Mu-opioid receptors in the paraventricular nucleus regulate ejaculatory behaviors via the sympathetic nerve system in male rats / 中华男科学杂志

Objective@#To explore the effects of the mu-opioid receptor (MOR) in the paraventricular nucleus (PVN) on the ejaculatory behaviors of male rats and its potential mechanisms.@*METHODS@#Male SD rats with normal ejaculation ability were mated with female ones in hormone-induced estrus. After bilateral PVN microinjection of D-Ala-2-Me-Phe-4-Gly-ol enkephalin (DAGO) or D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) with an inserted catheter, the male animals were observed for mount latency (ML), mount frequency (MF), intromission latency (IL), intromission frequency (IF), ejaculation latency (EL), ejaculation frequency (EF), post-ejaculation interval (PEI), and intromission ratio (IR). The lumbar sympathetic nerve activity (LSNA) of the rats was recorded using the PowerLab data acquisition hardware device, and the levels of norepinephrine (NE) in the peripheral plasma were measured by ELISA following microinjection of saline or different doses of DAGO or CTAP.@*RESULTS@#Neither CTAP nor DGAO significantly affected the ML of the male rats (P > 0.05). DGAO remarkably increased IF (P < 0.01) and MF (P < 0.01), prolonged IL (P < 0.01), EL (P < 0.01) and PEI (P < 0.01), and reduced EF (P <0.01) and IR (P < 0.05). On the contrary, CTAP markedly decreased IF (P < 0.01) and MF (P < 0.01), shortened IL (P < 0.01), EL (P < 0.01) and PFI (P < 0.01), and elevated EF (P < 0.01) and IR (P < 0.01). Additionally, DAGO decreased LSNA in a dose-dependent manner and reduced the NE level in the peripheral plasma. CTAP, however, not only offset the effects of DAGO on LSNA, but also significantly increased LSNA.@*CONCLUSIONS@#MOR in PVN inhibits ejaculatory behaviors in male rats by weakening LSNA, which has provided some theoretical evidence for the use of highly selective opioids in the treatment of premature ejaculation.

The effect of µ-opioid receptor activation on GABAergic neurons in the spinal dorsal horn

The superficial dorsal horn of the spinal cord plays an important role in pain transmission and opioid activity. Several studies have demonstrated that opioids modulate pain transmission, and the activation of µ-opioid receptors (MORs) by opioids contributes to analgesic effects in the spinal cord. However, the effect of the activation of MORs on GABAergic interneurons and the contribution to the analgesic effect are much less clear. In this study, using transgenic mice, which allow the identification of GABAergic interneurons, we investigated how the activation of MORs affects the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive afferent and GABAergic interneurons. We found that a selective µ-opioid agonist, [D-Ala², NMe-Phe⁴, Gly-ol]-enkephanlin (DAMGO), induced an outward current mediated by K⁺ channels in GABAergic interneurons. In addition, DAMGO reduced the amplitude of evoked excitatory postsynaptic currents (EPSCs) of GABAergic interneurons which receive monosynaptic inputs from primary nociceptive C fibers. Taken together, we found that DAMGO reduced the excitability of GABAergic interneurons and synaptic transmission between primary nociceptive C fibers and GABAergic interneurons. These results suggest one possibility that suppression of GABAergic interneurons by DMAGO may reduce the inhibition on secondary GABAergic interneurons, which increase the inhibition of the secondary GABAergic interneurons to excitatory neurons in the spinal dorsal horn. In this circumstance, the sum of excitation of the entire spinal network will control the pain transmission.

STEP signaling pathway mediates psychomotor stimulation and morphine withdrawal symptoms, but not for reward, analgesia and tolerance

