A patterned photostimulation microscope for studying neurons and microcircuitry

用于研究神经元和微电路的图案光刺激显微镜

• 批准号: 8052038
• 负责人: Thomas S Otis
• 金额: $ 60万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8052038
• 关键词: Biological; Brain Stem; Cells; Cellular biology; Cerebellar cortex structure; Collaborations; Contract Services; Coupling; Custom; Elements; Epithelium; Equipment; Exocytosis; Funding; Halorhodopsins; Image; Individual; Label; Laboratories; Light; Lighting; Measurement; Membrane Potentials; Microscope; Microscopy; Molecular; Nervous System Physiology; Neuronal Plasticity; Neurons; Neurotransmitters; Optical reporter; Optics; Pattern; Perfusion; Photons; Physiologic pulse; Physiological; Physiology; Principal Investigator; Proteins; Research; Resolution; Respiratory Center; Source; Specimen; Speed; Surveys; System; Technology; Temperature; Time; United States National Institutes of Health; Work; base; charge coupled device camera; design; detector; electrical measurement; instrument; instrumentation; medical schools; member; neurophysiology; novel; photo switch; relating to nervous system; research study; sensor; tool; two-photon; voltage

DESCRIPTION (provided by applicant): This application describes a multifunctional microscope system capable of patterned excitation of various optically sensitive targets. This custom-designed instrument will be used to explore cutting edge questions in neural cell biology, single neuron physiology, and microcircuit function. It will incorporate a pulsed-IR source for PMT-based two-photon imaging; in addition it will have another optical path allowing for patterned, single-photon illumination at various wavelengths (405, 473 and 594 nm). Patterned illumination will be accomplished with spatial light modulator (SLM) technology which permits user defined patterns of illumination to be delivered to the specimen plane (Lutz et al., 2008; Nikolenko et al., 2008). A high speed CCD camera will be used as an alternative detector for experiments in which high time resolution (i.e.> 1 kHz) optical measurements are necessary. This will allow optical measurements of electrical activity from many single neurons labeled with a novel voltage sensor developed by one of the group members. A survey of existing microscopy capabilities at UCLA demonstrates that no equipment with these capabilities is available; indeed two of the users have collaborations with overseas laboratories to enable the work. This instrument should allow experimental manipulation of several cutting edge optical tools with unparalleled spatial and temporal precision. These include: 1) photoactivatable or photoconvertible proteins (PA-GFP, Dendra2, DRONPA) useful for cell biological studies of nervous system function, 2) caged neurotransmitters that can be used to photostimulate individual neurons or circuits, 3) photosensitive channels (channel- and halorhodopsin) or reversible photo switches (PALs) to activate or inactivate specific circuit elements, and 4) a novel, ultra rapid optical reporter of membrane potential that enables simultaneous measurements of neural activity from many individual neurons. The application details how the instrument system will directly benefit more than 10 currently funded NIH projects on which the five major users serve as Principal Investigators. Research in these laboratories is directed at understanding fundamental issues in neurophysiology and neural cell biology including the molecular mechanisms involved in excitation-contraction coupling (Vergara), exocytosis (Schweizer), and neuronal plasticity (Martin) as well as aspects of microcircuit function in brainstem respiratory centers (Feldman), vestibular epithelium (Schweizer), and in cerebellar cortex (Otis). Various UCLA Departments and the UCLA School of Medicine will provide $92,922 in funding to enable the purchase of the system; they will also contribute more than $46,000 per year in ongoing funding to cover the service contract for the equipment. In addition, the user group will provide approximately $90,000 of electrophysiological, micro perfusion, and temperature control equipment so that neurophysiological experiments can be done in parallel with optical measurements under controlled physiological conditions. These pledges totaling well over $250,000 in institutional support are evidence of the scientific enthusiasm and intense need for this type of instrumentation at UCLA. PUBLIC HEALTH RELEVANCE: This grant would provide funding for a state-of-the-art microscope system incorporating advanced optical technology that enables parallel manipulation and/or measurement of neuronal activity within various brain microcircuits. The system will also permit sophisticated tracking and manipulation of optically-tagged signaling proteins with unprecedented precision in cell biological experiments. UCLA has a large and highly collaborative neuroscience research community and this equipment will accelerate progress in the laboratories of the NIH-funded major users whose research on basic mechanisms underlying disorders of breathing, movement, balance, and learning is currently funded by more than 15 PHS grants.

