Increasing of intrathecal CSF excitatory amino acids concentration following morphine challenge in morphine-tolerant rats

Excitatory amino acids (EAAs) are involved in the development of opioid tolerance. The present study reveals that an increasing of CSF EAAs concentration might be responsible for the losing of morphine's antinociceptive effect in morphine tolerant rats. Male Wistar rats were implanted with two intrathecal (i.t.) catheters and one microdialysis probe, then continuously infused i.t. for 5 days with saline (1 microl/h; control group), morphine (15 micrograms/h), the NMDA antagonist, MK-801 (5 micrograms/h), or morphine (15 micrograms/h) plus MK-801 (5 micrograms/h). Each day, tail-flick responses were measured; in addition, CSF dialysates were collected and CSF amino acids measured by high performance liquid chromatography using a fluorescence detector. Morphine started to lose its analgesic effect on day 2 and this effect was overcome by MK-801. The AD(50) (AD: analgesic dose) was 1.33 micrograms in control animals, 83.83 micrograms in morphine-tolerant rats (a 63-fold shift), and 11.2 micrograms (a 8.4-fold shift) in rats that had received MK-801 plus morphine. No significant differences were observed in CSF amino acid release between the groups from day 1 to day 5. On day 5, after basal dialysate collection, a 10-micrograms challenge of morphine was administered i.t., and CSF samples collected over the next 3 h. After morphine challenge, morphine-tolerant rats showed a significant increase in the release of glutamate and aspartate (131+/-9.5% and 156+/-12% of basal levels, respectively), and no antinociceptive effect in the tail-flick latency test, while MK-801/morphine co-infused rats showed no increase in morphine-induced EAA release and a partial antinociceptive effect (MPE=40%). The present study provides direct evidence for a relationship between EAA release and a lack of an antinociceptive response to morphine, and shows that the NMDA antagonist, MK-801, attenuates both of these effects.     

Supraspinal NMDA and non-NMDA receptors are differentially involved in the production of antinociception by morphine and beta-endorphin administered intracerebroventricularly in the formalin pain model

Our previous studies have demonstrated that supraspinal glutamate 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. The formalin pain test was used in the present study. Injection of mice with formalin solution (2%, 10 microl) into the hindpaw intraplantarly produced the first (0-5 min) and second (20-40 min) phases of formalin responses. The formalin responses in the both phases were attenuated dose-dependently by morphine (0.125-1 microg) or beta-endorphin (0.125-1 microg) administered i.c.v. 5 min before. The antinociceptive effect of morphine was slightly more potent in the second phase whereas the effect of beta-endorphin was more pronounced in the first phase. MK-801 (0.1-1 microg), a non-competitive NMDA receptor antagonist, and CNQX (0.05-0.5 microg), a non-NMDA antagonist, given i.c.v., produced antinociceptive effect in the both phases, but only in a partial manner. Both MK-801 (0.05 microg) and CNQX (0.01 microg), at the dose which had no intrinsic effect, reversed the antinociceptive effect of beta-endorphin (1 microg) observed during the second, but not the first, phase partially but significantly. However, the antinociceptive effect of morphine (1 microg) was not affected by the same dose of MK-801 or CNQX given i.c.v. Our results indicate that, at the supraspinal level, both NMDA and non-NMDA receptors are involved in the production of antinociception induced by supraspinally administered beta-endorphin, but not morphine, in the formalin pain model.     

The effect of cannabinoid receptor antagonism with SR141716A on antinociception induced by cocaine and the NMDA receptor antagonist,MK-801

