Changes in the Levels of p-ERK,p-CREB,and c-fos in Rat Mesocorticolimbic Dopaminergic System After Morphine-Induced Conditioned Place Preference: The Role of Acute and Subchronic Stress

ERK pathway plays a critical role in the cellular adaptive responses to environmental changes. Stressful conditions can induce the activation of activate ERK, and its downstream targets, CREB and c-fos, in neural cells. Exposure to opioids has the same effect. In this study, we investigated the effects of morphine-induced conditioned place preference (CPP) on p-ERK/ERK ratio, p-CREB/CREB ratio and c-fos level in the mesocorticolimbic dopaminergic system including the nucleus accumbens (NAc), amygdala (AMY), striatum (Str), and prefrontal cortex (PFC).Our aim was to determine if acute and subchronic stress would affect these alterations. Male Wistar rats were divided into two saline- and morphine-treated groups. Each group contained of control, acute stress, and subchronic stress subgroups. The CPP procedure was performed for all of the rats. We dissected out the NAc, AMY, Str, and PFC regions and measured the mentioned ratios and c-fos level by Western blot analysis. The results revealed that in saline-treated animals, all factors enhanced significantly after performing acute and subchronic stress while there was an exception in p-ERK/ERK ratio in the Str and PFC; the changes were not significant during acute stress. Conditioning score decreased after applying the subchronic but not acute stress. In morphine-treated animals, all factors were increased after application of acute and subchronic stress, and conditioning scores also decreased after stress. Our findings suggest that in saline- or morphine-treated animals, acute and subchronic stress increases p-ERK, p-CREB, and c-fos levels in the mesocorticolimbic system. It has been shown that morphine induces the enhancement of the mentioned factors; on the other hand, our result demonstrates that stress can amplify these changes.     

Dynamic Changes of Tyrosine Hydroxylase and Dopamine Concentrations in the Ventral Tegmental Area-Nucleus Accumbens Projection During the Expression of Morphine-Induced Conditioned Place Preference in Rats

Our previous study demonstrated that morphine dose- and time-dependently elevated dopamine (DA) concentrations in the nucleus accumbens (NAc) during the expression of morphine-induced conditioned place preference (CPP) in rats. However, still unknown are how DA concentrations dynamically change during the morphine-induced CPP test and whether tyrosine hydroxylase (TH) activity in the ventral tegmental area (VTA) plays a vital role in this process. In the present study, we measured dynamic changes in TH and phosphorylated TH serine 40 (pTH Ser(40)) and pTH Ser(31) proteins in the VTA, and DA concentrations in the NAc at 5 min intervals during a 30 min morphine-induced CPP test. Rats that underwent morphine-induced CPP training significantly preferred the morphine-paired chamber during the CPP expression test, an effect that lasted at least 30 min in the drug-free state. DA concentrations in the NAc markedly increased at 15 min when the rats were returned to the CPP boxes to assess the expression of preference for the previously drug-paired chamber. DA concentrations then declined 2 h after the CPP test. TH and pTH Ser(40) levels, but not pTH Ser(31) levels, in the VTA were enhanced during the CPP test. These results indicated that TH and the phosphorylation of TH Ser(40) in the VTA may be responsible for DA synthesis and release in the NAc during the behavioral expression of conditioned reward elicited by a drug-associated context.     

