The nucleus paragigantocellularis and opioid withdrawal-like behavior

Participation of the nucleus paragigantocellularis (PGi) in mediation of opioid withdrawal was examined in conscious, unrestrained, non-opioid-dependent rats, using electrical stimulation of the PGi. A characteristic series of behaviors, which resembled those seen during naloxone-precipitated withdrawal from dependence on the opioid agonist, butorphanol, was elicited during 30 min of PGi stimulation. Thus, the behavioral syndrome has been termed opioid withdrawal-like. Simultaneous microdialysis measurement of glutamate within the locus ceruleus indicated a positive correlation between extracellular glutamate concentrations and behavioral responses. Behavioral responses were inhibited by 50% during reverse dialysis perfusion of the locus ceruleus with the glutamate receptor antagonist, kynurenic acid, without any effect on glutamate concentrations. Thus, increases in locus ceruleus glutamate partially mediate opioid withdrawal-like behavior. Intracerebroventricular (i.c.v.) injections of the opioid antagonist, naloxone, or of the mu-selective (beta-funaltrexamine) or the delta-selective (naltrindole) opioid antagonists decreased, but did not abolish, stimulation-induced behavioral responses. Similar i.c.v. injections of the kappa-selective antagonist, nor-binaltorphimine, had no effect on behavioral responses to PGi stimulation. Activation of the PGi by electrical stimulation can elicit behaviors similar to those observed during opioid withdrawal. Moreover, additional levels of complexity are evident in the neuropharmacology of PGi stimulation-induced opioid withdrawal-like behavior.     

Purinoreceptors are involved in the control of acute morphine withdrawal.

The effects exerted by P1 and P2 purinoceptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 purinoceptor agonist, adenosine, was able dose-dependently to reduce morphine withdrawal whereas alpha,beta-methylene ATP (APCPP), a P2 purinoceptor agonist, increased morphine withdrawal. Caffeine, a P1 purinoceptor antagonist, was able significantly and in a concentration dependent manner to increase morphine withdrawal whereas quinidine, a P2 receptor antagonist, reduced it. The results of our experiments indicate that both P1 and P2 purinoceptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the purinergic system and opioid withdrawal.     

Physical dependence on physiologically released endogenous opiates

Mice which had been submitted to a chronic schedule of warm water swimming exhibited a naloxone precipiated withdrawal behaviour which was remarkably similar to that produced in mice following chronic morphine treatment. These results are consistent with the activation of endogenous opiates during swim stress in mice and present the possibility that opiate receptors are activated in a manner analogous to the repeated application of exogenous opiates, producing both tolerance and withdrawal-like behaviour.     

The NMDA antagonist dizocilpine (MK801) attenuates motivational as well as somatic aspects of naloxone precipitated opioid withdrawal.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine has recently been reported to antagonize certain overt withdrawal signs in morphine dependent rats. The purpose of the present study was to reassess this response and examine the effect of this drug in a model presumably reflective of the motivational impact of withdrawal using the place conditioning technique. Rats were made opiate dependent by the subcutaneous implantation of a 75 mg morphine pellet. Three-4 days later withdrawal was precipitated by naloxone 0.5 mg/kg. Dizocilpine (0.1-0.5 mg/kg) attenuated many of the subsequent behaviours elicited by naloxone, notably diarrhoea, mouth movements, paw shakes and ptosis. In a separate group of morphine dependent rats, naloxone (0.05 mg/kg) precipitated withdrawal produced a clear place aversion. This place aversion was blocked by dizocilpine (0.02-0.1 mg/kg) pre-treatment prior to conditioning. Therefore dizocilpine may modify both motivational and somatic aspects of opioid withdrawal.     

