• Energy Research

Environmental conditioning is a major trigger for relapse in abstinent addicts. We showed that activation of the neuropeptide S (NPS) system exacerbates reinstatement vulnerability to cocaine and alcohol via stimulation of the hypocretin-1/orexin-A (Hcrt-1/Ox-A) system.Combining pharmacologic manipulations with immunohistochemistry techniques, we sought to determine how NPS and Hcrt-1/Ox-A systems interact to modulate reinstatement of alcohol seeking in rats.Intrahypothalamic injection of NPS facilitated discriminative cue-induced reinstatement of alcohol seeking. This effect was blocked by the selective Hcrt-1/Ox-A antagonist SB334867 microinjected into the hypothalamic paraventricular nucleus (PVN) or into the bed nucleus of the stria terminalis (BNST) but not into the ventral tegmental area or the locus coeruleus. Combining double labeling and confocal microscopy analyses, we found that NPS-containing axons are in close apposition to hypothalamic Hcrt-1/Ox-A positive neurons, a significant proportion of which express NPS receptors, suggesting a direct interaction between the two systems. Retrograde tracing experiments showed that intra-PVN or intra-BNST red fluorobead unilateral injection labeled bilaterally Hcrt-1/Ox-A somata, suggesting that NPS could recruit two distinct neuronal pathways. Confirming this assumption, intra-BNST or PVN Hcrt-1/Ox-A injection enhanced alcohol seeking similarly to hypothalamic NPS injection but to a lesser degree.Results suggest that the Hcrt-1/Ox-A neurocircuitry mediating the facilitation of cue-induced reinstatement by NPS involves structures critically involved in stress regulation such as the PVN and the BNST. These findings open to the tempting hypothesis of a role of the NPS system in modulating the interactions between stress and environmental conditioning factors in drug relapse.

The neuropeptide S (NPS) system is characterized by a unique pharmacology because it has anxiolytic-like effects and promotes arousal and wakefulness. To shed light on this peptidergic system, we tested the sedative effect of the central depressants diazepam and ethanol on the loss of righting reflex in mice lacking the neuropeptide S receptor (NPSR), NPSR(-/-). Furthermore, we tested the effect of the intracerebroventricular (ICV) administration of NPS on the sedative effect of diazepam and ethanol in NPSR(-/-) and their wild type counterpart NPSR(+/+). Finally, we evaluated the effect of the pro-arousal neuropeptides CRF and Hcrt-1/Ox-A in NPSR-deficient mice. Contrary to our expectations, the results showed that the NPSR(-/-) were less sensitive to the hypnotic effects of both diazepam and ethanol compared with their wild type littermates. ICV NPS was able to attenuate the sedative effect of both alcohol and diazepam in wild type mice, but not in the NPSR(-/-) line. The administration of CRF and Hcrt-1/Ox-A, two classic pro-arousal peptides, elicited the same effects in both NPSR(-/-) and wild type mice, ruling out the possibility that adaptive mechanisms occurring at the level of these two systems could have occurred during NPSR(-/-) development to compensate for the lack of NPSR receptors. Our findings demonstrated that the deletion of NPSR leads to minor changes in the arousal behavior of mice. Moreover, we demonstrated that the deletion of NPSR did not lead to compensatory changes in the vigilance-promoting effects of the CRF and Hcrt-1/Ox-A systems.

The present study evaluated the consequences of perinatal Delta(9)-tetrahydrocannabinol (Delta(9)-THC) treatment (5 mg/kg/day by gavage), either alone or combined with ethanol (3% v/v as the only fluid available), on ethanol self-administration and alcohol-seeking behavior in rat adult offspring. Furthermore, the effect of the selective cannabinoid CB(1) receptor antagonist, SR-141716A, on ethanol self-administration and on reinstatement of ethanol-seeking behavior induced either by stress or conditioned drug-paired cues was evaluated in adult offspring of rats exposed to the same perinatal treatment. Lastly, microarray experiments were conducted to evaluate if perinatal treatment with Delta(9)-tetrahydrocannabinol, ethanol or their combination causes long-term changes in brain gene expression profile in rats. The results of microarray data analysis showed that 139, 112 and 170 genes were differentially expressed in the EtOH, Delta(9)-THC, or EtOH+Delta(9)-THC group, respectively. No differences in alcohol self-administration and alcohol seeking were observed between rat groups. Intraperitoneal (IP) administration of SR-141716A (0.3-3.0 mg/kg) significantly reduced lever pressing for ethanol and blocked conditioned reinstatement of alcohol seeking. At the same doses SR-141716A failed to block foot-shock stress-induced reinstatement of alcohol seeking. The results reveal that perinatal exposure to Delta(9)-THC ethanol or their combination results in evident changes in gene expression patterns. However, these treatments do not significantly affect vulnerability to ethanol abuse in adult offspring. On the other hand, the results obtained with SR-141716A emphasize that endocannabinoid mechanisms play a major role in ethanol self-administration, as well as in the reinstatement of ethanol-seeking behavior induced by conditioned cues, supporting the idea that cannabinoid CB(1) receptor antagonists may represent interesting agents for the pharmacotherapy of alcoholism.

