Previous anesthesia can temporarily overshadow the expression of a withdrawal syndrome in opiate dependent rats

We hypothesized that induction of opiate antagonist-precipitated withdrawal under anesthesia can decrease the expression of later withdrawal signs. Three groups of morphine-dependent rats were compared in different experimental conditions of withdrawal precipitation using naloxone. We showed that anesthesia can temporarily overshadow the expression of withdrawal signs, but that some signs can be delayed and increased in intensity. This can be explained by a parallel and temporary effect of anesthesia on arousal and pain threshold. This carries important implications on the use of anesthesia in detoxification procedures.     

Suppression of opiate withdrawal by cyclosporin A and dietary modification

It has been demonstrated in a murine model that a defined diet (Purina Basal Diet 5755) has immunosuppressive effects similar to cyclosporin A (CsA). It was also shown that CsA treatment in opiate dependent rats can attenuate the severity of opiate withdrawal. In this study, an opiate dependence model was established in Balb/c mice to assess the effects of the 5755 diet and CsA on morphine withdrawal - a CNS mediated phenomenon. Three groups of mice were used; a chow-fed control group (Purina 5008), a chow fed CsA treated group, and a group maintained on the 5755 diet. Morphine dependence was established by subcutaneous implantation of a 100 mg morphine base pellet under ether anesthesia. Seventy-two hours after pellet implantation, withdrawal was precipitated by a single injection of the opiate antagonist naloxone (2 mg/kg ip). Two indicators of withdrawal were assessed; jumping and diarrhea. The data demonstrated that both CsA and the 5755 diet resulted in significant attenuation of withdrawal symptoms with the 5755 diet being the most effective of the two. These findings suggest that immune modulation elicited by the 5755 diet and CsA treatment has a direct impact on the CNS opioid function.     

Effects of anaesthetic agents in interference of naloxone-induced opiate-withdrawal are dose-dependent in opiate-dependent rats

In opiate-dependent rats previous studies showed that anaesthetic agents, such as chloral hydrate, midazolam and ketamine interfere with naloxone-precipitated opiate withdrawal. Each anaesthetic induces a specific pattern of interference, indicating that the interference is agent-dependent. In order to further investigate these effects and highlight a potential pharmacological basis of opiate withdrawal interference through anaesthetic agents, we hypothesized that anaesthetic-mediated interference of opiate withdrawal is also dose-dependent. Three groups of rats were compared in an experimental procedure of rapid withdrawal induction by an antagonist under anaesthesia using sub-anaesthetic dosage of midazolam, ketamine or saline. We observed that sub-anaesthetic dosage of ketamine, or midazolam, interferes significantly with opiate withdrawal expression. This brings arguments in favour of a pharmacological basis underlying rapid antagonists induction in opiate dependent rats.     

Role of corticotropin-releasing factor receptor-1 in opiate withdrawal

Previous studies indicate that corticotropin-releasing factor (CRF) contributes to the anxiety-like and aversive states associated with drug-induced withdrawal. The present study extends this work by analyzing the CRF receptor subtype involved in withdrawal responses. First, the influence of a selective CRF receptor-1 (CRF-R1) antagonist, CP-154,526, on opiate withdrawal behavior was examined. Pretreatment with the CRF-R1 antagonist significantly attenuated several behavioral signs of naltrexone-induced morphine withdrawal, including writhing, chewing, weight loss, lacrimation, salivation, and irritability, measured during the first hour of withdrawal. Next the expression of CRF-R1 was determined as a second measure of the involvement of this receptor in opiate withdrawal. Naltrexone-induced morphine withdrawal resulted in down-regulation of CRF-R1 mRNA in several brain regions, including the frontal cortex, parietal cortex, striatum, nucleus accumbens, and amygdala, but not in the hypothalamus or periaqueductal gray. Expression of CRF-R2, the other major CRF receptor subtype, was not down-regulated significantly by withdrawal in any of the regions examined, although morphine alone significantly increased levels of this receptor subtype. Taken together, the behavioral and receptor regulation findings indicate that CRF-R1 is the primary mediator of the actions of the CRF system on opiate withdrawal, although it is possible that CRF-R2 contributes to the response.     