伏隔核壳部巴氯芬灌注阻断吗啡成瘾小鼠条件位置性偏爱记忆再巩固

氨基丁酸B型受体(GABAB受体)是治疗药物成瘾的潜在靶点,伏隔核壳部(nucleus accumbens shell, AcbSh)是成瘾环路的关键节点,但AcbSh GABA_B受体与记忆再巩固的关系尚不清楚。本文旨在探讨AcbSh微量灌注GABA_B受体激动剂巴氯芬(baclofen, BLF)对吗啡奖赏记忆再巩固及复吸行为的影响。建立吗啡条件位置性偏爱(conditioned place preference, CPP)小鼠模型,采用吗啡奖赏记忆提取激活实验,对比观察环境线索激活吗啡奖赏记忆后,双侧AcbSh灌注BLF对吗啡CPP、吗啡激发CPP重建以及自主活动量的影响。结果表明,吗啡奖赏记忆激活后,Acb Sh单次注入0.06nmol/0.2μL/侧或0.12nmol/0.2μL/侧BLF显著抑制吗啡CPP,且吗啡激发不能重建CPP,而0.01nmol/0.2μL/侧BLF灌注不能抑制吗啡CPP。激活后注入生理盐水及未激活组BLF灌注均未抑制CPP。无论是否激活吗啡奖赏记忆,BLF注入AcbSh都不影响小鼠自主活动。以上结果提示,AcbSh GABA_B受体参与了吗啡CPP的记忆再巩固。记忆激活后激动AcbSh GABA_B受体可通过阻断吗啡CPP的记忆再巩固,消除奖赏记忆,抑制复吸行为。     

Modification by the tissue plasminogen activator-plasmin system of morphine-induced dopamine release and hyperlocomotion, but not anti-nociceptive effect in mice

The extracellular serine protease tissue plasminogen activator (tPA) that converts plasminogen into plasmin is abundantly expressed throughout the central nervous system. We have recently demonstrated that the tPA-plasmin system participates in the rewarding and locomotor-stimulating effects of morphine by acutely regulating morphine-induced dopamine release in the nucleus accumbens (NAc). In the present study, we examined the effects of microinjections of plasminogen activator inhibitor-1 (PAI-1), tPA or plasmin into the NAc on morphine-induced dopamine release, hyperlocomotion and anti-nociceptive effects in ICR mice. A single morphine treatment resulted in an increase in protein levels of PAI-1 in the NAc. Microinjection of PAI-1 into the NAc dose-dependently reduced morphine-induced dopamine release and hyperlocomotion. In contrast, microinjection of tPA into the NAc significantly potentiated morphine-induced dopamine release and hyperlocomotion without affecting basal levels. Furthermore, microinjection of plasmin enhanced morphine-induced dopamine release, but did not modify the hyperlocomotion induced by morphine. The intracerebroventricular injection of PAI-1, tPA and plasmin at high doses had no effect on the anti-nociceptive effects of morphine. These results suggest that the tPA-plasmin system is involved in the regulation of morphine-induced dopamine release and dopamine-dependent behaviors but not the anti-nociceptive effects of morphine.     

Administration of the Glial Condition Medium in the Nucleus Accumbens Prolong Maintenance and Intensify Reinstatement of Morphine-Seeking Behavior

Accumulating evidence suggested that glial cells are involved in synaptic plasticity and behavioral changes induced by drugs abuse. The role of these cells in maintenance and reinstatement of morphine (MRP) conditioned place preference (CPP) remains poorly characterized. The aim of present study was to investigate the direct role of glial cells in nucleus accumbens (NAc) in the maintenance and reinstatement of MRP-seeking behavior. CPP induced with injection of MRP (5 mg/kg, s.c. for 3 days), lasted for 7 days after cessation of MRP treatment and priming dose of MRP (1 mg/kg, s.c.) reinstated the extinguished MRP-induced CPP. The astrocyte-conditioned medium (ACM) and neuroglia conditioned medium (NCM) exposed to MRP (10 and 100 µM) have been microinjected into the NAc. Intra-NAc administration of ACM during extinction period failed to change the maintenance of MRP-CPP, but MRP 100-treated ACM could slightly increase the magnitude of reinstatement. In contrast to ACM, intra-NAc administration of MRP 100-treated NCM caused slower extinction by 3 days and significantly increased the magnitude of reinstatement. Our findings suggest the involvement of glial cells activation in the maintenance and reinstatement of MRP-seeking behaviors, and provides new evidence that these cells might be a potential target for the treatment of MRP addiction.