In the rat, antinociception of supraspinal origin is observed in response to administration of cocaine or an antagonist of the NMDA receptor for glutamate. The current study was conducted to determine if endocannabinoids are involved in the antinociceptive effect of cocaine, or antagonism of NMDA receptor binding. Intraperitoneal (i.p.) administration to male rats of cocaine, or the NMDA receptor antagonist, MK-801, resulted in a significant antinociceptive response of supraspinal origin, as indicated by a significant increase in reaction time in the hot plate test of analgesia (increase in the amount of time before the animal reacted to the hot plate by licking its paws or jumping). Treatment with SR141716A, a specific antagonist of the cannabinoid (CB1) receptor, resulted in a complete reversal of cocaine-induced antinociception when administered at a dose of 5.0mg/kg. Although the 2.5 and 5.0mg/kg doses of SR141716A produced a significant reduction in the antinociceptive effect of MK-801, the effect was incomplete since the reaction time in the hot plate test remained greater than that observed in vehicle-treated controls. These findings suggest that activation of the CB1 receptor participates significantly in antinociception resulting from treatment with cocaine and with the NMDA receptor antagonist, MK-801. The partial reversal of the antinociceptive effect of MK-801 by CB1 receptor antagonism indicates other mediators of nociception, in addition to the endocannabinoids, appear to be active in the antinociceptive response to NMDA receptor antagonism.     

Intrathecal high-dose morphine induces spinally-mediated behavioral responses through NMDA receptors

Previous research has demonstrated that intrathecal i.t. morphine in a dose of 60.0 nmol into the spinal subarachnoid space of mice can evoke nociceptive behavioral responses consisting of a severe hindlimb scratching directed toward the flank followed by biting/licking of the hindpaw. The present study was undertaken to examine the involvement of spinal N-methyl-D-aspartate (NMDA) and opioid receptors on the behavioral responses evoked by high-dose i.t. morphine. Pretreatment with naloxone, an opioid receptor antagonist (1.0 and 4.0 mg/kg, s.c.), failed to reverse the morphine-evoked behavioral response, suggesting that the morphine effect is not mediated through the opioid receptors in the spinal cord. The morphine-induced behavior was dose-dependently inhibited by i.t. co-administration of the competitive NMDA receptor antagonists, D(-)-2-amino-5-phosphonovaleric acid (D-APV) (6.25-50.0 pmol) and 3-((+)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) (3.125-25.0 pmol). The characteristic behavior was also reduced by co-administration of (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptene-5,10-imine maleate (MK-801) (74.1-250 pmol), an NMDA ion-channel blocker. Ifenprodil, a competitive antagonist of the polyamine recognition site of NMDA receptor ion channel complex, produced a dose-related inhibitory effect on the behavioral response to i.t. morphine with less potency than the competitive and non-competitive antagonists examined. High doses of (+)-HA-966, a glycine/NMDA antagonist, induced a dose-dependent inhibition of morphine-induced response. The effective dose of i.t. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA receptor antagonist, needed to reduce the morphine-induced response, was approximately 10-fold greater than that of D-APV. These results suggest that spinal NMDA receptors, but not non-NMDA receptors, may be largely involved in elicitation of the behavioral episode following i.t. injection of morphine in mice.     

Effects of NMDA receptor blockade and nitric oxide synthase inhibition on the acute and chronic actions of Δ9-tetrahydrocannabinol in mice

The present studies examined the hypothesis that the N-methyl- d-aspartate (NMDA) receptor-nitric oxide (NO) pathway might be involved in the acute and chronic actions of Δ⁹-tetrahydrocannabinol (THC). The ability of dizocilpine (MK-801), a competitive NMDA receptor antagonist and N^G-monomethyl- l-arginine (l-NMMA), an inhibitor of NO synthase enzyme to modify the analgesic and hypothermic responses following the acute and chronic treatment of animals with THC was determined in male Swiss-Webster mice. Intraperitoneal administration of THC (5, 10 and 20 mg/kg) produced dose-dependent analgesic and hypothermic effects. MK-801 at 0.1 gg/kg i.p. attenuated the analgesic but not the hypothermic responses to THC (10 and 20 mg/kg, i.p.). The effects of various doses of MK-801 (0.03, 0.1 and 0.3 mg/kg, i.p.) on the analgesic and hypothermic responses to a 10 mg/kg, i.p. dose of THC was also determined. All the doses of MK-801 antogonized the analgesic but not the hypothermic effects of THC. The chronic treatment of animals with THC (10 mg/kg, i.p.) twice daily for 4 days produced tolerance to its analgesic and hypothermic effects. Pretreatment of animals with MK-801 (0.03–0.30 mg/kg, i.p.) did not affect the development of tolerance to the analgesic or the hypothermic action of THC. The pretreatment of animals with l-NMMA (2–8 mg/kg, i.p.), did not alter the analgesic or hypothermic effects of THC. Also, it did not modify the tolerance to its pharmacological actions. It is concluded that non-competitive antagonism of NMDA receptor by MK-801 selectively antagonized the analgesic action of THC and that the mechanisms in the analgesic response and tolerance to THC may be different. Finally, NO does not appear to be involved in the acute or chronic actions of THC.     