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.     

Receptor binding, antagonist, and withdrawal precipitating properties of opiate antagonists

A number of opiate antagonists and the dextro isomers of some of these drugs were studied for antagonism of acute opiate effects on ilea isolated from opiate-naive guinea pigs, precipitation of a withdrawal contraction of ilea isolated from morphine-dependent guinea pigs, precipitation of withdrawal in morphine-dependent rhesus monkeys and stereospecific displacement of 3H-etorphine binding to rat-brain membranes. With the exception of d-naloxone, all of the compounds displaced 3H-etorphine. With the exception of d-naloxone, nalorphine, and quaternary nalorphine, all of the antagonists caused a contraction of ilea isolated from morphine-dependent guinea pigs. Moreover, the IC 50 values of the compounds for displacing 3H-etorphine binding were well correlated with both their Ke values for antagonism in the ileum (r = 0.95) and with their EC 50 values for precipitating a contraction in this preparation (r = 0.92). Generally, the concentration of antagonist necessary to precipitate half maximal contracture was 30-fold greater than the Ke value of the antagonist. Most of the opiate antagonists also precipitated withdrawal when administered to morphine-dependent rhesus monkeys and their in vivo potencies were well correlated with their in vitro potencies in ileum (with Ke: r = 0.95; with EC 50: r = 0.99) and in displacing 3H-etorphine (r = 0.95). The quaternary derivative of naltrexone, however, was an effective opiate antagonist only in vitro, and was ineffective in precipitating withdrawal in morphine-dependent rhesus monkeys. These results suggest that the receptor sites labeled by 3H-etorphine are the same as those involved in antagonism of acute opiate actions and in precipitation of withdrawal.     

Opiate withdrawal induces dynamic expressions of AMPA receptors and its regulatory molecule CaMKIIalpha in hippocampal synapses

Adaptive changes in brain areas following drug withdrawal are believed to contribute to drug seeking and relapse. Cocaine withdrawal alters the expression of GluR1 and GluR2/3 subunits of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors in nucleus accumbens or amygdala, but the influence of drug withdrawal on hippocampus is little known. Here, we have examined the expression of GluR1 and GluR2/3 in hippocampal membrane and synaptic fractions following repeated morphine exposure and subsequent withdrawal. Repeated morphine exposure for 12 d increased GluR1 and GluR2/3 in synaptosome but not in membrane fraction. Interestingly, CaMKIIalpha, known to be able to regulate the function of AMPA receptors, was decreased in synaptosome but not in membrane fraction; pCaMKIIalpha, the phosphorylated form of CaMKIIalpha, was increased in both fractions. However, during opiate withdrawal, GluR1 was generally reduced while GluR2/3 was prominently increased in both fractions; pCaMKIIalpha was strongly decreased immediately after withdrawal, but detectably increased in late phase of morphine withdrawal in both fractions. Importantly, the opiate withdrawal-induced increase in GluR2/3 was dependent on the activation of glucocorticoid receptors and NMDA receptors, as it was prevented by the glucocorticoid receptor antagonist RU38486, or intrahippocampal injection of the NMDA receptor antagonist AP-5 or the antagonist to NR2B-containing NMDA receptors, Ro25-6981. These findings indicate that opiate withdrawal induces dynamic expression of GluR1 and GluR2/3 subunits of AMPA receptors in hippocampal synapses, possibly revealing an adaptive process of the hippocampal functions following opiate withdrawal.     

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.     

