Kinetic image cytometry assays for neuronal activity/toxicity

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 effectsof 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. PUBLIC HEALTH RELEVANCE In human patients treated with anti-cancer drugs, or suffering from inherited disorders, brainneuron function is lost, leading to impaired speech, understanding, and decision making. It is unclear why this occurs, no therapies have yet been found to improve brain function. In this project, we will develop methods to produce brain in a dish neuronal cultures that are very small (each dish features 96 individual cultures) along with a specialized microscope that will record the activity of the neurons in an automated manner. The methods developed in this project will enable neurological researchersat universities, pharmaceutical companies, and government agencies to test hundreds to thousands of compounds for potential toxic or beneficial effects on brain function.