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An increasing number of data show that exposure to mephedrone in adolescence can have long-lasting implication on brain activity and on peripheral organs/tissues. The aim of this study was to investigate whether adolescent exposure to mephedrone (10 mg/kg, i.p.) has influence upon the rewarding effect of morphine (5 mg/kg, i.p.) in adult rats. Thus, the adolescent rats (on the 30th PND) were treated with mephedrone for 7 consecutive days. When the animals were adult (on the 60th PND) the morphine-induced conditioned place preference (CPP) test was performed. After that, the level of DNA methylation in the striatum was investigated. DNA methylation is one of the epigenetic mechanisms which produces changes in the genome. These alterations may affect the phenotype, without effect on DNA sequences, and has influence on drug addiction. Additionally, in order to check the toxic properties of mephedrone on the peripheral organs, the histopathological examination of kidney and liver was carried out. The present experiments demonstrated that: 1) adolescent mephedrone exposure may intensify the rewarding effect of morphine in adult rats in the CPP test; 2) mephedrone may induce the alterations in DNA methylation in striatum of adult rats leading to changes in gene activity; 3) mephedrone may produce some retrogressive disturbances in kidney and liver, which confirms the toxic properties of this substance.

Current studies indicate that the cholinergic and opioid systems interact to modulate pain. In the present work, we investigated the influence of the cholinesterase inhibitors, donepezil (0.5; 1 or 3mg/kg, i.p.) and rivastigmine (0.03; 0.5 or 1mg/kg, i.p.), on the acute antinociceptive effects of morphine (5mg/kg, i.p.) in the hot plate test in mice. Herein, both inhibitors were found to enhance and prolong the analgesic effects of morphine without affecting latencies themselves. In an extension of this work, we determined which cholinergic receptors subtype mediates the enhancement of analgesic effects of morphine, following inhibition of cholinesterases. In this part of the study, scopolamine (0.5mg/kg, i.p.), a muscarinic cholinergic receptors antagonist, but not mecamylamine (3mg/kg, i.p.), a nicotinic cholinergic receptors antagonist, reversed the enhancing effects of donepezil (3mg/kg, i.p.) and rivastigmine (1mg/kg, i.p.) on the morphine antinociception. Moreover, both cholinesterase inhibitors attenuated the development of tolerance to the antinociceptive effects of morphine. In contrast, acute administration of donepezil (3mg/kg, i.p.) or rivastigmine (1mg/kg, i.p.) on the day of expression of tolerance, had no effect on the already developed morphine tolerance. What is more, in both set of experiments, rivastigmine was slightly more potent than donepezil due to the broader inhibitory spectrum of this drug on acetylcholine degradation. Thus, our results suggest that the cholinesterase inhibitors, donepezil and rivastigmine, may be administered with morphine in order to enhance the latter's analgesic effects for the treatment of acute and chronic pain.

The successful pharmacotherapy for drug addiction is a challenge for pharmacology in XXI century. We still do not have any registered medication for cocaine by FDA, alcohol therapies are not enough successful. High hopes concern metabotropic glutamate 7 receptor as a target for pharmacotherapy development of drug dependence. It is well documented the role of the mGluR7 in rewarding and motivational effects of cocaine and alcohol. The mGluR7 allosteric agonist AMN082 and antagonist MMPIP have become new tools in preclinical studies. AMN082 attenuated cocaine-enhanced electrical Brain Stimulation Reward (eBSR) in rats decreased cocaine self-administration, too, probably by counteracting cocaine-induced inhibition of the ventral pallidal GABA release. The mGluR7 activation reduced alcohol intake and preference in rats. The mGluR7 receptors take part in reinstatement of drug-seeking behavior after abstinence. The investigations showed that AMN082 suppressed cocaine-induced reinstatement of self-administrated rats via mGluR2/3 mechanism. It was mentioned also that mGluR7 allosteric agonist inhibited reinstatement of ethanol-induced CPP. Moreover, activation of the mGluR7 receptor exhibits less adverse reaction in comparison to other mGluRs and may give a promise for further development of drug dependence therapies in humans. However, some nonspecific effects of the mGluR7 in ethanol addiction have been recognized, so there are further investigations needed to explain the mechanisms of the mGluR7 action in rewarding and motivational effects of drugs.

Acetylcholine ACh, is involved in many CNS functions like sensory and motor processing, sleep, nociception, memory, mood, stress response, attention, arousal, motivation and reward. Due to the interaction with the dopaminergic reward system mainly in the ventral tegmental area (VTA), nucleus accumbens (NAc) and prefrontal cortex (PFC), it is believed that ACh plays a significant role on the initiation and maintenance of the drug addiction. Substances affecting the cholinergic system by increasing the levels of ACh, like cholinesterase and butyrylcholinesterase inhibitors (donepezil, rivastigmine, physostigmine, tacrine, galantamine), reduced the symptoms of addiction in a drug-induced CPP test as well as locomotor activity caused by morphine, cocaine and heroin. Unfortunately, donepezil failed to inhibit methamphetamine-CPP as well as the locomotor sensitization caused by this drug. For comparison, it has been indicated that mice lacking M5 receptor showed a reduction in cocaine self-administration and morphine-induced CPP. The stimulation of M4 receptor decreased DA release in NAc and the administration of non-selective antagonist of mAChRs, scopolamine decreased cocaine-induced locomotor activity in rats. Moreover, mecamylamine, a noncompetitve antagonist of nAChRs, prevented the increase in cocaine consumption as well as blocked cocaine and amphetamine-induced behavioral sensitization. Thus, drugs affecting cholinergic transduction could be approached as potential therapeutic agents for patients who abuse different psychoactive substances. These issues, however, require further studies.