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 kappa-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 kappa-opioid and N-methyl-D-aspartate (NMDA) receptor antagonists can modulate dynorphin toxicity, suggesting that dynorphin is acting (directly or indirectly) through kappa-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 kappa-opioid and NMDA receptors. To address this question, we isolated populations of neurons enriched in both kappa-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 microM), 2-amino-5-phosphopentanoic acid (100 microM), or 7-chlorokynurenic acid (100 microM)--suggesting that dynorphin A (1-13) was acting (directly or indirectly) through NMDA receptors. In contrast, cotreatment with (-)-naloxone (3 microM), or the more selective kappa-opioid receptor antagonist nor-binaltorphimine (3 microM), exacerbated dynorphin A (1-13)-induced neuronal loss; however, cell losses were not enhanced by the inactive stereoisomer (+)-naloxone (3 microM). Neuronal losses were not seen with exposure to the opioid antagonists alone (10 microM). 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 kappa-opioid receptors and suggests that kappa 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.     

Intrathecally administered big dynorphin,a prodynorphin-derived peptide,produces nociceptive behavior through an N-methyl-D-aspartate receptor mechanism

Intrathecal (i.t.) administration of big dynorphin (1-10 fmol), a prodynorphin-derived peptide consisting of dynorphin A and dynorphin B, to mice produced a characteristic behavioral response, the biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank, which peaked at 5-15 min after an injection. Dynorphin A produced a similar response, though the doses required were higher (0.1-30 pmol) whereas dynorphin B was practically inactive even at 1000 pmol. The behavior induced by big dynorphin (3 fmol) was dose-dependently inhibited by intraperitoneal injection of morphine (0.125-2 mg/kg) and also dose-dependently, by i.t. co-administration of D(-)-2-amino-5-phosphonovaleric acid (D-APV) (1-4 nmol), a competitive N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 (0.25-4 nmol), an NMDA ion-channel blocker, and ifenprodil (2-8 pmol), an inhibitor of the NMDA receptor ion-channel complex interacting with the NR2B subunit and the polyamine recognition site. On the other hand, naloxone, an opioid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), a non-NMDA glutamate receptor antagonist, 7-chlorokynurenic acid, a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex, [D-Phe(7),D-His(9)]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10376, a tachykinin NK2 receptor antagonist, had no effect. These results suggest that big dynorphin-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the NR2B subunit and/or the polyamine recognition site but not on the glycine recognition site, and does not involve opioid, non-NMDA glutamate receptor mechanisms or tachykinin receptors in the mouse spinal cord.     

Increases in protein kinase C gamma immunoreactivity in the spinal cord of rats associated with tolerance to the analgesic effects of morphine

