Amalia Fedeli

Variation at the rat Crhr1 locus and sensitivity to relapse into alcohol seeking induced by environmental stress

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.

Dysregulation of Nociceptin/Orphanin FQ Activity in the Amygdala Is Linked to Excessive Alcohol Drinking in the Rat

BACKGROUND Alcoholism is a complex behavioral disorder in which interactions between stressful life events and heritable susceptibility factors contribute to the initiation and progression of disease. Neural substrates of these interactions remain largely unknown. Here, we examined the role of the nociceptin/orphanin FQ (N/OFQ) system, with an animal model in which genetic selection for high alcohol preference has led to co-segregation of elevated behavioral sensitivity to stress (Marchigian Sardinian alcohol-preferring [msP]). METHODS The msP and Wistar rats trained to self-administer alcohol received central injections of N/OFQ. In situ hybridization and receptor binding assays were also performed to evaluate N/OFQ receptor (NOP) function in naïve msP and Wistar rats. RESULTS Intracerebroventricular (ICV) injection of N/OFQ significantly inhibited alcohol self-administration in msP but not in nonselected Wistar rats. The NOP receptor messenger RNA expression and binding was upregulated across most brain regions in msP compared with Wistar rats. However, in msP rats [(35)S]GTPgammaS binding revealed a selective impairment of NOP receptor signaling in the central amygdala (CeA). Ethanol self-administration in msP rats was suppressed after N/OFQ microinjection into the CeA but not into the bed nucleus of the stria terminalis or the basolateral amygdala. CONCLUSIONS These findings indicate that dysregulation of N/OFQ-NOP receptor signaling in the CeA contributes to excessive alcohol intake in msP rats and that this phenotype can be rescued by local administration of pharmacological doses of exogenous N/OFQ. Data are interpreted on the basis of the anti-corticotropin releasing factor (CRF) actions of N/OFQ and the significance of the CRF system in promoting excessive alcohol drinking in msP rats.

The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats

The intracerebroventricular administration of the 17 amino acid peptide nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor (previously referred to as ORL-1 or OP4 receptor), reduces voluntary 10% ethanol intake in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats. Studies aimed at the pharmacological characterization of the receptor, which mediates the effect, have shown that the C-terminal 13 amino acid sequence is crucial for activity and that the selective NOP receptor antagonist [Nphe(1)]N/OFQ(1-13)NH(2) blocks the effect of N/OFQ on ethanol drinking. In place conditioning studies, N/OFQ abolishes the conditioned place preference (CPP) induced by ethanol in msP rats, or by morphine in nonselected Wistar rats; these findings suggest that N/OFQ is able to abolish the rewarding properties of ethanol and morphine. Moreover, N/OFQ inhibits reinstatement of alcohol-seeking behavior induced to electric footshock stress, as well as reinstatement of alcohol-seeking behavior induced by ethanol-paired cues. Together, these findings suggest that N/OFQ and its receptor may represent an interesting target for pharmacological treatment of alcohol abuse.

Nociceptin/orphanin FQ acts as a functional antagonist of corticotropin-releasing factor to inhibit its anorectic effect.

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP opioid receptor (previously referred to as ORL1 or OP4 receptor), exerts a variety of behavioral effects. N/OFQ as well as the synthetic NOP receptor agonist Ro 64-6198 have been reported to possess antistress properties and to elicit a pronounced hyperphagic effect in freely feeding rats. These findings have raised our interest to investigate possible interactions in the control of ingestive behavior between N/OFQ and corticotropin-releasing factor (CRF), which is well known to be a major mediator of stress and to possess anorectic properties. These studies have shown that intracerebroventricular injections of N/OFQ or of Ro 64-6198 reverse the anorectic action evoked by intracerebroventricular administration of CRF. The anti-anorectic effect of N/OFQ or Ro 64-6198 is antagonized by the selective NOP receptor antagonist [Nphe1]N/OFQ1-13NH2, providing evidence that it is mediated by this receptor. The effect occurs at doses that are not hyperphagic per se and is clearly selective versus the anorectic action of CRF since N/OFQ or Ro 64-6198 do not influence the anorectic effect of Escherichia coli lipopolysaccharide (LPS). Neither N/OFQ nor Ro 64-6198 shows affinity for CRF receptors, suggesting that NOP receptor agonists might act as functional antagonists of CRF with regard to its anorectic action. Microinjection studies have revealed that the bed nucleus of the stria terminalis (BNST) is highly sensitive to the anorectic action of CRF, as well as to the anti-anorectic action of N/OFQ; pretreatment with 0.025-0.25 microg/site of N/OFQ into the BNST blocked the anorectic action of 0.1 microg/site of CRF given in the same area. On the other hand, intra-BNST microinjection of 0.025-0.25 microg/site of N/OFQ did not modify basal food intake. Thus, the BNST may be the site where the functional antagonism between N/OFQ and CRF takes place. These findings raise interest for the N/OFQ-NOP receptor system as a pharmacological target to block the anorectic effect of CRF. In comparison to CRF receptor antagonists, NOP receptor agonists may have the advantage of not inhibiting the hypothalamic-pituitary-adrenal (HPA) axis.

