Opiate-dependent Rats Research Articles

Chronic co-administration of nalbuphine attenuates the development of opioid dependence

Nalbuphine is an agonist of κ-opioid receptors and a partial agonist of μ-opioid receptors, which can stimulate κ-receptors and antagonize the acute rewarding effects of morphine. It is widely used either as an analgesic or as an adjuvant with morphine. This present study aimed to compare the acute and chronic effects of nalbuphine on the naloxone-precipitated opiate-withdrawal in rats. Male adult Wistar albino rats (150–175 g, n = 160) were made physically dependent by administrating increasing dose of morphine (5–25 mg/kg; i.p.). Motor activity was measured for 25 min at five-minute intervals on days 0, 1, 3, 5, and 6 using Activity Monitor (Coulbourn Instruments, Inc. USA) and True-scan software. The withdrawal was precipitated with intraperitoneal injections of naloxone (1 mg/kg) 4 h after the last injection of morphine. Somatic signs of withdrawal were scored using the global Gellert-Holtzman rating scale. Nalbuphine was co-administered acutely and chronically at various doses (0.1, 0.3, 1.0, and 3.0 mg/kg; i.p.) with morphine. In general, the opiate-dependent rats showed a significant increase in motor activity and Gellert-Holtzman score. Animals co-administered with chronic doses of nalbuphine showed a significant decrease in motor activity and naloxone-precipitated opiate withdrawal, but acute nalbuphine treatment did not attenuate the development of opioid dependence. These findings suggest that nalbuphine could be used as an effective pharmacological adjunct in the treatment of opioid addiction.

OR11-1 * ATTENUATION OF OPIATE WITHDRAWALS BY CO-ADMINISTRATION OF NALBUPHINE IN OPIATE DEPENDENT RATS

Introduction. Nalbuphine is a kappa-opiate agonist and Mu-opiate partial antagonist, stimulates kappa- receptors & antagonizes the acute reinforcing/rewarding effects of morphine and has been widely used as an analgesic or an adjuvant with morphine. The present study evaluated the effect of nalbuphine in opiate dependent rats. Method. Male adult Wistar albino rats (170-175gms, N = 164) were made physically dependent by administrating increasing dose of morphine over a period of 5 days (5-25 mg/kg, b.i.d., i.p.) followed by single dose of morphine on the 6th day. Withdrawals were precipitated with naloxone (1mg/kg, i.p.), 4 hours after last morphine injection. Somatic signs of withdrawals were scored by using the global Gellert-Holtzman rating scale. Nalbuphine was co-administered acutely and chronically in variable doses (0.1, 0.3, 1.0, 3.0 mg/kg, i.p.) with morphine and saline in experimental and control group respectively. Results. Animals pretreated with acute dose of nalbuphine did not produce any effect on the global withdrawal scale, whereas a significant decrease on the global withdrawal scale was observed with chronic co-administration of nalbuphine. Conclusion. These findings suggest that nalbuphine might be useful as pharmacological adjuncts in the treatment of opiate addiction (Supported by Indian Council of Medical Research, Govt. of India, New Delhi).

Asymmetric dimethylarginine (ADMA)—A modulator of nociception in opiate tolerance and addiction?

Nitric oxide (NO) is generated from l-arginine by NO synthases, of which three forms have been identified: endothelial, inducible and neuronal (eNOS, iNOS and nNOS, respectively). The l-arginine metabolite asymmetric dimethylarginine (ADMA) is a potent, noncompetitive inhibitor of nNOS, while its congener N G-monomethyl- l-arginine ( l-NMMA) is a less potent, competitive inhibitor. In rat neurons large amounts of ADMA are found, suggesting its importance in modulating neuronal activity. Humans generate approximately 300 μmol (∼60 mg) ADMA per day. It is released from myelin basic proteins that are highly expressed in neuronal tissue. ADMA is mainly degraded by the action of the enzyme dimethylarginine dimethylaminohydrolase (DDAH), which exists in two isoforms. DDAH1 is highly expressed in brain, suggesting specific function in this area. The presence of nNOS and DDAH1 in brain suggests that ADMA may have specific CNS activity and be more than an unregulated metabolite. Increased NO production—either prior to or concurrently with opioid administration—results in an enhanced rate and extent of development of tolerance to morphine in mice. NO produces an alteration in the μ-opioid receptor that increases constitutive receptor activity. It thereby reduces the ability of a selective μ-opioid agonist to activate the μ-opioid receptor; these in vitro molecular effects occur in a time course consistent with the in vivo development of antinociceptive tolerance in mice. Amongst many other synthetic NOS inhibitors of varying specificity, 7-nitroindazole (7-NI) has been shown to have a high affinity (IC 50 0.71 μM) to nNOS. Selective blockade of nNOS by 7-NI attenuated morphine withdrawal in opiate dependent rats, suggesting nNOS as a viable target for development of pharmacotherapies. We hypothesize that, by inhibiting nNOS and reducing NO levels, ADMA may decrease μ-opiate receptor constitutive activity, resulting in alteration of the analgesic dose–response curve of morphine.

