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N -methyl-D-aspartate receptor (NMDAR) antagonists have been found to be effective to inhibit morphine dependence. However, the discovery of the selective antagonist for NMDAR GluN2B with low side-effects still remains challenging. In the present study, we report a selective NMDAR GluN2B antagonist con-T[M8Q](a conantokin-T variant) that potently inhibits the naloxone-induced jumping and conditioned place preference of morphine-dependent mice at nmol/kg level, 100-fold higher than ifenprodil, a classical NMDAR NR2B antagonist. Con-T[M8Q] displays no significant impacts on coordinated locomotion function, spontaneous locomotor activity, and spatial memory mice motor function at the dose used. Further molecular mechanism experiments demonstrate that con-T[M8Q] effectively inhibited the transcription and expression levels of signaling molecules related to NMDAR NR2B subunit in hippocampus, including NR2B, p-NR2B, CaMKII-α, CaMKII-β, CaMKIV, pERK, and c-fos. The high efficacy and low side effects of con-T[M8Q] make it a good lead compound for the treatment of opiate dependence and for the reduction of morphine usage.

Opioid addiction is a chronic, relapsing disorder associated with persistent changes in brain plasticity. Reconfiguration of neuronal connectivity may explain heightened abuse liability in individuals with a history of chronic drug exposure. To characterize network-level changes in neuronal activity induced by chronic opiate exposure, we compared FOS expression in mice that are morphine-naïve, morphine-dependent, or have undergone 4 wk of withdrawal from chronic morphine exposure, relative to saline-exposed controls. Pairwise interregional correlations in FOS expression data were used to construct network models that reveal a persistent reduction in connectivity strength following opiate dependence. Further, we demonstrate that basal gene expression patterns are predictive of changes in FOS correlation networks in the morphine-dependent state. Finally, we determine that regions of the hippocampus, striatum, and midbrain are most influential in driving transitions between opiate-naïve and opiate-dependent brain states using a control theoretic approach. This study provides a framework for predicting the influence of specific therapeutic interventions on the state of the opiate-dependent brain., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

The paraventricular thalamic nucleus (PVT) is a brain region involved in regulating arousal, goal-oriented behaviors, and drug seeking, all key factors playing a role in substance use disorder. Given this, we investigated the temporal effects of administering morphine, an opioid with strongly addictive properties, on PVT neuronal function in mice using acute brain slices. Here, we show that morphine administration and electrophysiological recordings that occur during periods of animal inactivity (light cycle) elicit increases in PVT neuronal function during a 24-h abstinence time point. Furthermore, we show that morphine-induced increases in PVT neuronal activity at 24-h abstinence are occluded when morphine administration and recordings are performed during an animals' active state (dark cycle). Based on our electrophysiological results combined with previous findings demonstrating that PVT neuronal activity regulates drug-seeking behaviors, we investigated whether timing morphine administration with periods of vigilance (dark cycle) would decrease drug-seeking behaviors in an animal model of substance use disorder. We found that context-induced morphine-seeking behaviors were intact regardless of the time morphine was administered (e.g., light cycle or dark cycle). Our electrophysiological results suggest that timing morphine with various states of arousal may impact the firing of PVT neurons during abstinence. Although, this may not impact context-induced drug-seeking behaviors., (Copyright © 2019 McDevitt and Graziane.)

In opioid addiction, cues and contexts associated with drug reward can be powerful triggers for drug craving and relapse. The synapses linking ventral hippocampal outputs to medium spiny neurons of the accumbens may be key sites for the formation and storage of associations between place or context and reward, both drug-related and natural. To assess this, we implanted rats with electrodes in the accumbens shell to record synaptic potentials evoked by electrical stimulation of the ventral hippocampus, as well as continuous local-field-potential activity. Rats then underwent morphine-induced (10 mg/kg) conditioned-place-preference training, followed by extinction. Morphine caused an acute increase in the slope and amplitude of accumbens evoked responses, but no long-term changes were evident after conditioning or extinction of the place preference, suggesting that the formation of this type of memory does not lead to a net change in synaptic strength in the ventral hippocampal output to the accumbens. However, analysis of the local field potential revealed a marked sensitization of theta- and high-gamma-frequency activity with repeated morphine administration. This phenomenon may be linked to the behavioral changes-such as psychomotor sensitization and the development of drug craving-that are associated with chronic use of addictive drugs.

Methadone maintenance treatment (MMT) is the major tapering therapy for morphine addictive patients. There have gender differences reported in response to MMT. This study discovered that the estrogen-response element single nucleotide polymorphism (ERE-SNP; rs16974799, C/T) of cytochrome 2B6 gene (cyp2b6; methadone catabolic enzyme) responded differently to MMT dosing. Oestradiol was associated with high MMT dosing, high enantiomer (R- or S-) of 2-ethylidene-1,5-dimethyl-3,3-dipheny-pyrrolidine (EDDP; methadone metabolite) to methadone ratio and increased drug-seeking behaviour, implicating oestradiol-CYP-EDDP/methadone axis decreasing MMT efficacy. In mouse model, oestrogen mitigates methadone antinociceptive response, facilitates methadone catabolism and up-regulates methadone-associated metabolizing enzymes. Oestrogen also ablates chronic methadone administration-induced rewarding response. Mechanism dissection revealed the CC genotype of CYP2B6-ERE-SNP exerts higher ERE sequence alignment score, higher estrogenic response as compared to TT genotype. At last, preclinical study via targeting estrogen signal that tamoxifen (TMX; selective estrogen receptor modulator, SERM) could facilitate the tolerance phase rewarding response of methadone. Strikingly, TMX also reduces tapering/abstinence phases methadone liability in mice. In conclusion, this study demonstrates altering methadone metabolism through targeting estrogen signals might be able to free morphine addictive patients from the addiction of opioid replacement therapy. Therefore, the add-on therapy clinical trial introducing SERM in MMT regimen is suggested., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Affective memories associated with the negative emotional state experienced during opiate withdrawal are central in maintaining drug taking, seeking, and relapse. Nucleus accumbens (NAC) is a key structure for both acute withdrawal and withdrawal memories reactivation, but the NAC neuron coding properties underpinning the expression of these memories remain largely unknown. Here we aimed at deciphering the role of NAC neurons in the encoding and retrieval of opiate withdrawal memory. Chronic single neuron and local field potentials recordings were performed in morphine-dependent rats and placebo controls. Animals were subjected to an unbiased conditioned placed aversion protocol with one compartment (CS+) paired with naloxone-precipitated withdrawal, a second compartment with saline injection (CS-), and a third being neutral (no pairing). After conditioning, animals displayed a typical place aversion for CS+ and developed a preference for CS- characteristic of safety learning. We found that distinct NAC neurons code for CS+ or CS-. Both populations also displayed highly specific oscillatory dynamics, CS+ and CS- neurons, respectively, following 80 Hz (G80) and 60 Hz (G60) local field potential gamma rhythms. Finally, we found that the balance between G60 and G80 rhythms strongly correlated both with the ongoing behavior of the animal and the strength of the conditioning. We demonstrate here that the aversive and preferred environments are underpinned by distinct groups of NAC neurons as well as specific oscillatory dynamics. This suggest that G60/G80 interplay-established through the conditioning process-serves as a robust and versatile mechanism for a fine coding of the environment emotional weight.

