Involvement of spinal cord δ opiate receptors in the antinociception of gestation and its hormonal simulation

Physiological as well as hormone-simulated pregnancy (HSP) is associated with opioid-mediated elevations in maternal nociceptive thresholds. Previous reports from this laboratory have demonstrated the involvement of spinal cord κ opiate receptors in this phenomenon. The present study was undertaken in order to determine the exclusivity of this mediation. Intrathecal (i.t.) administration of the δ opiate receptor-selective antagonists naltrindole (NTI), 7-benzylidenenaltrexone (BNTX) or naltriben (NTB) substantially reduces nociceptive thresholds of gestation (day 20) and HSP (day 19). Hyperalgesic actions of these compounds following i.t. administration are not observed in non-pregnant or vehicle-treated control animals. These data indicate that δ opiate receptor activity is a prerequisite for the manifestation of a substantial portion of gestational and HSP analgesia. In contrast, i.t. application of the μ-selective antagonist d-Phe-Cys-Tyr-d-Trp-Arg-Thr-Pen-Thr-NH₂ (CTAP) has no effect on nociceptive thresholds of gestational day 20, as was previously demonstrated for HSP-induced antinociception. Thus, the potent spinal μ analgesic system does not participate in gestational or HSP analgesia. During physiological pregnancy, less robust constituents of intrinsic opioid pain-attenuating systems in the spinal cord (δ and κ opioid systems) are recruited to mediate the maternal antinociception of gestation. Furthermore, the ability of estrogen and progesterone to modulate spinal opioid antinociceptive activity emphasizes potential differences between men and women in their response to pain medication.     

Gestational and ovarian sex steroid antinociception: synergy between spinal κ and δ opioid systems

Pain thresholds are elevated during gestation and following the simulation of pregnancy blood levels of estrogen and progesterone (hormone simulated pregnancy; HSP). The analgesia associated with both conditions is opioid-mediated and results from the activation of spinal cord κ and δ (but not μ) opiate receptors. Blockade of spinal κ or δ opiate receptors, individually, can abolish the antinociception associated with either gestational day 20 or day 19 of HSP. Surprisingly, during either physiological pregnancy or HSP, the magnitude of reduction in the increment in jump thresholds following the combined intrathecal application of suboptimum concentrations of κ and δ antagonists is indistinguishable from that observed following their individual intrathecal application. These data indicate that gestational and ovarian sex steroid-induced antinociception is not simply the sum of the independent analgesic effects of spinal κ and δ opioid systems but requires their coincident activation. It is suggested that the synergy that has been reported following the exogenous intrathecal application of κ and δ opioids also occurs between their endogenous counterparts and underlies the intrinsic analgesia associated with each condition. Utilization of such a mechanism allows for significant physiological effects (analgesia) to be achieved with doses of relevant substrates (dynorphin and enkephalin) which alone would produce minimal receptor activation (and analgesia). This would minimize tolerance and dependence formation.     

Nociceptin/orphanin FQ into the rat periaqueductal gray decreases the withdrawal latency to heat and loading, an effect reversed by (Nphe(1))nociceptin(1-13)NH(2).

The present study investigated the effect of intraperiaqueductal grey injection of nociceptin/orphanin FQ (N/OFQ) and an antagonist (Nphe(1))nociceptin(1-13)NH(2) on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. N/OFQ (5 nmol) significantly decreased the nociceptive thresholds in both tests and 1, 5 and 10 nmol of (Nphe(1))nociceptin(1-13)NH(2) significantly reversed this effect in a dose dependent way. Our results demonstrate, that N/OFQ has a nociceptive action, possibly through inhibition of PAG neurons. This effect is blocked by the antagonist (Nphe(1))nociceptin(1-13)NH(2) probably via ORL1 receptors in the periaqueductal grey.     

Antagonism of ORLI receptor produces an algesic effect in the rat formalin test

The authors investigated the role of endogenously released nociceptin (also known as orphanin FQ) spinal and supraspinal nociceptive transmission during the rat formalin test by examining the effect of intrathecal and intracerebroventricular injection of J-113397, a non-peptidyl ORL1 receptor selective antagonist. When J-113397 was injected intrathecally or intracerebroventricularly 10 min before the formalin injection, it enhanced the agitation behavior induced by paw formalin injection. This suggested that paw formalin injection induced nociceptin release in the spinal cord and the supraspinal brain sites, that this endogenously released nociceptin produced an analgesic effect and that J-113397 antagonized this analgesic effect of nociceptin and produced an algesic effect in the rat formalin test.     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice.

