Naloxone-precipitated Withdrawal Jumping Research Articles

A limited access oral oxycodone paradigm produces physical dependence and mesocorticolimbic region-dependent increases in DeltaFosB expression without preference

The abuse of oral formulations of prescription opioids has precipitated the current opioid epidemic. We developed an oral oxycodone consumption model consisting of a limited access (4 h) two-bottle choice drinking in the dark (TBC-DID) paradigm and quantified dependence with naloxone challenge using mice of both sexes. We also assessed neurobiological correlates of withdrawal and dependence elicited via oral oxycodone consumption using immunohistochemistry for DeltaFosB (ΔFosB), a transcription factor described as a molecular marker for drug addiction. Neither sex developed a preference for the oxycodone bottle, irrespective of oxycodone concentration, bottle position or prior water restriction. Mice that volitionally consumed oxycodone exhibited hyperlocomotion in an open field test and supraspinal but not spinally-mediated antinociception. Both sexes also developed robust, dose-dependent levels of opioid withdrawal that was precipitated by the opioid antagonist naloxone. Oral oxycodone consumption followed by naloxone challenge led to mesocorticolimbic region-dependent increases in the number of ΔFosB expressing cells. Naloxone-precipitated withdrawal jumps, but not the oxycodone bottle % preference, was positively correlated with the number of ΔFosB expressing cells specifically in the nucleus accumbens shell. Thus, limited access oral consumption of oxycodone produced physical dependence and increased ΔFosB expression despite the absence of opioid preference. Our TBC-DID paradigm allows for the study of oral opioid consumption in a simple, high-throughput manner and elucidates the underlying neurobiological substrates that accompany opioid-induced physical dependence.

An auto real-time jump tagging system for exploring stereotyped jumping behavior in mice

The jump is one of the common stereotyped behavior in rodents which can be found in certain types of disease models, such as addiction. It can be easily identified by the human eye. However, it is difficult to be tagged in real-time by manual operation, which limits the detailed exploration of its neural mechanisms with the new techniques, such as fiber photometry recording. Here we introduced an auto real-time jump tagging system (Art-JT system) to record the jump based on online monitoring the pressure changes of the floor in which the mouse is free exploring. Meanwhile, the Art-JT system can send the digital signal of the jump timing to the external device for tagging the events in the fiber photometry system. We tested it with the mice induced by Naloxone precipitated withdrawal jumping and found that it could accurately record the jump events and provide several detailed parameters of the jump. We also confirmed that the jump was correlated with the medial prefrontal cortex and primary motor cortex neuronal activities by combining the Art-JT system, GCaMP6 mice, and fiber photometry system. Our results suggested that the Art-JT system may be a powerful tool for recording and analyzing jumps efficiently and helping us to understand stereotyped behavior.

Possible mechanism and potential application of anti-opioid effect of diazepam-binding inhibitor

AimsOur previous study has demonstrated that porcine diazepam-binding inhibitor (pDBI) and its active fragments, pDBI-16 and pDBI-19, have inhibition effect on morphine analgesia in mice. The present study aimed to investigate the underlying mechanism and potential application of this anti-opioid effect. Materials and methodsEffect of DBI on morphine analgesia was examined by the tail electric stimulation vocalization test. Complementary peptides and antiserum were used to further confirm the effect of DBI in morphine tolerance and dependence. Pharmacological and microinjection methods were used to investigate the underlying mechanism. Key findingsFirstly, pDBI administered either intracerebroventricularly or intravenously dose-dependently inhibited morphine analgesia, while blocking DBI-16 or DBI-19 by the complementary peptides for DBI-16 (CP-DBI-16) or DBI-19 (CP-DBI-19) potentiated it in mice. Secondly, explicit immunoexpression of DBI in the lateral habenular (LHb) was observed in naive rats, and intra-LHb injection of pDBI dose-dependently abolished analgesic effect produced by intra-periaqueductal gray (PAG) injection of morphine in rats. Thirdly, pretreatment with N-Methyl-d-Aspartate receptor (NMDAR) antagonist MK-801 or nitric oxide (NO) synthase inhibitor L-NAME abolished the inhibition effect of pDBI, pDBI-16 or pDBI-19 on morphine analgesia in mice. Finally, antiserum against DBI dose-dependently reversed analgesic tolerance induced by increasing doses of morphine twice daily for 13 days in mice, while CP-DBI-16 or CP-DBI-19 significantly inhibited naloxone-precipitated morphine withdrawal jumping in mice. SignificanceTaken together, our results demonstrated that NMDAR/NO signaling and LHb-PAG pathway are crucially involved in the anti-opioid effect of DBI, which could provide a potential biological target for opioid tolerance and dependence.

