Kratom Alkaloids, Natural and Semi-Synthetic, Show Less Physical Dependence and Ameliorate Opioid Withdrawal.

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea "kratom" and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n = 10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

Effects of processed Aconiti tuber on the extinction and reinstatement of morphine-induced conditioned place preference in rats.

AIM OF THE STUDY: To investigate the effect of processed Aconiti tuber (PAT) administered during or after the time of conditioned place preference (CPP) training on the extinction and reinstatement of morphine-priming CPP in rats. The dynorphin level in rats' nucleus accumbens (NAc) is detected as a target of the Dynorphin/Kappa Opioid Receptor (KOR) system for the possible mechanism. MATERIALS AND METHODS: Eight groups of rats were subcutaneously (s.c.) injected with morphine (10mg/kg) (on days 2,4,6,8) or saline (1ml/kg) (on days 3,5,7,9) alternately for 8 days. Five groups, including groups (Mor + Water, Mor + PAT (1.0/3.0g/kg) (S) and Sal + PAT(1.0/3.0g/kg)), were orally given distilled water or PAT 1.0 or 3.0 g/kg daily on days 1-8 during CPP training while other three groups, including groups (Sal + Water and Mor + PAT (1.0/3.0g/kg)(P), were given distilled water or PAT daily from day 10 until CPP was extinct. Morphine 1mg/kg (s.c.) was used to reinstate the extinct CPP and the CPP scores were recorded. The dynorphin concentration in nucleus accumbens (NAc) was assayed by radioimmunoassay after the last CPP measurement. RESULTS: 1) The CPP extinction shortened in Mor + PAT (1.0/3.0 g/kg) (S) groups but extended in Mor + PAT (1.0/3.0 g/kg)(P) groups. 2) Morphine-priming CPP did not change either in Mor + PAT (1.0/3.0 g/kg) (S) or Mor + PAT (1.0/3.0 g/kg)(P) groups. 3) The dynorphin concentration in NAc increased either in Mor + PAT (1.0/3.0 g/kg)(S) or Mor + PAT (1.0/3.0 g/kg)(P) groups. CONCLUSIONS: 1) PAT shortened the extinction from morphine induced CPP when administrated before CPP acquisition, whereas it extended the extinction when administrated after CPP formation. 2) PAT administrated during or after CPP training did not affect morphine-priming reinstatement of morphine induced CPP. 3) Dynorphin/KOR system might be a target to regulate morphine-induced CPP extinction but not reinstatement.

Circular RNA expression profiling in the nucleus accumbens: Effects of electroacupuncture treatment on morphine-induced conditioned place preference.

Electroacupuncture (EA) has been developed on the basis of traditional Chinese acupuncture. EA can suppress craving in opioid addicts and opioid-seeking responses in rodents. However, the molecular mechanism of EA on the rewarding properties of morphine and craving responses is not known. Here, we have applied a conditioned place preference paradigm in mice to measure morphine-induced rewarding effects along with EA treatment. Circular RNAs (circRNAs) can function as micro RNA (miRNA) sponges to effectively regulate gene expression levels. CircRNA profiling within the nucleus accumbens (NAc) was performed in EA-treated and sham-treated mice. Following RNAseq, data were analyzed by gene ontology (GO) and Kyoto Encyclopedia Genes and Genomes (KEGG) tools. We identified 112 significantly differentially expressed circRNAs, including 51 that were up-regulated and 61 that were down-regulated. Our bioinformatics analyses show that these differentially expressed circRNAs map into pathways that are mainly involved with renin secretion and the cGMP-PKG signaling. We further constructed a circRNA-miRNA network that predicts the potential roles of the differentially expressed circRNAs and the interaction of circRNAs with miRNAs. Our secondary sequencing and bioinformatics analysis in the NAc after EA treatment on morphine-induced CPP provides putative novel targets on molecular mechanisms involved in morphine reinforcement and possibly craving.

