Nazzareno Cannella

The role of hypocretin in driving arousal and goal-oriented behaviors

The hypocretins (Hcrts), also called orexins, are two neuropeptides secreted by a few thousand neurons restricted to the lateral hypothalamus. The Hcrt peptides bind to two receptors located in nuclei associated with diverse cognitive and physiological functions. Experimental evidence has demonstrated that the physiological roles of hypocretins extend far beyond its initial role in food consumption and has emerged as a key system in the fields of sleep disorders and drug addiction. Here, we discuss recent evidence demonstrating a key role of hypocretin in the motivation for reward seeking in general, and drug taking in particular, and we delineate a physiological framework for this peptidergic system in orchestrating the appropriate levels of alertness required for the elaboration and the execution of goal-oriented behaviors. We propose a general role for hypocretins in mediating arousal, especially when an organism must respond to unexpected stressors and environmental challenges, which serve to shape survival behaviors. We also discuss the limit of the current experimental paradigms to address the question of how a system normally involved in the regulation of vigilance states and hyperarousal may promote a pathological state that elicits compulsive craving and relapse to drug seeking.

Activation of Nuclear PPARγ Receptors by the Antidiabetic Agent Pioglitazone Suppresses Alcohol Drinking and Relapse to Alcohol Seeking

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 TZDs 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.

Persistent Increase of Alcohol-Seeking Evoked by Neuropeptide S: an Effect Mediated by the Hypothalamic Hypocretin System

The association of ethanols 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.

Neuropeptide S facilitates cue-induced relapse to cocaine seeking through activation of the hypothalamic hypocretin system

Drug addiction is a chronic relapsing disorder characterized by compulsive drug seeking and use. Environmental conditioning factors are among the major determinants of relapse in abstinent cocaine users. Here we describe a role of the neuropeptide S (NPS) system in regulating relapse. In rats with a history of cocaine self-administration, presentation of stimuli predictive of drug availability reinstates drug seeking, triggering relapse. Intracerebroventricular (ICV) injection of NPS increased conditioned reinstatement of cocaine seeking, whereas peripheral administration of the NPS receptor antagonist SHA 68 reduced it. Manipulation of the NPS receptor system did not modify cocaine self-administration. We also found that ICV NPS administration activates c-Fos expression in hypocretin-1/orexin-A (Hcrt-1/Ox-A) immunoreactive neurons in the lateral hypothalamus (LH) and in the perifornical area (PeF). Of note, intra-LH and intra-PeF administration of NPS increased conditioned reinstatement of cocaine responding, an effect that was selectively blocked with the Hcrt-1/Ox-A receptor selective antagonist SB334867. Finally, results showed that intra-LH injection of the NPS antagonist [D-Cys(tBu) (5)]NPS blocked cue-induced cocaine seeking, indicating a role for this system in the pathophysiology of drug relapse.

Activation of PPARγ by Pioglitazone Potentiates the Effects of Naltrexone on Alcohol Drinking and Relapse in msP Rats

BACKGROUND Pioglitazone is a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist used for the treatment of insulin resistance and type 2 diabetes. Previous studies conducted in our laboratory showed that activation of PPARγ by pioglitazone reduces alcohol drinking, stress-induced relapse, and alcohol withdrawal syndrome in rats. Pioglitazone was not able to prevent relapse elicited by alcohol cues. Conversely, the nonselective opioid antagonist naltrexone has been shown to reduce alcohol drinking and cue- but not stress-induced relapse in rodents. METHODS Based on these findings, this study was sought to determine the efficacy of pioglitazone and naltrexone combination on alcohol intake and relapse behavior. Genetically selected alcohol-preferring Marchigian Sardinian (msP) rats were used for the study. RESULTS Pioglitazone (10 and 30 mg/kg) and naltrexone (0.25 and 1.0 mg/kg) each individually reduced alcohol drinking in msP rats. The combination of the 2 drugs resulted in a more potent alcohol drinking reduction than single agents. Confirming previous studies, pioglitazone (10 and 30 mg/kg) significantly reduced relapse induced by the pharmacological stressor yohimbine (1.25 mg/kg) but not by cues predictive of alcohol availability. Conversely, naltrexone reduced reinstatement of drug seeking elicited by alcohol cues but not by yohimbine. CONCLUSIONS The drug combination was effective in reducing both relapse behaviors. These findings open new vistas in the use pioglitazone in combination with naltrexone for the treatment of alcoholism.

