Involvement of Activated Brain Stress Responsive Systems in Excessive and “Relapse” Alcohol Drinking in Rodent Models: Implications for Therapeutics

Abstract

Addictive diseases, including addiction to alcohol, pose massive public health costs. Addiction is a chronic relapsing disease caused by both the direct effects induced by drugs and persistent neuroadaptations at the molecular, cellular, and behavioral levels. These drug-type specific neuroadaptations are brought on largely by the reinforcing effects of drugs on the central nervous system and environmental stressors. Results from animal experiments have demonstrated important interactions between alcohol and stress-responsive systems. Addiction to specific drugs such as alcohol, psychostimulants, and opioids shares some common direct or downstream effects on the brain’s stress-responsive systems, including arginine vasopressin and its V1b receptors, dynorphin and the κ-opioid receptors, pro-opiomelanocortin/β-endorphin and the μ-opioid receptors, and the endocannabinoids. Further study of these systems through laboratory-based and translational research could lead to the discovery of novel treatment targets and the early optimization of interventions (for example, combination) for the pharmacologic therapy of alcoholism.

Introduction

The expanding literature has demonstrated that alcohol activates the brain’s stress-responsive systems, which contributes to excessive alcohol drinking and the development of alcoholism with the relapse of alcohol use. Several reviews from 2016 to 2018 of preclinical evidence from clinical trials have provided details on other important stress-responsive systems such as corticotrophin-releasing factor, neuropeptide Y, and glucocorticoid receptor (Koob and Mason, 2016; Mantsch et al., 2016; Blaine and Sinha, 2017; Mason, 2017; Pomrenze et al., 2017; Robinson and Thiele, 2017; Spierling and Zorrilla, 2017; Tunstall et al., 2017). The main focus in this mini-review is on important stress responsive systems that have yet to be reviewed such as arginine vasopressin/V1b receptors (in the section V1b Receptor and Arginine Vasopressin System) and pro-opiomelanocortin/β-endorphin (in the section POMC/β-Endorphin and μ-Opioid Receptor System). For two other stress-responsive systems, endocannabinoids/fatty acid amide hydrolase (Endocannabinoid System) and dynorphin/κ-opioid receptors (κ-Opioid Receptor and Dynorphin System), we examine controversies in the literature and the current state of the field for possible explanations (Parsons and Hurd, 2015; Chavkin and Koob, 2016; Anderson and Becker, 2017; Karkhanis et al., 2017; Tunstall et al., 2017). This mini-review provides an overview of the recent literature for these four stress-responsive systems in alcohol research, using laboratory-based animal models and clinical research to elucidate the biology of addictive diseases. We propose that translational bidirectional research will help refine future preclinical targets for the pharmacologic therapy of alcoholism.

V1b Receptor and Arginine Vasopressin System.

In the neurobiology of stress-related behaviors, increased arginine vasopressin (AVP) neuronal activity is involved in important steps in several rodent models (Griebel et al., 2002; Salome et al., 2006; Roper et al., 2011) and in humans (Katz et al., 2016; Ryan et al., 2017). Since the 1980s, evidence has emerged implicating AVP in the motivational properties of drugs of abuse (see van Ree et al., 1999). Systemic administration of desglycinamide-(Arg⁸)-vasopressin (DGAVP) reduced alcohol intake in studies exploring the role of AVP in rhesus monkeys (Kornet et al., 1991), and it also decreased alcohol intake in Brattleboro homozygote rats lacking vasopressin (Rigter and Crabbe, 1985) (Table 1).

TABLE 1.

Effects of AVP or V1b antagonists on alcohol-related behaviors

Subjects	Sex	Model	Effect	Reference
Brattleboro homozygote rats	Male	2-Bottle choice (24 h, 2%–10%)	Decrease (intake) by systemic (osmotic pump) DGAVP	Rigter and Crabbe (1985)
Rhesus monkeys	Male, female	Multiple-bottle choice (24 h,1%–8%)	Decrease (intake) by systemic (i.v.) DGAVP	Kornet et al. (1991)
Sardinian alcohol-preferring rats	Male	2-Bottle choice (24 h, 10%)	Decrease (intake and preference) by systemic (i.p.) SSR149415	Zhou et al. (2011)
Alcohol-dependent Wistar rats	Male	Operant self-administration (10%, 30 min)	Decrease (intake) by systemic (i.p.) or intracentral amygdala SSR149415	Edwards et al. (2012)
C57Bl/6J mice	Male, female	2-Bottle choice (24 h, every other day, 15%)	Decrease (intake and preference) by systemic (i.p.) SSR149415	Zhou et al. (2018)
Humans	Male, female	Phase 2, double-blind, placebo-controlled, randomized trial	Decrease (intake and relapse) by systemic (oral) ABT-436	Ryan et al. (2017)

ABT-436, V1b antagonist; DGAVP, desglycinamide-(Arg⁸)-vasopressin; SSR149415, V1b antagonist.

Of interest, after chronic exposure to an alcohol-containing diet, AVP mRNA levels were decreased in several stress-responsive brain regions of C57BL/6J mice, including the paraventricular nucleus (PVN), supraoptic nucleus, and bed nucleus of the stria terminalis (BNST) (Ishizawa et al., 1990; Gulya et al., 1991; Hoffman and Dave, 1991). Further studies demonstrated a decrease of the number of AVP-immunoreactive neurons and reduction of AVP mRNA levels in the hypothalamus after chronic alcohol consumption in both humans (Harding et al., 1996) and rats (Silva et al., 2002). In humans, abnormal levels of serum and urine AVP were found during alcohol withdrawal, particularly when the symptoms were severe (Eisenhofer et al., 1985; Trabert et al., 1992).

