Suppression of morphine withdrawal by electroacupuncture in rats: dynorphin and kappa-opioid receptor implicated

Our previous work has demonstrated that 100-Hz electroacupuncture (EA) or 100-Hz transcutaneous electrical nerve stimulation (TENS) was very effective in ameliorating the morphine withdrawal syndrome in rats and humans. The mechanism was obscure. (1) Rats were made dependent on morphine by repeated morphine injections (5-140 mg/kg, s.c., twice a day) for eight days. They were then given 100-Hz EA for 30 min 24 h after the last injection of morphine. A marked increase in tail flick latency (TFL) was observed. This effect of 100-Hz EA could be blocked by naloxone (NX) at 20 mg/kg, but not at 1 mg/kg, suggesting that 100-Hz EA-induced analgesia observed in morphine-dependent rats is mediated by kappa-opioid receptors. (2) A significant decrease of the concentration of dynorphin A (1-17) immunoreactivity (-ir) was observed in the spinal perfusate in morphine-dependent rats, that could be brought back to normal level by 100-Hz EA. (3) 100-Hz EA was very effective in suppressing NX-precipitated morphine withdrawal syndrome. This effect of EA could be prevented by intrathecal administration of nor-BNI (2.5 micrograms/20 microliters), a kappa-opioid receptor antagonist, or dynorphin A (1-13) antibodies (25 micrograms/20 microliters) administered 10 min prior to EA. In conclusion, while the steady-state spinal dynorphin release is low in morphine-dependent rats, it can be activated by 100-Hz EA stimulation, which may be responsible for eliciting an analgesic effect and ameliorating morphine withdrawal syndrome, most probably via interacting with kappa-opioid receptor at spinal level.     

Dynorphin A (1–13) Neurotoxicity in Vitro: Opioid and Non-Opioid Mechanisms in Mouse Spinal Cord Neurons

Dynorphin A is an endogenous opioid peptide that preferentially activates κ-opioid receptors and is antinociceptive at physiological concentrations. Levels of dynorphin A and a major metabolite, dynorphin A (1–13), increase significantly following spinal cord trauma and reportedly contribute to neurodegeneration associated with secondary injury. Interestingly, both κ-opioid and N-methyl- -aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through κ-opioid and/or NMDA receptor types. Despite these findings, few studies have systematically explored dynorphin toxicity at the cellular level in defined populations of neurons coexpressing κ-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both κ-opioid and NMDA receptors from embryonic mouse spinal cord and examined the effects of dynorphin A (1–13) on intracellular calcium concentration ([Ca²⁺]_i) and neuronal survival in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. At micromolar concentrations, dynorphin A (1–13) elevated [Ca²⁺]_i and caused a significant loss of neurons. The excitotoxic effects were prevented by MK-801 (Dizocilpine) (10 μM), 2-amino-5-phosphopentanoic acid (100 μM), or 7-chlorokynurenic acid (100 μM)—suggesting that dynorphin A (1–13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (−)-naloxone (3 μM), or the more selective κ-opioid receptor antagonist nor-binaltorphimine (3 μM), exacerbated dynorphin A (1–13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 μM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 μM). Thus, opioid receptor blockade significantly increased toxicity, but only in the presence of excitotoxic levels of dynorphin. This provided indirect evidence that dynorphin also stimulates κ-opioid receptors and suggests that κ receptor activation may be moderately neuroprotective in the presence of an excitotoxic insult. Our findings suggest that dynorphin A (1–13) can have paradoxical effects on neuronal viability through both opioid and non-opioid (glutamatergic) receptor-mediated actions. Therefore, dynorphin A potentially modulates secondary neurodegeneration in the spinal cord through complex interactions involving multiple receptors and signaling pathways.     

