Abbreviations

• P rats: Indiana alcohol Preferring rats

• CPP: conditioned place preference

• MOP: Mu-opioid receptor

• NAc: nucleus accumbens

• OFC: orbitofrontal cortex

• KOP: kappa-opioid receptor

• DOP: delta-opioid receptor

• NOP: nociceptin receptor

• DYN: dynorphin

• nor-BNI: nor-binaltorphimine

• CRH: corticotropin-releasing hormone

• BNST: bed nucleus of the stria terminalis

• JNK: c-Jun N-terminal kinase

• N/OFQ: nociceptin/orphanin FQ

• CeA: central nucleus of the amygdala

• GPCRs: G protein-coupled receptors

• HPA: hypothalamic-pituitary-adrenal

• SP: substance P

• NK: neurokinin

• VTA: ventral tegmental area

• (m)PFC: (medial) prefrontal cortex

• MSNs: medium spiny projection neurons

• ADE: alcohol deprivation effect

• COMT: Cathechol-O-Methyltransferase

• DLS: dorsolateral striatum

• iGluR: ionotropic glutamate receptors

• mGluR: metabotropic glutamate receptors

• BLA: basolateral amygdala

• DMS: dorsomedial striatum

• NMDAR: N-methyl-d-aspartate receptors

• AMPAR: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors

• OFC: orbitofrontal cortex

• PAM: positive allosteric modulator

• CaMKII: Ca²⁺/calmodulin-dependent protein kinase II

• NAM: negative allosteric modulator

• MPEP: 2-Methyl-6-(phenylethynyl)pyridine

• ERK_1/2: extracellular signal-regulated kinases ½

• CDPPB: 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide)

• LH: lateral hypothalamus

• mIPSC: miniature inhibitory postsynaptic current

• sP: sardinian alcohol preferring

1 INTRODUCTION

A main challenge of addiction treatment is to prevent relapse after patients achieve abstinence. Half a century ago, it was reported that more than 50% of newly abstinent patients with alcohol addiction (hereafter equated with alcoholism, alcohol dependence, or moderate – severe alcohol use disorder) relapse within three months (Hunt et al., 1971). Disappointingly, these numbers have remained largely unchanged over time (Sinha, 2011). In people suffering from alcohol addiction, stressful events, drug-associated cues and contexts, or re-exposure to a small amount of alcohol (“priming”, or “the first drink”) trigger a chain of behaviors that frequently culminates in relapse (Brownell et al., 1986; Hendershot et al., 2011). An urge to drink, or “craving”, is often (but not always) an antecedent of relapse (Wray et al., 2014). Its causal role for initiating substance use has long been debated (Tiffany, 1990), but research has shown that the magnitude of craving in response to triggers, assessed under controlled laboratory conditions, reliably predicts the risk of relapse in the subsequent months (Sinha et al., 2011).

1.1 Animal models for the study of alcohol seeking behavior

1.1.1 Alcohol seeking in reinstatement models to study craving and relapse

Since its introduction in a seminal study (de Wit & Stewart, 1981), reinstatement of drug seeking following extinction has become the most common model to study relapse in animals, and to investigate the underlying neural mechanisms (Epstein et al., 2006). To reinstate alcohol-seeking, it is first necessary to initiate robust and stable levels of alcohol self-administration. Operant self-administration, a workhorse of addiction research, poses some unique challenges when applied to alcohol. One of these is the aversive taste of high alcohol concentrations for most rodents, a phenomenon shared by humans with little or no experience of alcohol (Koob et al., 2003). Widely used protocols to overcome this barrier have required water deprivation, saccharin/sucrose fading (Samson, 1986; Samson et al., 1988), pre-exposure to alcohol in the homecage, or extended training to initiate the acquisition and maintenance of self-administration (Simms et al., 2010). Although effective, these procedures introduce a potential for confounds in alcohol self-administration studies, and even more so when examining reinstatement of responding. More recently, work from our lab and others has shown that robust and stable levels of alcohol self-administration can be achieved without resorting to these approaches (Augier et al., 2014, 2017; Puaud et al., 2018).

BOX . Mini-Dictionary of Terms

Operant self-administration

A procedure in which an animal is trained to perform an operant response (most of the time, pressing a lever or nose-poking) to obtain a reward, usually food pellets, or a drug solution that can be delivered orally in a drinking spout or intravenously depending of the drug studied and the method of administration chosen by the experimenter. In the vast majority of conditioning experiments, two levers (or two holes) are presented. Pressing on the "active" lever allows the animal to obtain the reward, whereas responding on the "inactive" lever has no behavioral consequences. This learning procedure is based on operant or Skinnerian conditioning. If the animal is able to learn the response/reward association and repeat it, its behavior is considered as reinforced and the drug is a reinforcer.