In several selectively bred alcohol-drinking rat lines, there are higher basal levels of AVP mRNA in the PVN of Indiana alcohol-preferring rats, Sardinian alcohol-preferring (sP) rats, and high-alcohol-drinking rats as compared with their respective alcohol-nonpreferring and low-alcohol-drinking counterparts (Hwang et al., 1998; Zhou et al., 2011). Higher basal AVP mRNA levels were also found in the medial amygdala of sP rats compared with Sardinian alcohol-nonpreferring (sNP) rats; chronic (>2 weeks) alcohol drinking reduced the AVP mRNA levels in the PVN and medial amygdala of sP rats (Zhou et al., 2011). Of interest, individual differences in AVP mRNA levels are positively associated with vulnerability to high alcohol drinking in C57BL/6J mice after acute stress (Nelson et al., 2018), so more studies are needed as individual vulnerability to drug relapse during abstinence is a key feature of drug addiction (Imperio et al., 2016; Sushchyk et al., 2016).

AVP binds to two G protein-coupled receptor subtypes in the brain: V1a and V1b. Both are expressed in the extended amygdala, with high concentrations in the central nucleus of amygdala (CeA), the BNST, and nucleus accumbens (NAc) (Veinante and Freund-Mercier 1997). Specifically, V1b receptors are mostly distributed in the PVN, hippocampus, and amygdala as well as the anterior pituitary (Lolait et al., 1995; Vaccari et al., 1998; Hernando et al., 2001; Young et al., 2006). In rodent models, many studies have suggested that augmented AVP/V1b activity in the amygdala plays a critical step in the stress-related behaviors. 1) After acute stress the rat amygdala shows increased levels of extracellular AVP (Wigger et al., 2004). 2) After acute withdrawal stress from drug exposure or by foot-shock stress after drug self-administration the rat amygdala shows increased levels of AVP mRNA (Zhou et al., 2005, 2008). 3) Activation of V1b receptors is involved in anxiety-like and depression-like behaviors (Griebel et al., 2002; Serradeil-Le Gal et al., 2002; Salome et al., 2006; Roper et al., 2011). SSR149415 [(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide], a highly selective nonpeptide antagonist for the V1b receptor, has anxiolytic-like and antidepressant-like properties (e.g., Overstreet and Griebel 2005).

One of the critical factors influencing individual vulnerability to drug relapse is atypical stress responsivity (Kreek and Koob, 1998; Zhou and Kreek, 2014). Using validated experimental models such as the forced swim and elevated plus maze tests, investigators have demonstrated anxiety-like and depression-like behaviors in rodents after chronic alcohol exposure, mostly during acute withdrawal (Colombo et al., 2006; Bell et al., 2012). As the high degree of anxiety-like and depression-like states is partially attenuated after voluntary alcohol drinking, rodents may drink alcohol to improve their emotional states (negative reinforcing mechanism) (Colombo et al., 2006; Bell et al., 2012; Pang et al., 2013).

In our alcohol study with sP rats, the V1b antagonist SSR149415 dose-dependently attenuated alcohol intake, suggesting that a V1b receptor-mediated mechanism is involved in modulating alcohol-drinking behaviors (Zhou et al., 2011). Importantly, SSR149415 reduces excessive alcohol self-administration in alcohol-dependent Wistar rats in a dose-dependent manner, without altering the alcohol drinking of nondependent rats (Edwards et al., 2012) (Table 1). Systemic administration of V1b antagonists blocks stress and drug priming-triggered seeking behavior (Zhou et al., 2008) and prevents the dysphoria induced by nicotine withdrawal (Qi et al., 2015) as well as nicotine-induced locomotor sensitization (Goutier et al., 2016). Therefore, the AVP/V1b system is a critical component of the negative reinforcing effects of alcohol, heroin, or nicotine, especially during drug withdrawal. In a recent phase 2, double-blind clinical trial (Table 1), pharmacologic blockade of the V1b receptor reduced alcohol consumption and the relapse rate in alcohol-dependent patients, especially those experiencing high stress (Ryan et al., 2017). As the V1b receptor is a feasible target in humans, there is translational potential for novel antialcoholism medications.

Stress increases secretion of corticotrophin-releasing factor (CRF) and AVP (the parvocellular division of the PVN) from terminals of the PVN into the pituitary portal circulation. The interaction between CRF and CRF1 receptors on corticotropes initiates the biosynthesis of pro-opiomelanocortin (POMC)–derived peptides and their release from the anterior pituitary (Vale et al., 1981). AVP from the parvocellular division of the PVN activating V1b receptors in the corticotropes enhances ACTH secretion from the anterior pituitary (Lolait et al., 1995; Aguilera and Rabadan-Diehl. 2000). However, AVP neurons in the magnocellular division of the PVN project to the posterior pituitary and then release AVP into the systemic circulation in response to stress. Both CRF/CRF1 receptor and AVP/V1b receptor systems are also mediators of the actions of central stress responsive systems, as both are widely distributed in the central nervous system (CNS) (Zhou et al., 1996; Roper et al., 2011).

Different from the hypothalamic CRF in response to acute cocaine use, several studies have shown that AVP in the PVN does not contribute to the acute stimulatory effects of alcohol on hypothalamic-pituitary-adrenal (HPA) activity (Rivier and Vale, 1988; Lee and Rivier, 1997). It is still not known whether the AVP/V1b receptor systems are specifically involved in the HPA modulation during acute or chronic withdrawal from alcohol exposure or after relapse-like drinking in rodent models, though AVP is potent modulator of HPA axis. While the activation of the PVN CRF contributes to the stimulating effect of acute alcohol on the HPA axis (e.g., Rivier and Vale, 1988), chronic alcohol exposure blunts the HPA hormonal response to alcohol, showing the development of HPA tolerance with either no change or an even decreased CRF mRNA level in the PVN (Zhou et al., 2000; Richardson et al., 2008).

Acute and protracted withdrawal from alcohol is coupled with decreased levels of both plasma corticosterone and hypothalamic CRF-like immunoreactivity in alcohol-dependent rats (Zorrilla et al., 2001). In humans, acute exposure to alcohol profoundly activates the HPA axis, and many alcoholics develop HPA tolerance after chronic alcohol exposure (Adinoff et al., 1990; Inder et al., 1995). In contrast, acute alcohol withdrawal transiently activates the HPA axis (Hundt et al., 2001; Zimmermann et al., 2003). Also, the noradrenergic system, the known key stress mediator involved in stress and anxiety responses (e.g., Tunstall et al., 2017), probably interacts with AVP and CRF systems to regulate alcohol drinking and HPA activity.

