Resolving neuronal circuits of emotional memory with confocal microscopy

用共焦显微镜解析情绪记忆的神经元回路

• 批准号: RTI-2023-00315
• 负责人: Rozeske, Robert
• 金额: $ 10.93万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Research Tools and Instruments
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=764220
• 关键词: Resolving; neuronal; circuits; emotional; memory

We request funds to address a critical gap in the research infrastructure at the University of Toronto Scarborough (UTSC) with the purchase of a laser scanning confocal fluorescence microscope for imaging mammalian tissue. Together our labs consist of 27 HQP that share a goal to understand how defined neuronal circuits give rise to emotional behaviour. At UTSC there is no core laser scanning confocal microscope available to our labs. Laser scanning confocal microscopes are located at other University of Toronto sites, but these locations charge $36 per hour and are over 30 km from the UTSC campus. Together these factors generate an unsustainable situation. If funds are not received our research programs will be severely impeded because either (1) we must travel long distances and pay exorbitant fees for offsite microscopes of (2) regress our research hypotheses to a level that is out of step with modern behavioural neuroscience and current publishing standards. The applicants require a confocal microscope because interpreting their experimental results hinges upon generating fine spatial resolution images of neuronal tissue that can disambiguate multiple fluorescent protein targets. The applicants share a conventional widefield fluorescence microscope that is unable to provide focused single cell resolution due to background fluorescence that arises from tissue outside of the field of view. This issue is compounded when imaging multiple fluorescent proteins in tissue that contains multiple cell layers and complex neuronal morphology. A laser scanning confocal microscope is the only method that can dissect neuronal circuits at this level because it optically sections neural tissue in the axial (depth) plane to create unambiguous, high-resolution, neuronal images. Optical sectioning technology is essential to correct the current misalignment between the specificity of our sophisticated neuronal manipulation and recording techniques, and our outdated current microscopy infrastructure. Reception of RTI funds will correct this disparity and allow us to perform pioneering research. Without a laser scanning confocal microscope on site, the breadth and impact of our research scope is restricted and diminished because we cannot validate transgenic, viral, and neural recording strategies at the levels currently required for publication in high-impact neuroscience journals. Instead, reception of a confocal microscope will (1) allow our research programs to discover activity-dependent, genetically-defined, and pathway-specific neuronal circuits that generate emotional behaviour, (2) modernize UTSC's research infrastructure to the standard of most Canadian neuroscience programs, (3) fill a technological gap that provides HQP with training and research opportunities using a fundamental microscopy method in the life sciences.

我们申请资金，以解决多伦多斯卡伯勒大学(UTSC)研究基础设施中的一个关键缺口，购买用于为哺乳动物组织成像的激光扫描共聚焦荧光显微镜。我们的实验室总共由27个HQP组成，他们都有一个共同的目标，那就是了解明确的神经元回路是如何引发情绪行为的。在UTSC，我们的实验室没有核心激光扫描共聚焦显微镜。激光扫描共聚焦显微镜位于多伦多大学的其他地点，但这些地点每小时收费36美元，距离多伦多大学校园超过30公里。这些因素加在一起，造成了一种不可持续的局面。如果没有收到资金，我们的研究计划将受到严重阻碍，因为(1)我们必须长途跋涉，支付昂贵的场外显微镜费用；(2)将我们的研究假设倒退到与现代行为神经科学和当前出版标准不同步的水平。申请者需要共聚焦显微镜，因为解释他们的实验结果取决于产生精细的神经元组织空间分辨率图像，可以消除多个荧光蛋白目标的歧义。申请人共享传统的广域荧光显微镜，由于视野外的组织产生的背景荧光，该显微镜不能提供聚焦的单细胞分辨率。当对包含多个细胞层和复杂神经元形态的组织中的多个荧光蛋白进行成像时，这个问题变得复杂起来。激光扫描共聚焦显微镜是唯一可以在这个水平上解剖神经元电路的方法，因为它在轴向(深度)平面上对神经组织进行光学切片，以创建明确、高分辨率的神经元图像。光学切片技术对于纠正目前我们复杂的神经元操作和记录技术的特殊性与我们过时的显微镜基础设施之间的错位是必不可少的。接受RTI资金将纠正这种差距，并使我们能够开展开创性的研究。如果现场没有激光扫描共聚焦显微镜，我们研究范围的广度和影响就会受到限制和影响，因为我们无法验证转基因、病毒和神经记录策略，目前要求在高影响力的神经科学期刊上发表。取而代之的是，接受共聚焦显微镜将(1)使我们的研究计划能够发现产生情绪行为的活动依赖、基因定义和途径特定的神经回路，(2)使UTSC的研究基础设施现代化，达到大多数加拿大神经科学计划的标准，(3)填补为HQP提供培训和研究机会的技术空白，使用生命科学的基础显微镜方法。