Morphine-induced analgesic tolerance, locomotor sensitization and physical dependence do not require modification of mu opioid receptor, cdk5 and adenylate cyclase activity

Acute morphine administration produces analgesia and reward, but prolonged use may lead to analgesic tolerance in patients chronically treated for pain and to compulsive intake in opioid addicts. Moreover, long-term exposure may induce physical dependence, manifested as somatic withdrawal symptoms in the absence of the drug. We set up three behavioral paradigms to model these adaptations in mice, using distinct regimens of repeated morphine injections to induce either analgesic tolerance, locomotor sensitization or physical dependence. Interestingly, mice tolerant to analgesia were not sensitized to hyperlocomotion, whereas sensitized mice displayed some analgesic tolerance. We then examined candidate molecular modifications that could underlie the development of each behavioral adaptation. First, analgesic tolerance was not accompanied by mu opioid receptor desensitization in the periaqueductal gray. Second, cdk5 and p35 protein levels were unchanged in caudate-putamen, nucleus accumbens and prefrontal cortex of mice displaying locomotor sensitization. Finally, naloxone-precipitated morphine withdrawal did not enhance basal or forskolin-stimulated adenylate cyclase activity in nucleus accumbens, prefrontal cortex, amygdala, bed nucleus of stria terminalis or periaqueductal gray. Therefore, the expression of behavioral adaptations to chronic morphine treatment was not associated with the regulation of micro opioid receptor, cdk5 or adenylate cyclase activity in relevant brain areas. Although we cannot exclude that these modifications were not detected under our experimental conditions, another hypothesis is that alternative molecular mechanisms, yet to be discovered, underlie analgesic tolerance, locomotor sensitization and physical dependence induced by chronic morphine administration.     

Association of morphine-induced antinociception with variations in the 5' flanking and 3' untranslated regions of the mu opioid receptor gene in 10 inbred mouse strains

OBJECTIVE: Genetic factors are hypothesized to be involved in interindividual differences in opioid sensitivity. Inbred mouse strains that are genetically different and isogenic within each strain are useful for elucidating the genetic mechanisms underlying the interindividual differences in opioid-induced analgesia. METHODS: We examined the effects of morphine in 10 inbred mouse strains, including wild-derived strains that have a wide range of genetic diversity, including BLG2, CHD, KJR, MSM, NJL, PGN2, and SWN. We also performed full sequencing of the 5' flanking region and exons of the mouse mu opioid receptor gene Oprm1 and analyzed the association between genotypes and phenotypes in these mice. RESULTS: The effects of morphine on locomotor activation and antinociception varied among the inbred strains. The nucleotide differences that cause amino acid substitutions were not found in the Oprm1 gene in the inbred strains analyzed in this study. In the 5' flanking region and 3' untranslated region of the Oprm1 gene, four highly variable regions containing novel short tandem repeat polymorphisms (GA, T, TA, and CA/CT) were identified. The GA, T, and TA repeat numbers were significantly associated with morphine-induced antinociception. CONCLUSION: These results suggest that the short tandem repeats in the 5' flanking and 3' untranslated regions of the mu opioid receptor gene are involved in interstrain differences in opioid sensitivity in mice. Wild-derived inbred mouse strains with different numbers of these repeats may be useful models for examining interindividual differences in opioid sensitivity.     

Antinarcotic effects of the standardized ginseng extract G115 on morphine

The study was undertaken to determine the antagonism of morphine analgesia by the standardized ginseng extract G115 from Panax ginseng, the inhibitory effects of orally administered G115 on the development of morphine-induced tolerance and physical dependence, the hepatic glutathione levels, the inhibitory effects of intraperitoneally administered G115 on the dopamine receptor supersensitivity, and the reverse tolerance to the locomotor accelerating effect of morphine. G115 significantly inhibits the development of morphine-induced tolerance and physical dependence, the hepatic glutathione level decrease induced by morphine multiple injections, the development of morphine-induced dopamine receptor supersensitivity, and reverse tolerance to the locomotor accelerating effect of morphine. It did not, however, antagonize morphine analgesia.     