An investigation of the role of kappa opiate receptor agonists in the initiation of feeding

A large body of evidence has suggested a role for the endogenous opiates and their receptors in the regulation of appetite. In this study we have examined the relative effects of ketocyclazocine (KC), cyclazocine and ethylketocyclazocine, all putative kappa opiate receptor agonists, and morphine, a putative mu receptor agonist, on food consumption. All the kappa agonists induced feeding when administered at 8 AM as did morphine. KC failed to induce feeding during the nocturnal feeding period (2000 and 0200 hours) and morphine suppressed feeding at these times. KC and morphine suppressed starvation induced feeding when food was made available immediately after injection and had no effect when food was presented 2 and 4 hours after injection. High doses of naloxone (5 mg/kg) suppressed KC induced feeding while actually enhancing high dose morphine (25 mg/kg) induced feeding. Repeated injections of KC or morphine for 5 days resulted in enhancement of the feeding response with initiation of feeding occuring earlier. Taken together with the studies showing that the endogenous kappa ligand, dynorphin, enhabces feeding the most parsimonious interpretation of these studies is that kappa agonists are endogenous initiators of feeding and that kappa receptors are maximally saturated at times of food deprivation and during spontaneous feeding. The mu (or one of the other) opiate receptors inhibit feeding due to their sedative effect and antagonism of this effect leads to enhancement of the feeding response. It is postulated that kappa opiate receptors represent an important component of the natural feeding drive.     

Adenosine receptors are involved in the control of acute naloxone-precipitated withdrawal: in vitro evidence

The effects exerted by adenosine A1 and A2 receptor agonists and antagonists on the acute opiate withdrawal induced by morphine were investigated in vitro. Following a 4 min in vitro exposure to morphine, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone. The P1 adenosine receptor agonist, adenosine, was able to reduce dose-dependently naloxone-precipitaded withdrawal. The same effect was induced by the adenosine A1 receptor agonist, N6-Cyclopentyladenosine (CPA) whereas the selective adenosine A2A receptor agonist CGS 21680 increased the naloxone-precipitated withdrawal phenomenon. Dipyridamole, a blocker of adenosine reuptake, induced a significant reduction of morphine dependence. Caffeine, an adenosine receptor antagonist, significantly increased the naloxone-precipitated withdrawal effect in a concentration dependent manner. The same effect was observed with 8-phenyltheophylline (8PT), an A1 adenosine receptor antagonist, whereas 3,7-dimethyl-1-propargylxanthine (DMPX), an A2 adenosine receptor antagonist, reduced the naloxone-precipitated withdrawal phenomenon. The results of our experiments indicate that both A1 and A2 adenosine receptor agonists and antagonists are able to influence opiate withdrawal in vitro, suggesting an important functional interaction between the adenosine receptors and opioid withdrawal.     

Effect of the opiate antagonist naloxone on the electrical activity of identified neurons in the edible snail

The opiate antagonist naloxone modifies the electric activity of some identified neurons of the Helix lucorum which have not been preliminary exposed to the effect of exogenous opioids. Some neurons are excited while others are inhibited by naloxone, and in both cases the reaction may have both a short and long latent period. The reactions depend on naloxone dose and become less expressed or are blocked when naloxone is administered together with the agonists of opiate receptor (morphine, D-Ala2, D-Leu5-enkephalin, bremazocine and beta-endorphin). Opioids alone do not produce any effect on neurons. The effect of naloxone on neurons is assumed to be a result of the elimination by the opiate antagonist of the tonic effect of endogenous opioids by their replacing on opiate receptors which are constantly stimulated by endogenous ligands.     

Ketazocines and morphine: effects on gastrointestinal transit after central and peripheral administration