Reinstatement

The gold standard of animal models to study drug relapse. Following drug self-administration acquisition, maintenance and subsequent extinction of the drug-associated responding, animals are tested for reinstatement of their drug seeking behavior induced by different kind of stressors (pharmacological, physical, and psychological), drug-priming, discrete cues or contextual cues.

Stress-induced reinstatement

In this variant of the reinstatement paradigm, laboratory animals are initially trained to self-administer a drug, for which delivery is paired with discrete cues (tone, cue light, noise of the injection pumps, smell of the alcohol solution). Operant responding (lever presses or nose pokes) is then extinguished in the presence of the drug-associated discrete cues.

Cue-induced reinstatement

Similarly to stress-induced reinstatement, animals are first trained to self-administer a drug in the presence of concomitant discrete cues. Their responses are consecutively extinguished in the absence of the cues previously associated with the drug. During reinstatement testing, reintroduction of these discrete cues precipitate relapse-like behavior as shown by increased responses on the lever associated with the drug.

Drug-induced reinstatement

Animals are similarly trained to self-administer a drug and drug delivery is paired with a discrete cue. Operant responding is then extinguished in the presence of the discrete cues. Once stable and low rate of responses is achieved, responding for the drug is reinstated by a unit dose of the drug previously self-administer (drug priming).

Once alcohol self-administration is acquired, the reinstatement procedures start with an extinction phase, in which the operant response that previously led to an alcohol delivery no longer has a programmed consequence. Following extinction training, responses on the alcohol-associated lever decrease to low levels or stop. Reinstatement of responding for alcohol under extinction conditions (i.e. in the absence of the reinforcer) can then be induced by triggers, with discrete cues and stress being most robust for alcohol (Figure 1). The rate of operant responding (i.e reinstatement) on the lever previously associated with alcohol delivery is taken as a measure of the animal's urge to obtain alcohol, a model of craving in patients.

FIGURE 1. Animal models for the study of alcohol seeking behavior.

Priming injections of alcohol can successfully reinstate responding in rats (Le et al., 1998), but are less commonly used. With alcohol, reinstatement is more robustly produced by discrete alcohol-associated cues (Sinclair et al., 2012) or contexts (Chaudhri et al., 2009; Hamlin et al., 2009) but the efficacy of these stimuli to trigger reinstatement may rely on the specific type of cue presented. For instance, exposure to olfactory but not auditory cues can trigger relapse in rodents (Katner et al., 1999). In addition, reinstatement of alcohol seeking was potentiated when the olfactory cue was combined to a discriminative visual cue, enduring resistance to extinction, specifically in genetically selected alcohol preferring P rats (Ciccocioppo et al., 2001). A robust alcohol reinstatement is also produced by physical stressors such as intermittent footshock (Le et al., ,1998, 2002; Le & Shaham, 2002) or pharmacological stressors such as the anxiogenic drug yohimbine (Cippitelli et al., 2010; Le et al., 2005). For example, Le and co-workers found in their seminal study that exposure during 5 and 15 min to intermittent footshock (0.5 s shock, intensity of 0.8 mA) potently reinstated responding for alcohol, but not sucrose. By contrast, a priming injection of a dose of 0.48 g/kg of alcohol only marginally reinstated responding (Le et al., 1998).

However, because stressors that precipitate relapse in patients with alcohol addiction are typically psychosocial, they differ from the type of triggers used in preclinical reinstatement studies. This may be a limitation (Bjorkqvist, 2001; Epstein et al., 2006; Katz & Higgins, 2003), and it cannot be excluded that molecular mechanisms identified in reinstatement studies may differ from those that promote relapse in humans for this reason. In an attempt to address this issue, a recent study used the resident-intruder paradigm to study the effect of social defeat stress on cocaine seeking (Manvich et al., 2016). When rats were re-exposed to cues predictive of psychosocial stress (olfactory cues that signaled sessions of defeat stress), they potently reinstated lever pressing for cocaine. Whether this observation would generalize to reinstatement of alcohol-seeking, and whether the neural mechanisms mediating reinstatement behavior differ between these types of stressors is an important question for future studies.

Although used less commonly, reinstatement can also be assessed using conditioned place preference (CPP) procedures (Mueller & Stewart, 2000), or the operant runway model of drug-self administration (Ettenberg et al., 1996; Geist & Ettenberg, 1990). These procedures have to our knowledge not been applied to alcohol seeking.

