Development of tolerance to the stimulatory effect of morphine in dog intestine

Intestinal motor responses to morphine and 5-hydroxytryptamine (5-HT) were measured in dogs injected daily for 16 days with morphine (stabilized at 30 mg/kg i.v.) and compared to responses obtained in control animals. Intestinal contractions produced by 10 and 20 μg/kg morphine were reduced in the morphine-treated animals. Responses to 5-HT did not differ between the two groups. The contractor dose-response curve for morphine, but not for 5-HT or bethanechol, was shifted markedly to the right in intestinal segments isolated from morphine-treated dogs. It is concluded that tolerance to the intestinal stimulatory effect of morphine occurs in the dog.     

Assessment of precipitated abstinence in morphine- dependent rats

An experimental model is described for quantifying the precipitated abstinence syndrome in morphine-dependent rats. Male rats were made dependent on morphine by subcutaneous implantation of a morphine pellet and the abstinence syndrome precipitated by intraperitoneal injection of naloxone hydrochloride. A ranking system, based on the median effective dose of naloxone for abstinence signs, quantitatively related the incidence of certain precipitated signs to the dose of naloxone. The time course for the development of dependence was shown to be maximal 70–74 h after pellet implantation. Food or water deprivation for 48 h dissociated the body weight loss during abstinence from the behavioral signs of precipitated withdrawal. Ganglionic blockade did not significantly modify abstinence behavior. An evaluative procedure which ranks abstinence signs is proposed for quantifying physical dependence on morphine.     

Influence of Plasma-protein Binding on Analgesic Effect of Methadone in Rats with Spontaneous Withdrawal

The effect of spontaneous withdrawal on α₁-acid glycoprotein (AAG) levels and methadone protein binding has been studied in the rat. Animals were made physically dependent on morphine by providing morphine HCl in drinking water for three weeks. The natural opiate withdrawal was induced in rats by substituting the morphine solution with drinking water. The severity of the abstinence syndrome was assessed at various time intervals. After 12 h of withdrawal, the animals showing abstinence signs and low morphine levels were injected with intravenous methadone (0.35 mg kg^?1) and the analgesic effect was measured by the tail-flick method and compared with animals receiving water. The oral administration of morphine produced an increase in AAG levels from 0.64 ± 005 g L^?1 in control animals to 1.47 ± 0.92 g L^?1 in experimental animals at the point of withdrawal and 1.21 ± 009 g L^?1 24 h after withdrawal. The percentage of methadone unbound was significantly lower in morphine-treated than in control animals. A significant correlation between AAG levels and percentage of methadone bound was observed. A parallel analgesic effect after intravenous methadone, as measured by AUC in the tail-flick test, was less in abstinence animals than in control (287.6 ± 24.8 compared with 401.0 ± 37.06 s min). We suggest that in the withdrawal syndrome an adjustment of methadone dose may be necessary because of changes in protein binding.     

Conditioned temperature effects using morphine as the unconditioned stimulus

The conditioning of body temperature changes using an injection of morphine sulphate as the conditioned stimulus was studied in 30 male Wistar rats. Three groups of animals received daily i.p. injections of either 5, 25, or an increasing dose to 200 mg/kg morphine; a fourth group received saline injections throughout. Rectal temperature was measured in three different environments five times during the day: in a neutral environment, the home cage; in a pre-injection environment, in which animals were placed for a period before the daily injection; and in an injection environment, in which animals remained after the injection. Conditioning trials were followed by a period of abstinence from morphine. Tests for conditioned effects were carried out both during conditioning and after the period of abstinence. During conditioning, animals in the morphine groups, when compared to the saline control animals, showed a conditioned anticipatory hypothermia in the preinjection environment that was opposite in direction to the unconditioned hyperthermia to morphine. In contrast, in the injection environment, animals in the morphine groups showed a conditioned hyperthermia when tested after the period of abstinence. These results suggest a complex interaction between the conditioned and unconditioned temperature responses to morphine.     