There is growing appreciation for the importance of gastrointestinal microbiota in many physiological and pathophysiological processes. While morphine and other narcotics are the most widely prescribed therapy for moderate to severe pain clinically, they have been noted to alter microbial composition and promote bacterial translocation to other tissues. Here we examined the pharmacodynamic properties of chronic morphine in mice following bacterial depletion with oral gavage of an antibiotic cocktail (ABX). ABX significantly reduced gut bacteria and prevented chronic morphine induced increases in gut permeability, colonic mucosal destruction, and colonic IL-1β expression. In addition, ABX prevented the development of antinociceptive tolerance to chronic morphine in both the tail-immersion and acetic acid stretch assays. Morphine tolerance was also reduced by oral vancomycin that has 0% bioavailability. These findings were recapitulated in primary afferent neurons isolated from dorsal root ganglia (DRG) innervating the lower gastrointestinal tract, wherein in-vivo administration of ABX prevented tolerance to morphine-induced hypoexcitability. Finally, though ABX repeatedly demonstrated an ability to prevent tolerance, we show that it did not alter susceptibility to precipitation of withdrawal by naloxone. Collectively, these finding indicate that the gastrointestinal microbiome is an important modulator of physiological responses induced by chronic morphine administration.

The GABAergic tail of the ventral tegmental area (tVTA), also named rostromedial tegmental nucleus (RMTg), exerts an inhibitory control on dopamine neurons of the VTA and substantia nigra. The tVTA has been implicated in avoidance behaviors, response to drugs of abuse, reward prediction error, and motor functions. Stimulation of the lateral habenula (LHb) inputs to the tVTA, or of the tVTA itself, induces avoidance behaviors, which suggests a role of the tVTA in processing aversive information. Our aim was to test the impact of aversive stimuli on the molecular recruitment of the tVTA, and the behavioral consequences of tVTA lesions. In rats, we assessed Fos response to lithium chloride (LiCl), β-carboline, naloxone, lipopolysaccharide (LPS), inflammatory pain, neuropathic pain, foot-shock, restraint stress, forced swimming, predator odor, and opiate withdrawal. We also determined the effect of tVTA bilateral ablation on physical signs of opiate withdrawal, and on LPS- and LiCl-induced conditioned taste aversion (CTA). Naloxone-precipitated opiate withdrawal induced Fos in μ-opioid receptor-positive (15%) and -negative (85%) tVTA cells, suggesting the presence of both direct and indirect mechanisms in tVTA recruitment during withdrawal. However, tVTA lesion did not impact physical signs of opiate withdrawal. Fos induction was also present with repeated, but not single, foot-shock delivery. However, such induction was mostly absent with other aversive stimuli. Moreover, tVTA ablation had no impact on CTA. Although stimulation of the tVTA favors avoidance behaviors, present findings suggest that this structure may be important to the response to some, but not all, aversive stimuli.

Morphine is the most potent analgesic for chronic pain, but its clinical use has been limited by the opiate's innate tendency to produce tolerance, severe withdrawal symptoms and rewarding properties with a high risk of relapse. To understand the addictive properties of morphine, past studies have focused on relevant molecular and cellular changes in the brain, highlighting the functional roles of reward-related brain regions. Given the accumulated findings, a recent, emerging trend in morphine research is that of examining the dynamics of neuronal interactions in brain reward circuits under the influence of morphine action. In this review, we highlight recent findings on the roles of several reward circuits involved in morphine addiction based on pharmacological, molecular and physiological evidences.

Background and Purpose: The endocannabinoid (eCB) system is involved in pathways that regulate drug addiction and eCB-mediated synaptic plasticity has been linked with addictive behaviours. Here, we investigated the molecular mechanisms underlying the changes in eCB-dependent synaptic plasticity in the nucleus accumbens core (NAcc) following short-term withdrawal from repeated morphine treatment., Experimental Approach: Conditioned place preference (CPP) was used to evaluate the rewarding effects of morphine in rats. Evoked inhibitory postsynaptic currents of medium spiny neurons in NAcc were measured using whole-cell patch-clamp recordings. Changes in depolarization-induced suppression of inhibition (DSI) in the NAcc were assessed to determine the effect of short-term morphine withdrawal on the eCB system. To identify the potential modulation mechanism of short-term morphine withdrawal on the eCB system, the expression of diacylglycerol lipase α (DGL-α) and monoacylglycerol lipase was detected by Western blot analysis., Key Results: Repeated morphine administration for 7 days induced stable CPP. Compared with the saline group, the level of DSI in the NAcc was significantly increased in rats after short-term morphine withdrawal. Furthermore, this increase in DSI coincided with a significant increase in the expression of DGL-α., Conclusions and Implications: Short-term morphine withdrawal potentiates eCB modulation of inhibitory synaptic transmission in the NAcc. We also found that DGL-α expression was elevated after short-term morphine withdrawal, suggesting that the eCB 2-arachidonyl-glycerol but not anandamide mediates the increase in DSI. These findings provide useful insights into the mechanisms underlying eCB-mediated plasticity in the NAcc during drug addiction., Linked Articles: This article is part of a themed section on Endocannabinoids. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.7/issuetoc., (© 2014 The British Pharmacological Society.)

Aim: Brain-derived neurotrophic factor (BDNF) plays an important role in learning and memory in multiple brain areas. In the present study, we investigated the roles of BDNF in aversive memories associated with conditioned drug withdrawal in acute morphine-dependent rats., Methods: Conditioned place aversion (CPA) was induced in male SD rats exposed to a single dose of morphine (10 mg/kg, sc) followed by naloxone (0.3 mg/kg, sc). In some rats, BDNF receptor antagonist K252a (8.5 ng per side) or BDNF scavenger TrkB-FC (0.65 μg per side) was bilaterally microinjected into amygdala before naloxone injection. BDNF mRNA and protein expression levels in amygdala were detected after the behavior testing., Results: CPA behavior was induced in rats by the naloxone-precipitated morphine withdrawal, which was accompanied by significantly increased levels of BDNF mRNA and protein in the amygdala. Bilateral microinjection of TrkB-FC or K252a into the amygdala completely blocked CPA behavior in the rats., Conclusion: Formation of aversive memories associated with conditioned drug withdrawal in acute morphine-dependent rats requires BDNF expression in the amygdala.

Addictive behavior is increasingly accepted as a drug-associated pathological memory in which the hippocampus is profoundly engaged. It has been well documented that adaptations of synaptic plasticity of excitatory transmission in the hippocampus may contribute to opioid addiction. However, it remains unknown whether and how adaptive changes of synaptic plasticity of inhibitory transmission in the hippocampus occurs during opioid abuse. Here, we reported that a single in vivo morphine exposure (SM) did not affect inhibitory long-term depression (I-LTD) in the hippocampus, compared with saline control; while repeated morphine exposure (RM) abolished this I-LTD. Interestingly, opioid withdrawal for 3-5 days after repeated (RMW), but not a single morphine exposure (SMW), largely enhanced I-LTD. More importantly, the I-LTD in single morphine treatment is dependent on presynaptic mechanism since it can be blocked by AM251, a selective cannabinoid receptor 1 antagonist. While the large I-LTD in RMW group is dependent on combinatorial presynaptic and postsynaptic mechanisms since it can be blocked by co-application of AM251 and L-type calcium channel blocker LaCl3. Thus, these results demonstrate that opioid use and withdrawal drive the dynamics of presynaptic and postsynaptic I-LTD expression in the hippocampus that may contribute to the persistent behavioral changes during opioid abuse.

