Kinetic image cytometry assays for neuronal activity/toxicity

描述(申请人提供)：该项目是Vala Sciences Inc.与桑福德·伯纳姆医学研究所的第一阶段合作项目，Vala Sciences Inc.是一家专门开发基于细胞的高含量/高通量分析的小企业，Sanford Burnham医学研究所是生物医学研究的全球领先者。该项目将开发一种分析方法(动态图像细胞测量仪-神经元，或KIC-神经元)，以量化测试化合物对神经元传递和利用光遗传学方法发展神经元细胞电路的影响。对于KIC-Neuron，原代大鼠海马神经元将以高通量形式(96或384孔)培养，然后与编码光激活通道视紫红质的Lenti病毒转导，并加载DNA荧光指示剂(以显示细胞核)和细胞内钙离子。这 将通过使用闪光使细胞去极化，这导致神经元培养中的神经传递和突触后神经元细胞内钙的升高，这是通过自动显微镜和图像分析技术进行量化的。在活性分析之后，细胞也被固定、标记和成像以寻找神经元生物标记物，这些生物标记物可以在逐个细胞的基础上与神经活动相关联。该测试系统将用于量化短期接触化合物的影响(如活性测试期间离子通道阻滞剂或神经递质受体拮抗剂的存在)，也将用于量化化合物对神经回路的潜在长期影响。后一种现象与“化学脑”状态有关，后者是癌症患者在化疗后出现的一种认知能力丧失，也可能与其他形式的记忆力丧失和痴呆症有关，包括阿尔茨海默氏症。包括美国环境保护局在内的政府机构、美国国立卫生研究院(NCI和NHCLBI)以及制药和学术研究人员都会对这种测试感兴趣。KIC-Neuron将被商业化，用于Vala的合同研究筛选，并开发成以套件、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.