Catecholaminergic activity and 3',5'-cyclic adenosine monophosphate levels in heart right ventricle after naloxone induced withdrawal

This study evaluated the adaptive changes in noradrenergic neurons and the concomitant production of cAMP during morphine dependence and withdrawal in the right ventricle of the rat. Rats were made dependent on morphine by morphine pellet implantation for 7 days. On the day of sacrifice animals received an acute injection of saline or naloxone (1 mg/kg s.c.) and were decapitated 30 min later. Pretreatment with propranolol 15 min prior to naloxone was conducted to evaluate the possible implication of beta-adrenoceptors. The contents of noradrenaline and dopamine and their metabolites were examined. After naloxone administration to morphine-dependent rats (withdrawal) there was a pronounced increase in the content of normetanephrine and 3,4-dihydroxyphenylacetic acid and increased noradrenaline and dopamine turnover. In addition cAMP levels were increased after naloxone administration to morphine-treated rats. Propranolol did not block the hyperactivity of catecholaminergic neurons or the enhancement of cAMP observed in the heart during withdrawal. The present results indicate that heart catecholaminergic neurons play a significant role in the alterations in heart functions during morphine abstinence syndrome and suggest that those alterations are mediated through cAMP.     

A dual action of valproic acid upon morphine analgesia and morphine withdrawal

Effects of valproic acid administration on morphine analgesia and on morphine tolerance and dependence were investigated in mice. Valproate increased the reaction time to thermal stimulation in naive animals. This effect was additive with morphine when valproate was administered shortly before the analgesic. However, an antagonism was observed if a 4-hour period elapsed between valproate and morphine administration. When administered to mice receiving a sustained release preparation of morphine, valproate antagonized the development of tolerance to morphine. Valproate elicited a dual action on the abstinence signs observed after naloxone administration in morphine-treated mice. The effect consisted in a reduction of abstinence behavior if the anticonvulsant was administered a few minutes before naloxone; the same treatment increased the severity of the abstinence behavior when valproate was injected 1 h before the precipitating dose of naloxone. In this latter schedule, concomitant administration of gamma-vinyl-GABA failed to reduce the severity of the convulsions observed during the abstinence syndrome. These results suggest that valproate is metabolized to a compound responsible for decreased analgesia and intensified withdrawal signs.     

Calcium channel blockers: effect on morphine-induced hypermotility

Acute morphine treatment has been shown to cause a uniform calcium depletion in various brain regions and to evoke hypermotility in mice. On the other hand, it has been reported previously that calcium channel blockers reduce the behavioral stimulation induced by different methods in mice, and it is known that these drugs increase the morphine analgesia and reduce the abstinence syndrome. The effect of calcium channel blockers, nifedipine and diltiazem, on the morphine- and amphetamine-induced hypermotility were evaluated. Mice activity was measured with photocell motility meters. The results show that neither nifedipine nor diltiazem decrease significantly the motility in control and amphetamine-treated mice; however, when they were administered to morphine-treated mice the hypermotility was significantly reduced. The mechanism responsible for this interference is still unknown.     

Effect of agmatine on long-term potentiation in morphine-treated rats

Agmatine is an endogenous amine derived from l-arginine that potentiates morphine analgesia and inhibits naloxone precipitated abstinent symptoms in morphine dependent rats. In this study, the effects of agmatine on long-term potentiation (LTP) in the lateral perforant path (LPP)-granule cell synapse of the rat dentate gyrus (DG) on saline or morphine-treated rats were investigated. Population spikes (PS), evoked by stimulation of the LPP, was recorded from DG region. Acute agmatine (2.5-10 mg/kg, s.c.) treatment facilitated hippocampal LTP. Acute morphine (30 mg/kg, s.c.) treatment significantly attenuated hippocampal LTP and agmatine (10 mg/kg, s.c.) restored the amplitude of PS that was attenuated by morphine. Chronic morphine treatment resulted in the enhancement of hippocampal LTP, agmatine co-administered with morphine significantly attenuated the enhancement of morphine on hippocampal LTP. Imidazoline receptor antagonist idazoxan (5 mg/kg, i.p.) reversed the effect of agmatine. These results suggest that agmatine attenuated the effect of morphine on hippocampal LTP, possibly through activation of imidazoline receptor.     

Morphine-like physical dependence: a pharmacologic method for drug assessment using the rat

Rats maintained on various dosage regimens of morphine showed dose related taste aversions to a saccharin solution offered to them upon withdrawal from the drug. Maximal saccharin aversions occurred between 72 and 96 hr after termination of morphine injections, and gradually returned to baseline preference levels after 14 days of morphine abstinence. The results were interpreted as suggesting that the morphine treated animals associated the aversive components of the morphine withdrawal syndrome with saccharin consumption, and manifested conditioned aversions to the saccharin solution which extinguished as the withdrawal syndrome subsided. The findings were further discussed with regard to the potential use of conditioned taste aversions in determining whether test compounds are capable of causing physical dependence in rats.     