Effects of Cannabinoid Type 2 Receptor Agonist AM1241 on Morphine-Induced Antinociception, Acute and Chronic Tolerance, and Dependence in Mice

Morphine is a potent opioid analgesic used to alleviate moderate or severe pain, but the development of drug tolerance and dependence limits its use in pain management. Previous studies showed that cannabinoid type 2 (CB2) receptor ligands may modulate opioid effects. However, there is no report of the effect of CB2 receptor agonist on acute morphine tolerance and physical dependence. We therefore investigated the effect of a CB2 receptor agonist (AM1241) on morphine-induced morphine tolerance and physical dependence in mice. Repeated coadministration of AM1241 (1 or 3mg/kg intraperitoneally) and morphine (10mg/kg subcutaneously) for 7days increased the mechanical paw withdrawal threshold in mice as measured by the von Frey filament test, and 3mg/kg AM1241 in combination with morphine increased the thermal paw withdrawal latency as measured by the hot-plate test. Combination with 3mg/kg AM1241 and morphine increased acute morphine antinociception. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced acute morphine tolerance, and 3mg/kg AM1241 reduced chronic morphine tolerance. Coadministration of 1 or 3mg/kg AM1241 and morphine reduced naloxone-precipitated withdrawal jumping, but not diarrhea. Coadministration of AM1241 and morphine did not inhibit spontaneous locomotor activity. Pretreatment with 3mg/kg AM1241 decreased the chronic morphine-induced Iba1 expression in spinal cord. Coadministration of AM1241 (3 mg/kg) reduced the production of interleukin-1β, tumor necrosis factor-α, and interleukin-6 induced by long-term and acute morphine treatment. Our findings suggest that the coadministration of the CB2 receptor agonist and morphine could increase morphine antinociception and reduce morphine tolerance and physical dependence in mice.Perspective:The combination of a CB2 agonist and morphine may provide a new strategy for better treatment of acute and chronic pain and prevention of opioid tolerance and dependence. This finding may also provide a clue for the treatment of opioid tolerance and dependence in clinics.

Attenuation of morphine-induced dependence and tolerance by ceftriaxone and amitriptyline in mice

IntroductionTolerance to and dependence on the analgesic effect of opioids is a pharmacological phenomenon that occurs after their prolonged administration. ObjectiveThe aim of this study was to evaluate the protective effects of ceftriaxone and amitriptyline on the development of morphine-induced tolerance and dependence. MethodsIn this study, 18 groups (9 groups each for tolerance and dependency tests) of mice (n = 8) received saline [10 mL/kg, intraperitoneally (i.p.)], morphine (50 mg/kg, i.p.), ceftriaxone (50 mg/kg, i.p., 100 mg/kg, i.p., and 200 mg/kg, i.p.), amitriptyline (5 mg/kg, i.p., 10 mg/kg, i.p., and 15 mg/kg, i.p.), or a combination of ceftriaxone (50 mg/kg, i.p.) and amitriptyline (5 mg/kg, i.p.) once per day for 4 days for investigation and comparison of the effects of ceftriaxone and amitriptyline on the prevention of dependency and tolerance to morphine. Tolerance was assessed with administration of morphine (9 mg/kg, i.p.) and using the hot plate test on the 5th day. In dependency tests, withdrawal symptoms were assessed on the 4th day for each animal 30 minutes after the administration of naloxone (4 mg/kg, i.p.; 2 hours after the last dose of morphine). ResultsIt was found that treatment with ceftriaxone or amitriptyline attenuated the development of tolerance to the antinociceptive effect of morphine and also reduced naloxone-precipitated withdrawal jumping and standing on feet. Furthermore, coadministration of ceftriaxone and amitriptyline at low doses (50 mg/kg, i.p. and 5 mg/kg, i.p., respectively) prior to morphine injection also decreased both morphine-induced tolerance and dependence. ConclusionResults indicate that the treatment with ceftriaxone and amitriptyline, alone or in combination, could attenuate the development of morphine-induced tolerance and dependence.

