Withdrawal Jumping Research Articles

Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis

Morphine is a frequently used analgesic that activates the mu-opioid receptor (MOR), which has prominent side effects of tolerance. Although the inefficiency of morphine in inducing the endocytosis of MOR underlies the development of morphine tolerance, currently, there is no effective therapy to treat morphine tolerance. In the current study, we aimed to develop a monoclonal antibody (mAb) precisely targeting MOR and to determine its therapeutic efficacy on morphine tolerance and the underlying molecular mechanisms. We successfully prepared a mAb targeting MOR, named 3A5C7, by hybridoma technique using a strategy of deoxyribonucleic acid immunization combined with cell immunization, and identified it as an immunoglobulin G mAb with high specificity and affinity for MOR and binding ability to antigens with spatial conformation. Treatment of two cell lines, HEK293T and SH-SY5Y, with 3A5C7 enhanced morphine-induced MOR endocytosis via a G protein-coupled receptor kinase 2 (GRK2)/β-arrestin2-dependent mechanism, as demonstrated by immunofluorescence staining, flow cytometry, Western blotting, coimmunoprecipitation, and small interfering ribonucleic acid (siRNA)-based knockdown. This mAb also allowed MOR recycling from cytoplasm to plasma membrane and attenuated morphine-induced phosphorylation of MOR. We established an in vitro morphine tolerance model using differentiated SH-SY5Y cells induced by retinoic acid. Western blot, enzyme-linked immunosorbent assays, and siRNA-based knockdown revealed that 3A5C7 mAb diminished hyperactivation of adenylate cyclase, the in vitro biomarker of morphine tolerance, via the GRK2/β-arrestin2 pathway. Furthermore, in vivo hotplate test demonstrated that chronic intrathecal administration of 3A5C7 significantly alleviated morphine tolerance in mice, and withdrawal jumping test revealed that both chronic and acute 3A5C7 intrathecal administration attenuated morphine dependence. Finally, intrathecal electroporation of silencing short hairpin RNA illustrated that the in vivo anti-tolerance and anti-dependence efficacy of 3A5C7 was mediated by enhanced morphine-induced MOR endocytosis via GRK2/β-arrestin2 pathway. Collectively, our study provided a therapeutic mAb, 3A5C7, targeting MOR to treat morphine tolerance, mediated by enhancing morphine-induced MOR endocytosis. The mAb 3A5C7 demonstrates promising translational value to treat clinical morphine tolerance.

Physiological dependence to mitragynine indicated by a rapid cross-dependence procedure with heroin-dependent mice.

The potential of mitragynine to produce physiological dependence (withdrawal) was assessed using a rapid assessment procedure with male ICR mice exposed to heroin-admixed food followed by naloxone (subcutaneously, s.c.) precipitation of withdrawal. Initial studies indicated that 3 days of exposure to 3.0 mg/g of heroin-admixed food followed by naloxone (0.6 mg/kg) reliably precipitated withdrawal jumping and weight loss. Lower concentrations of heroin-admixed food and lower doses of naloxone produced fewer withdrawal signs. A longer exposure to heroin-admixed food did not produce significantly greater amounts of jumping or weight loss. Further, these withdrawal signs were dose-dependently reversed by s.c. administration of heroin immediately following naloxone administration. Mitragynine (s.c.) also dose-dependently suppressed naloxone-precipitated withdrawal signs. Additionally, both jumping and weight loss were suppressed over a comparable range of mitragynine doses when administered by gavage with a noticeably, but not significantly, higher potency than with s.c. administration. The ED50 values for mitragynine for the suppression of withdrawal by any route (354-911 μmol/kg) were greater than the minimally effective dose that decreased locomotor activity (251 μmol/kg) and from 40- to 104-fold greater than those for heroin. The results suggest inherent opioid dependence liability of mitragynine. The in vivo potency relations between mitragynine and heroin are consistent with a conclusion of dependence-producing effects, indicated by the suppression of withdrawal, comparable to standard opioid μ-receptor agonists, differing primarily in terms of potency. The present paper provides a method for the rapid assessment of physiological dependence liability applicable to other kratom plant constituents or any potential opioid dependence-producing agents.

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.

