The development of morphine tolerance and dependence in rats with chronic pain

The development of tolerance and dependence to morphine injected onto the spinal cord was examined in a model of chronic pain following spinal cord injury in rats. Intrathecal morphine completely relieved the marked pain-like response of these rats to innocuous mechanical stimuli. The analgesic effect of morphine injected twice daily was, however, diminished within a few days. Tolerance to the antinociceptive effect of morphine assessed with the tail flick test also developed similarly in rats with chronic pain and in normal controls. Both groups exhibited similar signs of naloxone-precipitated withdrawal after 3 weeks of morphine treatment. The results suggest that the presence of chronic pain-like behavior did not prevent the development of morphine tolerance and dependence, even when morphine was used to treat the chronic pain itself.     

Toll-like receptor 4-mediated nuclear factor-κB activation in spinal cord contributes to chronic morphine-induced analgesic tolerance and hyperalgesia in rats

Nuclear factor kappa B（NF-κB） in the spinal cord is involved in pro-infl ammatory cytokine-mediated pain facilitation. However, the role of NF-κB activation in chronic morphine-induced analgesic tolerance and the underlying mechanisms remain unclear. In the present study, we found that the level of phosphorylated NF-κB p65（p-p65） was increased in the dorsal horn of the lumbar 4–6 segments after intrathecal administration of morphine for 7 consecutive days, and the p-p65 was co-localized with neurons and astrocytes. The expression of TNF-α and IL-1β was also increased in the same area. In addition, pretreatment with pyrrolidinedithiocarbamate（PDTC） or SN50, inhibitors of NF-κB, prevented the development of morphine analgesic tolerance and alleviated morphine withdrawal-induced allodynia and hyperalgesia. The increase in TNF-α and IL-1β expression induced by chronic morphine exposure was also partially blocked by PDTC pretreatment. In another experiment, rats receiving PDTC or SN50 beginning on day 7 of morphine injection showed partial recovery of the anti-nociceptive effects of morphine and attenuation of the withdrawal-induced abnormal pain. Meanwhile, intrathecal pretreatment with lipopolysaccharide from Rhodobacter sphae-roides, an antagonist of toll-like receptor 4（TLR4）, blocked the activation of NF-κB, and prevented the development of morphine tolerance and withdrawal-induced abnormal pain. These data indicated that TLR4-mediated NF-κB activation in the spinal cord is involved in the development and maintenance of morphine analgesic tolerance and withdrawalinduced pain hypersensitivity.     

Involvement of neuropeptide Y in the acute, chronic and withdrawal responses of morphine in nociception in neuropathic rats: Behavioral and neuroanatomical correlates

Although morphine is a potent antinociceptive agent, its chronic use developed tolerance in neuropathic pain (NP). Furthermore, opioid antagonist naloxone attenuated the antinociceptive effect of neuropeptide Y (NPY). The present study investigated the role of NPY and NPY Y1/Y5 receptors in acute and chronic actions of morphine in neuropathic rats using thermal paw withdrawal test and immunocytochemistry. In acute study, intracerebroventricular (icv) administration of morphine, NPY or NPY Y1/Y5 receptors agonist [Leu³¹, Pro³⁴]-NPY produced antinociception, whereas selective NPY Y1 receptors antagonist BIBP3226 caused hyperalgesia. While NPY or [Leu³¹, Pro³⁴]-NPY potentiated, BIBP3226 attenuated morphine induced antinociception. Chronic icv infusion of morphine via osmotic minipumps developed tolerance to its antinociceptive effect, and produced hyperalgesia following withdrawal. However, co-administration of NPY or [Leu³¹, Pro³⁴]-NPY prevented the development of tolerance and withdrawal hyperalgesia. Sciatic nerve ligation resulted in significant increase in the NPY-immunoreactive (NPY-ir) fibers in ventrolateral periaqueductal gray (VLPAG) and locus coeruleus (LC); fibers in the dorsal part of dorsal raphe nucleus (DRD) did not respond. While chronic morphine treatment significantly reduced NPY-ir fibers in VLPAG and DRD, morphine withdrawal triggered significant augmentation in NPY-immunoreactivity in the VLPAG. NPY-immunoreactivity profile of LC remained unchanged in all the morphine treatment conditions. Furthermore, removal of sciatic nerve ligation reversed the effects of NP, increased pain threshold and restored NPY-ir fiber population in VLPAG. NPY, perhaps acting via Y1/Y5 receptors, might profoundly influence the processing of NP information and interact with the endogenous opioid system primarily within the framework of the VLPAG.     