The aim of this study was to investigate whether the δ-opioid receptors are involved in the rewarding and reinstatement effect of cocaine in the conditioned place preference (CPP) test. Male Wistar rats were conditioned with cocaine (5 mg/kg) or saline in a biased CPP procedure. The intracerebroventricular (i.c.v.) administration of naltrindole (5 nmol), δ-opioid receptor antagonist but not β-funaltrexamine (5 nmol), or nor-binaltorphimine (10 nmol), μ-opioid and κ-opioid receptor antagonists, respectively reversed the expression of the cocaine CPP. The i.c.v. administration of new analogues of deltorphins with potent agonist activity at δ-opioid receptors, such as cyclo(N, N-carbonyl-D-Orn, Orn)deltorphin (DEL-6) at the dose of 10 and 20 nmol and deltorphin II N-(ureidoethyl)amide (DK-4) at the dose of 10 and 20 nmol reinstated the rewarding effect of cocaine after extinction sessions in the CPP test. Naltrindole (5 nmol, i.c.v.) abolished the reinstated effect of DK-4 (10 nmol). In addition, DEL-6 and DK-4 induce anxiolytic-like effects in the elevated plus-maze test. However, neither peptide given alone either produced a rewarding effect in the CPP test, or influenced the locomotor activity and motor coordination, thus suggesting that these effects of peptides did not influence the results obtained in the reinstatement procedure of CPP. In conclusion, our results show that δ-opioid receptors play a dominant role in cocaine reward and reinstatement of cocaine seeking behavior in the CPP test.

Cocaine abuse and dependence is a worldwide health problem. However, there are no currently approved medications to reduce cocaine abuse/relapse and toxicity. The aim of the present study was to test, whether group I metabotropic glutamate receptors (mGluRs) antagonists (mGluR1 and mGluR5) differentially regulate toxic versus behavioral effects of cocaine, both phenomena relevant to the psychopathology of cocaine addiction in humans. In the present study, we assessed the impact of mGluR1 antagonist-EMQMCM and mGluR5 antagonist-MTEP on the cocaine-induced lethality and the expression of sensitization to hyperlocomotor effect of cocaine in mice. Our study indicated that EMQMCM and MTEP, both substances at the doses of 5 and 10 mg/kg (but not 2.5 mg/kg), decreased cocaine-induced lethality produced by 75 mg/kg of cocaine, which was given acutely. The effect of EMQMCM was dose-dependent, and this compound at the dose of 10 mg/kg almost completely abolished the lethality induced by cocaine. MTEP reduced this cocaine effect at the doses of 5 and 10 mg/kg, equally. Furthermore, EMQMCM (1.25-5 mg/kg) at the doses of 2.5 and 5.0 mg/kg, and MTEP (2.5-10 mg/kg) only at the highest dose of 10 mg/kg, significantly reduced the expression of cocaine-induced (10 mg/kg) behavioral sensitization. Our results suggest that stimulation of mGluR1 and mGluR5 is involved in lethal effect of cocaine overdose and cocaine seeking behavior evaluated in behavioral sensitization test. However, the participation of mGluR1 in these cocaine effects seems to be dominant. Therefore, antagonists showing preferences towards mGluR1 might be useful in therapy of cocaine toxicity and abuse.

Much evidence indicates that endogenous opioid peptides are involved in effects caused by ethanol. The aim of the present study was to determine whether dansyl-PQR amide, a putative antagonist of receptors for an anti-opioid peptide—neuropeptide FF (NPFF) could affect anxiety-like behavior measured during withdrawal from acute-, and chronic ethanol administration in the elevated plus maze test in rats. Our study indicated that intracerebroventricular (i.c.v.) administration of dansyl-PQRamide (2.4 and 4.8 nmol) reversed anxiety-like behavior measured as a percent time spent in the open arms, and a percent open arm entries onto the open arms in the elevated plus-maze test in rats. These effects were inhibited by NPFF (10 and/or 20 nmol, i.c.v.) in the experiments performed during withdrawal from acute- and chronic ethanol administration. During withdrawal from acute ethanol, naloxone (1 mg/kg, i.p.), a nonselective opioid receptor antagonist, attenuated only an increased percent time spent in the open arms induced by dansyl-PQR amide (4.8 nmol). Dansyl-PQR amide, NPFF and naloxone given alone to naive rats did not have influence on spontaneous locomotor activity of animals. Furthermore, NPFF potentiated anxiety-like behavior during withdrawal from chronic, but not acute, ethanol administration in rats. Our data suggest that NPFF system is involved in regulation of affective symptoms of ethanol withdrawal. It seems that involvement of the NPFF system in ethanol withdrawal anxiety-like behavior is associated with regulation of the opioid system activity.

Using primary neuronal cell culture assays, combined with 2-D gel electrophoresis and capillary LC-MS, we identified differences in proteomes between control and morphine-treated cells. Statistically significant differences were observed among 26 proteins. Nineteen of them were up-regulated, while seven were down-regulated in morphine-treated cell populations. The identified proteins belong to classes involved in energy metabolism, associated with oxidative stress, linked with protein biosynthesis, cytoskeletal ones, and chaperones. The detected proteins demand further detailed studies of their biological roles in morphine addiction. It is crucial to confirm observed processes in vivo in order to reveal the nature and importance of the biological effect of proteome changes after morphine administration. Further investigations may lead to the discovery of new proteome-based effects of morphine on living organisms.

Neuropeptide FF (NPFF) has been described as an anti-opioid peptide. It plays a role in opioid antinociception, dependence and tolerance. Previous study has indicated that 1DMe ([D-Tyr 1 , (NMe)Phe 3 ]NPFF), a stable analog of NPFF, inhibits acquisition of the rewarding effect of morphine but not of ethanol in mice. The rewarding effects of these drugs were measured in the unbiased paradigm of conditioned place preference (CPP). The present study examines the influence of NPFF on the expression of morphine- and ethanol-induced CPP in the biased procedure in rats. Our experiments showed that NPFF, given intracerebroventricularly (i.c.v.) at the doses of 5, 10 and 20 nmol, inhibited the expression of morphine-induced CPP. NPFF gave itself, neither induced place preference nor aversion, although a tendency to aversive effect was seen at the highest dose of 20 nmol. NPFF did not indicate fear behavior in the elevated plus maze test, and did not disturb locomotor activity of rats. However, NPFF was unable to inhibit the expression of ethanol-induced CPP. Probably this effect is due to the fact that ethanol reward is a more complex process and apart from the role of opioids, there are other neurotransmitters also involved in this mechanism. These results suggest that NPFF is involved in the expression of morphine reward. Moreover, our study supports an anti-opioid character of this peptide.