Overexpression of type 7 adenylyl cyclase in the mouse brain enhances acute and chronic actions of morphine

The mechanisms by which morphine-induced analgesia and tolerance and physical dependence on morphine arise have been the subject of intense study, and much work has pointed to the involvement of cAMP-mediated events in the neuroadaptive phenomena leading to morphine tolerance and/or dependence. We overexpressed an opioid receptor-stimulatable form of adenylyl cyclase (type 7) in the central nervous system of mice and demonstrated significant effects of this manipulation on the animals' acute response to morphine, the development of morphine tolerance, and development of sensitization to morphine. Measurements of the acute analgesic response to morphine demonstrated that the ED(50) values for the transgenic mice were significantly lower than the ED(50) values determined for the "wild-type" animals. During chronic treatment with morphine, the transgenic mice developed tolerance more rapidly than the wild-type mice, and transgenic animals of the C57BL/6xSJL background showed a larger sensitization to morphine's effects on locomotor activity than did wild-type mice of the same background. These results indicated that cAMP-generating systems may simultaneously modulate the development of tolerance and sensitization. Interestingly, the signs of physical dependence on morphine in the transgenic mice did not differ from those in their wild-type litter mates, indicating that separate mechanisms may modulate opiate tolerance and opiate dependence.     

The effects of p-chlorophenylalanine on morphine analgesia,tolerance and dependence development in two strains of rats

The effects of p-chlorophenylalanine (p-CPA; 100 mg/kg/day for 3 days) on morphine analgesia and the development of tolerance and physical dependence were investigated in Sprague-Dawley (SD) and Fisher (F) strains of albino rats. Using a modified flinch-jump method to detect changes in reactivity to electric footshock, F strain rats were more reactive to the footshock than SD rats, but showed less relative increase in threshold (analgesia) than SD rats following various doses of morphine. Pretreatment with p-CPA attenuated significantly morphine analgesia in SD, but not F rats. In animals implanted subcutaneously with morphine pellets, p-CPA appeared to delay the development of tolerance to morphine in both strains of rats. Hyperalgesia and loss of body weight resulted from administration of naloxone to pellet-implanted rats and p-CPA pretreatment lessened these withdrawal effects significantly in SD rats only. These results emphasize the importance of strain differences in the study of morphine analgesia and development of tolerance and dependence. Assuming differences in the function of the serotonergic inhibitory system in the two strains of rats, these data provide general support for the involvement of brain 5-HT mechanisms in modulating, if not mediating the effects of morphine.     

Hypothalamic peptide hormone,prolyl-leucyl-glycinamide and analog,inhibit tolerance to the analgesic and locomotor depressant but not to the locomotor stimulant effects of morphine in the mouse

The effects of prolyl-leucyl-glycinamide (melanotropin release inhibiting factor, MIF) and cyclo(Leu-Gly) on development of tolerance to the analgesic, locomotor stimulant and depressant effects of morphine were investigated in the mouse. Mice were made tolerant to morphine by subcutaneous implantation of a pellet (each pellet contained 75 mg of morphine free base) for three days. Both MIF and cyclo(Leu-Gly) inhibited the development of tolerance to the analgesic response to a challenge dose of morphine in peptide-treated as compared to vehicle-treated morphine-tolerant mice. Morphine in a small dose (10 mg/kg) depressed spontaneous motor activity, while, in a larger dose (80 mg/kg), increased the motor activity. Implantation of a morphine pellet resulted in the development of tolerance to both the locomotor depressant and stimulant effects of morphine. Administration of MIF or cyclo(Leu-Gly) during induction of tolerance in doses (2 mg/kg/day for 3 days) that inhibited the development of tolerance to morphine-induced analgesia and locomotor depressant activity, did not alter the development of tolerance to the locomotor stimulant effect. These studies indicate that the development of tolerance to the analgesic and locomotor depreressant effect of morphine may involve similar mechanisms, whereas, tolerance to the locomotor stimulant effect of morphine may be mediated via a different mechanism.     

