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从机理上了解此类缺陷如何导致神经和感觉系统疾病至关重要。为了实现这一点，需要神经追踪试剂，其a）甚至可以在部分死亡的细胞中描绘神经纤维，和B）允许同时追踪胚胎、青少年和成年人中的多个神经束，对于粗纤维与细纤维以及有髓鞘纤维与无髓鞘纤维具有相同的分辨率。因为所有的神经细胞都有膜，即使关键基因的缺陷导致异常发育，亲脂性荧光染料也可以提供其他技术可能失败的信息。用这样的染料示踪受到耗时和繁琐的方法的限制，这主要是由于匹配差的光谱和扩散特性。项目？的长期目标是建立一个亲脂性荧光染料/报告分子家族，以最大限度地增加神经元束的数量，这些神经元束可以在单个固定和/或活标本中同时追踪。在第一阶段，一套新的亲脂性NeuroVue？开发了具有良好匹配的光谱和漫射性质的染料，并以简单使用的涂覆滤光片形式商业化。这些新产品可在标准共焦系统上进行多达3种颜色的研究，并在具有光谱检测/颜色分解功能的增强系统上进行多达5种颜色的研究。该SBIR第II阶段提案将添加紫色、近红外和时间分辨NeuroVue染料，以进一步增加可同时追踪的纤维数量，并创建专用探针组，用于i）检测发育中的神经元连接和ii）有髓鞘/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，以便能够使用标准共聚焦显微镜在需要至少4周扩散期的组织中同时追踪多达5个神经元束; 2）创建优化的染料对，以便能够使用标准共聚焦显微镜检测固定组织中的神经元邻近/连接形成（NeuroVue连接）;第三章扩展I期NeuroVue增强套件，以便使用增强型共聚焦系统在至少4周的扩散期内同时追踪至少8个神经元束，光谱检测/颜色分解、双光子激发和时间分辨荧光检测能力; 4）创建优化用于有髓鞘纤维（例如，来自成年小鼠的组织和潜在的固定的人类样本）的长期（至少6周扩散）研究的染料组，5）将来自特定目的1-4的最佳新染料商业化。（a）提供一系列匹配良好的神经示踪探头，使标准共聚焦系统能够进行至少5色神经示踪，先进共聚焦系统能够进行8色研究; B）显著减少追踪神经元回路所需的动物的成本和数量，以及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，并且在疾病、损伤或老化的情况下被重塑。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措施的重要见解。

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.