Chronic Morphine Pretreatment Research Articles

Modulation of miR-139-5p on chronic morphine-induced, naloxone-precipitated cAMP overshoot in vitro.

Chronic exposure to morphine can produce tolerance, dependence and addiction, but the underlying neurobiological basis is still incompletely understood. c-Jun, as an important component of the activator protein-1 transcription factor, is supposed to take part in regulating gene expression in AC/cAMP/PKA signaling. MicroRNA (miRNA) has emerged as a critical regulator of neuronal functions. Although a number of miRNAs have been reported to regulate the μ-opioid receptor expression, there has been no report about miRNAs to regulate chronic morphine-induced, naloxone-precipitated cAMP overshoot. Our results showed that chronic morphine pretreatment induced naloxone-precipitated cAMP overshoot in concentration- and time-dependent manners in HEK 293/μ cells. Chronic morphine pretreatment alone elevated both c-Jun protein and miR-139-5p expression levels, while dramatically artificial elevation of miR-139-5p inhibited c-Jun at the translational level. Furthermore, dramatically artificial upregulation of intracellular miR-139-5p limited chronic morphine-induced, naloxone-precipitated cAMP overshoot. These findings suggested that miR-139-5p was involved in regulating chronic morphine-induced, naloxone-precipitated cAMP overshoot in a negative feedback manner through its target c-Jun, which extends our understanding of neurobiological mechanisms underlying morphine dependence and addiction.

Opioids Enhance CXCL1 Expression and Function After Incision in Mice

Chronic opioid consumption increases postoperative pain. Epigenetic changes related to chronic opioid use and surgical incision may be partially responsible for this enhancement. The CXCL1/CXCR2 signaling pathway, implicated in several pain models, is known to be epigenetically regulated via histone acetylation. The current study was designed to investigate the role of CXCL1/CXCR2 signaling in opioid-enhanced incisional sensitization and to elucidate the possible epigenetic mechanism underlying CXCL1/CXCR2 pathway–mediated regulation of nociceptive sensitization in mice. Chronic morphine treatment generated mechanical and thermal nociceptive sensitization and also significantly exacerbated incision-induced mechanical allodynia. Peripheral but not central messenger RNA levels of CXCL1 and CXCR2 were increased after incision. The source of peripheral CXCL1 appeared to be wound area neutrophils. Histone H3 subunit acetylated at the lysine 9 position (AcH3K9) was increased in infiltrating dermal neutrophils after incision and was further increased in mice with chronic morphine treatment. The association of AcH3K9 with the promoter region of CXCL1 was enhanced in mice after chronic morphine treatment. The increase in CXCL1 near wounds caused by chronic morphine pretreatment was mimicked by pharmacologic inhibition of histone deacetylation. Finally, local injection of CXCL1 induced mechanical sensitivity in naive mice, whereas blocking CXCR2 reversed mechanical hypersensitivity after hind paw incision. PerspectivePeripheral CXCL1/CXCR2 signaling helps to control nociceptive sensitization after incision, and epigenetic regulation of CXCL1 expression explains in part opioid-enhanced incisional allodynia in mice. These results suggest that targeting CXCL1/CXCR2 signaling may be useful in treating nociceptive sensitization, particularly for postoperative pain in chronic opioid-consuming patients.

Differential patterns of neuronal activation in rostral versus caudal ventral tegmental area involved in behavioral sensitization induced by an escalating-dose morphine administration paradigm

The transition to addiction often involves a gradual process of escalated drug intake. The purpose of the present study was to characterize neuronal activation in the ventral tegmental area (VTA) and substantia nigra (SN) following chronic escalating-dose morphine exposure (days 1–7, 2mg/kg/d; days 8–21, beginning at 10mg/kg/d, increasing by 2mg/kg/d), with steady-dose morphine (2mg/kg/d, i.p., for 21 days) as the comparison. Using immunohistochemical double-staining for tyrosine hydroxylase (TH) and Fos, we found that the number of Fos+TH+ neurons in the rostral VTA and number of Fos+TH− neurons in the lateral SNr were significantly increased in escalating-dose morphine-treated rats compared with steady-dose morphine-treated rats and acute morphine-treated rats. Meanwhile, this increase was associated with robust expression of behavioral sensitization after a challenge with 10mg/kg morphine. The number of Fos+TH+ neurons was significantly increased by acute morphine in the caudal VTA and SNc, but this number did not increase further with morphine pretreatment. These results demonstrate that behavioral sensitization was associated with elevated activation of dopaminergic neurons in the rostral VTA and nondopaminergic neurons in the lateral SNr, which could only be induced by chronic escalating-dose morphine rather than chronic steady-dose morphine pretreatment.

