Long-term brain circuit imaging with chemical and optogenetic stimulation

主要研究者：Albrecht，Dirk R.提案编号：1605679该3年提案的重点是开发一种急需的微流体平台，用于长期，高分辨率光学成像和记录活体动物的刺激大脑活动。 现有的光学系统受到以下限制：1）相对强的激发光的要求，其导致光漂白和光毒性，这限制了实验的持续时间并阻碍刺激所研究的神经元回路;或2）可以在较低强度下与标准微流体刺激方法操作的光学系统的不兼容性。 该提案的中心目标是开发使用与低强度显微镜方法“选择性平面照明显微镜（SPIM）”兼容的光学折射率匹配材料的微流体装置。“初步结果表明，基于水凝胶的系统可以记录秀丽隐杆线虫数小时的神经反应，以及与同步光遗传（脉冲可见光）神经激活和读出的兼容性。 该系统将首次实现同时进行化学和光学刺激以及对正在研究的大脑回路的扰动，并在数小时内监测多个神经元的活动。 开发的方法将影响更广泛的神经科学界，其中神经成像是发育，结构和功能大脑研究的关键方法。 材料和硬件，包括微流体系统，将提供给研究和商业界。 更广泛的影响也通过一个全面的教育计划，包括创新的课程，在先进的生物医学成像集中在神经科学的应用和推广活动，以增加STEM代表性不足的学生和当地社区的参与，通过令人兴奋的，动手模块的夏季计划和科学演示实现。 感觉、记忆和行为在大脑神经元内以动态电化学模式编码。快速三维显微镜的最新进展使大量神经元的活动能够同时进行光学成像，在某些情况下几乎是生物体的整个大脑。这种系统有望彻底改变神经回路调节的研究，与稀疏的单神经元记录相比，不能捕捉回路中其他地方的神经动力学。然而，目前的共焦和结构化照明系统受到它们对相对强烈的激发光的要求的限制，从而导致限制实验持续时间的光漂白和光毒性，并且受到难以刺激所研究的神经元回路的限制。虽然光片或选择性平面照明显微镜（SPIM）捕获更多的发射光，因此在较低的激发强度下工作，但光学要求与标准的微流体刺激方法不兼容。因此，迫切需要SPIM兼容的微流体刺激方法，以能够长期、高分辨率地记录活体动物的受刺激脑活动。该提案的中心目标是开发使用与SPIM兼容的光学折射率匹配材料的微流体装置。初步结果显示了基于水凝胶的系统记录秀丽隐杆线虫数小时神经反应的可行性，以及与同时光遗传神经激活和读出的兼容性。具体目标是：1）开发diSPIM相容的样品固定和微流体刺激，2）感觉刺激的脑回路的多神经元成像以观察和研究感觉反馈，和3）光遗传学刺激的脑回路的多神经元成像以观察可逆回路扰动对同一动物中的整体神经活动的影响。 该提案的创新包括：1）鉴定与diSPIM相容的水凝胶澄清剂，其澄清活体动物但维持生物体健康和功能; 2）微流体设计，包括水凝胶-玻璃或水凝胶-硅酮混合物，其递送精确的化学浓度; 3）鉴定检测新化学刺激的感觉神经元; 4）识别感觉反馈并通过候选遗传突变体研究其调节; 5）同时光遗传学和化学刺激，同时监测多个感觉和中间神经元，以观察动态电路扰动期间的神经反应。 最终结果将是双视图倒置（diSPIM）系统的新配置和适用于细胞和小生物体嵌入的协议，以在化学和/或光遗传学刺激期间和之后长时间记录高分辨率，各向同性，荧光3-D体积图像。计划中的研究将提高对C语言电路计算的理解。当用天然感官刺激刺激时（例如，化学物质）和通过紧凑且明确定义的神经回路内的任意光遗传学刺激。开发的方法将影响更广泛的神经科学界，其中神经成像是发育，结构和功能大脑研究的关键方法。 材料和硬件，包括微流体系统，将提供给研究和商业界。 与拟议的研究相结合的是一个全面的教育计划，旨在培养和教育下一代跨学科科学家，特别是生物学家工程师，包括：1）先进生物医学成像的创新课程，重点是神经科学应用，2）通过研究和工程设计项目指导本科生和研究生，和3）外展，以增加STEM代表性不足的学生和当地社区的参与，通过令人兴奋的，动手模块的夏季计划和科学演示。

PI: Albrecht, Dirk R.Proposal #: 1605679The focus of this 3 year proposal is developing a much needed microfluidic platform for long-term, high resolution optical imaging and recording of stimulated brain activity in living animals. Existing optical systems are limited by 1) requirements of relatively intense excitation light that causes photobleaching and phototoxicity that limits the duration of the experiment and hinders stimulating the neuronal circuit under investigation or 2) by the incompatability of optical systems that can operate at lower intensities with standard microfluidic stimulation methods. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with a low intensity microscopy method, "selective plane illumination microscopy (SPIM)." Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic (pulsed visible light) neural activation and readout. The proposed system will for the first time enable simultaneous chemical and optical stimulation and perturbation of brain circuits under investigation, with multiple neurons monitored for activity over several hours. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Broader impact is also achieved through a comprehensive educational plan including innovative curricula in advanced biomedical imaging focused on neuroscience applications and outreach activities to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations. Sensation, memory, and behaviors are encoded in dynamic electrochemical patterns within neurons of the brain. Recent advances in fast three-3D microscopy have enabled the optical imaging of activity in large numbers of neurons at once, in some cases nearly the entire brain of an organism. Such systems promise to revolutionize the study of neural circuit regulation, compared with sparse single-neuron recordings that do not capture neural dynamics elsewhere