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Orexins (OX) and their receptors (OXR) modulate feeding, arousal, stress, and drug abuse. Neural systems that motivate and reinforce drug abuse may also underlie compulsive food seeking and intake. Therefore, the effects of GSK1059865 (5-bromo-N-[(2S,5S)-1-(3-fluoro-2-methoxybenzoyl)-5-methylpiperidin-2-yl]methyl-pyridin-2-amine), a selective OX(1)R antagonist, JNJ-10397049 (N-(2,4-dibromophenyl)-N'-[(4S,5S)-2,2-dimethyl-4-phenyl-1,3-dioxan-5-yl]urea), a selective OX(2)R antagonist, and SB-649868 (N-[((2S)-1-{[5-(4-fluorophenyl)-2-methyl-1,3-thiazol-4-yl]carbonyl}-2-piperidinyl)methyl]-1-benzofuran-4-carboxamide), a dual OX(1)/OX(2)R antagonist were evaluated in a binge eating (BE) model in female rats. BE of highly palatable food (HPF) was evoked by three cycles of food restriction followed by stress, elicited by exposing rats to HPF, but preventing them from having access to it for 15 min. Pharmacokinetic assessments of all compounds were obtained under the same experimental conditions used for the behavioral experiments. Topiramate was used as the reference compound as it selectively blocks BE in rats and humans. Dose-related thresholds for sleep-inducing effects of the OXR antagonists were measured using polysomnography in parallel experiments. SB-649868 and GSK1059865, but not JNJ-10397049, selectively reduced BE for HPF without affecting standard food pellet intake, at doses that did not induce sleep. These results indicate, for the first time, a major role of OX(1)R mechanisms in BE, suggesting that selective antagonism at OX(1)R could represent a novel pharmacological treatment for BE and possibly other eating disorders with a compulsive component.

Nociceptin/orphanin FQ (N/OFQ), the nociceptin opioid peptide (NOP) receptor ligand, increases feeding when injected centrally. Initial data suggest that N/OFQ blocks the development of a conditioned taste aversion (CTA). The current project further characterized the involvement of N/OFQ in the regulation of hunger vs. aversive responses in rats by employing behavioral, immunohistochemical, and real-time PCR methodology. We determined that the same low dose of the NOP antagonist [Nphe1]N/OFQ(1-13)NH2 delivered via the lateral ventricle diminishes both N/OFQ- and deprivation-induced feeding. This anorexigenic effect did not stem from aversive consequences, as the antagonist did not cause the development of a CTA. When [Nphe1]N/OFQ(1-13)NH2 was administered with LiCl, it moderately delayed extinction of the LiCl-induced CTA. Injection of LiCl + antagonist compared with LiCl alone generated an increase in c-Fos immunoreactivity in the central nucleus of the amygdala. The antagonist alone elevated Fos immunoreactivity in the paraventricular nucleus of the hypothalamus, nucleus of the solitary tract, and central nucleus of the amygdala. Hypothalamic NOP mRNA levels were decreased during energy intake restriction induced by aversion, as well as in non-CTA rats food-restricted to match CTA-reduced consumption. Brain stem NOP was upregulated only in aversion. Prepro-N/OFQ mRNA showed a trend toward upregulation in restricted rats ( P = 0.068). We conclude that the N/OFQ system promotes feeding by affecting the need to replenish lacking calories and by reducing aversive responsiveness. It may belong to mechanisms that shift a balance between the drive to ingest energy and avoidance of potentially tainted food.

The association of ethanol's reinforcing effects with specific environmental stimuli is thought to be a critical factor for relapse risk in alcoholism. This study examined in rats the effects of a newly deorphanized neuropeptide receptor and its cognate ligand, Neuropeptide S (NPS), on ethanol consumption and reinstatement of ethanol-seeking by environmental cues previously associated with ethanol availability. In the self-administration experiments, the stable response rates observed for ethanol reinforcement were not modified by intracerebroventricular (ICV) injection of NPS (1.0 and 2.0 nmol per rat). In the reinstatement experiments, ethanol-associated cues induced robust rates of ethanol seeking, which were highly resistant to extinction over repeated sessions of reinstatement testing. ICV NPS treatment (1.0, 2.0 and 4.0 nmol per rat) resulted in a significant increase of ethanol seeking elicited by ethanol-associated cues. In contrast, NPS did not affect the reinstatement of responding to water-paired stimuli. Site-specific NPS injection (0.1 and 0.5 nmol per rat) into the lateral hypothalamus also reinstated extinguished responding to ethanol. This effect was selectively blocked by pre-treatment with the hypocretin-1/orexin-A antagonist SB-334867 (10 mg/kg, i.p.). At the dose tested, SB-334867 did not modify alcohol reinstatement per se. These results provide the first demonstration that activation of NPS receptors in the LH intensifies relapse to ethanol-seeking elicited by environmental conditioning factors. This effect is selective, and is mediated by activation of LH hypocretin neurones. Based on the present findings, we also predict that antagonism at NPS receptors could represent a novel pharmacological approach to alcohol relapse treatment.

The influence of peripheral nociceptin/orphanin FQ (N/OFQ) on cold restraint-induced gastric mucosal damage in the rat was investigated. Exposure to cold-restraint for 3 and 4h caused the formation of hemorrhagic lesions in the glandular portion of the stomach. N/OFQ dose-dependently decreased lesion formation, in the range 0.03-1 microg/kg/h i.p. Its effect was reversed by the selective NOP receptor antagonist [Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 30 microg/kg/h ip. The selective NOP receptor agonist [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112), 0.01-0.3 microg/kg/h i.p., similarly reduced lesion formation. Light and scanning electron microscopy confirmed the protective activity of N/OFQ. Cold-restraint stress causes a reduction in mucus content and in adhering mucus layer, partly counteracted by N/OFQ. These results suggest that N/OFQ counteracts acute stress-induced gastric mucosal damage by interacting with NOP receptor and by influencing mucous cell activity.

Activation of the NOP receptor by its endogenous ligand nociceptin/orphanin FQ reduces ethanol intake in genetically selected alcohol preferring Marchigian Sardinian alcohol preferring (msP) rats. Here we evaluated whether buprenorphine, a partial agonist at micro-opioid and NOP receptors, would reduce ethanol consumption in msP rats via activation of NOP receptors.Marchigian Sardinian alcohol preferring rats trained to drink 10% alcohol 2 hours/day were injected with buprenorphine (.03, .3, 3.0, or 6.0 mg/kg intraperitoneally [IP]) 90 min before access to ethanol.Similar to prototypical micro-agonists, the two lowest doses of buprenorphine significantly increased ethanol consumption (p.01); in contrast, the two highest doses reduced it (p.05). Pretreatment with naltrexone (.25 mg/kg IP) prevented the increase of ethanol intake induced by .03 mg/kg of buprenorphine (p.001) but did not affect the inhibition of ethanol drinking induced by 3.0 mg/kg of buprenorphine. Conversely, pretreatment with the selective NOP receptor antagonist UFP-101 (10.0 or 20.0 microg/rat) abolished the suppression of ethanol drinking by 3.0 mg/kg of buprenorphine.Buprenorphine has dualistic effects on ethanol drinking; low doses increase alcohol intake via stimulation of classic opioid receptors, whereas higher doses reduce it via activation of NOP receptors. We suggest that NOP agonistic properties of buprenorphine might be useful in the treatment of alcoholism.

Central injection of Nociceptin/Orphanin FQ (N/OFQ), inhibits the anorectic effect of corticotropin-relasing factor (CRF) and stress in rats. Recently, Urocortin II (Ucn II) and Urocortin III (Ucn III), two selective CRF 2 receptor agonists, have been identified. Here, we investigated the effect of intracerebroventricular (ICV) injection of 0.25, 0.75, 1.50 or 3 nmol/rat of Ucn II or Ucn III on food and water intake in food deprived rats. The effect of N/OFQ on Ucn II and UCNIII-induced anorexia was also studied. Results showed a greater inhibition of food consumption by Ucn II than Ucn III. Pretreatment with N/OFQ (0.25–2.0 nmol/rat) did not block the effects of Ucn II and UCNIII. Conversely, injection of N/OFQ (0.25–2.0 nmol/rat) blocked the anorectic effect of CRF (0.1 nmol/rat). These findings suggest that N/OFQ selectively prevent the anorectic effect mediated by activation of the CRF 1 receptor system.

Alcoholism is a chronic relapsing disorder with substantial heritability. Uncovering gene–environment interactions underlying this disease process can aid identification of novel treatment targets. Here, we found a lowered threshold for stress-induced reinstatement of alcohol seeking in Marchigian–Sardinian Preferring (msP) rats genetically selected for high alcohol preference. In situ hybridization for a panel of 20 stress-related genes in 16 brain regions was used to screen for differential gene expression that may underlie this behavioral phenotype. An innate up-regulation of the Crhr1 transcript, encoding the corticotropin-releasing hormone receptor 1 (CRH-R1), was found in several limbic brain areas of msP rats genetically selected for high alcohol preference, was associated with genetic polymorphism of the Crhr1 promoter, and was accompanied by increased CRH-R1 density. A selective CRH-R1 antagonist (antalarmin, 10–20 mg/kg) was devoid of effects on operant alcohol self-administration in unselected Wistar rats but significantly suppressed this behavior in the msP line. Stress-induced reinstatement of alcohol seeking was not significantly affected by antalarmin in Wistar rats but was fully blocked in msP animals. These data demonstrate that Crhr1 genotype and expression interact with environmental stress to reinstate alcohol-seeking behavior.