P.1.c.001 The effects of two different types of chronic morphine pretreatment on anxiety and nociception in adolescence offspring

P.1.c.001 The effects of two different types of chronic morphine pretreatment on anxiety and nociception in adolescence offspring

Chronic morphine administration results in tolerance to delta opioid receptor-mediated antinociception

Delta opioid receptor agonists produce only a moderate degree of antinociception, possibly reflecting the predominantly intracellular location of delta opioid receptor. However, recent studies suggest that short term morphine pretreatment can increase delta opioid receptor-mediated antinociception by promoting the translocation of delta opioid receptor to the cell surface. Even more striking sensitization has been reported after long term morphine pretreatment and withdrawal in locomotor tests. In the present study we therefore examined the effects of longer term morphine pretreatment and withdrawal on delta opioid receptor-mediated antinociception in the formalin test. Male adult rats were pretreated daily with morphine (10 mg/kg s.c.) or saline for 10 days, and were tested acutely with the delta opioid receptor agonist [ d-Ala 2,Glu 4]-deltorphin (intrathecal) at 0, 7 and 14 days of withdrawal. Unexpectedly, chronic morphine pre-exposure resulted in tolerance to [ d-Ala 2,Glu 4]-deltorphin-induced antinociception, and this occurred at 0 and 7 but not 14 days of morphine withdrawal. Morphine challenge at withdrawal day 7 confirmed the presence of tolerance to the antinociceptive effects of this drug. Chronic morphine pretreatment also resulted in tolerance to the locomotor stimulant effect of [ d-Ala 2,Glu 4]-deltorphin (given i.c.v.), contrary to a previous report of sensitization. However, consistent with previous reports, short term (2 day) pretreatment with morphine did result in sensitization to [ d-Ala 2,Glu 4]-deltorphin. Subsequent in vitro analysis, using [ 125I][ d-Ala 2,Glu 4]-deltorphin or guanosine 5′(γ- 35S-thio) triphosphate autoradiography, did not reveal any changes in delta opioid receptor binding or function resulting from chronic morphine pretreatment. In conclusion, chronic morphine pretreatment caused tolerance to delta opioid receptor-mediated behavioral effects with no clear change at the receptor level.

Catecholaminergic activity and 3',5'-cyclic adenosine monophosphate concentrations in the right ventricle after acute and chronic morphine administration in the rat

We have examined possible regulation of norepinephrine and dopamine concentrations and turnover in the right ventricle of the rat after acute administration of saline i.p. or morphine 30 mg kg-1 i.p. to placebo (naïve) or morphine (tolerant) pretreated rats. We also assessed concentrations of 3',5'-cyclic adenosine monophosphate (cAMP) in the right ventricle after the same treatments. Concentrations of catecholamines and their metabolites in the heart were measured by high-pressure liquid chromatography with electrochemical detection (HPLC/DE). Concentrations of cAMP in the heart were measured by radioimmunoassay (RIA). Administration of morphine to naïve rats did not modify concentrations of norepinephrine (NE), normetanephrine (NMN) or NMN/NE ratio in the right ventricle. However, dopamine concentrations increased whereas dopamine turnover decreased. In addition, cAMP concentrations decreased after acute administration morphine to naïve rats. In rats pretreated with morphine chronically, there was an increase in norepinephrine concentrations with no change in normetanephrine concentrations or norepinephrine turnover after acute injection of morphine. In contrast, dopamine turnover increased in the tolerant groups after acute injection of saline or morphine compared with the nave group given morphine, indicating that tolerance develops to the acute effects of the opioid. Concentrations of cAMP increased after chronic morphine administration. Our results demonstrate that chronic morphine pretreatment leads to up-regulation of the cAMP system in the heart and suggest that this up-regulation may be involved in the cellular mechanisms implicated in the adaptive changes of dopaminergic neurones in the heart observed during chronic treatment with morphine.

Morphine-induced long-term sensitization to the locomotor effects of morphine and amphetamine depends on the temporal pattern of the pretreatment regimen.

The development of behavioural sensitization is thought to depend on the dose and temporal pattern of drug treatment. Previous studies have shown that two distinct morphine pretreatment regimens cause different long-term neuroadaptations in rat striatum. Therefore, in the present study the ability of these pretreatment regimens to induce long-term behavioural sensitization was investigated. One pretreatment regimen, termed "chronic", consisted of three daily injections, for 5 days, with escalating doses (10-50 mg/kg) of morphine, and the other, termed "intermittent", of 14 daily injections with morphine (10 mg/kg). Both intermittent and chronic morphine pretreatment caused sensitization to the locomotor effects of morphine, 3 weeks post-treatment, although the former induced a far greater level of sensitization. Moreover, 3 weeks post-treatment, intermittent, but not chronic, morphine pretreatment induced cross-sensitization to the locomotor stimulant effects of amphetamine. Behavioural sensitization following intermittent morphine pretreatment was clear-cut both 1 day and 3 weeks post-treatment, while after 9 weeks, the locomotor effects of morphine were still slightly augmented. It is concluded that intermittent morphine pretreatment is far more effective in inducing long-term behavioural sensitization than chronic morphine pretreatment.