     

The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats

Previous studies suggested that electroacupuncture (EA) can suppress opioid dependence by the release of endogenous opioid peptides. To explore the site of action and the receptors involved, we tried to inject highly specific agonists for μ-, δ- and κ-opioid receptors into the CNS to test whether it can suppress morphine-induced conditioned place preference (CPP) in the rat. Male Sprague–Dawley rats were trained with 4 mg/kg morphine, i.p. for 4 days to establish the CPP model. This CPP can be prevented by (a) i.p. injection of 3 mg/kg dose of morphine, (b) intracerebroventricular (i.c.v.) injection of micrograms doses of the selective μ-opioid receptor agonist DAMGO, δ-agonist DPDPE or κ-agonist U-50,488H or (c) microinjection of DAMGO, DPDPE or U50488H into the shell of the nucleus accumbens (NAc). The results suggest that the release of endogenous μ-, δ- and κ-opioid agonists in the NAc shell may play a role for EA suppression of opiate addiction.     

Effect of Yulangsan Polysaccharide on the Reinstatement of Morphine-Induced Conditioned Place Preference in Sprague–Dawley Rats

We previously reported that Yulangsan polysaccharide (YLSP), which was isolated from the root of Millettia pulchra Kurz, attenuates withdrawal symptoms of morphine dependence by regulating the nitric oxide pathway and modulating monoaminergic neurotransmitters. In this study, we investigated the effects and mechanism of YLSP on the reinstatement of morphine-induced conditioned place preference (CPP) in rats. A CPP procedure was employed to assess the behavior of rats, and indicators of serum and four brain regions (nucleus accumbens, ventral tegmental area, hippocampus and prefrontal cortex) were determined to explore its underlying mechanism. YLSP inhibited priming morphine-induced reinstatement of CPP in a dose-dependent manner. YLSP markedly reduced nitric oxide and nitric oxide synthase levels in the brain. Moreover, YLSP significantly decreased the dopamine and norepinephrine levels in the serum and brain. Furthermore, YLSP significantly decreased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) concentrations, inhibited the expression of dopamine D1 receptors and cAMP response element binding protein mRNA, and improved the expression of dopamine D2 receptor mRNA in the four brain regions. Our findings indicated that YLSP could inhibit the reinstatement of morphine-induced CPP possibly by modulating the NO-cGMP and D1R-cAMP signaling pathways.     

Possible involvement of protease-activated receptor-1 in the regulation of morphine-induced dopamine release and hyperlocomotion by the tissue plasminogen activator-plasmin system

We have previously demonstrated that tissue plasminogen activator (tPA)-plasmin system participates in the rewarding effect of morphine, by regulating dopamine release in the nucleus accumbens (NAc). However, it is unclear how plasmin increases the morphine-induced release of dopamine and hyperlocomotion. In the present study we investigated whether protease activated receptor-1 (PAR-1) is involved in the regulation of acute morphine-induced dopamine release by the tPA-plasmin system. Morphine significantly but transiently increased extracellular tPA activity in the NAc, which was completely blocked by naloxone. Microinjection of a PAR-1 antagonist, (tyr(-1))-thrombin receptor activating peptide 7, into the NAc significantly reduced morphine-induced dopamine release in the NAc and hyperlocomotion although the treatment had no effect on basal dopamine release and spontaneous locomotor activity. Furthermore, the PAR-1 antagonist blocked the ameliorating effect of plasmin on the defect of morphine-induced dopamine release in the NAc of tPA-deficient mice. In contrast, intracerebroventricular injection of the PAR-1 antagonist had no effect on the antinociceptive effects of morphine in mice. These results suggest that PAR-1 is a target for the tPA-plasmin system in the regulation of acute morphine-induced dopamine release in the NAc.     