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK1 receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [D-Phe7, D-His9] substance P (6-11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK2 receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and D(-)-2-amino-5-phosphonovaleric acid (D-APV), and L-NG-nitro arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK1 receptors in the spinal cord.     

Nociceptin/orphanin FQ into the rat periaqueductal gray decreases the withdrawal latency to heat and loading, an effect reversed by (Nphe)nociceptin(1–13)NH2

The present study investigated the effect of intraperiaqueductal grey injection of nociceptin/orphanin FQ (N/OFQ) and an antagonist (Nphe¹)nociceptin(1–13)NH₂ on the hindpaw withdrawal response to thermal and mechanical stimulation in rats. N/OFQ (5 nmol) significantly decreased the nociceptive thresholds in both tests and 1, 5 and 10 nmol of (Nphe¹)nociceptin(1–13)NH₂ significantly reversed this effect in a dose dependent way. Our results demonstrate, that N/OFQ has a nociceptive action, possibly through inhibition of PAG neurons. This effect is blocked by the antagonist (Nphe¹)nociceptin(1–13)NH₂ probably via ORL1 receptors in the periaqueductal grey.     

Involvement of tachykinin NK1 receptors in nociceptin-induced hyperalgesia in mice

Intrathecal (i.t.) injection of nociceptin at small doses (3.0 and 30.0 fmol) produced a significant hyperalgesic response as assayed by the tail-flick test. This hyperalgesic effect peaked at 15 min following i.t. administration of nociceptin (3.0 fmol) and returned to control level within 30 min. Hyperalgesia elicited by nociceptin was inhibited dose-dependently by i.t. co-administration of tachykinin NK₁ receptor antagonists, CP-99,994 and sendide. A significant antagonistic effect of [ -Phe⁷, -His⁹] substance P (6–11), a selective antagonist for substance P, was observed against the nociceptin-induced hyperalgesia. Pretreatment with i.t. substance P antiserum and i.t. capsaicin resulted in a complete block of the reduced threshold produced by nociceptin. The NK₂ receptor antagonist, MEN-10,376 and pretreatment with neurokinin A antiserum did not alter the behavioural effect of nociceptin. The N-methyl- -aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and (−)-2-amino-5-phosphonovaleric acid ( -APV), and -N^G-nitro arginine methyl ester ( -NAME), a nitric oxide synthase inhibitor, failed to inhibit nociceptin-induced hyperalgesia. The results obtained suggest that the hyperalgesic effect of nociceptin may be mediated through tachykinin NK₁ receptors in the spinal cord.     

Effects of nociceptin on cardiac norepinephrine and acetylcholine release evoked by ouabain

We investigated whether the novel peptide, nociceptin, modulates neuronal transmission at autonomic nerve endings. Using a cardiac dialysis technique, the effects of locally applied nociceptin on cardiac acetylcholine (ACh) and norepinephrine (NE) release were examined in anesthetized cats. Dialysis probes were implanted in the left ventricular wall, with the concentration of dialysate NE or ACh serving as an indicator of NE or ACh output at cardiac sympathetic or parasympathetic nerve endings. Locally applied ouabain evoked increases in NE and ACh output. Nociceptin suppressed the ouabain induced ACh increment. The ouabain induced NE release was not altered by nociceptin. However, in the presence of desipramine (a NE uptake inhibitor), nociceptin suppressed the ouabain-induced NE release. Inhibition by nociceptin of ouabain-induced release of NE or ACh was blocked by pretreatment with nocistatin (a nociceptin action blocking peptide). Nociceptin-induced inhibition of ACh or NE release is attributable to pre-synaptic modulation rather than a reversal of the ouabain effect. These findings demonstrate that nociceptin inhibits cardiac autonomic neurotransmission via a presynaptic opioid receptor-like1(ORL1) receptor.     

