Development of opiate tolerance in the chick embyro

Tolerance to morphine was produced in the chick embryo. Eggs were injected with morphine sulfate (MS) (20 mg/kg egg) or H₂O daily starting on incubation day 12. On day 16, embryo activities were recorded and eggs were injected with either MS or naloxone. Activity of H₂O-pretreated controls decreased after both MS and naloxone. Embryos treated with MS from incubation days 12–15 showed no activity change after morphine and responded to naloxone with increases in activity. Baseline rates of distress vocalizations (DV) of 1–2 day old chicks were not affected by MS pretreatment during incubation days 12–19. However, 1 mg/kg MS decreased the rate of DV of control chicks by 90% whereas MS-pretreated chicks were unaffected. At age 4–5 days, the baseline rate of DV and rate after MS were higher in MS-pretreated chicks. However, all chicks showed significant decreases in rate of DV after MS injection. Naloxone increased the rate of DV of paired 1–2 day old chicks, but response of MS-pretreated chicks was significantly greater than controls.     

Conditioned morphine withdrawal in the hamster

Conditioned withdrawal among golden Syrian hamsters was studied in two experiments. In experiment 1, morphine-pelleted (75 mg) hamsters were observed for signs of withdrawal (wet-dog shakes, etc.) before and after a naloxone (1 mg/kg) injection that was administered in a distinctive environment. Withdrawal signs that occurred in the distinctive environment before the naloxone injection were defined as anticipatory conditioned withdrawal responses. Two, 9, and 30 days following pellet removal, retention of conditioned withdrawal responding was assessed in the distinctive environment before and after a saline injection. Results indicated that: a) withdrawal intensity was a direct function of the number of implanted pellets; b) conditioned withdrawal occurred among animals withdrawn in the distinctive environment, but not among those withdrawn in the home cage; c) conditioned withdrawal was a non-monotonic function of precipitated withdrawal intensity; d) conditioned withdrawal was evident up to 30 days after pellet removal. In experiment 2, conditioned withdrawal was extinguished by repeated exposure to the distinctive environment in the absence of precipitated withdrawal. It was concluded that environmental stimuli associated with theabsence of morphine (i.e., precipitated withdrawal) elicit conditioned withdrawal. The results are compared to similar findings in rats and humans.     

Hyperglycemic suppression of morphine withdrawal signs in the rat

Male Sprague-Dawley rats maintained under controlled lighting and temperature conditions were used in this experiment. Morphine dependency was induced by giving increasing doses of morphine by intraperitoneal injection (IP group) or by the ingestion of morphine through drinking water (PO group). Animals were injected with 10, 20, 30 and 50 mg/kg morphine sulfate at days 1, 2, 3 and 4, respectively. Another group of animals received increasing concentrations of morphine through drinking water from 0.1, 0.2, 0.3 to 0.4 mg/ml at 48 h intervals. Morphine dependent animals were given naloxone by the intraperitoneal route to precipitate withdrawal. Glucose (3 g/kg or 10 g/kg) was given 10 min prior to the administration of naloxone to the respective groups. Another two groups of animals were made diabetic by the administration of streptozotocin. In one group, animals received increasing concentrations of 10, 20, 30 and 50 mg/kg morphine sulfate by the IP route at days 1, 2, 3 and 4, while the other group was not treated with morphine but was assessed for withdrawal signs to serve as the control. Withdrawal signs were assessed by observing the presence of diarrhea, tremor, piloerection, hunchbacked posture, teeth chattering, salivation, erection, restless activity, territorial exploring, irritability to handling, vocalization and jumping. Results obtained indicate that glucose administration at 10 g/kg abolished most of the withdrawal signs, and we were unable to induce the same degree of morphine dependency in diabetic animals as compared to the non-diabetic groups. It was concluded from this study that hyperglycemia could suppress morphine withdrawal signs.     