Chronic ethanol intake affects various organ systems of the body. The present study evaluated modifications of fatty acid concentrations both in brain and striated skeletal muscles of rats genetically selected for voluntary high ethanol intake. Three groups of rats were tracked for 10 weeks of access to ethanol only as fluid (group 1) to free choice of ethanol and water (group 2) or to water only (group 3). At the end of the period, the animals were sacrificed and their brain hippocampus and striated skeletal muscles were removed and fatty acid content of these tissues was determined. Long-chain fatty acid content increased in the hippocampus while it decreased in the striated skeletal muscles. Short chain fatty acid content decreased in the hippocampus while short chain fatty acid content increased in the striated skeletal muscles. The data show that brain and striated skeletal muscles differently modulate fatty acid content perhaps because these areas utilize different cell membrane functionality regulation systems.

Background: Hypericum perforatum extracts attenuate ethanol intake in alcohol-preferring rats. The opioid receptor antagonists, naloxone and naltrexone, reduce ethanol intake in rats and humans. The combination of different agents that reduce ethanol intake has been proposed as an approach to the pharmacotherapy of alcoholism. This study evaluated the effect on ethanol intake of the combined administration of a CO2H. perforatum extract and naloxone or naltrexone in genetically selected Marchigian Sardinian alcohol-preferring rats. Methods: Ten percent (v/v) ethanol intake was offered 2 hr per day at the beginning of the dark phase of the reverse light-dark cycle. H. perforatum CO2 extract was given intragastrically, 1 hr before access to ethanol. Naloxone or naltrexone was given by intraperitoneal injection 10 min before the extract. Results: H. perforatum CO2 extract reduced ethanol intake at 31 or 125 mg/kg, but not 7 mg/kg. These doses neither modified food or water intake during access to ethanol, nor reduce 0.2% saccharin intake. Naloxone reduced ethanol and food intake at 3 or 5 mg/kg, but not 1 mg/kg. When naloxone 1 mg/kg was combined with the three doses of H. perforatum CO2 extract, the attenuation of ethanol intake was more pronounced than that observed after the administration of the extract alone. Alcohol intake was also significantly reduced by 7 mg/kg of H. perforatum CO2 extract combined with naloxone 1 mg/kg. The combined treatments never modified the rat's locomotor activity nor the simultaneous intake of food, water or 0.2% saccharin. Naltrexone reduced ethanol intake at 1 and 3 mg/kg, but not at 0.5 mg/kg. When naltrexone 0.5 mg/kg was combined with H. perforatum CO2 extract 7 mg/kg, ethanol intake was markedly reduced. Conclusions: These findings provide evidence that H. perforatum CO2 extract and opiate receptor antagonists act synergistically to induce a pronounced and selective reduction of voluntary ethanol consumption in alcohol-preferring rats.

Leptin, a hormone secreted by the adipocytes and involved in feeding and energy balance control, has been proposed to modulate alcohol craving in mice and humans. This study evaluated whether leptin modulates alcohol intake in Marchigian Sardinian alcohol-preferring (msP) rats. Rats were offered 10% ethanol either 2h per day at the beginning of dark period of the 12:12h light/dark cycle, or 24h per day. Leptin was injected into the lateral ventricle (LV), the third ventricle (3V), or intraperitoneally (IP) once a day, 1h before the onset of the dark period. Neither acute nor chronic (9 days) leptin injections (1 or 8microg per rat) into the LV or 3V modified ethanol intake in male msP rats, offered ethanol 2h per day. Chronic LV injection of leptin (8 or 32 microg per rat in male rats and 8 or 16 microg per rat in female rats for 7 days), or chronic IP injections of leptin (1mg/kg in male rats for 5 days) failed to modify the intake of ethanol, offered 24h per day. Finally, chronic LV leptin injections (8 or 32 microg per rat for 12 days) did not modify ethanol intake in male msP rats, adapted to ad libitum access to ethanol and then tested after a 6-day period of ethanol deprivation. In contrast, in most of these conditions leptin significantly reduced food intake. These data do not support a role for leptin in alcohol intake, preference, or craving in msP rats.

Aims: Recent studies have shown that Hypericum perforatum extracts (HPE) inhibit ethanol intake in alcohol-preferring rats, but their mechanism of action is still unknown. HPE have been shown to bind at γ-aminobutyric acid (GABA)A and GABAB receptors, to inhibit GABA reuptake, to evoke GABA release from synaptosomes and to exert an anxiolytic effect that is blocked by the benzodiazepine antagonist flumazenil. Since GABA-ergic mechanisms are known to influence ethanol intake, the present study was aimed at investigating whether they might mediate the effect of a CO 2 Hypericum extract (HPCO 2 ) on ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. Methods: The GABAA receptor antagonist bicuculline and the GABAB receptor antagonists CGP-36742 and phaclofen were tested versus the effect of HPCO 2 on ethanol intake. Results: The results of the present study confirm that HPCO2, given by intragastric injection, markedly reduces ethanol intake in msP rats and its effect is behaviourally selective, since the same doses which inhibited ethanol intake did not modify the simultaneous intake of food or water. The GABAA receptor antagonist bicuculline, given by intraperitoneal (i.p.) injection at a dose of 2 mg/kg, which effectively antagonizes the effects of GABA A receptor agonists, did not modify the effect of HPCO 2 , 15 or 125 mg/kg. The GABA B receptor antagonists CGP-36742, given by i.p. injection at a dose of 100 mg/kg, and phaclofen, given by intracerebroventricular injection at a dose of 25 µg/rat, did not modify the inhibitory effect on alcohol intake induced by HPCO 2 , 15 or 125 mg/kg. The same doses of the two GABA B receptor antagonists induced a pronounced reduction of the effect of the GABAB receptor agonist bacoflen, given by i.p. injection at a dose of 5 mg/kg. Conclusions: These findings suggest that the inhibitory effects of HPE on ethanol intake are not mediated by GABA agonist actions.

Cannabis derivatives and alcohol are widely co-abused, particularly among adolescents. Since both ethanol and cannabinoids are known to impair learning and memory, the present study investigated in rats the effects of combined exposure to ethanol and Δ9-tetrahydrocannabinol (THC) in a memory task, the object recognition test. The results of the present study provide evidence that ethanol, voluntarily ingested in alcohol-preferring rats, and THC, given by intraperitoneal injection, have a synergic action to impair object recognition, when a 15-min interval was adopted between the sample phase and the choice phase of the test. Neither voluntary ethanol ingestion nor 2 or 5 mg/kg of THC were able per se to modify object recognition in these experimental conditions, but when voluntary ethanol ingestion was combined with administration of these doses of THC object recognition was markedly impaired. THC impaired object recognition only at the dose of 10 mg/kg, when its administration was not combined with that of ethanol. The selective cannabinoid CB1 receptor antagonist SR 141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1(2, 4-dichloro-phenyl)-4-methyl-1H-pyrazole carboxamide·HCl) at the dose of 1 mg/kg reversed the amnesic effect of THC, 10 mg/kg, suggesting that the effect is mediated by this receptor subtype. The synergism of ethanol and THC was not detected when an inter-trial interval of 1 min was adopted. The present findings are in keeping with the notion that Cannabis derivatives impair memory processes and provide evidence for a synergic action of THC and ethanol, thus emphasizing the risks consequent to their co-administration.

This study tested the effects of 8 days of subchronic administration of 3,4-methylene dioxymethamphetamine (MDMA) (5 mg/kg b.w.) on preprotachykinin A mRNA levels in discrete rat brain regions. In situ hybridization examined preprotachykinin A mRNA levels in the core and shell of the nucleus accumbens, the islands of Calleja, the olfactory tubercle, the dorsal and ventral caudate-putamen, the bed nucleus of the stria terminalis, the medial preoptic area, the medial habenular nucleus and in the postero-dorsal part of the medial amygdala. Higher levels of preprotachykinin A mRNA were found in the core and shell of the nucleus accumbens, in the islands of calleja, in the olfactory tubercle, in the bed nucleus of the stria terminalis, in the medial habenular nucleus and the postero-dorsal part of the medial amygdala, compared to control animals. Conversely, increased preprotachykinin A mRNA levels were observed in the dorsal and ventral caudate-putamen in MDMA treated when compared to control rats. In the social memory test, MDMA significantly impaired rats' short-term working memory. These results show that chronic exposure to MDMA strongly affects preprotachykinin A mRNA levels in discrete rat brain regions. These changes occur in experimental conditions in which working memory is markedly reduced, suggesting that changes in gene expression of tachykinin mechanisms may contribute to the effects of MDMA on memory function.