POMC/β-Endorphin and μ-Opioid Receptor System.

In the pituitary, ACTH is produced from the anterior lobe corticotrophs, whereas N-acetylated forms of β-endorphin and α-melanocyte-stimulating hormone are produced in the intermediate lobe melanotrophs. In the brain, the arcuate nucleus of the hypothalamus processes POMC to produce the potent opioid peptide β-endorphin as well as α-, β-, and γ-melanocortins (Ragavan et al., 1983; Rubinstein et al., 1996; Cowley et al., 2001; Romanova et al., 2015). Besides the arcuate nucleus, the POMC mRNA molecule has also been detected in much lower levels in several other mouse and rat brain regions, including the NAc, amygdala, hippocampus, and cerebral cortex (Civelli et al., 1982; Zhou et al., 1996, 2013b; Leriche et al., 2007; Bodnar, 2014; Granholm et al., 2017). Using the POMC-enhanced green florescent protein (eGFP) mice, we have found that POMC expression in POMC-eGFP neurons can be visualized by GFP immunohistochemistry, and a modest amount of POMC-eGFP neurons is present in both the shell and core subregions of the NAc (Zhou et al., 2013b). In the NAc, the amount of POMC mRNA is ∼10% of that detected in the hypothalamus, and the relatively low POMC mRNA signal in the NAc is correlated with the relatively small number of POMC-eGFP neurons in POMC-eGFP mice. Though alcohol drinking for more than 2 weeks increased POMC mRNA in the NAc shell (but not the core) of sP rats, it remains unclear whether the POMC mRNA in the extrahypothalamic regions (e.g., NAc shell and core) will be processed to melanocortins, β-endorphin, or other functional peptides.

In the rat anterior pituitary after acute or chronic alcohol administration, an increase, a decrease, or no change of the POMC mRNA levels as well as levels of POMC-derived peptides was reported (Gianoulakis et al., 1988; Winkler et al., 1995; Zhou et al., 2000, 2013b). Using a pituitary-specific deletion of the POMC gene in Tpit transgenic mice, our recent study found that the pituitary POMC deficiency did not change either alcohol drinking in a drinking-in-the-dark (DID) model (with a 4-hour limited access to alcohol in the dark cycle) or an alcohol-induced conditioned place preference (CPP) in male or female mice, suggesting that the pituitary POMC cells may not involve in the rewarding action or “binge” consumption of alcohol (Zhou et al., 2017c).

In the hypothalamus, POMC mRNA levels are either increased or decreased after acute or chronic alcohol (Angelogianni and Gianoulakis, 1993; Zhou et al., 2000, 2013b; Rasmussen et al., 2002; Navarro et al., 2013). C57BL/6 mice have high basal POMC mRNA levels in the hypothalamus with a high alcohol intake or preference as compared with the alcohol-avoiding DBA/2 mice with low alcohol intake or preference (Jamensky and Gianoulakis, 1999). In parallel, we found that the sP rats have higher basal POMC mRNA levels in the hypothalamus than sNP rats; chronic alcohol drinking for more than 2 weeks resulted in further increases in the hypothalamic POMC mRNA levels in sP rats (Zhou et al., 2013b). Considering the well-established role of β-endorphin in alcohol-drinking behaviors, the genetically determined POMC expression at basal levels and in response to alcohol may contribute to the high alcohol preference and/or consumption found in sP rats and C57BL/6J mice.

Activation of the μ-opioid receptor (MOP-r) by β-endorphin produces rewarding (Barson et al., 2011; Koch et al., 2015) and regulates NAc dopamine release (e.g., Spanagel et al., 1991). Alcohol or other drugs of abuse may release β-endorphin in the NAc (Olive et al., 2001; Marinelli et al., 2003; Roth-Deri et al., 2008), and the effects could be involved in the reinforcing actions and motivational behaviors of the drugs of abuse in rodents. Indeed, intracerebroventricular administration of β-endorphin induces CPP in rats (Amalric et al., 1987). Consistent evidence has been provided by numerous pharmacologic studies in rodents showing that opioid antagonists reduce alcohol consumption, reward, reinstatement of seeking behavior induced by cue, and “relapse” drinking. In human alcoholics, the opioid antagonist naltrexone decreases alcohol drinking, craving, and relapse (e.g., Brown and Holtzman, 1981; Hall et al., 2001; Liu and Weiss, 2002; Kuzmin et al., 2003; Pastor et al., 2011; Lukas et al., 2013; see also reviews by Gianoulakis, 1993; Herz, 1997; Le Merrer et al., 2009). MOP-r knockout mice show a decrease in alcohol drinking or self-administration (Roberts et al., 2000; Hall et al., 2001; Ben Hamida et al., 2017) (Table 2), which further indicates that the β-endorphin/MOP-r plays a functional role in the modulation of alcohol drinking.

TABLE 2.

Effects of genetic deletion of β-endorphin, POMC, and MOP-r on alcohol-related behaviors

Subjects	Sex	Model	Effect	Reference
β-Endorphin KO	Male + female	[1] 2-Bottle choice (24 h, 7%);	[1] Increase (intake and preference);	Grisel et al. (1999)
		[2] 2-Bottle choice (24 h, 10%)	[2] No difference (intake or preference)
β-Endorphin KO	Male	[1] 2-Bottle choice (24 h, 10%);	[1] No difference (intake);	Grahame et al. (2000)
		[2] 2-Bottle choice (2 h, 10%);	[2] Increase (intake);
		[3] ADE	[3] Increase (intake)
β-Endorphin KO	Male, female	2-Bottle choice (24 h, 16%);	Decrease (intake and preference) with sex difference	Racz et al. (2008)
Tpit KO mice with pituitary-specific POMC deletion	Male, female	1-Bottle (4 h, 15%) in DID	No difference in intake or preference in either sex	Zhou et al. (2017c)
nPE KO mice with hypothalamic-specific POMC deletion	Male, female	[1] 1-Bottle (4 h, 7.5%–30%) in DID;	Decrease (intake and preference) in all three models, with sex difference	Zhou et al. (2017c)
		[2] 2-Bottle choice (24 h, every other day, 7.5%–30%) in IA;
		[3] ADE
MOP-r KO	Male	[1] 30-min Operant self-administration (10%);	[1] Decrease (intake);	Roberts et al. (2000)
		[2] 2-Bottle choice (24 h, 10%)	[2] Decrease (intake and preference)
MOP-r KO	Male, female	[1] 2-Bottle choice (24 h, 2%–32%);	[1] Decrease (intake) with sex difference;	Hall et al. (2001)
		[2] CPP	[2] Decrease
MOP-r KO with striatum-specific deletion	Male	[1] 2-Bottle choice (24 h, 10%);	[1] Decrease (intake and preference);	Ben Hamida et al. (2018)
		[2] 2-Bottle choice (24 h, every other day, 10%) in IA;	[2] Decrease (intakeand preference);
		[3] CPP	[3] Decrease

KO, knockout; nPE, neuronal Pomc enhancers.