Implication of delta opioid receptor subtype 2 but not delta opioid receptor subtype 1 in the development of morphine analgesic tolerance in a rat model of chronic inflammatory pain

Opioids are well known for their robust analgesic effects. Chronic activation of mu opioid receptors (MOPs) is, however, accompanied by various unwanted effects such as analgesic tolerance. Among other mechanisms, interactions between MOPs and delta opioid receptors (DOPs) are thought to play an important role in morphine‐induced behavioral adaptations. Interestingly, certain conditions such as inflammation enhance the function of the DOP through a MOP‐dependent mechanism. Here, we investigated the role of DOPs during the development of morphine tolerance in an animal model of chronic inflammatory pain. Using behavioral approaches, we first established that repeated systemic morphine treatment induced morphine analgesic tolerance in rats coping with chronic inflammatory pain. We then observed that blockade of DOPs with subcutaneous naltrindole (NTI), a selective DOP antagonist, significantly attenuated the development of morphine tolerance in a dose‐dependent manner. We confirmed that this effect was DOP mediated by showing that an acute injection of NTI had no effect on morphine‐induced analgesia in naive animals. Previous pharmacological characterizations revealed the existence of DOP subtype 1 and DOP subtype 2. As opposed to NTI, 7‐benzylidenenaltrexone and naltriben were reported to be selective DOP subtype 1 and DOP subtype 2 antagonists, respectively. Interestingly, naltriben but not 7‐benzylidenenaltrexone was able to attenuate the development of morphine analgesic tolerance in inflamed rats. Altogether, our results suggest that targeting of DOP subtype 2 with antagonists provides a valuable strategy to attenuate the analgesic tolerance that develops after repeated morphine administration in the setting of chronic inflammatory pain.     

Analgesic tolerance to high-efficacy agonists but not to morphine is diminished in phosphorylation-deficient S375A μ-opioid receptor knock-in mice

Morphine is one of the most potent analgesic drugs. However, the utility of morphine in the management of chronic pain is limited by its rapid development of tolerance. Morphine exerts all of its pharmacological effects via the μ-opioid receptor. In many systems, tolerance is associated with phosphorylation and desensitization of G-protein-coupled receptors (GPCRs). In case of the μ-opioid receptor, phosphorylation occurs in an agonist-selective manner. High-efficacy agonists such as [d-Ala(2)-MePhe(4)-Gly-ol]enkephalin (DAMGO), fentanyl, or etonitazene stimulate the phosphorylation of both C-terminal threonine 370 (T370) and serine 375 (S375). In contrast, morphine promotes the phosphorylation of S375 but fails to stimulate T370 phosphorylation. Here, we have assessed the contribution of S375 phosphorylation to the development of antinociceptive tolerance to high- and low-efficacy μ agonists in vivo. We show that S375 phosphorylation of the μ-opioid receptor occurs in intact mouse brain in a dose-dependent manner after administration of morphine, fentanyl, or etonitazene. In knock-in mice expressing the phosphorylation-deficient S375A mutant of the μ-opioid receptor, morphine and fentanyl exhibited greater dose-dependent antinociceptive responses than in wild-type mice. However, acute and chronic tolerance to morphine was retained in S375A mutant mice. In contrast, antinociceptive tolerance after repeated subcutaneous application of etonitazene or repeated intracerebroventricular application of DAMGO was diminished. Thus, tolerance to μ agonists with different efficacies develops through distinct pathways. Whereas tolerance induced by DAMGO or etonitazene requires agonist-driven phosphorylation of S375, the development and maintenance of antinociceptive tolerance to morphine occurs independent of S375 phosphorylation.     

Glutamate transporter activation enhances nicotine antinociception and attenuates nicotine analgesic tolerance

Analgesic tolerance is partially mediated by enhanced glutamatergic transmission in the CNS. β-lactam antibiotics, through glutamate transporter subtype 1 (GLT-1) activation, reduce extracellular glutamate levels and attenuate tolerance to morphine analgesia in rats. Similar to opioids, nicotine has potent analgesic properties that are subject to tolerance. The purpose of this study was to evaluate the effects of ceftriaxone, a β-lactam antibiotic and GLT-1 activator on nicotine antinociception and its tolerance. Rats were pretreated for 5 days with ceftriaxone (200 mg/kg, intraperitoneally) before evaluating their analgesic response to nicotine (1.0 or 2.5 mg/kg, subcutaneously) for seven consecutive days using the tail-flick assay. Ceftriaxone-treated rats displayed an enhanced antinociceptive response to nicotine and unlike saline-injected controls, did not develop tolerance to nicotine's analgesic effects. These results suggest that GLT-1 transporter activation enhances and preserves nicotine antinociception and identify β-lactam antibiotics as potential complementary therapeutic agents for the treatment of chronic pain.     