Inhibition of morphine tolerance and dependence by diazepam and its relation to μ-opioid receptors in the rat brain and spinal cord

We have recently observed that concomitant administration of diazepam to morphine pellet implanted rats results in the inhibition of the development of morphine tolerance and dependence. We have now analyzed μ-opioid receptors in rats treated with morphine and diazepam for 5 days by using -DAMGO for binding studies. Male Sprague–Dawley rats were made tolerant and dependent by subcutaneous (s.c.) implantation of six morphine pellets (two pellets on the first day, and four on the second day). Diazepam (0.25 mg/kg b.wt) was injected once daily intraperitoneally (i.p.) for 5 days. Control rats were implanted with placebo pellets and injected once daily with saline or diazepam (i.p.). Animals were administered s.c. naloxone (10 mg/kg) to induce naloxone-precipitated withdrawal syndrome on the final day of the experiment (day 5). There was an up-regulation of μ-receptor (B_max increased) in the spinal cord of morphine tolerant (+139%) and dependent (+155%) rats compared to saline treated animals. Diazepam treatment abolished the up-regulation of μ-receptors in spinal cord of morphine treated rats. In the cortex, B_max was not affected in morphine tolerant or dependent rats but it decreased by 38% in morphine tolerant and 65% in morphine dependent rats treated with diazepam. The K_d of μ-receptors increased in the cortex, striatum and hypothalamus of morphine dependent rats. Diazepam treatment decreased the K_d of μ-receptors in the cortex of morphine tolerant and hypothalamus of morphine-dependent rats. These results suggest that diazepam treatment antagonizes the up-regulation of CNS μ-receptors observed in morphine tolerant rats. In addition, morphine tolerance and dependence may be associated with conversion of μ-opioid receptors to μ*-constitutive opioid receptors that are less active, and this conversion is prevented in the brain of animals treated with diazepam.     

Effects of morphine and morphine withdrawal on adrenergic neurons of the rat rostral ventrolateral medulla

In urethane anesthetized rats, iontophoretic application of morphine or α-methylnoradrenaline (α-MNE) inhibited (80–100%) the discharges of all putative adrenergic (C1) cells of the rostral ventrolateral medulla (RVLM). The effect of morphine was blocked selectively by naloxone while that of α-MNE was blocked selectively by theα₂-adrenergic antagonist idazoxan. Putative C1 cells were inhibited (75–100%) by low i.v. doses of clonidine (10–15 μg/kg). Most cells (7/10) were also inhibited by morphine i.v. (81% at 7 mg/kg). Two cells were slightly excited at doses below 2 mg/kg and inhibited at higher doses. Three cells were excited only. All effects of morphine i.v. were reversed by naloxone (1 mg/kg, i.v.). Intravenous administration of naloxone to morphine-dependent rats increased significantly the firing rate of all putative C1 adrenergic cells (from 5.8 ± 0.9 spikes/s to 12.3 ± 1.5 spikes/s;n = 8). During withdrawal these cells could still be inhibited (80–100%) by i.v. injection of clonidine (15 μg/kg). C-Fos expression induced by naltrexone-precipitated withdrawal was examined in the brainstem of freely moving morphine-dependent rats pretreated with clonidine or saline before injection of the opioid antagonist. The locus coeruleus (LC) of the same rats was examined for comparison. Morphine withdrawal without clonidine treatment significantly increased the number of Fos-like-immunoreactive (Fos-LIR) cells in the RVLM and LC. Clonidine pretreatment (1 mg/kg, i.p.) reduced the number of withdrawal-activated Fos-LIR cells in LC by 81%. In the RVLM this reduction averaged 37% for all cell types and 48% for C1 adrenregic cells. Further, a very large proportion of RVLM neurons that expressed c-Fos during morphine withdrawal (83%) were immunoreactive forα₂A-adrenergic receptors. This study suggests that, like noradrenergic cells of the LC, C1 adrenergic neurons of the RVLM are: (i) inhibited by both opiate andα₂-adrenergic receptor agonists; and (ii) activated during naloxone-precipitated morphine withdrawal, Since C1 cells are considered essential to sympathetic tone generation, their inhibition by morphine may contribute to the hypotensive effects of this opioid agonist in non-dependent individuals. Their excitation during opiate withdrawal may also contribute to the autonomic activation that characterizes this syndrome. Finally, inhibition of C1 cells by clonidine may contribute to the clinically recognized efficacy of this drug to attenuate autonomic signs of opiate withdrawal.     

