Electrophysiology has long been the gold standard for the characterization of electrical activity in excitable cells. Whole-cell voltage and current clamp have proven to be invaluable techniques to our understanding of ion channel biophysics and pharmacology. Mechanisms underlying intrinsic excitability of neurons and cardiomyocytes, and dynamics of neuronal networks in health and disease have also be elucidated using these electrophysiological approaches. However, with traditional electrophysiological techniques chemical access to the cell is required and it is difficult to assay electrical activity with high spatio-temporal accuracy in individual cells and larger populations of cells. The ability to measure activity from multiple cells simultaneously is critical for diseases such as epilepsy, which involves localized networks or may generalized to involve the entire brain. Additionally, traditional electrophysiological techniques require extensive training, and highly specialized equipment. Recent advances in the use of genetically encoded voltage indicators (GEVI) and genetically encoded calcium indicators (GECI) have allowed for the examination of excitable cells without the use of electrodes. This growing assortment of genetically encoded tools are available in an array of colors with varied sensitivity and kinetics. GEVIs such as ASAP1 and Ace2N-mNeon, and GECIs such as GCamp6F and JRGeco1a produce large voltage-dependent or calcium-dependent fluorescence changes with millisecond timescale kinetics that allow for highly accurate interrogation of electrical signals and calcium dynamics in excitable cells. This project will develop a mouse model that intergrates genetically encoded voltage indicators and calcium indicators into a Cre-reporter lines, to allow researchers to study electrical activity using optogentic techniques across a wide range of diseases.
Aim 1 will focus of the generation and benchmarking of plasmids encoding dual GEVI and GECI genes, while Aim 2 will be to generate a mouse model of the best GEVI/GECI combination identified in Aim 1, followed by validation and mapping of transgene expression and functional testing in a well characterized mouse model of epilepsy.
Developing technologies to study electrical activity in the brain and heart is critical for our understanding of both normal physiology and pathophysiology of many human diseases, including epilepsy. Existing techniques require extensive training and experience. This project will develop a novel tools, including a mouse model capable that will allow researchers to study electrical activity in the brain and heart cells across multiple fields of research.