BPR1M97, a dual mu opioid receptor/nociceptin-orphanin FQ peptide receptor agonist, produces potent antinociceptive effects with safer properties than morphine

There is unmet need to design an analgesic with fewer side effects for severe pain management. Although traditional opioids are the most effective painkillers, they are accompanied by severe adverse responses, such as respiratory depression, constipation symptoms, tolerance, withdrawal, and addiction. We indicated BPR1M97 as a dual mu opioid receptor (MOP)/nociceptin-orphanin FQ peptide (NOP) receptor full agonist and investigated the pharmacology of BPR1M97 in multiple animal models. In vitro studies on BPR1M97 were assessed using cyclic-adenosine monophosphate production, β-arrestin, internalization, and membrane potential assays. In vivo studies were characterized using the tail-flick, tail-clip, lung functional, heart functional, acetone drop, von Frey hair, charcoal meal, glass bead, locomotor activity, conditioned place preference (CPP) and naloxone precipitation tests. BPR1M97 elicited full agonist properties for all cell-based assays tested in MOP-expressing cells. However, it acted as a G protein-biased agonist for NOP. BPR1M97 initiated faster antinociceptive effects at 10 min after subcutaneous injection and elicited better analgesia in cancer-induced pain than morphine. Unlike morphine, BPR1M97 caused less respiratory, cardiovascular, and gastrointestinal dysfunction. In addition, BPR1M97 decreased global activity and induced less withdrawal jumping precipitated by naloxone. Thus, BPR1M97 could serve as a novel small molecule dual receptor agonist for antinociception with fewer side effects than morphine.This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.

In vivo Dependence Liability Assessment of Novel Fentanyl Analogues: Science to Guide drug Policy

The emergence of fentanyl analogues as drugs of abuse raises concern regarding their dependence liability. Following prolonged opioid administration, physical dependence leads to increased use, which facilitates the development of tolerance and the dangers of overdose. In animal models, opioid dependence can be inferred by assessing signs of withdrawal, with vertical jumping being the most sensitive and easily quantified in the mouse. In this study, we aimed to characterize the dependence liability of fentanyl, morphine, and six fentanyl analogues by assessing antinociception and withdrawal signs elicited by each compound in the mouse. First, to determine antinociceptive effects, we used a warm‐water tail withdrawal procedure using both 50°C and 55°C water. Latencies until mice removed their tails from the water were recorded, and fully effective doses were determined using a 10 second cut‐off to prevent tissue damage. Dose effect‐curves were generated to determine the potency (ED50) of each compound for eliciting antinociception. All analogues were less potent than fentanyl but more potent than morphine. Additionally, all test compounds were fully effective in both 50°C and 55°C water. Next, to assess withdrawal, we treated animals with 3 injections of morphine, fentanyl or the various fentanyl analogues spaced 12 hours apart. Following the first two injections on day 1, we precipitated withdrawal on day 2, 3 hours after the final injection of agonist, using the nonselective opioid antagonist naloxone. Withdrawal sessions lasted 20 minutes and the number of vertical jumps were recorded for each session. Antagonist dose‐effect curves for withdrawal jumps were generated first using an agonist dose one‐log unit higher than the fully effective antinociceptive dose and precipitating withdrawal with 3, 10, and 30 mg/kg of naloxone. Next, we used 30 mg/kg of naloxone to precipitate withdrawal using increasing unit doses of each test compoewund since maximal jumping was elicited using 30 mg/kg of naloxone. Agonist dose‐effect curves were generated starting with the lowest fully effective antinociceptive dose and increasing in half‐log units. Our results indicate that all of these fentanyl derivatives are capable of eliciting dependence and offer valuable insight into their abuse potential.Support or Funding InformationThese studies supported in part by DEA/NCTR (224‐16‐0505R),) and the University of Arkansas for Medical ScienVA (D‐15‐OD‐002cesThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

In vivo effects of μ-opioid receptor agonist/δ-opioid receptor antagonist peptidomimetics following acute and repeated administration.

