Sensory sensitivity testing

Different groups of animals were used for each experiment. Mice were counter-balanced into groups following the first basal test for mechanical thresholds. Mice were tested in a behavior room, separate from the vivarium, with low light (~35–50 lux) and low-noise conditions, between 09:00 and 16:00. Mice were habituated to the testing racks for 2 days before the initial test day, and on each subsequent test days were habituated for 20 min before the first measurement. The cephalic region was tested throughout this study, except for the RN-73 experiment, where the hind paw was tested. For cephalic measures mice were tested in four oz paper cups. The periorbital region caudal to the eyes and near the midline was tested. For experiments testing peripheral mechanical responses, the intraplantar region of the hindpaw was assessed. Testing of mechanical thresholds to punctate mechanical stimuli was tested using the up-and-down method. The selected region of interest was stimulated using a series of manual von Frey hair filaments (bending force ranging from 0.008 g to 2 g). A response of the head was defined as shaking, repeated pawing, or cowering away from the filament. In the hind paw, a response was lifting of the paw, shaking, or licking the paw after stimulation. The first filament used was 0.4 g. If there was no response a heavier filament (up) was used, and if there was a response a lighter filament (down) was tested. The up-down pattern persisted for four filaments after the first response. To decrease bias in testing, researchers were blinded to treatment groups at time of testing. While the same researcher performed both the mechanical threshold testing and injections these measures were recorded in different places and at separate time points.