Rationale: (1S,3aS)-8-(2,3,3,4,5,6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (Ro 64-6198), a non-peptidic agonist for the opioid receptor-like1 (ORL1) receptor, exhibits anxiolytic properties in stressful conditions. Objective: The present study was aimed at evaluating whether activation of ORL1 receptors by Ro 64-6198 may reverse the anorectic effect of restraint stress or intracerebroventricular (ICV) CRF injection. Methods: In body restraint experiments, 20-h food deprived rats were treated with intraperitoneal (IP) injection of Ro 64-6198 or vehicle. Ten minutes later, they were confined in cylindrical Plexiglas tubes for 60 min and then returned to their cage with food. In CRF experiments, 20-h food deprived rats were IP injected with Ro 64-6198 or vehicle. Ten minutes later, they received ICV CRF, 200 ng/rat or vehicle; food was offered after 20 min. Results: Intraperitoneal (IP) pretreatment with Ro 64-6198 reversed the hypophagic effect induced by both restraint or CRF; the effect was statistically significant at the three doses tested (0.3, 1.0 or 2.5 mg/kg). ICV administration of the selective ORL1 receptor antagonist [Nphe1]NC(1–13)NH2 (two injections of 33 or 66 µg/rat) abolished the effect of Ro 64-6198 on CRF-induced anorexia. In freely feeding rats, Ro 64-6198 significantly increased feeding at 2.5, but not at 0.3 or 1.0 mg/kg; in food deprived rats, Ro 64-6198 (0.3 or 1.0 mg/kg) did not modify food intake. Thus, reversal of stress- and CRF-induced anorexia by Ro 64-6198 can be evoked at doses lower than those that are hyperphagic. Ro 64-6198 (1 or 2.5 mg/kg) did not modify the anorectic effect of E. coli lipopolysaccharide, suggesting that its effect is selective for stress- or CRF-induced anorexia. Lastly, the benzodiazepine diazepam was unable to reduce the anorectic effect of CRF at the anxiolytic dose of 0.3 mg/kg, and partially reduced it at the hyperphagic dose of 1 mg/kg. Conclusions: The results of this study show that the non-peptidic ORL1 receptor agonist Ro 64-6198 markedly and selectively inhibits the anorectic effect of stress and CRF, and provide evidence that this effect is mediated by ORL1 receptors. Thus, Ro 64-6198 may represent an interesting tool for treatment of stress-induced anorexia.

The influence of the tachykinin NK3 receptor agonist, aminosenktide on the immobility in the forced swimming test was studied in mouse lines selectively bred for divergent magnitudes of stress-induced analgesia. The high analgesia (HA) line is known to display enhanced, and the low analgesia (LA) line displays reduced activity of the opioid system. Aminosenktide at doses of 125 μg/kg or 250 μg/kg intraperitoneally (IP) reduced, in naltrexone-reversible manner, the immobility more of opioid receptor-dense HA than of unselected mice, but was ineffective in the opioid receptor-deficient LA line. The effect of aminosenktide was quite similar to the antiimmobility action of desipramine (10 mg/kg IP), a prototypic antidepressant agent. None of the compounds increased animals’ locomotion as found with an open field test; therefore their antiimmobility effect cannot be attributed to a change in general motility. The results claim that aminosenktide causes an antidepressant effect, and endogenous opioids are involved in this process.

This study examined whether nociceptin/orphanin FQ (NC), the endogenous ligand of the opioid receptor-like1 (ORL1) receptor, can block drug-seeking behavior induced by foot-shock stress. Male Wistar rats were trained to operantly self-administer ethanol or cocaine, and then subjected to daily extinction training until responding ceased. Subsequent exposure to 15 min of intermittent footshock elicited robust reinstatement of responding at the previously drug-paired lever. NC (0.1-2.0 microg; i.c.v.) significantly inhibited the effects of footshock stress on ethanol- but not cocaine-seeking behavior. The results support the hypothesis that the NC system participates in the regulation of behavioral responses to stress, and that drugs interacting with NC receptors may have therapeutic potential for the treatment of stress-induced alcohol-seeking behavior and relapse.

Previous studies have suggested that imidazoline I(2) receptors play a role in feeding control in rats. The effect of subcutaneous (s.c.) injections of four novel imidazoline I(2) ligands, 2-naphthalen-2yl-4,5-dihydro-1H-imidazole hydrochloride (benazoline), 2-styryl-4,5-dihydro-1H-imidazole oxalate (tracizoline), o-nitro-tracizoline and o-methyl-tracizoline (metrazoline) on food intake during the light phase was now evaluated in freely feeding male Wistar rats. Their effect was compared to that of idazoxan, a high-affinity ligand at imidazoline I(2) binding sites, but also a potent alpha(2)-adrenoceptor antagonist. Compared to idazoxan, metrazoline exhibits a higher pK(i) for imidazoline I(2) binding sites in rat liver, while the other compounds have a slightly lower pK(i); on the other hand, the novel compounds have much lower affinity than idazoxan at alpha(2)-adrenoceptors. Idazoxan stimulated drinking at a dose as low as 1 mg/kg, and evoked feeding at a higher dose (30 mg/kg). The selective alpha(2)-adrenoceptor antagonist 2-methoxy-idazoxan (RX821002), with negligible affinity at imidazoline I(2) binding sites, significantly increased drinking but failed to stimulate feeding at doses of 10-50 mg/kg. Metrazoline induced hyperphagia and water drinking at doses of 50 mg/kg or higher. Its dipsogenic effect was secondary to the hyperphagic effect, since it was not observed in rats without access to food. Benazoline significantly increased feeding only in response to 30 mg/kg, but its effect was less pronounced than that of metrazoline. Tracizoline and o-nitro-tracizoline were inactive. Following injection into the lateral cerebroventricle at doses up to 100 microgram/rat, and into the third or fourth brain ventricle at doses up to 50 microgram/rat, neither idazoxan nor metrazoline induced hyperphagia. The present results support the idea that imidazoline I(2) ligands influence feeding in rats, and suggest that their site of action is not in the central nervous system. The finding that idazoxan elicits a more potent hyperphagic effect than metrazoline and benazoline, although its affinity for imidazoline I(2) binding sites is lower than that of metrazoline and similar to that of benazoline, raises the question whether its hyperphagic effect might also be due to interaction with other receptors.

Intracerebroventricular (ICV) injection of tachykinin (TK) NK-3 receptor agonists inhibits alcohol intake in genetically selected alcohol-preferring rats. The present study investigated the mechanism of action by which the selective TK NK-3 receptor agonist aminosenktide (NH 2 -SENK) attenuates ethanol intake in Marchigian Sardinian alcohol-preferring (msP) rats. The effect of NH 2 -SENK was studied by ICV injection in the conditioned taste aversion (CTA) and in the conditioned place preference (CPP) paradigms; moreover, the effect of NH 2 -SENK on blood alcohol levels (BAL) following intragastric ethanol administration was investigated. The ICV dose of 125 ng/rat of NH 2 -SENK, that markedly reduces ethanol intake, did not modify BAL, nor did it increase the CTA induced by intraperitoneal injection of ethanol, 1 g/kg body weight. These findings suggest that the effect of NH 2 -SENK on alcohol consumption is not related to modification of the pharmacokinetics of ethanol, nor to increase of the aversive properties of ethanol. On the other hand, the same ICV dose of NH 2 -SENK evoked a pronounced and statistically significant CPP. This finding indicates that the TK NK-3 receptor agonist NH 2 -SENK possesses rewarding properties in msP rats and suggests that its inhibitory effect on ethanol consumption may be due to substitution of the rewarding properties of ethanol, thus making its consumption redundant.

Tachykinin NK-3 receptor agonists reduce alcohol intake in alcohol-preferring rats; the nucleus basalis magnocellularis (NBM) is highly sensitive to their effect. Tachykinins and their receptors are widely distributed in gustatory pathways and NK-3 receptor agonists have been reported to modify taste reactivity to salt solutions in rats. The present study evaluated whether the TK NK-3 receptor agonist aminosenktide (NH2-SENK) influences taste reactivity to ethanol solutions. Genetically selected Marchigian Sardinian alcohol-preferring (msP) rats were employed. In response to the intraoral infusion (0.8 ml in 1 min) of 10, 20, 40, or even 60% ethanol solution, ethanol-naive rats showed a large number of ingestive reactions and a much lower number of aversive reactions. Two min before the intraoral infusion of 10 or 40% ethanol, NH2-SENK was injected either into the lateral ventricle (LV) or into the NBM. Doses of NH2-SENK that markedly reduce alcohol intake, 125 ng/rat into the LV or 5 ng/site into the NBM, did not modify the ingestive reactions and, in some instances, reduced the aversive reactions to ethanol solutions in ethanol-naive rats. Injections of 125 ng/rat into the LV failed to modify taste reactions in ethanol-experienced rats. The present results show that msP rats have an innate hedonic evaluation of ethanol solutions, even of high concentration. Moreover, they indicate that reduction of ethanol intake induced by TK NK-3 receptor agonists in alcohol-preferring rats does not depend on influences on gustatory processes.