Effects of chronic morphine pretreatment on amygdaloid kindling development, postictal seizure and suppression and benzodiazepine receptor binding in rats.

Effects of chronic morphine pretreatment on the development of amygdaloid kindling, seizure suppression and benzodiazepine (BDZ) receptor binding in rats were evaluated. The morphine-pretreated animals showed faster acquisition of seizure activity. Further evaluation of the postictal seizure suppression immediately after a fully kindled seizure demonstrated that morphine-pretreated rats had a decreased sensitivity to subsequent kindling stimulations. Twenty-four hours after the last electrical stimulation, saline-pretreated fully kindled rats showed enhanced BDZ receptor binding in dentate gyrus, and decreased binding in cingulate cortex ipsilateral to the stimulation site, compared to saline controls. Morphine-pretreated amygdala-kindled rats had significantly higher BDZ binding in piriform, entorhinal and sensorimotor cortices, basolateral and cortical amygdaloid nuclei, dentate gyrus, CAI-3 areas, substantia nigra pars reticulata and periaqueductal gray. The present study indicates that the previous experience with chronic morphine modifies the kindling process and that the enhanced BDZ receptor binding detected in our experiments may be involved in the enhanced postictal seizure suppression observed in these animals.

Morphine modulates IL-1 activation of fos in the rat hypothalamus

By using FOS proto-oncogene protein as a marker for neuronal activity induced by an extracellular stimulus, we have shown that several hypothalamic nuclei are sites of action for interleukin-1 (IL-1). These nuclei, including the paraventricular (PVN) and supraoptic (SON) nuclei, have relatively few IL-1 receptors. One of the possibilities for this dysjunction may be that IL-1 acts on receptors other than currently defined IL-1 receptors to induce FOS activation. Acute subcutaneous morphine treatment also increased FOS immunoreactivity in several hypothalamic nuclei including PVN and SON. Taken together, these results suggest the possibility of an interaction between morphine and IL-1 in the hypothalamic PVN and SON. The PVN and SON have been suggested to be involved in a neuroendocrine-immune axis. In this study we examined the effects of chronic morphine on IL-1 activation of FOS. The chronic morphine pretreatment consisted of 2 pellets on the 1st day and 4 pellets on the 2nd day, 75 mg morphine sulfate/pellet. This regimen has been shown to induce a high degree of tolerance and dependence, and it markedly decreased IL-1 activation of c-fos mRNA in the hypothalamus and FOS immunoreactivity in the hypothalamic PVN and SON nuclei. These data suggest that attenuation of IL-1 activation of FOS expression in the hypothalamus may be a mechanism by which morphine tolerance and dependence affects the neuro-endocrine-immune axis.

Naloxone-induced cardiovascular depression in rats that had received chronic morphine-treatment

1. Cardiovascular changes in response to intravenous injection of naloxone were studied in pentobarbitone-anaesthetized rats which had been given morphine in their drinking water for 1-21 days. The mechanisms of the observed changes were investigated in intact animals and in isolated hearts and tail arteries. 2. In rats that had received chronic morphine-treatment, intravenous administration of naloxone caused immediate decreases in blood pressure, heart rate, left ventricular pressure and dLVP/dtmax which were followed by the occurrence of atrial or ventricular extrasystoles and other signs of opiate withdrawal such as faecal passage and muscle twitching. 3. The intensities of the naloxone-precipitated cardiovascular changes were directly related to the duration of chronic morphine pretreatment, reaching statistically significant levels on day 2 or 3 and maximal levels on day 7 or 14. This phenomenon disappeared on days 3 to 14 following opiate withdrawal in animals which had been treated previously with morphine for 21 days. 4. Either atropine or clonidine pretreatment significantly prevented the occurrence of faecal passage or muscle twitching during naloxone-precipitated opiate withdrawal. However, clonidine, but not atropine or yohimbine, abolished the decreases in various haemodynamic parameters. The occurrence of cardiac extrasystoles was not affected. 5. In isolated heart or tail artery preparations from chronically morphine-treated rats, naloxone administration did not elicit reactions which differed from those of the preparations from naive animals. These findings suggest that under pentobarbitone anaesthesia, the cardiovascular systems of rats that had received chronic morphine treatment exhibit inhibitory, instead of excitatory, reactions to naloxone-precipitated opiate withdrawal.

