The mineralocorticoid receptor antagonist spironolactone reduces alcohol self-administration in female and male rats
Introduction
There is a wealth of literature examining the role of the hypothalamic-pituitary-adrenal (HPA) axis and corticosteroids in alcohol consumption, seeking, and dependence (Koenig and Olive, 2004; Vendruscolo et al., 2012; Vendruscolo et al., 2015). Within the HPA axis, cortisol (corticosterone in rodents) is one of the primary hormonal stress signals, and several studies in male adrenalectomized rats have shown that corticosterone moderates alcohol drinking (Fahlke et al., 1994a; Fahlke et al., 1995). To date, most studies of alcohol and the HPA axis have focused on the role of glucocorticoid receptors (GRs), which primarily bind corticosterone, in alcohol-related behaviors. For example, in preclinical studies of male rats, the GR and progesterone receptor antagonist mifepristone has been shown to block escalation of alcohol drinking following induction of dependence by chronic alcohol vapor exposure (Vendruscolo et al., 2012), and reduce alcohol consumption in a homecage limited-access two-bottle choice study (Koenig and Olive, 2004). Additionally, in alcohol-dependent male rats, GRs are downregulated in the prefrontal cortex (PFC), nucleus accumbens (NAc), and bed nucleus of the stria terminalis (BNST) during acute alcohol withdrawal, and upregulated in the NAc core, ventral BNST, and central amygdala (CeA) 3 weeks into abstinence (Vendruscolo et al., 2012). Overall these studies suggest that glucocorticoid signaling via the GR plays a dynamic role in both acute alcohol consumption and alcohol dependence, though it is important to note the bias towards utilizing male subjects in the literature as the HPA axis is known to be sexually dimorphic, both in normal and diseased states (Bangasser and Valentino, 2014). For example, in female mice with a history of predator stress and alcohol drinking GR is upregulated in the PFC during acute withdrawal, while males show no change (Finn et al., 2018).
Surprisingly, few alcohol-related studies have focused on the functional role of the mineralocorticoid receptor (MR). The MR has mainly been studied for its peripheral effects such as modulating fluid balance and blood pressure via its endogenous ligand, aldosterone, but is also known to modulate memory formation, fear extinction, and recall in male rats (Zhou et al., 2010; Dorey et al., 2011; Zhou et al., 2011; Ter Horst et al., 2012; Gomez-Sanchez and Gomez-Sanchez, 2014). Furthermore, there are sizeable sex differences in the role of MR in fear extinction, with female mice showing greater extinction deficits following MR deletion in the forebrain than male mice (Ter Horst et al., 2012). While the MR is traditionally thought of as a cytosolic ligand-dependent transcription factor that effects genomic changes on the time-scale of hours, recent studies have identified a membrane bound variant of the MR that can act on the time-scale of minutes (Karst et al., 2005; Khaksari et al., 2007; Dorey et al., 2011; Gomez-Sanchez and Gomez-Sanchez, 2014). In fact, the MR also binds corticosterone, and is expressed in brain regions generally associated with addiction such as the prefrontal cortex, hippocampus, and amygdala (Reul and de Kloet, 1986; Fuller et al., 2000). The MR has also been shown to mediate some responses to corticosterone that GR does not, such as modulating hippocampal glutamate signaling (Karst et al., 2005) and corticosterone-induced impairment of memory retrieval (Khaksari et al., 2007). Earlier studies reported the lack of modulatory effect of MR antagonism on alcohol drinking (Koenig and Olive, 2004; O'Callaghan et al., 2005) (see later discussion), and MR mRNA levels are not changed during acute withdrawal in alcohol dependent male rats (Vendruscolo et al., 2012). However, a recent multi-species study linked lower MR gene expression levels in the central amygdala (CeA) to higher alcohol drinking behavior in male primates with a history of alcohol consumption and more compulsive-like alcohol drinking in alcohol dependent male rats (Aoun et al., 2018). In male and female humans it was confirmed that higher levels of the MR ligand aldosterone correlated with higher alcohol craving in recovering alcohol use disorder (AUD) patients, and that non-abstinent patients had higher levels of aldosterone than abstinent patients (Leggio et al., 2008; Aoun et al., 2018). As such, there is growing evidence that MR signaling may play an important role in alcohol drinking behavior, and that there may be sex differences in this role, but it is unclear if this receptor may prove a potential therapeutic target.
One of the goals of the present study was to assess the role of MR signaling in the maintenance of alcohol self-administration using the MR antagonist spironolactone. Another goal of this study was to examine the effects of spironolactone on behavior under extinction conditions. To do this, probe extinction tests were used in which the cues associated with the reinforcer were presented, but alcohol delivery was withheld. This test allows for the examination of the persistence of responding in the presence of drug-associated cues, but absence of the primary reinforcer, which is an important feature of drug seeking behavior. Based on the literature, we hypothesized that MR antagonism would reduce alcohol self-administration and the persistence of non-reinforced alcohol responding. We also hypothesized that females would be more sensitive to this MR antagonism given greater behavioral response to MR knockout as well as documented HPA axis dimorphism (Ter Horst et al., 2012; Bangasser and Valentino, 2014). To test this hypothesis, male and female Long-Evans rats were trained to self-administer a sweetened alcohol solution (15% (v/v) alcohol + 2% (w/v) sucrose) and administered spironolactone prior to alcohol self-administration and probe extinction sessions. Furthermore, to explore the specificity of this effect to an alcohol reinforcer, spironolactone was tested in a separate group of male and female Long-Evans rats trained on sucrose self-administration. Together with recent studies implicating MR signaling in alcohol drinking behavior, these findings suggest that the MR may be an important avenue for research in the alcohol field.
Section snippets
Animals
38 adult Long-Evans rats (19 male/19 female) were single housed under a 12 h light/dark cycle (7am/pm). All experiments were conducted during the light cycle. Animals were continuously monitored and cared for by the veterinary staff of the UNC-Chapel Hill Division of Comparative Medicine. All procedures were carried out in accordance with the NIH Guide for Care and Use of Laboratory Animals and institutional guidelines. All protocols were approved by the UNC Institutional Animal Care and Use
Experiment 1: effect of spironolactone on maintenance of alcohol self-administration
The baseline alcohol lever responses (mean and S.E.M. of the two days prior to the initiation of spironolactone testing) was as follows: females 42.1 ± 3.51 and males 88.0 ± 9.53. Males had significantly higher responses (t(20) = 4.84, p < 0.001), but there was no significant difference in alcohol intake (females: 0.80 ± 0.06 g/kg; males: 0.93 ± 0.10 g/kg). Female rats had a higher locomotor rate (27.1 ± 1.40 beam breaks/min) than male rats (21.9 ± 0.10 beam breaks/min; t(20) = 2.92, p = 0.008).
Discussion
This study demonstrates that pharmacological antagonism of the MR with spironolactone reduces alcohol self-administration in both male and female rats, albeit at a higher dose in female rats. In male, but not female rats this reduction in self-administration was accompanied by a reduction in general locomotor activity during the self-administration session. Spironolactone also reduced the persistence of alcohol responding in the presence of alcohol-associated cues in female rats, but not male
Declarations of interest
None.
Acknowledgements
This work was supported in part by the National Institutes of Health [AA019682, AA026537], and by the Bowles Center for Alcohol Studies. VHM was supported by NS007431-18.
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