Extended Data Fig. 6. Genetic ablation of LPB→SNR neurons reduces behavioral responses to formalin-induced pain.

(a) Injection of retrogradely-transported pseudotyped equine infectious anemia virus expressing Cre-recombinase (RG-EIAVCre) into the right SNR and AAV carrying Cre-dependent Caspase 3 (CASP) or eYFP into the right LPB of C57BL/6 mice. 5 weeks later, mice received bilateral intraplantar injections of 1% formalin into the hind paws. (b) Comparison between animals in which SNR-projecting LPB neurons were genetically ablated using CASP (left) and animals that express a control vector (eYFP) in SNR-projecting LPB neurons (middle). Sections were stained using an eYFP antibody (green; scale bar 50 μm). CASP animals show significantly reduced number of eYFP-positive SNR-projecting LPB cells when compared to control animals (right; CASP: n = 5 mice, eYFP: n = 5 mice). (c) Mean total distance traveled was not significantly different between CASP and eYFP animals in the open field test (CASP: n = 10 mice, eYFP: n = 7 mice). (d) Number of licks in response to formalin injection in CASP mice (n = 10 mice) for the left (blue) and right (grey) hind paws (left). Mean number of licks during phase I and phase II of the formalin test for comparison of left and right hind paws (right). (e) Same as in (d) but for analysis of lick duration. (f,g) Same as in (d,e) but for eYFP control animals (n = 7 mice). (h) Comparison of mean total number of licks (left) and mean total lick duration (right) for CASP and eYFP mice for individual hind paws. Data represent mean ± SEM. Significance was calculated by means of unpaired t-test (a,b,d,e,f,g) and one-way RM ANOVA with Tukey’s post-hoc test (h). * p < 0.05, ** p < 0.01, *** p < 0.001.