Clonidine fails to modify dopaminergic neuronal activity during morphine withdrawal

RATIONALE: Cellular substrates of opiate withdrawal syndrome involve several brain areas, in particular the mesolimbic dopaminergic and noradrenergic systems, but the interactions between the two pathways remain unclear. OBJECTIVES: The aim of the present work was to investigate the effects of the alpha2-agonist clonidine on ventral tegmental area dopamine neurons during morphine withdrawal syndrome by recording their neuronal activity before and after the administration of low and relatively high doses of clonidine (from 5 to 100 microg/kg). METHODS: The spontaneous neuronal activity of meso-accumbens dopaminergic neurons, identified by antidromical stimulation from the nucleus accumbens, was recorded by use of in vivo extracellular single-unit recordings in control and morphine-withdrawn rats after chronic administration (15 days). RESULTS: Control rats showed a mean spontaneous firing frequency of 2.47+/-0.48 Hz, percentage of burst firing of 22+/-12 and an increase in firing after the administration of cumulative doses of clonidine (5, 10, 20, 40, 100 microg/kg). Conversely, both spontaneous firing rate (1.55+/-0.25 Hz) and the percentage of burst firing (5+/-2) were found to be significantly reduced in rats abstinent for 24 h, and increasing doses of clonidine did not re-establish electrophysiological activity observed in the controls. CONCLUSION: The results indicate that: 1) clonidine did not restore the decreased firing activity of DA neurons in morphine-withdrawn rats, and 2) high doses of clonidine increased firing in control rats but not in morphine-withdrawn rats.     

Excitatory amino acids and morphine withdrawal: differential effects of central and peripheral kynurenic acid administration

The non-selective excitatory amino acid antagonist kynurenic acid, which does not readily cross the blood-brain barrier, dose-dependently attenuated the behavioral signs of naltrexone-precipitated withdrawal in morphine-dependent rats following both intraventricular and subcutaneous administration. However, intraventricular and subcutaneous administration of kynurenic acid and different effects on individual withdrawal behaviors. Moreover, single unit recordings in anesthetized animals showed that intraventricular, but not subcutaneous, kynurenic acid administration attenuated the withdrawal-induced increased firing of locus coeruleus neurons. These studies indicate that: (1) both central and peripheral excitatory amino acid receptors may play an important role in opiate withdrawal; and (2) excitatory amino acid antagonist treatments might be developed to reduce opiate abstinence symptoms in man.     

The effects of morphine treatment and morphine withdrawal on the dynorphin and enkephalin systems in sprague-dawley rats

The effect of morphine tolerance and withdrawal on prodynorphin peptides was studied in relevant brain areas and in the pituitary gland of male Sprague-Dawley rats, and compared with effects on the proenkephalin-derived peptide Met-enkephalin. After 8 days of morphine injections (twice daily), dynorphin A and B levels increased in the nucleus accumbens and dynorphin A levels increased also in the striatum. Morphine treatment increased striatal Met-enkephalin. Leu-enkephalinArg⁶ levels were reduced in the ventral tegmental area (VTA). Morphine-treated rats had very low Leu-enkephalinArg⁶ levels in the hippocampus as compared to saline control rats. Comparison of the relative amounts of dynorphin peptides and the shorter prodynorphin-derived peptides, Leu-enkephalinArg⁶ and Leu-enkephalin, revealed a relative increase in dynorphin peptides versus shorter fragments in the nucleus accumbens, VTA and hippocampus. Morphine-tolerant rats had lower levels of dynorphin A in both lobes of the pituitary gland, whereas hypothalamic dynorphin levels were unaffected by morphine. Leu-enkephalinArg⁶ levels were reduced in the hypothalamus, but not changed in the pituitary gland. Naloxone-precipitated withdrawal accentuated the increase in dynorphin A and B levels in the accumbens and dynorphin A levels in the striatum, while inducing an increase in enkephalin levels in the accumbens and Met-enkephalin in the VTA. In the hippocampus, Leu-enkephalinArg⁶ levels remained low in the withdrawal state. The low dynorphin levels in the anterior part of the pituitary gland were reversed by naloxone, whereas the low dynorphin A levels in the neurointer-mediate lobe were even lower in the withdrawal state. In conclusion, morphine tolerance and withdrawal affected prodynorphin-derived peptides in areas related to central reward mechanisms, and in the pituitary gland. The dynorphin peptides and the LeuenkephalinArg⁶ fragment were not affected similarly, indicating an effect also on metabolic interconversion.     