Objective: To investigate the changes of telemetry electrical activity in the infralimbic cortex (IL) of morphine-dependent rats with extinguished drug-seeking behavior., Methods: SD rats were randomly divided into model group and control group and received operations of brain stereotaxic electrode embedding in the IL. The rats in the model group were induced to acquire morphine dependence and then received subsequent extinction training, and the changes of electrical activity in the IL were recorded with a physical wireless telemetry system., Results: In rats with morphine dependence, the time staying in the white box was significantly longer on days 1 and 2 after withdrawal than that before morphine injection and that of the control rats, but was obviously reduced on days 1 and 2 after extinction training to the control level. Compared with the control group, the morphine-dependent rats on day 2 following withdrawal showed significantly increased β wave and decreased δ wave when they stayed in the white box but significantly increased δ wave and decreased α wave and β wave when they shuttled from the black to the white box. On day 2 of extinction, the model rats, when staying in the white box, showed significantly decreased θ wave compared with that of the control rats group but decreased β wave and θ wave and increased δ wave compared with those in the withdrawal period. When they shuttled from black to white box, the model rats showed decreased δ wave and increased α wave and β wave compared with those in the withdrawal period., Conclusion: Morphine-dependent rats have abnormal changes of electrical activity in the IL in drug-seeking extinction to affect their drug-seeking motive and inhibit the expression and maintenance of drug-seeking behaviors.

Background: Heroin use among young women of reproductive age has drawn much attention around the world. Although methadone is widely used in maintenance therapy for heroin/morphine addiction, the long-term effects of prenatal exposure to methadone and preventative therapy remain unclear. For revealing this question, female pregnant Sprague-Dawley rats were sub-grouped to receive (1) vehicle, (2) methadone 5 mg/kg at embryonic day 3 (E3) and then 7 mg/kg from E4 to E20, (3) dextromethorphan (DM) 3 mg/kg, and (4) methadone + DM (the rats received methadone followed by DM treatment), subcutaneously, twice a day from E3 to E20. The body weight, natural withdrawal, pain sensitivity, ED50, conditioned place preference and water maze were conducted at different postnatal stages (P1 to P79) of offspring. The quantitative real-time RT-PCR and electrophysiology were also used to measure the gene expression of opioid receptors in the spinal cord and changes of LTP/LTD in the hippocampus, separately., Results: Prenatal exposure to methadone or DM did not affect survival rate, body weight, water maze and LTP or LTD of offspring. However, prenatal methadone significantly increased the withdrawal symptoms, pain sensitivity, addiction liability and decreased the mRNA expression of pain related opioid receptors. Co-administration of DM with methadone in the maternal rats effectively prevented these abnormalities of offspring induced by methadone., Conclusions: Our study clearly showed that co-administration of dextromethorphan with methadone in the maternal rats prevented the adverse effects induced by prenatal methadone exposure. It implies that dextromethorphan may have a potential to be used in combination with methadone for maintenance treatment in pregnant heroin-addicted women to prevent the adverse effects induced by methadone on offspring.

As long-term opioids are increasingly used for control of chronic pain, how pain affects the rewarding effect of opioids and hence risk of prescription opioid misuse and abuse remains a healthcare concern and a challenging issue in current pain management. In this study, using a rat model of morphine self-administration, we investigated the molecular mechanisms underlying the impact of pain on operant behavior of morphine intake and morphine seeking before and after morphine withdrawal. We found that rats with persistent pain consumed a similar amount of daily morphine to that in control rats without pain, but maintained their level-pressing behavior of morphine seeking after abstinence of morphine at 0.2 mg/kg, whereas this behavior was gradually diminished in control rats. In the central nucleus of amygdala (CeA), a limbic structure critically involved in the affective dimension of pain, proteins of GluA1 subunits of glutamate AMPA receptors were upregulated during morphine withdrawal, and viral knockdown of CeA GluA1 eliminated the morphine-seeking behavior in withdrawn rats of the pain group. Chromatin immunoprecipitation analysis revealed that the methyl CpG-binding protein 2 (MeCP2) was enriched in the promoter region of Gria1 encoding GluA1 and this enrichment was significantly attenuated in withdrawn rats of the pain group. Furthermore, viral overexpression of CeA MeCP2 repressed the GluA1 level and eliminated the maintenance of morphine-seeking behavior after morphine withdrawal. These results suggest direct MeCp2 repression of GluA1 function as a likely mechanism for morphine-seeking behavior maintained by long-lasting affective pain after morphine withdrawal., (Copyright © 2015 the authors 0270-6474/15/353689-12$15.00/0.)

The innate immune system modulates opioid-induced effects within the central nervous system and one target that has received considerable attention is the toll-like receptor 4 (TLR4). Here, we examined the contribution of TLR4 in the development of morphine tolerance, hyperalgesia, and physical dependence in two inbred mouse strains: C3H/HeJ mice which have a dominant negative point mutation in the Tlr4 gene rendering the receptor non-functional, and B10ScNJ mice which are TLR4 null mutants. We found that neither acute antinociceptive response to a single dose of morphine, nor the development of analgesic tolerance to repeated morphine treatment, was affected by TLR4 genotype. Likewise, opioid induced hyperalgesia and opioid physical dependence (assessed by naloxone precipitated withdrawal) were not altered in TLR4 mutant or null mice. We also examined the behavioural consequence of two stereoisomers of naloxone: (-) naloxone, an opioid receptor antagonist, and (+) naloxone, a purported antagonist of TLR4. Both stereoisomers of naloxone suppressed opioid induced hyperalgesia in wild-type control, TLR4 mutant, and TLR4 null mice. Collectively, our data suggest that TLR4 is not required for opioid-induced analgesic tolerance, hyperalgesia, or physical dependence.

Background and Purpose: The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [noradrenergic activity, induction of the hypothalamic-pituitary-adrenocortical (HPA) axis and activation of heat shock proteins (Hsps)]. Corticotropin-releasing factor (CRF) pathways are important mediators in the negative symptoms of opioid withdrawal. We performed a series of experiments to characterize the role of the CRF₁ receptor in the response of stress systems to morphine withdrawal and its effect in the heart using genetically engineered mice lacking functional CRF₁ receptors., Experimental Approach: Wild-type and CRF₁ receptor-knockout mice were treated with increasing doses of morphine. Precipitated withdrawal was induced by naloxone. Plasma adrenocorticotropic hormone (ACTH) and corticosterone levels, the expression of myocardial Hsp27, Hsp27 phosphorylated at Ser⁸², membrane (MB)- COMT, soluble (S)-COMT protein and NA turnover were evaluated by RIA, immunoblotting and HPLC., Key Results: During morphine withdrawal we observed an enhancement of NA turnover in parallel with an increase in mean arterial blood pressure (MAP) and heart rate (HR) in wild-type mice. In addition, naloxone-precipitated morphine withdrawal induced an activation of HPA axis and Hsp27. The principal finding of the present study was that plasma ACTH and corticosterone levels, MB-COMT, S-COMT, NA turnover, and Hsp27 expression and activation observed during morphine withdrawal were significantly inhibited in the CRF₁ receptor-knockout mice., Conclusion and Implications: Our results demonstrate that CRF/CRF₁ receptor activation may contribute to stress-induced cardiovascular dysfunction after naloxone-precipitated morphine withdrawal and suggest that CRF/CRF₁ receptor pathways could contribute to cardiovascular disease associated with opioid addiction., (© 2013 The British Pharmacological Society.)

Learned associations between environmental cues and morphine use play an important role in the maintenance and/or relapse of opioid addiction. Although previous studies suggest that context-dependent morphine treatment alters glutamatergic transmission and synaptic plasticity in the hippocampus, their role in morphine conditioned place preference (CPP) and reinstatement remains unknown. We investigated changes in synaptic plasticity and NMDAR expression in the hippocampus after the expression, extinction, and reinstatement of morphine CPP. Here we report that morphine CPP is associated with increased basal synaptic transmission, impaired hippocampal long-term potentiation (LTP), and increased synaptic expression of the NR1 and NR2b NMDAR subunits. Changes in synaptic plasticity, synaptic NR1 and NR2b expression, and morphine CPP were absent when morphine was not paired with a specific context. Furthermore, hippocampal LTP was impaired and synaptic NR2b expression was increased after extinction of morphine CPP, indicating that these alterations in plasticity may be involved in the mechanisms underlying the learning of drug-environment associations. After extinction of morphine CPP, a priming dose of morphine was sufficient to reinstate morphine CPP and was associated with LTP that was indistinguishable from saline control groups. In contrast, morphine CPP extinguished mice that received a saline priming dose did not show CPP and had disrupted hippocampal LTP. Finally, we found that reinstatement of morphine CPP was prevented by the selective blockade of the NR2b subunit in the hippocampus. Together, these data suggest that alterations in synaptic plasticity and glutamatergic transmission play an important role in the reinstatement of morphine CPP.