Agonists at μ-opioid receptors (μ-receptors) are used for pain management but produce adverse effects including tolerance, dependence and euphoria. The co-administration of a μ-receptor agonist with a δ-opioid receptor (δ-receptor) antagonist has been shown to produce antinociception with reduced development of some side effects. We characterized the effects of three μ-receptor agonist/δ-receptor antagonist peptidomimetics in vivo after acute and repeated administration to determine if this profile provides a viable alternative to traditional opioid analgesics. Three μ-receptor agonist / δ-receptor antagonist peptidomimetics, AAH8, AMB46 and AMB47, and morphine were evaluated for the development of tolerance and dependence after 5days of twice daily treatment with escalating doses of drug (10-50mg·kg-1 ). Antinociceptive effects were measured in the warm water tail withdrawal assay before and after repeated drug treatment. Physical dependence was evaluated by naltrexone-precipitated withdrawal jumping. The rewarding effects of AAH8 were evaluated using a conditioned place preference (CPP) assay with twice daily conditioning sessions performed for 5days. Morphine, AAH8, AMB47 and AMB46 all demonstrated acute antinociceptive effects, but repeated administration only produced tolerance in animals treated with morphine and AMB46. Injection of naltrexone precipitated fewer jumps in mice treated repeatedly with AAH8 as compared with morphine, AMB47 or AMB46. Conditioning with morphine, but not AAH8, produced significant CPP. AAH8 may be a better alternative than traditional opioid analgesics, producing antinociception with less development of tolerance and dependence and may be less rewarding than morphine.

Src‐dependent phosphorylation of μ‐opioid receptor at Tyr336 modulates opiate withdrawal

Opiate withdrawal/negative reinforcement has been implicated as one of the mechanisms for the progression from impulsive to compulsive drug use. Increase in the intracellular cAMP level and protein kinase A (PKA) activities within the neurocircuitry of addiction has been a leading hypothesis for opiate addiction. This increase requires the phosphorylation of μ‐opioid receptor (MOR) at Tyr336 by Src after prolonged opiate treatment in vitro. Here, we report that the Src‐mediated MOR phosphorylation at Tyr336 is a prerequisite for opiate withdrawal in mice. We observed the recruitment of Src in the vicinity of MOR and an increase in phosphorylated Tyr336 (pY336) levels during naloxone‐precipitated withdrawal. The intracerebroventricular or stereotaxic injection of a Src inhibitor (AZD0530), or Src shRNA viruses attenuated pY336 levels, and several somatic withdrawal signs. This was also observed in Fyn−/− mice. The stereotaxic injection of wild‐type MOR, but not mutant (Y336F) MOR, lentiviruses into the locus coeruleus of MOR−/− mice restored somatic withdrawal jumping. Regulating pY336 levels during withdrawal might be a future target for drug development to prevent opiate addictive behaviors.

Sophora alopecuroides L. var. alopecuroides alleviates morphine withdrawal syndrome in mice: involvement of alkaloid fraction and matrine.

Evaluation of the Sophora alopecuroides var. alopecuroides seed effects on morphine withdrawal syndrome in mice and determination of the alkaloid composition of the seed total extract. The effects of the seed total extract, alkaloid fraction and major compound matrine on the mice morphine withdrawal syndrome were compared to saline and methadone. Mice were made dependent on morphine by morphine sulfate injection 3 times a day for 3 days. The withdrawal jumping and diarrhea were induced by administration of naloxone 2 hr after the 10th injection of morphine sulfate on the day 4. The total extract (100, 200, 300 mg/kg), alkaloid fraction (5, 10, 20 mg/kg), matrine (5, 15, 30 mg/kg), methadone (10 mg/kg) or saline were injected 30 min before naloxone. All drugs were administered by subcutaneous injection. The total extract alkaloid composition was also determined by gas chromatography (GC) and GC-MS analysis. All doses of the total extract, alkaloid fraction and matrine as well as methadone decreased jumping and diarrhea significantly compared to the saline. The effects of the total extract and alkaloid fraction were not significantly different from methadone. But, there were significant differences between the effects of matrine and methadone. Matrine, cytisine, sophoridine, n-methyl cytisine, sophocarpine and sophoramine were the major alkaloids. There was no nicotine in the total extract. S. alopecuroides var. alopecuroides suppresses opioid withdrawal with efficacy comparable to methadone. Matrine may be one of the alkaloids responsible for the effect of the plant.

