Confocal calcium imaging and electrophysiology in live brain cells for neuroscience research at Université de Sherbrooke

我们的研究重点是脑干和脊髓细胞在运动控制中的作用（Ryczko博士），G蛋白偶联受体（GPCR）在疼痛调制中的作用（Sarret博士）和δ阿片受体（DORs）在嗅球中的作用（Gendron博士）。该提案旨在升级两种用于钙成像和膜片钳记录活脑准备的设置。所要求的设备将成为舍布鲁克大学（UdeS）神经科学研究的新电生理学/钙成像平台的核心。我们目前的设备（设置1）包括用于钙成像的落射荧光显微镜和用于膜片钳记录的电生理学装置。我们建议将这种显微镜升级为共聚焦技术。这需要激光光源和带有计算机和软件的共焦单元。使用我们目前的落射荧光显微镜，光散射阻止了从位于脑组织深处的细胞获得荧光信号。通过去除失焦光，共聚焦技术将使我们能够使用具有精确分辨率的荧光钙传感器轻松记录不同深度脑细胞的活动，并进行精确的光遗传学刺激。这将使我们能够确定招募和协调脑细胞活动的重要机制。我们的膜片钳记录也将从这次升级中受益。然后，我们将用贴片微量移液器在我们目前的系统无法达到的组织深度处靶向感兴趣的荧光细胞。此外，我们将回收设置1中的主要项目（高速摄像机，LED光源，计算机和软件），以构建第二个上荧光钙成像/膜片钳设置（设置2），这将非常适合在小鼠脑切片中记录。* 所要求的设备将使我们的记录能力增加一倍，并使我们的NSERC研究计划达到一个新的水平，使我们能够解决神经科学中精确的药理学和生理学机制问题。通过进入大脑和脊髓的深层，Ryczko博士将解码蝾螈运动系统中细胞募集的基本原理，蝾螈是一种可以在损伤后完全修复其神经系统的动物。Sarret博士将开发膜片钳电生理学和钙成像的应用，这将进一步巩固他对GPCRs在疼痛神经网络中作用的发现。Gendron博士将首次获得由作用于哺乳动物嗅球中DORs的化合物引起的细胞活动。* 该设置将为我们的机构带来第一个综合平台，结合共聚焦显微镜，钙成像和膜片钳电生理学，为学生提供最先进的神经科学技术的实践培训。

Our research focuses on the role of brainstem and spinal cells in locomotor control (Dr. Ryczko), the role of G-protein coupled receptors (GPCRs) in pain modulation (Dr. Sarret) and the role of delta opioid receptors (DORs) in the olfactory bulbs (Dr. Gendron). The proposal aims at upgrading two setups for calcium imaging and patch-clamp recordings in live brain preparations. The requested equipment will be the core of a new electrophysiology/calcium imaging platform for neuroscience studies at Université de Sherbrooke (UdeS).******Our current equipment (setup1) comprises an epifluorescent microscope for calcium imaging and an electrophysiology rig for patch-clamp recordings. We propose to upgrade this microscope to confocal technology. This requires a laser light source and a confocal unit with computer and software. With our current epifluorescent microscope, light scattering prevents the acquisition of fluorescent signals from cells located deep in the brain tissue. By removing out-of-focus light, confocal technology will allow us to record easily the activity of brain cells at different depths using fluorescent calcium sensors with exquisite resolution, and to perform precise optogenetic stimulations. This will allow us to identify important mechanisms underlying the recruitment and coordination of brain cell activities. Our patch-clamp recordings will also benefit from this upgrade. We will then target the fluorescent cells of interest with patch micropipettes at tissue depths that we cannot reach with our current system. In addition, we will recycle the main items from setup1 (high speed camera, LED light source, computer and software) to build a second epifluorescent calcium imaging/patch-clamp setup (setup2), which will be ideally suited for recordings in mouse brain slices. ***The requested equipment will double our capacity for such recordings and bring to a new level our NSERC research programs by enabling us to address precise pharmacological and physiological mechanistic questions in neuroscience. By having access to the deep layers of the brain and spinal cord, Dr. Ryczko will decode the principles underlying cell recruitment in the locomotor system of the salamander, an animal that can fully repair its nervous system after a lesion. Dr. Sarret will develop the use of patch-clamp electrophysiology and calcium imaging and this will fasten further his discoveries on the role of GPCRs in pain neural networks. Dr. Gendron will gain access for the first time to the cellular activities evoked by compounds acting on DORs in the olfactory bulbs of mammals. ***The setup will bring to our institution the first comprehensive platform combining confocal microscopy, calcium imaging and patch-clamp electrophysiology, providing hands-on training to the students with state-of-the-art neuroscience techniques.**