Tachykinins inhibit salt appetite when applied intracranially in a number of brain regions and may function as endogenous inhibitors of sodium intake. To test the hypothesis that induced increases in salt appetite might involve disinhibition via a reduction in endogenous tachykinin expression, we used a semi-quantitative in situ hybridization analysis to investigate changes in brain areas expressing preprotachykinin-A (PPT-A) and preprotachykinin-B (PPT-B) mRNAs of rats after 1 day of sodium depletion (1d Na dep). PPT-A mRNA levels were detected in neurons of the olfactory tubercle (Tu), the nucleus of the olfactory tubercle (LOT), the dorsal and ventral caudate-putamen (d-CPu and v-CPu), the bed nucleus of the stria terminalis (BNST), the medial preoptic area (mPOA), the habenula (Hb) and the postero-dorsal part of the amygdala (MePD). PPT-B mRNA levels were measured in fundus striati (FStr), d-CPu, v-CPu, BNST, mPOA, dorsomedial hypothalamic nucleus (DMD), arcuate nucleus (Arc), central amygdaloid nucleus (CeL), basolateral amygdaloid nucleus (BLV), LOT, Hb and basal nucleus of Meynert (B). 1d Na dep reduced by 33–61% the mean number of PPT-A grains/cell in Tu, LOT, d-CPu, BNST, mPOA, Hb and MePD compared to control animals. Levels of PPT-B mRNA were not reduced as much by 1d Na dep, although statistically significant reductions of 26, 34 and 17% were found in v-CPu, BNST and B, respectively. These findings, therefore, support the hypothesis that endogenous tachykinins exert an inhibitory influence over sodium appetite.

It is well-known that central administration of tachykinins (Tks) inhibit salt intake in rats. Recent studies have shown that conditions that arouse salt appetite, such as adrenalectomy and sodium depletion, induce a decrease in preprotachykinin-A (PPT-A) mRNA in discrete regions of the rat brain, suggesting that reduced levels of PPT-A mRNA in the brain may have a permissive role on the expression of salt appetite. It has also been shown that spontaneously hypertensive rats (SHR) show higher avidity for salty solutions than their normotensive control Wistar–Kyoto (WKY) rats. In this regard, the present study tested whether SHR and WKY rats differ in expression of the gene coding for PPT-A, the precursor for Tks peptides. Using semi-quantitative in situ hybridization histochemistry, we examined the level of PPT-A mRNA in discrete rat brain regions of SHR and WKY rats under no treatment, after 1 or 3 days of Na+ depletion. Levels of PPT-A mRNA were analysed in the olfactory tubercle (Tu), in the lateral olfactory tubercle (LOT), in the dorsal and ventral caudate putamen (d/v CPu), in the medial preoptic area (mPOA), in the bed nucleus of the stria terminalis (BNST), in the habenula (Hb) and in the postero-dorsal part of the amygdala (MePD). Semi-quantitative analysis of silver grains revealed a 27.5% lower expression of the PPT-A mRNA levels in SHR opposite to WKY rats under no treatment in v-CPu, mPOA, BNST and Hb. 1 day of Na+ depletion reduced PPT-A mRNA levels when opposite to Na+-repleted animals in Tu and mPOA in both SHR and WKY rats. On the other hand, when comparing SHR and WKY rats after 1 day of Na+ depletion, a 26% lower level of PPT-A mRNA was detected in Tu and d-CPu of SHR opposite to WKYrats whereas a 14% and an 18% lower level was detected in v-CPu and Hb, respectively. A lower expression of PPT-A mRNA in SHR compared to WKY rats was also found in BNST and MePD, although no statistical significance was detected in these two brain areas. In the last experiment, 3 days of Na+ depletion reduced PPT-A mRNA levels in mPOA while negligibly increased mRNA levels in d-CPu and v-CPu, in BNST, Hb and MePD, both in SHR and WKY rats. Conversely, when making comparisons between the two strains, a 35% lower level of PPT-A mRNA in SHR with respect to WKY rats was found after 3 days of Na+ depletion in d-CPu, v-CPu and mPOA. A lower gene expression, even though not statistically significant, was found in Tu, LOT, MePD. These findings show a consistent difference of PPT-A mRNA levels in discrete regions of the SHR brain opposite to WKY rats and confirm that 1 day of Na+ depletion reduces PPT-A mRNA in discrete brain regions. Since SHR are notoriously more salt-avid than WKY rats and Tks are potent inhibitors of sodium intake, the down-regulation of PPT-A mRNA may contribute to the higher natriophilia and, therefore, to the etiology of the hypertensive disease.

Ciccocioppo, R., I. Panocka, C. Polidori, G. De Caro, D. Regoli and M. Massi. Stimulation of tachykinin NK-3 receptors in the nucleus basalis magnocellularis reduces alcohol intake in rats. Peptides 18(9) 1349–1355, 1997.—Injections in the nucleus basalis magnocellularis (NBM) of the tachykinin (TK) NK-3 receptor agonist [Asp 5,6 ,MePhe 8 ]substance P(5–11), also referred to as amino-senktide (NH 2 -SENK), markedly reduced alcohol intake in genetically selected alcohol-preferring rats, offered 10% ethanol 2 h/day. The threshold dose in the NBM was 0.5 ng/site, while neither 1 nor 10 ng/rat of NH 2 -SENK inhibited alcohol intake following administration into the lateral ventricle. Injection of NH 2 -SENK, 25 ng/site, in the NBM did not modify water or food intake in water deprived rats, providing evidence for the behavioral selectivity of the effect on ethanol intake. The selective TK NK-3 receptor antagonist, R-820, injected in the NBM at the dose of 1000 ng/site 5 min before NH 2 -SENK 5 ng/site, significantly reduced the effect of NH 2 -SENK. The selective TK NK-1 receptor agonist [Sar 9 ,Met(O 2 ) 11 ]substance P inhibited alcohol intake following injection in the NBM only at 25 ng/site; but the same dose induced marked grooming and inhibited also water intake in water deprived rats. The present results confirm that TK NK-3, but not NK-1, receptor agonists selectively inhibit ethanol intake in alcohol-preferring rats and suggest that the NBM is a site of action for their effect.

The 5-HT2 receptor antagonist, ritanserin, reduces alcohol intake in rats and the nucleus accumbens (NAC) has been proposed as a site of action for the drug. Recent microdialysis studies have shown that acute subcutaneous (SC) administration of ritanserin increases extracellular 5-HT levels in the NAC. The present study evaluated, in genetically heterogeneous rats with developed preference for 3% ethanol, whether the attenuation of ethanol intake induced by ritanserin might be related to its effect on the synaptic availability of 5-HT in the NAC. Damaging 5-HTergic neurons by intracerebroventricular infusion of 5,7-dihydroxytryptamine (5,7-DHT) abolished the effect of ritanserin on ethanol consumption. Injections of the 5-HT3 receptor antagonist MDL 72222 into the NAC significantly reduced the inhibitory effect of SC injection of ritanserin, 1 mg/kg, and completely abolished the effect of ritanserin, 0.1 mg/kg. Subcutaneous injections of MDL 72222, 0.3 mg/kg 3 times/day, suppressed the effect of SC ritanserin, 0.1 mg/kg. The present findings, together with those of previous experiments showing that the tryptophan hydroxylase inhibitor p-chlorophenylalanine abolishes the effect of ritanserin, support the hypothesis that its effect on ethanol intake may be due to increased synaptic availability of 5-HT into the NAC.

The present study evaluated the effect of the selective 5-HT4 receptor antagonist, GR113808, on ethanol intake in alcohol-preferring rats. Rats were offered 10% ethanol 2 h/day. In the first experiment, rats had food and water ad lib and 10% ethanol was offered from 1800 to 2000 h. In the second experiment, food was freely available, 10% ethanol was offered 2 h/day, from 1800 to 2000 h, and water was offered for 4 h, from 1800 to 2200 h. In both experiments GR113808 was subcutaneously injected at doses of 1, 3, or 10 mg/kg for 4 consecutive days, 5 min before access to ethanol. From the first day of administration, GR113808 significantly reduced the volitional ethanol intake in water sated rats at the three doses tested. In water-deprived rats, it reduced ethanol intake at 3 and 10 mg/kg, without modifying total fluid and food intake. In both experiments the effect of GR113808 remained rather stable during the 4 days of administration. The present findings, showing that the 5-HT4 receptor antagonist, GR113808, selectively reduces ethanol intake in alcohol-preferring rats, suggest that 5-HT4 receptors may play a role in alcohol intake control.

