Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA.
Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA.
Methods Mol Biol. 2021;2191:221-233. doi: 10.1007/978-1-0716-0830-2_14.
All-optical methods of probing in vivo brain function are advantageous for their compatibility with automated microscopy and fast spatial targeting of neural circuit excitation and response. Recent advances in optogenetic technologies allow simultaneous light activation of specific neurons and optical readout of neural activity via fluorescent calcium reporters, providing an attractive opportunity for high-throughput screening assays that directly assess dynamic neural function in vivo. Here we describe a method to automatically record optogenetically activated neural responses in living, hydrogel-embedded organisms over many hours in a multiwell plate format. This method is suitable for screening the neural effects of hundreds of chemical compounds and assessing the time course of bioactivity over 12 h or more. As examples, we show the suppression of neural responses over time with various concentrations of two voltage-gated calcium channel blockers and a full-plate screen of 320 chemicals with positive and negative controls in a single experiment.
用于探测活体大脑功能的全光学方法具有与自动化显微镜兼容、快速靶向神经回路激发和响应等优点。光遗传学技术的最新进展允许通过荧光钙报告器对特定神经元进行同时光激活和神经活动的光学读出，为高通量筛选测定法提供了有吸引力的机会，该方法可直接评估体内动态神经功能。本文描述了一种方法，可在多孔板格式中以活的、水凝胶嵌入的生物体为对象，自动记录经光遗传学激活的神经反应，可持续数小时。该方法适用于筛选数百种化合物对神经的影响，并在 12 小时或更长时间内评估生物活性的时程。作为示例，我们展示了随着两种电压门控钙通道阻滞剂浓度的增加，神经反应随时间的抑制作用，以及在单个实验中对 320 种化学物质进行阳性和阴性对照的全板筛选。
