Central amygdala relaxin-3/relaxin family peptide receptor 3 signalling modulates alcohol seeking in rats.

BACKGROUND AND PURPOSE: Alcohol use disorders are a leading cause of preventable deaths worldwide, and stress is a major trigger of relapse. The neuropeptide relaxin-3 and its cognate receptor, relaxin family peptide receptor 3 (RXFP3), modulate stress-induced relapse to alcohol seeking in rats, and while the bed nucleus of the stria terminalis has been implicated in this regard, the central nucleus of the amygdala (CeA) also receives a relaxin-3 innervation and CeA neurons densely express RXFP3 mRNA. Moreover, the CeA is consistently implicated in both stress and addictive disorders. Yohimbine precipitates relapse-like behaviour in rodents, although exactly how yohimbine induces relapse is unknown, possibly by increasing stress levels and inducing heightened cue reactivity. EXPERIMENTAL APPROACH: In the current study, we examined the effects of yohimbine (1 mg·kg-1 , i.p.) on anxiety-like behaviour in alcohol-experienced rats. Furthermore, we assessed CeA neuronal activation following yohimbine-induced reinstatement of alcohol seeking and the role of the relaxin-3/RXFP3 signalling within the CeA in yohimbine-induced reinstatement to alcohol seeking. KEY RESULTS: Low-dose yohimbine was anxiogenic in rats with a history of alcohol use. Furthermore, yohimbine-induced reinstatement of alcohol seeking increased Fos activation in CeA corticotrophin-releasing factor, dynorphin and GABA neurons compared with naïve and vehicle controls. Bilateral intra-CeA injections of the selective RXFP3 antagonist, R3(B1-22)R, attenuated yohimbine-induced reinstatement of alcohol seeking. CONCLUSIONS: Collectively, these data suggest that the CeA is a node where yohimbine acts to induce reinstatement of alcohol seeking and implicate the relaxin-3/RXFP3 system within the CeA in this process.

Nucleus incertus corticotrophin-releasing factor 1 receptor signalling regulates alcohol seeking in rats.

Alcoholism is a chronic relapsing disorder, and stress is a key precipitant of relapse. The nucleus incertus (NI) is highly responsive to corticotrophin-releasing factor (CRF) and psychological stressors, receives a CRF innervation and expresses CRF1 and CRF2 receptor mRNA. Furthermore, the ascending NI relaxin-3 system is implicated in alcohol seeking in rats. Therefore, in alcohol-preferring rats, we examined the effect of bilateral injections into the NI of the CRF1 receptor antagonist, CP376395 or the CRF2 receptor antagonist, astressin-2B on yohimbine-induced reinstatement of alcohol seeking. Using quantitative PCR analysis of NI micropunches, we assessed the effects of chronic alcohol consumption on gene expression profiles for components of the relaxin-3 and CRF systems. Bilateral intra-NI injections of CP376395 (500 ng/0.25 µl) attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injections of astressin-2B (200 ng/0.25 µl) had no significant effect. In line with these data, CRF1 , but not CRF2 , receptor mRNA was upregulated in the NI following chronic ethanol intake. Relaxin family peptide 3 receptor mRNA was also increased in the NI following chronic ethanol. Our quantitative PCR analysis also identified CRF mRNA within the rat NI, and the existence of a newly identified population of CRF-containing neurons was subsequently confirmed by detection of CRF immunoreactivity in rat and mouse NI. These data suggest that NI neurons contribute to reinstatement of alcohol seeking, via an involvement of CRF1 signalling. Furthermore, chronic ethanol intake leads to neuroadaptive changes in CRF and relaxin-3 systems within rat NI.

The Nicotinic α6-Subunit Selective Antagonist bPiDI Reduces Alcohol Self-Administration in Alcohol-Preferring Rats.

