A comparison was made of spontaneous nociceptive behaviors elicited by subcutaneous injection of formalin (0.5-10.0%) into the plantar or dorsal surface of the right hindpaw in rats. In the present study, we also examined the effect of paw formalin injection on the release of nitric oxide (NO) metabolites (nitrite/nitrate) and glutamate from the spinal cord in anesthetized rats using a dialysis probe placed in the lumbar subarachnoid space. Two distinct quantifiable behaviors indicative of pain were identified by formalin injected into both regions of the paw. There were no significant alterations in the number of flinches during the early and late phases induced by different regions of formalin injection. However, the early phase licking/biting activity evoked by formalin injection into the plantar surface of the paw was significantly higher than that evoked by formalin injected into the dorsal region. The maximum effect in the early and late phases was produced by 5.0% formalin injection into the dorsal and plantar paw. At a higher concentration (10.0%) of formalin, nociceptive behavioral responses were decreased except for the late phase flinching when injected into the dorsal paw. Injections of formalin (5.0%) into both regions of the paw evoked a biphasic spinal release of nitrite/nitrate with a significant increase during the early phase (0-10 min) and the late phase (30-80 or 90 min). A higher concentration of formalin (10.0%) failed to produce a clear-cut release of nitrite/nitrate. A significant increase of glutamate was observed in the 0-10 min samples obtained after injection of formalin (5.0%) into the plantar and dorsal surface of the paw, whereas 0.5 and 10.0% formalin induced no substantial release. These results suggest that 5.0% formalin should be used when studying antinociceptive activity of NO- and N-methyl-D-aspartate-related compounds in the formalin test in rats. Formalin injection into the plantar surface of the paw might prove to be useful for evoking the licking/biting response, particularly in the early phase.

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Injection of high-dose of morphine into the spinal lumbar intrathecal (i.t.) space of rats elicits a nociceptive behavioural syndrome characterized by periodic bouts of spontaneous agitation and severe vocalization. The induced behavioural response such as vocalization and agitation was observed dose-dependently by i.t. administration of morphine (125-500 nmol). Pretreatment with naloxone (s.c. and i.t.), an opioid receptor antagonist, failed to reverse the morphine-induced behavioural response. The excitatory effect of morphine was inhibited dose-dependently by pretreatment with 3-((+)2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist and MK-801, a non-competitive NMDA receptor antagonist. The non-selective nitric oxide (NO) synthase inhibitor N(G)-nitro L-arginine methyl ester (L-NAME) inhibited dose-dependently the behavioural response to high-dose i.t. morphine (500 nmol), whereas D-NAME was without affecting the response to high-dose i.t. morphine. In the present study, we measured NO metabolites (nitrite/nitrate) in the extracellular fluid of rat dorsal spinal cord using in vivo microdialysis. The i.t. injection of morphine (500 nmol) evoked significant increases in NO metabolites and glutamate from the spinal cord. Not only NO metabolites but also glutamate released by high-dose morphine were reduced significantly by pretreatment with L-NAME (400 nmol). Pretreatment with CPP and MK-801 showed a significant reduction of the NO metabolites and glutamate levels elevated by high-dose i.t. morphine. These results suggest that the excitatory action of high-dose i.t. morphine may be mediated by an NMDA-NO cascade in the spinal cord.