Striatal-enriched protein tyrosine phosphatase (STEP) is abundantly expressed in the striatum, which strongly expresses dopamine and opioid receptors and mediates the effects of many drugs of abuse. However, little is known about the role of STEP in opioid receptor function. In the present study, we generated STEP-targeted mice carrying a nonsense mutation (C230X) in the kinase interaction domain of STEP by screening the N-ethyl-N-nitrosourea (ENU)-driven mutant mouse genomic DNA library and subsequent in vitro fertilization. It was confirmed that the C230X nonsense mutation completely abolished functional STEP protein expression in the brain. STEP(C230X−/−) mice showed attenuated acute morphine-induced psychomotor activity and withdrawal symptoms, whereas morphine-induced analgesia, tolerance and reward behaviors were unaffected. STEP(C230X−/−) mice displayed reduced hyperlocomotion in response to intrastriatal injection of the μ-opioid receptor agonist DAMGO, but the behavioral responses to δ- and κ-opioid receptor agonists remained intact. These results suggest that STEP has a key role in the regulation of psychomotor action and physical dependency to morphine. These data suggest that STEP inhibition may be a critical target for the treatment of withdrawal symptoms associated with morphine.

DAMGO modulates two-pore domain K⁺ channels in the substantia gelatinosa neurons of rat spinal cord

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K⁺ current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K⁺ channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K⁺ equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K⁺ channel) related acid-sensitive K⁺ channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K⁺ current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K⁺ channel (TASK1 and 3) in addition to inwardly rectifying K⁺ channel.

Opioid μ receptors mediate the stress-induced spatial reference memory impairment / 生理学报

Learning/memory impairment is one of the most serious problems induced by stress, and the underlying mechanisms remain unclear. Opiates and opioid receptors are implicated in multiple physiological functions including learning and memory. However, there is no clear evidence whether the endogenous opioid system is involved in the formation of the stress-induced spatial reference memory impairment. The aim of the present study was to evaluate the role of μ opioid receptor in the stress-induced spatial reference memory impairment by means of Morris water maze (MWM) test in a mouse elevated platform stress model. The mice were trained in the MWM for four trials a session for 4 consecutive days after receiving the elevated platform stress, and intracerebroventricular injection of μ opioid receptor agonist DAMGO, antagonist CTAP or saline. Retention of the spatial training was assessed 24 h after the last training session with a 60-s free-swim probe trial using a new starting position. The results showed that intracerebroventricular injection of μ opioid receptor agonist DAMGO but not antagonist CTAP before MWM training impaired the memory retrieval of mice. Elevated platform stress before MWM training also impaired memory retrieval, which could be reversed by pre-injection of CTAP, and aggravated by DAMGO. These results suggest that endogenous opioid system may play a crucial role in the formation of the stress-induced memory impairment.

µ-opioid receptors in the central nucleus of the amygdala regulate food rather than water intake in rats / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of µ-opioid receptors (µ-ORs) in the central nucleus of the amygdala (CeA) on feeding and drinking behaviors in rats and evaluate the role of glutamate signaling in opioid-mediated ingestive behaviors.</p><p><b>METHODS</b>Stainless steel cannulas were implanted in the unilateral CeA for microinjection of different doses of the selective µ-OR agonist DAMGO in satiated or water-deprived male SD rats. The subsequent food intake or water intake of the rats was measured at 60, 120, and 240 min after the injection. The rats receiving microinjections of naloxone (NTX, a nonselective opioid antagonist) or D-AP-5 (a selective N-methyl-D-aspartic acid-type glutamate receptor antagonist) prior to DAMGO microinjection were tested for food intake at 60, 120, and 240 min after the injections.</p><p><b>RESULTS</b>Injections of DAMGO (1-4 nmol in 0.5 µl) into the CeA significantly increased food intake in satiated rats, but did not affect water intake in rats with water deprivation. NTX (26.5 nmol in 0.5 µl) injected into the CeA antagonized DAMGO-induced feeding but D-AP-5 (6.3-25.4 nmol in 0.5 µl) injections did not produce such an effect.</p><p><b>CONCLUSION</b>µ-ORs in the CeA regulate food intake rather than water intake in rats, and the orexigenic role of µ-ORs is not dependent on the activation of the NMDA receptors in the CeA.</p>

Effects of Whole Body Irradiation on Morphine, DAMGO, DPDPE, U50,488H and beta-endorphin-Induced Antinociception