Elevated μ‐opioid receptor expression in the nucleus of the solitary tract accompanies attenuated withdrawal signs after chronic low dose naltrexone in opiate‐dependent rats

We previously described a decrease in withdrawal behaviors in opiate-dependent rats that were chronically treated with very low doses of naltrexone in their drinking water. Attenuated expression of withdrawal behaviors correlated with decreased c-Fos expression and intracellular signal transduction elements [protein kinase A regulatory subunit II (PKA) and phosphorylated cAMP response element binding protein (pCREB)] in brainstem noradrenergic nuclei. In this study, to determine whether similar cellular changes occurred in forebrain nuclei associated with drug reward, expressions of PKA and pCREB were analyzed in the ventral tegmental area, frontal cortex, striatum, and amygdala of opiate-treated rats that received low doses of naltrexone in their drinking water. No significant difference in PKA or pCREB was detected in these regions following drug treatment. To examine further the cellular mechanisms in noradrenergic nuclei that could underlie attenuated withdrawal behaviors following low dose naltrexone administration, the nucleus of the solitary tract (NTS) and locus coeruleus (LC) were examined for opioid receptor (OR) protein expression. Results showed a significant increase in muOR expression in the NTS of morphine-dependent rats that received low doses of naltrexone in their drinking water, and increases in muOR expression were also found to be dose dependent. Protein expression of muOR in the LC and deltaOR in either brain region remained unchanged. In conclusion, our previously reported decreases in c-Fos and PKA expression in the NTS following pretreatment with low doses of naltrexone may be partially explained by a greater inhibition of NTS neurons resulting from increased muOR expression in this region.

A pharmacological modulation of opiate withdrawal using an up-/down-regulation of the noradrenergic system in opiate-dependent rats

Chronic opioid exposure induces neuroadaptative changes in several brain systems. Amongst others the alpha adrenergic system appears to be extremely sensitive to opioid exposure and has, therefore, been proposed to play a key role in opiate withdrawal symptoms. In order to better understand the influence of the noradrenergic system in opioid withdrawal and be able to develop new therapeutic strategies, we studied the effect of pre-treatment with the alpha2 agonist (clonidine) and alpha2 antagonist (yohimbine) on naloxone-precipitated withdrawal in opiate-dependent rats. As is already known clonidine pre-treatment significantly enhances autonomic and behavioural signs of opioid withdrawal whereas yohimbine significantly attenuates them with dose-related effect. We also tested the effect of clonidine (0.1 mg/kg) during naloxone-precipitated opiate withdrawal in rats pre-treated with yohimbine (5 mg/kg) and we observed that yohimbine pre-treatment potentiates clonidine efficiency in decreasing opiate withdrawal signs. This study supports the possibility of using a noradrenergic antagonist in order to regulate adrenoreceptors chronically exposed to opioids, therefore interfering with the intensity of naloxone-precipitated opiate withdrawal and potentiating later effectiveness of noradrenergic agonists like clonidine. These results may have various applications in clinical opiate detoxification protocols and are discussed through an up-/down- regulation of adrenoreceptors.

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.

A specific limbic circuit underlies opiate withdrawal memories.

