Mice were anesthetized with isoflurane and sacrificed by decapitation and the TG or dura mater were removed and prepared for culture as previously described (24 (link)). For cultures with back-labelled TG neurons, TGs were taken 7 days following supra-dural application of the retrograde tracer, wheat germ agglutinin conjugated Alexa-Fluor 555 nm. After removal, tissue was placed in ice-cold Hanks balanced-salt solution (divalent free). Trigeminal tissue was dissociated enzymatically with collagenase A (1 mg/ml, 25 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 20 min at 37°C. Dural tissue was dissociated enzymatically with collagenase A (1 mg/ml, 30 min, Roche, Indianapolis, IN) and collagenase D (1 mg/ml, Roche, Indianapolis, IN) with papain (30 units/ml) for 25 min at 37°C. The tissues were then triturated through fire-polished Pasteur pipettes, and trigeminal cells were plated on poly-D-lysine (Becton Dickinson) and dural cells plated on untreated plates. After several hours at room temperature to allow adhesion, cells were placed in a room-temperature, humidified chamber in Liebovitz L-15 medium supplemented with 10% FBS, 10 mM glucose, 10 mM HEPES and 50 U/ml penicillin/streptomycin. Trigeminal cells were tested within 24 hours post plating and dural cell cultures were tested 2–4 days post plating.