Opioid receptors have been pharmacologically classified as micro, delta, kappa and epsilon. We have recently reported that the antinociceptive effect of morphine (a micro-opioid receptor agonist), but not that of beta-endorphin (a novel micro/epsilon-opioid receptor agonist), is attenuated by whole body irradiation (WBI). It is unclear at present whether WBI has differential effects on the antinociceptive effects of micro-, delta-, kappa- and epsilon-opioid receptor agonists. In our current experiments, male ICR mice were exposed to WBI (5Gy) from a 60Co gamma-source and the antinociceptive effects of opioid receptor agonists were assessed two hours later using the hot water (52degrees C) tail-immersion test. Morphine and D-Ala2,N-Me-Phe4,Gly-olenkephalin(DAMGO), [D-Pen2-D-Pen5]enkephalin (DPDPE), trans-3,4-Dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U50,488H), and beta-endorphin were tested as agonists for micro, delta, kappa, and epsilon-opioid receptors, respectively. WBI significantly attenuated the antinociceptive effects of morphine and DAMGO, but increased those of beta-endorphin. The antinociceptive effects of DPDPE and U50,488H were not affected by WBI. In addition, to more preciously understand the differential effects of WBI on micro- and epsilon-opioid receptor agonists, we assessed pretreatment effects of beta-funaltrexamine (beta-FNA, a micro-opioid receptor antagonist) or beta-endorphin1-27 (beta-EP1-27, an epsilon-opioid receptor antagonist), and found that pretreatment with beta-FNA significantly attenuated the antinociceptive effects of morphine and beta-endorphin by WBI. beta-EP1-27 significantly reversed the attenuation of morphine by WBI and significantly attenuated the increased effects of beta-endorphin by WBI. The results demonstrate differential sensitivities of opioid receptors to WBI, especially for micro- and epsilon-opioid receptors.

Investigation of Orphanin FQ-stimulated 35SGTPgammaS Binding in the Whole Brain of Mice: Does Orphanin FQ Have Anti-opioid Effect in the Level of Receptor-ligand Interaction and 35SGTPgammaS Activation? / 대한마취과학회지

BACKGROUND: This study was examined whether or not the orphanin FQ (OFQ)-stimulated [35S]GTPgammaS activity interact with DAMGO in the whole brain of mice. METHODS: ICR mice (male, n = 20, 20-25 g) were euthanized for the membrane preparations. In the agonist-stimulated [35S]GTPgammaS binding dose-response curves by OFQ, Ro-64-6198 and DAMGO, the EC50 (effective concentration 50, nM) and maximum stimulation (% over basal) were determined in the presence or absence of J-113397 (10 nM), a NOP (nociceptin-opioid peptide) receptor antagonist. OFQ (1micrometer), Ro-64-6198 (10micrometer), DAMGO (10micrometer) and their combination cocktail were used to determine the interaction between the NOP and MOP (micron-opioid peptide) receptor. RESULTS: The values of EC50 and maximum stimulation of [35S]GTPgammaS binding were as follows: OFQ (9.2 +/- 0.2 nM/17.9 +/- 0.1%), Ro-64-6198 (143.5 +/- 0.5 nM/18.1 +/- 0.4%), and DAMGO (680.6 +/- 0.7 nM/18.1 +/- 0.5%). J-113397 produced a 8.7 and 7.1 fold rightward shifting in the OFQ and Ro-64-6198-stimulated [35S]GTPgammaS binding dose-response curve respectively, but not in the DAMGO. OFQ combined with DAMGO-stimulated [35S]GTPgammaS binding had an additive effect, but not in the OFQ combined with Ro-64-6198. CONCLUSIONS: OFQ, Ro-64-6198 and DAMGO-stimulated [35S]GTPgammaS binding in the brain of mice has receptor selectivity. The [35S]GTPgammaS stimulation of OFQ and DAMGO had an additive effect rather than an anti-opioid effect on the level of intracellular signal transduction through agonist-stimulated [35S]GTPgammaS bindings.