Chromium picolinate reduces morphine-dependence in rats, while increasing brain serotonin levels.

Chromium is an essential trace element with anti-diabetic and anti-depressant effect; the latter is considered related to chromium properties of increasing brain serotonin. Cr3+ salts were shown to improve some forced swimming-parameters and to induce rewarding effects, which are additive to those of morphine, but Cr effect on addictive processes has not been tested. AIM: The present study aimed to assess chromium picolinate (CrPi) influence on morphine-dependence in rats. MATHERIAL AND METHODS: We used five groups of 10 rats. Groups 1 and 2 (controls) received saline, respectively CrPi, 0.01 mg/kg/day, for 10 days. In groups 3, 4 and 5 dependence was induced with progressively-increased morphine doses (from 5 - day 1-90 mg/kg/day - day 10, s.c.). Group 3 received only morphine, while groups 4 and 5 received CrPi, i.p., 10 and respectively 5 µg/kg/day, during the 10 days of dependence induction. On day 11, groups 3, 4, and 5 were administered 90 mg/kg morphine, and, 2 h later, all rats received naloxone, 2 mg/kg s.c., to precipitate withdrawal. We compared withdrawal intensity in group 3 vs. groups 4 and 5, assessing both individual symptoms and Gellert-Holtzman global withdrawal score. Upon rats sacrifice at the end of the experiments, brain serotonin (5HT) in certain areas and serum Cr were assessed. RESULTS: Some withdrawal signs were unequally influenced by CrPi: compulsive mastication was reduced by both CrPi doses (p < 0.05), while teeth chattering and grooming were significantly reduced only by the higher dose (p < 0.05). Withdrawal score was reduced by both CrPi doses: from 132.4 ±â€¯9.87 - group 3 to 122.2 ±â€¯6.47 - group 4 (p < 0.01 vs. group 3) and 124.1 ±â€¯8.41 - group 5 (p < 0.05 vs. group 3). CrPi reduction of withdrawal is accompanied by increased brain 5 H T, mainly in the prefrontal cortex (646.3 ±â€¯8.51 - group 3 vs. 661.5 ±â€¯14.63 - group 4, p < 0.01 and 660.7 ±â€¯14.01 pg/mg tissue - group 5, p < 0.05 vs. group 3). CrPi also increases brain 5 H T in non-dependent rats (prefrontal cortex: 541.6 ±â€¯31.80, group 1 and 565.5 ±â€¯16. 46 pg/mg tissue, group 2, p < 0.05). Administration of CrPi determined a dose-dependent increase of serum Cr. CONCLUSIONS: Our study evidenced a slight, but significant reduction of morphine dependence in rats induced by chromium picolinate, accompanied by increased brain serotonin. This might be considered a supplementary evidence for chromium anti-depressant effect and its serotonin-mediated mechanisms.

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.

Hyperbaric Oxygen Attenuates Withdrawal Symptoms by Regulating Monoaminergic Neurotransmitters and NO Signaling Pathway at Nucleus Accumbens in Morphine-Dependent Rats.

In this study, we examined whether hyperbaric oxygen (HBO2) plays a detoxification role in withdrawal symptoms in a morphine-dependent rat model. The model was established through injections of morphine at increasing doses for 7 days. Withdrawal symptoms were induced by naloxone injection on the 8th day. The detoxification effect of HBO2 was evaluated using the withdrawal symptom scores, biochemical indices and neurotransmitters. Compared with the model group, HBO2 therapy significantly attenuated the withdrawal symptom scores, body weight loss and the level of norepinephrine level, whereas it increased the dopamine level and tyrosine hydroxylase expression in the nucleus accumbens. Moreover, HBO2 therapy substantially alleviated the NO, NOS, cAMP, and cGMP levels. Our findings indicate that HBO2 can effectively alleviate withdrawal symptoms induced by morphine dependence, and these effects may be attributed to the modulation of monoaminergic neurotransmitters and the suppression of the NO-cGMP signaling pathway.