Effect of novel nociceptin/orphanin FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats

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 microg), UFP-102 (0.25 and 1.0 microg) or UFP-112 (0.01 and 0.05 microg), or with Ro 64-6198 (0.3 and 1.0 mg/kg) given intraperitoneally (i.p.) 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 microg dose) and UFP-102 (at the 0.25 microg 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. Pre-treatment with the selective mu-receptor antagonist naloxone (0.5 mg/kg, i.p.) 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 increased alcohol drinking, an effect probably induced by a residual agonist activity of this compound at mu-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.

The paraventricular nucleus of the hypothalamus is a neuroanatomical substrate for the inhibition of palatable food intake by neuropeptide S

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.

Effect of neuropeptide S receptor antagonists and partial agonists on palatable food consumption in the rat.

Neuropeptide S (NPS) is the endogenous ligand for the previously orphan G-protein-coupled-receptor, now termed NPS receptor (NPSR). NPS has both anxiolytic and pro-arousal properties and decreases food intake. In this work we use a rat model of palatable food intake to test in vivo different analogs of human NPS developed in our laboratories and characterized in previous in vitro experiments as partial agonists ([Ala(3)]NPS and [Aib(5)]NPS), or antagonists ([D-Cys((t)Bu)(5)]NPS and [(t)Bu-D-Gly(5)]NPS). Our results confirmed that intracerebroventricular (ICV) injection of NPS (1 nmol) decreases standard chow intake in food restricted rats as well as in freely feeding animals fed with standard or palatable food diets. [Aib(5)]NPS (30 and 60 nmol), like NPS, reduced palatable food intake, thus confirming in vivo its ability to activate NPSR. [Ala(3)]NPS (60 nmol) did not affect palatable food intake per se but blocked the anorectic effect of NPS, thus suggesting its ability to function as an antagonist in this model. Finally, [D-Cys((t)Bu)(5)]NPS (20-60 nmol) and [(t)Bu-D-Gly(5)]NPS (10-30 nmol), described in previous in vitro studies as pure NPSR antagonists, did not affect palatable food intake when given alone, but fully blocked the anorectic effect of NPS. These results provide an important characterization of the pharmacological properties of these NPS analogs in vivo. Of particular relevance are the data showing that [D-Cys((t)Bu)(5)]NPS and [(t)Bu-D-Gly(5)]NPS behave as pure antagonists at NPSR regulating food intake, indicating that these molecules are suitable tools for further investigation of the physiopharmacology of the NPS/NPSR system.

Lymphocyte DNA alteration by sub-chronic ethanol intake in alcohol-preferring rats

BACKGROUND Excessive alcohol consumption has been correlated with a higher susceptibility to infections among humans. Chromosome aberrations and other parameters have been suggested as useful biomarkers in assessing genetic damage due to ethanol intake. METHODS Genetically selected alcohol-preferring rats were given water, 10% ethanol and water or 10% ethanol alone for 3 months as fluid to drink. Food was available ad libitum for the entire period. At the end of the sub-chronic treatment their blood and liver were collected. All blood cells were counted and both lymphocytes and hepatocytes of all three groups were tested with the Comet assay to determine whether any DNA damage had occurred. RESULTS Only lymphocytes showed DNA damage, with differences among groups. The group that had only ethanol to drink showed greater lymphocyte DNA damage than the ethanol/water and water alone groups. On the other hand, hepatocyte DNA did not show any signs of damage. CONCLUSIONS Ten weeks of sub-chronic ethanol treatment produces small but significant damage to lymphocytes but not to hepatocytes, a result which confirms the observations of previous authors, and extends them even to a strain of rats genetically selected for high ethanol intake.

Activation of the nociceptin/orphanin FQ system is unable to reverse CRF2 receptor mediated anorexia in the rat.

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.

Leptin fails to reduce ethanol intake in Marchigian Sardinian 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.