Chronic morphine treatment induces over-expression of HSP70 in mice striatum related with abnormal ubiquitin-proteasome degradation

BackgroundIt has been shown that opioid dependence-related neuronal plasticity may rely not only on protein synthesis, but also on protein degradation, mainly mediated by ubiquitin-proteasome system (UPS). The aim of the present study was to determine the effect of morphine on the regulation of protein degradation in the brain and to determine which proteins are involved in the underlying mechanism. MethodsMice were given chronic morphine administration and the state of morphine dependence was confirmed by induction of naloxone-precipitated withdrawal jumping. The level of ubiquitinated proteins in the striatum and spinal cord of morphine-dependent mice was detected by Western blotting. One of the abnormal-ubiquitinated proteins in mice striatum was identified by electrospray ionization quadrupole time-of-flight tandem mass spectrometry and the result was further confirmed by Western blotting and immunofluorescence method. ResultsWe found that the expression of some ubiquitinated proteins was clearly decreased in the striatum of morphine-depnendent mice, but not in the spinal cord. And we identified a ubiquitinated protein (>79kDa) decreased in the striatum as heat shock cognate 70 protein, one member of the 70kDa family of heat shock proteins (HSP70). Moreover, we confirmed the level of HSP70 protein was significantly increased in mice striatum. ConclusionsThese data strongly suggest morphine-induced HSP70 overexpression in the striatum is closely related with its abnormal degradation by UPS and it seems to be an important mechanism associated with morphine dependence.

Phenylmethanesulfonyl fluoride, a serine protease inhibitor, suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice

We have previously shown that intracerebroventricular (i.c.v.) administration of cysteine protease inhibitors suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation (see (Tan-No, K., Sato, T., Shimoda, M., Nakagawasai, O., Niijima, F., Kawamura, S., Furuta, S., Sato, T., Satoh, S., Silberring, J., Terenius, L., Tadano, T., 2010. Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice. Neuropeptides 44, 279–283)). In the present study, we examined the effect of phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor, on naloxone-precipitated withdrawal jumping in morphine-dependent mice. The doses of morphine (mg/kg per injection) were subcutaneously given twice daily for 2days [day 1 (30) and day 2 (60)]. On day 3, naloxone (8mg/kg) was intraperitoneally administered 3h after the final injection of morphine (60mg/kg), and the number of jumps was immediately recorded for 20min. Naloxone-precipitated withdrawal jumping was significantly suppressed by i.c.v. administration of PMSF (4nmol), given 5min before each morphine treatment during the induction phase, with none given on the test day. The expression of tissue plasminogen activator (tPA), a serine protease that converts plasminogen to plasmin, in the prefrontal cortex was significantly increased in morphine-dependent and -withdrawal mice, as compared with saline-treated mice. Moreover, trans-4-(aminomethyl)-cyclohexanecarboxylic acid (300pmol), an antiplasmin agent, and (Tyr1)-thrombin receptor activating peptide 7 (0.45 and 2nmol), an antagonist of protease activated receptor-1 (PAR-1), significantly suppressed naloxone-precipitated withdrawal jumping. The present results suggest that PMSF suppresses naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of activities of tPA and plasmin belonging to the serine proteases family, which subsequently activates PAR-1.

Phenothiazine-type antipsychotics may attenuate naloxone-precipitated withdrawal jumping in morphine-dependent mice

ObjectivesWithdrawal of opioids is usually associated with intolerable aversive symptoms. Our objective was to evaluate the efficacy of phenothiazine-type antipsychotics for reducing withdrawal symptoms during morphine abstinence. MethodsAdult NRL mice were rendered physically dependent on morphine by escalating the doses of subcutaneous morphine for 3 days. Withdrawal jumping was precipitated by a subcutaneous injection of naloxone (50 mg/kg) on day 4. In study I, on an equimolar basis, we compared the efficacy of six phenothiazine antipsychotics in saline on reducing morphine withdrawal symptoms. One hour before naloxone injection, the mice were assigned to receive intramuscular (i.m.) saline or one of the six phenothiazine-type antipsychotics (0.3 μmol/kg). After naloxone injection, the tested mouse was immediately placed in a transparent acrylic cylinder, and the severity of withdrawal symptoms was assessed, via a computer connected to the floor of the cylinder, by counting the number of the withdrawal jumps over a 30-minute interval. In study II, we performed a dose–response test in these six phenothiazine antipsychotics (0.03, 0.3, and 3 μmol/kg, i.m., for each test drug) on the inhibition of naloxone-precipitated morphine withdrawal jumping. ResultsWe found that all six phenothiazine-type antipsychotics attenuated the morphine withdrawal jumps, as compared with saline (p < 0.05). The effect is dose-dependent, with the potency ranking order as follows: fluphenazine ＝ triflupromazine > chlorpromazine ＝ perphenazine > promazine ＝ thioridazine (p < 0.05). ConclusionsAll six phenothiazine-type antipsychotics could attenuate morphine withdrawal symptoms; in particular, fluphenazine and triflupromazine may potentially be the more appropriate candidates for the treatment of morphine withdrawal symptoms.

