To address mechanisms that enable the motor circuit to execute directional movement, we established a semiautomated in vivo calcium imaging system to identify activity patterns of the C. elegans motor circuit associated with directional movements (see Figure S1A available online; Experimental Procedures). Briefly, late larvae (L4) or adult animals expressing a genetic calcium sensor cameleon ( Miyawaki et al., 1997) in various motor circuit neurons were allowed to move and alter directions spontaneously on glass slides.

Fluorescent signals from the neuron soma were tracked over time; the intensity and positional change of the fluorescent objects provided indices for neuronal activity and the direction of movement, respectively. Calcium-insensitive Cabozantinib research buy cameleons served as negative controls for all reporters ( Figure 4 and Figure 6; Figure S3). We first examined the activity of AVA, AVE, and AVD premotor interneurons that were proposed to drive or modulate backing. Simultaneous imaging

of these tightly clustered neurons, which was only possible in animals with restricted movement (Experimental PF-02341066 supplier Procedures), revealed temporally correlated calcium profiles for AVA and AVE, indicating their coactivation and inactivation (Figure 1C; Figure S1B; Movie S1, part A). We did not detect activity in AVD (Figure 1C), which probably reflects their proposed role in touch-stimulated, instead of spontaneous, movement (Chalfie et al., 1985 and Wicks et al., 1996). To better correlate AVA and AVE activity with motion, we allowed animals to move more freely and imaged the interneuron pair as a single region of interest (ROI) (Experimental

Procedures). almost Consistent with previous reports for AVA (Ben Arous et al., 2010 and Chronis et al., 2007), the initiation of reversals (Figure 1D, dotted vertical lines) temporally correlated with a sharp increase of intracellular calcium in AVA/AVE (Figure 1D, upper trace, right). The period of gradual decline in the calcium transient correlated with continuous forward motion (Figure 1D; Movie S1, part B). Therefore, the activation of AVA/AVE is associated with backward motion. In contrast, the initiation of forward movements (Figure 1E, dotted vertical lines) generally corresponded with a calcium increase in AVB (Figure 1E, upper trace, right), the key premotor interneuron required for spontaneous forward movement (Chalfie et al., 1985 and Wicks et al., 1996), whereas a decrease of the calcium transient correlated with either a reduced forward velocity or reversals (Figure 1E), correlating AVB activation with forward motion. We could not record PVC, premotor interneurons that contribute to stimulated forward motion (Chalfie et al., 1985 and Wicks et al.