Objective: This study aimed to investigate the effects of addiction to morphine and nicotine as well as their withdrawal on both baroreflex sensitivity and blood pressure in hypertensive rats., Methods: In this experimental study 40 male rats were divided into two main groups as follows: in group I, hypertensive rats received saline for 8 weeks; in group II, hypertensive rats were treated with morphine and nicotine for 8 weeks. At the end of 8 weeks group II rats were divided into four sub-groups including, 3 sub-groups of those were put on drug withdrawal protocol. At the end of experiment, blood pressure, heart rate, plasma renin activity (PRA), serum NO concentration and baroreflex sensitivity (BRS) were measured., Results: RESULTS demonstrated that BP and BRS were significantly lower in addicted to morphine and nicotine hypertensive rats compared to control (p < 0.05). Addiction withdrawal (in morphine and nicotine withdrawal rats) completely reversed BP and BRS to the pre-addiction levels (p < 0.05). Withdrawal in the only nicotine treated group lowered BP and BRS compared to group that had received morphine and nicotine together (p < 0.05)., Conclusion: RESULTS of current study may propose simultaneous morphine and nicotine withdrawal can prevent cardiovascular complications raised due to withdrawal (Fig. 5, Ref. 58).

Regulator of G protein signaling 4 (RGS4) is a critical modulator of G protein-coupled receptor (GPCR)-mediated signaling and plays important roles in many neural process and diseases. Particularly, drug-induced alteration in RGS4 protein levels is associated with acute and chronic effects of drugs of abuse. However, the precise mechanism underlying the regulation of RGS4 expression is largely unknown. Here, we demonstrated that the expression of RGS4 gene was subject to regulation by alternative splicing of the exon 6. Transformer-2β (Tra2β), an important splicing factor, bound to RGS4 mRNA and increased the relative level of RGS4-1 mRNA isoform by enhancing the inclusion of exon 6. Meanwhile, Tra2β increased the expression of full-length RGS4 protein. In rat brain, Tra2β was co-localized with RGS4 in multiple opioid action-related brain regions. In addition, the acute and chronic morphine treatment induced alteration in the expression level of Tra2β in rat locus coerulus (LC) in parallel to that of RGS4 proteins. It suggests that induction of this splicing factor may contribute to the change of RGS4 level elicited by morphine. Taken together, the results provide the evidence demonstrating the function of Tra2β as a new mediator in opioid-induced signaling pathway via regulating RGS4 expression.

Recent evidence suggests that histone deacetylase (HDAC) inhibitors facilitate extinction of rewarding memory of drug taking. However, little is known about the role of chromatin modification in the extinction of aversive memory of drug withdrawal. In this study, we used conditioned place aversion (CPA), a highly sensitive model for measuring aversive memory of drug withdrawal, to investigate the role of epigenetic regulation of brain-derived neurotrophic factor (BDNF) gene expression in extinction of aversive memory. We found that CPA extinction training induced an increase in recruiting cAMP response element-binding protein (CREB) to and acetylation of histone H3 at the promoters of BDNF exon I transcript and increased BDNF mRNA and protein expression in the ventromedial prefrontal cortex (vmPFC) of acute morphine-dependent rats and that such epigenetic regulation of BDNF gene transcription could be facilitated or diminished by intra-vmPFC infusion of HDAC inhibitor trichostatin A or extracellular signal-regulated kinase (ERK) inhibitor U0126 (1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene) before extinction training. Correspondingly, disruption of the epigenetic regulation of BDNF gene transcription with U0126 or suppression of BDNF signaling with Trk receptor antagonist K252a or BDNF scavenger tyrosine kinase receptor B (TrkB)-Fc blocked extinction of CPA behavior. We also found that extinction training-induced activation of ERK and CREB and extinction of CPA behavior could be potentiated or suppressed by intra-vmPFC infusion of d-cycloserine, a NMDA receptor partial agonist or aminophosphonopentanoic acid, a NMDA receptor antagonist. We conclude that extinction of aversive memory of morphine withdrawal requires epigenetic regulation of BDNF gene transcription in the vmPFC through activation of the ERK-CREB signaling pathway perhaps in a NMDA receptor-dependent manner.

Background and Purpose: Recent evidence suggests that glucocorticoid receptor (GR) is a major molecular substrate of addictive properties of drugs of abuse. Hence, we performed a series of experiments to further characterize the role of GR signalling in opiate withdrawal-induced physical signs of dependence, enhanced noradrenaline (NA) turnover in the hypothalamic paraventricular nucleus (PVN) and tyrosine hydroxylase (TH) phosphorylation (activation) as well as GR expression in the nucleus of the solitary tract noradrenergic cell group (NTS-A₂)., Experimental Approach: The role of GR signalling was assessed by i.p. pretreatment of the selective GR antagonist, mifepristone. Rats were implanted with two morphine (or placebo) pellets. Six days later, rats were pretreated with mifepristone or vehicle 30 min before naloxone and physical signs of abstinence, NA turnover, TH activation, GR expression and the hypothalamus-pituitary-adrenocortical axis activity were measured using HPLC, immunoblotting and RIA., Key Results: Mifepristone alleviated the somatic signs of naloxone-induced opiate withdrawal. Mifepristone attenuated the increase in the NA metabolite, 3-methoxy-4-hydroxyphenylethylen glycol (MHPG), in the PVN, and the enhanced NA turnover observed in morphine-withdrawn rats. Mifepristone antagonized the TH phosphorylation at Ser³¹ and the expression of c-Fos expression induced by morphine withdrawal. Finally, naloxone-precipitated morphine withdrawal induced up-regulation of GR in the NTS., Conclusions and Implications: These results suggest that the physical signs of opiate withdrawal, TH activation and stimulation of noradrenergic pathways innervating the PVN are modulated by GR signalling. Overall, the present data suggest that drugs targeting the GR may ameliorate stress and aversive effects associated with opiate withdrawal., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

The adaptor protein R7 family binding protein (R7BP) modulates G protein coupled receptor (GPCR) signaling and desensitization by controlling the function of regulator of G protein signaling (RGS) proteins. R7BP is expressed throughout the brain and appears to modulate the membrane localization and stability of three proteins that belong to R7 RGS family: RGS6, RGS7, and RGS9-2. RGS9-2 is a potent negative modulator of opiate and psychostimulant addiction and promotes the development of analgesic tolerance to morphine, whereas the role of RGS6 and RGS7 in addiction remains unknown. Recent studies revealed that functional deletion of R7BP reduces R7 protein activity by preventing their anchoring to the cell membrane and enhances GPCR responsiveness in the basal ganglia. Here, we take advantage of R7BP knockout mice in order to examine the way interventions in R7 proteins function throughout the brain affect opiate actions. Our results suggest that R7BP is a negative modulator of the analgesic and locomotor activating actions of morphine. We also report that R7BP contributes to the development of morphine tolerance. Finally, our data suggest that although prevention of R7BP actions enhances the analgesic responses to morphine, it does not affect the severity of somatic withdrawal signs. Our data suggest that interventions in R7BP actions enhance the analgesic effect of morphine and prevent tolerance, without affecting withdrawal, pointing to R7BP complexes as potential new targets for analgesic drugs.