Co-administration of dextromethorphan with morphine attenuates morphine rewarding effect and related dopamine releases at the nucleus accumbens

Morphine is one of the most effective analgesics in clinic to treat postoperative pain or cancer pain. A major drawback of its continuous use is the development of tolerance and dependence. In our previous study we found that a widely used antitussive agent in clinics, dextromethorphan [(DM); also known as a non-competitive N-methyl-d-aspartate (NMDA) antagonist], could prevent the development of morphine tolerance. In the present study, we further investigated its effect on morphine addiction. Conditioned place preference (CPP) test and behavioral sensitization of locomotor activity were used to investigate the drug-seeking related behaviors, which were in correlation with psychological dependence. Our results showed that co-administered DM was able to abolish completely the CPP effect induced by morphine, but had no effect on morphine-induced behavioral sensitization. By employing the microdialysis technique in free-moving animals, we also determined the extracellular level of dopamine and serotonin metabolites in the shell region of the nucleus accumbens (NAc) in its response to morphine with/without DM. A significant increase in dopamine metabolites following morphine administration was demonstrated in the NAc. This increase by morphine could be attenuated by co-administered DM, whereas DM itself did not show any effect. Based on our results, it is speculated that DM may effectively attenuate morphine-induced psychological dependence. Neurochemical analysis revealed that the effect of DM could be through its action on the dopaminergic mesolimbic pathway, which could be activated by morphine and attributed to the cause of rewarding.     

Evidence for the dissociation of morphine analgesia, tolerance and dependence

A single large dose of morphine produced profound analgesia accompanied by the development of tolerance and physical dependence. The tolerance developed acutely within 24 h and was further intensified, reaching a peak on the 5th day, then gradually disappeared. Partial or complete masking of morphine analgesia by naloxone inhibited the development of the acute, but could not prevent the development of the delayed, tolerance. These results suggest there are two kinds of tolerance and that the analgesic effect is separate from tolerance. Similarly, treatment with morphine produced physical dependence which was precipitated by naloxone. Unlike tolerance, dependence did not develop when morphine analgesia was completely masked by naloxone. The findings provide for the dissociation or morphine analgesia, tolerance and dependence.     

Effects of nandrolone on acute morphine responses,tolerance and dependence in mice

Anabolic-androgenic steroid exposure has been proposed to present a risk factor for the misuse of other drugs of abuse. We now examined whether the exposure to the anabolic-androgenic steroid, nandrolone, would affect the acute morphine responses, tolerance and dependence in rodents. For this purpose, mice received nandrolone using pre-exposure (for 14 days before morphine experiments) or co-administration (1 h before each morphine injection) procedures. Nandrolone treatments increased the acute hypothermic effects of morphine without modifying its acute antinociceptive and locomotor effects. Nandrolone also attenuated the development of tolerance to morphine antinociception in the hot plate test, but did not affect tolerance to its hypothermic effects, nor the sensitisation to morphine locomotor responses. After nandrolone pre-exposure, we observed an attenuation of morphine-induced place preference and an increase in the somatic manifestations of naloxone-precipitated morphine withdrawal. These results indicate that anabolic-androgenic steroid consumption may induce adaptations in neurobiological systems implicated in the development of morphine dependence.     

High affinity binding of 2-chloroadenosine to rat brain synaptic membranes

Intracerebroventicular (i.c.v.) administration of subanalgesic doses of β- and leucibe-endorphin in mice 15 min before subcutaneous morphine injection, significantly enhanced the analgesic effects of the morphine as measured by the tail-flick assay. A similar effect was seen with levorphanol analgesia but no enhancement of leucine- or methionine-enkephalin analgesia by β-endorphin was observed. The same doses of the endorphins did not affect the development of single-dose morphine tolerance and dependence. Leucine-enkephalin failed to affect morphine analgesia, tolerance or dependence development, while low doses of methionine-enkephalin administered i.c.v. were observed to have an antagonistic effect on morphine analgesia without affecting tolerance or dependence development. Neither enkephalin had any effect on β-endorphin analgesia.     