Our previous studies have indicated a critical role of protein kinase C (PKC) in intracellular mechanisms of tolerance to morphine analgesia. In the present experiments, we examined (1) the cellular distribution of a PKC isoform (PKCγ) in the spinal cord dorsal horn of rats associated with morphine tolerance by utilizing an immunocytochemical method and (2) the effects of theN-methyl-d-aspartate receptor antagonist MK-801 on tolerance-associated PKCγ changes. In association with the development of tolerance to morphine analgesia induced by once daily intrathecal administration of 10 μg morphine for eight days, PKCγ immunoreactivity was clearly increased in the spinal cord dorsal horn of these same rats. Within the spinal cord dorsal horn of morphine tolerant rats, there were significantly more PKCγ immunostained neurons in laminae I–II than in laminae III–IV and V–VI. Such PKCγ immunostaining was observed primarily in neuronal somata indicating a postsynaptic site of PKCγ increases. Moreover, both the development of morphine tolerance and the increase in PKCγ immunoreactivity were prevented by co-administration of morphine with 10 nmol MK-801 between Day 2 and Day 7 of the eight day treatment schedule. In contrast, PKCγ immunoreactivity was not increased in rats receiving a single i.t. administration of 10 μg morphine on Day 8, nor did repeated treatment with 10 nmol MK-801 alone change baseline levels of PKCγ immunoreactivity. These results provide further evidence for the involvement of PKC in NMDA receptor-mediated mechanisms of morphine tolerance.     

Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance.

The nonopioid actions of spinal dynorphin may promote aspects of abnormal pain after nerve injury. Mechanistic similarities have been suggested between opioid tolerance and neuropathic pain. Here, the hypothesis that spinal dynorphin might mediate effects of sustained spinal opioids was explored. Possible abnormal pain and spinal antinociceptive tolerance were evaluated after intrathecal administration of [D-Ala(2), N-Me-Phe(4), Gly-ol(5)]enkephalin (DAMGO), an opioid mu agonist. Rats infused with DAMGO, but not saline, demonstrated tactile allodynia and thermal hyperalgesia of the hindpaws (during the DAMGO infusion) and a decrease in antinociceptive potency and efficacy of spinal opioids (tolerance), signs also characteristic of nerve injury. Spinal DAMGO elicited an increase in lumbar dynorphin content and a decrease in the mu receptor immunoreactivity in the spinal dorsal horn, signs also seen in the postnerve-injury state. Intrathecal administration of dynorphin A(1-17) antiserum blocked tactile allodynia and reversed thermal hyperalgesia to above baseline levels (i.e., antinociception). Spinal dynorphin antiserum, but not control serum, also reestablished the antinociceptive potency and efficacy of spinal morphine. Neither dynorphin antiserum nor control serum administration altered baseline non-noxious or noxious thresholds or affected the intrathecal morphine antinociceptive response in saline-infused rats. These data suggest that spinal dynorphin promotes abnormal pain and acts to reduce the antinociceptive efficacy of spinal opioids (i.e., tolerance). The data also identify a possible mechanism for previously unexplained clinical observations and offer a novel approach for the development of strategies that could improve the long-term use of opioids for pain.     

Inhibition of morphine analgesia by LPS: role of opioid and NMDA receptors and spinal glia

Intraperitoneal (i.p.) injection of toxins, such as the bacterial endotoxin lipopolysaccharide (LPS), is associated with a well-characterized increase in sensitivity to painful stimuli (hyperalgesia) [Watkins LR, Maier SF, Goehler LE. Immune activation: the role of pro-inflammatory cytokines in inflammation, illness responses and pathological pain states. Pain 1995;63:289-302. [53]] and a longer-lasting reduction in opioid analgesia (anti-analgesia) when pain sensitivity returns to basal levels [Johnston IN, Westbrook RF. Acute and conditioned sickness reduces morphine analgesia. Behav Brain Res 2003;142:89-97]. Here we show that this inhibition of morphine analgesia 24 h after a single i.p. injection of LPS involves mechanisms that contribute to illness-induced hyperalgesia and the development of analgesic tolerance to morphine. Specifically, morphine analgesia was restored if LPS was preceded by systemic administration of a non-competitive NMDA receptor antagonist (MK-801), spinal infusion of a glial metabolic inhibitor (fluorocitrate), or intracerebroventricular microinjection of an opioid receptor antagonist (naloxone). Morphine analgesia was also restored if MK-801 was administered after LPS. These results demonstrate that LPS recruits similar, if not the same mechanisms that reduce morphine tolerance following opiate administration: namely, stimulation of opioid and NMDA receptors and recruitment of spinal glia.     

