All behavioral tests were performed with an experimenter blind to genotype. The mice were 2 to 3 month-old males (20–30 g) that had been backcrossed to C57Bl/6 mice for at least six generations. All experiments were performed under the protocol approved by the Animal Care and Use Committee of Johns Hopkins University School of Medicine. 5 weeks old
MrgprA3GFP-Cre;
ROSA26DTR mice and
ROSA26DTR littermates were injected with diphtheria toxin (i.p., 40ug/kg, EMD Biosciences) twice separated by 72 hrs. Behavioral experiments were performed three weeks after the first toxin injection. The day before the behavioral tests, all animals were acclimated for at least 30 min to their testing environment. We housed 4–5 mice in each cage in the vivarium with 12 hr light/dark cycle and all the behavioral tests were performed in the morning.
Back injections and cheek injections of chemicals were performed as previously described
35 (link). Briefly, pruritic compounds were subcutaneously injected into the nape of the neck for back injection and the right cheek of the animal for the cheek injection after acclimatization. Behavioral responses were video recorded for 30 min. The video recording was subsequently played back in slow-motion and the number of bouts of scratching with the hindpaw and also, for the cheek, wiping with the forepaw, each directed toward the injection site, were counted.
In the dry skin model, the rostal back of the mice were treated twice daily with cutaneous application of acetone/ether (1:1) mixture followed by water. After a 6-day treatment, the mice showed robust spontaneous scratching and the treated skin area exhibited decreased stratum corneum hydration and increased trans-epidermal water loss, which mimic the symptoms of dry skin in patients
29 (link), 31 (link). In an allergy model of itch, 50 µg ovabumin dissolved in PBS was administered intraperitoneally together with 2 mg of Inject Alum twice separated by 10 days. One week after the second sensitization, 50 µg ovalbumin dissolved in saline was administered in the same manner as other pruritogens and scratching behavior was quantified.
For the hot plate test, a clear plexiglass cylinder was placed on the plate and the mice were placed inside the cylinder. The onset of brisk hindpaw lifts and/or flicking/licking of the hindpaw was assessed at different temperatures.
For the cold plate test, a ceramic plate on a bed of ice was cooled in a −20°C freezer. During the test, the plate was allowed to warm to 0°C as measured by two independent temperature probes. The onset of brisk hindpaw lifts and/or flicking/licking of the hindpaw was assessed.
For the tail immersion test, mice were gently restrained in a 50 ml conical tube into which the mice voluntarily entered. The protruding one third of the tail was then dipped into a water bath of varying temperatures. The latency to respond to the heat stimulus with vigorous flexion of the tail was measured.
For the Hargreaves test, mice were placed under a transparent plastic box (4.5 × 5 × 10 cm) on a glass floor. The infrared source was placed under the glass floor and the infrared light was delivered to the hindpaw. The latency for the animal to withdraw its hindpaw was measured.
For the Von Frey filament test, mice were placed under a transparent plastic box (4.5 × 5 ×10 cm) on a metal mesh. Von Frey filaments, each delivering a different bending force, were applied to the hind paw using the up-down method and the threshold force corresponding to 50% withdrawal determined.
For the chemically-induced pain test, an intraplantar injection was used to administer a total volume of 6 μl of capsaicin (1 mg in 10 ml saline/7% Tween-80), or formalin (2% formalin in saline). The time mice spent licking and flinching was measured for 15 minutes after capsaicin injection and for 1hr after formalin injection.
For the Rotarod test, each mouse was trained for 5 minutes at a constant speed of 4 rpm on the rotarod (Rotamex, Columbus Instruments). The first trial started at least 1 hour after training. Every day, each mouse received 3 trials, separated by 30 minutes, at speeds accelerating from 4 to 40rpm (with a 4 rpm increase every 30 s). Each mouse was tested for 3 consecutive days. The trial was finished when the mouse fell off the rotarod. The latency to falling off the rotarod was recorded and used in subsequent analyses.
Han L., Ma C., Liu Q., Weng H.J., Cui Y., Tang Z., Kim Y., Nie H., Qu L., Patel K.N., Li Z., McNeil B., He S., Guan Y., Xiao B., LaMotte R, & Dong X. (2012). A subpopulation of nociceptors specifically linked to itch. Nature neuroscience, 16(2), 174-182.