Noni (Morinda citrifolia L.) fruit extract attenuates the rewarding effect of heroin in conditioned place preference but not withdrawal in rodents.

The present study was designed to investigate the effect of a methanolic extract of Morinda citrifolia Linn. fruit (MMC) on the rewarding effect of heroin in the rat conditioned place preference (CPP) paradigm and naloxone-precipitated withdrawal in mice. In the first experiment, following a baseline preference test (preconditioning score), the rats were subjected to conditioning trials with five counterbalanced escalating doses of heroin versus saline followed by a preference test conducted under drug-free conditions (post-conditioning score) using the CPP test. Meanwhile, in the second experiment, withdrawal jumping was precipitated by naloxone administration after heroin dependence was induced by escalating doses for 6 days (3×/ day). The CPP test results revealed that acute administration of MMC (1, 3, and 5 g/kg body weight (bw), p.o.), 1 h prior to the CPP test on the 12th day significantly reversed the heroin-seeking behavior in a dose-dependent manner, which was similar to the results observed with a reference drug, methadone (3 mg/kg bw, p.o.). On the other hand, MMC (0.5, 1, and 3 g/kg bw, p.o.) did not attenuate the heroin withdrawal jumps precipitated by naloxone. These findings suggest that the mechanism by which MMC inhibits the rewarding effect of heroin is distinct from naloxone-precipitated heroin withdrawal.

Attenuation of Morphine-Induced Tolerance and Dependency by Pretreatment with Magnesium Sulfate and Amitriptyline in Male Mice

Clinical use of Morphine in pain management is a controversial issue and long-term exposure to opiates induces physical dependency and tolerance. The aim of this study was to evaluate the attenuation effects of Magnesium sulfate and Amitriptyline on the development of Morphine-induced dependency and tolerance. Methods: In this study different groups of mice were received saline (10 mL/kg,ip), Morphine (50 mg/kg,ip), Magnesium sulfate (20, 40 and 60 mg/kg,ip), Amitriptyline (5, 10 and 15 or 20 mg/kg,ip), or combination of Magnesium sulfate (20 mg/kg,ip) and Amitriptyline (5 mg/kg,ip) once a day for 4 and 10 continues days, respectively for investigation of the effects of Magnesium sulfate and Amitriptyline on the prevention of dependency and tolerance. Tolerance was assessed by administration of Morphine (9 mg/kg,ip) and using hot plate test on eleventh day. Withdrawal symptoms were assessed in fifth day by administration of naloxone (4 mg/kg,ip), 2 hrs after last dose of Morphine during 30 minutes for each animal. Results: It was found that pretreatment with Magnesium sulfate or Amitriptyline decreased the development of tolerance to the antinociceptive action of Morphine and also reduced naloxone-precipitated withdrawal jumps and standing on feet. Additionally, pretreatment by co-administration of Magnesium sulfate and Amitriptyline, before morphine administration, decreased the dependency and tolerance more significantly. Conclusion: From these results it could be concluded that pretreatment of Magnesium sulfate via blocking the N-methyl-D-Aspartate receptor-operated calcium channel and Amitriptyline by Glutamate transporter activation property alone or in combination could prevent the development of Morphine-induced dependency and tolerance.

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.

Differential effects of endocannabinoid catabolic inhibitors on morphine withdrawal in mice