Morphine tolerance and dependence in the nociceptin receptor knockout mice

Summary. Here we report the involvement of nociceptin receptor in tolerance to morphine-induced antinociception and in morphine dependence. There was no different nociceptive perception and antinociceptive effects of morphine between wild-type and the nociceptin receptor knockout mice. Tolerance to morphine (10 mg/kg)-induced antinociception was developed in both wild-type and the nociceptin receptor knockout mice after administration of morphine (10 mg/kg) twice a day for 5 days. When naloxone (5 mg/kg) was administered to mice treated with morphine repeatedly on the 6th day, morphine withdrawal syndrome was observed in both wild-type and the nociceptin receptor knockout mice, which were accompanied by the elevation of cyclic AMP levels. While naloxone benzoylhydrazone (1 mg/kg), a putative antagonist for nociceptin receptor/naloxone benzoylhydrazone-sensitive sites, also induced the morphine withdrawal signs in both wild-type and the nociceptin receptor knockout mice, the jumping signs in the nociceptin receptor knockout mice were less severe than those in wild-type mice. Treatment with naloxone benzoylhydrazone in morphine-dependent wild-type mice caused a significant increase in cyclic AMP levels in the thalamus while it had no effect in the nociceptin receptor knockout mice. The analysis of opioid mu-receptor binding showed no difference between wild-type and the nociceptin receptor knockout mice. These results suggest that the nociceptin receptor/naloxone benzoylhydrazone-sensitive sites contribute to the induction of morphine withdrawal syndrome in part. Furthermore, it is demonstrated that morphine withdrawal syndrome excepting jumping can be induced by naloxone benzoylhydrazone without any changes in the cyclic AMP levels in the thalamus. Received July 17, 2000; accepted July 11, 2001     

Effects of intrathecal nocistatin on the flexor reflex and its interaction with orphanin FQ nociceptin

We studied the effects of intrathecal (i.t.) nocistatin, a peptide identified from the precursor of orphanin FQ/nociceptin (OFQ) on the spinal nociceptive flexor reflex in decerebrate, spinalized, unanesthetized rats and its interaction with i.t. OFQ. Nocistatin induced a moderate, non-dose-dependent facilitation of the flexor reflex without producing reflex depression whereas i.t. OFQ induced a biphasic dose-dependent facilitatory and inhibitory effect. The facilitatory effect of low dose (0.55 pmol) OFQ was significantly increased by nocistatin. On the other hand, the duration, but not magnitude, of reflex depression induced by a high (550 pmol) dose of OFQ was significantly shortened by 5.5 nmol nocistatin. Thus, nocistatin interacts with OFQ in a complex fashion, increasing excitation and reducing inhibition. No evidence was obtained for an antinociceptive effect of nocistatin in rat spinal cord.     

Differential modulation by baclofen on antinociception induced by morphine and beta-endorphin administered intracerebroventricularly in the formalin test

Our previous studies have demonstrated that supraspinal GABAergic receptors are differentially involved in the antinociception induced by morphine and beta-endorphin given intracerebroventricularly (i.c.v.) in the tail-flick and hot-plate tests. These two models employed a phasic, thermal nociceptive stimulus. The present study was designed to examine the possible involvement of supraspinal GABAergic receptors in opioid-induced antinociception in the formalin test. Morphine (1 microg) and beta-endorphin (1 microg) given i.c.v. displayed the almost complete inhibitory effects against the hyperalgesic response in both phases. Muscimol (75-100 ng) and baclofen (5-10 ng) injected i.c.v. produced the hypoalgesic response in the both phases. The hypoalgesic response induced by muscimol and baclofen observed during the second phase was more pronounced than that observed during the second phase. Baclofen (2.5 ng), at the dose which did not affect the hyperalgesic response, resulted in a significant reversal of the i.c.v. administered beta-endorphin-induced hypoalgesic response observed during the second, but not the first, phase. However, the hypoalgesic response induced by i.c.v. administered morphine was not changed by the same dose of muscimol or baclofen injected i.c.v. Our results indicate that, at the supraspinal level, GABA(B)receptors appear to be involved in the modulation of antinociception induced by supraspinally administered beta-endorphin, but not morphine, in the formalin test model.     