Antinociception produced by receptor selective opioids. Modulation of supraspinal antinociceptive effects by spinal opioids

This study evaluated the antinociceptive effects produced when different combinations of supraspinal μ- and δ-opioid agonist were co-administered with spinal μ-, δ-, and κ-opioid agonist. Using the Randall-Selitto paw-withdrawal test, in the rat, changes in nociceptive thresholds were measured following co-administration of sequentially increasing i.c.b. doses of either DAMGO or DPDPE with a low-antinociceptive dose of intrathecal DAMGO, DPDPE, or U50,488H. Antinociceptive synergy (i.e. a more than additive antinociceptive effect) was demonstrated with all of the combinations tested except for supraspinal DPDPE co-administered with spinal DAMGO. The results of this study provide support for the suggestion that supraspinal and spinal antinociceptive mechanisms share, in part, common neural circuits. Marked differences in the overall magnitude of the antinociceptive effects produced by the various combinations of opioid agonists were demonstrated through a secondary analysis of the data. When sequentially increasing i.c.v. doses of DAMGO were administered, significantly larger increases in nociceptive thresholds were observed with co-administration of intrathecal injections of low antinociceptive doses of either DAMGO or U50,488H compared to DPDPE. In contrast, when DPDPE was administered supraspinally, the largest increases in nociceptive thresholds were demonstrated with co-administration of DPDPE at the spinal site. The results of the secondary analysis provide support for the hypothesis that descending antinociceptive control systems activated by supraspinal administration of selective μ- and δ-opioid agonists interact, differently, with spinal μ-, δ, and κ-opioidergic mechanisms.     

Sex differences in the inhibitory effects of the NMDA antagonist, MK-801, on morphine and stress-induced analgesia

We examined the effects of intraperitoneal administrations of the noncompetitive NMDA receptor antagonist, (+) MK-801, its inactive enantiomer, (-) MK-801, and the prototypic opiate antagonist, naloxone, on restraint- and morphine-induced analgesia in male and female deer mice, Peromyscus maniculatus. Both restraint (30 min) and morphine (1.0 mg/kg) induced significant analgesic responses with male mice displaying significantly greater levels of opioid-induced analgesia than female animals. These analgesic responses were completely blocked by, naloxone (1.0 mg/kg), significantly reduced by (+) MK-801 (0.25 mg/kg) and unaffected by (-) MK-801 (0.25 mg/kg) pretreatments. There were significant male-female differences in the inhibitory effects of (+) MK-801; the higher levels of morphine- and restraint-induced analgesia of the males were completely blocked, while the lower level analgesic responses of the females were significantly reduced, but not blocked, by (+) MK-801. These observations provide further evidence that NMDA receptors are involved in the mediation of endogenous and exogenous opioid analgesia and show that there are significant male-female differences in the inhibitory effects of (+) MK-801 on opioid-mediated analgesia.     

Supraspinal and systemic administration of the nicotinic-cholinergic agonist (+/-)-epibatidine has inhibitory effects on C-fiber reflexes in the rat

This study assessed the effects of the nicotinic agonist (+/-)-epibatidine (EPIB) on the C-fiber flexor reflex in the anesthetized rat. Electrical stimulation of the hindpaw produces a long latency (> 150 ms) C-fiber mediated electromyographic (EMG) burst in hindlimb flexor muscles. EPIB (0.01, 0.03 micromol/kg, i.p.) significantly reduced (p < 0.05) C-fiber -related EMG activity by 46 and 64%, respectively. This effect was similar to that produced by the opioid morphine (21.0 micromol/kg, i.p.) and the NMDA receptor antagonist MK-801 (3.0 micromol/kg, i.p.). Nicotinic receptor blockade with the antagonists mecamylamine (5.0 micromol/kg, i.p.) and chlorisondamine (23.0 nmol/rat, intracerebroventricular) attenuated the effects of systemic EPIB on the C-fiber reflex. EPIB injection (0.04 nmol/rat) into the nucleus raphe magnus significantly decreased C-fiber EMG activity by 67%, suggesting a supraspinal site of action. In contrast, EPIB (0.6 nmol/rat) administered into the lumbar spinal cord significantly increased the C-fiber reflex by 117%. In summary, systemic and supraspinal EPIB exerted an inhibitory effect on central pain transmitting pathways, while a stimulatory effect is elicited in the spinal cord. The inhibitory effects are consistent with the reported analgesic properties of EPIB. The excitatory effect may be related to the reported algogenic responses when EPIB is administered intrathecally.     

