Optimizing Tracers For Multicolor Neuronal Profiling

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了解这种缺陷如何导致神经和感觉系统疾病的机制至关重要。为了实现这一目标，需要神经示踪试剂，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，对粗与薄，有髓与无髓纤维具有相同的分辨率。因为所有的神经细胞都有细胞膜，即使关键基因的缺陷导致异常发育，亲脂性荧光染料可以提供其他技术可能无法提供的信息。这种染料的追踪受到耗时和繁琐的方法的限制，主要是由于光谱和扩散特性不匹配。这个项目吗?S的长期目标是建立一个亲脂性荧光染料/报告分子家族，以最大限度地在单个固定和/或活标本中同时追踪神经元束的数量。在第一阶段，一套新的亲脂性NeuroVue？染料具有良好匹配的光谱和扩散特性，并在一个简单易用的涂层过滤器格式开发和商业化。这些新产品可以在标准共聚焦系统上进行多达3种颜色的研究，在具有光谱检测/颜色解混能力的增强系统上进行多达5种颜色的研究。SBIR II期提案将添加紫色、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数量，并创建专门的探针集，用于i)检测发育中的神经元连接和II)髓鞘/成人组织中的长期神经追踪研究。II期的具体目标是：1)扩展I期NeuroVue标准染料组，使其能够同时追踪组织中多达5个神经元束，使用标准共聚焦显微镜需要至少4周的扩散期；2)创建优化的染料对，使用标准共聚焦显微镜（NeuroVue Connections）检测固定组织中的神经元接近/连接形成；3)扩展一期NeuroVue Enhanced set，使用增强型共聚焦系统，具有光谱检测/解色、双光子激发和时间分辨荧光检测功能，使其能够在至少4周的扩散期内同时追踪至少8个神经元束；4)为有髓纤维（例如，成年小鼠的组织和潜在的固定人类标本）的长期（至少6周的扩散）研究创造一套优化的染料；5)将来自Specific Aims 1-4的最佳新染料商业化。完成所提出的工作计划将：a)提供一系列匹配良好的神经示踪探针，使其能够在标准共焦系统上至少进行5色神经示踪，并在高级共焦系统上进行8色研究；b)显著降低追踪神经元回路所需动物的成本和数量，c)提供新的工具来研究细胞连接的发育如何受到突变的影响，以及在疾病、损伤或衰老的情况下如何重建。这项工作将为研究神经系统中的连接如何被遗传缺陷、疾病、创伤或衰老所改变提供新的工具。新技术将允许从更少的动物中获得更多的信息，利用现有的NIH投资开发数千种具有明确遗传缺陷的突变小鼠模型。这些工具和模型有望为神经科学界提供对发育中的大脑以及如何设计更有效的治疗干预的关键见解。

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.