Prevention of morphine dependence and tolerance by Nepeta menthoides was accompanied by attenuation of Nitric oxide overproduction in male mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Repeated administration of morphine for chronic pain leads to dependence and tolerance that limits clinical usage. Nepeta menthoides is commonly known as Iranian Ustukhuddoos and are administered in traditional medicine for gastrodynia, bone pain, blood depurative and restlessness. AIMS OF STUDY: To investigate the effects of Nepeta menthoides on expression and acquisition of morphine dependence and tolerance in mice with regard to oxidative stress. MATERIALS AND METHODS: Morphine dependence in mice was developed by administration of gradually increasing doses of morphine twice daily for 7 consecutive days. In experimental groups, administration of Nepeta menthoides (200 and 400mg/kg), methadone and their combination were performed 60min prior to each morphine injection (for acquisition) or the last injection of morphine on test day (for expression). Morphine tolerance was measured by the tail-immersion test before and after the administration of a single dose of morphine (100mg/kg; i.p.) on the test day (8th day). Morphine dependence was also evaluated by counting the number of jumps after the injection of naloxone (5mg/kg; i.p.). RESULTS: Nepeta menthoides, similar to methadone, significantly prevented the development (but not the expression) of morphine dependence, tolerance, and potentiated morphine antinociception and also reduced (23.23±1.15) Nitric oxide (NO) overproduction (35.23±3.36) (in compared with naloxone group (6.3±0.52)). However, single and repeated application of the extract could not change high single-dose morphine analgesia. CONCLUSION: It appears that Nepeta menthoides and methadone prevented morphine dependence and tolerance, partly through inhibition of the NO overproduction.

Protective effects of atorvastatin against morphine-induced tolerance and dependence in mice.

BACKGROUND: In this study, we evaluated the effects of atorvastatin, a lipid-lowering medication on morphine-induced tolerance and dependence in mice. METHODS: Tolerance was induced by subcutaneous administration of morphine (20mg/kg) to animals, twice a day for 9days. Atorvastatin was given at the doses of 5, 10 and 20mg/kg, 30min before each morphine administration, once daily for 9days. Hot plate test was employed to assess antinociceptive effect of morphine on days 1, 3, 5, 7 and 9. Dependence was evaluated by naloxone-precipitated withdrawal syndrome. We attempted to verify withdrawal regulation of induced nitric oxide synthase (iNOS), astroglia marker, glial fibrillary acidic protein (GFAP), ionized calcium-binding protein (Iba1), a microglia activation marker, a pro-inflammatory mediator, tumor necrosis alpha (TNF-α) and immune receptor, toll like receptor 4 (TLR-4) genes by real-time polymerase chain reaction (RT-PCR). Lipid peroxidation was estimated by assessing malondialdehyde (MDA) content in the spinal cord of animals. RESULTS: Tolerance to antinociceptive effects of morphine was observed on days 7 and 9. Decrease in morphine-induced antinociception was reversed by concomitant intraperitoneal administration of atorvastatin (10 and 20mg/kg). Atorvastatin (10 and 20mg/kg) mitigated naloxone-induced withdrawal parameters. Brain expression levels of TNF-α, GFAP, Iba1 and iNOS increased in morphine withdrawn animals which were attenuated by nine days treatment with atorvastatin. Increased MDA was also normalized in withdrawn animals received atorvastatin. CONCLUSION: Atorvastatin exhibits meaningful protective effects against both tolerance to antinociceptive effects of morphine and withdrawal-induced behavioral profile. Neuroprotective effects of atorvastatin is further supported via inhibition of glia activity and antioxidant effects.

Acupuncture Suppresses Morphine Craving in Progressive Ratio Through the GABA System.