The aim of the present study was to compare the influence of group I metabotropic glutamate receptor (mGluR) antagonists (mGluR1 and mGluR5) on the expression of sensitization to the locomotor effect of morphine. We also tested how these compounds affect the morphine withdrawal jumps in mice. In our study, the mGluR1 antagonist EMQMCM [3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate] and the mGluR5 antagonist MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine) were used. Sensitization was induced by five intraperitoneal (i.p.) injections of morphine at the dose of 10 mg/kg, every 3 days. Morphine dependence was induced by subcutaneous (s.c.) implantation of pellets containing 37.5 mg of morphine base for three days. Our data indicate that pretreatment with EMQMCM (5, 10, 20 mg/kg) and MTEP (5, 10 mg/kg) on the challenge day, inhibited the expression of sensitization to the locomotor effect of morphine in mice. Antagonists of both subtypes of the group I mGlurs given alone, did not modify the acute locomotor effect of morphine. On the other hand, EMQMCM did not attenuate the morphine withdrawal jumps precipitated by naloxone (4 mg/kg). The results suggest that both subtypes of the group I mGluRs (mGluR1 and mGluR5) take part in the expression of morphine sensitization processes but mGluR1 is not involved in the expression of morphine withdrawal jumps in mice. These findings may have implications for the treatment of opiate addiction in future.

Amphetamine (AMPH) induces deficits in cognition, and depressive-like behavior following withdrawal. The aim of the present study was to investigate whether pre-treatment with memantine (5mg/kg, i.p.), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, attenuates memory impairment induced by withdrawal from a 1 day binge regimen of AMPH (2mg/kg, four times every 2h, i.p.), in the novel object recognition test in rats. Herein, the influence of scopolamine (0.1mg/kg), an antagonist of the muscarinic cholinergic receptors, and the impact of MK-801 (0.1mg/kg), an antagonist of the NMDA receptors, on the memantine effect, were ascertained. Furthermore, the impact of memantine (5; 10; 20mg/kg, i.p.) was measured on depression-like effects of abstinence, 14 days after the last AMPH treatment (2mg/kg×1×14 days), in the forced swim test. In this test, the efficacy of memantine was compared to that of tricyclic antidepressant imipramine (10; 20; 30mg/kg, i.p.). Our study indicated that withdrawal from a binge regimen of AMPH impaired recognition memory. This effect was attenuated by administration of memantine at both 72h and 7 days of withdrawal. Moreover, prior administration of scopolamine, but not MK-801, decreased the memantine-induced recognition memory improvement. In addition, memantine reversed the AMPH-induced depressive-like behavior in the forced swim test in rats. The antidepressant-like effects of memantine were stronger than those of imipramine. Our study indicates that memantine constitutes a useful approach towards preventing cognitive deficits induced by withdrawal from an AMPH binge regimen and by depressive-like behavior during AMPH abstinence.

In the present study we investigated whether synthetic agonists of the nociceptin (NOP) receptors, Ro 64-6198 {(1 S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1 H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one} and Ro 65-6570 (8-acenaphthen-1-yl-1 -phenyl-1,3,8-triaza-spiro[4.5] decan-4-one), influence the expression of sensitization to the locomotor stimulant effect of morphine in mice. Sensitization was produced by five repeated administrations of morphine (10mg/kg, s.c.) at 3-day intervals. Seven days after the last morphine injection, Ro 64-6198 (1 and 3 mg/kg, i.p.) and Ro 65-6570 (3 and 6 mg/kg, i.p.) were given immediately before the challenge dose of morphine (10 mg/kg, s.c.). Both substances inhibited the expression of sensitization to the locomotor stimulant action of morphine. However, the selective NOP receptor antagonist, [Nphe 1 ]NC(1-13)NH 2 (30nmol, i.c.v.) did not reverse the inhibitory effect of the Ro-compounds. Therefore, our results suggest that the NOP receptor may not be critical for the influence of Ro-compounds on the morphine-induced sensitization, or the observed effect may be attributed to one functional subset of this receptor, stimulation of which is not blocked by [Nphe 1 ]NC (1-13)NH 2 .

It has been suggested that drugs modulating the glutamate/N-methyl-D-aspartate (NMDA) receptor system may be useful in the treatment of alcohol dependence. The effect of neramexane, a low-to-moderate affinity uncompetitive NMDA receptor antagonist, was examined on the development and expression of ethanol dependence (withdrawal-associated audiogenic seizures) and ethanol-induced conditioned place preference. Neramexane hydrochloride (3.5 mg/kg and higher) inhibited both the development and expression of ethanol dependence. Neramexane hydrochloride also inhibited the acquisition (1.75 mg/kg and higher) and expression (3.5 mg/kg and higher) of ethanol-induced place preference. Our data support therapeutic potential of neramexane as a treatment for alcohol abuse.

Our previous studies demonstrated that endogenous ligand of nociceptin (NOP) receptor, nociceptin(1-17) (also known as orphanin FQ), inhibits morphine-withdrawal syndrome measured as wet dog shakes in rats [Life Sci. 66 (2000) PL119]. This peptide is metabolized in the spinal cord, both in vitro and in vivo, to shorter fragments, including nociceptin(1-11) and nociceptin(1-6). These fragments, formed after cleavage by endogenous peptidase, are behaviorally active and modulate nociception in a bi-phasic process [Peptides 20 (1999) 239]. As these peptides induced transient naloxone-reversible analgesia in behavioral tests [Peptides 20 (1999) 239], in the present study we tested the influence of nociceptin(1-11) (10 and 20 microg) and nociceptin(1-6) (10, 20 and 40 microg) on the morphine-withdrawal syndrome in rats. Furthermore, the modified fragment of nociceptin(1-6) with an opioid-message domain achieved by replacement of Phe1 with Tyr was tested. Morphine-withdrawal syndrome was precipitated by the i.p. injection of naloxone hydrochloride (2 mg/kg), 72 h after implantation of morphine pellets. The wet-dog shakes were chosen for statistical analyses of the abstinence signs. The results show that nociceptin(1-11) and (1-6) attenuate this morphine-withdrawal symptom. The replacement of Phe1 with Tyr in nociceptin(1-6) fragment did not potentiate the influence of nociceptin(1-6) on wet dog shakes precipitated by naloxone in morphine-dependent rats.

The non-peptidergic opioid receptor-like 1 (ORL1, OP4) receptor ligand, Ro 64-6198 [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one], is a full agonist of the OP4 receptor. The aim of this study was to evaluate whether this compound influences morphine antinociception and dependence in mice. Ro 64-6198 inhibits the acute analgesic effect of morphine in the tail-immersion test, however, when given chronically during the acquisition of morphine dependence, development of this dependence is not prevented. The acute injection of Ro 64-6198 suppresses withdrawal escape jumps in morphine dependent mice, though this effect may be a result of the loss of locomotor activity induced by this compound and/or its myorelaxant action. The study provides evidence that stimulation of the OP4 receptor suppresses acute morphine antinociception, but is not sufficient to inhibit the development of morphine dependence in mice.

