An inverse correlation between AVA/AVE and AVB activation with the directionality of the movement of C. elegans implies their reciprocal activation. To test this possibility, we simultaneously imaged AVE and AVB, the only neuron pair that is spatially separated sufficiently to permit unambiguous tracking of calcium signals in animals with restricted

movement. Indeed, Lapatinib the calcium change in AVE was anticorrelated with the calcium change in AVB ( Figure 1F; Movie S1, part C). These results suggest that reciprocal activation and inactivation between the forward (AVB) and backward (AVA/AVE) premotor interneurons correlate with the directional movement of C. elegans. The C. elegans wiring diagram predicts that AVA/AVE and AVB innervate the A and B motoneurons, respectively, via chemical and/or electrical synapses ( White et al., 1976). We simultaneously imaged VB9 and VA8, two motoneurons that provide excitatory inputs onto adjacent ventral midposterior body musculature, as a proxy for the motoneuron output of forward and backward circuits ( Figure 1A). During episodes of continuous forward and backward movements, VB9 and VA8 motoneurons maintained a clear separation in their calcium levels (Figures 2A and 2B; Movie S1, part

D). Noticeably, a higher mean calcium level of VB9 (denoted by a red dotted line in Figure 2A) than of VA8 (denoted by a blue dotted line), referred to as the B > A state, coincided with continuous forward movement, whereas a higher mean activity level buy Linsitinib of VA8 than of VB9, referred to as the A > B state, coincided with backing (Figure 2A, lower trace). During continuous movement, regardless

of the directionality, both VA8 and VB9 often exhibited periodic, sometimes in-phase changes over their mean calcium level (Figure 2A, asterisks), whereas VB9 and DB6 and VA8 and DA6, the same class motoneuron pairs that input onto the opposite ventral and dorsal musculature (Figure 1A), tend to exhibit mostly out-of-phase already changes (Figure 2B, asterisks). The cause for these small calcium changes remains to be determined. On the other hand, directional changes (Figure 2A, denoted by dotted vertical lines) coincided with the large, reciprocal switches between the mean calcium level of VA8 and VB9 or the A > B and B > A states (Figure 2A, denoted by blue and red arrows; Figure S1D). Importantly, the transition from backward to forward motion was temporally correlated with a calcium rise in VB9 and a calcium decrease in VA8 (Figure 2C, left), whereas reversals temporally correlated with a reversed pattern (Figure 2C, right). Critically, the initiation of a reciprocal change in the A and B motoneuron activity temporally correlates with the initiation of directional change. It is noted that the initiation of directional change generally preceded the crossover between VA8 and VB9 calcium level.