Cigarettes and alcohol are the most abused substances in the world and are commonly co-abused. Nicotine primarily acts in the brain on nicotinic acetylcholine receptors (nAChR), which are also a target for alcohol. The alpha6 subunit of nAChR is expressed almost exclusively in the brain reward system and may modulate the rewarding properties of alcohol and nicotine. Recently, N,N-decane-1,10-diyl-bis-3-picolinium diiodide (bPiDI) was synthesized as a selective, brain penetrant α6 subunit antagonist that reduces nicotine self-administration. The current study aimed to examine the effects of bPiDI on alcohol self-administration in inbred alcohol-preferring (iP) rats. Adult, male iP rats were trained to self-administer alcohol or sucrose. Once stable responding was achieved, rats were injected with bPiDI (1, 3 mg/kg, i.p.) and tested for self-administration under fixed and progressive ratio schedules of reinforcement. They subsequently underwent extinction, in which no rewards or cues were presented in the operant chambers. Then, they were injected with bPiDI prior to testing for cue-induced reinstatement of reward seeking. bPiDI (3 mg/kg) significantly reduced alcohol self-administration in both fixed and progressive ratios without any effects on sucrose self-administration or locomotor activity. In contrast, bPiDI (3 mg/kg) did not inhibit cue-induced reinstatement of either alcohol or sucrose seeking. The results support the involvement of α6 containing nAChR in reinforcing effects of alcohol, but not relapse to alcohol-seeking, without any impact on responding for a natural reward or general activity. bPiDI may be a potential lead molecule for a therapeutic strategy to limit nicotine and alcohol consumption.

Nucleus incertus Orexin2 receptors mediate alcohol seeking in rats.

Alcoholism is a chronic relapsing disorder and a major global health problem. Stress is a key precipitant of relapse in human alcoholics and in animal models of alcohol seeking. The brainstem nucleus incertus (NI) contains a population of relaxin-3 neurons that are highly responsive to psychological stressors; and the ascending NI relaxin-3/RXFP3 signalling system is implicated in stress-induced reinstatement of alcohol seeking. The NI receives orexinergic innervation and expresses orexin1 (OX1) and orexin2 (OX2) receptor mRNA. In alcohol-preferring (iP) rats, we examined the impact of yohimbine-induced reinstatement of alcohol seeking on orexin neuronal activation, and the effect of bilateral injections into NI of the OX1 receptor antagonist, SB-334867 (n = 16) or the OX2 receptor antagonist, TCS-OX2-29 (n = 8) on stress-induced reinstatement of alcohol seeking. We also assessed the effects of orexin-A on NI neuronal activity and the involvement of OX1 and OX2 receptors using whole cell patch-clamp recordings in rat brain slices. Yohimbine-induced reinstatement of alcohol seeking activated orexin neurons. Bilateral NI injections of TCS-OX2-29 attenuated yohimbine-induced reinstatement of alcohol seeking. In contrast, intra-NI injection of SB-334867 had no significant effect. In line with these data, orexin-A (600 nM) depolarized a majority of NI neurons recorded in coronal brain slices (18/28 cells), effects prevented by bath application of TCS-OX2-29 (10 µM), but not SB-334867 (10 µM). These data suggest an excitatory orexinergic input to NI contributes to yohimbine-induced reinstatement of alcohol seeking, predominantly via OX2 receptor signalling.

Relaxin-3 receptor (RXFP3) signalling mediates stress-related alcohol preference in mice.

Stressful life events are causally linked with alcohol use disorders (AUDs), providing support for a hypothesis that alcohol consumption is aimed at stress reduction. We have previously shown that expression of relaxin-3 mRNA in rat brain correlates with alcohol intake and that central antagonism of relaxin-3 receptors (RXFP3) prevents stress-induced reinstatement of alcohol-seeking. Therefore the objectives of these studies were to investigate the impact of Rxfp3 gene deletion in C57BL/6J mice on baseline and stress-related alcohol consumption. Male wild-type (WT) and Rxfp3 knockout (KO) (C57/B6JRXFP3TM1/DGen) littermate mice were tested for baseline saccharin and alcohol consumption and preference over water in a continuous access two-bottle free-choice paradigm. Another cohort of mice was subjected to repeated restraint followed by swim stress to examine stress-related alcohol preference. Hepatic alcohol and aldehyde dehydrogenase activity was assessed in mice following chronic alcohol intake and in naive controls. WT and Rxfp3 KO mice had similar baseline saccharin and alcohol preference, and hepatic alcohol processing. However, Rxfp3 KO mice displayed a stress-induced reduction in alcohol preference that was not observed in WT littermates. Notably, this phenotype, once established, persisted for at least six weeks after cessation of stress exposure. These findings suggest that in mice, relaxin-3/RXFP3 signalling is involved in maintaining high alcohol preference during and after stress, but does not appear to strongly regulate the primary reinforcing effects of alcohol.