The present study investigated the effect of orphanin FQ/nociceptin (OFQ/N), the endogenous ligand of the opioid receptor-like 1 (ORL-1) receptor on the expression of cocaine-induced conditioned place preference (CPP) in rats. To extend this study, the new non-peptidic compound Ro 65-6570 (8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one), with agonist activity at ORL-1 receptors, was examined. The influence of both compounds on cocaine-induced hyperactivity was also studied. Our experiments indicated that intracerebroventricular (i.c.v.) injection of OFQ/N, at doses of 10 and 20 microg/rat, significantly suppressed the expression of cocaine-induced place preference. Ro 65-6570 (3 and 6 mg/kg, i.p.) did not change the effect of cocaine, although its acute injection in control rats significantly increased the time spent in the drug-associated compartment of the CPP apparatus. The substances exhibited opposite effects on cocaine-induced hyperactivity (OFQ/N suppressed it but Ro 65-6570 increased it). Our results suggest that the effect of OFQ/N on the expression of cocaine-induced CPP may be a result of its influence on dopamine (DA) neurotransmission in mesolimbic structures. Ro 65-6570 does not share this effect with OFQ/N.

Previous studies have indicated that metabotropic glutamate receptors 7 (mGluR7s) are involved in drug addiction. However, the role of these receptors in drug-induced behavioral sensitization is unknown. The aim of the present study was to determine whether systemic injection of AMN082, a selective mGluR7 allosteric agonist, reduces the cocaine- and morphine-induced hyperactivity and the development and expression of locomotor sensitization, and also affects the reciprocal cross-sensitization to the stimulant effect of cocaine and morphine in mice. AMN082 (1.25-10.0 mg/kg, i.p.) did not have an impact on locomotion of naive mice and did not affect the acute cocaine- or morphine-induced hyperactivity, except the dose of 10 mg/kg that suppressed the locomotor effect of both drugs. Repeated exposure to cocaine or morphine (10 mg/kg, 5× every 3 days) gradually increased locomotion during induction of sensitization and after 4 (cocaine) or 7 day (morphine) withdrawal phase when challenged with cocaine (10 mg/kg, i.p.) or morphine (10 mg/kg, i.p.) on day 17 or 20, respectively. Pretreatment of animals with the lower doses of AMN082 (1.25-5.0 mg/kg, i.p.), 30 min before every cocaine or morphine injection during repeated drug administration or before cocaine or morphine challenge, dose-dependently attenuated the development, as well as the expression of cocaine or morphine locomotor sensitization. AMN082 also inhibited the reciprocal cross-sensitization between these drugs. Prior to administration of MMPIP (10 mg/kg, i.p.), a selective mGluR7 antagonist reversed the inhibitory effect of AMN082 on the development or expression of cocaine or morphine sensitization. These data indicate that AMN082 attenuated the development and expression of cocaine and morphine sensitization, and the reciprocal cross-sensitization via a mechanism that involves mGluR7s. Thus, AMN082 might have therapeutic implications not only in the treatment of cocaine or opioid addiction but also in the treatment of cocaine/opioid polydrug-abusers.

The influence of systemic administration of cholinesterase inhibitors, donepezil and rivastigmine on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference (CPP) was examined in rats. Additionally, this study aimed to compare the effects of donepezil, which selectively inhibits acetylcholinesterase, and rivastigmine, which inhibits both acetylcholinesterase and butyrylcholinesterase on morphine reward. Morphine-induced CPP (unbiased method) was induced by four injections of morphine (5 mg/kg, i.p.). Donepezil (0.5, 1, and 3 mg/kg, i.p.) or rivastigmine (0.03, 0.5, and 1 mg/kg, i.p.) were given 20 min before morphine during conditioning phase and 20 min before the expression or reinstatement of morphine-induced CPP. Our results indicated that both inhibitors of cholinesterase attenuated the acquisition and expression of morphine CPP. The results were more significant after rivastigmine due to a broader inhibitory spectrum of this drug. Moreover, donepezil (1 mg/kg) and rivastigmine (0.5 mg/kg) attenuated the morphine CPP reinstated by priming injection of 5mg/kg morphine. These properties of both cholinesterase inhibitors were reversed by mecamylamine (3 mg/kg, i.p.), a nicotinic acetylcholine receptor antagonist but not scopolamine (0.5 mg/kg, i.p.), a muscarinic acetylcholine receptor antagonist. All effects of cholinesterase inhibitors were observed at the doses that had no effects on locomotor activity of animals. Our results suggest beneficial role of cholinesterase inhibitors in reduction of morphine reward and morphine-induced seeking behavior. Finally, we found that the efficacy of cholinesterase inhibitors in attenuating reinstatement of morphine CPP provoked by priming injection may be due to stimulation of nicotinic acetylcholine receptors.

Background Phosphorylation is a key process regulating a large number of fundamental biochemical reactions in living organisms. It is known that many mechanisms of response to chronic drugs administration are regulated by phosphorylation. It can be assumed that some of the phosphorylation sites are known, but they represent only a small fraction of the regulatory phosphorylation events in this system. Therefore, it is important to investigate protein phosphorylation with high-throughput methods such as mass spectrometry, that allow for efficient global analysis. The aim of this work was to develop a robust workflow for quantitative phosphoproteomic analysis, which operates in a semi-automatic manner. Methods The proposed approach consists of two methods of phosphopeptides enrichment (TiO 2 , IMAC), stable isotope methyl labeling, data-dependent mass spectrometry acquisition with simultaneous CID/ETD fragmentation, and data analysis platform based on Trans-Proteomic Pipeline. We have applied our method to analyze selected brain structures from rat involved in morphine dependence. Results We have identified and quantified number of phosphoproteins that were up- or down-regulated as a result of morphine treatment. Finally, we have applied a three-step filtration process to emerge the most regulated candidates. In parallel, all of the regulated proteins were annotated with GO terms to follow global trends of protein regulation. Conclusions The proposed MS-based workflow with following data analysis is efficient method for quantitative phosphoproteomic analysis: • Stable isotope methyl labeling introduces tags for quantitative analysis and in the same time increases specificity of phosphopeptides enrichment. • Utilization of two complementary methods for phosphopeptides enrichment (TiO 2 /IMAC) broadens the phosphoproteomic coverage. • Simultaneous CID/ETD fragmentation increases sequencing and phosphorylation sites mapping capabilities. • Data analysis with Trans-Proteomic Pipeline allows for optimization of FDR, and efficient quantitative analysis with ASAPRatio.

