S项目的长期目标是建立一系列亲脂性荧光染料/报告分子，以最大限度地增加在单个固定和/或活标本中可以同时追踪的神经束的数量。在第一阶段，一套新的亲脂性NeuroVue？具有良好匹配的光谱和扩散特性的染料被开发出来，并以简单易用的涂层滤光片形式进行商业化。这些新产品支持对标准共焦系统进行最多3种颜色的研究，对具有光谱检测/颜色分解功能的增强型系统进行最多5种颜色研究。这项SBIR第二阶段计划将添加紫色、近红外和时间分辨的NeuroVue染料，以进一步增加可同时追踪的纤维的数量，并创建专门的探针组，用于i)检测正在发展的神经元连接和ii)在有髓/成人组织中进行长期神经追踪研究。第二阶段的具体目标是：1)扩展第一阶段NeuroVue标准染料集，以便能够使用标准共焦显微镜同时追踪需要至少四周扩散周期的组织中的最多5个神经元束；2)创建优化的染料对，能够使用标准共焦显微镜(NeuroVue Connections)检测固定组织中神经元的接近/连接形成；3)扩展第一阶段NeuroVue增强集，使用具有光谱检测/颜色分解、双光子激发和时间分辨荧光检测能力的增强型共聚焦系统，在至少四周的扩散期内同时追踪至少8个神经纤维束；4)为有髓纤维的长期(至少6周扩散)研究(例如，成年小鼠的组织，可能还包括固定的人类标本)创造一套优化的染料组，5)将特定目标1-4的最佳新染料商业化。拟议工作计划的完成将：(A)提供一系列匹配良好的神经追踪探针，使标准共焦系统能够进行至少5色神经追踪，并对先进的共焦系统进行8色研究；(B)显著降低追踪神经元回路所需的动物数量和成本；以及(C)提供新的工具，以研究细胞连接的发育如何受到突变的影响，以及在疾病、损伤或衰老存在时如何重塑。这项工作将为研究神经系统中的连接如何因遗传缺陷、疾病、创伤或衰老而改变提供新的工具。新技术将允许从更少的动物中获得更多信息，利用NIH在开发数千个具有明确遗传缺陷的突变小鼠模型方面的现有投资。这些工具和模型有望为神经科学界提供有关大脑发育以及如何设计更有效的治疗干预措施的关键见解。