Effect of aciclovir on the development of the chick embryo in ovo

The effect of aciclovir (ACV) on embryonic development was investigated using the chick embryo in ovo and treatment during organogenesis. ACV 30–1000 g was applied as single doses prior to or after 24 h of incubation into the yolk sac, and ACV 3–100 g after 2, 3 or 4 days of incubation (DI) directly to the embryo. Data were evaluated after a total of 8 days of incubation. (1) A dose-related increase in the rate of abnormal development was found in the surviving embryos. Depending on the route of drug administration a dose of 300, respectively, 5 g ACV/egg had to be applied to induce 50% abnormal development. (2) Gross structural abnormalities of the surviving embryos mainly concerned the beak and the extremities. With the experimental set-up used a different pattern of abnormalities in the survivors after treatment at various stages could not be observed. The results are compared with data obtained with ACV in rodents in our laboratory. It is suggested that chick embryos are also capable of converting ACV into its triphosphate to interfere with DNA metabolism, probably through a chain break mechanism.Dedicated to Professor Gerhard Zbinden on the occasion of his retirement     

Naloxone-precipitated withdrawal reveals sensitization to neurotransmitters in morphine tolerant/dependent rats

Summary Morphine tolerant/dependent rats were tested for their sensitivity to putative neurotransmitters or other receptor agonists injected intracerebroventricularly (i.c.v.) during naloxone-precipitated withdrawal. Dopamine, apomorphine, clonidine and serotonin were found to reinitiate withdrawal jumping behaviour when injected 30 min after naloxone. Dopamine and apomorphine also reinitiated jumping, but of a lesser intensity, when injected 3 h after naloxone-precipitated withdrawal. I.c.v. injection of acetylcholine or prostaglandin E₁ failed to reinitiate withdrawal jumping. In addition, all the above substances failed to induce jumping behaviour in naive rats or in morphine tolerant/dependent rats before naloxone-precipitated withdrawal. Morphine tolerance and dependence therefore appears to be associated with changes in the sensitivity of the CNS to putative neurotransmitter substances. These changes are best demonstrated during the sudden termination of opiate action that is caused by administration of naloxone.     

Tachykinin antagonists inhibit the morphine withdrawal response in guinea-pigs

Summary This study was an investigation of the effects of the tachykinin antagonists, spantide and (d-Pro⁴, d-Trp^{7, 9, 10})substance P 4-11, injected intracerebroventricularly (ICV), on the locomotor and behavioural responses of guinea-pigs to substance P (SP) injected ICV and to naloxone-induced morphine withdrawal. SP, 50 nmol, produced increased locomotor activity and behaviour that mimicked the response induced by injection of naloxone hydrochloride, 15 mg/kg, in guinea-pigs treated 2 h previously with morphine sulphate, 15 mg/kg. Spantide or (d-Pro⁴, d-Trp^{7, 9, 10})SP4-11, 10 nmol, reduced the locomotor and behavioural responses to SP and to morphine withdrawal. The results support the suggestion that SP or a related tachykinin might be a mediator of the opioid withdrawal response in the central nervous system as has been proposed for the enteric nervous system.     

Influence of prazosin and clonidine on morphine analgesia,tolerance and withdrawal in mice

Rapid development of tolerance and dependence limits the usefulness of morphine in long-term treatment. We examined the effects of clonidine (₂-adrenoceptor agonist) and prazosin (₁-adrenoceptor antagonist) on morphine analgesia, tolerance and withdrawal. Morphine tolerance was induced using a 3-day cumulative twice-daily dosing regimen with s.c. doses up to 120 mg/kg. Tolerance was assessed on day 4, as loss of the antinociceptive effect of a test dose of morphine (5 mg/kg). After 10 h, morphine withdrawal was precipitated with naloxone (1 mg/kg). Prazosin had no analgesic effect alone but dose-dependently potentiated morphine analgesia in morphine-naive mice. Another ₁-adrenoceptor antagonist, corynanthine, had similar effects. Prazosin also increased the analgesic potency of the morphine test dose in morphine-tolerant mice. Naloxone-precipitated vertical jumping was not affected, but weight loss was reduced by prazosin. Acutely administered clonidine potentiated morphine analgesia and alleviated opioid withdrawal signs, as expected. We conclude that in addition to the already established involvement of ₂-adrenoceptors in opioid actions, also ₁-adrenoceptors have significant modulatory role in opioid analgesia and withdrawal.     