Actions of NMDA and cholinergic receptor antagonists in the rostral ventromedial medulla upon beta-endorphin analgesia elicited from the ventrolateral periaqueductal gray.

Analgesia elicited by morphine in the ventrolateral periaqueductal gray is mediated in part by NMDA and cholinergic receptors in the rostral ventromedial medulla because selective receptor antagonists applied to the latter structure reduced morphine analgesia elicited from the former structure. Previous studies have demonstrated that morphine and beta-endorphin employ different anatomical and neurochemical pathways in exerting their supraspinal analgesic effects. The present study evaluated whether pretreatment with either competitive (AP7, 3-10 microg) or non-competitive (MK-801, 3-10 microg) NMDA antagonists, or muscarinic (scopolamine, 5 microg) or nicotinic (mecamylamine, 1 microg) cholinergic antagonists administered into the rostral ventromedial medulla altered beta-endorphin (15 microg) analgesia elicited from the ventrolateral periaqueductal gray as measured by the tail-flick and jump tests in rats. Whereas AP7 produced minimal (11%) and transient (30 min) reductions in beta-endorphin analgesia on the jump test, MK-801 produced minimal (9%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Whereas mecamylamine failed to reduce beta-endorphin analgesia on either measure, scopolamine produced small (23%) and transient (30 min) reductions in beta-endorphin analgesia on the tail-flick test. Each of these antagonists administered into the rostral ventromedial medulla at comparable or lower doses virtually eliminated morphine analgesia elicited from the ventrolateral periaqueductal gray. The opioid mediation of beta-endorphin analgesia in the ventrolateral periaqueductal gray was confirmed by its sensitivity to naltrexone (1-20 microg) pretreatment into the same structure. These data provide further evidence for dissociations between the descending neuroanatomical and neurochemical circuitry mediating the supraspinal analgesic responses induced by morphine and beta-endorphin, and indicate that the latter response is mediated by either non-cholinergic and non-NMDA synapses within the rostral ventromedial medulla, and/or by brainstem sites outside of the rostral ventromedial medulla.     

Structure-activity analysis of dynorphin A toxicity in spinal cord neurons: intrinsic neurotoxicity of dynorphin A and its carboxyl-terminal, nonopioid metabolites

Dynorphin A [dynorphin A (1-17)] is an endogenous opioid peptide that is antinociceptive at physiological concentrations. Levels of dynorphin A increase markedly following spinal cord trauma and may contribute to secondary neurodegeneration. Both kappa opioid and N-methyl-d-aspartate (NMDA) receptor antagonists can modulate the effects of dynorphin, suggesting that dynorphin is acting through kappa opioid and/or NMDA receptor types. Despite these findings, few studies have critically examined the mechanisms of dynorphin A neurotoxicity at the cellular level. To better understand how dynorphin affects cell viability, structure-activity studies were performed examining the effects of dynorphin A and dynorphin A-derived peptide fragments on the survival of mouse spinal cord neurons coexpressing kappa opioid and NMDA receptors in vitro. Time-lapse photography was used to repeatedly follow the same neurons before and during experimental treatments. Dynorphin A caused significant neuronal losses that were dependent on concentration (> or = 1 microM) and duration of exposure. Moreover, exposure to an equimolar concentration of dynorphin A fragments (100 microM) also caused a significant loss of neurons. The rank order of toxicity was dynorphin A (1-17) > dynorphin A (1-13) congruent with dynorphin A (2-13) congruent with dynorphin A (13-17) (least toxic) > dynorphin A (1-5) ([Leu(5)]-enkephalin) or dynorphin A (1-11). Dynorphin A (1-5) or dynorphin A (1-11) did not cause neuronal losses even following 96 h of continuous exposure, while dynorphin A (3-13), dynorphin A (6-17), and dynorphin A (13-17) were neurotoxic. The NMDA receptor antagonist MK-801 (dizocilpine) (10 microM) significantly attenuated the neurotoxic effects of dynorphin A and/or dynorphin-derived fragments except dynorphin A (13-17), suggesting that the neurotoxic effects of dynorphin were largely mediated by NMDA receptors. Thus, toxicity resides in the carboxyl-terminal portion of dynorphin A and this minimally includes dynorphin A (3-13) and (13-17). Our findings suggest that dynorphin A and/or its metabolites may contribute significantly to neurodegeneration during spinal cord injury and that alterations in dynorphin A biosynthesis, metabolism, and/or degradation may be important in determining injury outcome.     