Attenuation of morphine tolerance development by electroconvulsive shock in mice

The development of tolerance to morphine has been proposed as being analogous to learning or memory, since the presentation of a novel stimulus (morphine) to an organism results in an altered response on subsequent presentations of the same stimulus. Electroconvulsive shock has been widely used to disrupt memory in animals and its effect on the development of tolerance to morphine was examined. Pretreatment of mice with six, progressively increasing intraperitoneal doses of morphine induced tolerance to an excitant action of morphine, shown by a marked reduction in the locomotor activity elicited by a subsequent intraperitoneal test dose of morphine. In three separate experiments, the administration of eleetroconvulsive shock 2–3 hr after each pretreatment with morphine attenuated the degree of tolerance developed. The eleetroconvulsive shock had no significant effect on the locomotor activity of non-tolerant mice tested under similar conditions in each experiment. In a single pilot study, electroconvulsive shock was found to reduce the frequency of jumping precipitated by an intraperitoneal dose of naloxone, suggesting a possible decrease in the degree of dependence. The present results are interpreted as supporting the view that the mechanisms of morphine tolerance may be similar to those involved in learning or memory.     

Increased analgesic tolerance to acute morphine in fosB knock-out mice: a gender study

The proteins of Fos family are a potential candidate to link molecular mechanisms of morphine action with behavioural effects such as morphine-induced reward, dependence and tolerance. We used both male and female mice lacking fosB gene to study its contribution to morphine effects. Morphine analgesia (tail-flick test) and hypothermia were studied using morphine at cumulative doses in morphine-naïve and morphine-tolerant (tolerance induced by 24 h prior 100 mg/kg morphine administration) mice. FosB −/− mice, as compared to fosB +/+ mice, developed enhanced tolerance to morphine-induced analgesia. No effects of genotype or gender on tolerance to morphine-induced hypothermia were observed. These results suggest that fosB may be involved in the development of tolerance to morphine analgesia but not hypothermia. The gender study implicates that lack of FosB proteins in female fosB −/− mice enhanced morphine analgesic potency. In conclusion, we show that fosB gene is important to analgesia but not hypothermia phenotype indicating its role in morphine effects.     

Effects of NRGC-lesion on the rates of the development of morphine tolerance and dependence in rats

The development of tolerance to and dependence on morphine over 1-8 days treatment with morphine were studied with time in rats in which the nucleus reticularis gigantocellularis (NRGC), including the nucleus reticularis paragigantocellularis, were electrically destructed. The NRGC of SD male rats was bilaterally lesioned (DC, 0.5 mA, 40 sec), and morphine analgesia was estimated by the tail flick method. Morphine analgesia in NRGC-destructed rats (D-rat) was reduced to about 50% of that in sham-operated rats (S-rat). The dose of morphine to produce equi-analgesia increased 2-21.8 times during 1-8 days treatment with morphine in S-rat. Throughout this period, ratios of the equi-analgesic dose in D-rat to that in S-rat were almost the same, i.e., the rate of tolerance development to morphine analgesia in D-rat was to the same degree as that in S-rat. Administration of naloxone after 1-6 days treatment with morphine elicited body weight loss and increase in plasma corticosterone (Pcs), degrees of which were dependent on the dose of naloxone or the period of morphine treatment. No difference in these abstinence signs were detected between S- and D-rat, i.e., the rate of development of dependence on morphine in D-rat was to the same degree as that in S-rat. These results suggest that the NRGC participates in the development of morphine analgesia, but does not participate in the development of tolerance to and dependence on morphine.     

Strain differences in the rewarding and dopamine-releasing effects of morphine in rats

Studies examining differential sensitivity to psychoactive drugs in mice suggest that genotype may play a critical role. Furthermore, an involvement of genotype in mediating individual differences in sensitivity to the rewarding effects of several drugs of abuse has also been postulated. The aim of this study was to examine the conditioned rewarding and dopamine-releasing effects of morphine in two outbred rat strains commonly used in addiction research. Additionally, the behavioural and neuroendocrine responses of these strains to the stress of novelty were also examined. Basal locomotor activity was higher in Wistar rats than Sprague-Dawley following exposure to a novel environment. In contrast, elevations in plasma corticosteroid levels following novelty exposure did not differ between the two strains. In a counterbalanced place preference conditioning procedure, increasing doses of morphine (1.0–10.0 mg/kg SC) produced significant conditioned place preferences (CPP) in both Wistar and Sprague-Dawley strains. However, Wistar rats required a significantly larger dose of morphine (5.0 mg/kg) to produce a significant CPP than the Sprague-Dawley rats. In the latter strain, CPP occurred with doses of 3.0 mg/kg and greater. In parallel microdialysis experiments, both strains showed significant dose-related increases in dopamine release in the nucleus accumbens following acute morphine challenge (1.0–10.0 mg/kg SC). Again in Wistar rats, a larger dose of morphine was necessary to produce a significant increase in comparison to Sprague-Dawley rats. These results show that genetically distinct rat strains can show differential sensitivity to opioids, more specifically to drug-seeking responses.     

