Behavioral, electrophysiological, and autoradiographic experiments were done to study the second nociceptive phase in the formalin test. In initial experiments, this second phase was attenuated by 1–10 mg of the NK-1 receptor antagonist CP-99,994, given subcutaneously 10, 30, or 60 min before formalin (
n= 8–10) and by 20 μg given intrathecally 20 min after formalin ( n= 13); the inactive isomer CP-100,263 was ineffective. In electrophysiological experiments on single dorsal horn neurons in vivo, the excitatory responses to subcutaneous formalin injection (50 μl, 2.5%) were attenuated by subsequent intravenously administration of the NK-1 receptor antagonist CP-96,345 (0.5 mg/kg; n= 8), given 35–40 min after formalin, but not by the inactive enantiomer CP-96,344 (0.5 mg/kg; n= 9). Finally, autoradiographic binding of exogenous [125I]BH-substance P in the lumbar cord was reduced at 5 and 25 min after formalin (50 μl, 1 or 5%), with an intermediate level of reduction at 12 min. These data are interpreted as evidence that the second phase of nociceptive scores in the formalin test is attributable at least partially to tonic activation of NK-1 receptors at the spinal level, whether because of a temporally limited release of substance P, for example only during the first phase, but a slow removal or breakdown of substance P, or, more likely, because of tonic release from primary afferents throughout the second phase. Irrespective of the mechanism, it can be concluded that at least some of the persistent nociceptive effects associated with peripheral inflammation, or at least those provoked by subcutaneous injection of formalin, are mediated via continuous activation of NK-1 receptors at the level of the spinal dorsal horn; this may relate directly to mechanisms underlying prolonged nociceptive pains in humans.