The mGluR2/3 Agonist LY379268 Induced Anti-Reinstatement Effects in Rats Exhibiting Addiction-like Behavior

Medication development for cocaine-addicted patients is difficult, and many promising preclinical candidates have failed in clinical trials. One reason for the difficulty in translating preclinical findings to the human condition is that drug testing is typically conducted in behavioral procedures in which animals do not show addiction-like traits. Recently, a DSM-IV-based animal model has been developed that allows studying the transition to an addiction-like behavior. Changes in synaptic plasticity are involved in the transition to cocaine addiction. In particular, it has been shown that metabotropic glutamate receptor 2/3 (mGluR2/3)-mediated long-term depression is suppressed in the prelimbic cortex in addict-like rats. We therefore hypothesized that cocaine-seeking in addict-like rats could be treated with an mGluR2/3 agonist. Indeed, addict-like rats that were treated systemically with the mGluR2/3 agonist LY379268 (0, 0.3, and 3 mg/kg) showed a pronounced reduction in cue-induced reinstatement of cocaine-seeking. In an attempt to dissect the role played by mGluR2 and mGluR3 in cue-induced reinstatement, we analyzed the mRNA expression patterns in several relevant brain areas but did not find any significant differences between cocaine addict-like and non-addict-like rats, suggesting that the behavioral differences observed are due to translational rather than transcriptional regulation. Another possibility to study the contributions of mGluR2 and mGluR3 in mediating addictive-like behavior is the use of knockout models. Because mGluR2 knockouts cannot be used in operant procedures due to motoric impairment, we only tested mGluR3 knockouts. These mice did not differ from controls in reinstatement, suggesting that mGluR2 receptors are critical in mediating addictive-like behavior.

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.

Neuropeptide S Receptor Gene Expression in Alcohol Withdrawal and Protracted Abstinence in Postdependent Rats

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.

Increased Perioculomotor Urocortin 1 Immunoreactivity in Genetically Selected Alcohol Preferring Rats

INTRODUCTION Urocortin 1 (Ucn 1) is an endogenous peptide related to the corticotropin-releasing factor (CRF). Ucn 1 is mainly expressed in the perioculomotor area (pIII), and its involvement in alcohol self-administration is well confirmed in mice. In other species, the relationship between the perioculomotor Ucn 1-containing population of neurons (pIIIu) and alcohol consumption needs further investigation. The pIII also has a significant subpopulation of dopaminergic neurons. Because of dopamines (DA) role in addiction, it is important to evaluate whether this subpopulation of neurons contributes to addiction-related phenotypes. Furthermore, the effects of gender on the relationship between Ucn 1 and tyrosine hydroxylase (TH) in pIII and alcohol preference in rats have not been previously assessed. METHODS To address these issues, we compared 2 Sardinian alcohol-preferring sublines of rats, a population maintained at the Scripps Research Institute (Scr:sP) and a population maintained at University of Camerino-Marchigian Sardinian preferring rats (msP), to corresponding nonselectively bred Wistar rats of both sexes. Ucn 1- and TH-positive cells were detected on coronal midbrain sections from 6- to 8-week-old alcohol-naïve animals using brightfield and fluorescent immunohistochemistry. Ucn 1- and TH-positive cells in pIII were counted in the perioculomotor area, averaged across 2 to 3 sets, and binned into 3 bregma levels. RESULTS Results demonstrated increased average counts of Ucn 1-positive cells in the middle bregma level in preferring male rats compared to Wistar controls and no difference in TH-positive cell counts in pIII. In addition, fluorescent double labeling revealed no colocalization of Ucn 1-positive and TH-positive neurons. Ucn 1 but not TH distribution was influenced by gender with female animals expressing more Ucn 1-positive cells than male animals in the peak bregma level. CONCLUSIONS These findings extend previous reports of increased Ucn 1-positive cell distribution in preferring lines of animals. They indicate that Ucn1 contributes to increased alcohol consumption across different species and that this contribution could be gender specific. The results also suggest that Ucn1 regulates positive reinforcing rather than aversive properties of alcohol and that these effects could be mediated by CRF(2) receptors, independent of direct actions of DA.

The role of the neuropeptide S system in addiction: Focus on its interaction with the CRF and hypocretin/orexin neurotransmission

Recent behavioral, pharmacological and molecular findings have linked the NPS system to drug dependence. Most of the evidence supports the possibility that increased NPS activity may contribute to shaping vulnerability to addiction, especially relapse. However, data suggesting that the anxiolytic-like properties of NPS may have protective effects on addiction have been also published. In addition, evidence from conditioned place preference experiments, though not unequivocal, suggests that NPS per se is devoid of motivational properties. Intriguingly, several effects of NPS on drugs of abuse appear to be mediated by downstream activation of brain corticotrophin releasing factor (CRF) and hypocretin-1/orexin-A (Hcrt-1/Ox-A) systems. The major objective of the present article is to review the existing work on NPS and addiction. Particular attention is devoted to the interpretation of findings revealing complex neuroanatomical and functional interactions between NPS, CRF, and the Hcrt-1/Ox-A systems. Original data aimed at shedding light on the role of NPS in reward processing are also shown. Finally, existing findings are discussed within the framework of addiction theories, and the potential of the NPS system as a treatment target for addiction is analyzed.