Cold stimulation triggered ATP release from SCAP, and conditioned supernatant activated cultured TG neurons. (A) SCAP (5 × 105 cells) were placed in PCR tubes and maintained at 37 °C for 10 min or exposed to 5 cycles of 37 °C for 1 min and 45 s and 15 °C for 5 s. Tubes were immediately centrifuged at 2000 rpm for 2 min, and supernatant aliquots were used for ATP quantification. Cold stimulation caused an increase in relative ATP levels in conditioned supernatant (n = 7–8/group, **p < 0.01). (B, C) Conditioned supernatant from SCAP maintained at 37 °C was unable to activate acutely (24 h) cultured TG neurons, while conditioned supernatant after cold stimulation to SCAP were able to activate six out of eight neurons tested. (D) Representative trace of inward current generated from conditioned supernatant after cold stimulation to SCAP. Data were analyzed by unpaired Student’s t-test. Error bars are SEM.