1.1.2 Compulsive alcohol seeking

Alcohol seeking and taking that becomes “compulsive”, i.e. continues despite negative consequences, is a hallmark of alcohol addiction (Corbit et al., 2012; Everitt & Robbins, 2016; Koob & Volkow, 2010; Wagner & Anthony, 2002). Understanding the transition from controlled to compulsive alcohol use is a critical challenge for addiction research. Most preclinical alcohol studies have focused on compulsive alcohol taking, as assessed by the persistence of animals to drink alcohol despite adulteration with the bitter tastant, quinine (Wolffgramm, 1991; Wolffgramm & Heyne, 1995), or more recently, their persistence to self-administer alcohol in operant procedures despite adverse consequences such as an electric footshock delivered contingently with the alcohol (Augier et al., 2018; Seif et al., 2013). Compulsive alcohol seeking in the absence of alcohol, preceding actual intake (Everitt & Robbins, 2005) has only recently begun to be studied.

In a recent paper, the authors adapted procedures previously developed to study cocaine-seeking in rats (Pelloux et al., 2007; Vanderschuren & Everitt, 2004), and used these to disentangle alcohol seeking from alcohol taking using Indiana alcohol-preferring P rats (Giuliano et al., 2018, 2019).

The protocol used to study compulsive seeking in these experiments can be divided into four main phases (Figure 1a). First, rats undergo Pavlovian conditioning (1), in which they are trained to associate a 20s cue-light, which serves a conditioned stimulus, with the availability of a 15% alcohol solution. Next, during the taking phase (2), one of the two levers is randomly assigned as a “taking lever”, and operant responses on this lever are reinforced with the delivery of alcohol, together with presentation of the cue light. During the seeking-taking phase (3), the other lever serves as the “seeking lever”, and operant responses on this lever under a random interval schedule (with interval length progressively increased from 5 to 60 s) lead to the presentation of the taking lever, while the seeking lever is retracted. Similar to phase ii, pressing on the taking lever is now reinforced with a delivery of alcohol, together with activation of the cue light. After this, both levers retract, and rats need to re-initiate this chained schedule of reinforcement in order to drink more alcohol. Finally, during the last phase (4), the seeking-taking chain schedule becomes punished. Rats now receive mild footshocks (0.25 increased to 0.45 mA over daily sessions), randomly delivered on 30% of the trials. Using this protocol, a cluster analysis identified three subgroups of rats. Following the introduction of unpredictable punishment associated with the seeking lever, 34% of the population showed punishment-resistant alcohol seeking, whereas 30% of the rats markedly reduced their responses on the seeking lever. The rest of the animals (36%) were classified as intermediate, and partially suppressed alcohol seeking behavior (Giuliano et al., 2018).

Finally, an alternative approach to study both unpunished and punished alcohol seeking in preclinical models has been provided by multicriteria paradigm. Based on the seminal work of Deroche-Gamonet and co-workers (Deroche-Gamonet et al., 2004), alcohol seeking has been recently studied in a multisymptomatic addiction model that characterizes addiction-prone phenotype in rats, derived from the DSM-IV/5 diagnostic criteria of addiction (Domi et al., 2019; Jadhav et al., 2017). Alcohol-seeking was measured during “no-drug” periods as a progressive daily increase in seeking when responding for alcohol was neither reinforced by conditioned stimuli nor alcohol delivery. Over time, only one-third of rats developed persistence in alcohol seeking. In one of these papers (Domi et al., 2019), alcohol seeking despite a punishment was also assessed. Rats were punished with a 0.3 mA footshock that anticipated alcohol taking response. Punishment-resistant alcohol seeking was observed only in a subset of individuals, confirming the inter-individual vulnerability to develop alcohol addictive behaviors.

2 NEUROBIOLOGICAL MECHANISMS MEDIATING ALCOHOL SEEKING

2.1 Section I: opioid systems and alcohol seeking

In reviewing the neurobiology of alcohol seeking, opioid systems offer a useful starting point, since the opioid antagonist naltrexone and its structural analog nalmefene are clinically approved treatments for alcohol addiction. Meta-analysis of randomized controlled trials robustly show that naltrexone reduces relapse to heavy drinking (Jonas et al., 2014; Mann et al., 2013). In contrast with acamprosate, naltrexone is not effective for maintaining abstinence. Its clinical profile thus indicates an ability to block the progression from a slip, in which alcohol is sampled, to relapse and heavy drinking. This closely parallels blockade of priming-induced reinstatement in rats, which has also been reported with naltrexone (Le et al., 1999). Furthermore, meta-analysis of human laboratory studies supports an ability of naltrexone to suppress cue-induced craving (Hendershot et al., 2017). This is presumably related to the observation that, in patients with alcohol addiction, elevations of striatal mu-opioid receptors (MOP) correlate with subjective cravings 1 to 3 weeks into abstinence (Heinz et al., 2005), while, in social drinkers, alcohol administration results in release of endogenous opioids in the nucleus accumbens (NAc) and orbitofrontal cortex (OFC) (Mitchell et al., 2012). MOP activation is likely to promote and modulate alcohol-induced dopamine release in humans (Ramchandani et al., 2011). These and other data provide some degree of support for a predictive validity of preclinical reinstatement models, and may offer opportunities for reverse-translational research strategies (Venniro et al., 2020). It is, however, clear that the degree of predictive validity of these models may vary on a system-by-system basis (see below).