POMC neurons in the hypothalamus, the main region producing β-endorphin in the brain, may contribute to alcohol consumption. It is not clear, however, whether there is an involvement of β-endorphin in regulation of alcohol drinking, as studies using β-endorphin-deficient mice have shown inconsistent results (Grisel et al., 1999; Grahame et al., 2000; Racz et al., 2008) (Table 2). A limitation of this global β-endorphin knockout mouse model is that it does not allow for clarification of which specific regions of POMC cells (e.g., hypothalamus or possible pituitary) are involved in alcohol-drinking behaviors. Recently, the neuronal Pomc enhancers (nPE1 and nPE2) that are necessary for POMC expression specifically in hypothalamic arcuate neurons have been identified. The simultaneous transcriptional interference of Pomc enhancer function by insertion of a neomycin selection cassette in the enhancer vicinity abolishes POMC gene expression in the hypothalamic arcuate nucleus of transgenic mice, while leaving normal levels of POMC expression in the pituitary cells (Bumaschny et al., 2012).

Therefore, to determine the role of hypothalamic POMC neurons in alcohol-drinking behaviors, we have used transgenic mice with a region-specific POMC deficiency resulting from selective deletion of Pomc enhancers (Lam et al., 2015). Specifically, in mice of both sexes we determined the effect of tissue-specific Pomc gene manipulation on 1) binge drinking in a DID model (Rhodes et al., 2005), 2) acquisition and escalation of excessive alcohol drinking in a chronic intermittent access (IA) model (Wise, 1973; Simms et al., 2008; Hwa et al., 2011), and 3) relapse drinking in an alcohol-deprivation effect (ADE) model (Holter and Spanagel, 1999; Heyser et al., 2003). The wild-type mice exposed to DID rapidly established stable alcohol drinking behaviors, with more intake in females, whereas the hypothalamic POMC–deficient mice of both sexes had lower alcohol intake and preference. Though the hypothalamic POMC–deficient mice showed less saccharin intake and preference than the wild-type mice, there was no genotype difference in sucrose intake or preference. After 3 weeks of IA, the wild-type mice gradually escalated to high alcohol intake and preference, with more intake in females; the hypothalamic POMC–deficient mice showed less escalation. Of interest, pharmacologic blockade of MOP-r with naltrexone (NTN) dose-dependently reduced intake in the wild-type mice but had blunted effect in the hypothalamic POMC–deficient mice. The wild-type mice of both sexes displayed significant relapse-like ADE drinking, with more pronounced ADE in females; the hypothalamic POMC–deficient mice showed no ADE in either sex. Our results suggest an involvement of neuronal POMC/β-endorphin in the regulation of binge drinking, excessive drinking, and relapse, possibly through a hypothalamic-mediated mechanism, and with sex differences (Zhou et al., 2017c) (Table 2). Consistently, mice lacking MOP-r have shown reduced excessive alcohol drinking (Ben Hamida et al., 2017).

Consistent with previous studies in mice (Hall et al., 2001; Racz et al., 2008; Hwa et al., 2011; Yoo et al., 2012) and rats (recently reviewed by Becker and Koob (2016)), we have confirmed sex differences in alcohol drinking, with higher alcohol intake in females. The genotype differences in alcohol intake between hypothalamic POMC–deficient and wild-type mice are much greater in females than in males. The POMC deficiency affects female mice more strongly than males, suggesting that POMC may influence alcohol consumption in a sex-specific manner (Zhou et al., 2017c). Our results are in line with earlier studies that demonstrated decreased alcohol intake in β-endorphin and MOP-r knockout mice with more notable differences in females (Hall et al., 2001; Racz et al., 2008) (Table 2). Sex differences have also been observed in a human genetic study, which showed that the Pomc two-marker haplotype is associated with alcoholism only in women (Racz et al., 2008). These results also contribute to the idea of sex differences in opioid regulation of alcohol dependence (Becker and Koob, 2016).

Activation of POMC neurons affects food intake (which is increased and decreased by endorphin and melanocortins, respectively) especially at the onset of the dark cycle in mice (Mercer et al., 2013), so we purposely monitored drinking activity in the IA model during the 24-hour cycle with three time points: the first 4-hour dark cycle, the second 4-hour dark cycle, and the whole light cycle. Both male and female mice displayed escalated alcohol intake after 3 weeks of chronic IA exposure, mainly occurring at the first 4-hour dark cycle (25%–30% in total daily intake), without much change in the other two time periods (Zhou et al., 2017a,c). Of interest, in both sexes the hypothalamic POMC–deficient mice displayed lower alcohol intake than the wild-type mice during the first 4-hour dark cycle (Zhou et al., 2017c), suggesting a potential contribution of hypothalamic POMC to the genetically determined tendency of hypothalamic POMC–deficient mice toward reduced alcohol consumption, with the potential influence of clock genes as found in other studies with alcohol (Spanagel et al., 2005; Agapito et al., 2010; Partonen, 2015).

