Inflammatory pain measurements (formalin test)

Mice were placed in a clear enclosure for a 10-min habituation period. Mice were subcutaneously injected in the dorsal hindpaw with 2% formalin (20 μL). Mice were monitored for time spent licking the injected paw, and number of lick bouts, for 1 h post-injection by researchers blinded to experimental condition. All sessions were video-recorded. The time spent paw licking was recorded for 1 h and analyzed during the inflammatory phase (15–45 min).

Mice received optogenetic stimulation of AgRP→PBN neurons beginning 10 min prior to formalin injection and lasting throughout the formalin test. Paw licking was recorded for 1 h post formalin injection. The inflammatory phase of the formalin assay (15–45 min) was analyzed.

Sham- and capsaicin-treated mice were injected with formalin in the dorsal hindpaw. Paw licking was recorded for 1 h post formalin injection. The inflammatory phase of the formalin assay (15–45 m) was presented.