BackgroundInhibition of endocannabinoid catabolic enzymes fatty acid amide hydrolase (FAAH) and/or monoacylglycerol lipase (MAGL) reduces somatic morphine withdrawal signs, but its effects on aversive aspects of withdrawal are unknown. The present study investigated whether Δ9-tetrahydrocannabinol (THC), the MAGL inhibitor JZL184, the FAAH inhibitor PF-3845, or the dual FAAH/MAGL inhibitor SA-57 would reduce acquisition of morphine withdrawal-induced conditioned place avoidance (CPA) and jumping. MethodsMice were implanted with placebo or 75mg morphine pellets, 48h later injected with naloxone or saline and placed in the conditioning apparatus, and assessed for CPA at 72h. Subjects were also observed for jumping behavior following naloxone challenge. ResultsNaloxone (0.056mg/kg) produced robust CPA in morphine-pelleted, but not placebo-pelleted, mice. Morphine pretreatment prevented the occurrence of withdrawal CPA and withdrawal jumping, while clonidine (an α2 adrenergic receptor agonist) only blocked withdrawal CPA. THC, JZL184, and SA-57 significantly reduced the percentage of mice that jumped during the conditioning session, but did not affect acquisition of withdrawal CPA. PF-3845 did not reduce morphine withdrawal CPA or jumping. Finally, neither THC nor the endocannabinoid catabolic enzyme inhibitors in non-dependent mice elicited a conditioned place preference or aversion. ConclusionsThese findings suggest that inhibiting endocannabinoid catabolic enzymes reduces somatic morphine withdrawal signs, but not aversive aspects as inferred in the CPA paradigm. The observation that non-dependent mice administered inhibitors of endocannabinoid degradation did not display place preferences is consistent with the idea that that endocannabinoid catabolic enzymes might be targeted therapeutically, with reduced risk of abuse.

Activation of Spinal EphrinBs/EphBs Contributes to Morphine Dependence and Tolerance

Background: Previous studies have demonstrated that spinal ephrinBs/EphBs system plays a critical role in the development and maintenance of chronic pain, and neuropathic pain and opiate dependence may share some common mechanisms. The aim of this study was to further explore the role of the spinal ephrinBs/EphBs system in opiate dependence and tolerance.Methods: Adult male kunming mice were randomly allocated into treatment group and control group. Four different models including acute/chronic morphine dependence and acute/chronic morphine tolerance were set up in treatment group and control group individually. Withdrawal syndromes were precipitated by naloxone (4mg/kg, intraperitoneal), the inhibitory or reversal role of EphB1- Fc in acute/chronic morphine or tolerance was assessed by injecting 0.5 μg/5 μl EphB1- Fc intrathecally prior to the treatment of morphine. In control group, equal value of vehicle (saline) was substituted for morphine. In behavior part, morphine physical dependence was assessed by withdrawal jumping counting; and morphine induced antinociceptive tolerance was evaluated by paw latency in hot-plate test. P-ERK and C-Fos expression in the spinal cord were detected by western blot and immunohistochemistry individually.Results: Acute and chronic morphine treatment increased the expression of spinal ephrinB1, which was further increased by acute morphine precipitated by administration of naloxone; however, chronic morphine precipitated by administration of naloxone decreased the expression of ephrinB1, which was significantly higher than that in saline group. Intrathecal pretreatment of EphB1-Fc inhibited or reversed morphine tolerance-induced antinociceptive tolerance and naloxone-precipitated jumping, which was accompanied with the decreased expression of spinal Fos protein and p-ERK.Conclusions: These results demonstrated that activation of spinal ephrinBs/EphBs contributed to morphine dependence and tolerance. Citation: He Liu, Di-Yang Ling, Yan Yuan, Xue- Hai Guan, Jia- Ping Ruan, Hong-Xing Zhang, et al. Activation of spinal ephrinBs/EphBs contributes to morphine dependence and tolerance. J Anesth Perioper Med 2014; 1: 79-89. doi: 10.24015/JAPM.2014.0013This is an open-access article, published by Evidence Based Communications (EBC). This work is licensed under the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium or format for any lawful purpose. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Hyperbaric oxygen treatment suppresses withdrawal signs in opioid‐dependent mice (659.12)

HBO2 therapy has been approved by the Food and Drug Administration for limited clinical conditions (Gesell, The Hyperbaric Oxygen Therapy Committee Report: Indications and Results, 12th Ed, 2008). It has been reported that HBO2 treatments reduce opioid withdrawal in human subjects (Epifanova, Anesteziol Reanimatol, May‐June 1995 issue, 34‐39). However, no further research has been conducted to investigate the mechanism of HBO2 in reducing opioid withdrawal. The purpose of this research was to determine whether HBO2 treatment could suppress signs of withdrawal in opioid‐dependent mice. Male NIH Swiss mice received repeated s.c. injections of morphine sulfate or saline twice a day for 4 days. The daily dose of morphine sulfate was increased progressively from 50 mg/kg on day 1 to 125 mg/kg on day 4. On day 5, opioid withdrawal was precipitated by intraperitoneal injection of 5.0 mg/kg naloxone. Withdrawal signs in mice, including jumping, forepaw tremor and wet dog shakes, were recorded for 30 min and scored. Sixty min prior to the naloxone injection, different groups of mice received a 30‐min or 60‐min HBO2 treatment at 3.5 atmospheres absolute. The results show that HBO2 treatment caused a dose‐dependent reduction in withdrawal jumping, forepaw tremor and wet dog shakes in opioid‐dependent mice.Grant Funding Source: Supported by NIH Grant AT‐007222 and the Allen I. White Distinguished Professorship.