Neonatal animal models of opiate withdrawal

The symptoms of opiate withdrawal in infants are defined as neonatal abstinence syndrome (NAS). NAS is a significant cause of morbidity in term and preterm infants. Factors, such as polysubstance abuse, inadequate prenatal care, nutritional deprivation, and the biology of the developing central nervous system contribute to the challenge of evaluating and treating opiate-induced alterations in the newborn. Although research on the effects of opiates in neonatal animal models is limited, the data from adult animal models have greatly contributed to understanding and treating opiate tolerance, addiction, and withdrawal in adult humans. Yet the limited neonatal data that are available indicate that the mechanisms involved in these processes in the newborn differ from those in adult animals, and that neonatal models of opiate withdrawal are needed to understand and develop effective treatment regimens for NAS. In this review, the behavioral and neurochemical evidence from the literature is presented and suggests that mechanisms responsible for opiate tolerance, dependence, and withdrawal differ between adult and neonatal models. Also reviewed are studies that have used neonatal rodent models, the authors' preliminary data based on the use of neonatal rat and mouse models of opiate withdrawal, and other neonatal models that have been proposed for the study of neonatal opiate withdrawal.     

Long-term treatment with SR141716A, the CB1 receptor antagonist, influences morphine withdrawal syndrome

The role of the cannabinoid system in morphine withdrawal was examined through long-term CB1 receptor antagonist administration in morphine pellet implanted rats. SR141716A chronic treatment (5mg/kg i.p. twice a day for four days) did not influence the development of tolerance to the morphine analgesic effect but significantly reduced the intensity of naloxone-induced opiate withdrawal in tolerant rats: Specifically there was a significant reduction in the number of digging, teeth chattering and penile licking and the incidence of diarrhoea while other signs such as writhing, head dog shakes and rearing were unaffected. These results suggest that the pharmacological treatment with SR141716A could be of some interest in ameliorating opiate withdrawal syndrome.     

Morphine withdrawal causes subsensitivity of adrenergic receptor response

To determine whether the hyperactivity of noradrenergic neurons that occurs during opiate withdrawal might be associated with a reciprocal alteration in noradrenergic receptor response, isoproterenol-stimulated adenylate cyclase activity was examined in the cerebella of morphine-addicted monkeys at two time periods: a) prior to withdrawal, and b) during late withdrawal. Compared to controls, the chronically-addicted group showed a significant increase in maximal enzyme velocity, a finding consistent with the observed hypoactivity of noradrenergic neurons which occurs during opiate administration. In contrast, the morphine withdrawal group demonstrated a significant decrease in enzyme activity. Piperoxan, known to mimic the effects of withdrawal in causing noradrenergic hyperactivity, also decreased enzyme activity. In no group was there a change in K_a for isoproterenol. These findings, indicating that opiate withdrawal is associated with a subsensitivity of noradrenergic receptor response, suggest a possible etiology for certain of the physiological changes of noradrenergic hypoactivity seen during the secondary (or protracted) abstinence syndrome.     

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.     

Cyclosporine alters opiate withdrawal in rodents

Opiates exert numerous effects on all levels of the central nervous system with tolerance, physical dependence and withdrawal being characteristics of this drug class. The degree of dependence is directly correlated to the intensity of withdrawal. Therefore, success in modifying the withdrawal syndrome may shed light on the dynamics of opiate addiction. The present study demonstrates that cyclosporine, a widely used immunosuppressive drug, considerably modified the behavioral signs of a naloxone-induced abstinence syndrome in morphine-addicted rats. In previous experiments, alpha-interferon has shown similar results. The similarity in actions of these two immunomodulator drugs is discussed and we suggest that opiate addiction may involve the immune system.     

Mitragynine attenuates withdrawal syndrome in morphine-withdrawn zebrafish

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway.     

