Long-term brain circuit imaging with chemical and optogenetic stimulation

PI: Albrecht, Dirk r .提案#:1605679这个为期3年的提案的重点是开发一个急需的微流控平台，用于长期，高分辨率光学成像和记录活体动物受刺激的大脑活动。现有的光学系统受到以下限制：1)需要相对强烈的激发光，这会导致光漂白和光毒性，从而限制了实验的持续时间，阻碍了对所研究的神经元回路的刺激；2)光学系统与标准微流体刺激方法不兼容，可以在较低强度下工作。本提案的中心目标是使用光学折射率匹配的材料与低强度显微镜方法“选择性平面照明显微镜（SPIM）”兼容，开发微流体装置。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，并且可以同时进行光遗传（脉冲可见光）神经激活和读出。该系统将首次能够同时对正在研究的大脑回路进行化学和光学刺激和扰动，并在几个小时内监测多个神经元的活动。所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社区。通过全面的教育计划，包括以神经科学应用为重点的先进生物医学成像创新课程和推广活动，通过令人兴奋的暑期课程和科学示范实践模块，增加stem学生和当地社区的参与，也取得了更广泛的影响。感觉、记忆和行为都是在大脑神经元的动态电化学模式中编码的。快速三维显微镜技术的最新进展使人们能够同时对大量神经元的活动进行光学成像，在某些情况下，几乎可以对生物体的整个大脑进行成像。这种系统有望彻底改变神经回路调节的研究，相比之下，稀疏的单神经元记录不能捕捉到回路中其他地方的神经动力学。然而，目前的共聚焦和结构照明系统受到其对相对强烈的激发光的要求的限制，导致光漂白和光毒性，限制了实验的持续时间，并且难以刺激被研究的神经元回路。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，但光学要求与标准微流体刺激方法不兼容。因此，迫切需要一种与spim兼容的微流控刺激方法，以实现对活体动物受刺激大脑活动的长期、高分辨率记录。本提案的中心目标是使用与SPIM兼容的光学折射率匹配材料开发微流控器件。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，并且可以同时进行光遗传神经激活和读取。具体目标是：1)开发与dispim兼容的样品固定和微流体刺激；2)感觉刺激脑回路的多神经元成像以观察和研究感觉反馈；3)光遗传刺激脑回路的多神经元成像以观察可逆回路扰动对同一动物整体神经活动的影响。该提案的创新包括：1)鉴定与diSPIM兼容的水凝胶包封剂，使活体动物固定但保持生物体的健康和功能；2)微流体设计，包括水凝胶-玻璃或水凝胶-硅酮混合物，提供精确的化学浓度；3)识别检测新化学刺激的感觉神经元；4)通过候选基因突变体识别感觉反馈及其调控研究；5)同时进行光遗传和化学刺激，同时监测多个感觉神经元和中间神经元，观察动态电路扰动时的神经反应。最终结果将是一种新的双视图倒置（diSPIM）系统和方案，适用于细胞和小生物的嵌入，在化学和/或光遗传刺激期间和之后长时间记录高分辨率，各向同性，荧光三维体积图像。计划中的研究将提高对秀丽隐杆线虫在受到自然感官刺激（例如，化学物质）和在紧凑且定义明确的神经回路中受到任意光遗传刺激时的电路计算的理解。所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社区。与拟议的研究相结合的是一个全面的教育计划，旨在培训和教育下一代跨学科科学家，特别是生物学工程师，包括：1)以神经科学应用为重点的先进生物医学成像创新课程；2)通过研究和工程设计项目指导本科生和研究生；3)通过令人兴奋的暑期课程和科学演示实践模块，增加stem学生和当地社区的参与。

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.