The influence of orphanin FQ/nociceptin (OFQ/N) on the morphine-withdrawal symptom was investigated. Withdrawal syndrome was induced in the morphine-dependent rats by an intraperitoneal (i.p.) injection of 2 mg/kg naloxone hydrochloride--an opioid receptors antagonist. Wet-dog shakes were used as a measure of the abstinence syndrome. Intraventricular injections of OFQ/N (5-20 microg/animal) caused significant inhibition of the withdrawal signs at doses between 15-20 microg, in the morphine-dependent rats. OFQ/N alone did not change behavior of the morphine-dependent animals. The obtained results indicate that OFQ/N can inhibit the morphine withdrawal symptoms induced by naloxone.

Metabolism of orphanin FQ/nociceptin (OFQ/N) was studied in the spinal cord of rats. The heptadecapeptide was efficiently cleaved by a neutral serine endopeptidase, thus releasing the major metabolite, OFQ/N (1–11) , further truncated to the final product, OFQ/N (1–6) . Biologic activity of this latter fragment was tested in vivo, after intracerebroventricular and intrathecal injections. Hexapeptide exhibited a bi-phasic effect, causing antinociception up to 10 min after injection, followed by a hyperalgesia. The analgesic effect was blocked by naloxone and hyperalgesia was inhibited by NMDA and NMDA/glycine site antagonists. The results indicate that shorter nociceptin fragments still possess their biologic activity though possibly acting via receptors other than ORL1.

The ethanol-like discriminative stimulus properties of a novel NMDA glycine receptor antagonist, L-701,324 ((7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2-(1H)-quinolone), a polyamine receptor antagonist, eliprodil, and a non-competitive NMDA receptor antagonist, MK-801 (dizocilpine), were examined in rats trained to discriminate ethanol from vehicle in a two-lever discrimination procedure. In rats trained to discriminate ethanol from vehicle, L-701,324 and MK-801 substituted for ethanol in a dose-dependent fashion with a complete substitution noted following administration of 7.5 mg/kg L-701,324 and 0.2 mg/kg MK-801, respectively. Full substitution for ethanol was achieved with no alteration in the rate of responding. In contrast, administration of eliprodil (in doses up to 5 mg/kg) showed only a partial, but not dose-dependent, substitution for ethanol. These findings indicate that a reduction of NMDA receptor activity, produced either via a blockade of non-competitive NMDA recognition sites or of NMDA/glycine-sensitive regulatory sites, had discriminative stimulus properties that are similar to those produced by ethanol. Furthermore, the observation that the NMDA/glycine receptor antagonist, L-701,324, was a more effective substitute for ethanol than was the polyamine antagonist, eliprodil, suggests that several NMDA receptor subunits, and thus not only NMDAR2B receptor subunits, are of importance for the discriminative stimulus effects of ethanol.

NPFF precursor, pro-NPFF(A) contains three known bioactive sequences: NPFF (FLFQPQRF-NH(2)), neuropeptide AF (NPAF; AGEGLSSPFWSLAAPQRF-NH(2)) and neuropeptide SF (NPSF; SLAAPQRF-NH(2)). The key-feature of these fragments is their common PQRF-amidated sequence at their C termini. Here, we evaluated the biological activity of two other sequences derived from the mouse NPFF(A) precursor, that does not have PQRF-amidated C-terminus. One peptide was residing between positions 85 and 99 in the mice pro-NPFF(A). This peptide was referred to as neuropeptide SA (NPSA; SAWGSWSKEQLNPQA), assigned due to its flanking amino acids. Another sequence used in the experiments was N-terminal fragment of NPSA, here referred to as neuropeptide SS (NPSS; SAWGSWS). These two peptides, classified as crypteins, were synthesized and tested in the hot-plate and tail immersion tests in mice for their pharmacological activity in morphine-induced antinociception. The effects of both crypteins were compared to NPFF. Our experiments indicated that both crypteins inhibited morphine antinociception and their effects were reversed by RF9, an antagonist of NPFF receptors. These data show that NPSA and NPSS possess NPFF-like anti-opioid activity in these behavioral tests.

Many data indicate that endogenous opioid system is involved in amphetamine-induced behavior. Neuropeptide FF (NPFF) possesses opioid-modulating properties. The aim of the present study was to determine whether pharmacological modulation of NPFF receptors modify the expression of amphetamine-induced conditioned place preference (CPP) and amphetamine withdrawal anxiety-like behavior, both processes relevant to drug addiction/abuse. Intracerebroventricular (i.c.v.) injection of NPFF (5, 10, and 20 nmol) inhibited the expression of amphetamine CPP at the doses of 10 and 20 nmol. RF9, the NPFF receptors antagonist, reversed inhibitory effect of NPFF (20 nmol, i.c.v.) at the doses of 10 and 20 nmol and did not show any effect in amphetamine- and saline conditioned rats. Anxiety-like effect of amphetamine withdrawal was measured 24h after the last (14 days) amphetamine (2.5mg/kg, i.p.) treatment in the elevated plus-maze test. Amphetamine withdrawal decreased the percent of time spent by rats in the open arms and the percent of open arms entries. RF9 (5, 10, and 20 nmol, i.c.v.) significantly reversed these anxiety-like effects of amphetamine withdrawal and elevated the percent of time spent by rats in open arms at doses of 5 and 10 nmol, and the percent of open arms entries in all doses used. NPFF (20 nmol) pretreatment inhibited the effect of RF9 (10 nmol). Our results indicated that stimulation or inhibition of NPFF receptors decrease the expression of amphetamine CPP and amphetamine withdrawal anxiety, respectively. These findings may have implications for a better understanding of the processes involved in amphetamine dependence.