Acetylcholine release from the brain of unanaesthetized cats following habituation to morphine and during precipitation of the abstinence syndrome

The rate of release of central acetylcholine (ACh) was determined in unanaesthetized cats by perfusing the sensorimotor cortex or the lateral ventricles with a neostigmine-containing solution by means of push-pull cannulae.Cats were made physically dependent on morphine over a 10-day schedule of daily increasing doses of the narcotic. No difference in both cortical and ventricular ACh release was noted between these morphine-dependent animals (determined following the last dose of morphine-20 mg/kg i.p.) and the same animals prior to the injection of any morphine. When the abstinence syndrome was precipitated in these habituated animals (0.2 mg/kg of naloxone, i.p.), both cortical and ventricular ACh release was enhanced, coincident with behavioural agitation of the animal. While the behavioural symptoms of the abstinence syndrome persisted, this increased release of ACh was only of short duration.In non-dependent cats, a 1-h localized perfusion of the sensorimotor cortex with a morphine-containing solution (10^–4 or 5×10^–4 g/ml) had no effect on either cortical ACh release or the behaviour of the animal.These results raise questions concerning the hypothesis that a cholinergic mechanism governs the occurrence of the morphine abstinence syndrome in cats.     

Effects of methylnaltrexone on guinea pig gastrointestinal motility

The purpose of the present study was to compare the effects of methylnaltrexone (MNTX), a peripherally acting μ opioid receptor (μOR) antagonist, on gastrointestinal (GI) motility in naïve vs. opiate chronically treated guinea pigs in vitro and in vivo. We have used the electrically stimulated muscle twitch contractions of longitudinal muscle-myenteric plexus (LMMP) preparations and total GI transit as measure of GI motility. In LMMP preparations of naïve guinea pigs, MNTX (1–30 μM) induced a significant, dose–response reduction of morphine-induced inhibition of electrically stimulated muscle twitch contractions, with an IC₅₀ of 9.4 10^?8M. By contrast, MNTX abolished the inhibitory effect of acute morphine at any concentrations tested (1–30 μM) in the guinea pigs chronically treated with opiates. In vivo, MNTX (10–50 mg s.c.) did not affect GI transit in naïve guinea pigs when administered acutely or for five consecutive days, but reversed the GI transit delay induced by chronic morphine treatment. These findings show that MNTX is effective in reversing opiate-induced inhibition of GI motility acting at peripheral μ opioid receptors, but does not exert a pharmacologic effect on GI transit in the absence of opiate stimulation.     

Conditioned place preference: no tolerance to the rewarding properties of morphine

The effect of repeated morphine administration on conditioned place preference (CPP) using a novel treatment schedule, i.e., drug treatment was always contingent with the conditioned environmental stimuli, was investigated. We also examined whether changes in the μ- and κ-opioid receptor binding occurred in the brain of morphine-treated animals. Intraperitoneal (i.p.) administration of morphine (2 and 10 mg/kg) induced a place preference after 8 daily conditioning trials (4 morphine injections on alternate trials), the level of preference being the same with the two doses of the opiate. No change in place preference was observed in the morphine-treated rats at 2 mg/kg, when animals were further trained up to a total of 32 conditioning trials (16 morphine injections). Conversely, after 20 conditioning trials (10 morphine injections), a stronger CPP response developed in the morphine-treated rats at 10 mg/kg. Signs of morphine withdrawal were never detected in morphine-treated rats during the experiment. Loss of body weight (index of opiate dependence) was not observed either 24 h or 48 h after the last morphine administration. μ- and κ-opioid receptor density and affinity were not affected by repeated morphine administrations at either dose. The results demonstrate that no tolerance develops to the rewarding properties of morphine. Indeed, a sensitisation effect may occur at increasing doses of the opiate. Furthermore, changes in the rewarding effect of morphine are not dependent upon alterations in opioid receptors involved in the reinforcing mechanisms. Received: 31 October 1996 / Accepted: 7 February 1997     

The Effects of Maternally–administered Morphine on Rat Foetal Development and Resultant Tolerance to the Analgesic Effect of Morphine