Effects of lesions of the ventromedial hypothalamus on naloxone-induced morphine withdrawal in rats

A previous report indicated that lesions of the hypothalamic ventromedial nucleus suppressed autonomic signs of withdrawal from morphine. The present experiment was designed to determine whether such lesions suppressed a second class, skeletal responses of withdrawal, as well. Naloxone-precipitated withdrawal was compared in previously-addicted VMN-lesioned and sham animals, and drugnaive VMN-lesioned and sham animals. The lesion failed to suppress any signs of withdrawal in the addicted lesioned animals as compared with the shams. An attempt was made to explain the discrepancy between these and earlier published results on the basis of differing experimental designs. Implications of these results for possible dissociation of tolerance and dependence effects or possible plasticity of the brain in regard to drug effects were discussed. Further experiments were proposed in which time of lesion is manipulated in relation to the induction of dependence.     

Drugs affecting brain dopamine interfere with the effect of Z-prolyl-D-leucine on morphine withdrawal

The dipeptide Z-prolyl-D-leucine (Z-Pro-D-Leu) has been demonstrated to inhibit the development of tolerance to and dependence on morphine in the mouse. Since the dipeptide affects dopamine (DA) utilization in the terminal regions of the mesolimbic and nigrostriatal DA-ergic projections, the question has been studied of whether DA-ergic mechanisms are involved in the action of Z-Pro-D-Leu on morphine withdrawal. Both inhibition of tyrosine hydroxylase by alpha-methyl-p-tyrosine (alpha-MPT) and inhibition of DA receptors by pimozide interfere with the effect of Z-Pro-D-Leu on naloxone-precipitated morphine withdrawal. Inhibition of serotonin (5-HT) synthesis by DL-p-chlorophenylalanine (PCPA), on the other hand, does not modify the effect of the dipeptide. The results argue for a role of DA-ergic mechanisms in the effect of Z-Pro-D-Leu on the development of morphine dependence.     

Cardiovascular changes during morphine withdrawal in the rat: Effects of clonidine

Arterial blood pressure and heart rate were measured in unrestrained rats as an index of the autonomic component of the morphine withdrawal syndrome. Physical dependence was produced by a constant infusion of morphine at increasing doses over 7 days. Signs of physical dependence observed during abrupt withdrawal included classical behavioral symptoms such as withdrawal body shakes (WBS) and increased autonomic responsiveness which was indicated by a sustained increase in mean arterial pressure (MAP) up to 23 mmHg. Injection of naloxone in morphine dependent rats also evoked a dose-related increase in MAP to about 40 mmHg. The antiwithdrawal effects of clonidine were tested in this model pretreating dependent rats with this agent (6–60 μg/kg). Clonidine inhibited the pressor response produced by naloxone by 23–60%. These findings indicate that the increase in MAP during opiate withdrawal provides an objective and quantitative index of the intensity of the narcotic withdrawal syndrome in dependent rats.     

Effects of clonidine and morphine on opioid withdrawal in rhesus monkeys

Rhesus monkeys undergoing opioid withdrawal either due to withholding morphine administration for 14 h or due to administration of naloxone, were treated with either morphine or clonidine. Morphine eliminated all of the withdrawal signs that developed when morphine was withheld for 14 h. Clonidine also eliminated some but not all signs that developed when morphine was withheld. The frequencies of individual signs prior to drug administration were directly related to the minimal doses necessary to eliminate signs for morphine but not for clonidine. Morphine also eliminated most of the signs precipitated by naloxone, whereas clonidine did not eliminate as many of the naloxone-precipitated signs. Additionally, some of the naloxone-precipitated signs that were eliminated by clonidine were not eliminated by morphine. The present results are consistent with clinical findings indicating an efficacy of clonidine in the treatment of opioid withdrawal through a non-opioid mechanism.     