Neuropeptide γ (NPγ) is a 21 aminoacid peptide belonging to the tachykinin (TK) family and including neurokinin A (NKA) in its C -terminal sequence. NPγ possesses higher affinity than NKA for central NK-2 receptors; it shows lower affinity for NK-1 receptors, however, it potently stimulates salivary secretion, which is mediated by NK-1 receptor activation. Pulse intracerebroventricular (pICV) injection of TKs selectively inhibits water intake in rats. Our studies have suggested that NK-1 receptors may mediate the inhibition of angiotensin II-induced drinking, while NK-2 receptors that of drinking induced by cell dehydration. The present study evaluated the effect of pICV injections of NPγ on water intake in rats. The injection of NPγ, 8–250 ng/rat, markedly inhibited angiotensin II-induced drinking, and its effect was blocked by the NK-1 receptor antagonist WIN 62577. NPγ potently inhibited also drinking induced by SC hypertonic NaCl load or water deprivation. The threshold dose for these effects was 31 ng/rat. Also carbachol-induced drinking was inhibited, but at higher doses. On the other hand, NPγ did not modify food intake in food deprived rats or 0.1% saccharin intake in water and food sates rats, at the same doses effective on drinking. Present findings support the idea that TKs selectively inhibit water intake in rats and are in keeping with our hypothesis that NK-1 and NK-2 receptors mediate, respectively, inhibition of angiotensin II- and cell dehydration-induced drinking. In comparison to the nonmammalian TKs eledoisin and physalaemin, which proved to be the most potent antidipsogens in rats among the TKs so far tested, NPγ shows similar spectrum of antidipsogenic action and, on a molar basis, similar potency of action. These findings suggest that NPγ may be the most important mammalian TK in the control of water intake in the rat.

The present study investigated the effect of the central injection of selective tachykinin (TK) agonists on the need-free intake of 0.9% NaCl in rats. Isotonic NaCl was offered for 60 min (between 1800 and 1900 h); water was offered for 4 h (between 1800 and 2200 h). The TK agonists were injected into the third ventricle just before access to fluids. The NK3-selective agonists [Asp5,6, MePhe8]substance P(5–11) and Succ[Asp6,MePhe8]substance P(6–11), as well as the NK1-selective agonist [Sar9, Met(O2)11]substance P, markedly reduced salt intake, the threshold dose for their effects being 5 ng/rat. The NK2-selective agonist GR64349 reduced salt intake only at 500 ng/rat. At the dose of 31.2 ng/rat, neither the NK1 nor the NK3 agonists inhibited water intake, when water was the only fluid offered (between 1800 and 2200 h), or modified food intake, but also the need-free food-deprived rats. The present study shows that a) TKs inhibit not only the need-induced salt intake, but also the need-free intake of isotonic saline, b) this effect is behaviorally selective, and c) the effect is apparently mediated by NK1 and NK3 receptors. The finding that TKs suppress salt intake in a large variety of experimental conditions supports the idea that the antinatriorexic effect of TKs is independent of the physiological and hormonal status of the animal. It is hypothesized that TKs might modify taste sensitivity or the hedonic evaluation of the salty taste.

The present study evaluated the effect of tachykinin agonists, selective for different neurokinin (NK) receptors, on alcohol intake in genetically selected, Sardinian alcohol-preferring rats. Tachykinins were given by intracerebroventricular injection just before access to fluids. In rats offered both water and 8% ethanol 2 h/day, the NK3 selective agonists [Asp5.6,MePhe8]substance P(5-11), Suc[Asp6,MePhe8]substance P(6-11), and [MePhe7]neurokinin B markedly suppressed alcohol intake. The NK1 selective agonist [Sar9,Met(O2)11]substance P and the NK2 selective agonist GR 64349 did not At doses that inhibited alcohol intake, the NK3 agonists did not modify water intake; total fluid intake was significantly reduced only following 500 ng/rat of [Asp5.6,MePhe8]substance P(5-11). When rats were given a longer access to fluids (8% alcohol for 2 h, but water for 4 h), again, NK3 agonists suppressed alcohol intake, but not total fluid intake. Moreover, NK3 agonists did not modify solid food intake in food-deprived rats, nor water intake in water-deprived rats, when alcohol was not available. These findings indicate that NK3 agonists inhibit alcohol intake in Sardinian alcohol-preferring rats and that their effect is behaviorally selective. They also suggest that central NK3 receptors may be involved in alcohol intake control in rats.

A(2A) adenosine receptors (A(2A)ARs) have been proposed to be involved in drug addiction; however, preclinical studies about the effects of A(2A)AR ligands on alcohol consumption have provided inconsistent results.The present study evaluated the effect of intraperitoneal injections of the A(2A)AR antagonist ANR 94, and the A(2A)AR agonists CGS 21680 and VT 7 on voluntary drinking and operant self-administration of 10% ethanol in Marchigian Sardinian alcohol-preferring (msP) rats.Voluntary ethanol drinking was increased by ANR 94 in acute and subchronic experiments, while it was reduced by A(2A)AR agonists. The effect of CGS 21680 was abolished by a low dose of ANR 94, confirming its mediation by A(2A)ARs. Ethanol self-administration was reduced by CGS 21680 and VT 7, while ANR 94 slightly but significantly increased it. Blood alcohol levels were not modified by A(2A)AR agonists, indicating that their effect is not related to ethanol pharmacokinetics. The effect of VT 7 on ethanol drinking was behaviourally selective; ethanol and food intake were reduced, but water intake was increased, and total fluid intake was not different from that of controls. Moreover, VT 7 did not affect locomotor activity. CGS 21680 (0.1 mg/kg) did not modify total fluid intake, but 0.2 and 0.3 mg/kg reduced total fluid intake and locomotor activity.These results provide evidence that A(2A)AR agonists reduce ethanol consumption in msP rats, which represent an animal model of alcohol abuse related to stress, anxiety and depression. A(2A)ARs may represent a potential target for treatment of alcohol abuse.

Alcohol withdrawal refers to a cluster of symptoms that may occur from suddenly ceasing the use of alcohol after chronic or prolonged ingestion. These symptoms make alcohol abstinence difficult and increase the risk of relapse in recovering alcoholics. In previous studies, we demonstrated that treatment with Nociceptin/orphanin FQ (N/OFQ) significantly reduces alcohol consumption and attenuates alcohol-seeking behavior induced by environmental conditioning factors or by stress in rats. In this study, we evaluated whether activation of brain NOP receptors may also attenuate alcohol withdrawal signs in rats.For this purpose, animals were subjected to a 6-day chronic alcohol intoxication (by intragastric administration), and at 8, 10, and 12 hours following cessation of alcohol exposure, they were treated intracerebroventricularly (ICV) with N/OFQ (0.0, 1.0, and 3.0 μg/rat). Somatic withdrawal signs were scored after ICV treatment. In a subsequent experiment, to evaluate N/OFQ effects on alcohol withdrawal-induced anxiety, another group of rats was subjected to ethanol intoxication and after 1 week was tested for anxiety behavior in the elevated plus maze (EPM). In the last experiment, an additional group of rats was tested for anxiety elicited by acute ethanol intoxication (hangover anxiety). For this purpose, animals received an acute dose (3.0 g/kg) of 20% alcohol and 12 hour later were tested in the EPM following ICV N/OFQ (0.0, 1.0, and 2.0 μg/rat).Results showed that N/OFQ significantly reduced the expression of somatic withdrawal signs and reversed anxiety-like behaviors associated with both chronic and acute alcohol intoxication. N/OFQ did not affect anxiety scores in nondependent animals.These findings suggest that the N/OFQ-NOP receptor system may represent a promising target for the development of new treatments to ameliorate alcohol withdrawal symptoms.

Aims Mucosal mast cells (MMC) are mediators of the stress responses in the gastrointestinal tract. We examined the effect of acute cold-restraint stress and of the neuropeptide nociceptin/orphanin FQ (N/OFQ), implicated in the modulation of stress responses, on MMC density in the rat colon. Main methods Stress was induced by restraining the animals into individual cages at 3 °C for 3 h. N/OFQ and the selective N/OFQ peptide (NOP) receptor antagonist, UFP-101, were infused subcutaneously via Alzet osmotic minipumps. Segments of the distal colon were collected. MMCs were identified immunohistochemically with a monoclonal antibody to rat mast cell protease (RMCP) II and with a rabbit polyclonal antibody to CD117/c-kit receptor. Key findings Acute stress caused a decrease in the density of MMCs in the rat colonic mucosa. Short-term peripheral infusion of N/OFQ (0.1 to 10 μg/kg/h for 4 h) caused a dose-related reduction of MMC density. Peak reduction occurred after the 4-h infusion of N/OFQ, 1 μg/kg/h. Reduction was maintained after the 52-h infusion period and declined following 7 and 14 days of infusion. The infusion of N/OFQ (1 μg/kg/h for 4 h) in rats exposed to acute stress caused a decrease in MMC density comparable to that obtained with the single treatments. UFP-101, at the doses of 1 and 10 μg/kg/h, which itself had no significant effect on MMC density, when concurrently infused in stress-exposed rats, abolished the stress-induced decrease of MMC density. Significance Present results indicate that the peripheral N/OFQ-NOP system is involved in stress-induced reduction of MMC density.