Opioid systems comprise a vast and complex landscape of neuropeptide ligand families (endorphins, dynorphins, enkephalins, and the related non-opioid peptide nociceptin), as well as their receptor families [MOP, kappa-opioid (KOP), delta-opioid (DOP) receptors; the nociceptin receptor (NOP), while related, does not bind to opioid ligands]. Opioid systems, and their multitude of roles in addictive disorders have been the subject of multiple excellent reviews [e.g. (Lutz & Kieffer, 2013)], including reviews that have specifically summarized the role of opioid systems in alcohol addiction (Nutt, 2014). Opioid receptors are Gi-coupled, highly expressed in brain areas of importance for reinforcing properties of drugs, and are involved in the regulation of both unconditioned and conditioned behavioral effects of alcohol. For instance, stimulation of MOP regulates the positive reinforcing effects of alcohol, whereas activation of KOP mediates aversive and dysphoric aspects of alcohol effects. In the following, we describe the involvement of the opioid receptor subtypes with a focus on alcohol seeking in laboratory animals (see Table 1 for a summary).

TABLE 1. Compounds targeting the opioid system in alcohol seeking behavior

2.1.1 MOP receptors and alcohol seeking

In a parallel to human findings, the non-selective opioid antagonist, naltrexone was first shown to suppress reinstatement of alcohol seeking triggered by a priming dose of alcohol (Le et al., 1999). In a further parallel to human observations, MOP blockade results in reduced incentive motivational (anticipatory) responses for alcohol and reinstatement of alcohol seeking by alcohol-associated stimuli. Naltrexone, at doses that are likely to predominantly act through MOP blockade (0.25, 1.0 mg/kg), selectively inhibits cue- but not stress-induced reinstatement in Wistar rats (Liu & Weiss, 2002). It has also been reported that both naltrexone and the selective of MOP antagonist, naloxonazine, (1–15 mg/kg) inhibits cue-induced alcohol-seeking (Ciccocioppo et al., 2002). In agreement with findings in rats, naltrexone (0.32–3.2 mg/kg) has also been shown to reduce motivation to drink in the presence of alcohol-related cues in baboons (Kaminski et al., 2012). On the other hand, naltrexone, even when given at higher doses (3 and 10 mg/kg) during extinction had minimal effects on subsequent sensitivity to alcohol cues and alcohol consumption (Ciccocioppo et al., 2002). Furthermore, naltrexone given during repeated alcohol cue exposure does not alter the subsequent incentive value of alcohol cues presented in its absence, or enhance exposure-induced extinction, a procedure that parallels clinical cue-exposure therapy (Williams & Schimmel, 2008).

The selective and potent MOP antagonist GSK1521498 has been shown to reduce both alcohol drinking and cue-induced alcohol seeking in alcohol-preferring P rats (Giuliano et al., 2015). GSK1521498 has also been evaluated in a model of compulsive alcohol seeking that relies on a chained seeking-taking schedule (see Section 1.1.2). GSK1521498 reduced alcohol seeking under non-punished conditions both in rats previously identified as compulsive, and those that were not. However, the degree with which seeking behavior was suppressed was greater in the compulsive rats, potentially suggesting that the therapeutic value of GSK1521498 may be particularly pronounced in individuals with a higher degree of alcohol addiction severity (Giuliano et al., 2018). In contrast with naltrexone, GSK1521498 is selective for MOP, and lacks partial agonist activity (Nathan et al., 2012).

Collectively, preclinical data strongly support MOP-blockade as a treatment to prevent alcohol craving and relapse, in agreement with clinical findings. Clinical effect sizes achieved through this mechanism are, however, modest (Del Re et al., 2013). It is unclear whether more selective MOP antagonists have a potential to improve outcomes beyond what is achieved with currently approved medications, since near complete MOP blockade can be achieved using naltrexone (Lee et al., 1988), and the duration of occupancy can be further improved using nalmefene (Ingman et al., 2005).

