A relationship between the aldosterone-mineralocorticoid receptor pathway and alcohol drinking: preliminary translational findings across rats, monkeys and humans

Abstract

Aldosterone regulates electrolyte and fluid homeostasis through binding to the mineralocorticoid receptors (MRs). Previous work provides evidence for a role of aldosterone in alcohol use disorders (AUDs). We tested the hypothesis that high functional activity of the mineralocorticoid endocrine pathway contributes to vulnerability for AUDs. In Study 1, we investigated the relationship between plasma aldosterone levels, ethanol self-administration and the expression of CYP11B2 and MR (NR3C2) genes in the prefrontal cortex area (PFC) and central nucleus of the amygdala (CeA) in monkeys. Aldosterone significantly increased after 6- and 12-month ethanol self-administration. NR3C2 expression in the CeA was negatively correlated to average ethanol intake during the 12 months. In Study 2, we measured Nr3c2 mRNA levels in the PFC and CeA of dependent and nondependent rats and the correlates with ethanol drinking during acute withdrawal. Low Nr3c2 expression levels in the CeA were significantly associated with increased anxiety-like behavior and compulsive-like drinking in dependent rats. In Study 3, the relationship between plasma aldosterone levels, alcohol drinking and craving was investigated in alcohol-dependent patients. Non-abstinent patients had significantly higher aldosterone levels than abstinent patients. Aldosterone levels positively correlated with the number of drinks consumed, craving and anxiety scores. These findings support a relationship between ethanol drinking and the aldosterone/MR pathway in three different species.

Figure 1. Study 1 in monkeys: Experimental timeline

(A). All monkeys were trained during the baseline phase to participate in awake blood collection from the home cage and to operate an operant panel. Schedule-induced polydipsia began with a 1.5 g/kg volume equivalent of water, after which ethanol (4% w/v) was introduced and the daily dose increased each 30-days. Following ethanol induction, all animals had 22-h/day access to ethanol and water for approximately 14 months. Aldosterone was assayed from samples collected during baseline and after the first and second 6 months of self-administration as indicated by the arrows. Samples for blood ethanol concentration were collected throughout the experiment and are indicated by vertical lines. Plasma aldosterone levels before ethanol access (baseline), and following 6 and 12 months of voluntary ethanol self-administration (B). There was a significant effect of experimental phase (F_2,23 = 6.19, **p = 0.0071). Tukey post hoc comparisons revealed that aldosterone was significantly higher at both 6 months (**p = 0.0075) and 12 months (**P = 0.0048) of self-administration, but no differences were found between 6 months and 12 months (p = 0.99). Results are expressed as m±s.d.

Figure 2. Study 1: correlations between average ethanol intake (g/kg/day) and the mineralocorticoid nuclear receptor gene NR3C2 expression in the central nucleus of the amygdala (A, B) in rhesus macaques

Ethanol intake is shown as an average during the second 6 months block (A) and over the entire 12 months period (B) of ethanol self-administration. There was a significant correlation between the expression of NR3C2 in the CeA and ethanol intake at both the second block of 6 months (A; r² = 0.48; p = 0.008) and the overall 12 months (B; r² = 0.33; p = 0.042).

Figure 3. Study 2 in rats: experiment timeline (top panel) and behavioral findings in dependent and nondependent rats based on Nr3c2 expression levels in the central nucleus of the amygdala (CeA) and prefrontal cortex (PFC)

Top panel: The rats were tested in the elevated plus maze prior to any exposure to ethanol to measure baseline anxiety-like behavior. The same rats were then trained in daily 30-min sessions to lever press for access to ethanol (10%, w/v, 0.1 ml per delivery) and assigned to two groups: dependent (n = 16; chronically exposed to ethanol vapor) and nondependent (n = 18; exposed to air without ethanol) rats. They were tested under a fixed-ratio 1 (FR1) schedule of reinforcement to measure “intake,” a progressive ratio (PR) schedule to measure “motivation” for ethanol, and tested for the persistence to drink ethanol adulterated with quinine (used herein as an index of “compulsive drinking”). At the end of the behavioral experiments, we measured Nr3c2 mRNA levels in the PFC and CeA. Bottom panel: The behavior and Nr3c2 mRNA levels data have been previously published[26]. Here, we present a new statistical analysis that has not been previously published. A PCA revealed the occurrence of two main factors for each dependent and nondependent group that were interpreted to reflect “intake/motivation” and “anxiety/compulsive drinking” (A and D; see Figure S1 for additional results). The Nr3c2 expression levels in the CeA were significantly correlated with “anxiety/compulsive drinking” in dependent (B), but not in nondependent rats (E). No significant correlations were found for the PFC data for either dependent (C) or nondependent (F) rats. Ethanol drinking tests and Nr3c2 level measurements were conducted during acute alcohol withdrawal in the dependent rats.

Figure 4. Study 3 in humans: Plasma aldosterone levels at Week 12 in abstinent versus non-abstinent alcohol-dependent patients

Patients who did not maintain abstinence had significantly higher aldosterone levels compared to abstinent patients (F_1,213 = 4.53, *P = 0.047). Results are expressed as m±s.e.m.

Figure 5. Study 3 in humans: Correlations between plasma aldosterone levels at Week 12 and alcohol craving scores in alcohol-dependent patients

There were significant positive correlations between plasma aldosterone levels and the craving scales examined, i.e. Obsessive Compulsive Drinking Scale (OCDS) total score (r² = 0.21, p = 0.02; A), the obsessive ODS subscale (r² = 0.19, p = 0.02; B) and the compulsive CDS subscale (r² = 0.19, p = 0.02; C). Correlation between plasma aldosterone levels at Week 12 and state anxiety measured by the State and Trait Inventory (STAI) Y1 scale in alcohol-dependent patients. There was a significant positive correlation between plasma aldosterone levels and state anxiety levels (r² = 0.19, p = 0.03; D).