The mu agonist, morphine, and the prototype kappa agonists, ketocyclazocine and ethylketocyclazocine (EK), were studied for their effects on gastrointestinal transit. Following s.c. administration, both morphine (0.3-3 mg/kg) and ketocyclazocine (0.3-10 mg/kg) antagonized transit of an opaque marker through the small intestines of mice. Morphine (0.1-1 microgram) was also effective after intracerebroventricular (icv) administration in mice whereas ketocyclazocine (0.3-30 micrograms) was not. Similarly, while both morphine (0.3-5 mg/kg) and EK (0.6-10 mg/kg) slowed transit after s.c. injection to rats, only morphine (1-10 micrograms), but not EK (0.3-300 micrograms), was active following icv administration. Icv infusion of the mu benzomorphan, phenazocine (10-100 micrograms), slowed transit in a dose-related manner. These results indicate that there may be an anatomically distinct distribution of receptors for benzomorphan kappa agonists in both the mouse and rat, with these opiate receptors not being located near the lateral cerebral ventricles. The difference in efficacy between morphine and ketazocines in slowing gastrointestinal transit after icv administration to rodents suggests that (a) inactivity in this endpoint is a characteristic of benzomorphan kappa compounds and (b) the model may serve as a useful screen when establishing in vivo profiles of kappa agonists in mice and rats.     

Is morphine dependence mediated exclusively by the Mu receptor?

Regional cerebral glucose utilization (RCGU) and behavior during naloxone precipitated withdrawal were studied in rats made dependent on either sufentanil, a highly selective mu opiate agonist, or morphine. Changes in RCGU during withdrawal in 23 of 24 anatomically related limbic and brainstem structures were indistinguishable between rats dependent on morphine or sufentanil. Linear regression analysis indicated that RCGU changes during withdrawal in these two groups were highly correlated (r=0.95). Withdrawal behaviors (autonomic signs of withdrawal, jumps, weight loss, and incidence of diarrhea) were likewise indistinguishable between the two groups. The correlations of both these measurements of dependence suggest that chronic occupation of mu opiate receptors alone is sufficient to produce dependence that is indistinguishable from morphine dependence.     

Long-term enhancement of morphine hypalgesia as a result of periodic blockade of opiate receptors using naloxone

A significant enhancement of the analgetic effect of morphine (6 mg/kg, subcutaneously; tail withdrawal reflex at 60 degrees C) was observed in rats 3-4 hours after single naloxone (1 mg/kg) administration. Periodical naloxone injection (0.5 mg/kg, subcutaneously, 3 times per day at 3.5-hour intervals for 3 days) led to a prominent and long-term (testing on the 20th and 105th hour after the last naloxone administration) enhancement of morphine analgesia (2.6 mg/kg subcutaneously) and insignificant inhibition of stress analgesia during two-hour immobilization of animals. These modifications of morphine and stress analgetic effects are considered a result of adaptive changes of opiate receptors after their blockade.     

Participation of lymphoid cells in the withdrawal syndrome of opiate dependent rats

Treatment of rats with 500 Rads whole-body ionizing irradiation prior to chronic administration of morphine reduced the severity of the naloxone induced withdrawal signs. In contrast, adoptive transfer of 2-6 X 10(8) lymphoid cells to irradiated rats prior to chronic morphine treatment completely restored the ability to manifest the withdrawal signs precipitated by naloxone. These observations offer the possibility that the immune system participates in opiate addiction.     

Magnetic fields inhibit opioid-mediated ‘analgesic’ behaviours of the terrestrial snail,Cepaea nemoralis

1. The terrestrial snail, Cepaea nemoralis, when placed on a warmed surface (40 degrees C) displays a thermal avoidance behaviour that entails an elevation of the anterior portion of the fully extended foot. The latency of this nociceptive response was increased by the prototypical mu and specific kappa opiate agonists, morphine and U-50, 488H, respectively, in a manner indicative of anti-nociception and the induction of 'analgesia'. Pretreatment with the prototypical opiate antagonist, naloxone, blocked the morphine- and reduced the U-50, 488H-induced analgesia. Naloxone had no effects on the thermal response latencies of saline treated animals. 2. Exposure to either cold (7 degrees C) or warm (38 degrees C) temperature stress increased the nociceptive thresholds of Cepaea in a manner indicative of the induction of 'stress-induced analgesia'. The warm stress-induced analgesia was opioid mediated, being blocked by naloxone, whereas, the cold stress-induced analgesia was insensitive to naloxone. 3. Exposure for 15-30 min to 0.5 Hz weak rotating magnetic fields (1.5-8.0 G) significantly reduced the analgesic effects of the mu and kappa opiate agonists in a manner similar to that observed with naloxone. The magnetic stimuli also inhibited the endogenous opioid mediated warm stress-induced analgesia and significantly reduced the cold stress-induced analgesia. The magnetic stimuli had no evident effects on the nociceptive responses of saline-treated animals. The dihydropyridine (DHP) and non-DHP calcium channel antagonists diltiazem, verapamil. and nifedipine differentially and significantly reduced, while the DHP calcium channel agonist, BAY K8644, significantly enhanced the inhibitory effects of the magnetic fields on morphine-induced analgesia.     