2.1.2 KOP receptors and alcohol seeking

KOPs and their endogenous ligand dynorphin (DYN) play a critical role in stress-reactivity and negative emotionality in addictive disorders, including alcohol addiction (Bruchas et al., 2010). Prolonged alcohol exposure in rats induces long-term neuroadaptation in the KOP/DYN system, resulting in negative affective-like states that promote excessive drinking presumably through negative reinforcement (Walker & Koob, 2008). Increased KOP sensitivity, together with a hypodopaminergic state in the NAc, is a key mechanism in mediating the aversive properties of alcohol withdrawal (Rose et al. 2016), because an increased activity of DYN/KOP during protracted abstinence may contribute to a negative emotional state that facilitates alcohol seeking, KOP antagonists may have a potential to become useful therapeutics in alcohol addiction (Drews & Zimmer, 2010).

In rats, the prototypical KOP antagonist nor-binaltorphimine (nor-BNI) has been shown to suppress stress-induced alcohol seeking by both a physical stressor (footshock) and the pharmacological stressor yohimbine (Funk et al., 2014; Harshberger et al., 2016). Conversely, activation of KOP receptors using systemic administration of the prototypical KOP agonist U50,488 reinstates alcohol seeking, and this is blocked by nor-BNI. Reinstatement triggered by KOP activation in this study was also blocked by pretreatment with the corticotropin-releasing hormone type-1 (CRH1; see Section 2.2.1) receptor antagonist antalarmin, indicating that DYN acts upstream of CRH to produce stress-induced reinstatement (Funk et al., 2014). nor-BNI has also been shown to reduce U50,488-induced reinstatement of alcohol seeking when injected in the bed nucleus of the stria terminalis (BNST; 4 μg/side) in Long Evans rats, suggesting BNST as a key player in DYN/KOP mechanisms of stress-induced alcohol seeking (Crowley et al., 2016; Erikson et al., 2018; Le et al., 2018).

The KOP antagonist nor-BNI has also been shown to block cue-induced reinstatement of alcohol seeking (Funk et al., 2014). We have also previously shown that JDTic (Carroll et al., 2004), a first-generation non-peptide selective KOP antagonist, blocked withdrawal-induced anxiety-like behavior and reduced cue-induced reinstatement in Wistar rats (Schank et al., 2012). In another study, conducted in female alcohol preferring P rats, JDTic dose dependently (1, 3, or 10 mg/kg) reduced relapse-like responding tested in the Pavlovian Spontaneous Recovery test (Deehan et al., 2012). Because of the complex actions of nor-BNI and JDTic discussed below, it is unclear how these effects are related to their acute KOP blockade.

Based on these and other findings, KOP blockade has been considered as a mechanism with therapeutic potential in alcohol addiction, but KOP antagonists with properties making them suitable candidates for clinical development have been lacking. Presumably because of phosphorylation of c-Jun N-terminal kinase (JNK), nor-BNI has effects that last long after it has dissociated from the receptor, resulting in complex pharmacokinetic and pharmacodynamic properties. JDTic has a similarly complex pharmacology, related to non-competitive effects likely to be mediated by modulation of JNK signaling (Bruchas et al., 2007), and was terminated from clinical development because of cardiac toxicity (Buda et al., 2015). A new generation of KOP antagonists may finally allow their evaluation for clinical efficacy. A representative of this generation is CERC-501 (Rorick-Kehn et al., 2014), a novel and a selective KOP antagonist that has been found safe in Phase 1 in both healthy and cocaine dependent subjects (Lowe et al., 2014; Naganawa et al., 2016; Reed et al., 2017).

We have evaluated CERC-501 in a battery of preclinical tests to assess its potential as a clinical candidate in alcohol addiction (Domi et al., 2018). At an oral dose of 10 mg/kg, CERC-501 fully reversed the anxiogenic effects of alcohol withdrawal, and blocked stress-induced reinstatement of alcohol seeking. These effects were highly specific behaviorally, since the same dose did not affect cue-induced reinstatement or nicotine induced escalated-drinking (Domi et al., ,2020, 2021). These findings are in agreement with the hypothesis that KOP activation is primarily associated with negative emotional states, and their ability to promote alcohol seeking and taking. The profile of CERC-501 complements that of naltrexone, which selectively inhibits cue-induced but not stress-induced reinstatement (Liu & Weiss, 2002). Combining KOP and MOP antagonism in clinical treatment therefore appears to be an attractive strategy. The preclinical safety profile of CERC-501 is promising for clinical development, since it did not affect the sedative properties of alcohol, its metabolism or general locomotor activity.