The effect of morphine tolerance dependence and abstinence on immunoreactive dynorphin (1–13) levels in discrete brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

The effect of morphine tolerance dependence and protracted abstinence on the levels of dynorphin (1–13) in discrete brain regions, spinal cord, pituitary gland and peripheral tissues was determined in male Sprague-Dawley rats. Of all the tissues examined, the highest level of dynorphin (1–13) was found to be in the pituitary gland. Among the brain regions and spinal cord examined, the levels of dynorphin (1–13) in descending order were: hypothalamus, spinal cord, midbrain, pons and medulla, hippocampus, cortex, amygdala and striatum. The descending order for the levels of dynorphin (1–13) in peripheral tissues was: adrenals, heart and kidneys. In morphine tolerant rats, the levels of dynorphin (1–13) increased in amygdala but were decreased in pons and medulla. In morphine abstinent rats, the levels of dynorphin (1–13) were increased in amygdala, hypothalamus and hippocampus. The levels of dynorphin (1–13) were increased in pituitary but decreased in spinal cord and remained so even during protracted abstinence. The levels of dynorphin (1–13) in the peripheral tissues of morphine tolerant rats were unaffected. However, in the heart and kidneys of morphine abstinent rats, the levels of dynorphin (1–13) were increased significantly. It is concluded that both morphine tolerance and abstinence modify the levels of dynorphin (1–13) in pituitary, central and peripheral tissues. Morphine abstinence differed from non-abstinence process in that there were additional changes (increases) in the levels of dynorphin (1–13) in brain regions (hypothalamus and hippocampus) and peripheral tissues (heart and kidneys) and may contribute to the symptoms of the morphine abstinence syndrome. The lower levels of dynorphin (1–13) in spinal cord may be responsible for the potentiation of morphine effects by κ-opiate agonist in morphine tolerant dependent rodents.     

μ-, δ-, κ- and ε-Opioid receptor modulation of the hypothalamic-pituitary-adrenocortical (HPA) axis: subchronic tolerance studies of endogenous opioid peptides

In opiate-naive rats, the endogenous opioid peptides, β-endorphin, dynorphin(1–13) and Met---Enk---Arg---Phe (MEAP) and the synthetic enkephalin analogue -Ala²- -Leu⁵-Enk (DADLE) potently stimulated plasma corticosterone in a dose-dependent, naloxone-reversible manner. To characterize their in vivo affinities, the effects of these peptides on plasma corticosterone release were tested in rats made tolerant to morphine, U50488H, DADLE/morphine or β-endorphin. These cross-tolerance studies showed that dynorphin and MEAP exerted their action on plasma corticosterone release at κ-opioid receptors. The action of DADLE occurred at δ-opioid receptors, while the action of β-endorphin occurred principally at another receptor site. These results indicate that there is independent modulation of the hypothalamic-pituitary-adrenal axis by endogenous opioid peptides at μ-, δ- and κ-opioid receptors. In addition, there may be modulation by β-endorphin at a separate site that we suggest could be a central ε-receptor site. This cross-tolerance paradigm, using a neuroendocrine model, provides in vivo evidence for the action of centrally active endogenous opioid peptides at multiple and independent opioid receptors.     

The unconditioned fear produced by morphine withdrawal is regulated by μ- and κ-opioid receptors in the midbrain tectum

We have recently shown that morphine withdrawal sensitizes the neural substrates of fear in the midbrain tectum structures—the dorsal periaqueductal gray (dPAG) and inferior colliculus (IC). In the present study, we investigated the role of μ- and κ-opioid receptors in the mediation of these effects. Periadolescent rats chronically treated with morphine (10 mg/kg; s.c.) twice daily for 10 days were implanted with an electrode glued to a guide-cannula into the dPAG or the IC. Forty-eight hours after the interruption of this treatment, the effects of intra-dPAG or intra-IC microinjections of [D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephalin (DAMGO; 0.6 and 1 nmol/0.2 μl) – a selective μ-receptor agonist – or nor-binaltorphimine (BNI; 2.5 and 5 μg/0.2 μl) – a selective κ-receptor antagonist with tardive action – on the freezing and escape thresholds determined by electrical stimulation of the dPAG and the IC were examined. For both structures, morphine withdrawal produced pro-aversive effects. DAMGO and BNI had antiaversive effects when injected into the dPAG and IC of non-dependent rats. In morphine-withdrawn rats, only BNI continued to promote antiaversive effects in both structures. Whereas DAMGO lost its antiaversive efficacy when injected into the dPAG, only its highest dose promoted antiaversive effects in the IC of morphine-withdrawn rats, suggesting the development of an apparent tolerance. Thus, the enhanced reactivity of the midbrain tectum in morphine-withdrawn periadolescent rats may be due, at least partially, to an impairment of the inhibitory influence of mechanisms mediated by μ-receptors on the neural substrates of fear in this region.     