Withdrawal and bidirectional cross-withdrawal responses in rats treated with adenosine agonists and morphine

The aim of this study was to investigate whether the A1/A2 receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the selective A1 agonist, N6-cyclopentyladenosine (CPA), induced physical dependence by quantifying specific antagonist-precipitated withdrawal syndromes in conscious rats. In addition, the presence of bidirectional cross-withdrawal was also investigated. The agonists were administered s.c. to groups of rats at 12 h intervals. Antagonists were administered s.c., 12 hours after the last dose, followed by observation and measurement of faecal output for 20 min. NECA (4 x 0.03 mg kg(-1), s.c) and CPA (4 x 0.03, 0.1 and 0.3 mg kg(-1), s.c.) induced physical dependence, as shown by the expression of a significant withdrawal syndrome when challenged with the adenosine A1/A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX, 0.1 mg kg(-1), s.c.) and the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPDPX, 0.1 mg kg(-1), s.c.) respectively. The syndromes consisted of teeth chattering and shaking behaviours shown to occur in morphine-dependent animals withdrawn with naloxone viz, paw, body and 'wet-dog' shakes, but with the additional behaviours of head shaking and yawning. In further contrast to the opiate withdrawal syndrome, no diarrhoea occurred in the groups of animals treated with adenosine agonists and withdrawn with their respective antagonists. Bidirectional cross-withdrawal syndromes were also revealed when naloxone (3 mg kg(-1), s.c.) was administered to adenosine agonist pre-treated rats and adenosine antagonists were given to morphine pre-treated rats. This study provides further information illustrating that close links exist between the adenosine and opiate systems.     

Presynaptic Dopaminergic Function in the Nucleus Accumbens Following Chronic Opiate Treatment and Precipitated Withdrawal

Naloxone treatment at three days following implantation of pellets containing morphine base increased uptake of tritiated dopamine by the nucleus accumbens but did not alter efflux of tritiated dopamine by the nucleus accumbens or tritiated norepinephrine by the hippocampus. At six days following placement of pellets containing morphine base, withdrawal score was increased after treatment with either saline or naloxone, indicating that animals were undergoing spontaneous opiate withdrawal. Fractional efflux of tritiated dopamine was decreased at this time point following intermittent stimulation with 317 and 1000 M 4-aminopyridine, for striatal slices obtained from animals pretreated with either saline or naloxone. For the nucleus accumbens at six days after placement of morphine pellets, similar decreases in the efflux of tritiated dopamine were only observed in slices obtained from naloxone treated animals. Fractional dopamine efflux was also diminished after in vitro exposure to rising concentrations of 4-aminopyridine, amphetamine, or cocaine for tissue obtained from the nucleus accumbens, but not for slices from the striatum at six days following morphine pellet implantation. In conclusion, deficits in dopamine efflux by the nucleus accumbens occur at a time when animals are undergoing spontaneous opiate withdrawal at six days following morphine pellet implantation, but do not occur at an earlier time point when withdrawal is precipitated by naloxone treatment. These deficits are apparent for brain slices obtained from the striatum or nucleus accumbens after exposure to rising concentrations of different in vitro treatments, with tissue obtained from the nucleus accumbens being more sensitive than the striatum to dopamine efflux produced by a wider range of treatments.     