Studies of mu-, kappa-, and delta-Opioid Receptor Densities and G Protein Activation in Cerebral Membranes of Mice / 대한마취과학회지

BACKGROUND: The aim of this study was to investigate the relative densities of mu-, kappa-, and delta-opioid receptors (MOR, KOR, and DOR) and [35S]GTPgammaS binding as stimulated by full agonists in mouse cerebral membranes METHODS: Naive mice (ICR, male, n = 20) were euthanized for cerebral membrane preparation. For saturation binding, [3H]DAMGO, [3H]U69593, and [3H]DPDPE were used to determine the binding parameters [Bmax (femtomoles per milligram)/Kd (nanomolar)]. And, for [35S]GTPgammaS stimulation binding, DAMGO, U69593, and SNC80 were used to determine EC50 (nanomolar) and maximum stimulation (% over basal) for MOR, KOR, and DOR, respectively. The Ke values of the corresponding selective antagonist, naloxone (20 nM), nor-BNI (3 nM), and naltrindole (3 nM) were also calculated. RESULTS: The values of Bmax and Kd for saturation binding were as follows: [3H]DAMGO (MOR; 56.4/0.92), [3H]U69593 (KOR; 23.6/1.66), and [3H]DPDPE (DOR; 71.4/3.12), and the relative proportions of MOR, KOR, and DOR were 37.3, 15.6, and 47.2%. The EC50, maximum stimulation, and the Ke value of [35S]GTPgammaS binding were as follows: DAMGO (MOR; 215.3/18.3/2.10), U69593 (KOR; 38.5/8.9/0.32), and SNC80 (DOR; 84.3/28.3/0.36). Bmax and maximum [35S]GTPgammaS binding stimulation were linearly correlated (r = 0.99). CONCLUSIONS: The densities of three opioid receptors were found to be significantly different in mouse cerebral membrane. The amount of G protein activated by agonists were found to be directly proportional to relative receptor density.

Change of Hyperexcitability of Hippocampus by Cyclosporin A and Its Modulatory Action by Fentanyl

Cyclosporin A is used to treat patients with immune-mediated diseases, chronic diseases requiring organ transplantation, or malignancies. These conditions often require higher cyclosporin A doses, which may be toxic to the central nervous system. Fentanyl is also used in clinical conditions that have a risk of hypoxic neurosusceptiblity, which suggests that the drug may be a neuroprotective agonist against brain ischemic injury. Fentanyl is an opioid agonist and appears to play an important role in regulating the excitability of the hippocampus under electroconvulsion. In this study, the effects of fentanyl on modulating cyclosporin A-induced neurotoxicity was investigated. Treatment with 3 micrometer of cyclosporin A was found to reduce the electroconvulsive activity threshold. Fifty ng/mL of fentanyl reduced the electroconvulsive activity, and 1 micrometer of DAGO ([D-Ala2, N-Me-Phe4, Gly-ol]-enkephalin) also decreased the electroconvulsive activity. Fifty ng/mL of fentanyl was also found to reduce cyclosporin A-induced electroconvulsive activity. Although cyclosporin A neurotoxicity may be observed in various conditions, the opioid effect of neuroprotection may be involved in an interrelated mechanism. The exogenous opioid agonist suppressed cyclosporin A-induced electroconvulsive activity. Furthermore, there may be a functional anticonvulsant effect on cyclosporin A-induced neurotoxicity with an increased opioid agonist concentration.

Inhibitory Mechanism of Periaqueductal Gray Matter on Neuropathic Pain in Rat

OBJECTIVE: Using Lee et al (1996) model, we assessed the effect of opioid within the PAG on the manifestations of the neuropathic pain, and we studied the effects of naloxone on the analgesic effects of opioid. METHOD: Under pentobarbital anesthesia, male Sprague-Dawley rats were implanted with cannula in the ventral (n=10) and dorsal (n=6) PAG after the unilateral tibial and sural nerves were ligated and cut, leaving the common peroneal nerve intact. Pain sensitivity was assessed using the von Frey filament (8 mN) and acetone applied to the sensitive area for 1 week postoperatively. Rats with neuropathic pain were intracerebrally microinjected with DAMGO (0.1microgram/5microliter) and enkephaline (20microgram/5microliter) into the ventral and dorsal PAG and the pain sensitivity was assessed. Naloxone was injected to assess the observed change of pain sensitivity. RESULTS: Intracerebral microinjection of DAMGO and enkephaline into the ventral PAG, but not the dorsal PAG, increased the pain threshold which was reversed by naloxone. CONCLUSION: The results suggest that stimulation of the ventral PAG in neuropathic rats may reduce neuropathic pain via opioid-mediating pathway of the descending pain inhibition system.