Background: Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal., Methods: Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h⁻¹) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg⁻¹) was performed to assess morphine dependence., Results: In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose-response curve, as measured by per cent maximal possible effect (MPE), such that the AD₅₀ was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal., Conclusions: We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.

Unlabelled: Opioids produce analgesic effects, and extended use can produce physical dependence in both humans and animals. Dependence to opiates can be demonstrated by either termination of drug administration or through precipitation of the withdrawal syndrome by opiate antagonists. Key features of the opiate withdrawal syndrome include hyperalgesia, anxiety, and autonomic signs such as diarrhea. The rostral ventromedial medulla (RVM) plays an important role in the modulation of pain and for this reason, may influence withdrawal-induced hyperalgesia. The mechanisms that drive opiate withdrawal-induced hyperalgesia have not been elucidated. Here, rats made dependent upon morphine received naloxone to precipitate withdrawal. RVM microinjection of lidocaine, kynurenic acid (excitatory amino acid antagonist) or YM022 (CCK2 receptor antagonist) blocked withdrawal-induced hyperalgesia. Additionally, these treatments reduced both somatic and autonomic signs of naloxone-induced withdrawal. Spinal application of ondansetron, a 5HT3 receptor antagonist thought to ultimately be engaged by descending pain facilitatory drive, also blocked hyperalgesia and somatic and autonomic features of the withdrawal syndrome. These results indicate that the RVM plays a critical role in mediating components of opioid withdrawal that may contribute to opioid dependence., Perspective: Manipulations targeting these descending pathways from the RVM may diminish the consequences of prolonged opioid administration-induced dependence and be useful adjunct strategies in reducing the risk of opioid addiction., (Copyright © 2011 American Pain Society. Published by Elsevier Inc. All rights reserved.)

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines. In vivo administration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca(2+) and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the μ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba(2+)-sensitive basal K(+) currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.

Addicts repeatedly relapse to drug seeking even after years of abstinence, and this behavior is frequently induced by the recall of memories of the rewarding effects of the drug. Established memories, including those induced by drugs of abuse, can become transiently fragile if reactivated, and during this labile phase, known as reconsolidation, can be persistently disrupted. Here we show that, in rats, a morphine-induced place preference (mCPP) memory is linked to context-dependent withdrawal as disrupting the reconsolidation of the memory leads to a significant reduction of withdrawal evoked in the same context. Moreover, the hippocampus plays a critical role in linking the place preference memory with the context-conditioned withdrawal, as disrupting hippocampal protein synthesis and cAMP-dependent-protein kinase A after the reactivation of mCPP significantly weakens the withdrawal. Hence, targeting memories induced by drugs may represent an important strategy for attenuating context-conditioned withdrawal and therefore subsequent relapse in opiate addicts.

Background: Abuse of addictive substances is a serious problem that has a significant impact on areas such as health, the economy, and public safety. Heroin use among young women of reproductive age has drawn much attention around the world. However, there is a lack of information on effects of prenatal exposure to opioids on their offspring. In this study, an animal model was established to study effects of prenatal exposure to opioids on offspring., Methods: Female pregnant Sprague-Dawley rats were sub-grouped to receive (1) vehicle, (2) 2-4 mg/kg morphine (1 mg/kg increment per week), (3) 7 mg/kg methadone, and (4) 3 mg/kg buprenorphine, subcutaneously, once or twice a day from E3 to E20. The experiments were conducted on animals 8-12 weeks old and with body weight between 250 and 350 g., Results: Results showed that prenatal exposure to buprenorphine caused higher mortality than other tested substance groups. Although we observed a significantly lower increase in body weight in all of the opioid-administered dams, the birth weight of the offspring was not altered in all treated groups. Moreover, no obvious behavioral abnormality or body-weight difference was noted during the growing period (8-12 weeks) in all offspring. When the male offspring received morphine injection twice a day for 4 days, the prenatally opioid-exposed rats more quickly developed a tolerance to morphine (as shown by the tail-flick tests), most notably the prenatally buprenorphine-exposed offspring. However, the tolerance development to methadone or buprenorphine was not different in offspring exposed prenatally to methadone or buprenorphine, respectively, when compared with that of the vehicle controlled group. Similar results were also obtained in the female animals., Conclusions: Animals prenatally exposed to morphine, methadone, or buprenorphine developed tolerance to morphine faster than their controlled mates. In our animal model, prenatal exposure to buprenorphine also resulted in higher mortality and much less sensitivity to morphine-induced antinociception than prenatal exposure to morphine or methadone. This indicates that buprenorphine in higher doses may not be an ideal maintenance drug for treating pregnant women. This study provides a reference in selecting doses for clinical usage in treating pregnant heroin addicts.

Preventing and reversing opioid dependence continues to be a clinical challenge and underlying mechanisms of opioid actions remain elusive. We report that matrix metalloproteinase-9 (MMP-9) in the spinal cord contributes to development of physical dependence on morphine. Chronic morphine exposure and naloxone-precipitated withdrawal increase activity of spinal MMP-9. Spinal inhibition or targeted mutation of MMP-9 suppresses behavioral signs of morphine withdrawal and the associated neurochemical alterations. The increased MMP-9 activity is mainly distributed in the superficial dorsal horn and colocalized primarily with neurons and small numbers of astrocytes and microglia. Morphine exposure and withdrawal increase phosphorylation of NR1 and NR2B receptors, ERK1/2, calmodulin-dependent kinase II, and cAMP response element binding proteins; and such phosphorylation is suppressed by either spinal inhibition or targeted mutation of MMP-9. Further, spinal administration of exogenous MMP-9 induces morphine withdrawal-like behavioral signs and mechanical allodynia, activates NR1 and NR2 receptors, and downregulates integrin-beta1, while a function-neutralizing antibody against integrin-beta1 suppresses MMP-9-induced phosphorylation of NR1 and NR2B. Morphine withdrawal-induced MMP-9 activity is also reduced by an nNOS inhibitor. Thus, we hypothesize that spinal MMP-9 may contribute to the development of morphine dependence primarily through neuronal activation and interaction with NR1 and NR2B receptors via integrin-beta1 and NO pathways. The other gelatinase, MMP-2, is not involved in morphine dependence. Inhibiting spinal MMP-9 or MMP-2 reduces chronic and/or acute morphine tolerance. This study suggests a novel therapeutic approach for preventing, minimizing, or reversing opioid dependence and tolerance.

Chronic morphine drives adaptations in synaptic transmission thought to underlie opiate dependence. Here we examine the role of micro-opioid receptor (MOR) trafficking in one of these adaptations, specifically, changes in GABA transmission in the ventral tegmental area (VTA). To address this question, we used a knock-in mouse, RMOR (for recycling MOR), in which genetic change in the MOR promotes morphine-induced receptor desensitization and endocytosis in GABA interneurons of the VTA. In wild-type mice (postnatal days 23-28) chronic morphine (10 mg/kg, s.c., twice daily for 5 d), induced a cAMP-dependent increase in the probability of GABA release onto VTA dopamine neurons. The increased GABA release frequency correlated with physical dependence on morphine measured by counting somatic signs of morphine withdrawal, such as, tremors, jumps, rears, wet-dog shakes, and grooming behavior precipitated by subcutaneous administration of naloxone (NLX) (2 mg/kg). This adaptation in GABA release was prevented in RMOR mice given the same morphine treatment, implicating MOR trafficking in this morphine-induced change in plasticity. Importantly, treatment with the cAMP activity inhibitor rp-cAMPS [(R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium] (50 ng/0.5 microl), directly to the VTA, attenuated somatic withdrawal signs to systemic morphine produced by intra-VTA NLX (500 ng/0.5 microl), directly tying enhanced cAMP-driven GABA release to naloxone-precipitated morphine withdrawal in the VTA.