Characterization of mechanical withdrawal responses and effects of μ-, δ- and κ-opioid agonists in normal and μ-opioid receptor knockout mice

Clinical and experimental observations suggest that opiates can exert different influences on the perception of stimuli from distinct sensory modalities. Thermally-induced nociception is classically responsive to opiate agonists. μ-Opioid receptor-deficient transgenic mice are more sensitive to thermal nociceptive stimuli and morphine fails to attenuate the nociceptive responses to thermal stimuli in these animals. To enhance our understanding of opiate influences on mechanical sensitivity, we have examined withdrawal responses to a sequence of ascending forces of mechanical stimuli in mice with normal (wild type), half-normal (heterozygous) and absent (homozygous) μ-opioid receptor levels. We report data from mice examined without drug pretreatment or following pretreatment with morphine, the selective κ-opioid agonist, U50488H, and the selective δ-opioid agonist, DPDPE. Saline-pretreated mice of each genotype displayed similar, monotonically increasing frequency of withdrawal responses to the graded stimuli. Subcutaneously administered morphine produced a dose-dependent reduction in withdrawal responses in wild type and heterozygous mice, but had no significant effect in homozygous mice. Intraventricular administration of DPDPE also reduced the frequency of paw withdrawal (FPW) in wild type mice, but not in homozygous mice. In contrast, systemic U50488H produced a dose-dependent attenuation of paw withdrawal in both wild type and homozygous mice. These findings suggest that (1) interactions of endogenous peptides with μ-opioid receptors may not play a significant role in the response to mechanical stimuli in drug-free animals, and (2) deficiency of μ-opioid receptors has no functional consequence on the response to the prototypical κ-opioid receptor agonist, but decreases responses to the prototypical μ- and δ-opioid receptor agonists.     

The effect of U-50,488H tolerance-dependence and abstinence on the levels of dynorphin (1–13) in brain regions, spinal cord, pituitary gland and peripheral tissues of the rat

Male Sprague-Dawley rats were rendered tolerant to and physically dependent on U-50,488H, a κ-opiate agonist, by injecting 25 mg/kg of the drug intraperitoneally twice a day for 4 days. Two sets of rats were used. Rats labeled as tolerant-dependent were injected with U-50,488H (25 mg/kg) 1 h before sacrificing on day 5, whereas the abstinent rats were sacrificed on day 5 without the injection of U-50,488H. Of all the tissues examined, the pituitary gland had the highest level of dynorphin (1–13), whereas the heart had the lowest level. The levels of dynorphin (1–13) increased in the hypothalamus, hippocampus and pons/medulla of U-50,488H tolerant-dependent rats, whereas in abstinent rats the levels of dynorphin (1–13) were elevated only in the midbrain. The levels of dynorphin (1–13) in the pituitary gland of U-50,488H tolerant-dependent or abstinent rats were unchanged. In peripheral tissues, the levels of dynorphin (1–13) in the heart of U-50,488H tolerant-dependent rats were increased. In the abstinent rats they were elevated in the adrenals, spleen, and the heart but were decreased in the kidneys. Compared to morphine tolerant-dependent and abstinent rats, significant differences in the levels of dynorphin (1–13) in tissues of 50,488H tolerant-dependent and abstinent rats were observed and may explain many pharmacological differences in the μ- and κ-opiate induced tolerance-dependence and abstinence processes.     