β-Endorphin is critically involved in the regulation of HPA activity. In both animal and human studies, it has been demonstrated that endogenous β-endorphin has tonic inhibition of the HPA axis by acting on the MOP-r (e.g., Kreek and Koob, 1998; Wand et al., 2002; Zhou et al., 2017c). NTN is a clinical MOP-r antagonist in the treatment of alcoholism (O’Malley et al., 1992; Volpicelli et al., 1992). As β-endorphin exerts tonic inhibition of CRF in the PVN (central part of the HPA axis), NTN blocks MOP-r, disinhibits the inhibition of the CRF, and then acutely and persistently activates the HPA axis (O’Malley et al., 2002). In a human study, the NTN-treated group showed higher plasma ACTH and cortisol levels than the placebo-treated group. Of great interest, the alcohol-craving levels in both groups were negatively correlated with the plasma cortisol levels. As the first human laboratory study, the results clearly demonstrated that modest activation of the HPA axis by NTN contributed to either the suppression of alcohol craving or the reduction in alcohol drinking (O’Malley et al., 2002). Other studies in humans support for this finding (e.g., Schuckit, 1994).

The potential role of endogenous ACTH and melanocortins in the brain (encoded by the Pomc gene) in the regulation of alcohol-related behavior is not clear. Recent pharmacologic studies have demonstrated that specific melanocortin 4 receptor (MC4) agonists significantly decrease alcohol binge-like drinking in a DID model as well as reduce appetitive and consumption behaviors (Olney et al., 2014; Sprow et al., 2016). In contrast, another study found that MC4 receptor antagonists in the ventral tegmental area reduced alcohol self-administration in rats (Shelkar et al., 2015), suggesting that endogenous melanocortins and MC4 activation mediate the alcohol-reinforcing effect.

Endocannabinoid System.

The endocannabinoid (eCB) system contains endogenous cannabinoids (including anandamide [AEA] and 2-arachidonoyl glycerol) and cannabinoid receptors (CB1 and CB2). In rodents, pharmacologic studies have demonstrated that specific blockade of CB1 receptors decreases alcohol drinking, blocks the motivation to consume alcohol, and reduces alcohol seeking, suggesting that the eCB/CB1 system is important in mediating the positive reinforcing properties and consumption of alcohol (Arnone et al., 1997; Colombo et al., 1998; Gallate and McGregor, 1999; McGregor et al., 2005). Furthermore, CB1 knockout mice show reduced alcohol drinking or preference and alcohol reward (Hungund et al., 2003; Wang et al., 2003; Naassila et al., 2004; Houchi et al., 2005). Therefore, during early stages of alcohol drinking, increased eCB/CB1 activity may promote alcohol reward and then enhance alcohol intake (Manzanares et al., 1999).

After chronic alcohol exposure and protracted withdrawal, however, there may be an eCB/CB1 signaling deficiency, which could also increase alcohol intake via the negative reinforcement mechanism. This idea is supported by several findings. 1) In rats, down-regulation of CB1 expression and function was observed during protracted alcohol withdrawal (Mitrirattanakul et al., 2007; Varodayan et al., 2016). 2) In human imaging studies, decreased CB1 availability was observed in heavy-drinking alcoholics, which persisted into abstinence (Hirvonen et al., 2013; Ceccarini et al., 2014). 3) In alcohol-dependent human patients, a lowered plasma AEA level was found during recent abstinence (Mangieri et al., 2009).

AEA-dependent signaling is regulated by an enzyme involved in AEA catabolism: fatty acid amide hydrolase (FAAH) (Cravatt et al., 1996, 2001). Numerous studies have demonstrated that AEA is involved in the behavioral effects of alcohol. FAAH knockout mice show a resultant increase in AEA levels (Cravatt et al., 2001) and increased alcohol consumption and preference (Basavarajappa et al., 2006; Blednov et al., 2007) (Table 3). In human genetic studies, increased alcohol abuse and dependency are associated with the FAAH C385A polymorphism (increased eCB activity due to impaired FAAH function) (e.g., Sipe et al., 2002; Sloan et al., 2018) (Table 3). Consistently, we found increased alcohol consumption in knock-in mice with human FAAH C385A (Zhou et al., 2016), with reduced anxiety-like behavior (Dincheva et al., 2015).

TABLE 3.

Effects of genetic mutation or deletion of FAAH gene and of FAAH inhibitors on alcohol-related behaviors

Subjects	Sex	Model	Effect	Reference
FAAH KO	Male, female	2-Bottle choice (24 h, 12%–20%)	Increase (intake and preference), with sex difference	Basavarajappa et al. (2006)
FAAH KO	Male, female	[1] 2-Bottle choice (24 h, 3%–15%);	[1] Increase (intake and preference, with sex difference;	Blednov et al. (2007)
		[2] CPP	[2] No effect
FAAH KO mice	Male, female	2-Bottle choice (24 h, 3%–12%)	Increase (intake and preference by systemic (i.p.) URB597, with sex difference	Blednov et al. (2007)
C57Bl/6J mice	Male, female	[1] 1-Bottle (4 h, 15%) in DID for 3 wk;	Decrease (intake and preference) by systemic (i.p.) URB597 in IA and ADE (but not DID) models, with no sex difference	Zhou et al. (2017d)
		[2] 2-Bottle choice (24 h, every other day, 7.5%–30%) in IA;
		[3] ADE
Marchigian Sardinian alcohol-preferring rats	Male	Operant self-administration (10%, 30 min)	Decrease (intake) by intracentral and basolateral amygdala URB597	Stopponi et al. (2018)
FAAH C385A Knock-in mice	Male	1-Bottle (4 h, 15%) in DID for 4 days and 2-bottle choice (4 h, 15% vs. water) on day 5	Increase (intake and preference)	Zhou et al. (2016)
Human FAAH C385A SNP	Male, female	2119 Patients	Association with street drug use and problem drug/alcohol use	Sipe et al. (2002)
Human FAAH C385A SNP	Male, female	1434 European Americans with AD diagnosis	Association with probability and severity of alcohol dependence	Sloan et al. (2018)

AD, alcohol dependence; KO, knockout; SNP, single-nucleotide polymorphisms; URB597, FAAH inhibitor.