Opiate addiction is a chronic, relapsing behavioral disorder where learned associations that develop between the abused opiate and the environment in which it is consumed are brought about through Pavlovian (classical) conditioning processes. However, the signaling mechanisms/pathways regulating the mechanisms that underlie the responses to opiate-associated cues or the development of sensitization as a consequence of repeated context-independent administration of opiates are unknown. In this study we examined the phosphorylation levels of various classic signaling molecules in brain regions implicated in addictive behaviors after acute and repeated morphine administration. An unbiased place conditioning protocol was used to examine changes in phosphorylation that are associated with (1) the expression of the rewarding effects of morphine and (2) the sensitization that develops to this effect. We also examined the effects of a delta-receptor antagonist on morphine-induced conditioned behavior and on the phosphorylation of classic signaling molecules in view of data showing that blockade of delta-opioid receptor (deltaOR) prevents the development of sensitization to the rewarding effects of morphine. We find that CREB phosphorylation is specifically induced upon the expression of a sensitized response to morphine-induced conditioned behavior in brain areas related to memory consolidation, such as the hippocampus and cortex. A similar effect is also observed, albeit to a lesser extent, in the case of the GluR1 subunit of AMPA glutamate receptor. These increases in the phosphorylation levels of CREB and pGluR1 are significantly blocked by pretreatment with a deltaOR antagonist. These results indicate a critical role for phospho-CREB, AMPA, and deltaOR activities in mediating the expression of a sensitized response to morphine-dependent conditioned behavior.

Chronic opiate exposure induces neurochemical adaptations in the noradrenergic system. Enhanced responsiveness of the hypothalamo-pituitary-adrenal axis after morphine withdrawal has been associated with hyperactivity of ascending noradrenergic input from the nucleus of the solitary tract (NTS-A(2)) cell group to the hypothalamic paraventricular nucleus (PVN). This study addressed the role of morphine withdrawal-induced corticosterone (CORT) release in regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis in adrenalectomized (ADX) rats supplemented with low CORT pellet (ADX plus CORT). Present results show that in sham-ADX rats, noradrenergic neurons in the NTS-A(2) became activated during morphine withdrawal, as indicated by increased TH mRNA expression. However, this induction of TH expression is not detected in ADX plus CORT rats that are unable to mount CORT secretory response to morphine withdrawal. Total TH protein levels were elevated in the NTS-A(2) from sham-operated rats during morphine dependence and withdrawal, whereas we did not find any alteration in ADX plus CORT animals. Furthermore, high levels of TH phosphorylated (activated) at Ser31 (but not at Ser40) were found in the A(2) area from sham-morphine withdrawn rats. Consistent with these effects, we observed an increase in the enzyme activity of TH in the PVN. However, induction of morphine withdrawal to ADX plus CORT animals did not alter the phosphorylation (activation) of TH in NTS-A(2) and decreased TH activity in the PVN. These results suggest the existence of a positive reverberating circle in which elevated glucocorticoids during morphine abstinence play a permissive role in morphine withdrawal-induced activation of noradrenergic pathway innervating the PVN.

The Gbetagamma subunit has been implicated in many downstream signaling events associated with opioids. We previously demonstrated that a small molecule inhibitor of Gbetagamma-subunit-dependent phospholipase (PLC) activation potentiated morphine-induced analgesia (Bonacci et al., 2006). Here, we demonstrate that this inhibitor, M119 (cyclohexanecarboxylic acid [2-(4,5,6-trihydroxy-3-oxo-3H-xanthen-9-yl)-(9Cl)]), is selective for mu-opioid receptor-dependent analgesia and has additional efficacy in mouse models of acute tolerance and dependence. When administered by an intracerebroventricular injection in mice, M119 caused 10-fold and sevenfold increases in the potencies of morphine and the mu-selective peptide, DAMGO, respectively. M119 had little or no effect on analgesia induced by the kappa agonist U50,488 or delta agonists DPDPE or Deltorphin II. Similar results were obtained in vitro, as only activation of the mu-opioid receptor stimulated PLC activation, whereas no effect was seen with the kappa- and delta-opioid receptors. M119 inhibited mu-receptor-dependent PLC activation. In studies to further explore the in vivo efficacy of M119, systemic administration M119 also resulted in a fourfold shift increase in potency of systemically administered morphine. Of particular interest, M119 was also able to attenuate acute, antinociceptive tolerance and dependence in mice treated concomitantly with both M119 and morphine. These studies suggest that small organic molecules, such as M119, that specifically regulate Gbetagamma subunit signaling may have important therapeutic applications in enhancing opioid analgesia, while attenuating the development of tolerance and dependence.

C57BL/6J (B6) and DBA/2J (D2) mice differ in behaviors related to substance abuse, including voluntary morphine consumption and preference in a two-bottle choice paradigm. Two major quantitative trait loci (QTL) for morphine consumption and preference exist between these strains on chromosomes (Chrs.) 6 and 10 when the two-bottle choice involves morphine in saccharin vs quinine in saccharin. Here, we report the refinement of the Chr. 10 QTL in subcongenic strains of D2.B6-Mop2 congenic mice described previously. With these subcongenic mouse strains, we have divided the introgressed region of Chr. 10 containing the QTL gene(s) into two segments, one between the acromere and Stxbp5 (in D2.B6-Mop2-P1 mice) and the other between marker D10Mit211 and marker D10Mit51 (in D2.B6-Mop2-D1 mice). We find that, similar to B6 mice, the D2.B6-Mop2-P1 congenic mice exhibit a strong preference for morphine over quinine, whereas D2.B6-Mop2-D1 congenic mice avoid morphine (similar to D2 mice). We have also created a line of double congenic mice, B6.D2-Mop2.Qui, which contains both Chr. 10 and Chr. 6 QTL. We find that they are intermediate in their morphine preference scores when compared with B6 and D2 animals. Overall, these data suggest that the gene(s) involved in morphine preference in the morphine-quinine two-bottle choice paradigm are contained within the proximal region of Chr. 10 (which harbors Oprm1) between the acromere and Stxbp5, as well as on distal Chr. 6 between marker D6Mit10 and the telomere.

Objective: To observe the effects of gamma-aminobutyric acid (GABA) on the electric activities of pain-excited neurons (PEN) in nucleus accumbens (NAc) in central nervous system (CNS) of morphine-dependent rats., Methods: After GABA or the GABA(A)-receptor antagonist, bicuculline (Bic), was injected into cerebral ventricles or NAc, right sciatic nerve was stimulated by electrical pulses, which was considered as traumatic pain stimulation. Extracellular recordings methods were used to record the electric activities of PEN in NAc., Results: When GABA was injected into intracerebroventricle (ICV) as well as NAc, it could decrease the pain-evoked discharge frequency and prolong the latency of PEN. Bic could interdict the above effects of GABA on the electric activities of PEN., Conclusion: Exogenous GABA might have an inhibitory effect on the central pain adjustment. Furthermore, GABA and GABA(A) receptor participate and mediate the traumatic information transmission process in CNS.

Aim: To determine whether testosterone is involved in morphine withdrawal syndrome (WS)., Methods: In order to induce dependency, rats were treated with subcutaneous injection of morphine (days 1-2, 5 mg/kg; days 3-5, 7.5 mg/kg; days 6-8, 10 mg/kg), and after the last dose of morphine (day 8) WS was induced by intraperitoneal injection of naloxone (1 mg/kg). Wet dog shake (WDS), abdomen writhing (AW), and jumps (J) were recorded as indicators of WS., Results: The severity of WDS, AW, and J in male rats was greater than that in females. Accordingly, in 4-week castrated and flutamide-treated (10 mg/kg/day for 8 days, i.p.) male rats, WDS, AW, and J were significantly decreased compared to male control rats. Testosterone replacement therapy (10 mg/kg/day for 8 days, i.m.) in 4-week castrated rats restored the severity of WDS, AW, and J behaviors to the level of non-castrated male rats, whereas testosterone potentiated the WDS behavior in non-castrated male rats., Conclusion: It can be concluded that testosterone might be effectively involved in morphine WS., ((c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.)