Acamprosate produces its anti-relapse effects via calcium.

Alcoholism is one of the most prevalent neuropsychiatric diseases, having an enormous health and socioeconomic impact. Along with a few other medications, acamprosate (Campral-calcium-bis (N-acetylhomotaurinate)) is clinically used in many countries for relapse prevention. Although there is accumulated evidence suggesting that acamprosate interferes with the glutamate system, the molecular mode of action still remains undefined. Here we show that acamprosate does not interact with proposed glutamate receptor mechanisms. In particular, acamprosate does not interact with NMDA receptors or metabotropic glutamate receptor group I. In three different preclinical animal models of either excessive alcohol drinking, alcohol-seeking, or relapse-like drinking behavior, we demonstrate that N-acetylhomotaurinate by itself is not an active psychotropic molecule. Hence, the sodium salt of N-acetylhomotaurinate (i) is ineffective in alcohol-preferring rats to reduce operant responding for ethanol, (ii) is ineffective in alcohol-seeking rats in a cue-induced reinstatement paradigm, (iii) and is ineffective in rats with an alcohol deprivation effect. Surprisingly, calcium salts produce acamprosate-like effects in all three animal models. We conclude that calcium is the active moiety of acamprosate. Indeed, when translating these findings to the human situation, we found that patients with high plasma calcium levels due to acamprosate treatment showed better primary efficacy parameters such as time to relapse and cumulative abstinence. We conclude that N-acetylhomotaurinate is a biologically inactive molecule and that the effects of acamprosate described in more than 450 published original investigations and clinical trials and 1.5 million treated patients can possibly be attributed to calcium.

Relaxin-3/RXFP3 system regulates alcohol-seeking.

Relapse and hazardous drinking represent the most difficult clinical problems in treating patients with alcohol use disorders. Using a rat model of alcohol use and alcohol-seeking, we demonstrated that central administration of peptide antagonists for relaxin family peptide 3 receptor (RXFP3), the cognate receptor for the highly conserved neuropeptide, relaxin-3, decreased self-administration of alcohol in a dose-related manner and attenuated cue- and stress-induced reinstatement following extinction. By comparison, RXFP3 antagonist treatment did not significantly attenuate self-administration or reinstatement of sucrose-seeking, suggesting a selective effect for alcohol. RXFP3 is densely expressed in the stress-responsive bed nucleus of the stria terminalis, and bilateral injections of RXFP3 antagonist into the bed nucleus of the stria terminalis significantly decreased self-administration and stress-induced reinstatement of alcohol, suggesting that this brain region may, at least in part, mediate the effects of RXFP3 antagonism. RXFP3 antagonist treatment had no effect on general ingestive behavior, activity, or procedural memory for lever pressing in the paradigms assessed. These data suggest that relaxin-3/RXFP3 signaling regulates alcohol intake and relapse-like behavior, adding to current knowledge of the brain chemistry of reward-seeking.

Maternally administered sustained-release naltrexone in rats affects offspring neurochemistry and behaviour in adulthood.