A comparison of the effects of digoxin, ouabain and milrinone on naloxone-precipitated withdrawal syndrome in mice

Modulation of Na+, K+-ATPase activity by acute and chronic opiates has been established for many years. However, the effects of digoxin, a putative inhibitor of Na+, K+-ATPase, on naloxone-precipitated morphine withdrawal syndrome are unknown. In the present study, a digoxin dose-response curve was conducted to observe the effects on naloxone-precipitated withdrawal and locomotor activity in mice. Higher doses of digoxin (1.0 and 2.5mg/kg) inhibited locomotor activity and naloxone-precipitated withdrawal jumping and weight loss, while lower doses of digoxin (0.1 and 0.25mg/kg) inhibited withdrawal weight loss precipitated by naloxone without affecting locomotor activity and naloxone-precipitated withdrawal jumping. To explore the possible mechanisms underlying this behavior, another Na+, K+-ATPase inhibitor ouabain, which does not cross the blood brain barrier, and another cardiotonic drug milrinone, a non-inhibitor of Na+, K+-ATPase, were also included in the present study. Both milrinone and ouabain inhibited, in a dose-dependent manner, naloxone-precipitated weight loss while neither affected naloxone-precipitated withdrawal jumping nor locomotor activity in mice. These results indicate that both the cardiotonic effects and central inhibition of Na+, K+-ATPase contribute to the inhibitory effects of digoxin on morphine withdrawal syndrome in mice.

Suppressive effects by cysteine protease inhibitors on naloxone-precipitated withdrawal jumping in morphine-dependent mice

The effects of various protease inhibitors on naloxone-precipitated withdrawal jumping were examined in morphine-dependent mice. The doses of morphine were subcutaneously given twice daily for 2 days (day 1, 30 mg/kg; day 2, 60 mg/kg). On day 3, naloxone (8 mg/kg) was intraperitoneally administered 3h after final injection of morphine (60 mg/kg), and the number of jumping was immediately recorded for 20 min. Naloxone-precipitated withdrawal jumping was significantly suppressed by the intracerebroventricular administration of N-ethylmaleimide (0.5 nmol) and Boc-Tyr-Gly-NHO-Bz (0.4 nmol), inhibitors of cysteine proteases involved in dynorphin degradation, 5 min before each morphine treatment during the induction phase, with none given on the test day, as well as by dynorphin A (62.5 pmol) and dynorphin B (250 pmol). However, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, and captopril, an angiotensin-converting enzyme inhibitor, caused no changes. The present results suggest that cysteine protease inhibitors suppress naloxone-precipitated withdrawal jumping in morphine-dependent mice, presumably through the inhibition of dynorphin degradation.

Morphine induces conditioned place preference behavior in histidine decarboxylase knockout mice

Pharmacological and lesion studies have shown that histamine exerts inhibitory effects on morphine-induced reward-seeking behavior. The present study was designed to further investigate the involvement of endogenous histamine in morphine-induced reward-seeking behavior using histidine decarboxylase gene knockout (HDC-KO) mice. Conditioned place preference (CPP) was present in both wild-type (WT) and HDC-KO mice treated with 5 or 10 mg/kg morphine. HDC-KO mice showed stronger morphine-induced CPP as compared with WT mice. Meanwhile, morphine significantly increased dopamine level in the VTA and NAc, especially in HDC-KO mice. However, the extinction of CPP is similar between WT and HDC-KO mice. Moreover, naloxone-precipitated withdrawal jumping was markedly decreased in HDC-KO mice. These results indicate that endogenous histamine inhibits the development, but not the extinction, of morphine-induced CPP and reduces morphine withdrawal sign, probably through modulating dopaminergic activity in the brain.