The Effect of Mu Agonist on Seizure in Hippocampus

PURPOSE: Mu agonists can be used in clinical conditions where threr is a risk of hypoxic neurosusceptibility. Fentanyl, one of mu agonists may serve as neuroprotection from brain ischemic injury and protects against some kinds of brain injury and appears to play an important role in the regulation of brain excitability, especially in hippocampus during seizure. We investigated the effects of mu agonists - fentanyl and DAGO([D-Ala2, N- Me-Phe4, Gly-ol]-enkephalin) on electroconvulsive seizures. METHODS: The study was done with Sprague-Dawley rats(7-30 days), weighting from 20.4 g to 78.7 g. The animal were kept in-groups with mother rat in cages, and had free access to food and tap water. The temperature of the animal room is room temperature. Hippocampal slices were taken. Hippocampal slices were exposed to DAGO. Then, we began to record electrical activity of slices every 10 minutes in artificial cerebrospinal fluid. We observed the frequency and duration of electrical activity. RESULTS: The mean duration and frequency of fentanyl 50 ng/mL-treated ictal activity was 23.1+/-2.4 seconds and 85.98.1(n=7). These results were significant differences compared with control. The mean duration and frequency in fentanyl 50 ng/mL-treated interictal activity was 518.5+/-64.0 seconds, 132.0+/-14.0(n=5). There were also significant differences in the duration and frequency of onset in fentanyl 50 ng/mL-treated interictal activity compared with control showing interictal activity. The mean duration of latency time of onset in fentanyl 50 ng/mL-treated interictal activity was 143.0+/-11.3 seconds(n=5), and there was a significantly different latency of onset time in fentanyl 50 ng/mL-treated interictal activity compared with control showing interictal activity. The mean duration and frequency of DAGO 1M-treated ictal activity was 12.72.8 seconds and 87.3+/-11.1(n=7). These results were significant differences compared with control. The mean duration and frequency in DAGO 1M-treated interictal activity was 464.6+/-42.5 seconds, 64.2+/-23.0(n=5). There were also significant differences in the duration and frequency of onset in DAGO 1M-treated interictal activity compared with control showing interictal activity. The mean duration of latency time of onset in DAGO 1 microM-treated interictal activity was 28.49.4 seconds(n=5), and there was a significantly different latency of onset time in DAGO 1 microM- treated interictal activity compared with control showing interictal activity. CONCLUSION: Fentanyl 50 ng/mL showed a decrease of electroconvulsive seizures, so did DAGO 1 microM. These exogenous mu agonists suppress electroconvulsive seizures, and with increased levels of mu agonists, there may be a functional anticonvulsant effect through mu agonists, there may be a functional anticonvulsant effect through mu receptors. Also, these seem to modulate the convulsions. The study suggests that mu agonists may play a role in the pathogenesis of electroconvulsive seizures and shows a positive clue to the relationship between anticonvulsive effects and mu agonists.

The inhibitory effect of opioid on the hyperpolarization-activated cation currents in rat substantia gelatinosa neurons

The action of opioid on the hyperpolarization-activated cation current (Ih) in substantia gelatinosa neurons were investigated by using whole-cell voltage-clamp recording in rat spinal brain slices. Hyperpolarizing voltage steps revealed slowly activating currents in a subgroup of neurons. The half-maximal activation and the reversal potential of the current were compatible to neuronal Ih. DAMGO (1 muM), a selective-opioid agonist, reduced the amplitude of Ih reversibly. This reduction was dose-dependent and was blocked by CTOP (2 muM), a selective mu-opioid antagonist. DAMGO shifted the voltage dependence of activation to more hyperpolarized potential. Cesium (1 mM) or ZD 7288 (100 muM) blocked Ih and the currents inhibited by cesium, ZD 7288 and DAMGO shared a similar time and voltage dependence. These results suggest that activation of mu-opioid receptor by DAMGO can inhibit Ih in a subgroup of rat substantia gelatinosa neurons.