Priming enhances endotoxin-induced thermal hyperalgesia and mechanical allodynia in rats

Central inflammation is an integral component and contributor of the pathology of many debilitating diseases and has been shown to produce spontaneous pain and hyperalgesia. Recently, administration of lipopolysaccharide (LPS) into the lateral ventricle of rats was shown to elicit both thermal hyperalgesia and tactile allodynia [K. Walker, A. Dray, M. Perkins, Hyperalgesia in rats following intracerebroventricular administration of endotoxin: effect of bradykinin B₁ and B₂ receptor antagonist treatment, Pain 65 (1996) 211–219]. In this study, we have replicated the LPS model with some adaptations and correlated the nociceptive behaviors with an increased expression of activated macrophages in the central nervous system. We also examined the effects of priming on LPS-induced decreases in thermal nociceptive thresholds and mechanical response thresholds following either central or peripheral administration. Intracerebroventricular (i.c.v.) administration of LPS (0.2 μg/rat) did not alter either thermal (hot plate) or mechanical (von Frey filaments) thresholds compared to baseline values in the first few hours after injection. However, priming rats by pretreating with i.c.v. LPS (0.2 μg) 24 h prior to testing with i.c.v. LPS (0.2 μg) produced significant mechanical allodynia and thermal hyperalgesia. The mechanical allodynia had an onset of 80 min after injection and a duration of 5 h. A similar time course was observed for thermal hyperalgesia, although its expression was less pronounced. Immunohistochemical studies indicated an increased expression of activated macrophages in the brain parenchyma of primed rats but not in unprimed rats. Intraperitoneal (i.p., 2 mg/kg) administration of LPS had no significant effect on either thermal or mechanical thresholds in the first few hours after injection; however, priming rats via i.p. (0.2 mg/kg) or i.c.v. (0.2 μg) LPS produced a reduction in both thermal nociceptive thresholds and mechanical response thresholds in rats given a subsequent i.p. injection of LPS. This study demonstrates that priming is an effective protocol for the induction of central inflammation and increases the duration of these behaviors after i.c.v. administration.     

Is substance P a primary afferent neurotransmitter for nociceptive input? IV. 2-Amino-5-phosphonovalerate (APV) and [D-Pro2,D-Trp7,9]-substance P exert different effects on behaviors induced by intrathecal substance P, strychnine and kainic acid

Intrathecal (i.t.) pretreatment with 2-amino-5-phosphonovalerate (APV), an NMDA receptor antagonist, diminished scratching with the hindlimbs, vocalization, biting or licking, and myoclonic twitches produced by i.t. strychnine, kainic acid, or morphine in rats. APV did not diminish these behaviors when produced by i.t. substance P (SP). The SP analogue [D-Pro2,D-Trp7,9]-substance P (DPDT) failed to affect any behaviors produced by all of these compounds at non-paralytic doses. At these doses, neither APV nor DPDT changed the thermal and mechanical pain thresholds. These results indicate that SP does not produce scratching at the primary afferent synapse in the spinal cord, that hindlimb scratching in rats is neither necessary nor sufficient to infer the presence of pain in rats, and that SP may not be involved uniquely, if at all, in the transmission of nociceptive information at the primary afferent synapse.     

Differential effects of specific δ and κ opioid receptor antagonists on the bidirectional dose-dependent effect of systemic naloxone in arthritic rats, an experimental model of persistent pain

In an attempt to determine the opioid receptor class(es) which underly the two opposing effects of naloxone in models of persistent pain, we tested the action of the selective δ antagonist naltrindole, and that of the κ antagonist MR-2266 on the bidirectional effect of systemic naloxone in arthritic rats. As a nociceptive test, we used the measure of the vocalization thresholds to paw pressure. The antagonists were administered at a dose (1 mg/kg i.v. naltrindole, 0.2 mg/kg i.v. MR-2266), without action per se, but which prevents the analgesic effect of the δ agonist DTLET (3 mg/kg, i.v.) or the κ agonist U-69, 593 (1.5 mg/kg, i.v.) respectively, and does not influence the effect of morphine (1 mg/kg i.v.) or the μ agonist DAMGO (2 mg/kg, i.v.) in these animals. In arthritic rats injected with the δ antagonist, the paradoxical antinociceptive effect produced by 3 μg/kg i.v. naloxone was not significantly modified (maximal vocalization thresholds (% of control) were 146 ± 9% versus 161 ± 7% in the control group). By contrast, the hyperalgesic effect produced by 1 mg/kg i.v. naloxone was significantly reduced (maximal vocalization thresholds were87 ± 4% versus 69 ± 5% in the control group). In rats injected with the κ antagonist, the antinociceptive effect of the low dose of naloxone was almost abolished (mean vocalization thresholds were 115 ± 3% versuss 169 ± 7%) whereas the hyperalgesic effect of naloxone 1 mg/kg i.v. was not significantly modified (mean vocalization thresholds =70 ± 3% and 65 ± 3%, respectively). Based on these results, the possible role of each receptor subtype in the putative control exerted by endogenous opioid substances on nociceptive messages in pathological conditions is discussed.     

