4-OHT preparation and delivery

We dissolved 10 mg of 4-OHT (Sigma, Cat# H6278-10MG) in 500 µL ethanol (100%) (20 mg/mL stock) first by vortexing and then sonicating. We then add autoclaved corn oil (1:4) to dissolve 4-OHT (previously heated to 45°C) to 5 mg/mL and sonicate until solution cloudiness clears. As a final step, vacuum centrifuge for 10 min to evaporate the alcohol from the final injection solution. Male FosTRAP (FosCreER+/-, FosCreER+/-, Ai9+/-) mice were used. Mice were single housed and gently handled for 7–10 days prior to the experiment to minimize the unwanted labeling of neurons associated with stress of handling. On the experiment day, mice were given 4-OHT 20 mg/kg in their homecage environment. 60 min post 4-OHT, we injected either saline or chloroquine (200 µg/50 µL) subcutaneously in the nape of the neck to TRAP neurons that are activated by pruritic stimuli. In FosCreER+/-, Ai9 ±mice, robust tdTomato expression was seen 1 week post TRAPing. In the FosCreER ± mice injected with the optogenetic or chemogenetic constructs, robust labeling was seen 4 weeks post TRAPing. All the TRAPs for behavioral experiments were performed between October and March, between 9.00 am and 1.00 pm.