(A) Expression of the genetically-encoded calcium indicator, GCaMP3 in DVB neurons (vjIs58). Top: diagram of the DVB neuron in the tail region. Synapse means the neuromuscular junction where the DVB neuron innervates enteric muscles. Middle and Bottom: two snapshots from a real-time imaging video of wild type animals showing an increase in fluorescence in synaptic region of DVB neurons, as indicated by the white arrow, right before the Exp step. (B) A representative trace of the GCaMP3 fluorescence in the synaptic region of DVB neurons in wild type animals showing DVB neurons are rhythmically activated during three consecutive defecation cycles. Note that the cycle length is longer than 50 seconds, likely due to unc-13(s69) mutation, which was used to immobilize the worms for calcium imaging (See Materials and Methods for details). (C) Representative traces of the GCaMP3 fluorescence in the synaptic region of DVB neurons in worms with the indicated genotypes. The observed pBoc step and Exp step are indicated by arrows and arrowheads, respectively. (D) Classification of the different patterns of pBoc, fluorescent spikes in DVB and Exp in worms with the indicated genotypes. vjIs76 was used for the PKA[DN] strain. In (D), wild type: 23 cycles in 11 animals; unc-25: 44 cycles in 11 animals; aex-2: 44 cycles in 11 animals; PKA[DN]: 30 cycles in 12 animals.