Resolving neuronal circuits of emotional memory with confocal microscopy

我们请求资金来解决多伦多大学斯卡伯勒分校（UTSC）研究基础设施的关键缺口，购买用于哺乳动物组织成像的激光扫描共聚焦荧光显微镜。我们的实验室共有27个HQP，他们的共同目标是理解已定义的神经回路是如何引起情绪行为的。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，为异地显微镜支付高昂的费用；(2)我们的研究假设将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。申请人需要一个共聚焦显微镜，因为解释他们的实验结果取决于生成精细的神经组织空间分辨率图像，可以消除多个荧光蛋白目标的歧义。申请人共用一个传统的宽视场荧光显微镜，由于视野外组织产生的背景荧光，该显微镜无法提供聚焦的单细胞分辨率。当在含有多个细胞层和复杂神经元形态的组织中成像多个荧光蛋白时，这个问题变得更加复杂。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回路的方法，因为它在轴向（深度）平面上对神经组织进行光学切片，以产生明确的、高分辨率的神经元图像。光学切片技术对于纠正当前复杂的神经元操作和记录技术的特异性与我们过时的当前显微镜基础设施之间的错位至关重要。接受RTI基金将纠正这种差距，使我们能够进行开创性的研究。如果现场没有激光扫描共聚焦显微镜，我们的研究范围的广度和影响就会受到限制和削弱，因为我们无法验证转基因、病毒和神经记录策略，这些策略目前需要在高影响力的神经科学期刊上发表。相反，共聚焦显微镜的接收将(1)允许我们的研究项目发现活动依赖的，基因定义的，以及产生情感行为的通路特定的神经元回路，(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项目的标准，(3)填补技术空白，为HQP提供使用生命科学基本显微镜方法的培训和研究机会。

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.