Tolerance to the pain-relieving effects of opiates limits their clinical use. Although morphine tolerance is associated with desensitization of mu-opioid receptors, the underlying cellular mechanisms are not understood. One problem with the desensitization hypothesis is that acute morphine does not readily desensitize mu-opioid receptors in many cell types. Given that neurons in the periaqueductal gray (PAG) contribute to morphine antinociception and tolerance, an understanding of desensitization in PAG neurons is particularly relevant. Opioid activity in the PAG can be monitored with activation of G-protein-mediated inwardly rectifying potassium (GIRK) currents. The present data show that opioids have a biphasic effect on GIRK currents in morphine tolerant rats. Opioid activation of GIRK currents is initially potentiated in morphine (EC(50)=281 nM) compared to saline (EC(50)=8.8 microM) pretreated rats as indicated by a leftward shift in the concentration-response curve for met-enkephalin (ME)-induced currents. These currents were inhibited by superfusion of the mu-opioid receptor antagonist beta-funaltrexamine (beta-FNA) suggesting that repeated morphine administration enhances agonist stimulation of mu-opioid receptor coupling to G-proteins. Although supersensitivity of mu-opioid receptors in the PAG is counterintuitive to the development of tolerance, peak GIRK currents from tolerant rats desensitized more than currents from saline pretreated rats (56% of peak current after 10 min compared to 15%, respectively). These data indicate that antinociceptive tolerance may be triggered by enhanced agonist potency resulting in increased desensitization of mu-opioid receptors.

The mechanisms underlying responses to drugs of abuse have been widely investigated; however, less is known about pathways normally protective against the development of drug reinforcement. These pathways are also important since they may regulate individual differences in vulnerability to addiction. The neuropeptide galanin and its binding sites are expressed in brain areas important for drug reward. Previous studies have shown that centrally infused galanin attenuates morphine place preference and peripheral injection of galnon, a galanin agonist, decreases opiate withdrawal signs. The current studies in galanin knockout (GKO) mice examined the hypothesis that galanin is an endogenous negative regulator of opiate reward and identified downstream signaling pathways regulated by galanin. We show that GKO mice demonstrate increased locomotor activation following morphine administration, which is inhibited by acute administration of galnon. GKO mice also show enhanced morphine place preference, supporting the idea that galanin normally antagonizes opiate reward. In addition, morphine-induced ERK1/2 phosphorylation was increased in the VTA of both wild-type and GKO mice, but only the GKO mice showed increases in ERK1/2 and CREB phosphorylation in the amygdala or nucleus accumbens. Furthermore, a single systemic injection of galnon in GKO mice was sufficient to reverse some of the biochemical changes brought about by morphine administration. These data suggest that galanin normally attenuates behavioral and neurochemical effects of opiates; thus, galanin agonists may represent a new class of therapeutic targets for opiate addiction.

As functional changes in L-type high voltage-gated calcium channels (HVCCs) are recognized to be one of the major neurochemical modifications occurring in brains of animals with morphine physical dependence, this study attempts to examine whether regional difference in the expressions of HVCC subunits are produced in the brains under such pathological conditions. Scatchard analysis of [(3)H]PN200-110 binding showed increased Bmax values in the cerebral cortex and the mesolimbic region including the nucleus accumbence, which are brain regions participating in the development of morphine physical dependence, but not in the cerebellum. In the former two brain regions, alpha1C and alpha1D subunits of L-type HVCCs and alpha2/delta1 subunit increased, although decreases of alpha1B and alpha2/delta1 subunits were observed in the cerebellum. A single dose of morphine did not change the expression of any of the HVCC subunits. These results indicate that the increased L-type HVCC subunits in the cerebral cortex and mesolimbic region participate in the development of morphine physical dependence.

Objective: To investigate the influence of dopamine (DA) and DA receptor's antagonist on the transmission of noxious information in the central nervous system of normal rats or morphinistic rats., Methods: The influence of DA on the electric activity of the pain-excited neuron (PEN) in the caudate nucleus (Cd) of normal rats or morphinistic rats was recorded after the sciatic nerve was noxiously stimulated., Results: DA shortened the average latency of the evoked discharge of PEN in the Cd of normal rats, indicating that DA could increase the activity of PEN and pain sensitivity in normal rats. This effect could be inhibited by Droperidol. DA increased the average latency of the evoked discharge of PEN in the Cd of morphinistic rats, indicating that DA could inhibit the activity of PEN and pain sensitivity in morphinistic rats., Conclusion: The responses to painful stimulation were completely opposite between normal rats and morphinistic rats after the intracerebroventricular injection of DA.

Opiate dependence develops due to changes in intracellular signaling caused by long-term exposure to morphine. Here we investigated changes in the mRNA expression of the main classes of G-protein alpha (Galpha) subunits in various brain regions in morphine-dependent rats. Rats received increasing doses of morphine, 10-50 mg/kg, b.i.d., for 14 days. G-protein alpha-subunit mRNA expression was determined shortly following the conclusion of chronic morphine administration (2 h after the final dose) and during withdrawal (48 h after the final dose). Significant changes in mRNA expression for Galpha subunits were observed in several brain areas during withdrawal, while the changes were much less evident or absent 2 h after the final drug injection. Changes in mRNA expression were particularly evident in the nucleus accumbens (increases in Galpha(12), Galpha(q), Galpha(11), and Galpha(o) during withdrawal, increase in Galpha(i) and decrease in Galpha(s) just following treatment). The direction of the changes, which were not all significant, for Galpha(12), Galpha(q), and Galpha(11) was generally consistent in the amygdala and prefrontal cortex; changes in G proteins coupled to the adenylyl cyclase cascade were less consistent. These results suggest that morphine dependence leads to alterations in intracellular signaling, which are reflected in changes in the expression of genes encoding various G proteins. The results may explain why signs of opiate dependence are not expressed during chronic administration of morphine, but only after cessation of the treatment.

Objective: To evaluate the effects of Caulis Sinomenii and sinomenine on conditioned place preference (CPP) induced by morphine and brain histamine level in mice., Methods: Sixty mice were randomized into 6 equal groups and morphine (Mor) was injected subcutaneously (9 mg/kg) for 6 consecutive days to induce CPP using a shuttle box. Since the 4th day of training, the mice in 5 of the groups were treated for 3 consecutive days with Caulis Sinomenii (10 g/kg), sinomenine (60 mg/kg), diphenhydramine (30 mg/kg), CP48/80 (5 mg/kg) and L-histidine (750 mg/kg) in addition to morphine (9 mg/kg) treatment, respectively, leaving the other group with exclusive morphine treatment. Another 10 mice received saline injection to serve as saline control group. The content of histamine (HA) in the mouse brain was measured by fluorospectrophotometry., Results: In morphine group, the mice showed significantly extended stay in morphine-paired compartment whose HA content in the brain was markedly increased (P<0.01). Treatment with Caulis Sinomenii and sinomenine resulted in significantly reduced time of stay in morphine-paired compartment and brain HA level (P<0.01)., Conclusion: CPP induced by morphine in mice is associated with increased HA level in the brain. Caulis Sinomenii and sinomenine can suppress the acquisition of place preference induced by morphine and modulate HA level in the central nervous system in morphine-dependent mice.