Effect of precipitated morphine withdrawal on post-translational processing of prothyrotropin releasing hormone (proTRH) in the ventrolateral column of the midbrain periaqueductal gray

We have demonstrated that during opiate withdrawal, preprothyrotropin releasing hormone (preproTRH) mRNA is increased in neurons of the midbrain periaqueductal gray matter (PAG) while the concentration of TRH remained unaltered, suggesting that the processing of proTRH may be different in this region of the brain. The aim of the present study was to determine which of the proTRH-derived peptides are affected by opiate withdrawal in the PAG. These changes were compared to other TRH-containing areas such as the hypothalamic paraventricular nucleus (PVN), median eminence (ME) and the lateral hypothalamus (LH). Control and morphine-treated rats 24 h following naltrexone-precipitated withdrawal were decapitated and the brain microdissected. Pooled samples from each animal group were acid extracted, and peptides were electrophoretically separated then analyzed by specific radioimmunoassay. Opiate withdrawal caused a significant change in the level of some post-translational processing products derived from the TRH precursor. In the PAG, opiate withdrawal resulted in an accumulation of the intervening preproTRH(83-106) peptide from the N-terminal side of the prohormone, while the levels of the C-terminal preproTRH(208-285) peptide were reduced, with no change in preproTRH(25-50) or TRH, itself, as compared to control animals. Immunohistochemical analysis also showed significant increases in cellular preproTRH(83-106) peptide immunolabeling in the PAG. Opiate withdrawal in the lateral hypothalamus, unlike from the PAG, was accompanied by an increase in the concentration of TRH. In addition, western blot analysis showed that during opiate withdrawal, the mature form of the prohormone convertase 2 (PC2) increased only in PAG as compared with their respective controls. Thus, these results demonstrate a region-specific regulation of TRH prohormone processing in the brain, which may engage PC2, further suggesting a role for specific proTRH-derived peptides in the manifestations of opiate withdrawal.     

Naltrexone precipitated opiate withdrawal in methadone addicted human subjects: Evidence for noradrenergic hyperactivity

The noradrenergic alpha-2 receptor agonist, clonidine, suppresses many of the effects of opiate withdrawal in both humans and other animals and this is consistent with the data indicating important interactions of opiate and noradrenergic systems in brain. This evidence supports the hypothesis that central noradrenergic hyperactivity is involved in the expression of major signs and symptoms of the opiate withdrawal syndrome, but to date clinical studies have not provided biochemical data consistent with this idea. In order to assess whether naltrexone precipitated opiate withdrawal in methadone addicted human subjects is associated with changes in noradrenergic function, a double-blind study was completed in which 15 methadone-dependent subjects received naltrexone and 8 subjects received placebo. Signs and symptoms of the opiate abstinence syndrome increased significantly in the 15 subjects who received naltrexone compared with the 8 who received placebo. Plasma concentrations of free MHPG also increased significantly in those subjects who took naltrexone compared to the placebo treated subjects. In addition, withdrawal signs and symptoms were significantly correlated with plasma MHPG concentration. Since several compounds which suppress central noradrenergic activity also reduce the severity of the withdrawal syndrome, the noradrenergic hyperactivity suggested by the present clinical study may be a functional mechanism for at least part of the opiate withdrawal syndrome.     

An analysis of GABA receptor changes in the discrete regions of mouse brain after acute and chronic treatments with morphine

Abstract: The effects of morphine on the affinity and distribution of GABA receptors in the mouse regions (striatum, medulla, diencephalon, cortex, and cerebellum) were investigated in relation to: (a) acute administration, (b) chronic administration (tolerance), (c) precipitated withdrawal by naloxone, an opiate antagonist, and (d) abrupt withdrawal for 8 and 24 h. The alterations in the affinity as reflected by the dissociation constant (K_D) and the number of receptors (B_max) in the synaptic membranes obtained from controls and various treatments were determined by radioligand binding assay using [³H]muscimol as a ligand. Significant changes were observed in striatum, medulla, and diencephalon, whereas other regions including whole brain exhibited marginal changes. In general the number of GABA receptors increased after tolerance development, which upon abrupt withdrawal returned to control levels except in the case of naloxone-induced precipitated withdrawal. The affinity changes in different regions were diverse in nature and were not evident in the whole brain membranes. These results indicate that: (a) the regional alterations in the affinity and distribution of GABA receptors may play a role in the induction, maintenance, and regression of morphine tolerance; (b) abrupt withdrawal and antagonist precipitated withdrawal affect the GABA system differently, (c) chronic morphine treatment appears to influence the GABA receptors in the cerebellum, a region generally known for its lack of opiate receptors.     