Background Pioglitazone and rosiglitazone belong to the class of thiazolidinediones (TZDs). They were first developed as antioxidants and then approved for the clinical treatment of insulin resistance and Type 2 diabetes. TZDs bind with high affinity and activate peroxisome proliferator-activated receptor-gamma (PPARγ) receptors, which in the brain are expressed both in neurons and in glia. Methods We evaluated the effect of PPARγ activation by TZDs on alcohol drinking, relapse-like behavior, and withdrawal in the rat. We also tested the effect of TZDs on alcohol and saccharin self-administration. Results We showed that activation of PPARγ receptors by pioglitazone (0, 10, and 30 mg/kg) and rosiglitazone (0, 10 and 30 mg/kg) given orally selectively reduced alcohol drinking. The effect was blocked by pretreatment with the selective PPARγ antagonist GW9662 (5 μg/rat) given into the lateral cerebroventricle, suggesting that this TZD's effect is mediated by PPARγ receptors in the central nervous system. Pioglitazone abolished reinstatement of alcohol seeking, a relapse-like behavior, induced by yohimbine, a pharmacologic stressor, but did not affect cue-induced relapse. In the self-administration experiments, pioglitazone reduced lever pressing for alcohol but not for saccharin. Finally, pioglitazone prevented the expression of somatic signs of alcohol withdrawal. Conclusions These findings provide new information about the role of brain PPARγ receptors and identify pioglitazone as candidate treatments for alcoholism and possibly other addictions.

Pregabalin (Lyrica™) is a structural analogue of γ-aminobutyric acid (GABA) approved by FDA for partial epilepsy, neuropathic pain and recently generalized anxiety disorder. While the exact cellular mechanism of action of pregabalin is still unclear, evidence from several studies suggests that it reduces excitatory neurotransmitter release and postsynaptic excitability. Based on these mechanisms we sought interesting to evaluate the effect of pregabalin on alcohol-abuse-related behaviours. For this purpose, using genetically selected alcohol-preferring Marchigian Sardinian (msP) rats, we evaluated the effect of pregabalin on alcohol drinking and relapse to alcohol seeking elicited by environmental conditioning factors or stress. Our results showed that treatment with pregabalin (0, 10, 30 and 60 mg/kg) given orally selectively reduced home cage alcohol drinking in msP rat. This effect was confirmed in self-administration experiments where pregabalin (0, 10 and 30 mg/kg) significantly reduced operant responding for alcohol but not for food. Using alcohol reinstatement models we also found that pregabalin (0, 10 and 30 mg/kg) abolished seeking behaviour elicited by the pharmacological stressor yohimbine as well as cues predictive of alcohol availability. Results demonstrate that pregabalin may have potential in the treatment of alcohol addiction.

Several prazosin-related compounds have been synthesized and evaluated for their blocking activity toward alpha-adrenoreceptors. The structural modification performed on the prazosin structure included the replacement of the piperazine ring with 2,3-dialkylpiperazine or 1,2-cyclohexanediamine moieties to characterize a lipophilic binding pocket in the alpha 1-adrenoreceptor surface. Cyclohexanediamine derivatives 3-6 were almost devoid of potency and selectivity, whereas dialkylpiperazine compounds 7-14 showed high affinity and selectivity toward alpha 1-adrenoreceptors. The cis derivative 13 (cyclazosin) was the most potent and selective with an alpha 1/alpha 2 selectivity ratio value of 7800. The particular trend of antagonist activity within cis/trans stereoisomeric compounds not only supports the presence of a lipophilic binding area on alpha 1-adrenoreceptor surface but also suggests that the lipophilic pocket is endowed with a well-defined size and spatial orientation. The most active compound of the series, 13, was tested also in vivo for antihypertensive activity on spontaneously hypertensive rats. It showed an interesting long-lasting hypotensive effect, very similar to that of doxazosin, which was statistically significant 12 h after oral administration.

The present study evaluated the sensitivity of spontaneously hypertensive (SHR) and of Wistar Kyoto (WKY) rats to the hypotensive effect of tachykinins (TKs). Eledoisin, substance P, and the NK-1-selective agonist [Sar 9 ,Met(O 2 ) 11 ]substance P evoked a smaller hypotensive response in SHR than in WKY rats. The hypotensive effect of NKA was slightly smaller in SHR, but no significant strain difference was observed. The NK-2-selective agonist [βAla 8 ]NKA(4–10) was a very weak hypotensive agent in WKY rats, while being completely inactive in SHR. The NK-3-selective agonists [Asp 5,6 ,MePhe 8 ]substance P(5–11) and [MePhe 7 ]NKB did not modify blood pressure in both strains. Heart rate was essentially unmodified following the NK-3 agonists, while it was increased after injection of substance P, [Sar 9 ,Met(O 2 ) 11 ]substance P, and neurokinin A, the increase being greater in WKY than in SHR. Surprisingly, eledoisin increased heart rate in SHR, but not in WKY rats, despite the greater hypotensive effect elicited in the latter strain. The present results confirm that the hypotensive effect of peripheral TKs is mediated by NK-1 receptors and show that SHR are less sensitive than WKY rats to this effect.

The endogenous neuropeptide nociceptin/orphanin FQ (N/OFQ) modulates behavioral and gastrointestinal responses to stress. Mucosal mast cells (MMCs) are primary mediators of stress-related responses in the gastrointestinal tract. We investigated the influence of N/OFQ and of the N/OFQ peptide (NOP) receptor antagonist, UFP-101, on MMCs in the rat gastric fundus. N/OFQ was infused subcutaneously for 52 h at 0.1, 1 and 10 μg/kg/h and at 1 μg/kg/h for 4h, 52 h, 7 days and 14 days via Alzet osmotic minipumps. Density of MMCs and connective tissue mast cells (CTMCs) was assessed histochemically and immunohistochemically. Activation and location of MMCs were assessed by transmission electron microscopy. Contacts between MMCs and nerve elements were assessed by double immunofluorescence. N/OFQ (1 μg/kg/h) and UFP-101 (10 and 30 μg/kg/h) were infused subcutaneously in the absence and presence of acute cold-restraint stress and density of MMCs was assessed. Peripheral N/OFQ dose-dependently increased the density of MMCs, while not influencing CTMCs. The increasing effect was maintained up to 14 days following continuous infusion, while after termination of the 4-h infusion, the effect declined rapidly. The peptide promoted the activation of MMCs and their migration from the lamina propria toward the epithelial layer. The association between MMCs and nerve fibers was time-dependently down-regulated following N/OFQ infusion. The stress-induced hyperplasia of MMCs was not influenced by N/OFQ and abolished by UFP-101. UFP-101 alone was ineffective. The present results suggest that endogenous N/OFQ could be considered a potential component of the circuit neuropeptides-mast cells-stress.

The present study investigated the effect on water and food intake in the rat of the intracerebroventricular (ICV) injection of neuropeptide K (NPK), the N-terminally extended form of neurokinin A. NPK inhibited water deprivation-induced water intake even at 31.2 ng/rat. At higher doses, it inhibited also water intake induced by ICV angiotensin II or by subcutaneous hypertonic NaCl, and food-associated drinking, the threshold dose being 125 ng/rat. In response to 125 ng/rat, food intake following 16 h food deprivation was not reduced. NPK inhibited food intake only at 500 ng/rat, a dose that evoked excessive grooming in treated animals. Thus NPK is a potent inhibitor of water deprivation-induced drinking and at higher doses it exerts a general antidipsogenic effect towards several dipsogenic determinants, without affecting food intake. On the other hand, it inhibits food intake only at high doses, 500 ng/rat or more, but this inhibition might be just related to the intense grooming evoked. The effects of NPK on ingestive behavior are markedly different from those of neurokinin A, which selectively inhibits osmotic drinking and food-associated drinking. These differences suggest that NPK itself may exert its effects on the central nervous system, not necessarily through the conversion to neurokinin A.

Neuropeptide S (NPS) is a recently discovered neurotransmitter that binds to its cognate G-protein coupled receptor, NPSR. Previous studies have shown that central administration of this peptide induces anxiolytic-like effects, promotes arousal and inhibits feeding in the same dose range. In the present study, we sought to investigate further the unique physiopharmacological profile of the NPS system by characterizing its effects on palatable food consumption in rats and comparing it with the effect of the classical anxiolytic benzodiazepine midazolam. The results demonstrated that midazolam (5.0 or 10.0 mg ⁄kg) increases palatable food consumption, while intracerebroventricular (ICV) administration of NPS markedly reduces it. The anorectic effect of NPS (0.1‐ 1.0 nmol per rat, ICV) was prevented by ICV pretreatment with the NPSR antagonist [d-Cys(tBU) 5 ]NPS (20.0‐60.0 nmol per rat). Pretreatment with the nonselective corticotrophin-releasing factor receptor (CRF) antagonist alpha-helical CRF 9‐41 (6.25 and 12.5 nmol per rat) completely reversed the hypophagic action of CRF (0.4 nmol per rat, ICV) but did not prevent the anorectic effect of ICV NPS (1.0 nmol per rat). Brain site-specific microinjection experiments revealed that NPS markedly inhibits palatable food intake if administered into the paraventricular nucleus of the hypothalamus (PVN). A similar but smaller and shorter lasting reduction of feeding was observed following intra-lateral hypothalamus administration, whereas no effect was observed following injection into the central amygdala. The present study demonstrates that NPS evokes a potent inhibition of palatable food consumption and that the PVN is an important site of action for its effect.

