Supplementary Figure 4: Laminar distribution of mCherry expression and lack of expression in dorsal root ganglia in Pdyn^Cre and nNOS^CreERT2 mice that had received intraspinal injections of AAV2.flex.hM3Dq-mCherry.

A-D show transverse sections through the rostral part of the L3 segment of a Pdyn^Cre mouse (A,B) and through the caudal part of L3 of a nNOS^CreERT2 mouse (C,D). In both genotypes, mCherry-positive cells form a dense band in the superficial dorsal horn, and there are scattered neurons in deeper laminae of the dorsal horn, consistent with the distribution of dynorphin- and nNOS-expressing neurons. Images to the right (B, D) show mCherry-immunoreactivity superimposed on a dark-field image, which indicates the location of the gray matter. Similar results were obtained from 5 animals of each genotype. E-J show scans of L4 dorsal root ganglia from the right side (ipsilateral to the intraspinal injection) of Pdyn^Cre (E-G) and nNOS^CreERT2 (H-J) mice. In each case, immunostaining for mCherry is shown on the left, a dark-field image in the middle and a merged image on the right. No mCherry-positive cells were observed in the DRGs in either strain. Scale bar = 200 μm. Similar results were obtained from 4 nNOS^CreERT2 animals and from 2 Pdyn^Cre animals. The time spent licking the calf in response to intradermal injection of chloroquine (100 μg) was reduced following chemogenetic activation (CNO) in Pdyn^Cre mice (K), whereas there was no effect on responses in nNOS^CreERT2 animals (L). Significant differences were assessed using two-sided unpaired Student’s t-tests (t₂₁ = 2.82, *p = 0.0103; ns not significant, t₂₃ = 1.2, p = 0.2422). Data represent means ± SEM (n=11, 12, 12, and 13 animals, for Pdyn^Cre treated with CNO and vehicle and for nNOS^CreERT2 treated with CNO and vehicle, respectively).