Kinetic image cytometry assays for neuronal activity/toxicity

描述（由申请人提供）：拟议项目是Vala Sciences Inc（一家专门开发基于细胞的高含量/高通量检测的小型企业）与Sanford Burnham医学研究所（生物医学研究的全球领导者）之间的I期STTR合作。该项目将开发一种测定法（动力学图像细胞仪-神经元，或KIC-神经元），以利用光遗传学方法量化测试化合物对神经元传递和神经元细胞回路发育的影响。对于KIC-神经元，原代大鼠海马神经元将以高通量形式（96或384孔）培养，然后用编码光激活通道视紫红质的慢病毒转导，并加载DNA的荧光指示剂（以可视化细胞核）和细胞内钙。这 将通过使用闪光使细胞去极化，这导致神经元培养物中的神经传递和突触后神经元中细胞内钙的升高，这通过自动显微镜和图像分析技术进行定量。在活性测定之后，还对细胞进行固定、标记和成像以获得神经元生物标志物，其可以在逐个细胞的基础上与神经活性相关联。该试验系统将用于定量短期暴露于化合物的影响（如活性试验期间存在离子通道阻滞剂或神经递质受体拮抗剂），以及定量化合物对神经回路的潜在长期影响。后一种现象与“化学脑”状况有关，这是癌症患者在化疗后发展的认知能力丧失，并且可能与其他形式的记忆丧失和痴呆症有关，包括阿尔茨海默氏症。该分析将引起政府机构的兴趣，包括美国环境保护署和NIH研究所（NCI和NHCLBI）以及制药和学术研究人员。KIC-NEURON将被商业化用于Vala的合同研究筛选，并开发成一条以套件、软件和仪器为特色的产品线。

DESCRIPTION (provided by applicant): The proposed project is a Phase I STTR collaboration between Vala Sciences Inc, a small business that specializes in the development of cell-based high content/high throughput assays, and the Sanford Burnham Medical Research Institute, a worldwide leader in biomedical research. The project will develop an assay (Kinetic Image Cytometer-Neuron, or KIC-Neuron) to quantify the effect of test compounds on neuronal transmission and the development of neuronal cell circuits utilizing optogenetic methods. For KIC-Neuron, primary rat hippocampal neurons will be cultured in a high throughput format (96 or 384 well), then transduced with lenti-viruses encoding light-activated channel rhodopsin, and loaded with fluorescent indicators of DNA (to visualize the nuclei) and intracellular calcium. This will enable depolarization of the cells via use of a flash of light, which leads to neurotransmission across the neuronal cultures and elevation of intracellular calcium in post-synaptic neurons, which is quantified via automated microscopy and image analysis techniques. Following the activity assay, the cells are also fixed, labeled, and imaged for neuronal biomarkers, which can be correlated on a cell-by-cell basis to neuroactivity. The assay system will be used to quantify the effects of short -term exposure to compounds (such as the presence of ion channel blockers or antagonists of neural transmitter receptors during the activity assay), and also to quantify potential long-term effects of compounds on the neural circuits. The latter phenomenon is relevant to the "Chemo-brain" condition, which is a loss cognitive ability that develops in cancer patients following chemotherapy, and may be related to other forms of memory loss and dementia, as well, including Alzheimer's. The assay will be of interest to government agencies including the US Environmental Protection Agency, and NIH institutes (NCI and NHCLBI) as well as to both pharmaceutical and academic researchers. KIC-NEURON will be commercialized for Vala's contract research screening as well as developed into a product line featuring kits, software, and instrumentation.