High-Frequency Stimulation of Nucleus Accumbens Changes in Dopaminergic Reward Circuit

Deep brain stimulation (DBS) of the nucleus accumbens (NAc) is a potential remedial therapy for drug craving and relapse, but the mechanism is poorly understood. We investigated changes in neurotransmitter levels during high frequency stimulation (HFS) of the unilateral NAc on morphine-induced rats. Sixty adult Wistar rats were randomized into five groups: the control group (administration of saline), the morphine-only group (systematic administration of morphine without electrode implantation), the morphine-sham-stimulation group (systematic administration of morphine with electrode implantation but not given stimulation), the morphine-stimulation group (systematic administration of morphine with electrode implantation and stimulation) and the saline-stimulation group (administration of saline with electrode implantation and stimulation). The stimulation electrode was stereotaxically implanted into the core of unilateral NAc and microdialysis probes were unilaterally lowered into the ipsilateral ventral tegmental area (VTA), NAc, and ventral pallidum (VP). Samples from microdialysis probes in the ipsilateral VTA, NAc, and VP were analyzed for glutamate (Glu) and γ-aminobutyric acid (GABA) by high-performance liquid chromatography (HPLC). The levels of Glu were increased in the ipsilateral NAc and VP of morphine-only group versus control group, whereas Glu levels were not significantly changed in the ipsilateral VTA. Furthermore, the levels of GABA decreased significantly in the ipsilateral NAc, VP, and VTA of morphine-only group when compared with control group. The profiles of increased Glu and reduced GABA in morphine-induced rats suggest that the presence of increased excitatory neurotransmission in these brain regions. The concentrations of the Glu significantly decreased while the levels of GABA increased in ipsilateral VTA, NAc, and VP in the morphine-stimulation group compared with the morphine-only group. No significant changes were seen in the morphine-sham stimulation group compared with the morphine-only group. These findings indicated that unilateral NAc stimulation inhibits the morphine-induced rats associated hyperactivation of excitatory neurotransmission in the mesocorticolimbic reward circuit.     

Reversal of cocaine-conditioned place preference through methyl supplementation in mice: altering global DNA methylation in the prefrontal cortex

Analysis of global methylation in cells has revealed correlations between overall DNA methylation status and some biological states. Recent studies suggest that epigenetic regulation through DNA methylation could be responsible for neuroadaptations induced by addictive drugs. However, there is no investigation to determine global DNA methylation status following repeated exposure to addictive drugs. Using mice conditioned place preference (CPP) procedure, we measured global DNA methylation level in the nucleus accumbens (NAc) and the prefrontal cortex (PFC) associated with drug rewarding effects. We found that cocaine-, but not morphine- or food-CPP training decreased global DNA methylation in the PFC. Chronic treatment with methionine, a methyl donor, for 25 consecutive days prior to and during CPP training inhibited the establishment of cocaine, but not morphine or food CPP. We also found that both mRNA and protein level of DNMT (DNA methytransferase) 3b in the PFC were downregulated following the establishment of cocaine CPP, and the downregulation could be reversed by repeated administration of methionine. Our study indicates a crucial role of global PFC DNA hypomethylation in the rewarding effects of cocaine. Reversal of global DNA hypomethylation could significantly attenuate the rewarding effects induced by cocaine. Our results suggest that methionine may have become a potential therapeutic target to treat cocaine addiction.     

Proteomic analysis of rat prefrontal cortex in three phases of morphine-induced conditioned place preference