Compulsive drug-seeking behavior and its renewal in former drug addicts is promoted by several situations, among which reactivation of drug withdrawal memories plays a crucial role. A neural hypothesis is that such memories reactivate the circuits involved in withdrawal itself and promote a motivational state leading to drug seeking or taking. To test this hypothesis, we have analyzed the neural circuits and cell populations recruited when opiate-dependent rats are reexposed to stimuli previously paired with withdrawal (memory retrieval) and compared them with those underlying acute withdrawal during conditioning (memory formation). Using in situ hybridization for c-fos expression, we report here that reexposure to a withdrawal-paired environment induced conditioned c-fos responses in a specific limbic circuit, which can be partially dissociated from the structures involved in acute withdrawal. At the amygdala level, c-fos responses were doubly dissociated between the central and basolateral (BLA) nuclei, when comparing the two situations. Detailed phenotypical analyses in the amygdala and ventral tegmental area (VTA) show that specific subpopulations in the BLA are differentially involved in the formation and retrieval of withdrawal memories, and strikingly that a population of VTA dopamine neurons is activated in both situations. Together, this indicates that withdrawal memories can drive activity changes in specific neuronal populations of interconnected limbic areas known to be involved in aversive motivational processes. This first study on the neural substrates of withdrawal memories strongly supports an incentive-motivational view of withdrawal in opiate addiction that could be crucial in compulsive drug seeking and relapse.

Cannabinoid receptor antagonist reduces heroin self-administration only in dependent rats

Functionality of the endogenous cannabinoid system undergoes relevant changes in reward-related brain areas in animal models of opiate addiction. By using a limited access heroin self-administration paradigm we show that cannabinoid CB 1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A, 0.03–3.0 mg/kg) suppresses heroin self-administration only in opiate-dependent rats but not in non-dependent animals. These results further support the study of cannabinoid CB 1 receptor antagonists for the treatment of opiate addiction.

Interference with withdrawal signs of naloxone-induced opiate withdrawal under anesthesia is anesthetic-specific in opiate-dependent rats

We hypothesized that interference of opiate antagonist-precipitated withdrawal signs under anesthesia is anesthetic-specific. Three groups of morphine-dependent rats were compared in different experimental conditions using a protocol of rapid withdrawal induction by an antagonist under anesthesia. We observed that ketamine and midazolam have different effects on the expression of withdrawal. This brings specific insights into the pharmacological basis of therapy with induction of opiate antagonist.

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.

Perinatal Δ9-tetrahydrocannabinol exposure reduces proenkephalin gene expression in the caudate-putamen of adult female rats

Perinatal Δ 9-tetrahydrocannabinol ( Δ 9-THC) exposure in rats affects several behavioral responses, such as opiate self-administration behavior or pain sensitivity, that can be directly related to changes in opioidergic neurotransmission. In addition, we have recently reported that the administration of naloxone to animals perinatally exposed to Δ 9-THC produced withdrawal responses, that resemble those observed in opiate-dependent rats. The purpose of the present study was to examine the basal opioid activity in the brain of adult male and female rats that had been perinatally exposed to Δ 9-THC. To this aim, proenkephalin mRNA levels were measured, by using in situ hybridization histochemistry, in the caudate-putamen, nucleus accumbens, central amygdala and prefrontal cingulate cortex. The results showed a marked reduction in proenkephalin mRNA levels in the caudate-putamen of Δ 9-THC-exposed females as compared to oil-exposed females, whereas no changes were observed between Δ 9-THC- and oil-exposed males. There were no differences in proenkephalin mRNA levels in the nucleus accumbens, central amygdala and prefrontal cingulate cortex between males and females perinatally exposed to Δ 9-THC and their respective controls, although a certain trend to decrease was observed in Δ 9-THC-exposed females. In summary, perinatal exposure to Δ 9-THC exposure decreased proenkephalin gene expression in the caudate-putamen of adult rats, although this effect exhibited a marked sexual dimorphism since it was only seen in females. This result is in agreement with a previous observation from our laboratory that females, but not males, that had been perinatally exposed to Δ 9-THC, self-administered more morphine in adulthood. This suggests that low levels of proenkephalin mRNA may be used as a predictor of greater vulnerability to opiates.