Enhancement of spatial attention in nociceptin/orphanin FQ receptor-knockout mice

We isolated genes for the opioid receptor homologue MOR-C, namely nociceptin receptor (designated alternatively as orphanin FQ receptor) and generated nociceptin receptor-knockout mice. Previously, we have reported that the nociceptin system appears to participate in the regulation of the auditory system. However, the behavior of the nociceptin receptor-knockout mice has yet to be fully characterized. In the present study, we investigated changes in several behavioral performances in mice which lack nociceptin receptor. Nociceptive thresholds of nociceptin receptor-knockout mice were unchanged in the hot-plate and electric foot-shock tests as well as tail-flick and acetic acid-induced writhing tests compared to those of wild-type mice. The nociceptin receptor-knockout mice did not show any behavioral changes in the elevated plus-maze task. Surprisingly, in the water-finding test, the nociceptin receptor-knockout mice showed an enhanced retention of spatial attention (latent learning) compared to wild-type mice. In a biochemical study, dopamine content in the frontal cortex was lower in nociceptin receptor-knockout mice than wild-type mice. These results suggest that nociceptin receptor plays an important role in spatial attention by regulating the dopaminergic system in the brain.     

Effects of pre-emptively administered nociceptin on the development of thermal hyperalgesia induced by two models of experimental mononeuropathy in the rat

Pre-emptive analgesia is thought to be produced by the prevention of spinal facilitation evoked by nociceptive input to the spinal cord. Opioid receptor-like 1 (ORL1) receptor agonist has been reported to inhibit the development of spinal facilitation. We investigated the effect of nociceptin, an ORL1 receptor agonist, on the development of thermal hyperalgesia and the expression of Fos-like immunoreactivity (Fos-LI) in the spinal dorsal horn induced by two neuropathic pain models, the chronic constriction injury model and the partial sciatic nerve injury model. Chronic constriction injury is created by placing four loosely tied ligatures around the right sciatic nerve. Partial sciatic nerve injury was created by tight ligation of one third to one half of the right sciatic nerve. All drugs were injected intrathecally 10 min before the nerve injury. The anti-hyperalgesic effect of drugs was evaluated by the measurement of the paw withdrawal latency (PWL) against thermal nociceptive stimulation. The PWLs of the injured paws were measured 7, 14 and 21 days after the nerve injury. Expression of Fos-LI was examined 2 h after the nerve injury. Intrathecal injection of nociceptin significantly delayed the development of thermal hyperalgesia and decreased the expression of Fos-LI induced by chronic constriction injury, but not that induced by partial sciatic nerve injury. These data indicate that pre-emptive administration of nociceptin might be one strategy for the prevention of the development of neuropathic pain.     

Differential modulation by baclofen on antinociception induced by morphine and β -endorphin administered intracerebroventricularly in the formalin test

Our previous studies have demonstrated that supraspinal GABAergic receptors are differentially involved in the antinociception induced by morphine and β-endorphin given intracerebroventricularly (i.c.v.) in the tail-flick and hot-plate tests. These two models employed a phasic, thermal nociceptive stimulus. The present study was designed to examine the possible involvement of supraspinal GABAergic receptors in opioid-induced antinociception in the formalin test. Morphine (1 μg) and β-endorphin (1 μg) given i.c.v. displayed the almost complete inhibitory effects against the hyperalgesic response in both phases. Muscimol (75–100 ng) and baclofen (5–10 ng) injected i.c.v. produced the hypoalgesic response in the both phases. The hypoalgesic response induced by muscimol and baclofen observed during the second phase was more pronounced than that observed during the second phase. Baclofen (2.5 ng), at the dose which did not affect the hyperalgesic response, resulted in a significant reversal of the i.c.v. administered β-endorphin-induced hypoalgesic response observed during the second, but not the first, phase. However, the hypoalgesic response induced by i.c.v. administered morphine was not changed by the same dose of muscimol or baclofen injected i.c.v. Our results indicate that, at the supraspinal level, GABA_Breceptors appear to be involved in the modulation of antinociception induced by supraspinally administered β-endorphin, but not morphine, in the formalin test model.     