Groups of 6–9 pregnant (sperm = Day 1) Sprague-Dawley (Simonsen) rats were injected with morphine sulphate, 20 mg/kg subcutaneously, on gestational days 2–5, 7–9 or 11–13. Pups delivered by Caesarian section on gestational day 22 were essentially free of any observable teratogenic changes including bone defects. However, offspring of morphine-treated rats (20 mg/kg/day subcutaneously on gestational days 17, 18, 19 and 20) which were allowed to litter normally exhibited on day 12–13 a significantly (P < 0.05) decreased response to a nocioceptive (55° surface) stimulus 30 min. following an injection of morphine sulphate 0.45 mg/kg subcutaneously (offspring of morphine-treated mothers, 16.8 sec. ± 2.3 S.E.M. versus control offspring, 25.2 sec. ± 1.7 S.E.M.). On days 21–22 of age larger doses of morphine were required to elicit an analgesic response in the young. Following an injection of morphine sulphate, 10 mg/kg subcutaneously, the maternal animals also showed significant reduction in reaction time 30 minutes later (treated, 7.6 sec. ± 0.6 S.E.M. versus control, 16.1 sec. ± 0.6 S.E.M.) and 60 minutes later (treated, 7.5 sec. ± 0.9 S.E.M. versus control, 20.8 sec. ± 2.9 S.E.M.) as compared to paired waterinjected control pregnant animals.     

Di-n-propylacetate-induced abstinence behaviour as a possible correlate of increased GABA-ergic activity in the rat

Administration of di-n-propylacetate (DPA), an inhibitor of SSA-dehydrogenase, produces in naive rats abstinence behaviour which can be blocked by morphine and bicuculline and may be useful as a behavioural correlate of increased GABA-ergic activity. The usefulness of this model has been demonstrated by studying the effec of bicuculline, picrotoxin, strychnine, morphine, aminooxyacetic acid, 3-mercaptopropionate and thiosemicarbazide on DPA-induced abstinence behaviour. Behaviour was suppressed both by bicuculline or picrotoxin, while the selective glycine antagonist strychnine was ineffective. A comparable syndrome could not be evoked by treatment with aminooxyacetic acid, a GABA-transaminase inhibitor, indicating that the effect of DPA was not caused by inhibition of this enzyme. Instead, aminooxyacetic acid suppressed the DPA-induced abstinence behaviour, suggesting that two GABA-ergic systems with opposite effects on behaviour can be distinguished. The syndrome was also suppressed by convulsant doses of 3-mercaptopropionate, while thiosemicarbazide was ineffective. Abstinence behaviour was further suppressed by morphine with an ED₅₀ of 0.5 mg/kg and this action could be clearly separated from its depressant effect on locomotor activity in non-treated animals. These results suggest that morphine receptors may be involved in DPA-induced abstinence behaviour. Based on these experiments a model has been proposed for GABA-ergic terminals being under the inhibitory influence of GABA-ergic autoreceptors. It is proposed that DPA-induced abstinence behaviour may be useful as a model of increased GABA-ergic activity to aid study of the regulation and properties of the GABA-ergic system in vivo.     

Effect of morphine on the brain histamine levels in stress-exposed rats

The effect of morphine on brain histamine levels in stress-exposed rats was investigated. It seemed that the brain histamine level was not directly affected by stressors. However, morphine induced a sharp rise in the brain histamine levels in both morphine-treated and morphine-treated stressed groups. This effect of morphine was reversed by naloxone.     

Comparison of abstinence syndromes following chronic administration of mu and kappa opioid agonists in the rat

Abstinent states were compared following chronic administration of mu and kappa opioid agonists, morphine and ethylketocyclazocine, respectively. Rats were prepared with chronic EEG and EMG electrodes and indwelling IV cannulae. One group of rats was chronically administered IV morphine, while a second group received chronic injections of IV ethylketocyclazocine. Morphine abstinence was associated with suppression of REM sleep occurrences, increases in number of wet-dog shakes, and a decline in EEG spectral power during slow-wave sleep episodes. In contrast, the ethylketocyclazocine abstinence syndrome included minor abstinence signs. Differences in abstinent states between morphine and ethylketocyclazocine indicate the involvement of separate receptor populations in the process of dependence on morphine and ethylketocyclazocine.     