Suppression of withdrawal jumping: Comparison of narcotic tolerance and dependence

The median and maximal suppressive doses (SD₅₀ and SD₉₅) of methadone and morphine for abrupt withdrawal jumping were determined in male ICR mice (25–30 g) rendered tolerant to and physically dependent on morphine after implantation with a morphine pellet for 3 days. Three separate groups of mice were then given one of the following treatments for 5 successive days: SD₉₅ methadone subcutaneously (s.c.) once daily, SD₉₅ methadone per os (p.o.) once daily, and SD₉₅ morphine s.c. every 8 hours. Respective controls received either water p.o. or saline s.c. daily. The SD₉₅ for each narcotic was found to suppress withdrawal jumping equally (by 80%) each day during the 5-day administration period. However, at the end of that time, only the morphine group demonstrated significant analgetic tolerance to both s.c. morphine and methadone (a three-fold elevation), and detectable physical dependence as evidenced by naloxone-precipitated withdrawal jumping. Thus, daily doses of an opiate sufficient to suppress abrupt withdrawal jumping in morphine-dependent animals may not necessarily maintain the animals' tolerance and physical dependence on morphine. It is concluded that the sites for the expression of acute opiate analgesia and withdrawal jumping are not those involved with the development of tolerance and physical dependence.     

Morphine analgesia and tolerance in the tail-flick and formalin tests: Dose-response relationships

The dose-response relationships for morphine analgesia were studied in morphine-tolerant and non-tolerant rats using two pain tests: tail-flick test which measures the threshold for an escape response, and the formalin test which assesses the behavioral response to continuous pain generated in injured tissue. The effects of prior experience with both pain tests on tolerance were also examined. In the formalin test, effective analgesia was obtained in non-tolerant rats at doses that produce minimal depression of locomotor behavior. Morphine tolerance was produced by 20 daily injections of morphine with increments that reached 16 mg/kg, a dose over the LD₁₀₀ for barrier sustained Long Evans rats. This dose regimen produced a 1.8-fold increase in the ED₅₀ in the tail-flick test and a 2.7-fold increase in the formalin test. Daily experience of the pain test, as well as the morphine regimen produced a 4.8-fold increase in the ED₅₀ in the tail-flick test but did not effect the potency of morphine in the formalin test. The magnitude of tolerance in the absence of daily behavioral testing is consistent with recent clinical reports that little tolerance occurs after prolonged administration of morphine in cancer patients and that tolerance is not an important consideration in the management of pain.     

Modification of morphine-induced analgesia, tolerance and dependence by bromocriptine

The effect of two doses of bromocriptine, a dopamine agonist, on morphine-induced analgesia, tolerance and dependence was investigated in mice. Bromocriptine at doses of 0.04 and 0.08 mg/kg did not affect the baseline tail flick latency of mice but potentiated the morphine analgesia. Pretreatment of mice with 5 mg/kg of sulpiride, a D-2 antagonist, not only blocked the effect of 0.08 mg/kg of bromocriptine but also antagonized the morphine analgesia. Control animals given daily injections of 10 mg/kg of morphine rapidly developed tolerance to the analgesic effect. A combined treatment of bromocriptine with morphine given daily suppressed the development of tolerance to morphine analgesia. However, development of tolerance to morphine analgesia was not significantly modified in the animals treated daily with bromocriptine (0.08 mg/kg) plus sulpiride (5 mg/kg). Acute dependence was induced by the administration of 100 mg/kg of morphine. The administration of bromocriptine 30 min before naloxone significantly decreased the ED₅₀ value for naloxone for inducing jumping in mice. Coadministration of sulpiride and bromocriptine attenuated the ability of bromocriptine to potentiate the withdrawal syndrome of morphine dependence. The results indicate that bromocriptine potentiates morphine analgesia, suppresses the development of tolerance to morphine analgesia but exacerbates opiate withdrawal signs in morphine-dependent mice. These effects of bromocriptine appear to be mediated via D-2 receptors.     