Pharmacological characterization of the ameliorating effect on short-term memory impairment and antinociceptive effect of KT-90 in mice

(−)-3-Acetyl-6β-acetylthio-N-cyclopropylmethyl-normorphine (KT-90) is a synthesized compound that binds to μ-, δ- and κ-opioid receptors in vitro. KT-90 induces analgesia in the tail-flick test and this effect is antagonized by nor-BNI, a selective κ-opioid receptor antagonist. However, lower doses of KT-90 antagonize morphine-induced analgesia. We reported that κ-opioid receptor agonists such as U-50,488H and dynorphin A (1-13), improved scopolamine-induced impairment of learning and memory in mice and/or rats. In this study, the effects of KT-90 were investigated in an acetic acid-induced writhing test and scopolamine-induced memory impairment test using spontaneous alternation performance in a Y-maze. Male ddY mice were treated with scopolamine (1.65 μmol/kg, s.c.) 30 min before the behavioral test. KT-90 (0.07–2.35 μmol/kg, s.c.) was injected 30 min before testing. In the writhing test, the antinociceptive effect of KT-90 (0.71 μmol/kg) was completely antagonized by a selective μ-opioid receptor antagonist, β-funaltrexamine (10.2 nmol/mouse, i.c.v.) and partially antagonized by nor-BNI (4.9 nmol/mouse, i.c.v.), but it was not antagonized by a selective δ-opioid receptor antagonist, naltrindole (9.1 pmol/mouse, i.c.v.). KT-90 significantly improved the impairment of spontaneous alternation induced by scopolamine. The ameliorating effect of KT-90 was not antagonized by nor-BNI, but was almost completely antagonized by a selective σ receptor antagonist, NE-100 (2.6 μmol/kg, i.p.). These results suggested that the KT-90-induced antinociceptive effect was mediated by μ- and partially by κ-opioid receptors, and the KT-90-induced improvement in scopolamine-induced impairment of spontaneous alternation was mediated mainly via σ receptors.     

Streptozotocin-induced diabetes selectively alters the potency of analgesia produced by μ-opioid agonists, but not by δ- and κ-opioid agonists

To investigate the possible mechanisms of the alterations in morphine-induced analgesia observed in diabetic mice, we examined the influence of streptozotocin-induced (STZ-induced) diabetes on analgesia mediated by the different opioid receptors. The antinociceptive potency of morphine (10 mg/kg), administered s.c., as determined by both the tail-pinch and the tail-flick test, was significantly reduced in diabetic mice as compared to that in controls. Mice with STZ-induced diabetes had significantly decreased sensitivity to intracerebroventricularly (i.c.v.) administered μ-opioid agonists, such as morphine (10 μg) and [d-Ala², N-Me Phe⁴,Gly-ol⁵]enkephalin (DAMGO, 0.5 μg). However, i.c.v. administration of [d-Pen^2,5]enkephalin (DPDPE, 5 μg), a δ-opioid agonist, and U-50,488H (50 μg), a κ-opioid agonist, produced pronounced antinociception in both control and diabetic mice. Furthermore, there were no significant differences in antinociceptive potency between diabetic and control mice when morphine (1 μg), DAMGO (10 μg), DPDPE (0.5 μg) or U-50,488H (50 μg) was administered intrathecally. In conclusion, mice with STZ-induced diabetes are selectively hyporesponsive to supraspinal μ-opioid receptor-mediated antinociception, but they are normally responsive to activation of δ- and κ-opioid receptors.     

The effects of morphine on carrageenin-induced spinal c-Fos expression are completely blocked by β-funaltrexamine, a selective μ-opioid receptor antagonist

We have demonstrated that pre-administered morphine (3 mg/kg, i.v.) decreased spinal c-Fos expression induced 2 h after intraplantar carrageenin (55 ± 5% reduction, P < 0.0001). These effects were completely blocked by pre-administered β-funaltrexamine (10 mg/kg, i.v., 24 h prior to stimulation) a selective long-lasting μ-opioid receptor antagonist. In conclusion, these results clearly demonstrate that the effects of morphine on noxiously-evoked spinal c-Fos expression are essentially mediated via μ-opioid receptors.     