Similarities and differences between d-ALA2 MET5 enkephalin amide and morphine in the induction of tolerance to their effects on catalepsy and on dopamine metabolism in the rat brain

Summary Rats were made tolerant to morphine or to DALA, a synthetic analogue of met-enkephalin, by prolonged exposure to these compounds. Tolerance was assessed by evaluating the resistance of the treated rats to present catalepsy after an acute dose of the opiates. Both morphine and DALA induced tolerance and cross-tolerance to the cataleptic effect. Acute administration of morphine and DALA increased the concentration of DOPAC in striatum, limbic area and s.nigra of control rats. This increase was not present when morphine was given acutely to chronically morphine-treated rats, indicating that these animals were tolerant to this effect. Chronically morphine-treated rats given DALA presented partial tolerance to the biochemical effect of the peptide in limbic area and in s.nigra but not in striatum, indicating that only in certain areas was crosstolerance produced by chronic morphine. When DALA was administered at different doses to chronically DALA treated rats, the peptide induced rise in DA catabolite was similar to that produced in control animals, so clearly there was no tolerance to this biochemical effect. In these animals cross tolerance to morphine's effect on DA metabolism was present in s.nigra but not in the other two areas, indicating that s.nigra is particularly sensitive to opiate-induced tolerance on DA metabolism.Supported by CNR-ROME Grant no. CT81.00258.04     

Long-term effects of morphine on the electroencephalogram and behavior of the rat

Adult female Sprague-Dawley rats were given 10 mg/kg i.p. injections of morphine sulfate twice weekly, at 84-h intervals, over a period of three weeks. A control group of rats was simultaneously treated with equivalent volumes of isotonic saline. The animals were then prepared with cortical and temporalis muscle electrodes. Ten days after the last injection of morphine or of saline, they were placed in individual cages for recording of the EEG and the EMG and both groups were given i.p. a 10 mg/kg test dose of morphine. In the saline-treated rats, high voltage EEG slow bursts in association with stuporous behavior appeared almost immediately after injection and prevailed for 60–90 min. This phase was followed by continuous EEG and behavioral arousal for another period of 60–90 min, after which sleep appeared. Administration of the 10 mg/kg test dose of morphine to the rats having prior morphine exposure resulted in a much shorter initial period of EEG and behavioral stupor and a longer secondary phase of EEG and behavioral arousal. The duration of the entire morphine effect as determined by the latency to sleep onset, however, was the same in the saline-treated and morphine-treated groups of rats. These results support the assumption that long-term alterations in the function of the CNS occur not only after morphine addiction, but also after only exposure to morphine.Supported by NIMH Grant MH 16 693.     

Intestinal effects of 5-hydroxytryptamine and morphine in guinea pigs, dogs, cats and monkeys

Intestinal introluminal pressure was measured in vivo in anesthetized guinea pigs, dogs, cats ad monkeys. In guinea pigs, but not in the other species, the intestinal stimulatory effect of 5-hydroxytryptamine was antagonized by morphine. The 5-HT-blocking action of morphine in intestine seems to be unique to the guinea pig.     

Sensitivity of morphine-tolerant rats to muscarinic and dopaminergic agonists: Relation to tolerance or withdrawal

Male hooded Wistar rats were treated chronically once daily with morphine sulfate (20 mg/kg SC) after completing a session in an operant chamber. Periodical challenges with morphine (8 mg/kg) prior to the operant session established that tolerance development was virtually complete within 10 days. The morphine-treated rats were more sensitive to the behavioral suppressing effects of apomorphine, a dopamine agonist, and pilocarpine, an acetylcholine agonist, when administered in the tolerant state (morphine given 30 min prior to operant session), regardless of whether they were administered peripherally or directly into the striatum. Conversely, the morphine-treated rats were less sensitive to both agonists when administered in the withdrawal state (morphine given 24 h prior to the operant session). In animals undergoing a similar regimen of chronic morphine treatment, receptor binding studies revealed a lowered affinity (Higher K _D apparent) for the dopamine receptor in the striatum of morphine-withdrawn rats, using ³H-spiroperidol as the ligand. The morphine-withdrawn rats also appeared to have fewer muscarinic cholinergic receptors (lower B _max), using ³H-QNB as the ligand. The also had a lower concentration of membrane-bound phosphodiesterase modulator protein. In general, no significant differences were observed for the above parameters in the morphine-tolerant rats. These behavioral and neurochemical studies are consistent with the view that morphine-tolerant rats are supersensitive to dopamine and acetylcholine agonists and morphine-withdrawn rats are subsensitive.