Differential responses to morphine-induced analgesia in the tail-flick test

We compared acute and chronic antinociceptive effects of morphine in animals with high reactivity (HR) vs. low reactivity (LR) to novelty. Antinociception was assessed by tail-flick test. Rats were i.p. injected with either saline or morphine (1.5 or 3mg/kg) every 12h for 7 days according to the treatment group. On day 1 of the experiment, LR animals in the 1.5mg/kg morphine group showed significantly higher tail-flick latency than HR. Moreover, significant tolerance to the antinociceptive effects of morphine at the used doses was observed in LR but not HR animals. However, effects of chronic morphine treatment on tail-flick latency in rat groups with similar morphine-induced acute antinociception were undistinguishable. The difference in tail-flick latency between HR and LR rats observed after acute 1.5mg/kg morphine injection was eliminated if beta-funaltrexamine (3mg/kg, i.p.) was administered 24h before the test, an indication that mu opioid receptors are responsible for the difference observed. Studies to anatomically characterize the difference in the acute analgesic effect of morphine in HR vs. LR animals did not however yield any significant difference in mu opioid receptor mRNA levels in locus coeruleus (LC), ventral periaqueductal gray (vPAG), nucleus raphe magnus (NRM) and nucleus reticularis paragigantocellularis (NRPG) between these two groups of animals. In conclusion, our results show that differences in novelty-seeking behavior can predict inter-individual variability in morphine-induced antinociception in rats. Such variability is dependent upon activation of mu opioid receptors, but does not correlate with mu opioid receptor expression in LC, vPAG or ventral medulla.     

Contribution of acid sphingomyelinase in the periaqueductal gray region to morphine-induced analgesia in mice

Opioids are the most widely used drugs for long-term pain management, but their use is limited by the development of antinociceptive tolerance. The present study investigated the role of ceramide production through acid sphingomyelinase (ASM) activation in the periaqueductal gray region, a brain region implicated in opioid analgesia and tolerance. Morphine treatment was found, using immunohistochemistry, to increase ASM expression and intracellular ceramide in the periaqueductal gray 30 min after an acute injection (10 mg/kg). The effects of acute morphine treatment on ASM expression and ceramide generation in the periaqueductal gray region were completely blocked by pretreatment with naloxone and by silencing the ASM gene by plasmid-mediated transfection of ASM shRNA. In chronic morphine pellet-implanted mice, ASM expression and ceramide generation in the periaqueductal gray region were also significantly increased. Functionally, selective silencing of the ASM gene by local ASM shRNA transfection reduced the analgesic response to acute morphine, but the data on the effect of ASM shRNA on the development of antinociceptive tolerance were inconclusive. These data provide evidence that ASM activation and ceramide generation in the periaqueductal gray region play a major role in the antinociceptive mechanism of morphine.     

Development of tolerance to the antinociceptive effect of systemic morphine at the lumbar spinal cord level: a c-Fos study in the rat

The development of tolerance to the antinociceptive effects of morphine was investigated in rats using carrageenin-induced spinal c-Fos expression. We took advantage of this technique to especially study, at the cellular level, in freely moving animals, the development of tolerance based on the visualization of dorsal horn spinal cord neurons which play a major role in nociceptive processes. Two hours after intraplantar injection of carrageenin (6 mg/150 μl of saline), c-Fos-like immunoreactivity (FLI) was observed predominantly in the superficial and deep laminae of the dorsal horn in segments L4 and L5 of the spinal cord. In naive rats, acute intravenous morphine (3 mg/kg, i.v.) reduced the number of superficial and deep FLI neurons; 49% and 59% reduction respectively (p<0.0001 for both). In morphine-pretreated rats (daily administration of subcutaneous morphine: 1, 3, 5, 10, 20 or 40 mg/kg once a day for 4 days), antinociceptive tolerance tested on day 5 by acute morphine (3 mg/kg, i.v.) was manifest in those groups pretreated with the highest doses of morphine (10, 20 or 40 mg/kg). From regression analysis, it appeared that tolerance to the antinociceptive effect of morphine developed progressively as a function of the chronic morphine dose used on neurons involved in spinal nociceptive processes (superficial and deep dorsal horn neurons). Similarly, in rats pretreated with 10 mg/kg of morphine over 1, 2, 3 or 4 days, tolerance progressively developed, for both spinal neuronal populations, as a function of the duration of the pretreatment. These results are discussed in the context of the several possible sites of action of morphine.     

