Meeting Abstract
Optogenetics is the process by which a light-operated ion channel is expressed in neurons to enable photon-activation of action potential firing in neurons. This project identified mechanisms of innovation in the usage of optogenetics in selective activation of neural networks. Building on the work done previously with ChrimsonR, a red-shifted variant of channelrhodopsin-2 identified by the Boyden lab from a novel microbe, we identified and optimized expression of the protein. Using epifluorescent and confocal microscopy, we compared GFP expression between original and optimized constructs of the plasmid by transiently transfecting the plasmid into HT22 cells, a murine neuronal cancer cell line. Unlike several other promoters we concurrently tested, we find that the CaMKII-alpha promoter does not express efficiently in the undifferentiated HT22 cells. We have analyzed the effect of additives required for differentiation to enhance expression from the CaMKII-alpha promoter. Optogenetic stimulation during electrophysiology confirmed expression of ChrimsonR, and evaluated the efficacy of the new plasmid. Adeno-associated virus produced with the new ChrimsonR plasmid was purified using an iodixanol density gradient centrifugation, dialysis, and anion-exchange chromatography. The purified adeno-associated virus will be surgically injected into mice to test the plasmids efficacy in vivo. Thus, we have created an optimized optogenetics vector that will enable optical control of two different inputs.