Effect of morphine on dynorphin B and GABA release in the basal ganglia of rats

In vivo microdialysis was used to study the effects of systemic, as well as intracerebral administration of morphine and naloxone on dynorphin B release in neostriatum and substantia nigra of rats. The release of dopamine (DA), γ-aminobutyric acid (GABA), glutamate (Glu) and aspartate (Asp) was also investigated. Systemic injection of morphine (1 mg/kg s.c.) induced long-lasting increases in extracellular dynorphin B and GABA levels in the substantia nigra, whereas DA, Glu and Asp levels, measured in the same region, were not significantly affected. No effect on striatal neurotransmitter levels was observed following systemic morphine administration. Local perfusion of the substantia nigra with morphine (100 μM) through the microdialysis probe also increased nigral dynorphin B and GABA levels. Perfusion of the neostriatum with morphine (100 μM) significantly increased GABA and dynorphin B levels in the ipsilateral substantia nigra, but no effect was observed locally. Naloxone blocked the effect of systemic morphine administration on nigral dynorphin B and GABA release, already at a dose of 0.2 mg/kg s.c. Naloxone alone, given either systemically (0.2–4 mg/kg s.c.) or intracerebrally (1–100 μM), did not affect dynorphin B or amino acid levels, either in neostriatum or in substantia nigra. However, naloxone produced a concentration-dependent increase in DA levels. The present results indicate that systemic morphine administration stimulates the release of dynorphin B in the substantia nigra, probably by activating the μ-subtype of opioid receptor, since the effect of morphine on nigral dynorphin B and GABA was antagonized by a low dose of naloxone. The increase in extracellular DA levels produced by high concentrations of naloxone, both in neostriatum and substantia nigra, indicates a disinhibitory effect of this drug on DA release, probably via a non-μ subtype of opioid receptors located on nigro-striatal DA neurones.     

The anxiolytic effect of acute ethanol or diazepam exposure is unaltered in μ-opioid receptor knockout mice

Previous researchers demonstrate an opioidergic involvement in the anxiolytic and rewarding actions of ethanol and diazepam. Therefore, to further characterize the role of the opioid system in the anxiolytic action of ethanol and diazepam, normal (C57BL/6J), hybrid (B6129F1) and μ-opioid receptor knockout mice were given i.p. ethanol (0, 1.0 or 1.6 g/kg) or diazepam (1.5 mg/kg). The anxiolytic properties of these agents were then tested in the elevated plus-maze. Additional ethanol-treated μ-opioid receptor knockout mice (1 g/kg) were pretreated with the κ-opioid receptor antagonist nor-BNI (0 or 3 mg/kg) to assess the involvement of κ-opioid activity in ethanol’s anxiolytic actions. The anxiolytic action of ethanol and diazepam in the μ-opioid receptor knockout mouse did not differ from the effects obtained in normal mice and pretreatment with nor-BNI did not significantly attenuate ethanol’s actions in μ-opioid receptor knockout mice. Thus, the anxiolytic actions of ethanol and diazepam appear to be independent of opioid system activity in the μ-opioid receptor knockout mouse.     

Evidence for roles of κ-opioid and NMDA receptors in the mechanism of action of ibogaine

Ibogaine, a putatively anti-addictive alkaloid, binds to κ-opioid and NMDA receptors. In the present study we investigated the roles of κ-opioid and NMDA actions in mediating ibogaine's (40 mg/kg, i.p.) behavioral and neurochemical effects in rats. A combination of a κ-opioid antagonist (norbinaltorphimine, 10 mg/kg, s.c.) and a NMDA agonist (NMDA, 20 mg/kg, i.p.) partially prevented ibogaine-induced inhibition of intravenous morphine self-administration and ibogaine-induced antagonism of morphine-induced locomotor stimulation. The combination, as well as norbinaltorphimine and NMDA alone, blocked the acute effects of ibogaine on dopamine release and metabolism in the striatum. The data suggest that both κ-opioid agonist and NMDA antagonist actions of ibogaine contribute to its putative anti-addictive effects.     

