Von Frey test

Hindlimb withdrawal reaction to light touch was measured in conscious, unrestrained mice, as described elsewhere (72). Briefly, mice were allowed to acclimate to an observation chamber for 30 min before testing. Paw withdrawal to pressure applied at the plantar surface was measured using von Frey filaments applied sequentially in an up-down protocol as originally described for rats (73) and subsequently modified for use in mice (72). Filaments were applied for 1 s at 1-s intervals with 5-min break between each set of stimulations, with five stimulations per filament. Response frequency for each filament was recorded, and 50% threshold was calculated using Hill equation (Origin 2017, OriginLab).