Naltrexone is not recommended during pregnancy. However, sustained-release naltrexone implant use in humans has resulted in cases of inadvertent foetal exposure. Here, we used clinically relevant dosing to examine the effects of maternally administered sustained-release naltrexone on the rat brain by examining offspring at birth and in adulthood. Maternal treatment (naltrexone or placebo implant) started before conception and ceased during gestation, birth or weaning. Morphometry was assessed in offspring at birth and adulthood. Adult offspring were evaluated for differences in locomotor behaviour (basal and morphine-induced, 10 mg/kg, s.c.) and opioid neurochemistry, propensity to self-administer morphine and cue-induced drug-seeking after abstinence. Blood analysis confirmed offspring exposure to naltrexone during gestation, birth and weaning. Naltrexone exposure increased litter size and reduced offspring birth-weight but did not alter brain morphometry. Compared to placebo, basal motor activity of naltrexone-exposed adult offspring was lower, yet they showed enhanced development of psychomotor sensitization to morphine. Developmental naltrexone exposure was associated with resistance to morphine-induced down-regulation of striatal preproenkephalin mRNA expression in adulthood. Adult offspring also exhibited greater operant responding for morphine and, in addition, cue-induced drug-seeking was enhanced. Collectively, these data show pronounced effects of developmental naltrexone exposure, some of which persist into adulthood, highlighting the need for follow up of humans that were exposed to naltrexone in utero.

The orexin1 receptor antagonist SB-334867 dissociates the motivational properties of alcohol and sucrose in rats.

A role for orexin A in mediating the primary and conditioned reinforcing effects of alcohol has been established. It is unclear however whether the contribution of orexins to alcohol reward occurs independently of effects on appetite and feeding, and whether orexins regulate the motivation to consume alcohol compared to other rewards. To examine this further here we investigate the effect of the orexin(1) receptor antagonist, SB-334867, on self-administration of alcohol (10% v/v) under both fixed (FR) and progressive ratio (PR) schedules of reinforcement, and whether this differs from the motivation to administer a natural food reward, sucrose (0.2-0.7% w/v) in alcohol preferring (iP) rats. SB-334867 treatment significantly reduced responding for both alcohol and sucrose under a FR3 schedule; however, at the same dose, reduced responding and break point for ethanol, but not sucrose, under a PR schedule. These findings for the first time implicate a role for orexins in the motivation to self-administer alcohol and suggest that this may occur independent of any generalized effect on appetitive drive.

Quantification of phosphorylated cAMP-response element-binding protein expression throughout the brain of amphetamine-sensitized rats: activation of hypothalamic orexin A-containing neurons.

In the present study, using rats, we have examined acute, contextual, and sensitized patterns of activated or phosphorylated cAMP response element-binding protein (pCREB) expression in parallel, assaying across multiple nuclei that have been implicated in addiction. The paradigm used included a comparison of pretreatment dose of amphetamine upon patterns of cellular activation, following rechallenge. Because efferent orexinergic projections synapse on many targets through the mammalian brain, including mesotelencephalic regions and limbic systems involved in drug reward and reinforcement, we examined for coexpression of pCREB or c-Fos double labeling within orexin A-immunopositive neurons following sensitization. Acute challenge with amphetamine (1.5 mg/kg i.p.) resulted in an increase in the number of pCREB-immunoreactive (-IR) cells within the substantia nigra but a decrease of pCREB-IR cells in the central and medial subnuclei of the amygdala. Contextual re-exposure to the drug treatment environment altered pCREB expression, particularly in the basal ganglia and hypothalamus, although these effects were dictated by pretreatment dose of amphetamine. Sensitization to amphetamine resulted in robust increases in pCREB-IR cell numbers in the basal ganglia and lateral septum of rats that had been pretreated with high-dose (10 mg/kg i.p.) but not low-dose (2 mg/kg i.p.) amphetamine, despite a similar behavioral response. Orexin A-containing cells in the hypothalamus of sensitized rats did not coexpress pCREB; however, these cells double-labeled for c-Fos and orexin A. These data suggest that orexinergic neurons are activated during the expression of behavioral sensitization, although in a heterogenous manner with regard to afferent topologies and functional roles in the nervous system.