The 17-amino acid peptide nociceptin/orphanin FQ (N/OFQ) plays a role in the regulation of stress responses and of emotional disorders. The objective of this study is to evaluate whether long-term peripheral N/OFQ could dose- and time-dependently influence the responses to repeated cold-restraint stress on the rat gastric and colonic mucosa. Rats were exposed to cold-restraint stress for 3h per day for 1, 2 and 3 consecutive days. N/OFQ was administered at doses of 0.1, 1 and 10 microg/kg/h via Alzet osmotic minipumps. In the gastric fundus, N/OFQ exerted dose-dependent beneficial effects against acute and repeated stress but, after prolonged treatment, became damaging in non-stressed rats. In the distal colon, N/OFQ exerted a protective effect against damage by acute and repeated stress with no influence on epithelial integrity in non-stressed rats. In both regions, the peptide itself dose- and time-dependently reduced intraepithelial mucins. The reduction in mucin content caused by stress was effectively counteracted by N/OFQ, 0.1 microg/kg/h, in the distal colon only. N/OFQ did not modify basal mucosal cell proliferation. The peptide at 0.1 and 1 microg/kg/h had no influence while at 10 microg/kg/h abolished stress-induced increase in cell proliferation. The present results provide evidence that N/OFQ is implicated in the regulation of resting and stress-challenged mucosal integrity and activity of mucin-producing cells.

Aim: Adipose tissue has been the object of intense research in the field of obesity and diabetes diseases in the last decade. Examination of adipocyte-secreted peptides led to the identification of a unique polypeptide, resistin (RSTN), which has been suggested as a link between obesity and diabetes. RSTN plays a clearly documented role in blocking insulin (INS)-induced hypoglycaemia. As brain injection of INS affects feeding behaviour, we studied the possible interaction between INS and RSTN in food-deprived rats, measuring effects on food intake. In addition, we examined how RSTN might affect neuropeptide Y (NPY)-induced feeding, as studies have shown that rat RSTN can interfere with the NPY system. Methods: Overnight food-deprived rats were injected into the third brain ventricle (3V) with either INS (10 or 20 mUI), RSTN (0.1–0.4 nmol/rat), or saline before access to food. Another group of rats was injected into the 3V with RSTN alone, NPY alone or RSTN plus NPY. Their food intake and body weight were measured. Results: Our results confirm the hypophagic effect of RSTN on food deprivation-induced food intake, and more importantly, show that RSTN neither potentiates nor blocks the effects of INS on food intake, but does reduce the hyperphagic effect of NPY. Conclusion: The observation that RSTN does not modify feeding INS-induced hypophagia, but does influence NPY-induced feeding, points to the possibility that RSTN may be involved in control of food intake through an NPY-ergic mechanism as INS.

The present study was designed to assess the antidepressant effects of UFP-101, a selective nociceptin/orphanin FQ peptide (NOP) receptor antagonist, in a validated animal model of depression: the chronic mild stress (CMS). UFP-101 (5, 10 and 20 nmol/rat; i.c.v., once a day for 21 days) dose- and time-dependently reinstated sucrose consumption in stressed animals without affecting the same parameter in non-stressed ones. In the forced swimming test, UFP-101 reduced immobility of stressed rats from day 8 of treatment. After a 3-week treatment, rats were killed for biochemical evaluations. UFP-101 abolished increase in serum corticosterone induced by CMS and reverted changes in central 5-HT/5-HIAA ratio. The behavioural and biochemical effects of UFP-101 mimicked those of imipramine, the reference antidepressant drug, administered at the dose of 15 mg/kg (i.p.). Co-administration of nociceptin/orphanin FQ (5 nmol/rat, from day 12 to 21) prevented the effects of UFP-101. Brain-derived neurotrophic factor mRNA and protein in hippocampus were not reduced by CMS nor did UFP-101 modify these parameters. This study demonstrated that chronic treatment with UFP-101 produces antidepressant-like effects in rats subjected to CMS supporting the proposal that NOP receptors represent a candidate target for the development of innovative antidepressant drugs.

The 5-HT 2 antagonist ketanserin inhibited salt appetite induced by subchronic deoxycorticosterone acetate (DOCA) treatment, as well as salt induced by sodium depletion (which is governed by the synergy of aldosterone and angiotensin in the brain). The effect of ketanserin was more evident following intraperitoneal than intracerebroventricular injection. On the other hand, ketanserin did not inhibit either salt intake induced by intracranial renin injection, or the need-free salt intake of the multidepleted female rat, which is not dependent on the renin-angiotensin-aldosterone system. These findings suggest that the antinatriorexic action of ketanserin is selective for the mineralcorticoid mechanisms controlling salt appetite. Ritanserin, too, a potent 5-HT 2 antagonist showing a different receptor selectively profile from that of ketanserin, suppressed DOCA-induced salt appetite, thus supporting the involvement of 5-HT receptors in the antinatriorexic action. DOCA treatment alters serotonin metabolism in the central nervous system and it has been proposed that changes in 5-HT metabolism may be important in the genesis of DOCA-induced hypertension. The present results indicate that ketanserin inhibits DOCA-induced salt appetite and suggest that serotoninergic mechanisms might be involved in the elicitation of mineral-ocorticoid-induced salt appetite.

The present study evaluated the effect of the intracerebroventricular injection of the tachykinins, substance P, neurokinin A and [Asp 5,6 ,MePhe 8 ]substance P(5–11) (also referred to as NH 2 -senktide), on the alcohol intake of genetically selected, alcohol-preferring rats. Animals were offered both water and 8% ethanol 2 h/day; tachykinins were administered just before access to fluids. Neurokinin A and substance P did not modify alcohol intake at doses up to 1000 and 2000 ng/rat, respectively. On the other hand, NH 2 -senktide potently suppressed alcohol intake at doses of 31.2–500 ng/rat. At the same doses, however, it did not signi0icantly affect water intake. This finding suggests that its effect on alcohol intake might be rather selective and not due to general impairment of the behavior. Activation of tachykinin NK-3 receptors, for which NH 2 -senktide is a highly selective agonist, produces angiotensin II release in the brain; however, the effect of NH 2 -senktide on alcohol intake is probably not mediated by angiotensin II, as suggested by the fact that it is not modified by captopril pretreatment.

The present study investigated the effect of selective muscarinic antagonists on natriuresis, kaliuresis and antidiuresis induced by intracerebroventricular (i.c.v.) injection of carbachol in the rat. The muscarinic antagonists were given by i.c.v. injection 1 min before carbachol (1 μg/rat). 4-Diphenylacetoxy-N-methyl-piperidine methiodide (4-DAMP), a rather selective M1 and M3 receptor antagonist, was the most potent inhibitor of carbachol-induced natriuresis, kaliuresis and antidiuresis, its ID50 being respectively 0.12,0.04 and 0.56 nmol/rat. Pirenzepine, a selective m1 antagonist, potently inhibited the above mentioned carbachol effects, its 1050 being 1.85, 3.25 and 1.49 nmol/rat, respectively. On the other hand, the M2-selective antagonist methoctramine and the M3-selective antagonist p-fluoro-nexahydro-sila-difenidol were very weak inhibitors. Methoctramine at doses up to 60 nmol/rat produced non statistically significant inhibition of carbachol-induced natriuresis, kaliuresis and antidiuresis. Parafluoro-hexahydro-sila-diphenidol showed an ID50 of 64.4 nmol/rat on carbachol-induced natriuresis, while at the maximum dose employed, 100 nmol/rat, the inhibition of carbachol-induced kaliuresis and antidiuresis was lower than 50%. The rank order of potency of the antagonists tested proved to be related to their pA2 values for muscarinic M1 receptors, suggesting that this receptor subtype mediates the central effects of cholinergic mechanisms on water and electrolyte excretion.

Nociceptin/orphanin FQ (N/OFQ) has been proposed to be a functional antagonist of corticotropin-releasing factor (CRF) in relation to its anti-stress action and its ability to antagonize the anorectic effect of CRF in rats without exhibiting affinity for CRF receptors. The bed nucleus of the stria terminalis (BST) is highly sensitive to the inhibitory effect of N/OFQ on CRF-induced anorexia.The present study was aimed at further evaluating the role of the BST in the functional antagonism between N/OFQ and CRF by examining it at molecular level and in the context of CRF-induced anxiety in the rat.First, in situ hybridization experiments investigated the expression of the pro-N/OFQ precursor and of NOP receptors in several brain areas 6 h after injection of CRF (0.2 and 1 microg/rat) into the lateral cerebroventricle (LV). Second, the elevated plus maze test was used to evaluate whether N/OFQ, injected into the BST (0.05 and 0.5 microg/rat) or into the LV (0.5, 1.8, and 2.4 microg/rat), inhibits the anxiogenic-like effect evoked by LV injection of CRF (1 microg/rat) in rats.The in situ hybridization study showed that LV injection of CRF 1 microg/rat increases NOP receptor expression in the BST, while no change of the N/OFQ precursor was observed. On the other hand, N/OFQ injection into the BST blocks the anxiogenic effect of CRF at doses lower than those required by LV injection (0.5 vs 1.8 microg/rat, respectively).These data provide further support for the hypothesis that N/OFQ may behave as functional antagonist of CRF and suggest that this antagonism may occur within the BST.

