Optical Imaging of Calcium Neurotechnique Transients in Neurons and Pharyngeal Muscle of C. elegans

Summary omy of C. elegans presents a number of difficulties for using traditional dyes for calcium imaging. The relatively Electrophysiology and optical indicators have been impermeant cuticle prevents uptake of bath-applied used in vertebrate systems to investigate excitable dye, and the hydrostatic skeleton makes dissection and cell firing and calcium transients, but both techniques microinjection difficult. Although large cells, such as oohave been difficult to apply in organisms with powerful cytes and intestinal cells, have been successfully loaded reverse genetics. To overcome this limitation, we ex- with calcium-sensitive dye through microinjection, neupressed cameleon proteins, genetically encoded cal- rons and even many muscle cells are too small to reliably cium indicators, in the pharyngeal muscle of the load in this way. Moreover, even when successful, minematode worm Caenorhabditis elegans. In intact croinjection has been limited to single cells (Dal Santo transgenic animals expressing cameleons, fluores- et al., 1999). cence ratio changes accompanied muscular contrac- To circumvent these problems, we are using the caltion, verifying detection of calcium transients. By com- cium indicator protein cameleon (Miyawaki et al., 1997) paring the magnitude and duration of calcium influx to image calcium transients in intact C. elegans. Camelin wild-type and mutant animals, we were able to de- eons are composed of four domains: cyan fluorescent termine the effects of calcium channel proteins on protein (CFP), calmodulin, M13 (a calmodulin binding pharyngeal calcium transients. We also successfully domain), and yellow fluorescent protein (YFP). In low used cameleons to detect electrically evoked calcium calcium concentrations, calmodulin is not bound to transients in individual C. elegans neurons. This tech- M13, and the protein is in a poor configuration for fluonique therefore should have broad applications in ana- rescence resonance energy transfer (FRET) between lyzing the regulation of excitable cell activity in geneti- CFP and YFP. Excitation of CFP leads primarily to CFP cally tractable organisms. emission, with lesser energy transfer to YFP and YFP emission. Upon an increase in calcium concentration,