It is desirable in behavioral studies to record the activity of neurons and muscles, but this has traditionally been difficult in C. elegans. We are using a transfectable calcium indicator, cameleon [Miyawaki et al., Nature 388:882], to overcome this barrier. To develop the technique, we focused on the pharyngeal muscle. The pharynx has been previously characterized behaviorally and electrically, primarily by the Avery lab, and is large enough to simplify imaging. In addition, calcium influx in muscles causes contraction, which provides an independent measure of activity. Using the
myo-2 promoter, we expressed cameleon in the pharynx and recorded distinctive calcium transients coupled to muscle contraction in intact animals. The duration of these transients varied in mutants of
egl-19, a voltage-gated calcium channel, consistent with previous electrical recordings [Lee et al., EMBO 16:1066]. In mutants of
unc-36, a channel-associated
a2 subunit, we found that transients were substantially increased in magnitude, suggesting an inhibitory role for the wild-type UNC-36 protein. This was surprising as coexpression studies of vertebrate homologues in Xenopus oocytes had found that the
a2 had many relatively subtle effects, including increasing expression levels of the pore-forming subunit. We are currently extending our study of muscular calcium transients to investigate the effects of neurotransmitters and mutations on the vulval muscles and their role in egg-laying behavior. In addition, we expressed cameleon using the pan-neuronal promoter
unc-119. We provided direct electrical stimulation through an extracellular electrode inserted through the cuticle and positioned near the nerve ring. Transients were observed coupled to the stimulation, indicating that calcium influx through voltage-gated calcium channels can be observed in neurons using this method. This raises the possibility of constructing a functional map of neuronal connectivity by electrically stimulating single neurons and recording the activity of their synaptic partners using cameleon.