Dual effect of dynorphin A on single-unit spike potentials in rat trigeminal nucleus

The amygdala is known as a site for inducing analgesia, but its action on the trigeminal nucleus has not been known well. Little information is available on the effect of dynorphin on NMDA receptor-mediated electrophysiological events in the trigeminal nucleus. The purpose of this study was to investigate the changes in the single neuron spikes at the trigeminal nucleus caused by the amygdala and the action of dynorphin on the trigeminal nucleus. In the present study, extracellular single unit recordings were made in the dorsal horn of the medulla (trigeminal nucleus caudalis) and the effects of microiontophoretically applied compounds were examined. When (D-Ala2, N-Me-Phe4, Glys5-ol)enkephalin (DAMGO, 10-25 mM), a mu-opioid receptor agonist, was infused into the amygdala, the number of NMDA-evoked spikes at the trigeminal nucleus decreased. However, the application of naloxone into the trigeminal nucleus while DAMGO being infused into the amygdala increased the number of spikes. Low dose (1 mM) of dynorphin in the trigeminal nucleus produced a significant decrease in NMDA-evoked spikes of the trigeminal nucleus but the NMDA-evoked responses were facilitated by a high dose (5 mM) of dynorphin. After the kappa receptors were blocked with naloxone, dynorphin induced hyperalgesia. After the NMDA receptors were blocked with AP5, dynorphin induced analgesia. In conclusion, dynorphin A exerted dose-dependent dual effects (increased & decreased spike activity) on NMDA-evoked spikes in the trigeminal nucleus. The inhibitory effect of the dynorphin at a low concentration was due to the activation of kappa receptors and the excitatory effect at a high concentration was due to activation of NMDA receptors in the trigeminal neurons.

DAMGO, a micro-opioid agonist and cholecystokinin-octapeptide have dual modulatory effects on capsaicin-activated current in rat dorsal root ganglion neurons

Capsaicin, a pungent ingredient of hot pepper, elicits an intense burning pain when applied cutaneously and intradermally. Activation of capsaicin-gated channel in. C-type dorsal root ganglion (DRG) neurons produces nonselective cationic currents. Although electrophysiological and biochemical properties of capsaicin-activated current (ICAP) were studied, the regulatory mechanism and intracellular signaling pathway are still unclear. In the present study, we investigated the modulations of ICAP by DAMGO (micro-opioid agonist) and cholecystokinin octapeptide (CCK-8). In 18 out of 86 cells, the amplitude of ICAP was significantly increased by DAMGO and completely reversed after washout, while ICAP was decreased by DAMGO in 25 cells. In 43 cells, DAMGO had no effect on ICAP. Mean action potential duration was significantly different between 'increased-by-DAMGO' group and 'decreased-by-DAMGO' group. Mean amplitudes of IH were not significantly different between both groups. CCK-8 reversibly enhanced the amplitude of ICAP (5/13). DAMGO also increased ICAP amplitude significantly in the same cells. The amplitude of ICAP was increased in additive manner by combined applications of DAMGO and CCK-8 in these cells. These results suggest that DAMGO and CCK-8 can either increase or decrease ICAP presumably depending on the subtypes of DRG cells and classified by electrophysiological properties.

Effects of intrathecal L-and N-type calcium channel blockers on the antinociception evoked by opioid agonists in the rat tail flick test

The effects of intrathecal administration of nimodipine or omega-conotoxin GVIA(L- and N-type calcium channel blockers, respectively) alone or followed by DAMGO, DADLE or bremazocine (mu-, delta- and kappa- opioid agonists, respectively) were studied on the rat tail flick test. The N- (16 to 64 pmoles), but not the L-type blocker (60 to 240 pmoles) produced a dose and time-dependent increase in the latency for the tail-flick reflex. DAMGO (30 to 120 pmoles) or bremazocine (190 to 560 pmoles), but not DADLE (50 to 200 pmoles), produced a dose-dependent increase in the latency for the tail-flick reflex. The effect of the highest dose of DAMGO was smaller, while the effects of DADLE and bremazocine were not changed after nimodipine (60 pmoles). The effects of DADLE were significantly potentiated, while the effects of DAMGO and bremazocine were not changed after omega-conotoxin GVIA (16 pmoles). The intrathecal administration of an N-type calcium channel blocker with a delta-opioid agonist seems to be the most effective combination to produce antinociception in the rat tail flick test.