Pattern of neural activation following yohimbine-induced reinstatement of alcohol seeking in rats.

Alcohol use disorders represent an extensive socioeconomic burden, yet effective treatment options are suboptimal. A major hurdle in treating alcohol use disorders is the high rate of relapse. Stress is a major factor that promotes relapse in abstinent drug users; therefore, understanding neural mechanisms that underpin the effects of stress on alcohol seeking is critical. In rodent models of stress-induced relapse, the α2 -adrenoceptor antagonist, yohimbine, is a widely used chemical stressor to elicit reinstatement of drug/alcohol seeking. However, the exact mechanism how yohimbine precipitates reinstatement of alcohol seeking and the pattern of neural activation associated with yohimbine-induced reinstatement is poorly understood. Therefore, we counted Fos-protein positive nuclei across 42 brain regions in alcohol-experienced alcohol preferring rats that received either yohimbine in the home-cage (1 mg/kg i.p.) or following yohimbine-induced reinstatement of alcohol seeking. The number of Fos-protein positive nuclei was increased in the prefrontal cortex and extended amygdala after home-cage yohimbine compared to naïve- and vehicle-treated rats. Yohimbine-induced reinstatement increased the number of Fos-protein expressing nuclei in multiple other regions including the thalamus, hypothalamus and hippocampus. We then examined inter-regional correlations in Fos-protein expression for all 42 brain regions, which showed Fos expression was more strongly positively correlated following yohimbine-induced reinstatement of alcohol seeking, compared to home-cage yohimbine. These data suggest low-dose yohimbine in a non-drug-associated context activates stress/impulsivity centres within the brain, whereas yohimbine in the drug-associated context recruits additional brain regions to drive alcohol seeking.

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.

Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.

Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.

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.