Receptor-related interactions of opiates with PGE-induced adenylate cyclase in brain

The inhibition by opiates of the PGE2-induced formation of cAMP in slices from rat brain striatum was investigated. A maximal, 3.5-fold increase over the basal level of cAMP was obtained with an EC50 for PGE2 of 3 microM. Opiate agonists of both mu and kappa type were inhibitory. The IC50 values for morphine, levorphanol and ethylketocyclazocine (EKC) were 110 nM, 80 nM and 25 nM, respectively. These values were similar to the potencies of the compounds in displacing stereospecifically bound 3H-etorphine in rat brain membranes. As evidenced by the inactivity of dextrorphan, the inhibition of PGE2-dependent cAMP formation was stereospecific. Also ineffective were the opiate antagonists naloxone, naltrexone and MR 2266. These compounds did, however, reverse the inhibition by agonists, displaying thereby selectivity toward the putative mu and kappa opiates. Thus, the inhibition by morphine was antagonized to a greater degree by naloxone than by MR 2266, and the action of EKC was blocked more effectively by MR 2266 relative to naloxone.     

Catatonic or hypotonic immobility induced in mice by intracerebroventricular injection of mu or kappa opioid receptor agonists as well as enkephalins or inhibitors of their degradation

The intracerebroventricular injections in mice of the mu receptor agonists morphine and fentanyl induced an immobility state (the animals staying motionless with the head down on a 45° inclined plane) which was apparently hypertonic (catatonia ?) or at least enabled them to remain hanging on a horizontal wire with their forepaws. In similar conditions, injections of the kappa receptor agonists ketocyclazocine and bremazocine induced an immobility state which was hypotonic, in contrast with the preceding one. In a similar way to the mu agonists, Met-enkephalin or Leu-enkephalin injected i.c.v. in association with the inhibitor of enkephalinase thiorphan induced an apparently hypertonic immobility which was easily antagonized by naloxone. The association of thiorphan with bestatin ( an inhibitor of aminopeptidases involved in enkephalins inactivation ) produced similar results. In contrast, the hypotonic immobility induced by the kappa receptor agonists required relatively high doses of naloxone to be antagonized. The opiate antagonist MR 2266 antagonized equipotent doses of all the above mentioned agents with a similar efficacy. From these data it is suggested that enkephalins could induce an apparently tonic immobility by stimulating mu receptors and that endogenous enkephalins could be involved in a tonic mediation modulating the locomotor activity or regulating the muscular tone.     

A study of the actions of naloxone and morphine on gastric acid secretion and gastric mucosal damage in the rat

There exists a considerable controversy in the literature with regard to the effect of either opiate receptor blockade or that of morphine in different gastric and intestinal ulcer models in the rat. We performed experiments to evaluate the effects of naloxone and morphine on gastric acid secretion and gastric mucosal damage in different experimental models of gastric mucosal injury, namely in indomethacin-, HCl (0.6N)- and ethanol (96%)-models. We found that: 1) 10 mg/kg naloxone ip given twice, effectively protected gastric mucosa against indomethacin (30 mg/kg ip) and against the acid-dependent injury caused by 0.6 N HCl (1 mL ig), but not against the non acid-dependent injury caused by 96% ethanol (1 mL ig); 2) morphine (10 + 10 mg/kg ip) increased ulcers in the HCl-model, but had no effect in the two other models; 3) this ulcer-aggravating effect of morphine in the HCl-model was blocked by pretreatment of 2 mg/kg ip naloxone; and 4) both naloxone (5 + 5 and 10 + 10 mg/kg ip) significantly decreased gastric acid secretion in 1-h pylorus ligated rats. We conclude that: 1) naloxone dose-dependently protects against the indomethacin- and HCl-, but not against the ethanol-induced gastric mucosal damage; 2) morphine aggravates the HCl-induced ulcerogenesis; and 3) both opiod receptor agonist and antagonist decrease gastric acid secretion.     

