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A time-sampling method that allows up to eight rats to be tested simultaneously in the formalin test is described and compared to the continuous rating method. Time sampling the behavioural response to formalin every 1 or 2 min produces scores that are essentially identical to continuous rating for both the formalin concentration effect relationship and the morphine dose effect relationship, with no loss of statistical power. The most important advantage of the method is that it allows data on other aspects of the rats’ behaviour, such as behavioural state and the side effects of drugs to be scored during the formalin test. Formalin injection produces a dose-dependent decrease in locomotor and exploratory activity. The activity pattern of rats is normalized at morphine doses that produce about a 50% reduction in pain, while morphine doses high enough to completely suppress the pain response are accompanied by considerable sedation. The use of the jackknifing procedure to obtain unbiased estimates of the variability of parameters estimated from dose effect relationships is also described.

The formalin test is increasingly used as a model of injury-produced pain but there is no generally accepted method of pain rating. To examine the properties of various pain rating methods we established dose-response relations for formalin injected in the plantar surface of one hind paw, and the analgesic effects of morphine and amphetamine using the most frequently reported behavioural measures of pain (favouring, lifting, licking and flinching/shaking of the injured paw) and combinations of these. Licking, elevation and favouring of the injected paw showed a biphasic response at all formalin doses. Flinching varied in form across the time course of formalin, and the biphasic nature of the behaviour was not as apparent. In untreated rats all these behaviours were infrequent. Flinching and favouring were increased after injection of local anaesthetic into the paw but remained negligible relative to the effect of formalin. Grooming other than that directed to the injected paw was elevated in a dose-dependent manner by formalin. Intercorrelations between the behaviours were different for the initial response and the second phase. Correlational analysis indicated that no single behavioural measure was a strong predictor of formalin, morphine and amphetamine dose. A simple sum of time spent licking plus elevating the paw, or the weighted pain score of Dubuisson and Dennis (1977), were superior to any single measure (r ranging from 0.75 to 0.86). Addition of flinching and favouring to the combined pain score using multiple regression did not increase variance explained. Depending on the measure used, a sedative dose of pentobarbital produced apparent analgesia, hyperalgesia or no effect. The interphase depression of pain, as well as the analgesic effects of morphine and amphetamine, were all associated with increased motor activation. Power analysis indicated that using a moderate dose of formalin and a combined pain score gave the greatest power to detect differences in pain. It was also found that pain scores increase with ambient temperature and that rat strains may differ in formalin pain sensitivity.

Subcutaneous injection of dilute formalin induces pain in humans and behaviors indicative of pain in animals. The formalin test, which is based on these observations, is now widely used as a model of pain produced by tissue injury, but the neural mechanisms of pain and analgesia in this test have not been identified. Rats with transections of the brain rostral or caudal to the pons show behavioral reactions to formalin similar to those of normal rats, although the temporary abatement of pain 10-15 min after formalin is absent in transected animals. Doses of morphine that suppress the behavioral response to formalin in normal rats are not antinociceptive in the formalin test in decerebrate rats although sedation, catalepsy and inhibition of the tail-flick reflex still occur. These results suggest that the response to formalin is organized in the brain stem but the antinociceptive effect of morphine in this test is mediated by the diencephalon or forebrain.

We have examined the effects of muscimol and bicuculline microinjected in the rostral ventromedial medulla (RVM) on motor function and on nociception in three pain tests. In Exp. 1 microinjection of muscimol (6.25-400 ng in 1 microl) in the RVM dose-dependently decreased pain threshold of rats and the ED(50) for muscimol was the same in both the hot plate and tail immersion pain tests. In the hot plate test, but not in the tail immersion test, paw withdrawal latencies increased again with high doses of muscimol (75-400 ng). High doses also produced catalepsy. Exp. 2 examined the effects of muscimol (50 ng) and bicuculline (50 ng) over a range of formalin concentrations (0.25-4%) in the formalin test. Muscimol increased responsiveness to formalin and reduced the slope of the formalin dose-response relation. Bicuculline decreased responses to formalin and reduced the slope of the formalin dose-response relation. It is suggested that RVM cells with inhibitory projections to the dorsal horn are not subject to strong GABAergic influence under mild noxious stimulation. RVM cells are thus active, and spinal dorsal horn relay neurons are inhibited. On the other hand, intense noxious peripheral stimulation may stimulate the release of GABA onto RVM cells, which in turn shuts off descending inhibitory fibers to allow transmission of nociceptor input through the dorsal horn.

There has been conflicting evidence concerning the role of descending bulbospinal influences on pain and opioid analgesia in the formalin test. We examined the effect of lesions of the dorsolateral funiculus (DLF) on dose-effect relations for formalin and morphine in the formalin test. Experiment 1 showed that DLF lesions reduced the effect of 5 mg/kg morphine on pain in the tail-flick test, and eliminated morphine's effect on pain produced by 2.5% formalin. When lower concentrations of formalin were used, DLF lesions produced hyperalgesia, indicated by a left shift (1.7-fold) of the formalin concentration-response curve. In experiment 2, DLF lesions increased the pain produced by 1.25% formalin and shifted the dose-response relation for the effect of morphine on the second phase of the pain response produced by 1.25% formalin to 2.5-fold higher doses. The data show that DLF lesions increase sensitivity to the pain-inducing effect of formalin, and this accounts for a substantial component of the effect of DLF lesions on morphine analgesia.

Electrical stimulation of dorsal and dorsolateral periaqueductal gray (PAG) and internal capsule (IC) sites in the rat elicited tail flick and formalin test stimulation-produced analgesia (SPA). SPA from PAG sites was associated with aversion. SPA from IC sites was associated with aversion, generalized seizures and catalepsy. Ventrobasal nucleus of thalamus (VB) stimulation did not elicit analgesia or aversion but did induce behavior characteristic of limbic seizures. A sub-anesthetic dose of sodium pentobarbital (20 mg/kg) suppressed IC stimulation-produced generalized seizures and catalepsy, and attenuated, but did not eliminate, tail flick test analgesia. These data suggest that SPA from IC sites in the rat is partially confounded with reduced responsivity. The hypothesis that SPA associated with aversion may represent a form of stress-induced analgesia is discussed.