Previous studies revealed that acupuncture suppressed both morphine self-administration and morphine-seeking behavior after abstinence. Based on these results, this study examined whether acupuncture attenuated morphine-craving under a progressive ratio (PR) schedule and investigated the possible neuronal mechanism. Male Sprague-Dawley rats were trained to self-administer morphine (0.5 mg/kg) at a fixed ratio for 9 days, and rats who achieved stable infusion were switched to a PR schedule. When animals had taken no more morphine for 1 hour, the number of infusions was defined as the break point (BP). After PR training, animals that had established a stable BP received acupuncture the next day. Acupuncture was applied for 1 minute immediately before the test session. Bicuculline (1.0 mg/kg) and SCH 50911 (2.0 mg/kg) were given 30 minutes prior to acupuncture. The c-Fos levels in the ventral tegmental area (VTA) and the nucleus accumbens (NAc) were examined. Acupuncture at SI5 reduced the BP significantly. Moreover, the effects of acupuncture were blocked by either bicuculline or SCH 50911. Immunofluorescence revealed that acupuncture at SI5 decreased c-Fos expressions in the VTA and the NAc. This study demonstrates that acupuncture at SI5 is effective for the treatment of morphine-craving and that this effect is mediated via the GABA pathway.

Morphine causes persistent induction of nitrated neurofilaments in cortex and subcortex even during abstinence.

Morphine has a profound role in neurofilament (NF) expression. However, there are very few studies on the fate of NFs during morphine abstinence coinciding with periods of relapse. Mice were treated chronically with morphine to render them tolerant to and dependent on morphine and sacrificed thereafter while another group, treated similarly, was left for 2 months without morphine. A long-lasting alteration in the stoichiometric ratio of the three NFs was observed under both conditions in both the cortex and subcortex. Morphine abstinence caused significant alterations in the phosphorylated and nitrated forms of the three NF subunits. Nitrated neurofilament light polypeptide chain (NFL) was significantly increased during chronic morphine treatment which persisted even after 2 months of morphine withdrawal. Mass spectrometric analysis following two-dimensional gel electrophoresis (2DE)-gel electrophoresis of cytoskeleton fractions of both cortex and subcortex regions identified enzymes associated with energy metabolism, cytoskeleton-associated proteins as well as NFs which showed sustained regulation even after abstinence of morphine for 2 months. It is suggestive that alteration in the levels of some of these proteins may be instrumental in the increased nitration of NFL during morphine exposure. Such gross alteration in NF dynamics is indicative of a concerted biological process of neuroadaptation during morphine abstinence.

Sympathetic activity induced by naloxone-precipitated morphine withdrawal is blocked in genetically engineered mice lacking functional CRF1 receptor.

There is large body evidence indicating that stress can lead to cardiovascular disease. However, the exact brain areas and the mechanisms involved remain to be revealed. Here, we performed a series of experiments to characterize the role of CRF1 receptor (CRF1R) in the stress response induced by naloxone-precipitated morphine withdrawal. The experiments were performed in the hypothalamic paraventricular nucleus (PVN) ventrolateral medulla (VLM), brain regions involved in the regulation of cardiovascular activity, and in the right ventricle by using genetically engineered mice lacking functional CRF1R levels (KO). Mice were treated with increasing doses of morphine and withdrawal was precipitated by naloxone administration. Noradrenaline (NA) turnover, c-Fos, expression, PKA and TH phosphorylated at serine 40, was evaluated by high-performance liquid chromatography (HPLC), immunohistochemistry and immunoblotting. Morphine withdrawal induced an enhancement of NA turnover in PVN in parallel with an increase in TH neurons expressing c-Fos in VLM in wild-type mice. In addition we have demonstrated an increase in NA turnover, TH phosphorylated at serine 40 and PKA levels in heart. The main finding of the present study was that NA turnover, TH positive neurons that express c-Fos, TH phosphorylated at serine 40 and PKA expression observed during morphine withdrawal were significantly inhibited in CRF1R KO mice. Our results demonstrate that CRF/CRF1R activation may contribute to the adaptive changes induced by naloxone-precipitated withdrawal in the heart and in the brain areas which modulate the cardiac sympathetic function and suggest that CRF/CRF1R pathways could be contributing to cardiovascular disease associated to opioid addiction.