Morphological alterations of synapse are found after morphine administration, suggesting that regulation of synaptic plasticity may be one of the mechanisms of neuroadaptation in addiction. However, the molecular basis underlying the abnormal synapse morphological and physiological changes in the morphine-induced dependence, withdraw, and relapse is not well understood. As prefrontal cortex (PFC) is one of the most important brain regions, which provides executive control over drug use and is severely impaired in many addicts, systematic analysis of the biochemical and molecular alteration of synaptic fraction of PFC in morphine-induced neuroadaptation is necessary. In this study, differential protein expression profiling of synaptic fraction of rat PFC based on morphine-induced conditioned place preference (CPP) model was performed with two-dimensional gel electrophoresis (2-DE). Our results showed that a total of 80 proteins were differentially expressed by 2-DE analysis during three phases of CPP assay. Of them, 58 were further identified by mass spectrometry. These proteins were classified into multiple categories, such as energy metabolism, signal transduction, synaptic transmission, cytoskeletal proteins, chaperones, and local synaptic protein synthetic machinery according to their biological functions. Our study provides a global view of synaptic-related molecular networking in PFC under morphine-induced dependence, withdraw, and relapse, indicative of a concerted biological process in neuroadaptation under chronic morphine exposure.     

Orexin A in the ventral tegmental area induces conditioned place preference in a dose-dependent manner: Involvement of D1/D2 receptors in the nucleus accumbens

It has been shown that orexin A in the ventral tegmental area (VTA) is necessary for development of morphine place preference. Additionally, D1 and D2 dopamine receptors in the nucleus accumbens (NAc) have critical roles in motivation and reward. However, little is known about the function of orexin in conditioned place preference (CPP) in rats and involvement of D1/D2 receptors in the NAc. In the present study, we investigated the effect of direct administration of orexin A into the VTA, and examined the role of intra-accumbal dopamine receptors in development (acquisition) of reward-related behaviors in the rats. Adult male Wistar rats were unilaterally implanted by two separate cannulae into the VTA and NAc. The CPP paradigm was used, and, conditioning score and locomotor activity were recorded by Ethovision software. The results showed that unilateral intra-VTA administration of orexin A (27, 53 and 107ng/0.3μl saline) during conditioning phase induced CPP in a dose-dependent manner. The most effective dose of intra-VTA orexin-A in eliciting CPP was 107ng. However, intra-NAc administration of SCH 23390 (0.25, 1 and 4μg/0.5μl saline), a D1 receptor antagonist, and sulpiride (0.25, 1 and 4μg/0.5μl DMSO), a D2 receptor antagonist, inhibited the development of orexin-induced CPP. The inhibitory effect of D2 but not D1 receptor antagonist was exerted in a dose-dependent manner. It is supposed that the activation of VTA dopaminergic neuron by orexin impresses the D2 receptors more than D1 receptors in the NAc.     

Enhanced CREB and DARPP-32 phosphorylation in the nucleus accumbens and CREB, ERK, and GluR1 phosphorylation in the dorsal hippocampus is associated with cocaine-conditioned place preference behavior

Environment-induced relapse is a major concern in drug addiction because of the strong associations formed between drug reward and environment. Cocaine-conditioned place preference is an ideal experimental tool to examine adaptations in the molecular pathways that are activated upon re-exposure to an environment previously paired with drug reward. To better understand the mechanism of cocaine-conditioned place preference we have used western blot analysis to examine changes in phosphorylation of cAMP-response element binding protein (CREB), dopamine- and cyclic AMP-regulated phosphoprotein 32 (DARPP-32), extracellular signal-regulated kinase (ERK) and GluR1, key molecular substrates altered by cocaine, in the nucleus accumbens (NAc) and dorsal hippocampus (DHC) of C57BL/6 mice. Our studies revealed that re-exposing mice to an environment in which they were previously given cocaine resulted in increased levels of Ser133 phospho-CREB and Thr34 phospho-DARPP-32 with a corresponding decrease in Thr75 phospho-DARPP-32 in the NAc. In DHC there were increased levels of phospho-CREB, Thr183/Tyr185 phospho-ERK, and Ser845 phospho-GluR1. These data suggest that the formation of contextual drug reward associations involves recruitment of the DHC-NAc circuit with activation of the DARPP-32/CREB pathway in the NAc and the ERK/CREB pathway in the DHC.     