2.1.3 DOP receptors and alcohol seeking

In contrast with the rich literature on MOP and KOP, few studies have characterized the role of DOP on alcohol seeking behavior. Some data suggest that DOP may be involved in cue-induced reinstatement of alcohol-seeking. For example, the δ selective antagonist naltrindole, at a dose of 5 mg/kg (i.p.) selectively inhibited alcohol-seeking induced by alcohol-related environmental stimuli (Ciccocioppo et al., 2002). In agreement with this result, naltrindole, but not the MOP antagonist CTOP potently suppressed both cue-induced and context-induced reinstatement of alcohol seeking (Marinelli et al., 2009). Finally, the DOP antagonist SoRI-9409 effectively and dose-dependently reduces yohimbine stress-induced reinstatement of alcohol-seeking in rats (Nielsen et al., 2012).

2.1.4 NOP receptors in alcohol seeking

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is the endogenous ligand for the NOP. The N/OFQ-NOP system is involved in modulation of pain processing, affective states, and other physiological functions such as neuroendocrine and immune response (Bodnar, 2013; Valentino & Volkow, 2018). It has also been the subject of extensive investigation in models of addictive disorders, including alcohol. Originally, the overarching hypothesis was that NOP activation attenuates multiple measures of motivation for addictive drugs. This hypothesis has subsequently become complicated by observations that similar effects are also produced by NOP antagonists, making it unclear whether agonists or antagonists are most likely to offer opportunities to develop medications for addictive disorders (Ciccocioppo et al., 2019).

NOP activation, whether by nociceptin itself, peptide analogues, or small-molecule non-peptide agonists, has been shown to reduce expression of alcohol withdrawal signs, relapse after alcohol deprivation, and stress-induced reinstatement of alcohol seeking. This has been observed both in non-dependent Wistar rats, and, to an even higher extent, following a history of dependence (Economidou et al., 2011; de Guglielmo et al., 2015; Kuzmin et al., 2007; Martin-Fardon et al., 2000). Central administration of nociceptin also suppressed cue-induced alcohol seeking in alcohol preferring msP rats (Ciccocioppo et al., 2004). Electrophysiological studies have shown that, in the central nucleus of the amygdala (CeA), alcohol induces more pronounced changes of the N/OFQ-NOP system in alcohol dependent and msP rats compared to non-selected, naïve rats (Herman et al., 2013). In addition, msP rats show an innate over-expression of CRH1 receptors, driven mainly by two single nucleotide polymorphisms at CRHR1 gene locus (Hansson et al., 2006). These findings provide an important link between the innate dysregulation of CRH with the N/OFQ-NOP system and excessive drinking (Martin-Fardon et al., 2010).

We have also shown that the potent, brain-penetrant small-molecule NOP agonist, SR-8993 (1, 3 mg/kg), is able to reverse acute alcohol withdrawal-induced anxiety, and attenuate both stress- and cue-induced relapse to alcohol seeking in Wistar rats (Aziz et al., 2016). Paradoxically, similar findings have been obtained with an orally available small-molecule NOP antagonist, LY2940094 (3, 10 mg/kg). Using this antagonist and alcohol preferring msP rats, it was shown that blockade of NOP can also prevent alcohol taking (Borruto et al., 2020) and stress-induced reinstatement to alcohol seeking. LY2940094 also blocked alcohol-induced dopamine release in the NAc (Rorick-Kehn et al., 2016).

In an attempt to reconcile these paradoxical findings, it has been hypothesized that NOP receptors undergo rapid desensitization in response to activation by agonists (Toll et al., 2016). This is potentially consistent with observations that systemic treatment with the NOP agonist, MT-7716, which suppressed both cue- and stress-induced reinstatement of alcohol, gradually reduced alcohol drinking with an effect persisting also after discontinuation of the drug (Ciccocioppo et al., 2014). Exogenous administration of NOP agonists may thus down-regulate NOP transmission through receptor desensitization, and result in an antagonist-like effect.

It is thus presently unclear whether targeting the NOP system is a fruitful avenue for developing alcohol addiction medications, and if so, whether agonists or antagonists would be the preferred strategy. Ultimately, human data are needed to provide answers to these questions. To date, the only human data available come from a small, 8-week proof-of-concept study with LY2940094. These are inconclusive, as the study was negative for its primary endpoint of number of drinks per day, but did show significant effects in several secondary analyses, including the objective biomarker of alcohol consumption, gamma-glutamyl transferase (Post et al., 2016).