Disruption of the CRF/CRF1 receptor stress system exacerbates the somatic signs of opiate withdrawal

Escape from the extremely stressful opiate withdrawal syndrome may motivate opiate seeking and taking. The corticotropin-releasing factor receptor-1 (CRF1) pathway mediates behavioral and endocrine responses to stress. Here, we report that genetic inactivation (CRF1-/-) as well as pharmacological antagonism of the CRF/CRF1 receptor pathway increased and prolonged the somatic expression of opiate withdrawal. Opiate-withdrawn CRF1-/- mice also showed aberrant CRF and dynorphin expression in the paraventricular nucleus of the hypothalamus (PVN) and the striatum, indicating profound impairments in stress-responsive brain circuitry. Intake of nonstressful amounts of corticosterone effectively reduced the exaggerated somatic reactions of CRF1-/- mice to opiate withdrawal. Exogenous corticosterone also restored "wild-type-like" patterns of CRF and dynorphin gene expression in the PVN and the striatum of opiate-withdrawn CRF1-/- mice, respectively. The present findings unravel a key role for the hypothalamus-pituitary-adrenal (HPA) system and brain extra-hypothalamic CRF/CRF1 receptor circuitry in somatic, molecular, and endocrine alterations induced by opiate withdrawal.     

Opiate withdrawal increases ornithine decarboxylase activity which is otherwise unaltered in brains of dependent chicken fetuses

We have used the developing chicken to determine if ornithine decarboxylase (ODC) activity is altered in fetuses chronically exposed to the opiate N-desmethyl-l-alpha-acetylmethadol (NLAAM) or rendered abstinent by acute injection of naloxone (Nx). Exposure to NLAAM from day 3 of embryogenesis did not significantly change brain ODC activity in 15, 17 or 19-day-old fetuses. Acute treatment of 17-day-old fetuses with a motility suppressant dose of NLAAM did not differentially affect ODC activity in NLAAM-dependent fetuses, but an additional treatment with Nx, which precipitated withdrawal, resulted in a significant increase in ODC activity in this group. We conclude that withdrawal can alter fetal ODC activity which otherwise appears normal, even though fetuses have been chronically exposed to and dependent upon an opiate.     

Pharmacological aspects of shaking behavior produced by TRH, AG-3-5, and morphine withdrawal

In virtually all fur-coated and feathered animals, shaking movements of the body, similar to that made by a dog when wet, occur in response to irritation of the skin or in response to sensations of intense cold. Vigorous shaking movements occur in rats undergoing opiate withdrawal. I was led by this observation to investigations on the pharmacology of agents that stimulate or inhibit shaking. Thyrotropin-releasing hormone, injected centrally at submicrogram doses, produced in nondependent, barbiturate-anesthetized animals, shaking behavior identical in its general features to that of morphine withdrawal. AG-3-5 (1-[2-hydroxyphenyl]-4[3-nitrophenyl]-1,2,3,6-tetrahydropyrimidine-2-one), another chemical stimulant of shaking, produced specific sensations of cold in man by a peripheral site of action. In this context, it should be noted that sensations of cold, and the associated emotional discomfort, are conspicuous symptoms of opiate withdrawal in man. Shaking movements elicited by a variety of stimuli were inhibited by central administration of nanomolar doses of drugs that act as agonists on opiate, muscarinic, and alpha-adrenergic receptors. These observations may provide information on a) the identity of substances in brain that, when released, provoke opiate withdrawal signs and symptoms; b) the chemical nature of substances that stimulate peripheral cold receptors; and c) the pharmacologic classification of centrally acting agents that attenuate withdrawal and produce antinociception.