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.

Hydrogen Sulfide Attenuates Opioid Dependence by Suppression of Adenylate Cyclase/cAMP Pathway

The best-established mechanism of opioid dependence is the up-regulation of adenylate cyclase (AC)/cAMP pathway, which was reported to be negatively regulated by hydrogen sulfide (H2S), a novel endogenous neuromodulator. The present study was, therefore, designed to determine whether H2S is able to attenuate the development of opioid dependence via down-regulating AC/cAMP pathway. We demonstrated that application of sodium hydrosulphide (NaHS) and GYY4137, two donors of H2S, significantly alleviated naloxone-induced robust withdrawal jumping (the most sensitive and reliable index of opioid physical dependence) in morphine-treated mice. Repeated treatment with NaHS inhibited the up-regulated protein expression of AC in the striatum of morphine-dependent mice. Furthermore, NaHS also attenuated morphine/naloxone-elevated mRNA levels of AC isoform 1 and 8, production of cAMP, and phosphorylation of cAMP response element-binding protein (CREB) in mice striatum. These effects were mimicked by the application of exogenous H2S or over-expression of cystathione-β-synthase, an H2S -producing enzyme, in SH-SY5Y neuronal cells on treatment with [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-Enkephalin, a selective μ-opioid receptor agonist. Blockade of extracellular-regulated protein kinase 1/2 (ERK1/2) with its specific inhibitor attenuated naloxone-induced CREB phosphorylation. Pretreatment with NaHS or stimulation of endogenous H2S production also significantly suppressed opioid withdrawal-induced ERK1/2 activation in mice striatum or SH-SY5Y cells. H2S treatment is important in prevention of the development of opioid dependence via suppression of cAMP pathway in both animal and cellular models. Our data suggest a potential role of H2S in attenuating the development of opioid dependence, and the underlying mechanism is closely related to the inhibition of AC/cAMP pathway.

A dose–response study on the efficacy of tricyclic antidepressants on reducing morphine-withdrawal symptoms

BackgroundAcute detoxification may lead to withdrawal syndrome. The syndrome is sufficiently aversive in those who are morphine-dependents and thus it hinders abstinence. The opioids are most often used clinically to lighten this syndrome. Here, we evaluated the effects of tricyclic antidepressants (TCAs) in treating physical dependence to opioids upon acute detoxification in mice. Materials and methodsAdult NRL mice were rendered physically dependent on morphine by increasing daily doses of subcutaneous morphine for 3 days and precipitated withdrawal jumping by subcutaneous naloxone on Day 4. The mice were then assigned to receive intramuscular saline or one of the five TCAs 1 hour prior to creating naloxone-precipitated withdrawal. The withdrawal jumping frequency in 30 minutes was counted after naloxone-precipitated withdrawal. ResultsOur results (the newest findings) based on the equimolar dose (30 μmol/kg), showed that the severity of physical dependence on morphine could be attenuated with less intramuscular TCAs [percent maximal possible effect (MPE) < 50). Amoxapine, protriptyline, amitriptyline, clomipramine, and trimipramine produced a mean percent MPE (43, 18, 37, 45, and 36, respectively); amoxapine, protriptyline, amitriptyline, and clomipramine, also produced a dose-related effect on attenuating the severity of morphine dependence, but not trimipramine. A higher dose had a stronger effect. ConclusionAmoxapine, trimipramine, protriptyline, amitriptyline, and clomipramine could have a lightening effect on physical dependence on morphine.