An optogenetic platform for high-throughput interrogation of cellular basis of physiological function in zebrafish

我们将利用最近的技术突破，使细胞功能与动物的生理和行为直接联系起来。确定多细胞生物体中细胞活性的结果传统上受到无法直接操纵感兴趣的细胞的阻碍，特别是在活体动物中。大多数研究细胞功能的方法，如药物或电刺激，由于广泛的脱靶效应而存在缺陷。因此，使用这些技术，我们理解细胞功能如何与生理和行为相关的能力是有限的。这些限制现在可以通过使用对特定波长的光做出反应的蛋白质来克服。照亮表达这些光敏蛋白质的细胞可以立即塑造细胞的活性，同时不影响其他细胞。通过遗传控制哪些细胞表达光敏分子，一种称为光遗传学的方法，我们可以直接影响细胞活性。这种方法在具有透明动物的生命阶段的物种中特别有效，因此光很容易传输到活组织，就像斑马鱼幼虫一样。斑马鱼的光遗传学研究，其中生理或行为的变化可以直接归因于光诱导的细胞活性变化，导致我们对动物生理学的理解取得了重要进展。 渥太华大学的一个多学科研究小组在研究斑马鱼的细胞功能和动物生理学和行为方面具有专业知识，他们将组装一个专门用于研究斑马鱼的光遗传学系统。该系统将由两个平台组成。第一个平台由显微镜和光图案化装置组成，以选择性刺激斑马鱼幼虫特定区域内的靶细胞。光遗传学刺激的结果将由足够快和灵敏的相机监测，以检测细胞信号传导中的甚至微小和快速的变化。该平台将使我们能够在微观水平上研究细胞活动对动物生理学的作用。第二个平台将允许同时观察和分析多达96只斑马鱼幼虫的光遗传学激活的行为后果，从而大大提高研究成果。该平台的另一个组成部分将使研究幼虫阶段的光遗传学刺激如何影响成虫阶段的行为成为可能。该系统将使研究人员能够对特定细胞类型在许多重要生物过程中的作用进行基本的了解。光遗传学正在迅速成为生理学研究的标准技术，我们的HQP将为生物学，生物化学，生物医学工程，生态学和生理学等领域所需的尖端培训提供宝贵的机会。

We will take advantage of recent technological breakthroughs that now enable cellular function to be directly linked to animal physiology and behaviour. Determining the outcome of cell activity in multicellular organisms traditionally has been hampered by an inability to directly manipulate cells of interest, particularly in living animals. Most methods of studying cell function such as pharmacological agents or electrical stimulation are flawed owing to widespread off-target effects. Therefore, our ability to understand how cell function relates to physiology and behaviour is limited using these techniques. These limitations can now be overcome through the use of proteins that respond to light of specific wavelengths. Illuminating cells expressing these light-sensitive proteins can instantly shape the activity of the cells, while leaving other cells unaffected. By genetically controlling which cells express the light-sensitive molecules, an approach termed optogenetics, we can directly influence cell activity. The approach is particularly powerful in species with life stages where animals are transparent, so that light is readily transmitted to living tissue, as is the case for larval zebrafish. Optogenetics studies in zebrafish where changes in physiology or behaviour can be directly attributed to light-induced changes in cell activity have led to important advances in our understanding of animal physiology. A multidisciplinary team of researchers at the University of Ottawa with expertise in studying cell function and animal physiology and behaviour in zebrafish will assemble an optogenetics system specialized for the study of zebrafish. This system will be comprised of two platforms. The first platform consists of a microscope and a light patterning device to enable the selective stimulation of targeted cells within specific regions of zebrafish larvae. The outcome of optogenetic stimulation will be monitored by a camera sufficiently fast and sensitive to detect even minute and rapid changes in cell signalling. This platform will allow us to study the role of cell activity on animal physiology at the microscopic level. A second platform will allow simultaneous observation and analysis of the behavioral consequences of optogenetic activation in up to 96 larval zebrafish at a time, dramatically increasing research output. Another component of this platform will enable the study of how optogenetic stimulation at larval stages shapes behaviour in adult stages. The proposed system will allow researchers to make fundamental insights into the roles of specific cell types on many important biological processes. Optogenetics is rapidly become a standard technique for physiological studies and our HQP will be provided with an invaluable opportunity for the cutting edge training needed for fields such as biology, biochemistry, biomedical engineering, ecology, and physiology.