in the circuit. However, current confocal and structured illumination systems are limited by their requirements of relatively intense excitation light, causing photobleaching and phototoxicity that limits the duration of an experiment, and by difficulty in stimulating the neuronal circuit under investigation. While light sheet or selective plane illumination microscopy (SPIM) captures more emission light and therefore operates at lower excitation intensity, optical requirements are incompatible with standard microfluidic stimulation methods. Therefore, there exists an urgent need for SPIM-compatible microfluidic stimulation methods to enable long-term, high resolution recording of stimulated brain activity in living animals. The central aim of this proposal is to develop microfluidic devices using optical index-matched materials compatible with SPIM. Preliminary results show feasibility of hydrogel-based systems to record neural responses in C elegans for hours, as well as compatibility with simultaneous optogenetic neural activation and readout. Specific objectives are: 1) development of diSPIM-compatible sample immobilization and microfluidic stimulation, 2) multi-neuronal imaging of sensory-stimulated brain circuits to observe and study sensory feedback, and 3) multi-neuronal imaging of optogenetically-stimulated brain circuits to observe the effect of reversible circuit perturbations on ensemble neural activity in the same animal. Innovations of the propose include: 1) identification of hydrogel encapsulants compatible with diSPIM that immobilize living animals but maintain organism health and function; 2) microfluidic designs, including hydrogel-glass or hydrogel-silicone hybrids, that deliver precise chemical concentrations; 3) identification of sensory neurons detecting novel chemical stimuli; 4) identification of sensory feedback and study of its regulation via candidate genetic mutants; 5) simultaneous optogenetic and chemical stimulation while monitoring multiple sensory and interneurons, to observe neural responses during dynamic circuit perturbation. The end result will be a new configuration of the dual-view inverted (diSPIM) system and protocols suitable for the embedding of cells and small organisms to record high-resolution, isotropic, fluorescent 3-D volumetric images for long time periods during and after chemical and/or optogenetic stimulation. The studies planned will improve the understanding of circuit computation in C. elegans when stimulated with natural sensory stimuli (e.g., chemicals) and by arbitrary optogenetic stimulation within a compact and well-defined neural circuit. The methods developed will impact the broader neuroscience community, in which neural imaging is a critical method for developmental, structural, and functional brain studies. Materials and hardware, including microfluidic systems, will be made accessible to the research and commercial community. Integrated with the proposed research is a comprehensive educational program toward training and educating the next generation of interdisciplinary scientists, particularly biologist-engineers, including: 1) innovative curricula in advanced biomedical imaging with focus on neuroscience applications, 2) mentoring undergraduate and graduate students through research and engineering design projects, and 3) outreach to increase involvement of STEM-underrepresented students and local communities, through exciting, hands-on modules for summer programs and scientific demonstrations.