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

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

Comparison of locomotor activity between offspring groups in response to a novel environment.

<p>Locomotor activity is shown on days 1–3, after saline or morphine administration on days 4–8 (10 mg/kg, s.c.), and after a final morphine challenge on day 16 (5 mg/kg, s.c.). A: Total activity scores in the vertical plane and B: in the horizontal plane, reveal that habituation was greater among naltrexone-exposed offspring. C: Total horizontal locomotor activity did not differ between groups treated with saline and challenged with morphine (saline-morphine). D: Total horizontal locomotor activity did not differ between groups treated with morphine, although sensitization was expressed in both groups after challenge with morphine (morphine-morphine). E: Analysis of data expressing total morphine-induced locomotor activity as a function of basal locomotor activity on day 3 revealed significantly greater activity values for naltrexone-exposed offspring, indicating an increased development of sensitization. Data are expressed as vertical plane entries (A) or horizontal distance moved in centimetres (B, C, D and E) and represent the mean ± SEM (one-way ANOVA: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001; days 1–3 (A and B): placebo: n = 37, naltrexone: n = 53; days 4–16 (C): placebo: n = 12, naltrexone: n = 19; days 4–16 (D and E): placebo: n = 20, naltrexone: n = 26).</p

Comparison of naltrexone- and placebo-exposed offspring to progressive ratio morphine self-administration.

<p>Morphine self-administration under a progressive ratio (PR) schedule of 3–4 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052812#pone-0052812-t001" target="_blank">Table 1</a> for response requirement) at a dose of 0.3 mg/kg/infusion. Data from the PR3–4 session are expressed as total active lever presses (A), breakpoint, defined as the final ratio completed within the 2 h session (B), and number of drug infusions (C) and represent the mean ± SEM (unpaired t-test, * <i>p</i><0.05, naltrexone versus placebo (n = 7). D: Cumulative response record for the PR3-4 session divided into 10 minute time bins. Data are expressed as number of active lever presses and represent mean ± SEM (two-way ANOVA, * <i>p</i><0.05, ** <i>p</i><0.01, naltrexone versus placebo (n = 7) for each time bin).</p

The effects of maternal naltrexone, or placebo, implants on offspring.

<p>The effects of maternal implant treatments during pregnancy on total litter size (observed live and still births), sex ratio of litters and term of pregnancy. Term refers to the length of pregnancy, measured in days. IQR = inter-quartile range. The mean and standard deviation (StdDev) are also shown (one-way ANOVA: ** <i>p</i><0.01, naltrexone versus placebo).</p

Cohort 2 offspring body and brain morphometric measurements following maternal treatment with naltrexone or placebo implants.

<p>Morphometric effects of maternal treatments during pregnancy and/or weaning on offspring body parameters (body weight, foot and head lengths), at birth and in adulthood (at 8 weeks-of-age). Morphometric effects on brain parameters (brain [cerebral+cerebellar] weight, volume and length, and cerebral length, height and width) are shown for adult male and female offspring and all values represent the mean ± SEM. Using one-way ANOVA: *** <i>p</i><0.001, significant difference for naltrexone versus placebo; <b>†</b><i>p</i> = 0.04, trend for difference between naltrexone versus placebo; § <i>p</i><0.01, significant difference for male versus female (within naltrexone or placebo group).</p

Timeline and paradigm for behavioural sensitization experiment: offspring from cohort 2.

<p>Treatments are shown (habituation, daily injection with morphine [10 mg/kg] or saline, period of abstinence, challenge with morphine [5 mg/kg] or saline injection, brains fresh frozen for neurochemistry experiments), with corresponding day since start of locomotor sensitization testing, and number of days for each treatment (i.e. duration).</p

Comparison of naltrexone- and placebo-exposed offspring to cue-induced drug-seeking.

<p>Cue-induced drug seeking (cues present but no drug infusions;1 h) after an abstinence period of 6 weeks with comparison between offspring groups (placebo, naltrexone). A: Cumulative response record for cue-induce drug-seeking divided into 10 minute time bins. Data are expressed as number of active lever presses and represent mean ± SEM (two-way ANOVA, * <i>p</i><0.05, naltrexone (n = 9) versus placebo (n = 7) for each time bin). B: Total active and inactive lever presses during the drug-seeking session (represented by white bars, placebo: n = 9, naltrexone: n = 7) as compared to lever presses during 7 days of stable responding under a fixed-ratio 1 (represented by black bars, placebo: n = 12, naltrexone: n = 9). Data expressed as number of lever presses and represent mean ± SEM (one-way ANOVA: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, naltrexone versus placebo).</p

Free naltrexone levels in dams and offspring with naltrexone implant.

<p>Temporal profiles of free naltrexone concentration are shown in offspring blood or amniotic fluid (A, cohort 1) or maternal blood (A and B, cohorts 1, 2 & 3.) after maternal administration with a naltrexone implant (25 mg, s.c.). A<i>:</i> Temporal profiles of free naltrexone concentration in offspring blood (GD18 [purple] and Birth [blue]) or amniotic fluid (GD13 [yellow] and GD15 [green]) or maternal blood (black) after maternal administration with a naltrexone implant (25 mg, s.c.). Cohort 1 maternal and offspring rats were sampled throughout gestation. Exponential lines of best fit are shown for maternal rats (y = 6.76e^−0.07x, filled line) and offspring (y = 9.45e^−0.09x, dashed line). Offspring levels correlated with maternal levels (Pearson Correlation [ = 0.75] significant at 0.001 level; N = 15); and both maternal (<i>p</i><0.01; R² = 0.62) and offspring (<i>p</i><0.05; R² = 0.55) levels correlated with the number of days post-implant. To allow appropriate comparison with offspring, unadjusted maternal data are presented here. C: conception; GD: gestation day; B: birth. B<i>:</i> Maternal naltrexone-blood levels prior to conception, at birth and at weaning from cohorts 1 (closed circles), 2 (open circles) and 3 (crosses) were associated with days post-implant (<i>p</i><0.01, N = 54; mean ± SEM: 2.86±0.62 ng/ml). A polynomial line of best fit indicated a moderate level of association (R² = 0.68). C: conception, B: birth, W: weaning.</p