Acute and chronic heroin dependence in mice: Contribution of opioid and excitatory amino acid receptors

Opioid and excitatory amino acid receptors contribute to morphine dependence, but there are no studies of their role in heroin dependence. Thus, mice injected with acute or chronic heroin doses in the present study were pretreated with one of the following selective antagonists: 7-benzylidenenaltrexone (BNTX), naltriben (NTB), nor-binaltorphimine (nor-BNI; δ 1, δ 2, and κ opioid receptors, respectively), MK-801, or LY293558 (NMDA and AMPA excitatory amino acid receptors, respectively). Naloxone-precipitated withdrawal jumping frequency, shown here to be a reliable index of heroin dependence magnitude, was reduced by BNTX or NTB in mice injected with both acute and chronic heroin doses. In contrast, nor-BNI did not alter jumping frequencies in mice injected with an acute heroin dose but significantly increased them in mice receiving chronic heroin injections. Continuous MK-801 or LY293558 infusion, but not injection, reduced jumping frequencies during withdrawal from acute heroin treatment. Their delivery by injection was nonetheless effective against chronic heroin dependence, suggesting mechanisms not simply attributable to NMDA or AMPA blockade. These data indicate that whereas δ 1, δ 2, NMDA, and AMPA receptors enable acute and chronic heroin dependence, κ receptor activity limits the dependence liability of chronic heroin. With the exception of δ 1 receptors, the apparent role of these receptors to heroin dependence is consistent with their contribution to morphine dependence, indicating that there is substantial physiological commonality underlying dependence to both heroin and morphine. The ability of κ receptor blockade to differentially alter acute and chronic dependence supports previous assertions from studies with other opioids that acute and chronic opioid dependence are, at least in part, mechanistically distinct. Elucidating the substrates contributing to heroin dependence, and identifying their similarities and differences with those of other opioids such as morphine, may yield effective treatment strategies to the problem of heroin dependency.

Enkephalin release promotes homeostatic increases in constitutively active mu opioid receptors during morphine withdrawal

We previously demonstrated that naloxone administration produces a robust conditioned place aversion (CPA) in opiate-naive rodents by blocking the action of enkephalins at μ opioid receptors (MORs). The aversive response to naloxone is potentiated by prior exposure to morphine. Morphine-induced MOR constitutive activity is hypothesized to underlie this enhanced effect of naloxone, an inverse agonist at the MOR. We sought additional evidence for the role of constitutively active MORs in this morphine-induced enhancement using the pro-enkephalin knockout (pENK −/ −) mouse, which is devoid of naloxone CPA in the morphine-naive state. Naloxone, but not the neutral antagonist, 6-β-naloxol, produced CPA and physical withdrawal signs in pENK −/ − mice when administered 2 h, but not 20 h, after morphine administration. Naloxone-precipitated physical withdrawal signs were attenuated in the pENK −/ − mice relative to wild-type (WT) animals. In both WT and pENK −/ − mice, naloxone-precipitated withdrawal jumping was greatest when naloxone was administered 2 h after morphine treatment and diminished at 3 h, in agreement with previous estimates of the time course for morphine-induced MOR constitutive activity in vitro. However, naloxone regained an ability to precipitate physical withdrawal in the WT, but not the pENK −/ − mice when administered 4.5 h after morphine administration. Taken together, the data suggest that a compensatory increase in enkephalin release during spontaneous morphine withdrawal promotes a second period of MOR constitutive activity in WT mice that is responsible for the enhanced naloxone aversion observed in such animals even when naloxone is administered 20 h after morphine. The endogenous enkephalin system and MOR constitutive activity may therefore play vital roles in hedonic homeostatic dysregulation following chronic opiate administration.

Inhibition of the spinal phosphoinositide 3-kinase exacerbates morphine withdrawal response

The present study investigates the roles of the spinal phosphoinositide 3-kinase (PI3K) signaling pathway in naloxone-precipitated withdrawal in acute and chronic morphine-dependent mice. There are two principal findings: (1) intrathecal pretreatment with wortmannin or LY294002, two structurally unrelated PI3K inhibitors, produced a dose-dependent increase of naloxone-precipitated withdrawal jumping, which was accompanied by an increased expression of spinal Fos protein in acute and chronic morphine-dependent mice; and (2) the expression of spinal p110γ, the catalytic subunit PI3K, in the membrane fraction was significantly down-regulated by naloxone-precipitated withdrawal in acute and chronic morphine-dependent mice. This study provides new evidence showing that inactivation of the PI3K signaling pathway in the spinal cord may be involved in the expression of morphine withdrawal.