Attenuation of withdrawal-induced hyperactivity of locus coeruleus neurones by inhibitors of nitric oxide synthase in morphine-dependent rats

Electrophysiological, biochemical, and behavioural studies have suggested that opiate withdrawal is mediated, at least in part, by a hyperactivity of locus coeruleus (LC) neurones. The aim of this study was to evaluate, using single-unit extracellular recordings, the role of NO in the opiate withdrawal-induced hyperactivity of LC neurones in anaesthetized rats. In animals chronically treated with morphine (5 days), administration of naloxone caused an increase in the spontaneous firing rate of LC cells. Acute pretreatment with the nitric oxide synthase (NOS) inhibitor N G-nitro- l-arginine methyl ester (30 mg kg −1 i.p.) attenuated some signs of opiate withdrawal (total score reduced by 55%), and also the withdrawal-induced hyperactivity of LC neurones (hyperactivity reduced by ∼50%). Acute pretreatment with 7-nitro indazole (50 mg kg −1 i.p.), a selective inhibitor of neuronal NOS, caused a complete blockade of the withdrawal-induced hyperactivity of LC neurones. Application of 7-nitro indazole (30 μM) in the vicinity of the LC also caused a reduction (of ∼60%) in the withdrawal-induced hyperactivity of LC cells. Intravenous administration of these NOS inhibitors (after naloxone challenge) did not produce comparable changes in the LC cell firing activity. 7-Nitro indazole failed to affect the development of tolerance of the LC to the morphine effect in opiate-dependent rats (i.e. morphine dose-effect curves were shifted to the right by morphine treatments to a similar degree in vehicle- and 7-nitro indazole-pretreated rats). The present data suggest that opiate withdrawal might be mediated by nitric oxide acting as an intermediate messenger in the LC.

Regulation of G protein-coupled receptor kinase 2 in brains of opiate-treated rats and human opiate addicts.

The effects of opiate drugs (heroin, morphine, and methadone) on the levels of G protein-coupled receptor kinase 2 (GRK2) were studied in rat and human brain frontal cortices. The density of brain GRK2 was measured by immunoblot assays in acute and chronic opiate-treated rats as well as in opiate-dependent rats after spontaneous or naloxone-precipitated withdrawal and in human opiate addicts who had died of an opiate overdose. In postmortem brains from human addicts, total GRK2 immunoreactivity was not changed significantly, but the level of the membrane-associated kinase was modestly but significantly increased (12%) compared with matched controls. In rats treated chronically with morphine or methadone modest increases of the enzyme levels (only significant after methadone) were observed. Acute treatments with morphine and methadone induced dose- and time-dependent increases (8-22%) in total GRK2 concentrations [higher increases were observed for the membrane-associated enzyme (46%)]. Spontaneous and naloxone-precipitated withdrawal after chronic morphine or methadone induced a marked up-regulation in the levels of total GRK2 in the rat frontal cortex (18-25%). These results suggest that GRK2 is involved in the short-term regulation of mu-opioid receptors in vivo and that the activity of this regulatory kinase in brain could have a relevant role in opiate tolerance, dependence, and withdrawal.

Role of Morphine Glucuronide Metabolites in Morphine Dependence in the Rat

Concentrations of morphine and its 3- and 6-glucuronide metabolites (M3G and M6G) in plasma, brain, and urine of rats exposed to morphine for either 24 or 48 h were measured using high-performance liquid chromatography. In another group of morphine-treated rats, the intensity of naloxone-precipitated withdrawal behaviours was monitored at 24 and 48 h. The behavioural effects of M3G in opiate-naive and opiate-dependent rats were also investigated. Morphine was present in plasma, urine, and brain at 24 and 48 h, whereas M3G was detected in plasma and urine only. M6G was not present in detectable quantities in either plasma, urine, or brain. Although plasma concentrations of M3G were similar in both time groups, rats treated for 48 h had significantly larger quantities of M3G in their urine than did the other treatment groups. The incidence of withdrawal behaviour was significantly higher in animals exposed to morphine for 48 h than in those with only 24 h of exposure. M3G had no behavioural effects in the opiate-naive rats and did not precipitate an opiate-abstinence syndrome in morphine-dependent rats. From these results, it was concluded that although M3G is the major product formed by morphine breakdown in rats, it is unlikely that it is involved in the development of morphine dependence in this species.