Evidence for opiate-activated NMDA processes masking opiate analgesia in rats

The acute interaction between opioid receptors and N-methyl-D-aspartate (NMDA) receptors on nociception was examined in rats using tail-flick and paw-pressure vocalisation tests. When injected at various times (1 to 6 h) after morphine (5 to 20 mg/kg, i.v.) or fentanyl (4x40 microgram/kg, i.v.), the opioid receptor antagonist naloxone (1 mg/kg, s.c.) not only abolished the opiate-induced increase in nociceptive threshold, but also reduced it below the basal value (hyperalgesia). The noncompetitive NMDA receptor antagonist MK-801 (0.15 or 0.30 mg/kg, s.c.) prevented the naloxone-precipitated hyperalgesia and enhanced the antinociceptive effects of morphine (7.5 mg/kg, i.v.) and fentanyl (4x40 microgram/kg, i.v.). These results indicate that the antinociceptive effects of morphine and fentanyl, two opiate analgesics widely used in humans in the management of pain, are blunted by concomitant NMDA-dependent opposing effects which are only revealed when the predominant antinociceptive effect is sharply blocked by naloxone. This study provides new rationale for beneficial adjunction of NMDA receptor antagonists with opiates for relieving pain by preventing pain facilitatory processes triggered by opiate treatment per se.     

Acute interactive effects of MK-801 and morphine on cortical EEG and EEG power spectra in rats

The interaction between MK-801 and morphine-induced effects on cortical electroencephalography (EEG) was investigated. Rats were administered one of five MK-801 doses (IP) prior to morphine (IV). MK-801 dose-dependently increased morphine-induced global spectral power, duration of morphine-induced EEG bursts and latency to sleep onset, and decreased morphine-induced mean frequency, mobility, complexity, and edge frequency. MK-801 pretreatment shifted the relative distribution of total power to the left. Significant interaction effects were found for all spectral parameters except peak frequency. A second group of rats was administered MK-801 prior to an increasing cumulative morphine dose. MK-801 increased maximal morphine effects on all spectral parameters except peak frequency. The results are in agreement with those of recent analgesia and in vitro studies in spinal neurons, and support observations of a synergistic interaction between effects of NMDA antagonism and morphine. These data further suggest that the component of cortical EEG that is produced by mu-opioid- and NMDA-receptor interactive effects may be dominated by an inhibitory effect of morphine on NMDA receptor activity.     

Region-specific changes in NMDA receptor mRNA induced by chronic morphine treatment are prevented by the co-administration of the competitive NMDA receptor antagonist LY274614

