Among the various types of pain models, subcutaneous injection of diluted formalin solution into animal hindpaw causes tissue inflammation leading to nociceptive behaviors such as licking, biting and scratching the injected paw. The nociceptive behaviors revealed in the formalin test are more complex and highly organized rather than a simple reflex test such as tail-flick test, because of the involvement of supraspinal processing of pain tranmisstion. Moreover the biphasic behavioral responses of formalin test, representing the direct effect on nociceptors and the inflammatory nociceptive responses, makes it possible to consider the delicate central pain modulatory system. Thus the subcutaneous intraplantar injection of formalin is one of the best models in studying central processes of pain sensation. Therefore, we attempted to characterize the possible supraspinal mechanism of pain sensation in the mouse formalin-injected pain model. First, we examined the effect of the subcutaneous pretreatment of formalin into both hind paws of mice on the antinociception induced by the intracerebroventricularly or intrathecally administration of beta-endorphin using the tail-flick test. Pretreatment with formalin for 5 h had no affect on the i.c.v. administered beta-endorphin-induced tail-flick response. However, pretreatment with formalin for 40 h attenuated the tail-flick inhibition induced by i.c.v. administered beta-endorphin. This antinociceptive tolerance to i.c.v. beta-endorphin continued up to 1 week, but to a lesser extent. Pretreatment with formalin for 5 and 40 h significantly reduced the i.t. beta-endorphin-induced inhibition of the tail-flick response, which continued up to 1 week. The s.c. formalin treatment increased the hypothalamic pro-opiomelanocortin (POMC) mRNA level at 2 h, but this returned to the basal level after 40 h. It also has been found that the supraspinal CTX- and PTX- sensitive G-proteins may-be involved in an opioid-induced antinociception in the formalin test. Morphine and 分-endorphin given i.c.v. displayed near-maximal inhibitory effects against the formalin response in the first and the second phases. CTX- pretreated i.c.v. produced antinociceptive effects in both phases of the formalin responses. Its effect was more pronounced in the first phase. However, PTX injected i.c.v. produced the antinociceptive effect only in the first, but not the second, phase. Both CTX and PTX, at the dose which had no intrinsic effect, significantly reversed the p-endorphin-induced antinociceptive effect observed during the second, but not the first, phase. However, the antinociceptive effect by morphine failed to be affected by the same dose of treatment with CTX or Moreover, we demonstrated that the blockade of opioid receptor can induce antinociception in the formalin test. We found that naltrexone (a nonselective opioid antagonist) injected intraperitoneally, intrathecally and intracerebroventricularly inhibited nociceptive behaviors only during the second phase but not during the first phase. Administration of unaltrexamine, naltrindole and nor-binaltorphimine, which are selective ? 一, 公 一 and k -opioid antagonists, respectively, also produced antinociception during the second phase. Additionally, we examined the involvement of the descending monoaminergic systems in the naltrexone-induced antinociception in the formalin test. Interestingly, pretreatment with 5,7-dihydroxytryptamine, but not N- (2-ch3oroethyl)~N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin, 20 mg it), reversed the naltrexone-induced antinociception during the second phase. In addition, it has been characterized possible molecules involving supraspinal nociceptive processings in the hypothalamus. We examined POMC mRNA and 及一endorphin expression in mouse hypothalamus after subcutaneous pretreatment of formalin. The time-course study showed that POMC mRNA level significantly increases 2 hr after subcutaneous formalin injection. The expression level of POMC mRNA was increased about 2-fold at 2 hr after subcutaneous formalin administration. We found using in situ hybridization that the POMC mRNA after subcutaneous formalin injection is located at the arcuate nucleus of the hypothalamus. In the same manner endophin immunoreactivity was also increased in hypothalamic arcuate nucleus. In the hypothalamus, the expression level of phosphorylated ERK protein was increased within 30 min after formalin injection and remained at high level up to 10 hr. We found using an immunohistochemistry study that increased phospho-ERK protein after intraplantar formalin injection is mainly located at the arcuate nucleus of hypothalamus. In addition, the expression level of phospho-CaMKII protein was increased within 2 hr after formalin injection, but decreased at 10 hr, in the hypothalamic arcuate nucleus regions. However, formalin-induced POMC mRNA expression was significantly reduced by 10 min pretreatment with intracerebroventricular PD98059 (MAPK pathways inhibitor) and KN93 (pCaMKII inhibitor). Furthermore, phosphorylation of kB protein was increased at 2 hr and remained at high level up to 10 hr after formalin injection in the hypothalamus of mice. Using confocal immunofluorescence study, we confirmed that cells contain 兵-endorphin after s.c. formalin injection also express p-ERK, p-CaMKII and p-I/cB immunoreactivity in hypothalamic arcuate nucleus. In these series of studies, it is strongly suggested that the supraspinal processes of pain induced by subcutaneous formalin injection is closely related to the POMC mRNA expression in arcuate nucleus of the hypothalamus. Furthermore, p-ERK，p-CaMKII and p-IkB may play important roles in the regulation of hypothalamic POMC gene expression induced by subcutaneous formalin injection.