Interrogation of Downstream Calcium Responses via Optogenetic and Mechanical Stimulation in C. elegans

Abstract

Optogenetic methods have contributed substantially to the experimental analysis of neural function and behavior. In the nematode Caenorhabditis elegans, a commonly used model organism for studying neural circuits, optogenetic stimulation of several neuromuscular networks has induced identical behavioral responses to those resulting from mechanical stimulation. This has lead to the acceptance of optogenetic methods as physiologically relevant stimuli. Despite the similarities in macroscopic phenomenology, the calcium signal in downstream neurons resulting from optogenetic stimulation has yet to be investigated and compared to the calcium signal in downstream neurons resulting from environmental stimulation. The optogenetic channel, channelrhodopsin-2, will be expressed under the control of the mec-4 sensory neuron promoter in C. elegans. Additionally, a synthetic calcium sensor, RCaMP1h, will be expressed separately under the control of a specific downstream interneuron promoter, dbl-1, in order to compare the calcium signaling in this interneuron, AVA, resulting from optogenetic and mechanical stimulation. Using software we developed, these freely swimming worms will be tracked and optogenetically stimulated using a light source localized to the sensory neurons of interest. Traditional mechanical stimulation with an eyelash will be carried out in tandem. The downstream calcium responses observed during each stimulation will be compared to determine if the optogenetic stimulation resulted in the same physiological response as mechanical stimulation. This study will provide insight into the relevance of optogenetics as a physiological probe of neural networks.