Figure 7. DP-ilp7 activity and sNPF signaling are required for C4da neuron presynaptic facilitation and A08n neuron responses.

(a–b) Calcium responses visualized in C4da axon terminals expressing GCaMP6m after von Frey filament stimulation in semi-intact larval preparations. Baseline (F₀) and maximum responses (F_max) in C4da neuron terminals are shown for (a) control (ctl) and for ilp7 neuron inactivation by Kir2.1. Responses to local von Frey filament stimulation are restricted to a single VNC hemisegment corresponding to the stimulation site on the body wall. Scale bar: 10 µm. (a’) Maximum responses (ΔF_max/F₀) were plotted showing that ilp7 neuron inactivation reduces C4da neuron presynaptic calcium influx (n=12 each, p<0.05, Mann-Whitney test). (b,b’) Calcium responses in C4da axon terminals after mechanical stimulation are reduced in sNPF heterozygous animals and significantly rescued by sNPF expression in ilp7 neurons. (n=10 each, p<0.05, Mann-Whitney test). (c–e) Calcium responses in A08n somata expressing GCaMP6m (82E12-LexA) after mechano-nociceptive stimulation. (c) Baseline (F₀) and maximum responses (F_max) in A08n neurons are shown for control (ctl) and C2/3da, C4da, or ilp7 neuron specific inactivation by Kir2.1 expression. Scale bars: 10 µm. (c’) Maximum responses (ΔF_max/F₀) were plotted showing that A08n neuron calcium responses are strongly reduced upon inactivation of C2/3, C4da or ilp7 neurons (n=as indicated, p<0.05, Mann-Whitney test). (d,e) Baseline (F₀) and maximum responses (F_max) in A08n neurons are shown for control (ctl), RNAi mediated knockdown of sNPF-R and expression of a dominant negative sNPF-R in (d) C2/3da neurons or (e) C4da neurons. (d’,e’) Maximum responses (ΔF_max/F₀) were plotted showing that inactivation of sNPF signaling in either sensory subset significantly impairs A08n neuron responses (n=as indicated, p<0.05, Mann-Whitney test).