UC Berkeley

Optogenetics, the ability to control cells with light, has revolutionized neuroscience by endowing researchers with hyper precise tools which can dissect neural circuits. However, the use of optogenetic effectors is not limited to academic studies on neurons. This dissertation examines two alternative applications of optogenetics: to study glia-neuron interactions and to therapeutically restore light sensitivity to degenerate retinas. In order express these optogenetic effectors, Adeno-Associated Virus (AAV) was used to deliver transgenes.

Over the course of these studies, the ability to specifically target certain types of cells became paramount. Specifically, special AAV capsid variants had to be used in order to transduce Müller glia, the principal glia cell of the retina. The capsid variants, 7m8 and ShH10, are not specific for Müller cells, so different glial promoters were investigated. Two promoters evaluated, GLAST and gfaABC1D, had unexpected expression profiles. Although GLAST is a Müller cell specific protein, the GLAST promoter was unable to restrict transgene expression to Müller glia. However, the smaller the gfaABC1D promoter was able to completely restrict transgene expression to retinal glia in both health and disease.

With new methods to selectively transduce Müller glia, their role in glutamate uptake was investigated. The electrogenic transporters used to transport glutamate against its concentration gradient require a hyperpolarized membrane potential made possible through high expression of potassium channels. If glutamate uptake was dependent on Müller cell membrane potential, then transient depolarization of the Müller glia should inhibit glutamate uptake. To transiently depolarize Müller cells, they were targeted to express a bistable channelrhodopsin2 (ChR2) mutant (C128S/D156A, “BiChR2”). The effect of BiChR2 induced Müller glia depolarization on the retinal light response was investigated by examining the electroretinogram (ERG). While there were no significant changes to the ON-bipolar cell generated ERG B-wave, subtle temporal changes in arose that should be investigated further.

Optogenetics have a therapeutic potential to restore vision in degenerate retinas. People with late stage retinal degenerative disease lose sight as a consequence of photoreceptor death. However, other retinal cells remain. Endowing the surviving retinal cells with the ability to sense light through optogenetics could restore sight. The effectiveness of optogenetics tools used for vision restoration were analyzed based on their light and temporal sensitivity.