Anatomical and pharmacological specificity of the rewarding effect elicited by microinjections of morphine into the nucleus accumbens of mice

RATIONALE: The involvement of nucleus accumbens (NAc) in initiating opiate-induced reward has been difficult to demonstrate in rats, and has not been studied in mice. OBJECTIVES: To determine whether a reward-sensitive strain of mice (BALB/c) would self-administer morphine directly into the NAc or sub-regions of the dorsal striatum. METHODS: BALB/c mice were unilaterally implanted with a guide-cannula above either the NAc, the anterior caudate putamen, or the posterior caudate putamen. On each experimental day, a stainless-steel injection cannula was inserted into the guide cannula to test the capacity for morphine self-administration (6.5 pmol or 65 pmol/50 nl) using a spatial discrimination task in a Y maze. RESULTS: Only the ventro-medial NAc group discriminated between the arm enabling a microinjection of morphine and the neutral arm. Once self-administration had been acquired, the effects of a pretreatment with two doses of the opiate antagonist naloxone (0.4 mg/kg or 4 mg/kg) were tested. Both doses slightly disrupted self-administration on the first 2 days. Only subjects receiving the 4-mg/kg dose exhibited an extinction of self-administration, related to an increasing number of jump attempts; none of the other opiate withdrawal-associated signs were observed. Self-administration was reinstated when naloxone was replaced with saline. CONCLUSIONS: (1) Medio-ventral NAc is involved in acute rewarding effects of opiates in mice. (2) Neither anterior nor posterior dorsal striatum seem to participate in these effects. (3) NAc is involved in jumping caused by naloxone-induced extinction, a behavior presumably revealing an aversive state associated with the unexpected suppression of reward.     

Increase of tyrosine hydroxylase levels and activity during morphine withdrawal in the heart

Our previous studies have shown an enhanced activity of the noradrenergic pathways innervating the heart in rats withdrawn from morphine. However, the possible adaptive changes that can occur in these pathways during morphine dependence are not known. We studied the alterations in tyrosine hydroxylase (the rate-limiting enzyme in catecholamines biosynthesis) and tyrosine hydroxylase activity in the heart (right and left ventricle) during morphine withdrawal. In the same paradigm, we measured Fos expression as a marker of neuronal activation and the normetanephrine/noradrenaline ratio (an index of noradrenaline turnover). We evaluated the levels of tyrosine hydroxylase and Fos by quantitative Western blot analysis, and noradrenaline turnover using high-performance liquid chromatography (HPLC). Dependence on morphine was induced by a 7-day s.c. implantation of morphine pellets. Morphine withdrawal was precipitated on day 8 by an injection of naloxone (5 mg/kg s.c.). The results show a significant increase in tyrosine hydroxylase levels and activity in the right and left ventricle 30 or 90 min after naloxone precipitated withdrawal in parallel with an increase in noradrenaline turnover. Morphine withdrawal also induced an increase in the Fos expression, which indicates an activation of cardiac cellular activity. Our results suggest that an increase in tyrosine hydroxylase protein levels and tyrosine hydroxylase enzyme activity might contribute to the enhanced noradrenergic activity in the heart in response to morphine withdrawal.     

Involvement of the midbrain tectum in the unconditioned fear promoted by morphine withdrawal