Protective effect of bupropion on morphine tolerance and dependence in mice

Possible reversal of morphine-induced tolerance and dependence by bupropion was studied in mice. A 10-day repeated injection regimen was followed to induce morphine tolerance and dependence. Bupropion (2 and 5 mg/kg) per se, when chronically administered for 9 days, failed to produce any significant change in tail-flick latency compared with the control mice. Chronic administration of bupropion (2 or 5 mg/kg) during the induction phase (days 1-9) delayed the development of tolerance to the antinociceptive action of morphine and also reversed naloxone- (2 mg/kg) precipitated withdrawal jumps. On the other hand, acute administration of bupropion (2 or 5 mg/kg) on day 10, i.e., during the expression phase of morphine dependence, reduced the incidence of naloxone-precipitated withdrawal jumps without affecting tolerance to the analgesic effect. In conclusion, results of the present study suggest the potential use of bupropion in tolerance and dependence.

Possible mechanisms of action in quercetin reversal of morphine tolerance and dependence.

In an earlier study, we reported the ability of quercetin to reverse the development of morphine tolerance and dependence in mice. In the present study we have attempted to explore the possible involvement of nitric oxide (NO) system in quercetin reversal of morphine tolerance and dependence in mice. Co-administration of L-N(G)-nitro arginine methyl ester (L-NAME) or quercetin with morphine during the induction phase (days 1-9) delayed the development of tolerance to the antinociceptive action of morphine and also reversed naloxone precipitated withdrawal jumps. L-Arginine administration during the induction phase enhanced the development of tolerance to the antinociceptive effect of morphine but had no effect on the naloxone-precipitated withdrawal jumps. During the expression phase (day 10) acute administration of quercetin or L-NAME reversed, whereas L-arginine facilitated naloxone- precipitated withdrawal jumps in morphine-tolerant mice, but none of these drugs affected the nociceptive threshold in morphine-tolerant mice. Further, co-administration of quercetin or L-NAME with L-arginine during the induction phase antagonized the latter effects on the development of morphine tolerance. Also, prior administration of quercetin or L-NAME reversed the L-arginine potentiation of nalaxone-precipitated withdrawal jumps in morphine-tolerant mice. The results of the present study suggest that quercetin reversal of morphine tolerance and dependence may involve its ability to suppress nitric oxide synthase (NOS) activity.

Naloxone-precipitated withdrawal jumping in 11 inbred mouse strains: evidence for common genetic mechanisms in acute and chronic morphine physical dependence

Physical dependence is a widely known consequence of morphine intake. Although commonly associated with prolonged or repeated morphine administration, withdrawal symptoms can be elicited even after a single prior morphine exposure. What remains contentious is the extent to which physical dependence following acute and chronic morphine treatment is mediated by common physiological substrates and, accordingly, represent distinct syndromes. The genetic relationship between acute and chronic morphine dependence was thus presently studied by comparing mice of 11 inbred strains (129P3, A, AKR, BALB/c, C3H/He, C57BL/6, CBA, DBA/2, LP, SJL, and SWR) for naloxone-precipitated withdrawal jumping responses using three subcutaneous morphine administration paradigms: acute (single injection) or chronic (three daily morphine injections for 4 days) injection, or chronic infusion (7 days via implanted osmotic minipumps). Although there were differences in the magnitude of withdrawal jumping between the three different morphine administration paradigms, large and significant strain differences were observed for each. In addition, the same strains were unusually sensitive or, conversely, altogether refractory to withdrawal jumping across all morphine treatment conditions. Overall, strain jumping means between acute and chronic dependence paradigms displayed a high degree of genetic correlation ( r=0.87–0.95). The significant correlation between chronic morphine injection and continuous morphine infusion discounts the possible confounding effect of contextual learning and spontaneous withdrawal between chronic injections on the assessment of naloxone-precipitated withdrawal. Substantial heritability was also observed for acute and both paradigms of chronic dependence, with estimates ranging from h 2=0.53 to 0.70. The present demonstration of a strong genetic correlation between physical dependence to morphine following acute and chronic treatment implies that genes associated with variable sensitivity in the two traits are the same, and is suggestive of shared physiological substrates. The data also demonstrate that the differential genetic liability to morphine physical dependence begins with, and is predicted by, the first morphine exposure.