An-jun-ning, a traditional herbal formula, attenuates spontaneous withdrawal symptoms via modulation of the dopamine system in morphine-dependent rats.

BACKGROUND: The dopamine system, which is involved in drug dependence, can be damaged by opioid abuse. However, current clinical medicines cannot reverse these damages in the brain, which are believed to be a key reason for the high relapse rate after abstinence treatment. This study aimed to investigate the effects of An-jun-ning (AJN), a commercial traditional Chinese medicine formula used for the treatment of opioid addiction, on the dopamine system in morphine-dependent rats and to explore the possible mechanism underlying its therapeutic effects. METHODS: The morphine dependence model was obtained through injections of morphine at increasing doses for 8 days. The AJN pre-treatment group was administered AJN 30 min before each morphine administration, and the AJN post-treatment groups were treated with AJN for 10 days after withdrawal. Spontaneous withdrawal symptoms (wet dog shakes, and episodes of writhing) were observed after withdrawal. Autoradiography study and/or immunohistochemical staining were used to examine the levels of dopamine transporter (DAT), dopamine D2 receptor (D2R) and tyrosine hydroxylase (TH). RESULTS: (1) Pre-treatment with AJN attenuates wet dog shakes and episodes of writhing to approximately 50% or less of those observed in the morphine group (p < 0.01). (2) AJN post-treatment dose-dependently reduced the number of wet dog shakes (p < 0.01), and the episodes of writhing (p < 0.01). (3) Pre-treatment with AJN effectively interdicted the morphine-induced decreases in the levels of DAT, D2R, and TH in the striatum (p < 0.01) such that they remained at nearly normal levels. (4) Post-treatment with AJN restored DAT and D2R to the normal levels (p < 0.01) and the level of TH to 87% of normal in the striatum. CONCLUSIONS: AJN can effectively alleviate opioid withdrawal symptoms and preserve or restore the DAT, D2R, and TH levels in the striatum. The mechanism underlying the effect of AJN on withdrawal symptoms may be related to the modulation of the dopamine system by AJN. These results suggest that AJN may help to prevent relapse in opioid dependence treatment.

A Scutellaria baicalensis radix water extract inhibits morphine-induced conditioned place preference.

CONTEXT: Scutellaria baicalensis Georgi (Lamiaceae) has been used as a traditional herbal preparation for the treatment of neuropsychiatric disorders in Asian countries for centuries. OBJECTIVE: To evaluate the effects of S. baicalensis on morphine-induced drug dependence in rats. MATERIALS AND METHODS: In order to evaluate the effect of S. baicalensis and baicalin on morphine-induced dependence-like behavior, a water extract of S. baicalensis [500 mg/kg, intraperitoneally (i.p.)] or baicalin (50 mg/kg, i.p., a flavonoid found in S. baicalensis) was administered prior to morphine injection [5 and 2.5 mg/kg, respectively, subcutaneously (s.c.)] to rats for 8 and 4 d, respectively. Morphine-induced conditioned place preference was assessed by measuring the time spent in a drug-paired chamber. The effect of S. baicalensis on dopamine receptor supersensitivity (locomotor activity) and dopamine agonist-induced climbing behavior due to a single apomorphine treatment (2 mg/kg, s.c.) was also measured. RESULTS: At 50 mg/kg, a water extract of S. baicalensis decreased morphine (5 mg/kg)-induced conditioned place preference by 86% in rats. Apomorphine (2 mg/kg)-induced locomotor activity (dopamine receptor supersensitivity) in rats and climbing behavior in mice were attenuated after pretreatment with 500 mg/kg of S. baicalensis water extract by 41% and 56%, respectively. In addition, baicalin-reduced morphine-induced conditioned places preference by 86% in rats at 50 mg/kg. DISCUSSION AND CONCLUSION: These results suggest that S. baicalensis can ameliorate drug addiction-related behavior through functional regulation of dopamine receptors.