Comparative effects of N and MK-801 on the abstinence syndrome in morphine-dependent mice

The effects of N^{G-monomethyl-l-arginine} (l-NMMA), an inhibitor of nitric oxide (NO) synthase and MK-801, an NMDA receptor antagonist on abrupt and naltrexone-precipitated abstinence symptoms were determined in male Swiss-Webster mice rendered dependent on morphine by subcutaneous implantation of a pellet containing 75 mg of morphine base for 3 days. Mice which served as controls were implanted with placebo pellets. Six hours after pellet removal, mice were injected intraperitoneally with either the vehicle or MK-801 (0.03, 0.1 and 0.3 mg/kg). Thirty minutes later the animals were injected with naltrexone subcutaneously (50 μg/kg) and the intensity of abstinence symptoms were determined. Of the three doses of MK-801 used, only 0.1 mg/kg dose inhibited the jumping behavior precipitated by naltrexone in morphine-dependent mice. Whereas the lower dose (0.03 mg/kg) of MK-801 increased, the higher doses of MK-801 (0.1 and 0.3 mg/kg) displayed a decrease in the formation of fecal boli. Administration of MK-801 did not affect the body weight loss observed during abrupt withdrawal (induced by removal of the pellets) in morphine-dependent mice. MK-801 at 0.1 mg/kg dose further decreased the body temperature during abrupt withdrawal in morphine-dependent mice. Other two doses of MK-801 (0.03 and 0.3 mg/kg) did not modify the hypothermia observed during abrupt morphine withdrawal. On the other hand, l-NMMA (0.02 to 4.0 mg/kg) injected intraperitoneally 15 min prior to the naltrexone administration blocked the stereotyped jumping response in a dose-dependent manner. Higher doses of l-NMMA 2.0 and 4.0 mg/kg also decreased the number of fecal boli formation. l-NMMA (0.2 to 4.0 mg/kg) also significantly reduced the abrupt withdrawal-induced body weight loss in morphine-dependent mice. Thus MK-801 has very little effect, which is not dose-dependent, on abrupt and antagonist-precipitated withdrawal in morphine-dependent mice. However, the l-NMMA has more profound dose-dependent effects on both the abrupt and antagonist-precipitated withdrawal in morphine-dependent mice. It is concluded that the inhibitors of NO synthase may be more beneficial than NMDA receptor antagonists in managing the symptoms of morphine abstinence syndrome.     

κ-opioid receptor mediated antinociception in rats is dependent on the functional state of dihydropyridine-sensitive calcium channels

The modulatory effect of the dihydropyridine Ca²⁺ channel antagonist nimodipine on the analgesic action of the κ-opioid receptor agonist U-69,593 was analyzed using the tail-flick test in rats. The antinociceptive effect of U-69,593 (0.25–4 mg/kg) was antagonized by L-type Ca²⁺ channel blockade with nimodipine (200 μg/kg, i.p.), the ED₅₀ being increased from 1.4 to 7.3 mg/kg. On the contrary, when an increase in the density of these channels was induced by means of chronic and simultaneous treatment with nimodipine (1 μg/h, 7 days) and sufentanil (2 μg/h, 8 days), the analgesic effect of U-69,593 was potentiated by 5-fold. Our results suggest a functional coupling between κ-opioid receptors and L-type Ca²⁺ channels in nociception.     

Involvement of μ- and δ-opioid receptors in the ethanol-associated place preference in rats exposed to foot shock stress

The purpose of this study was to establish the ethanol-induced place preference in rats exposed to foot shock stress using the conditioned place preference paradigm. We also investigated the role of the endogenous opioid system in the development of the ethanol-induced place preference. The administration of ethanol (300 mg/kg, i.p.) with foot shock stress, but not without such stress, induced a marked and significant place preference. Naloxone (1 and 3 mg/kg, s.c.), a non-selective opioid receptor antagonist, significantly attenuated the ethanol-induced place preference. Moreover, the selective μ-opioid receptor antagonist β-funaltrexamine (3 and 10 mg/kg, i.p.) and selective δ-opioid receptor antagonist naltrindole (1 and 3 mg/kg, s.c.), but not the selective κ-opioid receptor antagonist nor-binaltorphimine (1 and 3 mg/kg, i.p.), significantly attenuated the ethanol-induced place preference. Furthermore, 150 mg/kg ethanol (which tended to produce a place preference, although not significantly) combined with each dose (that did not produce a place preference) of the μ-opioid receptor agonist morphine (0.1 mg/kg, s.c.) or selective δ-opioid receptor agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydroquinolino [2,3,3-g] isoquinoline (TAN-67; 20 mg/kg, s.c.), but not the selective κ-opioid receptor agonist trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzenacetamide methanesulfonate (U50,488H; 1 mg/kg, s.c.), produced a significant place preference. These data indicate that stress may be important for development of the rewarding effect of ethanol, and that μ- and δ-opioid receptors may be involved in the rewarding mechanism of ethanol under stressful conditions.     

