Hot Plate and Tail Flick Assay

Both assays investigated the latency of response to heat-induced pain. In the hot plate assay, each mouse was gently placed into a cylindrical enclosure onto the heated platform maintained at 52.5 °C. Each trial lasted 60 s to minimize tissue damage. The first occurrence of hindpaw licking, withdrawal, or jumping was recorded as response to heat-induced pain (Espejo et al, 1994). In the tail flick assay, each mouse was placed in a restraint tube and their tail placed over the test apparatus approximately 2–3 cm from the end. The latency to remove the tail from the apparatus was measured. The intensity of the heated lamp stimulus was set such that the baseline latency for tail removal was 3–5 s. For each time point, two readings were taken 1 cm apart on the tail and averaged together. Animals were tested prior to CNO injection, 30 min following injection, and 1 h following injection. Each trial lasted no more than 10 s to maximize behavioral responses and minimize tissue damage. During the first day of testing, animals were placed into the restraint tube, without any habituation to the apparatus, and tested immediately on the tail flick. To ensure that stress associated with the restraint tube did not block any analgesic effects of Gq DREADD activation, animals underwent two more training sessions in which saline injections were administered prior to animals being placed in the restraint tube and analgesia was tested. The following day, animals underwent test sessions with CNO injections prior to analgesia testing (no differences to initial test, data not shown).