Optogenetic Manipulation Of Neural Activity In Freely Moving Caenorhabditis elegans

Abstract

We present an optogenetic illumination system that is capable of real-time light delivery with high spatial resolution to specified cellular targets in freely moving C. elegans. In our system, a tracking microscope and high-speed video camera records the posture and motion of an unrestrained worm expressing Channelrhodopsin-2 or Halorhodopsin/NpHR in specific cell types. Custom image processing software analyzes the position of a worm within each video frame, and then rapidly estimates the locations of targeted cells. The software then instructs a digital micromirror device to illuminate targeted cells with laser light of the appropriate wavelengths to stimulate or inhibit activity. Since each cell in an unrestrained worm is a rapidly moving target, our imaging and analysis system operates at high speed \((\sim 50\) frames per second) to provide high spatial resolution \((\sim 30 \mu m)\). To demonstrate the accuracy, flexibility, and utility of our system, we present optogenetic analyses of the worm motor circuit, egg-laying circuit, and mechanosensory circuits that were not possible with previous methods.