Increased stress responsivity and persistent negative affective symptoms, such as anxiety and depression, are observed during alcohol withdrawal, and the severity may be associated with alcohol-relapse susceptibility (Koob and Kreek, 2007; Koob and Volkow 2010). The eCBs have considerable modulatory effects on the extended amygdala and corticostriatal circuitries, and stress disrupts these eCB-enriched regions that are involved in emotional control (Serrano et al., 2012; Dincheva et al., 2015; Morena et al., 2016). Pharmacologic and genetic manipulations (knockout or knock-in) of FAAH are found to alter anxiety-like and depression-like behaviors (Kathuria et al., 2003; Bortolato et al., 2007; Moreira et al., 2008; Gunduz-Cinar et al., 2013; Carnevali et al., 2015). Therefore, increased anxiety and depression are associated with the relatively deficient eCB function. Thus, impaired eCB activity may contribute to the negative affective states and increased stress responsivity that underlie the negative reinforcement mechanisms driving alcohol drinking by dependent individuals, which may also contribute to alcohol relapse after abstinence (Parsons and Hurd, 2015).

Though FAAH inhibition had been found to decrease the anxiety-like behaviors that are present during alcohol withdrawal (Cippitelli et al., 2008), no studies had tested the effect of FAAH inhibitors on alcohol drinking during withdrawal. We hypothesized that FAAH inhibition would enhance eCB signaling and then reduce the negative effect of alcohol withdrawal, which might reduce excessive and relapse drinking. To explore its potential for its therapeutic agent for alcoholism, we have investigated whether URB597 ([3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate), a selective FAAH inhibitor, alters alcohol drinking in mice during acute or chronic withdrawal from 3-week chronic IA excessive alcohol drinking (Zhou et al., 2017d). We also have investigated the pharmacologic effects of URB597 as a clinical FAAH inhibitor on the ADE. Mice were allowed to access to alcohol after 1 week of abstinence; after acute withdrawal from chronic IA, pretreatment with URB597 reduced alcohol intake and preference in both male and female mice. This effect was mediated through CB1 receptors. Of interest, the ADE can be prevented with an effective dose of URB597 via either a single- or multiple-dosing regimen, with no tolerance after 1 week of the multidosing regimen. At the most effective dose for reducing alcohol intake, URB597 had no effect on sucrose or saccharin preference in alcohol-naïve mice but increased the sucrose preference in mice after alcohol withdrawal (Zhou et al., 2017d).

In previous work, URB597 was found to increase the sucrose preference in stress-exposed animals, probably due to its “antidepression” properties (Bortolato et al., 2007; Rademacher and Hillard, 2007). Consistent with studies on cocaine, nicotine, and opioid seeking behavior (Panlilio et al., 2013; Sloan et al., 2017), our findings showed initial, promising data indicating that FAAH inhibitors decreases alcohol excessive drinking and relapse drinking in both male and female mice. Consistently, a new report has demonstrated that the CeA of alcohol-preferring rats is involved in the URB597 effect on reducing alcohol drinking (Stopponi et al., 2018) (Table 3).

Together, these results clearly suggest that the inhibition of FAAH plays a critical role in regulating alcohol drinking and related behaviors. Therefore, FAAH inhibitors with improved pharmacokinetics (long-lasting in vivo bioactivity, such as URB597) (Fegley et al., 2005; Basavarajappa et al., 2014) and with no rewarding effect (Gobbi et al., 2005) have the potential to become useful compounds for treating alcoholism (Zhou et al., 2017d; Stopponi et al., 2018).

κ Opioid Receptor and Dynorphin System.

Activation of the κ-opioid receptor (KOP-r)/dynorphin system is involved in aversive, dysphoria-like, and depression-like behaviors. For example, in dynorphin knockout mice, the aversive behaviors triggered by repeated forced swim or foot-shock stress are blocked by KOP-r antagonists or are absent (Land et al., 2008). Further study using an optogenetic approach has demonstrated that dynorphin/KOP-r in the NAc shell plays a functional role in aversive behaviors (Al-Hasani et al., 2015).

The dysphoric properties of chronic stress are encoded by dynorphin acting on KOP-r in specific stress-related brain regions, as dynorphin-dependent KOP-r activation by stress is found in these brain regions (including the basolateral amygdala, NAc, dorsal raphe, and hippocampus). Together, the dynorphin/KOP-r system is a key mediator of stress-induced aversion, dysphoria, and anxiety- and depression-like behaviors (Butelman et al., 2012; Lalanne et al., 2014).

Like stressors, KOP-r agonists stimulate HPA activity in rats, and the selective KOP-r antagonist nor-binaltorphimine (nor-BNI) blocks the stimulatory effects of KOP-r agonists on the HPA axis (e.g., Laorden and Milanes, 2000; Pascoe et al., 2008). Consistent with the early evidence that KOP-r/dynorphin regulates the HPA axis, our studies confirmed that blockade of KOP-r with nor-BNI prevents the ACTH and corticosterone increases that are induced by acute stress (Allen et al., 2013; Zhou et al., 2013c). In humans, KOP-r agonists or partial agonists increase plasma ACTH and cortisol levels (Ur et al., 1997; Schluger et al., 1998), and the short-acting KOP-r antagonist LY2456302 (4-[4-[[(2S)-2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methyl]phenoxy]-3-fluorobenzamide) does not cause aversive effects or HPA activity (Reed et al., 2018).

KOP-r/dynorphin activation is associated with the negative reinforcement aspects of alcohol addictions. It has been found that selective blockade of KOP-r attenuates excessive drinking and stress- or cue-induced alcohol-seeking in mice and rats (Walker and Koob, 2008; Sperling et al., 2010; Deehan et al., 2012; Schank et al., 2012; Funk et al., 2014; Rorick-Kehn et al., 2014; Anderson et al., 2016; Zhou et al., 2017a; but also see Mitchell et al., 2005; Sirohi et al., 2016). In line with these pharmacologic results, alcohol drinking is decreased in KOP-r knockout mice (Kovacs et al., 2005). These findings provide support for the critical involvement of the KOP-r/dynorphin system in the process of alcohol addiction, though the literature is not consistent (Table 4).

TABLE 4.