Involvement of Hippocampal D1-Like Dopamine Receptors in the Inhibitory Effect of Cannabidiol on Acquisition and Expression of Methamphetamine-Induced Conditioned Place Preference

Cannabidiol (CBD) is a non-psychotomimetic compound with strong potential to decrease the psychostimulant’s rewarding effect with unclear receptors. Furthermore, as a part of the reward circuit, the hippocampus plays a crucial role in regulating the reward properties of drugs as determined by conditioned place preference (CPP). In the current research, CPP was used to evaluate the role of intra-CA1 microinjection of D1-like dopamine receptor antagonists in CBD's inhibitory effect on the acquisition and expression phases of methamphetamine (METH). Animals were treated by METH (1 mg/kg; sc) in a five-day schedule to induce CPP. To find out the impact of D1-like dopamine receptor antagonist, SCH23390, in the CA1 on the inhibitory influence of CBD on the acquisition of METH, the rats received intra-CA1 administration of SCH23390 (0.25, 1, and 4 µg/0.5 µl) following ICV treatment of CBD (10 µg/5 µl) over conditioning phase of METH. Furthermore, animals were given SCH23390 in the CA1 ensuing ICV microinjection of CBD (50 µg/5 µl) in the expression phase of METH to rule out the influence of SCH23390 on the suppressive effect of CBD on the expression of METH CPP. Intra-CA1 microinjection of SCH23390 abolished CBD's suppressive impact on both METH-induced CPP phases without any side effect on the locomotion. The current research disclosed that CBD inhibited the rewarding characteristic of METH via D1-like dopamine receptors in the CA1 region of the hippocampus.

     

Chronic deep brain stimulation in the rat nucleus accumbens and its effect on morphine reinforcement.

In order to explore a novel method for the treatment of drug abuse, we evaluated the effect of chronic deep brain stimulation (DBS) of the rat nucleus accumbens (NAc) on morphine reinforcement, using a DBS apparatus and an implant method we developed. Thirty-two adult rats weighing 240-260 g were divided into three groups, which included a DBS group (n = 10, administration of surgery, morphine and DBS), a sham DBS group (n = 12, administration of surgery and morphine) and a control group (n = 10, administration of physiological saline). The DBS electrode was stereotaxically implanted into the core of unilateral NAc and connected to an implantable pulse generator. Then, they were fixed to the rat skull. One week later, the rats in each group were intraperitoneally injected with morphine at an increasing dose (10-60 mg/kg) once daily. The rats in the DBS group were administered a 130-Hz high-frequency stimulation (HFS) once daily. A 900-second conditioned place preference (CPP) paradigm was used for determining the effect of electrical stimulation on morphine reinforcement in rats. The data showed that 7-10 days later, the preference score of the DBS group was significantly lower than that of the sham DBS group. The results suggest that chronic HFS of the rat NAc can block CPP induced by morphine and attenuate morphine reinforcement.     

Morphine and Cocaine Exert Common Chronic Actions on Tyrosine Hydroxylase in Dopaminergic Brain Reward Regions

We studied levels of tyrosine hydroxylase immunoreactivity and phosphorylation state in the ventral tegmental area (VTA) and nucleus accumbens (NAc) in an effort to understand better the mechanisms by which these brain reward regions are influenced by opiates and cocaine. In the VTA, chronic, but not acute, administration of either morphine or cocaine increased levels of tyrosine hydroxylase immunoreactivity by 30-40%, with no change observed in the relative phosphorylation state of the enzyme. In the NAc, chronic, but not acute, morphine and cocaine treatments decreased the phosphorylation state of tyrosine hydroxylase, without a change in its total amount. In contrast, morphine and cocaine did not regulate tyrosine hydroxylase in the substantia nigra or caudate/putamen, brain regions generally not implicated in drug reward. Morphine and cocaine regulation of tyrosine hydroxylase could represent part of a common biochemical basis of morphine and cocaine addiction and craving.