The midbrain tectum structures, dorsal periaqueductal gray (dPAG) and inferior colliculus (IC), are involved in the organization of fear and anxiety states during the exposure to dangerous stimuli. Since opiate withdrawal is associated with increased anxiety in both humans and animals, this study aimed to investigate the possible sensitization of the neural substrates of fear in the midbrain tectum and its influence on the morphine withdrawal-induced anxiety. For the production of drug withdrawal, rats received morphine injections (10 mg/kg; s.c.) twice daily during 10 days. Forty-eight hours after the interruption of the chronic treatment, independent groups were probed in the elevated plus-maze and open-field tests. Additional groups of animals were implanted with a bipolar electrode into the dPAG or the IC and submitted to the electrical stimulation of these structures for the determination of the freezing and escape thresholds after 48 h of withdrawal. Our results showed that the morphine withdrawal promoted clear-cut levels of anxiety without the somatic signs of opiate withdrawal. Moreover, morphine-withdrawn rats had an increase in the reactivity to the electrical stimulation of the dPAG and the IC. These findings suggest that the increased anxiety induced by morphine withdrawal is associated with the sensitization of the neural substrates of fear in the dPAG and the IC. So, the present results give support to the hypothesis that withdrawal from chronic treatment with morphine leads to fear states possibly engendered by activation of the dPAG and IC, regardless of the production of somatic symptoms.     

Effects of morphine withdrawal on food competition hierarchies and fighting behavior in rats

The effects of chronic morphine administration and morphine withdrawal on competitive and fighting behaviors in rats were studied in a novel paradigm. Fixed-ratio 20 (FR-20) lever-pressing for food presentation was stabilized before placing pairs of rats, matched by response rate and body weight, in the behavioral testing chambers. Initially, overt fighting occurred, but in 60% of the pairs it dissipated within several sessions, accompanied by the development of stable dominant-submissive responding hierarchies. Invariably, the dominant rat in the initial fighting bouts became the dominant responding rat. Chronic morphine administration and subsequent withdrawal in one or both members of the pairs led to disruptions or reversals in the distribution of responding and to small increases in fighting. These effects were seen as early as 12 h after the second dose of morphine and, in some pairs, lasted at least 28 days after morphine was withdrawn. The results from experiments in which only one rat was made dependent show that both the withdrawn dominant rats and the withdrawn submissive rats contributed to the behavioral changes observed in the pairs during withdrawal. Differential food deprivation in pairs not exposed to morphine did not change the dominant-submissive hierarchies. Significant changes in the distribution of FR-20 responding in the paired condition were evident after FR-20 responding in the unpaired condition returned to control rates. It is therefore concluded that change in so-called appetite for food is not an important determinant of the hierarchical changes observed during protracted withdrawal.     

Aggression during morphine withdrawal: Effects of method of withdrawal,fighting experience,and social role

Offensive and defensive components of aggressive behavior were determined in resident and intruder mice. Withdrawal aggression was measured after the removal of a subcutaneous morphine pellet or after precipitation by naloxone in naive mice and after removal of a morphine pellet in mice with prior fighting experience. In naive mice, removal of a morphine pellet led to increases in attack bites and threats but naloxone-precipitated withdrawal led to decreases in these behaviors and to increases in defensive posturing, escape attempts and vocalizations. Prior fighting experience abolished the enhanced attack behaviors of resident mice following morphine pellet removal, but led to heightened defensive behavior in intruder mice. The behavior of intruder mice appeared more sensitive to naloxone administration than the behavior of resident mice; naloxone influenced not only intruder defensive behavior, but also other non-aggressive behaviors. The social role of the drug recipient and his prior history of aggressive behavior are important determinants of morphine and naloxone effects on aggression.     

Morphine withdrawal in mice selectively bred for differential sensitivity to ethanol

Mice which have been selectively bred for differences in sensitivity to acute doses of alcohol have also been shown to differ in severity of seizures upon withdrawal from chronic alcohol administration. We investigated the responsiveness of these mice to withdrawal from chronic morphine treatment. Mice were made dependent on morphine via pellet implantation, and withdrawal was precipitated with naloxone challenge. Mice which are less sensitive to the hypnotic effects of ethanol (short sleep: SS) displayed more jumping and wet dog shakes during withdrawal than did the more senstive long sleep (LS) mice. In addition, the amount of jumping was dependent on the dose of naloxone in both lines. Differences between lines in naloxone precipitated withdrawal may reflect differences in alterations in extrapyramidal dopaminergic activity, but other substrates for the observed differences cannot be discounted. Finally, the observed difference between SS and LS mice in severity of morphine withdrawal parallels the previously reported difference between these lines in seizure severity during withdrawal from alcohol.