2.2 Section II: other peptides involved in alcohol seeking

2.2.1 Corticotropin-releasing Hormone (CRH)

CRH, a 41 amino acid peptide best known for its role as the hypothalamic releasing factor for the adrenocorticotropic hormone, is also widely distributed outside the hypothalamus. Its biology and role in alcohol-related behaviors have been the subject of multiple reviews [e.g. (Heilig & Koob, 2007; Heinrichs & Koob, 2004; Zorrilla et al., 2013)]. In addition to high densities of CRH neurons within the paraventricular nucleus of the hypothalamus, CRH-positive cells are also present in structures involved in alcohol seeking, including CeA and BNST. Actions of CRH are mediated through two subtypes of Gs-coupled G protein-coupled receptors (GPCRs). Behavioral stress responses, including stress-induced alcohol seeking, are predominantly mediated by CRH1 receptors in CeA and BNST. Effects of CRH2 activation are less clear, but are commonly opposite to those of CRH1. Similar to many neuropeptide systems, CRH1 signaling that mediates behavioral stress responses is an “alarm system” that is quiescent under a wide range of conditions, but becomes activated in the presence of uncontrollable stress.

Blockade of CRH signaling robustly blocks stress-induced alcohol seeking, while leaving cue-induced relapse-like behavior unaffected. This was first demonstrated with intracerebral administration of the non-selective peptide antagonist D-Phe CRF12–41, as well as systemic administration of the selective small molecule CRH1 antagonist CP-154 526; both these approaches blocked stress-induced reinstatement of alcohol seeking. This study also demonstrated a central, hypothalamic-pituitary-adrenal (HPA) axis independent mediation CRH1 antagonism on stress-induced reinstatement, since adrenalectomy did not influence its ability to block reinstatement (Le et al., 2000). A subsequent study in rats with a history of alcohol dependence showed a dissociation between effects on stress- and cue-induced reinstatement, in which CRH antagonism was selective for reinstatement induced by stress (Liu & Weiss, 2002). Following a history of alcohol dependence, expression of CRH and its CRH1 receptor is up-regulated within the CeA (Sommer et al., 2008), and this is accompanied by a markedly increased sensitivity to blockade of stress-induced reinstatement by CRH antagonism (Gehlert et al., 2007).

Collectively, these and other data consistently show that in rodents, CRH1 receptors selectively mediate stress- but not cue-induced reinstatement, and that a recruitment of the CRH system following a prolonged history of alcohol dependence renders animals particularly sensitive to blockade of relapse-like behavior by CRH1 antagonism. This research predicted that preclinical findings with CRH1 antagonists would translate into suppression of stress-induced craving in people with alcohol addiction, an established biomarker that predicts clinical relapse (Sinha et al., 2011). The arrival of small-molecule CRH1 antagonists that were safe and well-tolerated in humans subsequently allowed an evaluation of this hypothesis.

Unfortunately, available studies do not find support for human translation of the preclinical findings (Kwako et al., 2015; Schwandt et al., 2016). These results may not be conclusive (Pomrenze et al., 2017; Shaham & de Wit, 2016), but it is noteworthy that both studies went to great length to ensure target engagement through the use of biomarkers, and that one of them used verucerfont, a “fast-on, slow-off” CRH1 type of CRH1 antagonist thought to be particularly effective to achieve a functional blockade of CRH1 receptors (Zorrilla et al., 2013). Combined with the failures of CRH1 antagonists on multiple other stress-related psychiatric indications (Binneman et al., 2008; Coric et al., 2010; Dunlop et al., 2017), and their termination in clinical development, it is in our view unlikely that this mechanism can be resurrected for treatment of alcohol addiction.

2.2.2 Substance P (SP) and its neurokinin 1 (NK1) receptor

SP is an 11 amino acid peptide that belongs to the tachykinin family, which also includes neurokinin A (NKA) and neurokinin B (NKB) (Pennefather et al., 2004). Tachykinins exert their effects through three receptor subtypes, NK1-3, among which SP preferentially binds to the NK1 receptor, while the NK2 receptors is preferentially activated by NKA, and the NK3 receptor by NKB. NK1 receptors are Gs/q-coupled GPCRs, are located in a range of brain regions involved in both appetitive and aversive behaviors, modulate behavioral responses to stress, and regulate several alcohol-related behaviors (Schank & Heilig, 2017).