Effects of KOP-r agonists or antagonists on alcohol-drinking behaviors

Subjects	Sex	Model	Effect	Reference
Wistar rats	Male	4-Bottle choice (24 h, 5%–20%)	Increase (intake and preference) by systemic (minipump) enadoline; no change by systemic (i.p.) nor-BNI	Holter et al. (2000)
Lewis rats	Male	2-Bottle choice (2 h, 10%)	Decrease (intake) by systemic (i.p.) U50,488	Lindholm et al. (2001)
Lewis rats	Male	2-Bottle choice (24 h, 10%)	Increase (intake) by systemic (s.c.) nor-BNI	Mitchell et al. (2005)
C57Bl/6J mice	Male	2-Bottle choice (24 h, 3%–10%)	Increase (intake) by systemic (i.p.) U50,488; decrease (intake) by systemic (i.p.) nor-BNI in stressed mice	Sperling et al. (2010)
C57Bl/6J mice	Male	2-Bottle choice (2 h, 10%–15%)	Increase (intake and preference) by systemic (i.p.) U50,488	Rose et al. (2016)
C57Bl/6J mice	Male	1-Bottle (1 h, 15%)	Increase (intake) by systemic (i.p.) U50,488; decrease (intake) by systemic (i.p.) LY2444296 in stressed mice	Anderson et al. (2016)
C57Bl/6J mice	Male, female	[1] 1-Bottle (4 h, 15%) in DID for 3 wk;	Decrease (intake and preference) by systemic (i.p.) MSB in IA (but not DID) model, with no sex difference	Zhou et al. (2017a)
		[2] 2-Bottle choice (24 h, every other day, 7.5%–30%) in IA	Decrease (intake) by systemic (i.p.) nor-BNI in IA model, with sex difference

Enadoline, k agonist; LY2444296, k antagonist; MSB, κ agonist; nor-BNI, KOP-r antagonist; U50,488, KOP-r agonist [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide].

There are sex differences in dynorphin/KOP-r systems (Chartoff and Mavrikaki, 2015) and alcohol-drinking behavior (Becker and Koob, 2016). Indeed, we have observed a reduction of alcohol drinking with the selective KOP-r antagonist nor-BNI (slow onset and extraordinarily long-lasting effect; Horan et al., 1992) in male mice while the same nor-BNI treatment has had no effect on alcohol drinking in female mice (Zhou et al., 2017a).

Microdialysis studies have demonstrated that acute alcohol increases the extracellular levels of dynorphin A1-8 in the CeA and NAc, two brain regions known to play important roles in the regulation of alcohol consumption (Marinelli et al., 2006; Lam and Gianoulakis, 2011). Dynorphin mRNA levels in the CeA are increased in rats after acute alcohol withdrawal from multiple binge administrations of alcohol (D’Addario et al., 2013). The CeA is one of critical brain regions mediating depression-like and anxiety-like behaviors (Shippenberg et al., 2007; Knoll and Carlezon, 2010), and it is a possible site for the potential interaction of alcohol and the KOP-r/dynorphin. In fact, in sP rats after a large amount of alcohol drinking, an increase in dynorphin mRNA levels is found in the CeA. Therefore, the KOP-r/dynorphin involved in neuronal structures related to stress responsivity (e.g., CeA) is activated after high levels of alcohol consumption in sP rats (Zhou et al., 2013a).

Chronic intermittent alcohol vapor exposure in alcohol-dependent Wistar rats has further confirmed that there are increases in dynorphin peptide levels and KOP-r signaling in the CeA (Kissler et al., 2014). This enhanced KOP-r/dynorphin activity in the CeA may present a homeostatic adaptation of the CNS after chronic alcohol consumption or in the negative affective state during alcohol withdrawal. Further work has found that KOP-r activation inhibits both GABAergic synaptic responses and alcohol effects in the CeA (and BNST), and regulates GABA release (Li et al., 2012; Kang-Park et al., 2013). In the NAc shell, KOP-r blockade also reduces alcohol self-administration in alcohol-dependent rats (Nealey et al., 2011).

On the basis of these data, increased levels of KOP-r/dynorphin in the CeA and NAc have been confirmed as playing a functional role in the regulation of the negative affective state and/or reward after alcohol exposure or withdrawal (Shippenberg et al., 2007; Wee and Koob, 2010).

Early work found that “classic” KOP-r agonists attenuated alcohol drinking and alcohol CPP (Lindholm et al., 2001; Logrip et al., 2009), but they also produced sedation and dysphoria—side effects that limited their potential for clinical use (e.g., Morani et al., 2009). The development of new KOP-r agonists with reduced side effects may produce useful compounds for the treatment of alcoholism. Rapidly growing research has focused on identifying functionally selective (biased) KOP-r full agonists or partial agonists for the development of antiaddictive compounds (Maillet et al., 2015; Simonson et al., 2015; White et al., 2015; Brust et al., 2016; Schattauer et al., 2017; Townsend et al., 2017; Zhou et al., 2017a).

For a good example, Mesyl Salvinorin B (MSB), an analog of salvinorin A, is a potent KOP-r full agonist with fewer side effects (sedation and dysphoria) compared with other classic KOP-r agonists (Simonson et al., 2015; Zhou et al., 2017a). We have further examined the pharmacologic effects of MSB on excessive or relapse drinking in mice to determine its potential for development as an antirelapse compound for alcoholism. Acute administration of MSB significantly reduced both excessive drinking in an IA model and relapse drinking in a mouse ADE model in a dose-dependent manner (Zhou et al., 2017a; 2018b). Nalfurafine, a clinically available G-biased KOP-r agonist (Schattauer et al., 2017), also decreases excessive alcohol drinking with few side effects in mice (Zhou and Kreek, 2018). These promising in vivo results indicate that biased KOP-r full agonists may offer novel approaches to treat alcoholism without the traditional dysphoric properties of classic KOP-r agonists.

