(A) Snapshots of the pattern of GCaMP signal recorded from motoneurons of control animals at 0 min (a), 10 min (b), and at around 20 min (c,d) after in vitro challenge with 600 nM ETH. (B) Corresponding recording of GCaMP signal, color-coded according to regions indicated in (Ab and Ac): (a) 'P' (posterior, black trace; cf., Ab); (b): 'L' (left, blue trace; cf., Ac); (c): 'C' (center, green trace; cf., Ac); (d) 'R' (right, red trace; cf., Ac). (e) Expanded segment of recording (recordings for 'P', 'L', 'C' and 'R' regions superimposed) during 18–20 min period (indicated by small bar beneath time axis of (d)). Note the alternating activity in 'L' and 'R' regions. (C) (a) Motoneuron activity patterns (recordings for 'P', 'L', 'C' and 'R' regions superimposed) from animals that express ETHR RNAi in CCAP neurons; (b) Expanded segment of recording shown in (a) (recordings for 'P', 'L', 'C' and 'R' regions superimposed) during 42–48 min period (indicated by small bar beneath time axis in (a)). Note that 'L' and 'R' activity no longer alternate (compare with Be). Zero min indicates time of ETH challenge in all records. Genotypes: Controls (A,B): CCAP>GCaMP (Ccap-GAL4 + UAS-GCaMP); ETHR knockdown in CCAP neurons (C): CCAP+MN>GCaMP+ETHR RNAi (MN: C164 motoneuron GAL4; see Materials and methods). Note that this genotype also knocks down ETHR expression in motoneurons (MNs). Nevertheless, knockdown of ETHR only in MNs had only a slight effect on ecdysis behavior (cf. Figure 2), suggesting that most defects observed here were due to knockdown of ETHR in CCAP neurons. In all experiments using RNAi, its effectiveness was boosted by including a UAS-dcr2 transgene.