Automated Paw Withdrawal Threshold Measurement

Paw withdrawal thresholds to mechanical stimulation were measured in grams (g) using an electronic von Frey anesthesiometer (IITC model Alemo 2390–5, Woodland Hills, CA, United States) as described previously (Slivicki et al., 2016 (link)). Mice were placed on an elevated metal mesh table where they were habituated under individual, inverted plastic cages for at least 30 min prior to testing. Following the cessation of exploratory behaviors, a force was applied to the midplantar region of the hind paw with a semiflexible tip connected to the anesthesiometer. Mechanical stimulation was terminated when the mouse withdrew its paw from the mesh surface. The threshold for paw withdrawal was determined in duplicate in each paw; responsiveness in each paw was subsequently averaged into a single determination for each animal.