Acupuncture at HT7 suppresses morphine self-administration at high dose through GABA system.

In the previous study, acupuncture at HT7 has shown to attenuate the self-administration of morphine at a low dose (0.1mg/kg). In this study, it was further investigated whether acupuncture at HT7 could attenuate the morphine self-administration at a high dose (0.5mg/kg). Male Sprague-Dawley rats weighing 270-300g were used. After surgery of catheterization, animals were trained to self-administer morphine solution (0.5mg/kg) using daily 1h session under fixed ratio 1 schedule for 3 weeks. Animals that had shown stable morphine-taking (establish baseline: variation less than 20% of the mean of three consecutive days) were subjected to the acupuncture treatment. Bicuculline and SCH 50911 were used to investigate the possible relation between the effect of acupuncture and the GABA receptor system. Acupuncture at HT7, but not at control acupoint, LI5, suppressed spontaneous morphine-taking behavior significantly. In addition, the effect of acupuncture was blocked by both GABA receptor antagonists. The results of this study suggest that acupuncture at HT7 suppresses morphine-taking behavior through the mediation of GABA receptor system.

Yulangsan polysaccharide attenuates withdrawal symptoms and regulates the NO pathway in morphine-dependent rats.

Yulangsan polysaccharide (YLSP) has been utilized as a phytomedicine to managing nervous dysfunction in China. Thus, this study aimed to evaluate the potential YLSP-mediated detoxification role against morphine dependence in rats. The results indicated that the morphine dependence model significantly increased withdrawal symptoms, levels of NO and NOS (P<0.05). Furthermore, monoaminergic neurotransmitters, including DA and NE, were detected at elevated levels in the ventral tegmental area (VTA), hippocampus (HIP) and prefrontal cortex (PFC), respectively, while the level of DA was decreased and NE was increased in the nucleus accumbens (NAc). Conversely, YLSP administration significantly reversed naloxone-induced withdrawal symptoms, expression of brain NO and NOS, and monoaminergic neurotransmitters (P<0.05). Interestingly, YLSP shows an even more effective trend in attenuating withdrawal symptoms than does clonidine, although without a significant difference. These findings indicate that YLSP attenuation of the naloxone-induced withdrawal symptoms of morphine dependence may be mediated by regulation of the NO pathway and modulation of monoaminergic neurotransmitters.

[Inhibition of morphine intake by antibodies to serotonin-modulating anticonsolidation protein in model of self-administration in rats].

The article concerns study of effects of polyclonal antibodies to serotonin-modulating anticonsolidation protein (SMAP) being in direct dependence on serotonin level and providing intracellular transduction of serotonergic signal, on positive reinforcement effect of morphine in rats. The task was formed in Wistar male rats in the model of morphine self-administration as a result of pressing of one of two levers attached to the wall, joined to the pump delivering each time 100 µg of morphine directly into the vena jugularis. In the 1st series of studies brain cingulate cortex and hypothalamus were taken from the rats achieved stable level of morphine intake and SMAP level was measured with indirect immune-enzyme assay. It was shown that in the morphine-self-injected rats SMAP level in the cingulate cortex is significantly upregulated (p = 0.01), while in the hypothalamus it was left unchanged. In the 2nd series of studies the rats with stable level of morphine intake were administered intraperitoneally with anti-SMAP rabbit polyclonal antibodies (experimental group) or non-immune γ-globulins (control group). Soon after antibodies administration the animals of the experimental group demonstrated manifold decrease of morphine intake lasted for 8 days (p < 0.008), whereas it did not change in the controls. SMAP upregulation in the brain cingulate cortex in the rats with stable morphine intake, obviously, indicates to its engagement in positive reinforcement effect of morphine. Blockade of SMAP activity with anti-SMAP antibodies in the nerve cells induced sharp decrease of morphine intake due to disturbances of transduction through intracellular serotonin's signal channels.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal.