Central overexpression of angiotensin AT(1A) receptors prevents dopamine D(2) receptor regulation of alcohol consumption in mice.

BACKGROUND: While angiotensin receptors are found on the soma and terminals of dopaminergic neurons, controversy surrounds the potential role of angiotensin in alcohol consumption. METHODS: Using a transgenic mouse with a brain-specific overexpression of angiotensin AT(1A) receptors (NSE-AT(1A) mice), we have examined the role of angiotensin in alcohol consumption and alcohol-induced regulation of the dopaminergic system. RESULTS: The functional relevance of the overexpressed AT(1A) receptors was confirmed by an exaggerated rehydration response following 24-hour dehydration. NSE-AT(1A) mice showed a high preference for alcohol (similar to wild-type mice); yet, raclopride treatment had no effect on alcohol consumption in NSE-AT(1A) mice, while significantly reducing consumption in wild-type mice. In contrast, NSE-AT(1A) mice showed enhanced sensitivity to raclopride compared with wild types in terms of D(2) receptor up-regulation within the ventral mesencephalon. In addition, striatal D(2) receptors in NSE-AT(1A) mice were sensitive to up-regulation by chronic alcohol consumption. CONCLUSIONS: Collectively, these data imply that while expression of angiotensin AT(1A) receptors on striatal neurons has no impact upon basal alcohol consumption or preference, AT(1A) receptors do modulate the sensitivity of dopamine D(2) receptors to regulation by alcohol and the ability of a D(2) receptor antagonist to reduce consumption.

Assessing appetitive and consummatory phases of ethanol self-administration in C57BL/6J mice under operant conditions: regulation by mGlu5 receptor antagonism.

RATIONALE: The development of mouse models of ethanol consumption and ethanol-seeking behavior is of particular importance in understanding the underlying mechanisms of drug abuse because these models can enable an analysis of an effect of specific genotype on drug-seeking behavior and the interaction of potential therapeutics with genotype. However, there are some limitations with present models, notably the inability to examine appetitive and consummatory behavior separately. MATERIALS AND METHODS: In the present study, C57BL/6 mice were trained to self-administer 10% ethanol in a modified operant protocol that allowed a clear delineation of consummatory and appetitive phases. The utility of this procedure was confirmed with the use of the metabotropic glutamate 5 (mGlu5) receptor antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP). RESULTS: Limited-access consumption during the dark phase of the light-dark cycle with intermittent access (every second or third day) led to a high level of consumption by the mice. MTEP caused a dose-dependent decrease in both the consumption of ethanol and the appetitive response for ethanol. Furthermore, this effect was unrelated to any effect of MTEP on locomotor activity. CONCLUSIONS: The model provides a useful paradigm for examining both the appetitive and consummatory phases of ethanol consumption in mice; furthermore, the data indicate mGlu5 receptors are involved in both phases.

The GABA(B) receptor allosteric modulator CGP7930, like baclofen, reduces operant self-administration of ethanol in alcohol-preferring rats.

GABA systems have been implicated as targets for ethanol at the cellular, molecular and behavioural level. The present study was designed to further examine the potential of the GABA(B) receptor as a target for regulating operant alcohol responding. Given that the prototypic agonist, baclofen, reduces the self-administration of alcohol, we hypothesized that the GABA(B) receptor allosteric modulator, CGP7930, might have similar actions but a reduced side-effect profile. In this context, inbred alcohol-preferring (iP) rats were trained to respond for 10% v/v ethanol in a fixed ratio paradigm; all drug testing was performed under an FR3 schedule. Both baclofen and CGP7930 independently reduced voluntary responding for 10% ethanol in a dose-related manner. Neither drug impacted upon responding for water. A combination of subthreshold doses of baclofen and CGP7930 was also able to reduce operant responding for ethanol, suggesting that CGP7930 is indeed acting to facilitate GABA(B) receptor-mediated signalling in this paradigm. These data demonstrate the potential of positive allosteric modulators of metabotropic GABA(B) receptors to regulate alcohol responding.