The steady-state mRNA levels of the NMDA receptor NR1 subunit were determined by a quantitative solution hybridization assay in selected CNS regions associated with antinociception in the rat. Tissues were obtained by microdissection from rats treated chronically with morphine alone or in combination with LY274614, a competitive NMDA receptor antagonist. Morphine treatment for 7 days resulted in the development of tolerance to morphine's analgesic effect and produced a significant decrease in the steady-state NR1 mRNA levels in the spinal cord dorsal horn (by 16%), and an elevation in nucleus raphe magnus and medial thalamus (by 26 and 38%, respectively). The NR1 mRNA levels were unchanged in the lateral paragigantocellular nucleus, locus coeruleus, periaqueductal grey, and sensorimotor cortex. NMDA receptor binding in the spinal cord measured with [3H]MK-801 was reduced approximately 50% by chronic morphine treatment. Co-administration of LY274614 (s.c. at 24 mg/kg/24 h via an osmotic pump) not only attenuated the development of morphine tolerance but also prevented the changes in the NR1 mRNA levels induced by chronic morphine administration. Neither a 7-day infusion of LY274614 nor an acute injection of morphine (10 mg/kg, s.c.) changed the NR1 mRNA levels. These results suggest that changes in the expression of the NR1 mRNA induced by chronic morphine in three CNS regions involved in antinociception are associated with the development of morphine tolerance and in the spinal cord, morphine tolerance is associated with the downregulation of NMDA receptors.     

MK-801 prevents the development of behavioral sensitization during repeated morphine administration

Acute administration of morphine (10 mg/kg) to rats elicited an increase in locomotion that became sensitized upon repeated treatment over 14 days. Administration of the noncompetitive N-methyl-D-aspartate receptor (NMDA) antagonist MK-801 (0.1 or 0.25 mg/kg) prior to each morphine injection prevented the development of behavioral sensitization to morphine, an effect that persisted even after a 7-day withdrawal from repeated treatment. Sensitization was also prevented by coadministration of the competitive NMDA receptor antagonist CGS 19755 (10 mg/kg). In contrast, acute pretreatment with MK-801 did not alter the response of sensitized rats to morphine challenge, indicating that MK-801 does not prevent the expression of sensitization. When administered alone, MK-801 produced stereotyped movements at moderate doses (0.25 rng/kg) and horizontal locomotion at higher- doses, (0.5 mg/kg). Repeated administration of 0.25 mg/kg MK-801 elicited sensitization to its own locomotor stimulatory effects, such that this dose became capable of eliciting horizontal locomotion. Sensitization was not seen during repeated administration of 0.1 mg/kg MK-801 or 10 mg/kg CGS 19755, although both of these pretreatments did produce a sensitized response to subsequent challenge with 0.25 mg/kg MK-801. This effect was enhanced by coadministration of morphine, even though repeated administration of morphine alone failed to sensitize rats to MK-801 challenge. These results suggest a complex interplay between NMDA and opioid receptors, such that NMDA antagonists prevent morphine sensitization while morphine enhances the ability of NMDA antagonists to elicit sensitization to their own locomotor stimulatory effects. © 1994 Wiley-Liss, Inc.     

Involvement of NMDA receptors in morphine state-dependent learning in mice

In the present study, the effects of intracerebroventricular (i.c.v.) injection of NMDA receptor agonist and antagonist on impairment of memory formation and the state-dependent learning by morphine have been investigated in mice. Pretraining administration of morphine (5 mg/kg; s.c.) decreased the learning of one-trial passive avoidance task. Pretest administration of morphine (5 mg/kg) induced state-dependent learning acquired under pretraining morphine influence. Pretest administration of NMDA receptor agonist, L-glutamate (0.00001 and 0.0001 and 0.001 microg/mouse, i.c.v.) following pretraining saline treatment did not affect retention. Amnesia induced by pretraining morphine was significantly reversed by pretest administration of L-glutamate (0.0001 and 0.001 microg/mouse, i.c.v.). Pretest administration of noncompetitive NMDA receptor antagonist, MK-801 (0.5, 1, and 2 microg/mouse, i.c.v.) significantly impaired memory formation. Amnesia induced by pretraining morphine was increased by pretest administration of MK-801 (2 microg/mouse, i.c.v.). Pretest coadministration of L-glutamate (0.0001 and 0.001 microg/mouse, i.c.v.) or MK-801 (0.5, 1, and 2 microg/mouse, i.c.v.) with morphine (5 mg/kg, s.c.) increased and decreased morphine state-dependent learning, respectively. The results suggest that NMDA receptors are involved in morphine state-dependent learning in mice.