Modification of the effects of naloxone in morphine-dependent mice

Mice were rendered dependent on morphine by mixing morphine with their food (2 mg/g) for three days. Increasing doses of naloxone precipitated dose-dependent withdrawal reactions such as weight loss and jumping. These withdrawal reactions were antagonized by morphine pretreatment. Effects of morphine, such as increased locomotor activity, inhibition of intestinal transport, and analgesia were antagonized by naloxone in both non-dependent and dependent subjects. The antagonist actions of naloxone were increased in dependent subjects; lower doses of naloxone were sufficient to antagonize effects of morphine. The present results confirm earlier studies indicating that precipitation of withdrawal can be antagonized by morphine pretreatment suggesting that withdrawal reactions are due to actions of naloxone at the same receptor at which opioid agonists act. The increased antagonist potency of naloxone in dependent subjects extends earlier results obtained with analgesic effects to several other agonist effects of morphine and is consistent with the interpretation that exposure to an opioid agonist induces a change in the conformation of opioid receptors.     

Hyperalgesia during naloxone-precipitated withdrawal from morphine is associated with increased on-cell activity in the rostral ventromedial medulla

Hyperresponsiveness to noxious stimulation (hyperalgesia) is observed with naloxone-precipitated morphine withdrawal in several experimental models, and may be due to changes in central nervous system neurons. Previous studies have demonstrated that certain neurons in the rostral ventromedial medulla (on-cells) discharge just prior to nocifensive withdrawal reflexes and are inhibited by morphine. Because the tail flick latency (TFL) is shorter when on-cells are active, it has been proposed that on-cells facilitate nocifensive reflexes. The present study examined the hypothesis that the hyperalgesia observed following naloxone-precipitated withdrawal from morphine is caused by increased on-cell discharge. Rats were maintained in a lightly anesthetized state with chloral hydrate. Administration of saline (1.25 cc, i.v.) or morphine sulfate (1.25 mg/kg, i.v.) was followed by naloxone (1.0 mg/kg, i.v.). On- and off-cell activity was continuously recorded and was correlated with TFL and paw withdrawal threshold (PWT). As previously reported, morphine increased off-cell activity, blocked on-cell activity, and suppressed the tail flick and paw withdrawal reflexes. When naloxone was given after morphine, TFL and PWT were reduced to values significantly below baseline (hyperalgesia). Both spontaneous and reflex-related on-cell activity increased to levels greater than the premorphine baseline. Spontaneous off-cell activity decreased abruptly to near zero when morphine was followed by naloxone. Linear regression analysis during the hyperresponsive state revealed a significant correlation between increased on-cell activity and reduced TFL, but not between decreased off-cell activity and TFL. These findings are consistent with the hypothesis that on-cells facilitate spinal nocifensive reflexes, and that the naloxone-precipitated hyperalgesia is at least in part accounted for by increased on-cell activity. A neural model of opiate dependence, tolerance, and withdrawal is proposed.     

Dorsal noradrenergic bundle lesions fail to alter opiate withdrawal or suppression of opiate withdrawal by clonidine

Previous work has shown that clonidine effectively supresses many of the signs of opiate withdrawal. The present study was designed to test the hypothesis that the supression of opiate withdrawal by clonidine is mediated by forebrain noradrenergic projections of the locus coeruleus. Two groups of 24 rats each were subjected to either a 6-hydroxydopamine lesion of the dorsal noradrenergic bundle (Lesion group) or a sham, vehicle injection (Sham group). All rats were made dependent on morphine by subcutaneous implantation of one 75 mg silastic morphine pellet for three days followed by 3 more days with two additional 75 mg pellets. Following removal of the morphine pellet, withdrawal was precipitated in all rats by subcutaneous injection of 4 mg/kg of naloxone. Pretreatment 10 min. before withdrawal with clonidine (0.1 or 0.2 mg/kg) produced a significant attenuation of withdrawal signs as compared to saline injected rats; this effect was equally significant in both sham and lesion groups. Lesions of the locus coeruleus had no effect on withdrawal, nor did they affect the ameliorating action of clonidine. These results substantiate the observation that clonidine can effectively attenuate signs of opiate withdrawal in the rat, but fail to support the hypothesis that these effects are mediated by the forebrain projections of the locus coeruleus.