The present study investigated the sensitivity of the medial region of the amygdala to the antinatriorexic action in the rat of the tachykinins eledoisin, substance P, neurokinin A and [Asp5,6, MePhe8] substance P(5-11) (also referred to as amino-senktide; NH2-SENK), which is a highly selective agonist for NK-3 receptors. The results obtained show that only the potent NK-3 agonists eledoisin and NH2-SENK inhibit salt appetite when injected into the medial region of the amygdala. Eledoisin and NH2-SENK inhibited salt appetite induced by sodium depletion, that has been proven to be governed by the synergism of angiotensin and aldosterone. They inhibited also salt appetite evoked by central renin injection, that is due to production of angiotensin II. On the other hand, eledoisin and NH2-SENK did not inhibit salt appetite evoked by subcutaneous deoxycorticosterone treatment. These findings suggest that the medial region of the amygdala is a site of action for the antinatriorexic effect of tachykinins and that their action at this site is mediated by NK-3 receptors. Moreover, our results show that in the medial amygdala, the antinatriorexic action of tachykinins appears to be directed toward the angiotensinergic component of the neural mechanism for salt appetite.

This study investigated the sensitivity of spontaneously hypertensive rats (SHR) and of Wistar Kyoto rats (WKR) to the antidipsogenic action of the tachykinin eledoisin (ELE). Drinking was evoked by: (a) intracerebroventricular (i.c.v.) injection of angiotensin II, (b) subcutaneous (s.c.) administration of hypertonic NaCl (1.5 M; 1 ml/100 g b.wt.) or (c) 18 h of water deprivation with free access to food. In accordance with previous studies, the dipsogenic effect of all three treatments was exaggerated in the SHR. And when treated with i.c.v. ELE (12.5–25 ng/rat) they were far less sensitive than WKR to its antidipsogenic action on angiotensin-induced drinking. Smaller differences in strain sensitivity were also observed for the effect of ELE on cell dehydration- and on water deprivation-induced drinking, but only at the dose of 200 and 50 ng/rat, respectively. The different sensitivity of the SHR to the antidipsogenic effect of ELE supports the idea that tachykininergic mechanisms for control of water intake are differently regulated in the SHR than they are in the normotensive WKR.

The endocannabinoid system mediates in the pharmacological actions of ethanol and genetic studies link endocannabinoid signaling to alcoholism. Drugs activating cannabinoid CB1 receptors have been found to promote alcohol consumption but their effects on self-administration of alcohol are less clear because of the interference with motor performance. To avoid this problem, a novel pharmacological approach to the study of the contribution of the cannabinoid system in alcoholism may be to use drugs that locally amplify the effects of alcohol on endogenous cannabinoids. In the present study we addressed this model by studying the effects of the anandamide transport inhibitor N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404) on both ethanol self-administration and reinstatement of alcohol-seeking behavior in rats. The results show that AM404 significantly reduced ethanol self-administration in a dose-dependent manner but failed to modify reinstatement for lever pressing induced by the stimulus associated with alcohol. This effect was not due to a motor depressant effect and was not related to a decrease in general motivational state, as it was not effective in other reward paradigms such as lever pressing for a saccharin solution. The mechanism of action of AM404 does not involve cannabinoid CB1 receptors as the CB1-selective antagonist SR141716A did not block the reduction of ethanol self-administration induced by the anandamide uptake blocker. Moreover, 3-(1,1-dimethylheptyl)-(-)-11-hydroxy-delta 8-tetrahydrocannabinol (HU-210), a classical cannabinoid receptor agonist, did not affect ethanol self-administration. The effects of AM404 are not mediated by either vanilloid VR1 receptors or cannabinoid CB2 receptors because it is not antagonized by either the VR1 receptor antagonist capsazepine or the CB2 antagonist AM630. These results indicate that AM404 may be considered as an innovative approach to reduce alcohol consumption.

Alcoholic patients and experimental animals exposed to ethanol display biochemical signs of oxidative damage, suggesting a possible role of free radicals in causing some of the toxic effects of alcohol. The ester derivative, ethyl pyruvate (EP) is stable in solution and should function as an antioxidant and energy precursor. In the present study, the effect of ethanol intake on plasma membrane fluidity, lipid oxidation and antioxidant enzyme activities (GPx, CAT and SOD) were first evaluated. Secondly, the consequences of ethyl pyruvate treatment on the physico-chemical properties of erythrocyte plasma membranes were investigated. The results obtained demonstrate that ethanol induces an increase in lipid peroxidation, a reduction of GPx activity and fluidity in the hydrophilic-hydrophobic region of the bilayer, moreover an increase of fluidity in hydrophobic part of the plasma membrane was measured. When rats were treated with ethyl pyruvate a partially protective effect can be observed for the hydrophilic-hydrophobic region tested by Laurdan, while EP cannot restore the DPH anisotropy values to the control values. In summary, our data indicate that treatment with EP can only partially reduce ethanol plasma membrane perturbation. Since this study shows an ethyl pyruvate dose-dependent effect, it is important to consider the amount of EP required to maintain the right level of membrane fluidity and polarity. These results could be interesting in order to investigate if EP, due to its radical scavenging effect, can prevent oxidative damage induced by ethanol intake and can protect against injure related with ethanol intake.

Alcohol consumption for long periods negatively influences physiological functions of many cells, and leads to organ damage. Reactive oxygen and nitrogen species produced by ethanol metabolism cause adverse effects that might be alleviated by simultaneous treatment with various antioxidants. Here, the ability of ethyl pyruvate (EP) to reduce ethanol-induced oxidative stress was evaluated. Chemiluminescence studies show that EP has a higher capacity than pyruvate to scavenge hydrogen peroxide and superoxide anions. In order to evaluate whether EP can exert a protective effect against ethanol, rats were offered 10% ethanol in drinking burettes, containing or not different concentrations of EP (0.3%, 1% and 3%). The comet assay was employed to quantify the alcohol-induced DNA damage in rat lymphocytes. This test is a promising tool for the estimation of DNA damage at the single cell level. A significant protective effect of EP was observed in rat groups treated with this antioxidant, compared with those drinking only ethanol. Since EP has been shown to decrease the expression of numerous pro-inflammatory mediators, the monocyte respiratory burst was evaluated. The activation of monocyte NADPH oxidase by phorbol esters (PMA) showed that superoxide anion production was higher in the ethanol group than in the control group. The presence of EP considerably reduced superoxide anion production. In conclusion, hypotheses on possible mechanisms of action of EP on rat white blood cells are proposed.

Activation of the NOP receptor by the endogenous ligand nociceptin/orphanin FQ (N/OFQ) reduces alcohol consumption in genetically selected alcohol preferring Marchigian Sardinian (msP) rats. The present study evaluated the effect of three newly synthesized peptidergic and one brain-penetrating heterocyclic NOP receptor agonists on alcohol drinking in the two bottle choice paradigm. MsP rats were intracerebroventricularly (ICV) injected with the NOP receptor agonists OS-462 (0.5 and 1.0 μg), UFP-102 (0.25 and 1.0 μg) or UFP-112 (0.01 and 0.05 μg), or with Ro 64-6198 (0.3 and 1.0 mg/kg) given intraperitoneally (IP), and tested for 10% alcohol consumption. Results showed decreased alcohol consumption after treatment with all three peptidergic NOP receptor agonists (OS-462, UFP-102 and UFP-112). OS-462 (at the 1.0 μg dose) and UFP-102 (at the 0.25 μg dose) induced a significant increase in food intake as well. Surprisingly, Ro 64-6198 was ineffective at the 0.3 mg/kg dose whereas it increased ethanol and food consumption at the 1.0 mg/kg dose. Interestingly, pre-treatment with the selective μ-receptor antagonist naloxone (0.5 mg/kg, IP) reduced these effects of 1.0 mg/kg of Ro 64-6198. These findings confirm that activation of brain NOP receptors reduces alcohol drinking in msP rats and demonstrate that OS-462, UFP-102 and UFP-112 act as potent NOP receptor agonists. On the other hand, Ro 64-6198 was not effective nor did it increase alcohol drinking, an effect probably induced by a residual agonist activity of this compound at μ-opioid receptors. Overall, the results indicate that OS-462, UFP-102 and UFP-112 may represent valuable pharmacological tools to investigate the functional role of the brain N/OFQ system.