We have recently demonstrated that allyphenyline, behaving as α2C-adrenoceptor/serotonin 5-HT1A receptor agonist and α2A-adrenoceptor antagonist, in mice enhanced morphine analgesia, attenuated morphine withdrawal symptoms, showed significant antidepressant-like activity and was devoid of sedative side effects. Opioid and alcohol withdrawal shares several common neurobiological and molecular mechanisms. Therefore, in this study we expanded our analysis of the pharmacological properties of allyphenyline by investigating its ability to prevent the expression of somatic withdrawal signs, anxiety-like behavior and hyperlocomotion associated with chronic ethanol intoxication. Rats were subjected to induction of ethanol dependence via repeated daily intragastric ethanol (20%) administration for 4 consecutive days. Twelve hours after the last alcohol administration, somatic alcohol withdrawal signs were scored. Results revealed a significant expression of physical withdrawal signs that were not affected by intraperitoneal (i.p.) administration of allyphenyline at the doses of 0.05, 0.275 and 0.5 mg/kg. In contrast, allyphenyline (0.05 and 0.275 mg/kg i.p.) significantly reduced hyperanxiety-like behavior observed 6 days after alcohol intoxication as measured using the defensive burying test. Allyphenyline also reduced open field hyperlocomotor activity associated with alcohol withdrawal. Notably, the anxiolytic effect of the compound, as well as the already reported antidepressant action, was observed at very low doses, suggesting the involvement of its α2C-adrenoceptor/serotonin 5-HT1A receptor agonism. Therefore, the present investigation suggests that allyphenyline might represent an interesting pharmacological tool to investigate the potential of compounds exhibiting α2C-adrenoceptor/serotonin 5-HT1A receptor agonism and α2A-adrenoceptor antagonism in the treatment of hyperanxiety and hyperlocomotion occurring during alcohol withdrawal in dependent subjects.

A large body of evidence has shown that the Corticotropin Releasing Factor (CRF) system, which plays a key role in stress modulation, is deeply involved in relapse to alcohol seeking induced by exposure to stressful events such as foot shock or yohimbine injections. Exposure to environmental cues is also known to be a trigger for alcohol relapse, nevertheless, the relationship between the relapse evoked by the cue-induced model and the CRF stress systems remains unclear. The purpose of this study was to evaluate, in male Wistar rats, the involvement of the CRF system and Hypothalamic-Pituitary-Adrenal (HPA) axis in relapse induced by environmental cues. Antalarmin, a selective CRF1 receptor antagonist, Metyrapone, a corticosterone (CORT) synthesis inhibitor and CORT were evaluated for their effects on the reinstatement test in a cue-induced relapse model. Antalarmin (20mg/kg) blocked relapse to alcohol seeking induced by environmental cues. Metyrapone (50 and 100mg/kg) also blocked relapse in Wistar rats but only at the highest dose (100mg/kg). Corticosterone had no effect on relapse at the doses tested. The results obtained from this study suggest that the CRF stress system and the HPA axis are involved in cue-induced alcohol relapse.

Prenatal alcohol exposure (PAE) induces a wide range of neurodevelopmental disabilities that are grouped under the term 'fetal alcohol spectrum disorders' (FASD). The effects of PAE on brain development are dependent on complex neurochemical events, including modification of AMPA receptors (AMPARs). We have recently found that chronic ethanol (EtOH) exposure decreases AMPA-mediated neurotransmission and expression through the overexpression of the specific microRNA (miR)137 and 501-3p, which target GluA1 AMPA subunit, in the developing hippocampus in vitro. Here, we explored how PAE mice may alter AMPAergic synapses in the hippocampus, and its effects on behavior.To model PAE, we exposed C57Bl/6 pregnant mice to 10 % EtOH during during the first 10 days of gestation (GD 0-10; equivalent to the first trimester of pregnancy in humans). AMPA subunits postsynaptic expression in the hippocampus, electrical properties of CA1 neurons, memory recognition, and locomotor functions were then analyzed in adolescent PAE-exposed offspring.PAE adolescent mice showed dysregulation of AMPAergic neurotransmission, and increased miR 501-3p expression, associated with a significant reduction of spontaneous AMPA currents and intrinsic somatic excitability. In addition, PAE reduced the phosphorylation of AMPAR-containing GluA1 subunit, despite an increase in its total levels. Of note, the total levels of GluA2 and GluA3 AMPA receptors were enhanced as well. Consistently, at behavioral level, PAE reduced object recognition without altering locomotor activity.Our study shows that PAE leads to dysfunctional formation of AMPAergic synapses that could be responsible for neurobehavioral impairments, contributing to the understanding of the pathogenesis of FASD.

Background:  Alcoholism is a chronic disease characterized by frequent intoxications followed by withdrawal episodes and relapse to alcohol use. Neuroplastic changes associated with these intoxication and withdrawal cycles are thought to play a key role in disease progression. Recently, it has been shown that neuropeptide S (NPS), a newly deorphanized neuropeptide receptor system, facilitates relapse to alcohol seeking in laboratory animals. Given that a history of ethanol intoxication may increase vulnerability to alcohol addiction, we sought to determine whether NPS receptor (NPSR) gene expression is altered during withdrawal.Methods:  Rats were subjected to 1 week of intoxication by oral alcohol administration. NPSR gene expression was analyzed by in situ hybridization in rats 12 hours and 7 days after the last alcohol administration. To investigate the functional significance of NPSR system adaptation following protracted withdrawal 7 days after intoxication, we tested the anxiolytic‐like properties of NPS in nondependent and postdependent rats using the shock probe defensive burying test (DB).Results:  At both time points, increased NPSR gene expression was observed in several brain areas, including the endopiriform nucleus, the motor cortex, and the medial amygdaloid nucleus. Moderate increases in gene expression were also found in the lateral hypothalamus, paraventricular nucleus, basolateral and central amygdala. Differences from control animals were more pronounced after 7 days of abstinence. The upregulation of the NPSR system at this time point was confirmed by functional data indicating that intracerebroventricular (ICV) NPS administration (0.0, 0.3, and 0.1 nmol/rat) elicits more pronounced anxiolytic effects in postdependent animals than in controls subjected to the electric shock probe DB test.Conclusions:  Neuropeptide S receptor mRNA expression is increased in different brain areas of postdependent rats; as shown in the DB test, this expression change is functionally relevant.

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.