The endogenous brain serotonin (5-HT) system is believed to have an important modulatory influence in mediating drug reward and seeking mechanisms. Data from preclinical behavioral studies have provided emerging evidence that 5-HT(6) receptors, among other 5-HT receptors, may play a significant role in the mechanisms of action of psychostimulant addicted drugs. The aim of the present study was to investigate whether the selective pharmacological blockade or activation of 5-HT(6) receptors altered the maintenance of cocaine self-administration, reinstatement of cocaine-seeking behavior following an extinction of cocaine self-administration or cocaine-evoked conditioned place preference in rats. We also evaluated the effects of 5-chloro-N-(4-methoxy-3-piperazin-1-ylphenyl)-3-methyl)-2-benzothiophene-sulfonamide (SB 271046, a 5-HT(6) receptor antagonist) or N-1-(6-chloroimidazo-[2,1-b]-[1,3]thiazole-5-sulfonyl)tryptamine (WAY 181187, a potent 5-HT(6) receptor agonist) on locomotor activity in rats. Our results indicate that SB 271046 (1-10 mg/kg) altered cocaine-maintained self-administration as well as cocaine-evoked reinstatement of cocaine seeking and expression of cocaine place preference in rats.We also demonstrate that pharmacological stimulation of 5-HT(6) receptors by WAY 181187 (3-30 mg/kg) attenuated the expression of cocaine conditioned place preference but not cocaine self-administration and reinstatement of cocaine seeking. WAY 181187 at the highest dose used (30 mg/kg) reduced basal locomotor activity. Despite current results, the precise function and therapeutic relevance of 5-HT(6) receptors need further clarification.

Glutamine synthetase is a key enzyme which has a regulatory role in the brain glutamate pool. According to previously published proteomic analysis, it was shown that the expression level of this enzyme is affected by morphine administration. In our study, we examined the activity of glutamine synthetase in various structures of rat brain (cortex, striatum, hippocampus and spinal cord) that are biochemically and functionally involved in drug addiction and antinociception caused by morphine. We were not able to observe any significant changes in the enzyme activity between morphine-treated and control samples despite previously reported changes in the expression levels of this enzyme. These findings stressed the fact that changes observed in the expression of particular proteins during proteomic studies may not be correlated with its activity.

Astrocytes are supportive cells, necessary for ensure optimal environment for neural cells functioning. They are involved in extracellular K+ level regulation and neurotransmitters removal. They are also dependent for myelination and synapses formation. They may make a contribution in signal propagation in the central nervous system, for example, through Ca2+ signaling. With the use of neonatal pure astrocyte cell culture, we investigated changes in astrocyte's proteomes under the influence of morphine. We found 10 major proteins, which show different expression between physiological cell culture and morphine treatment. With 2D gel electrophoresis and nanoLC-ESI-MS/MS, we identified proteins and characterized their potential role in morphine dependence. Observed differences were also confirmed by Western blotting. Our data suggests a role for astrocytes in the formation of the morphine dependence at the molecular level. This finding may support interpretation of causes of morphine dependence formation based only on behavioral data.

The aim of the study was to evaluate whether the newly synthesized analog of enkephalin, cyclo[N(epsilon),N(beta)-carbonyl-D-Lys(2), Dap(5)] enkephalinamide (cUENK6), a highly potent mu- (guinea pig ileum assay) and delta-receptors (mouse vas deferens assay) ligand, induces an antinociceptive effect in the hot-plate test and tail-immersion test after intracerebroventricular administration. Our study indicated that this peptide at the dose of 0.25 nmol produced comparable but at the dose of 0.5 nmol stronger than morphine (13 nmol), antinociceptive effect in both tests. Furthermore, rats with developed tolerance to morphine indicated cross-tolerance to antinociceptive effects of cUENK6. The antinociceptive effects of cUENK6 and morphine were inhibited by non-selective opioid receptor antagonist--naloxone. More detailed study indicated that the delta-opioid receptor antagonist - naltrindole very strongly and, to the lower extent, mu-opioid antagonist - beta-funaltrexamine (beta-FNA), inhibited antinociceptive effect of cUENK6 in the tail-immersion test. Nor-binaltorphimine (nor-BNI), a kappa-opioid receptor antagonist, did not influence this effect. These data suggest the dominant role of delta-opioid receptors as compared with mu-receptors in mediation antinociceptive effect of cUENK6. Furthermore, we found that cUENK6 is much more effective in inhibiting pain in the hot-plate (ED(50)=0.0792 nmol) than in the tail-immersion (ED(50)=0.3526 nmol) test. However, cUENK6 at the antinociceptive doses induced hypolocomotion, and although this effect is observed after administration of opioid agonists in rats as a one phase of their biphasic action (inhibition followed by activation), in our study it was not naloxone-reversible. Therefore, our study suggests that not only opioid receptors may be involved in behavioral effects of cUENK6.

The aim of the present study was to determine whether various glutamate receptor antagonists could affect ethanol withdrawal-induced anxiety-like behavior measured in the elevated plus-maze test in rats. In our study, memantine (8 and 12 mg/kg), a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist, did not show any effect on ethanol withdrawal anxiety. Acamprosate (NMDA and metabotropic glutamate5 (mGlu5) receptor antagonist), at a dose of 400 mg/kg showed anxiolytic-like effect, thus increasing the percent of time spent in open arms and open arms entries. Antagonists of group I mGlu receptors, such as MTEP ([(2-methyl-1,3-thiazol-4-yl) ethynyl] pyridine, mGlu5 receptor) or EMQMCM (3-ethyl-2-methyl-quinolin-6-yl-(4-methoxy-cyclohexyl)-methanone methanesulfonate, mGlu1 receptor), caused similar effects to acamprosate. In contrast to acamprosate and MTEP, EMQMCM (5 mg/kg) elevated the ethanol withdrawal-induced decrease in locomotion. When given alone to the saline-treated group, EMQMCM indicated anxiolytic-like effect. Our results imply a crucial role of mGlu5 receptor in an anxiety-like effect of ethanol withdrawal because MTEP (a selective mGlu5 receptor antagonist) and acamprosate (which also indirectly inhibits mGlu5 receptor) attenuated ethanol withdrawal anxiety-like behavior without influence on ethanol withdrawal hypolocomotion and did not show any effect in the saline-treated groups. However, difference in anxiolytic-like potency between both these group I mGlu receptors antagonists may be due to the recent experimental design. Therefore, taking into account a positive correlation between ethanol withdrawal-induced anxiety and relapse to ethanol drinking, our results suggest that mGlu receptor antagonists of group I (similarly to acamprosate) could prevent relapse to drinking and, therefore they might be useful in therapy of alcoholism.

