Hargreaves' paw withdrawal test. Mice were placed on a 3/16th-in. thick glass floor within small (9 cm L × 5 cm W × 5 cm H) Plexiglas cubicles and allowed a habituation period of 120 min. A focused, high-intensity projector lamp beam (IITC Model 336 Plantar Test and Tail-flick Analgesia Meter, Woodland Hills, CA, USA) was shone from below onto the mid-plantar surface of the hindpaw (Hargreaves et al. 1988). The beam was aligned to the mid-plantar surface of the left paw with the projector lamp set to 10% idle intensity (II10). The lamp was then switched to 25% active intensity (AI25) and the latency to respond with withdrawal of the paw from the light or licking of the paw was recorded using the internal timer. To avoid tissue damage, if no response occurred by 30 seconds, the lamp was returned to the idle intensity and removed from the paw. This process was repeated for all mice in the enclosure, and then migrated to the right hindpaw, following an intra-trial period of at least 5 min. Mice were tested for three to six trials depending on the variance observed on each paw, and the three tightest latencies averaged.