描述（由申请人提供）：本申请描述了一种能够对各种光学敏感目标进行图案化激发的多功能显微镜系统。这种定制设计的仪器将用于探索神经细胞生物学、单神经元生理学和微电路功能中的前沿问题。它将采用脉冲红外源，用于基于 PMT 的双光子成像；此外，它还有另一个光路，可实现各种波长（405、473 和 594 nm）的图案化单光子照明。图案照明将通过空间光调制器 (SLM) 技术来实现，该技术允许将用户定义的照明图案传递到样本平面（Lutz 等人，2008 年；Nikolenko 等人，2008 年）。高速 CCD 相机将用作需要高时间分辨率（即 > 1 kHz）光学测量的实验的替代探测器。这将允许对许多单个神经元的电活动进行光学测量，这些神经元标记有由小组成员之一开发的新型电压传感器。对加州大学洛杉矶分校现有显微镜功能的调查表明，没有具有这些功能的设备可用；事实上，其中两个用户与海外实验室合作来完成这项工作。 该仪器应该能够以无与伦比的空间和时间精度对多种尖端光学工具进行实验操作。这些包括：1）可光激活或光转换蛋白（PA-GFP、Dendra2、DRONPA）可用于神经系统功能的细胞生物学研究，2）可用于光刺激单个神经元或电路的笼状神经递质，3）光敏通道（通道-和盐视紫红质）或可逆光电开关（PAL）来激活或失活特定的电路元件，以及4）一种新型的、超快速的膜电位光学报告器，可以同时进行测量来自许多单个神经元的神经活动。 该申请详细介绍了该仪器系统将如何直接使目前资助的 10 多个 NIH 项目受益，其中五个主要用户担任首席研究员。这些实验室的研究旨在了解神经生理学和神经细胞生物学的基本问题，包括参与兴奋-收缩耦合（Vergara）、胞吐作用（Schweizer）和神经元可塑性（Martin）的分子机制以及脑干微电路功能的各个方面呼吸中枢（Feldman）、前庭上皮（Schweizer）和小脑皮质（Otis）。 加州大学洛杉矶分校各个系和加州大学洛杉矶分校医学院将提供 92,922 美元的资金来购买该系统；他们还将每年提供超过 46,000 美元的持续资金，用于支付设备的服务合同。此外，用户组还将提供约9万美元的电生理学、微灌注和温度控制设备，以便在受控生理条件下可以与光学测量并行进行神经生理学实验。这些承诺提供总计超过 250,000 美元的机构支持，证明了加州大学洛杉矶分校对此类仪器的科学热情和强烈需求。 公共健康相关性：这笔赠款将为最先进的显微镜系统提供资金，该系统采用先进的光学技术，能够并行操作和/或测量各种大脑微电路内的神经元活动。该系统还将允许在细胞生物实验中以前所未有的精度复杂地跟踪和操纵光学标记的信号蛋白。加州大学洛杉矶分校拥有庞大且高度协作的神经科学研究社区，该设备将加速 NIH 资助的主要用户实验室的进展，这些用户对呼吸、运动、平衡和学习障碍的基本机制的研究目前由超过 15 个 PHS 资助补助金。

项目成果

Ectopic Expression of α6 and δ GABAA Receptor Subunits in Hilar Somatostatin Neurons Increases Tonic Inhibition and Alters Network Activity in the Dentate Gyrus.

肺门生长抑素神经元中 α6 和 α GABAA 受体亚基的异位表达增加了强直抑制并改变了齿状回的网络活动。

• 发表时间: 2015-12-09
• 作者: Tong, Xiaoping;Peng, Zechun;Zhang, Nianhui;Cetina, Yliana;Huang, Christine S;Wallner, Martin;Otis, Thomas S;Houser, Carolyn R
• 通讯作者: Houser, Carolyn R