Polyarthritis-associated changes in the opioid control of spinal CGRP release in the rat

As a model of chronic inflammatory pain, Freund's adjuvant-induced polyarthritis has been shown to be associated with marked alterations in the activity of opioid- and calcitonin gene-related peptide (CGRP)-containing neurons in the dorsal horn of the spinal cord in rats. Possible changes in the interactions between these two peptidergic systems in chronic inflammatory pain were investigated by comparing the effects of various opioid receptor ligands on the spinal outflow of CGRP-like material (CGRPLM) in polyarthritic and age-paired control rats. Intrathecal perfusion of an artificial cerebrospinal fluid in halothane-anaesthetized animals allowed the collection of CGRPLM released from the spinal cord and the application of opioid receptor ligands. The blockade of κ-opioid receptors similarly increased CGRPLM release in both groups of rats as expected of a κ-mediated tonic inhibitory control of CGRP-containing fibres in control, as well as in polyarthritic rats. In contrast, the higher increase in CGRPLM outflow due to the preferential blockade of μ opioid receptors by naloxone in polyarthritic rats as compared to non-suffering animals supports the idea of a reinforced μ opioid receptor-mediated tonic inhibitory control of CGRP-containing fibres in rats suffering from chronic pain. Even more strikingly, the differences observed in the effects of ∂-opioid receptor ligands on CGRPLM outflow suggest that ∂ receptors are functionally shifted from a participation in a phasic excitatory control in non-suffering rats to a tonic inhibitory control in polyarthritic rats. These data indicate that agonists acting at the three types of opioid receptors all exert a tonic inhibitory influence on CGRP-containing nociceptive primary afferent fibres within the spinal cord of polyarthritic rats. Such a convergence probably explains why morphine and other opioids are especially potent to reduce pain in subjects suffering from chronic inflammatory diseases.     

Etorphine elicits anomalous excitatory opioid effects on sensory neurons treated with GM1 ganglioside or pertussis toxin in contrast to its potent inhibitory effects on naive or chronic morphine-treated cells

The ultra-potent opioid analgesic, etorphine, elicits naloxone-reversible, dose-dependent inhibitory effects, i.e. shortening of the action potential duration (APD) of naive and chronic morphine-treated sensory dorsal root ganglion (DRG) neurons, even at low (pM-nM) concentrations. In contrast, morphine and most other opioid agonists elicit excitatory effects, i.e. APD prolongation, at these low opioid concentrations, require much higher (ca. 0.1–1 μM) concentrations to shorten the APD of naive neurons, and evoke only excitatory effects on chronic morphine-treated cells even at high > 1–10 wM concentrations. In addition to the potent agonist action of etorphine at μ-, δ- and κ-inhibitory opioid receptors in vivo and on DRG neurons in culture, this opioid has also been shown to be a potentantagonist of excitatory μ-, δ- and κ-receptor functions in naive and chronic morphine-treated DRG neurons. The present study demonstrates that the potent inhibitory APD-shortening effects of etorphine still occur in DRG neurons tested in the presence of a mixture of selective antagonists that blocks all μ-, δ- and κ-opioid receptor-mediated functions, whereas addition of the epsilon (ε)-opioid-receptor antagonist, β-endorphin(1–27) prevents these effects of etorphine. Furthermore, after markedly enhancing excitatory opioid receptor functions in DRG neurons by treatment with GM1 ganglioside or pertussis toxin, etorphine showsexcitatory agonist action onnon-μ-/δ-/κ-opioid receptor functions in these sensory neurons, in contrast to its usual potent antagonist action on μ-, δ- and κ-excitatory receptor functions in naive and even in chronic morphine-treated cells which become supersensitive to the excitatory effects of μ-, δ- and -opioid agonists. This weak excitatory agonist action of etorphine on non-μ-/δ-/κ-opioid receptor functions may account for the tolerance and dependence observed after chronic treatment with extremely high doses of etorphine in vivo.