Analgesic activity of ZC88, a novel N-type voltage-dependent calcium channel blocker, and its modulation of morphine analgesia, tolerance and dependence

ZC88 is a novel non-peptide N-type voltage-sensitive calcium channel blocker synthesized by our institute. In the present study, the oral analgesic activity of ZC88 in animal models of acute and neuropathic pain, and functional interactions between ZC88 and morphine in terms of analgesia, tolerance and dependence were investigated. In mice acetic acid writhing tests, ZC88 (10-80 mg/kg) administered by oral route showed significant antinociceptive effects in a dose-dependent manner. The ED50 values of ZC88 were 14.5 and 14.3 mg/kg in male and female mice, respectively. In sciatic nerve chronic constriction injury rats, mechanical allodynia was ameliorated by oral administration of ZC88 at doses of 14, 28 and 56 mg/kg, suggesting ZC88 relieved allodynic response of neuropathic pain. When concurrently administered with morphine, ZC88 (20-80 mg/kg) dose-dependently potentiated morphine analgesia and attenuated morphine analgesic tolerance in hot-plate tests. ZC88 also prevented chronic exposure to morphine-induced physical dependence and withdrawal, but not morphine-induced psychological dependence in conditioned place preference model. These results suggested that ZC88, a new non-peptide N-type calcium channel blocker, had notable oral analgesia and anti-allodynia for acute and neuropathic pain. ZC88 might be used in pain relief by either application alone or in combination with opioids because it enhanced morphine analgesia while prevented morphine-induced tolerance and physical dependence.     

Effects of nor-binaltorphimine on the development of analgesic tolerance to and physical dependence on morphine.

The effects of a highly selective kappa antagonist, nor-binaltorphimine (nor-BNI), on the development of tolerance to morphine analgesia and physical dependence on morphine were examined. Pretreatment with nor-BNI (5 mg/kg s.c.) 2 h prior to injection of morphine or a selective kappa agonist, U-50,488H, significantly antagonized the analgesic effect of U-50,488H, but not morphine analgesia in mice. The development of tolerance to morphine analgesia was significantly potentiated by pretreatment of mice with nor-BNI 2 h prior to morphine treatment during chronic morphine treatment for 5 days. Additionally, the pretreatment with nor-BNI during chronic treatment with the high dose of morphine for 5 days significantly potentiated the naloxone-induced body weight loss in morphine-dependent mice and rats. These findings suggest that inactivation of the kappa opioid system may potentiate the development of tolerance to morphine analgesia in mice and may aggravate the naloxone-precipitated body weight loss in morphine-dependent mice and rats.     

The biochemical analysis of methadone modulation on morphine-induced tolerance and dependence in the rat brain

We have recently demonstrated that the combination of methadone and morphine enhances the ability of morphine to induce mu-opioid peptide (MOP) receptor endocytosis. As a result, rats receiving both drugs show reduced morphine tolerance and dependence. In the present study, we identify the biochemical basis for the protective effect of the drug combination. In rats treated with morphine alone, the inhibitory effect of DAMGO on forskolin-stimulated adenylyl cyclase activity was significantly reduced in a brain-region-selective manner. Importantly, these reductions were prevented in animals receiving the drug combination. We found that these changes were not due to alterations in MOP receptor density, or MOP receptor-G protein coupling, as no significant change in these parameters was observed. Together these data demonstrate that neither changes in receptor number nor function are required for morphine tolerance and dependence. Rather, brain-region-selective changes in adenylyl cyclase signal transduction are critical, and both these biochemical changes and the behavioral effects are prevented by facilitating endocytosis of the MOP receptor.