Involvement of endogenous cholecystokinin in tolerance to morphine antinociception in the nucleus accumbens of rats

Cholecystokinin (CCK) is an endogenous anti-opioid peptide in the central nervous system. The present study investigated the effects of endogenous CCK on tolerance to morphine antinociception in the nucleus accumbens (NAc) of rats. Chronic administration of morphine to NAc induced marked tolerance to antinociception. Intra-NAc administration of the CCK2 receptor antagonist LY225910 inhibited not only the development but also the expression of chronic morphine-induced antinociceptive tolerance. However, intra-NAc injection of LY225910 did not influence the antinociception induced by intra-NAc administration of morphine in the intact rats. The results indicate that endogenous CCK plays an important role in morphine-induced antinociceptive tolerance in the NAc of rats.     

Ziconotide: a review of its pharmacology and use in the treatment of pain

Ziconotide is a powerful analgesic drug that has a unique mechanism of action involving potent and selective block of N-type calcium channels, which control neurotransmission at many synapses. The analgesic efficacy of ziconotide likely results from its ability to interrupt pain signaling at the level of the spinal cord. Ziconotide is a peptidic drug and has been approved for the treatment of severe chronic pain in patients only when administered by the intrathecal route. Importantly, prolonged administration of ziconotide does not lead to the development of addiction or tolerance. The current review discusses the various studies that have addressed the in vitro biochemical and electrophysiological actions of ziconotide as well as the numerous pre-clinical studies that were conducted to elucidate its antinociceptive mechanism of action in animals. In addition, this review considers the pivotal Phase 3 (and other) clinical trials that were conducted in support of ziconotide’s approval for the treatment of severe chronic pain and tries to offer some insights regarding the future discovery and development of newer analgesic drugs that would act by a similar mechanism to ziconotide but which might offer improved safety, tolerability and ease of use.     

Dexamethasone mimics the inhibitory effect of chronic pain on the development of tolerance to morphine analgesia and compensates for morphine induced changes in G proteins gene expression

It is previously reported that the HPA axis plays role in the inhibitory effect of pain on tolerance development to analgesic effect of opioids. The present study was designed to investigate whether the chronic co-administration of dexamethasone as a glucocorticoid is also able to prevent or reverse analgesic tolerance to morphine and to compare the expression of G(alphai/o) and G(beta) subunits of G proteins in the context of chronic dexamethasone, development of morphine tolerance and their combination. Analgesic tolerance to morphine was induced by chronic intraperitoneally (i.p.) administration of morphine 20 mg/kg to male Wistar rats weighing 200-240 g within 4 consecutive days and analgesia was assessed using tail-flick test. Chronic dexamethasone was applied using 4 daily i.p. injections. Lumbar spinal tissues were assayed for the expression of G(alphai/o) and G(beta) proteins using "semiquantitative PCR" normalized to beta-actin gene expression. Results showed that chronic administration of dexamethasone could reduce and reverse the development of tolerance in rats that received chronic i.p. injections of morphine. Chronic administration of dexamethasone significantly increased the expression of G(alphai/o), while chronic administration of morphine did not change its expression. The expression of G(beta), however, was increased after the chronic administration of morphine, but did not change after the administration of chronic dexamethasone. None of these increases were observed when morphine and dexamethasone were co-administered. We conclude that the development of tolerance to analgesic effect of morphine could be prevented and reversed by dexamethasone co-administration. The increase in G(alphai/o) genes expression produced by chronic dexamethasone may facilitate the opioid signaling pathway and compensate for morphine-induced tolerance.     

Morphine fails to produce tolerance when administered in the presence of formalin pain in rats

The present study examined the development of tolerance to morphine analgesia under conditions in which morphine was administered in the presence or absence of pain induced by subcutaneous injection of 50 μl of 2.5% formalin into the hind paw of rats. Animals were injected with morphine (25 mg/kg, i.p.) or saline for 3 consecutive days either in the presence of pain (10 min after formalin injection) or in the absence of pain (6 h prior to formalin injection). On the 4th day, tolerance to the analgesic effect of test doses of morphine (6 or 10 mg/kg) was assessed in the formalin and tail-flick tests, respectively. Significant tolerance in both tests was observed in animals receiving morphine in the absence of pain during the tolerance induction period, but not in animals receiving morphine in the presence of pain.     