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15-30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls.

Gene expression profiling following short-term and long-term morphine exposure in mice uncovers genes involved in food intake.

Addictive drugs including opioids activate signal transduction pathways that regulate gene expression in the brain. However, changes in CNS gene expression following morphine exposure are poorly understood. We determined changes in gene expression following short- and long-term morphine treatment in the hypothalamus and pituitary using genome-wide DNA microarray analysis and confirmed those alterations in gene expression by real-time reverse transcriptase polymerase chain reaction (RT-PCR) analysis. In the hypothalamus, short-term morphine administration up-regulated (at least twofold) 39 genes and down-regulated six genes. Long-term morphine treatment up-regulated 35 genes and down-regulated 51 genes. In the pituitary, short-term morphine administration up-regulated 110 genes and down-regulated 29 genes. Long-term morphine treatment up-regulated 85 genes and down-regulated 37 pituitary genes. Microarray analysis uncovered several genes involved in food intake (neuropeptide Y, agouti-related protein, and cocaine and amphetamine-regulated transcript) whose expression was strongly altered by morphine exposure in either the hypothalamus or pituitary. Subsequent RT-PCR analysis confirmed similar regulation in expression of these genes in the hypothalamus and pituitary. Finally, we found functional correlation between morphine-induced alterations in food intake and regulation of genes involved in this process. Changes in genes related to food intake may uncover new pathways related to some of the physiological effects of opioids.

[Study on the detoxification of alcohol extracts from orientvine and its effective component on withdrawal syndromes of morphine].

OBJECTIVE: To observe the effect of alcohol extracts from orientivne and its effective component sinomenine on withdrawal syndromes and neurotransmitter of morphine-abstinent mice and on intracellular calcium level in morphine-dependent neuronal-cells line. To study the detoxification of alcohol extracts from orientvine and sinomenine on morphine-dependent animal and explore the mechanism of its effect. METHODS: The effect of alcohol extracts from orientivne and sinomenineon on abstinent syndromes was observed by experiment study on morphine-dependent ex vivo ileum from guinea pigs and morphine-dependent mice. The morphine-dependent model mice were established by injection on dosage increasing by degrees. The hypothalamic monomine neurotransmitters such as NA, DA, 5-HT were tested by fluorospectrophotometry. Morphine-dependent cell line was established by administering morphine at different doses into the culture medium. The cells were stained with fluo-3 and the intracellular calcium level was measured by flow cytometry. RESULTS: Alcohol extracts from orientvine and sinomenine could alleviate withdrawal contractile response of morphine-dependent ex vivo ileum from guinea pigs and withdrawal syndromes of morphine-dependent mice, decrease the concentration of the neurotransmitters, and elevate the intracellular calcium level and inhibit the decreasing of Ca2+ induced by naloxone. CONCLUSIONS: Alcohol extracts from orientvine and sinomenine have some effects on withdrawal syndromes which may be related to inhibiting neurotransmitters and the regulation of intracellular calcium concentration.

[Anti-alcoholic and anti-narcotic action of Methylobacteriim extorquens UCM B-3368].

The paper deals with action efficiency of microbial biomass on characteristic indicators at alcohol and morphine organism intoxication. The investigated microbial biomass affects the regulatory biochemical and physiological systems in experimental animals, normalizes activity of alcohol dehydrogenase and aldehide dehydrogenase, as well as the content of dophamine, disturbed under the effect of alcohol and morphine. Thus, the organism intoxication decreases. Except for the specific action, the above microbial biomass can be a source of protein, aminoacids, vitamins, microelements. So, the microbial preparation, made on its basis, can be used for the treatment of alcohol and morphine dependence in a form of biologically active dope. Thus the microbial drug intended for treatment of alcohol and opium dependence has been developed. One of its action mechanisms is based on the microorganisms capacity to transform alcohols and aldehides, owing to availability of alcohol and aldehide dehydrogenase, other its action mechanisms are at the stage of investigation.