(A) Schematic of the circuit. HSN (green) and VC (blue) motor neurons synapse onto the vm2 muscle postsynaptic termini (center of schematic). The uv1 neuroendocrine cells (pink) extend processes (grey) along the vulval slit and vm2 postsynaptic terminus. (B–E) Individual video frames of the GCaMP5:mCherry fluorescence ratio showing active state Ca2+ transients in HSNs (B), VCs (C), and vulval muscles during twitching (D) and egg-laying behaviors (E). Arrowheads, HSN and VC presynaptic termini; asterisks, cell bodies; scale bar, 10 µm. (F) 30 min recordings of HSN, VC, and vulval muscle activity (left panel), showing distinct active (yellow) and inactive (grey) egg-laying behavior states, with expanded timescale of one active state at right. Arrowheads show egg-laying events. (G) Scatter plots and median HSN, VC, and vulval muscle (vm) inter-transient intervals during egg-laying inactive (–, filled circles) and active (+, open circles) states. Asterisks indicate significant differences (p<0.0001). (H) Relationship between Ca2+ transient amplitude and egg release. Scatter plots and medians of normalized amplitude with (+; open circles) and without (–; closed circles) egg release. Also shown is the percent of total transients that accompanied egg release. (I) Timing of HSN, VC, and vulval muscle Ca2+ transients and egg release. Shown at top is a curve of the median of Ca2+ from HSN (green), VC (blue), and vulval muscles (orange) from normalized ∆R/R traces (with the peak Ca2+ set to 100%) synchronized to the moment of egg release (0 s, arrowhead and dotted line). Bars indicate 20% change in median GCaMP5/mCherry ratio. The timing of the HSN Ca2+ peak is significantly different from that of the VCs and vulval muscles (p<0.0001). Shown at bottom is a trace of median vulval muscle size. Bar shows a 5% change in median object size based on mCherry fluorescence.