Recently, we reported the discovery of a novel amino acid sequence derived from the NPFF precursor NAWGPWSKEQLSPQA, which blocked the expression of conditioned place preference induced by morphine and reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. Here, we name it as NPNA (Neuropeptide NA from its flanking amino acid residues). The synthetic peptide influenced the expression of mRNA coding for Gα(i1), (i2), and (i3) subunits. The results provide further evidence that yet another bioactive sequence might be present within the NPFF precursor.

The effects of various protease inhibitors on the development of antinociceptive tolerance to morphine were examined in mice. Intrathecal (i.t.) administration of morphine (0.01–1nmol) produced a dose-dependent and significant antinociceptive effect in the 0.5% formalin test. When the doses of morphine (mg/kg, s.c. per injection) were given as pretreatment twice daily for two days [first day (30) and second day (60)], i.t. administration of morphine (0.1nmol) was inactive due to antinociceptive tolerance on the third day. Tolerance to i.t. morphine was significantly suppressed by the i.t. injection of N -ethylmaleimide or Boc-Tyr-Gly-NHO-Bz, inhibitors of cysteine proteases involved in dynorphin degradation, as well as by dynorphin A, dynorphin B and (−) U-50,488, a selective κ-opioid receptor agonist. On the other hand, amastatin, an aminopeptidase inhibitor, phosphoramidon, an endopeptidase 24.11 inhibitor, lisinopril, an angiotensin-converting enzyme inhibitor, and phenylmethanesulfonyl fluoride, a serine protease inhibitor, were inactive. These results suggest that cysteine protease inhibitors suppress the development of morphine tolerance presumably through the inhibition of dynorphin degradation.

The endogenous brain opioid system is believed to play an important role in mediating reward mechanisms. Opioid innervation is high in many limbic regions and reinforcing actions of many drugs of abuse, including cocaine, are thought to be mediated via endogenous opioid system. The aim of the present study was to indicate whether the anti-opioid peptide, neuropeptide FF (NPFF; FLFQPQRF-NH2) was able to modify the rewarding effect of cocaine (5 mg/kg) measured in the expression of conditioned place preference (CPP) test in rats and the expression of sensitization to hyperlocomotor effect of cocaine (10 mg/kg) in mice. Our results indicate that NPFF (5, 10, and 20 nmol) given intracerebroventricularly (i.c.v.) inhibited the expression of cocaine-induced CPP at the dose of 10 nmol (P0.01) and 20 nmol (P0.001). Moreover, NPFF inhibited the expression of cocaine-induced sensitization to its hyperlocomotor effect at the dose of 20 nmol (P0.05) and acute hyperlocomotor effect of cocaine at doses of 5 nmol (P0.01), 10 nmol (P0.01), and 20 nmol (P0.05). Our study suggests that NPFF may participate in a rewarding effect of cocaine measured in the CPP paradigm. On the other hand, our experiments indicate that NPFF is involved in the mechanism of expression of sensitization to cocaine hyperlocomotion but this effect seems to be non-specific because NPFF also inhibited the acute hyperlocomotor effect of cocaine.

Neuropeptide FF (NPFF) precursors from different species contain at least three known neuropeptides, i.e. FF (FLFQPQRF-NH(2)), AF (AGEGLSSPFWSLAAPQR-NH(2)) and SF (SLAAPQRF-NH(2)). We demonstrate that the rat NPFF precursor contains another bioactive sequence, NAWGPWSKEQLSPQA, spanning between positions 85 and 99. Synthetic NPFF precursor (85-99) (10 and 20 nmol, i.c.v.) blocked the expression of conditioned place preference induced by morphine (5 mg/kg, s.c.). This peptide alone (10 and 20 nmol, i.c.v.) had no influence on the baseline latency of a nociceptive reaction but reversed the antinociceptive activity of morphine (5 mg/kg, s.c.) in the tail-immersion test in rats. These data suggest the existence of a novel bioactive cryptic peptide within an already known NPFF precursor.

This review provides an outline of the most important proteomic applications in the study of neurodegenerative disorders including Alzheimer's (AD), Parkinson's (PD), Huntington's (HD), and prion diseases, and also discusses advances in cancer and addiction. One of the scopes is to illustrate the potential of proteomics in the biomarkers discovery of these diseases. Finally, this article comments the advantages and drawbacks of the most commonly used techniques and methods for samples preparation.

Glutamate is involved in learning and memory processes, and chronic ethanol administration induces up-regulation of glutamate neurotransmission. PAM mGluR5, ADX-47273, potentiates mGlu5 function and can facilitate learning in spatial tasks. The aim of the present study was to investigate, whether ADX-47273 ameliorates reversal learning and memory impaired by …

Neuropeptide FF (NPFF) has been characterized as an endogenous anti-opioid peptide because its intraventricular injection (icv) reversed morphine- and stress-induced analgesia, and precipitates withdrawal syndrome in morphine-dependent rats. The role of NPFF in other aspects of drug dependence is unknown. Therefore, the aim of this study was to determine NPFF influence on the expression of sensitization to the morphine-induced hyperlocomotion. As the opioid system plays a role in ethanol effects, the influence of NPFF on the expression of sensitization to hyperlocomotor effect of ethanol was also investigated. Our study indicated that acute administration of NPFF (5, 10, 20nmol, icv) inhibited the expression of morphine-induced sensitization at doses of 10 (P

Although a series of proteins in the brain have been shown to be qualitatively or quantitatively dysregulated following morphine administration, a systematic proteomic study has not been carried out so far. We therefore aimed to show the effect of morphine on protein levels in the rat brain. For this purpose rats were given a morphine base in subcutaneously placed pellets and subsequently the cerebral cortex, hippocampus and striatum were taken for proteomic studies after three days. Extracted proteins were run on two-dimensional gel electrophoresis, scanned and quantified by specific software. Proteins with significantly different levels were analysed by mass spectrometry (MALDI-TOF-TOF). Twenty-six proteins were found to be differentially expressed and were unambiguously identified. Dysregulated proteins were from several protein pathways and cascades including signaling, metabolic, protein handling, antioxidant and miscellaneous classes. These findings represent an initial approach to the generation of a 'morphinome' and may form the basis for further protein chemical studies as a valuable analytical tool. Moreover, the study reveals morphine-regulated proteins in different brain areas and indicates the pathways involved following morphine administration in the rat, the main species for pharmacological studies in the field.