Spinal kappa-opiate receptor involvement in the analgesia of pregnancy: effects of intrathecal nor-binaltorphimine, a kappa-selective antagonist

In a variety of laboratory animals as well as humans, pregnancy has been associated with an activation of a maternal opioid system(s) and with a concomitant elevation in the threshold for maternal responsiveness to aversive stimuli. This analgesia is mediated via the activation of spinal opiate receptors and does not require an intact peripheral opioid system(s). The recently developed kappa-selective opioid antagonist, nor-binaltorphimine (nor-BNI), significantly reduces the threshold for reflexive jumping in response to electric foot shock when administered to the lumbar intrathecal (i.t.) space of pregnant rats (day 20 of gestation). In contrast, i.t. nor-BNI when administered to non-pregnant rats as well as systemic (i.p.) administration of an intrathecally effective dose of nor-BNI to pregnant rats is without effect on the jump thresholds. These data indicate that the kappa-type of opiate receptor mediates, at least in part, the analgesia observed during gestation, thus providing an important physiological function for this receptor type.     

Sex-related hormonal influences on pain and analgesic responses

Considerable evidence indicates sex-related differences in pain responses and in the effectiveness of various analgesic agents. Specifically, females are at greater risk for experiencing many forms of clinical pain and are more sensitive to experimentally induced pain relative to males. Regarding analgesic responses, nonhuman animal studies indicate greater opioid analgesia for males, while a limited human literature suggests the opposite. Though the mechanisms underlying these effects remain unclear, the influence of gonadal hormones on nociceptive processing represents one plausible pathway whereby such sex differences could emerge. The present article reviews the complex literature concerning sex steroid effects on pain responses and analgesia. First, nonhuman animal research related to hormonal effects on nociceptive sensitivity and analgesic responses is presented. Next, human studies regarding gonadal hormonal influences on experimental pain responses are reviewed. Several potential mechanisms underlying hormonal effects on nociceptive processing are discussed, including hormonal effects to both peripheral and central nervous system pathways involved in pain transmission. Finally, based on these findings we draw several conclusions and make specific recommendations that will guide future research as it attempts to elucidate the magnitude and importance of sex-related hormonal effects on the experience of pain.     

Intrathecal melatonin increases the mechanical nociceptive threshold in the rat

The aim of this investigation was to determine whether intrathecal (i.t.) administration of monodose melatonin provides an increase in mechanical nociceptive threshold in the rat. Twenty-four male Wistar rats were randomly assigned to four groups. Each animal was anesthetized, and a catheter was placed intrathecally via the cisterna magna. The study groups were: untreated controls (n=6); melatonin only (MEL, n=6); melatonin + luzindole (MEL+LZN, n=6); and melatonin + naloxone (MEL+NLX, n=6). Measurements of mechanical nociceptive threshold were made using an electronic algometer. Each animal was tested prior to injection and at 10, 20, 30 and 40 min after injection. In the MEL group, the mean nociceptive thresholds at all post-injection time points were significantly higher than the baseline value (p<0.05 for all). In the control and MEL+LZN groups, none of the four mean nociceptive thresholds recorded after i.t. injection was significantly different from the baseline value (p>0.05 for all). In the MEL+NLX group, the mean nociceptive thresholds at 20, 30 and 40 min post-injection were all significantly lower than the baseline value (p<0.05 for all). Comparison among the group nociceptive thresholds at baseline revealed no significant differences, and the same was true at 10 min after i.t. injection. At the 20, 30 and 40 min stages, the threshold in the MEL group was significantly higher than the threshold in the control group. The results indicate that i.t. injection of melatonin produces a time-dependent increase in mechanical nociceptive threshold in the rat and that the mechanism that underlies these effects involves both melatonin and opioid receptors.