Nimodipine reduction of naltrexone-precipitated locus coeruleus activation and abstinence behavior in morphine-dependent rats

Single-unit recordings in anesthetized animals revealed that the L-type calcium channel antagonist, nimodipine (10 mg/kg), attenuated the activation of locus coeruleus neurons produced by naltrexone in opiate-dependent rats. Nimodipine also dose-dependently (1 mg/kg, 10 mg/kg, IP) inhibited the display of naloxone-precipitated withdrawal behaviors with a time course similar to that of nimodipine effects on the locus coeruleus. These data suggest that nimodipine may suppress opiate withdrawal via noradrenergic mechanisms.

Elevated basal firing rates and enhanced responses to 8-Br-cAMP in locus coeruleus neurons in brain slices from opiate-dependent rats

Extracellular, single unit activity was recorded in noradrenergic neurons of the nucleus locus coeruleus (LC) in brain slices prepared from rats treated chronically with morphine. In contrast to previous reports, basal firing rates of LC neurons were 2-fold higher in slices from opiate-dependent animals compared to controls and they remained elevated for at least 7 h. In neurons from dependent animals the maximal excitation in response to 8-bromoadenosine 3′:5′-cyclic monophosphate (8-Br-cAMP), but not the EC 50, was found to be substantially greater than in controls. This result parallels biochemical evidence of an up-regulation of the cAMP pathway in the LC of opiate-dependent animals. There was no difference in the response to glutamate between cells from dependent and control animals. We conclude that an increase in basal firing rate, possibly mediated by an up-regulation of the intrinsic cAMP pathway, contributes to the hyperactivity of the LC during opiate withdrawal in vivo.

Failure of cholecystokinin to precipitate withdrawal in morphine-treated rats

In a test of the possible antagonistic interaction between cholecystokinin (CCK) and morphine, morphine-dependent rats were injected with one of three doses of CCK or with naloxone immediately following the consumption of a novel saccharin solution. Whereas opiate-dependent rats injected with the opiate antagonist naloxone acquired an aversion to the saccharin solution (and displayed a dramatic weight loss), CCK was without effect. These data were discussed in relation to the possible pharmacological antagonism between CCK and the opiates.

Reversal of naloxone-precipitated hyperalgesia in opiate dependent rats by lidocaine microinjection in the rostral ventromedial medulla: Evidence for a pain facilitating system

Reversal of naloxone-precipitated hyperalgesia in opiate dependent rats by lidocaine microinjection in the rostral ventromedial medulla: Evidence for a pain facilitating system

Withdrawal-induced activation of locus coeruleus neurons in opiate-dependent rats: attenuation by lesions of the nucleus paragigantocellularis

Single unit activity was recorded in the locus coeruleus (LC) of anesthetized, morphine-dependent rats during naltrexone-precipitated withdrawal. As has been reported previously, LC neurons displayed a strong withdrawal-induced activation of firing rate. Radio-frequency lesions of the nucleus paragigantocellularis (PGi), a major LC afferent, greatly attenuated withdrawal-induced activation of neurons in the LC ipsilateral but not contralateral to the PGi lesion. Lesions of the prepositus hypoglossi,]another major LC afferent, did not prevent the withdrawal-induced activation of LC neurons. Kynurenic acid, a non-selective excitatory amino acid antagonist known to block PGi-induced excitations of LC neurons, also blocked the withdrawal-induced activation of LC neurons. These studies indicate that withdrawal-induced activation of the LC in opiate-dependent rats is mediated at least in part by afferents from the PGi which utilize an excitatory amino acid transmitter.

Nucleus accumbens as a substrate for the aversive stimulus effects of opiate withdrawal

Previous studies from our laboratory using methylnaloxonium, a hydrophilic antagonist, showed that opiate receptors in the region of the nucleus accumbens are important for the acute reinforcing effects of heroin in non-dependent rats. A similar increased sensitivity to the response disruptive effects of intracerebrally injected methylnaloxonium in opiate dependent rats was observed in a fixed-ratio (FR) baseline of operant behaviors. These results suggest that the same opiate receptors in the region of the nucleus accumbens important for the positive reinforcing stimulus properties of opiates may also be responsible for the response disruptive, aversive stimulus properties of opiate withdrawal. These results also suggest that the neural substrates of some aspects of dependence may be partly related to those of the reinforcing effects of opiates. In particular, it is hypothesized that "euphoria" and "dysphoria" induced by opiates may reflect opponent motivational processes operating at a cellular level within the nucleus accumbens.