The effects of simultaneous administration of alpha(2) -adrenergic agents with L-NAME or L-arginine on the development and expression of morphine dependence in mice.

Both alpha(2)-adrenoceptors and the L-arginine/nitric oxide (NO) pathway have been implicated in the modulation of morphine dependence. This study examined the effects of simultaneous administration of the alpha(2)-adrenoceptor agonist clonidine or the antagonist yohimbine together with the NO precursor L-arginine or the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) on the induction and expression of morphine dependence as assessed by naloxone-precipitated withdrawal jumping and diarrhoea. Male NMRI mice weighing 20-30 g were used. In the induction phase, clonidine (0.01-0.1 mg/kg) intensified and yohimbine (0.5-2 mg/kg) attenuated the degree of morphine dependence. Yohimbine reversed the effect of clonidine. L-NAME (5 and 10 mg/kg) did not affect the development of morphine dependence, but significantly potentiated the effects of both subeffective (0.01 mg/kg) and effective (0.03 mg/kg) doses of clonidine. L-Arginine did not alter morphine dependence but inhibited the effect of clonidine. The effects of yohimbine in the induction phase were attenuated by L-NAME, but were not significantly affected by L-arginine. In the expression phase, clonidine attenuated and yohimbine intensified the signs of dependence. The effect of clonidine was inhibited by yohimbine. In the expression phase, L-NAME attenuated the withdrawal syndrome at 10 mg/kg and showed potentiation with clonidine in suppressing withdrawal signs. L-Arginine did not alter morphine dependence, but at 20 mg/kg inhibited and at 100 mg/kg potentiated the attenuating effect of clonidine on the expression of withdrawal syndrome. The effect of yohimbine on the expression phase was also attenuated by L-NAME, but was not significantly affected by L-arginine. In conclusion, alpha(2)-adrenergic and NO pathways seem to be functionally linked in the modulation of opioid dependence.

Reversal of Morphine Tolerance and Dependence by Passiflora incarnata – A Traditional Medicine to Combat Morphine Addiction

This paper describes the use of Passiflora incarnata Linneaus in reversing the development of dependence and tolerance of morphine in mice. A fraction (BZF) derived from the methanol extract of P. incarnata that had exhibited good anxiolytic activity, delayed the development of tolerance to the analgesic effect of morphine when administered at 10, 50 and 100 mg/kg doses along with 10 mg/kg dose of morphine for 9 days. A single dose of P. incarnata bioactive fraction (BZF) also decreased the naloxoneprecipitated withdrawal jumps in mice that had already been rendered tolerant due to chronic treatment with 10mg/kg of morphine.

Morphine tolerance and dependence in nociceptin/orphanin FQ transgenic knock-out mice.

It has been hypothesized that morphine tolerance and dependence in mice following chronic exposure may reflect increased compensatory activity of antiopioid systems. The endogenous peptide nociceptin/orphanin FQ has been shown to have anti-opioid effects, for example antagonizing morphine analgesia. Moreover, chronic morphine administration increases synthesis of the peptide, and morphine tolerance and dependence can be attenuated or reversed by antagonists and agonists of the nociceptin/orphanin FQ receptor, respectively. The present study seeks to confirm a role for nociceptin/orphanin FQ in opioid tolerance and dependence by comparing morphine ED(50) values and naloxone-precipitated withdrawal jumping in mice homozygous (knock-out) and heterozygous for a null mutation of the Npnc1 gene encoding the nociceptin/orphanin FQ propeptide, and their wild type littermates, following chronic morphine exposure. Relative to morphine-naive control mice, significant rightward shifts in the morphine dose-response curve, resulting in increased morphine ED(50) values (approximately two to three-fold), was observed for all genotypes following three days of repeated systemic morphine injections. However, no differences between genotypes in the magnitude of tolerance were observed. In contrast, knock-out mice displayed significantly increased naloxone-precipitated withdrawal jumping relative to heterozygous and wild-type mice following implantation with a morphine pellet (25mg) for 72h. Use of nociception/orphaninFQ transgenic knock-out mice thus demonstrate the differential involvement of nociceptin/orphanin FQ in morphine tolerance and dependence.