Changes in preganglionic sympathetic nerve function following chronic morphine treatment in rats

1. The effects of acute or chronic morphine treatment on the changes in blood pressure and pulse rate in response to ganglionic stimulation or blockade and to vagal stimulation, and of isolated atria to field stimulation or noradrenaline, were studied. 2. In pithed rats, intravenously injected hexamethonium significantly depressed the blood pressure responses to sympathetic nerve stimulation. The ganglionic blocking effects of hexamethonium were significantly greater in chronically morphine-treated rats, but were not significantly affected by acute morphine administration in naive animals. 3. Intravenous administration of nicotine dose-dependently increased blood pressure and pulse rate. The magnitudes of these changes were not significantly affected by acute or chronic morphine pretreatment. 4. Studies with rat isolated atrial preparations revealed that the changes in atrial contractile rate and force in response to noradrenaline or field stimulation were not influenced by either acute or chronic morphine treatment. 5. Cervical vagal stimulation produced voltage- or frequency-dependent decreases in pulse rate and blood pressure. The responses were not significantly affected by chronic morphine treatment. 6. These findings suggest that the site of the changes in sympathetic function following prolonged exposure to the opiate appears to be on the preganglionic nerve fibres.

Effects of acute and chronic morphine treatment on methadone analgesia and metabolism

Morphine sulfate (5 mg/kg s.c.) given 30 min prior to administration of methadone prolonged methadone analgesia and increased the brain level of methadone measured 60, 120 and 180 min after administration of methadone. Rats rendered tolerant to morphine analgesia by subcutaneous implantation of two pellets, each containing 75 mg of morphine base, for 1–3 days showed cross-tolerance to methadone analgesia regardless of the presence or absence of morphine pellets. Decreases in the brain concentrations of methadone measured at 60 and 120 min time points accompanied the decreased analgesia. Neither acute nor chronic morphine pretreatment affects the biotransformation of methadone. The results suggest that the cross-tolerance to methadone analgesia seen in chronic morphine-implanted rats was partly associated with a decrease in the brain concentration of methadone occurring by a mechanism not directly related to a change in the biotransformation of methadone. In view of the known inhibitory effect of chronic morphine pretreatment on drug metabolism, our findings might demonstrate a unique phenomenon between morphine and methadone.

Inhibition by β-endorphin of isoprenaline-induced vasopressin release and its reversal by naloxone

The effect of β-endorphin on isoprenaline-induced vasopressin release was investigated in rats. β-Endorphin (0.29 nmol i.c.v. or 2.9 nmol i.v.) suppressed markedly the increase in plasma vasopressin concentrations in response to isoprenaline without altering the concomitant increase in plasma renin concentration or fall in mean arterial blood pressure. Naloxone (1 mg/kg) prevented this inhibition as did chronic morphine pretreatment. These results show that β-endorphin can inhibit isoprenaline-induced vasopressin release by acting on opiate receptors.

Opiate antagonist-receptor interaction unchanged by acute or chronic opiate treatment

The apparent pA 2 of naloxone, a measure of the apparent receptor affinity for the antagonist, was estimated in naive rats, in rats after acute and various schedules of chronic morphine pretreatment, and in rats during abrupt withdrawal. The results were adjusted to compensate for the amount of pretreatment drug in the rat brains as, otherwise, such a drug could have inflated the apparent pA 2 values (see appendix). After this adjustment, a tendency of the apparent pA 2 values to be increased after chronic pretreatment disappeared. The apparent pA 2 of naloxone in acutely pretreated rats was also unchanged from that in naive rats, while the results in abruptly withdrawn rats were ambiguous. Thus, no change in the affinity of the antagonist for opiate receptors was found and theories suggesting that a conformational change takes place in the opiate receptors during the development of tolerance/dependence could not be supported.

The effects of microiontophoretically applied morphine and transmitter substances in rats during chronic treatment and after withdrawl from morphine.

The effects of microiontophoretically applied acetylcholine, noradrenaline, 5-hydroxytryptamine and morphine were studied on single brain stem neurones of rats during chronic morphine pretreatment and 24 h after its withdrawal. No significant changes were observed in the initial spontaneous neuronal firing rate or in the qualitative or quantitative effects of acetylcholine, noradrenaline or 5-hydroxytryptamine. However, in chronically treated animals there was a significant decrease in the number of neurones excited by morphine or showing tachyphylaxis to morphine on repeated microiontophoretic applications.We suggest that some of the cellular central nervous system changes which occur during chronic morphine treatment are reflected by the decrease in sensitivity of neurones to morphine excitation.