Many studies have demonstrated that classic KOP-r agonists increase alcohol drinking (Rose et al., 2016) and induce alcohol-seeking behavior in a reinstatement model (Funk et al., 2014) and relapse drinking in a ADE model (Hölter et al., 2000; see an update review by Anderson and Becker, 2017). Therefore, our new data that the KOP-r full agonist MSB reduces, rather than triggers, relapse-like drinking present an opposite scenario. After chronic excessive alcohol consumption, the endogenous dynorphin (a G-protein- and β-arrestin-dependent agonist; Maillet et al., 2015; White et al., 2015) and KOP-r systems are activated in several neuronal structures. Either the increased release of dynorphin (Marinelli et al., 2006) or the enhanced KOP-r activity (Rose et al., 2016) produces sedation, dysphoria, and anxiety- and depression-like behaviors that may drive excessive and relapse drinking (Tunstall et al., 2017). In support of this concept, the dynorphin levels and KOP-r activity are found to be increased in the rat CeA after chronic alcohol exposure (D’Addario et al., 2013; Zhou et al., 2013a; Kissler et al., 2014). Indeed, preclinical studies have demonstrated that the activation of p38 mitogen-activated protein kinase to stress-mediated dynorphin/KOP-r stimulation is linked to the β-arrestin-mediated transduction pathway (Bruchas et al., 2007, 2010). Unlike dynorphin, however, MSB does not induce sedation or anhedonia in rats or mice (Simonson et al., 2015; Zhou et al., 2017a), and could act as a G-protein-dependent (biased) agonist, which was suggested by our recent report (Simonson et al., 2015). Nalfurafine, acting as a biased KOP-r agonist, could possibly compete with excessive dynorphin to bind the KOP-r, thereby reducing β-arrestin signaling. This could be responsible, at least in part, for reducing excessive alcohol intake, as nalfurafine reverses the dynorphin-enhanced dysphoria and anxiety- or depression-like behavior during alcohol withdrawal.

Together, these studies support the notion that biased KOP-r agonists exhibit different molecular, cellular, and behavioral properties than classic KOP-r agonists (Che et al., 2018). Our study is in line with the growing research into the development of biased KOP-r ligands for antiaddictive compounds (Maillet et al., 2015; White et al., 2015; Brust et al., 2016; Townsend et al., 2017).

Conclusion and Future Directions

As presented in this mini-review, substantial progress has been made in our understanding how alcohol exposure disrupts the CNS stress-responsive systems to modulate alcohol taking and seeking behaviors in several selective animal models. It has been well known that the MOP-r/POMC and KOP-r/dynorphin endogenous opioid systems play critical roles in alcohol addiction, and specific alterations of their expression levels or receptor activity may affect stress responsivity and contribute to vulnerability to developing alcohol dependency or relapse. Other stress-responsive systems discussed here (including the V1b receptor with AVP, and FAAH with eCBs) are also potentially involved in alcohol addiction, as new evidence has emerged in recent studies.

Combination medications targeting multiple neurotransmitter pathways may show increased efficacy over the traditional single-medication strategy. As discussed previously, pharmacologic and neurobiologic studies have provided strong supportive findings; many stress-responsive systems, including CRF, neuropeptide Y, and glucocorticoid receptor, are profoundly disrupted after chronic alcohol exposure. Further studies on combination medications are needed to develop more effective new pharmacotherapies for treating alcoholism.

Although NTN is more effective in individuals with alcoholism who have MOP-r variant A118G (Bond et al., 1998; Bart et al., 2004; Kreek and LaForge, 2007; Anton et al., 2008), the single-target pharmacotherapy has relatively modest therapeutic value, which suggests the need for better efficacy (Müller et al., 2014). By targeting multiple neurotransmitters implicated in different components of alcohol addiction, combination medications are expected to have greater efficacy than single-medication therapy (Karoly et al., 2015; Zhou and Leri, 2016). Combinations of NTN with other compounds have several precedent in rodent models, such as acamprosate (Heyser et al., 2003) and prazosin (Froehlich et al., 2013).

Consistently, our recent studies in mouse alcohol-escalation drinking models have suggested that the combination of KOP-r agonist MSB, V1b antagonist SSR149415, or bupropion with NTN may be more efficacious in treating alcoholism than NTN alone (Zhou et al., 2017a, 2018a). 1) The effect of combined, low-dose administration of MSB/NTN, SSR149415/NTN, or bupropion/NTN on alcohol drinking is greater than that of either drug alone. 2) The combinations show persistent effects after repeated administration. In support of this idea, the effective medication nalmefene is a MOP-r antagonist plus a KOP-r partial agonist (Bart et al, 2005), targeting both MOP-r and KOP-r pathways and possibly synergistically reducing alcohol consumption.

Indeed, most drugs tested for alcoholism treatment—topiramate, varenicline, and gabapentin—target multiple systems (Karoly et al., 2015). Multiple targeting may have great advantages for treating alcoholism as a multigenic disease. Therefore, we propose that the combination drugs may prove more effective than drugs that are highly selective for a single target.

Abbreviations

ADE

alcohol-deprivation effect

AEA

anandamide

AVP

arginine vasopressin

BNST

bed nucleus of the stria terminalis

CB

cannabinoid receptors

CeA

central nucleus of amygdala

CNS

central nervous system

CPP

conditioned place preference

CRF

corticotrophin-releasing factor

DID

drinking-in-the-dark

eCB

endocannabinoids

eGFP

enhanced green florescent protein

FAAH

fatty acid amide hydrolase

HPA

hypothalamic-pituitary-adrenal

IA

intermittent access drinking

KOP-r

κ-opioid receptor

LY2456302

4-[4-[[(2S)-2-(3,5-dimethylphenyl)pyrrolidin-1-yl]methyl]phenoxy]-3-fluorobenzamide

MC4

melanocortin 4 receptor

MOP-r

μ-opioid receptor

MSB

mesyl salvinorin B

NAc

nucleus accumbens

nor-BNI

nor-binaltorphimine

NTN

naltrexone

POMC

pro-opiomelanocortin

PVN

paraventricular nucleus

sNP

Sardinian alcohol-nonpreferring

sP

Sardinian alcohol-preferring rats

SSR149415

(2S,4R)-1-[5-chloro-1-[(2,4-dimethoxyphenyl)sulfonyl]-3-(2-methoxy-phenyl)-2-oxo-2,3-dihydro-1H-indol-3-yl]-4-hydroxy-N,N-dimethyl-2-pyrrolidine carboxamide

U50,488

(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide

URB597

[3-(3-carbamoylphenyl)phenyl] N-cyclohexylcarbamate

Authorship Contributions

Wrote or contributed to the writing of the manuscript: Zhou, Kreek.