Formalin injection assay

Analgesia to formalin injection was carried out as described previously⁶⁵. Mice were first habituated for 20 min to the testing environment which included a home cage without bedding, food, and water. After habituation, vehicle (n = 6), morphine (10 mg/kg, n = 7), or PZM21 (40 mg/kg, n = 7) was injected subcutaneously. This was followed by injection of 20 μl of 1% formalin in 0.9% saline under the skin of the dorsal surface of the right hindpaw. Animals were returned to their home cage and behavioural responses were recorded for one hour. Nociception was estimated by measuring the cumulative time spent by animals licking the formalin-injected paw. As opioids classically display two phases of analgesic action, nociceptive behaviour was measured during both the early phase (0 to 5 min) and the late phase (20 to 30 min). In Fig. 4, an asterisk indicates a significant difference between drug and vehicle (P < 0.05 calculated using a one-way ANOVA with Bonferroni correction).