Tolerance to morphine microinjections in the periaqueductal gray (PAG) induces tolerance to systemic, but not intrathecal morphine

The acquisition and retention of tolerance to the antinociceptive effect of supraspinal morphine on the tail withdrawal reflex was assessed in rats implanted with unilateral cannulae in the periaqueductal gray (PAG). Development of tolerance to daily microinjections of morphine was indicated by the return of the tail flick response within 4 days, followed by the recovery of analgesic sensitivity one week later. After tolerance had developed, the effect of an acute systemic (1.5-4.5 mg/kg) or intrathecal (5-15 micrograms) morphine injection was determined. 'Cross-tolerance' was observed between systemic and supraspinal morphine but not between intrathecal and supraspinal morphine. The data indicate that tolerance to chronic intracerebral morphine produces the same behavioral consequences as tolerance to systemic morphine.     

Pharmacodynamics of morphine-induced neuronal nitric oxide production and antinociceptive tolerance development

Elevated nitric oxide (NO) production has been implicated in the development of morphine antinociceptive tolerance. This study was conducted to establish the temporal relationship between morphine-induced increases in neuronal NO and loss of pharmacologic activity. Five groups of rats equipped with microdialysis probes in the jugular vein and hippocampus received an intravenous infusion of saline or morphine (0.3, 1, 2, or 3 mg/kg/h) for 8 h. Morphine concentrations in the blood and hippocampal microdialysate were determined by LC/MS-MS; NO production was quantified with an amperometric sensor implanted in the contralateral hippocampus. Antinociceptive effect was monitored at selected time points during and following infusion by electrical stimulation vocalization. The data were fit with a pharmacokinetic/pharmacodynamic model to obtain parameters governing morphine disposition, stimulation of NO production, antinociception, and antinociceptive tolerance development. An additional three groups of rats were pretreated with l-arginine, the NO precursor (100, 300, or 500 mg/kg/h for 8 h), to elevate NO concentrations prior to morphine infusion. Morphine administration resulted in a dose-dependent increase in NO production; the time course of altered NO production coincided with the development of antinociceptive tolerance. l-arginine pretreatment initially enhanced morphine-induced analgesia early in the morphine infusion. However, this NO-associated increase in opioid response dissipated rapidly due to a dominant NO-induced loss of antinociception. Pharmacodynamic modeling suggested that this latter effect was consistent with a hyperalgesic response. These data define a strong, time-dependent relationship between morphine-induced stimulation of NO production and tolerance development, identify specific NO-induced alterations in nociceptive processing after morphine administration, and indicate that NO is a key mediator of antinociceptive tolerance development.     

Inhibition of neuronal nitric oxide synthase attenuates the development of morphine tolerance in rats

Our previous results have shown the involvement of nitric oxide in acute opioid desensitization of mu-opioid receptors in vitro. In the present study, we investigated the effect of repeated administration of 7-nitroindazole (7-NI; 30 mg/kg/12 h, i.p., 3 days), an inhibitor of neuronal nitric oxide synthase in vivo, on mu-opioid receptor tolerance induced by subchronic treatment with morphine in rats. The inhibitory effect of the opioid agonist Met5-enkephalin (ME) on the cell firing rate was evaluated by single-unit extracellular recordings of noradrenergic neurons in the locus coeruleus from brain slices, and the antinociceptive effect of morphine was measured by tail-flick techniques. In morphine-treated animals, concentration-effect curves for ME in the locus coeruleus were shifted by 5-fold to the right as compared to those in sham-treated animals, which confirmed the induction of mu-opioid receptor tolerance. However, tolerance to ME in morphine-treated rats was fully prevented by co-administration of 7-NI when compared to the vehicle-morphine group. Likewise, the antinociceptive effect of morphine was reduced in morphine-treated animals as compared to the sham group, whereas the antinociceptive tolerance was partially prevented by co-administration of 7-NI in morphine-treated rats (when compared to the vehicle-morphine group). Finally, 7-NI administration in sham-treated rats failed to change the effect induced by ME on the locus coeruleus or by morphine in the tail-flick test as compared to vehicle groups. These results demonstrate that subchronic administration of a neuronal inhibitor of nitric oxide synthase attenuates the development of morphine tolerance to the cellular and analgesic effects of mu-opioid receptor agonists.     

