Long-term brain circuit imaging with chemical and optogenetic stimulation

通过化学和光遗传学刺激进行长期脑回路成像

• 批准号: 1605679
• 负责人: Dirk Albrecht
• 金额: $ 31.48万
• 依托单位: Worcester Polytechnic Institute
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1605679&HistoricalAwards=false
• 关键词: Long; term; brain; circuit; imaging

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.

PI：Albrecht，Dirk R.Proposal#：1605679这项为期3年的计划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脑活动。现有的光学系统受到以下因素的限制：1)对相对较强的激发光的要求，这会导致光漂白和光毒性，从而限制了实验的持续时间，并阻碍了对被研究神经元电路的刺激；2)可以在较低强度下运行的光学系统与标准的微流控刺激方法不兼容。这项提议的中心目标是开发使用光学折射率匹配材料的微流体设备，该材料与低强度显微镜方法相兼容，即“选择性平面照明显微镜(SPIM)”。初步结果表明，基于水凝胶的系统可以记录线虫数小时的神经反应，并与同步光遗传(脉冲可见光)神经激活和读出兼容。拟议的系统将首次能够同时进行化学和光学刺激以及对正在研究的大脑电路进行扰动，并在几个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，在神经科学界，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，包括微流控系统，将向研究和商业界开放。还通过全面的教育计划实现了更广泛的影响，包括以神经科学应用为重点的高级生物医学成像方面的创新课程，以及通过暑期项目和科学演示的激动人心的动手模块，增加STEM代表不足的学生和当地社区的参与的外联活动。感觉、记忆和行为是以大脑神经元内的动态电化学模式编码的。快速三维显微镜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。与稀疏的单神经元记录相比，这种系统有望给神经电路调节的研究带来革命性的变化，因为稀疏的单神经元记录没有捕捉到电路中其他地方的神经动态。然而，目前的共聚焦和结构照明系统受到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，以及难以刺激所研究的神经元电路。虽然薄片或选择性平面照明显微镜(SPIM)捕获了更多的发射光，因此在较低的激发强度下工作，但光学要求与标准的微流控刺激方法不兼容。因此，迫切需要SPIM兼容的微流控刺激方法，以实现对活体动物刺激的脑活动的长期、高分辨率记录。这项提议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控器件。初步结果表明，基于水凝胶的系统可以记录线虫数小时的神经反应，并与同步光遗传神经激活和读出兼容。具体目标是：1)开发与DiSPIM兼容的样品固定化和微流控刺激，2)感官刺激脑回路的多神经元成像，以观察和研究感觉反馈，以及3)光遗传刺激脑回路的多神经元成像，以观察可逆电路扰动对同一动物整体神经活动的影响。这项建议的创新包括：1)鉴定与diSPIM兼容的水凝胶包封剂，它可以固定活动物，但保持生物体的健康和功能；2)微流控设计，包括水凝胶-玻璃或水凝胶-硅胶杂化，提供精确的化学浓度；3)鉴定感觉神经元，检测新的化学刺激；4)鉴定感觉反馈，并通过候选基因突变研究其调节；5)同时监测多个感觉神经元和中间神经元的光遗传和化学刺激，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倒置(DiSPIM)系统和协议，适用于细胞和小生物的嵌入，以在化学和/或光遗传刺激期间和之后的长时间段内记录高分辨率、各向同性、荧光的三维体积图像。计划中的研究将提高对线虫在自然感觉刺激(例如，化学物质)刺激和紧凑且定义明确的神经回路中的任意光遗传刺激时电路计算的理解。开发的方法将影响更广泛的神经科学界，在神经科学界，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，包括微流控系统，将向研究和商业界开放。与拟议的研究整合在一起的是一项全面的教育计划，旨在培训和教育下一代跨学科科学家，特别是生物学家和工程师，包括：1)以神经科学应用为重点的高级生物医学成像的创新课程，2)通过研究和工程设计项目指导本科生和研究生，以及3)通过暑期项目和科学演示的激动人心的动手模块，扩大STEM代表性不足的学生和当地社区的参与。