Synthetic derivative of C-terminal fragment of CART (55–102) with reduced thiol groups, [Abu 86,94 ]CART (85–102) red , given together with amphetamine (5 mg/kg, s.c.) or cocaine (15 mg/kg, s.c.), reversed hyperlocomotion induced by these drugs at a dose of 0.1 μg but not at a higher dose. In the cerebral cortex homogenate, [Abu 86,94 ]CART (85–102) red was nonspecifically cleaved from N- and C-termini. This peptide contains two chemically blocked Cys residues, and two others in reduced form. Concomitant with cleavage, rapid cyclization occurred. The newly formed cyclic peptides were stable. The cyclic peptide [Abu 86,94 ]CART (85–102) ox failed to inhibit amphetamine- and cocaine-induced locomotor activity. The ability to inhibit the locomotor-stimulant activity of amphetamine was retained in [Abu 86,88,94,101 ]CART (85–102), in which all Cys were replaced with 2-aminobutyric acid to prevent their pairing. Disulfide bridge formation may be an interesting mechanism that prevents proteolysis of [Abu 86,94 ]CART (85–102) red and terminates its ability to reverse amphetamine-induced hyperlocomotion.

The aim of this study was to reveal potential markers associated with drug dependence, using the proteomic approach. Gels containing samples derived from morphine-treated and control animals were compared and analyzed. Inspection of protein profiles, following TCA/acetone precipitation and the use of nano-scale liquid chromatography coupled to tandem mass spectrometry, allowed for identification of eleven potential dependence markers, mainly cytoplasmic and mitochondrial enzymes, e.g. proteins that belong to GTPase and GST superfamilies, ATPase, asparaginase or proteasome subunit p27 families.

Cortical cells in primary culture contain active proteolytic enzymes capable of cleaving nociceptin/orphanin FQ (N/OFQ) and dynorphins to shorter, bioactive fragments. Cells were divided into subpopulations (neurons, astrocytes and oligodendrocytes), and the metabolic pathways in particular populations of the cells were investigated. Processing of N/OFQ and dynorphins in the cytosolic fractions differed significantly. The proposed cell model can be used for the more detailed studies on the central nervous system proteomics.

Nociceptin (also called orphanin FQ), a 17-amino-acid peptide, is the natural ligand of the nociceptin opioid peptide (NOP) receptor. This peptide shows similarities, in its structure, to opioid peptides, mainly to dynorphin A. However, unlike opioid peptides, it does not produce a conditioned place preference or aversion but inhibits rewarding effect of drugs of abuse. The present study was designed to examine the ability of nociceptin to block the acquisition of amphetamine-induced place preference, and the development of amphetamine-induced sensitization to stereotypy in rats. Our experiments indicated that repeated administration of nociceptin at increasing doses during conditioning significantly attenuated the reinforcing effect of amphetamine in conditioned place preference paradigm. Nociceptin did not change the acute effect of amphetamine-induced stereotypy but prevented the development of sensitization to stereotypy measured on the challenge day. Our results suggest the involvement of nociceptin in long-lasting neuronal adaptation after repeated amphetamine treatment.

Cysteine proteinase found in the spinal cord of rat, called nociceptin-converting enzyme (NCE), is competitively inhibited by dynorphin A and its fragment des-[Tyr 1 ]-DYN A. This proteinase converts orphanin FQ/nociceptin (OFQ/N) to two major fragments: OFQ/N(1-11) and further OFQ/N(1-6) with analgesic properties. Dynorphin A at the concentration of 10 μM increases K M from 15.0 to 55.9 μM. The calculated K i for this interaction was estimated at 3.7 μM. This observation may suggest an interaction between opioid and nociceptive systems which may be affected by the balance between opioid and antiopioid systems. This balance between particular OFQ/N sequences that are derived from the same precursor and regulated by proteinases may play an important role in pain. Interestingly, dynorphin B does not reveal a similar action on the NCE.

A C-terminal analog of the hexapeptide orphanin FQ/nociceptin-(1–6), [Ala 6 ]-orphanin FQ/nociceptin-(1–6), and a pentapeptide orphanin FQ/nociceptin-(1–5) were tested in vivo for their analgesic/hyperalgesic activity in the hot-plate test with rats. Replacement of the C-terminal glycine by l -alanine (Phe-Gly-Gly-Phe-Thr-Ala) in orphanin FQ/nociceptin-(1–6) abolished the hyperalgesic potency of native orphanin FQ/nociceptin-(1–6) (Phe-Gly-Gly-Phe-Thr-Gly), but analgesic activity was retained and was diminished by naloxone. Removal of the C-terminal amino acid (glycine or alanine) from orphanin FQ/nociceptin-(1–6) caused a significant loss of analgesic activity. It is anticipated that glycine plays a crucial role in the biphasic activity of orphanin FQ/nociceptin-(1–6). This may suggest the existence of a mechanism for terminating the biological action of orphanin FQ/nociceptin.

Numerous data indicate that noncompetitive and competitive N -methyl- d -aspartate (NMDA) receptor antagonists inhibit the development of physical dependence on opioids when these substances are administered together, and NMDA receptor antagonists are used at lower range of doses. Higher doses of these antagonists can enhance some opioid-induced effects. The present study extends these findings to the effects of NMDA/glycine (glycine B ) site antagonists. Wistar rats were rendered dependent on morphine by implantation of morphine pellets. Both of the glycine B site antagonists used, 7-chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2( H )-quinolone (L-701,324; 2.5 and 5.0 mg/kg) and 5,7-dichlorokynurenic acid (5,7-DCKA; 25, 50, and 100 mg/kg), suppressed the expression of morphine withdrawal syndrome estimated as wet dog shakes. Furthermore, L-701,324 (2.5 and 5 mg/kg), given twice a day during the development of morphine dependence, attenuated the development of morphine dependence, and the results were comparable to those obtained after administration of noncompetitive NMDA receptor antagonist — MK801 (0.1 mg/kg). Our data suggest that glycine B site antagonists may attenuate wet dog shakes (withdrawal) and the development of dependence, both being induced by chronic morphine administration in rats.

Nociceptin-orphanin FQ (OFQ/N) is a newly discovered peptide involved in pain transmission. The method is described to identify metabolic pathway of this neuropeptide in the spinal cord of rats using capillary size-exclusion liquid chromatography coupled to electrospray ionization mass spectrometry. The applied technique is rapid and selective, and allows for simultaneous measurement and quantitation of several fragments in the incubation mixture.