Interaction and regulatory functions of μ- and δ-opioid receptors in nociceptive afferent neurons

μ-opioid receptor (MOR) agonists such as morphine are powerful analgesics used for pain therapy. However, the use of these drugs is limited by their side-effects, which include antinociceptive tolerance and dependence. Earlier studies reported that MOR analgesic tolerance is reduced by blockade of δ-opioid receptors (DORs) that interact with MORs. Recent studies show that the MOR/DOR interaction in nociceptive afferent neurons in the dorsal root ganglion may contribute to morphine analgesic tolerance. Further analysis of the mechanisms for regulating the trafficking of receptors, ion channels and signaling molecules in nociceptive afferent neurons would help to understand the nociceptive mechanisms and improve pain therapy.     

Lewis and Fischer rat strains display differences in biochemical, electrophysiological and behavioral parameters: studies in the nucleus accumbens and locus coeruleus of drug naive and morphine-treated animals

In previous studies, we demonstrated that tyrosine hydroxylase and neurofilament proteins are regulated by chronic morphine and chronic cocaine treatments in the ventral tegmental area in Sprague-Dawley rats and that the imbred Lewis and Fischer 344 rat strains, under drug-naive conditions, show different levels of these proteins specifically in this brain region. In the current study, we compared Lewis and Fischer rats with respect to levels of adenylate cyclase, cyclic AMP-dependent protein kinase and G-proteins in the nucleus accumbens (NAc) and locus coeruleus (LC), brain regions in Sprague-Dawley rats where these proteins are regulated by chronic exposure to morphine or to cocaine. We found that levels of adenylate cyclase and cyclic AMP-dependent protein kinase activity are higher in the NAc and LC of Lewis rats compared to Fischer rats, whereas levels of G_iα and G_β were lower. These strain differences were not seen in several other brain regions analyzed and no strain differences were detected in levels of other G-protein subunits. Lewis and Fischer rats also differed in the ability of chronic morphine to regulate adenylate cyclase and cyclic AMP-dependent protein kinase in the NAc and LC. In the NAc, chronic morphine increased levels of the two enzymes in the Fischer strain only, whereas in the LC chronic morphine increased levels of the enzymes in both strains, with more robust effects seen in the Lewis rat. To understand possible physiological consequences of these strain differences in the cyclic AMP pathway, we studied LC neuronal activity under basal and chronic morphine-treated conditions. LC neurons of Lewis rats showed higher spontaneous firing rates in brain slices in vitro than those of Fischer rats and also showed greater morphine-induced increases in responsiveness to bath-applied 8-bromo-cyclic AMP. These electrophysiological findings are generally consistent with the biochemical observations. Moreover, Lewis and Fischer rats displayed very different opiate withdrawal syndromes, with different types of behaviors elicited upon precipitation of opiate withdrawal with the opiate receptor antagonist, naltrexone. The possible relationship between these behavioral findings and the biochemical and electrophysiological data is discussed. These studies provide further support for the possibility that Lewis and Fischer rat strains provide a useful model system in which some of the genetic factors that contribute to drug-related behaviors can be investigated.     

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.     

Involvement of protein kinase C in morphine tolerance at spinal levels of rats

The present study was performed to investigate the possible role of protein kinase C (PKC) in morphine tolerance at spinal levels of rats. Intrathecal injection of 10 μg of morphine induced increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats. After intrathecal injections of 10 μg of morphine (twice a day) lasted for 5 days, the antinociceptive effects induced by intrathecal injections of morphine decreased significantly in rats. Interestingly, we found that there were significant increases in the content of PKC in the dorsal horn of the spinal cord and the dorsal root ganglion, but not in the ventral horn of the spinal cord, in rats with morphine tolerance determined by Western blot, suggesting that PKC is involved in morphine tolerance at spinal levels of rats. Furthermore, our results demonstrated that chronic intrathecal injection of the PKC inhibitor significantly inhibited the development of morphine tolerance. Moreover, we found that the maintenance of morphine tolerance was blocked by intrathecal administration of a PKC inhibitor in rats, and the inhibitory effects of the PKC inhibitor on morphine tolerance lasted for more than two days. Taken together, the present study clearly showed that PKC is involved in morphine tolerance at the spinal level of rats and that intrathecal administration of a PKC inhibitor can block the development and maintenance of morphine tolerance.