Background: Although dopamine transporter (DAT) is essential for addiction, the effect of additive drugs on DAT function is still controversial, especially for opiates. We investigated the functional changes of dopamine transporter in striatum of rhesus monkeys during acute morphine injection and its abstinence., Methods: Four rhesus monkeys, 6 to 9 years old, two male and two female, were examined for 12 days. Single photon emission computed tomography (SPECT) was performed with (99)T(cm)-TRODAT-1 as the radiopharmaceutical dopamine transporter agent during different stages of acute morphine injection and its abstinence. The ratios of SPECT signal between striatum and cerebellum (ST/CB) were calculated., Results: The ST/CB ratio declined significantly on the first day of morphine injection and continued declining with more morphine injections. After abstinence, the ratio increased with time, but was still significantly lower on the 5th day of abstinence than the normal level., Conclusions: In rhesus monkey, acute morphine injection has both rapid and lasting effects on DAT by downregulating its function. The decline was partially reversible following morphine abstinence. The results suggest that striatum is one effective target of morphine and that the DAT function in striatum is one indicator for morphine addiction.

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.

Substantial evidence indicates that the ventral tegmental area (VTA) of the mesocorticolimbic dopaminergic (DA) system has a key role in mechanisms of opiate dependence. Although DA neurons have been studied extensively, little is known about their activity and their response to acute morphine during morphine dependence. We recorded the activity of VTA DA neurons in five groups of anesthetized rats: drug-naive (naive) rats, morphine-dependent [(MD) implanted with pellets] rats, and three groups of withdrawn rats. Withdrawals either were precipitated by naltrexone or occurred spontaneously 24 h or 15 d after pellet removal. We confirmed that acute morphine in naive rats produced a marked increase in the firing of VTA DA neurons. We also found that the basal firing rate of VTA DA neurons was markedly higher in MD than in naive rats; however, in MD rats, acute morphine failed to increase DA activity. We confirmed inhibition of VTA DA activity in MD rats in response to precipitated withdrawal; however, this inhibition resulted only in a normalization of the firing rate to that of naive animals. In rats that had spontaneous withdrawal after 24 h or 15 d, the activity of VTA DA neurons was similar to that of naive rats, and an acute injection of morphine failed to alter their activity. Our results indicate that VTA DA neurons show long-lasting tolerance to the acute effect of morphine after withdrawal. These findings show that VTA DA neural activity is unlikely to be a factor in the altered behavioral responses that occur with acute morphine or naltrexone administration after chronic opiate exposure.

The purpose of this study was the evaluation of clinical heroin symptoms and buprenorphine drug addiction in the withdrawal period with the purpose of their comparison, study of parameters of bone metabolism in the both groups. In the study group were included 40 patients with heroin and 27 with buprenorphine addiction in the period of abstinence. Our investigations have shown, that in the both groups, among clinical symptoms ossalgias, arthralgias and mialgias attributes to the expressed dysfunction of vegetative system, were most prominent. Decrease of sexual functions was found in half of inspected patients. Biochemical investigations have shown intensive clearance of calcium with the urine that indicates intensifying resorbtion processes in the bone tissue. Symptoms of hypogonadism were accompanied by the decrease of the level of testosterone in the blood. Parameters of mineral consistency of the bone tissue was decreased both in patients with heroin and buprenorphine addiction.

In human addicts, craving and relapse are frequently evoked by the recall of memories connected to a drug experience. Established memories can become labile if recalled and can then be disrupted by several interfering events and pharmacological treatments, including inhibition of protein synthesis. Thus, reactivation of mnemonic traces provides an opportunity for disrupting memories that contribute to pathological states. Here, we tested whether the memory of a drug experience can be weakened by inhibiting protein synthesis after the reactivation of its trace. We found that an established morphine conditioned place preference (mCPP) was persistently disrupted if protein synthesis was blocked by either anisomycin or cycloheximide after the representation of a conditioning session. Unlike other types of memories, an established mCPP did not become labile after contextual recall, but required the concomitant re-experience of both the conditioning context and the drug. An established mCPP was disrupted after the conditioning session if protein synthesis was blocked selectively in the hippocampus, basolateral amygdala, or nucleus accumbens but not in the ventral tegmental area. This disruption seems to be permanent, because the preference did not return after further conditioning. Thus, established memories induced by a drug of abuse can be persistently disrupted after reactivation of the conditioning experience.

Changes of intestinal motility and transit produced by tolerance to and dependence upon morphine have been partly attributed to peripheral mechanisms. We evaluated the effect of chronic peripheral morphine administration and peripheral mu-receptor blockade on vagal afferent activity (VAA) and c-Kit positive intramuscular cells of Cajal (ICCs). Ten rats were subjected to chronic subcutaneous morphine infusion for 72 h with subsequent VAA recording. Potential frequency was evaluated within recordings before and after mu receptor blockade by (D)-Phe -Cys -Tyr -(D)-Trp -Orn -Thr -Phe -Thr (CTOP) i.p. injections. Afterwards the rats were sacrificed and intramuscular c-Kit antigen expression was assessed by image analysis within removed fragments of duodenum and ascending colon. An equal group of rats served as a control for VAA and c-Kit expression. Analysis of VAA revealed similar frequencies of potentials in morphine tolerant / dependent rats before CTOP and in the controls. CTOP increased potential frequency in the morphine group which effect was visible mostly within the first 20 minutes (p=0.01). The morphine infused animals presented also higher c-Kit expression in both the duodenum (p<0.001) and the ascending colon (p<0.001) in comparison to the control group. Results of our study may indicate the involvement of both the intestinal wall and the long vago-vagal reflexes in tolerance to and dependence upon opioids.

We have previously shown that abrupt withdrawal (AW) from morphine induces greater than 80% immunosuppression in murine spleen cells, as assessed by the capacity to mount an in vitro plaque-forming cell response to sheep red blood cells. Present studies about the mechanisms of immunosuppression following AW showed that addition of highly enriched (CD11b+) splenic macrophages (obtained by cell sorting or magnetic separation) from AW mice to cultures of normal, unfractionated spleen cells suppressed immune responses. Further, addition of highly enriched (CD19+) B cells (but not T cells) from AW mice to normal cells was also immunosuppressive. B cells from AW mice were also able to inhibit the proliferative response of normal spleen cells to concanavalin A but not to lipopolysaccharide. Overall, the data suggest that immunosuppression by AW spleen cells is a result of active suppression by macrophages and B cells.

The purpose of the present study is to compare the capacity of opioid antagonists to elicit withdrawal jumping in mice following two acute pretreatment doses of the opioid agonist morphine. Antagonists that precipitate vigorous withdrawal jumping across both morphine treatment doses are hypothesized to be strong inverse agonists at the mu-opioid receptor, whereas antagonists that elicit withdrawal jumping in mice treated with the high but not the low dose of morphine are hypothesized to be weak inverse agonists. Male, Swiss-Webster mice (15-30 g) were acutely treated with 56 or 180 mg kg(-1) morphine 4 h prior to injection with naloxone, naltrexone, diprenorphine, nalorphine, or naloxonazine. Vertical jumping, paw tremors, and weight loss were recorded. Naloxone, naltrexone, and diprenorphine produced withdrawal jumping after 56 and 180 mg kg(-1)morphine pretreatment. Nalorphine and naloxonazine produced moderate withdrawal jumping after 180 mg kg(-1) morphine pretreatment, but failed to elicit significant withdrawal jumping after 56 mg kg(-1) morphine pretreatment. Nalorphine and naloxonazine blocked the withdrawal jumping produced by naloxone. All antagonists produced paw tremors and weight loss although these effects were generally not dose-dependent. Taken together, these findings reveal a rank order of negative intrinsic efficacy for these opioid antagonists as follows: naloxone=naltrexone> or =diprenorphine>nalorphine=naloxonazine. Furthermore, the observation that nalorphine and naloxonazine blocked the naloxone-induced withdrawal jumping provides additional evidence that nalorphine and naloxonazine are weaker inverse agonists than naloxone.