A challenge for preclinical studies on the role of NK1 receptors is a limited sequence homology and ligand affinity profile between human and rodent NK1 receptors, which limits the utility of NK1 antagonists developed for human use for studies in rodents (Schank & Heilig, 2017). This was overcome through the synthesis of L822429, an NK1 antagonist specifically developed to possess high affinity at rat NK1 receptors (Ebner et al., 2004). Using this molecule as a tool, we found that systemic blockade of NK1 receptors blocks stress-induced reinstatement, an effect with high behavioral specificity, as the same dose of the antagonist left cue-induced reinstatement unaffected (Schank et al., 2011). In alcohol preferring P rats, NK1 expression in CeA is elevated because of a gene sequence variant enriched in this line (Schank et al., 2013). P rats show an increased sensitivity to reinstatement of alcohol seeking by the pharmacological stressor yohimbine, which is suppressed by intra-CeA infusion of L822429. Conversely, viral over-expression of NK1 receptors in the CeA of Wistar rats increases their sensitivity to yohimbine-induced reinstatement (Nelson et al., 2019). In addition, recent findings indicate that the role of NK1 receptors in promoting stress-induced alcohol seeking in the CeA may be related to the fact that activation of these receptors by SP increases GABA-release in the CeA, and that this effect is up-regulated following a history of dependence [(Khom et al., 2020); see Section 2.5]. Collectively, these findings show that NK1 receptors in the CeA promote sensitivity to stress-induced relapse, as well as other alcohol-related behaviors.

Based on preclinical findings, we evaluated the NK1 antagonist LY686017 in an academic experimental medicine study, carried out in recently detoxified patients with alcohol addiction. This study used stress-induced craving and brain responses to negative emotional stimuli as biomarkers, and found that LY686017 suppressed both (George et al., 2008). A subsequent Phase 2 study was carried out by Eli Lilly, and has not been published (NCT00805441). In contrast with the laboratory study, this study was carried out in unselected patients, who overall had a low level of anxiety, and was negative on the primary outcome. However, several secondary analyses suggested a signal for efficacy.

Development of NK1 antagonists was in part driven by the discovery of their potential as antidepressant medications (Kramer et al., 1998). Following inconsistent results in subsequent depression trials, development of NK1 antagonists for stress-related psychiatric disorders was discontinued throughout the pharmaceutical industry. It was only later that a key factor behind the inconsistent results was identified. In contrast with most GPCR antagonists, for which central receptor occupancy >90% is typically sufficient for therapeutic efficacy, robust effects of NK1 antagonists require a near complete blockade (Ratti et al., 2013; Rupniak & Kramer, 2017). In our view, it is therefore a possibility that NK1 antagonism remain a viable therapeutic mechanism in alcohol addiction, if delivered using a highly brain penetrant medication, administered at adequate doses, to anxious alcohol addicted patients. Unfortunately, this proposition may never be evaluated.

2.3 Section III: the role of dopaminergic neurotransmission in alcohol seeking

Alcohol activates dopaminergic neurons in the ventral tegmental area (VTA) resulting in increased dopamine release in forebrain cortico-limbic regions, including the NAc and medial prefrontal cortex (mPFC) (Di Chiara & Imperato, 1985; Ding et al., 2011; Gessa et al., 1985). The reinforcing effects of alcohol are in part dependent on this dopamine release, and inhibition of dopamine receptors reduces both alcohol self-administration (Ding et al., 2015; Engleman et al., 2020) and reinstatement of drug-seeking behaviors (Marinelli et al., 2003; McFarland et al., 2004). Alcohol-induced dopamine release also induces neuroplasticity which may further promote the development of addiction (Ma et al., 2018). Neuroplastic changes in reward- and memory-related circuits mediated by dopamine may further produce a hypofunctioning mPFC, resulting in diminished impulse control and increased vulnerability to drug relapse (Koob & Volkow, 2016; Langleben et al., 2008; Trantham-Davidson et al., 2014). Overall, the role of dopaminergic neurotransmission is dependent on the brain region studied, how long alcohol has been consumed, and the kind of alcohol seeking behavior that is monitored (see Table 2 for a summary).

TABLE 2. Compounds targeting the dopaminergic neurotransmission in alcohol seeking behavior

Dopamine elicited responses are mediated through five GPCRs. Based on sequence homology, and biological responses, these are divided into a D1-like and the D2-like family. The D1-like receptor family consists of dopamine D1 and dopamine D5 receptors, which share over 80% sequence homology within the transmembrane domains, but only 50% overall homology at the amino acid level (Sidhu, 1998). The D2-like family consists of dopamine D2, D3 and D4 receptors; the transmembrane regions of D3 and D4 receptors share 75% and 53% sequence homology with the D2 receptor, respectively (Sokoloff et al., 1992). The dopamine D1 and D2 receptors are the most abundant subtypes, and are highly expressed in reward-related brain areas. Although most studies have focused on the role of dopamine D1 and D2 receptors in mediating addictive properties of alcohol, D3, D4, and D5 subtypes may also have specific roles in regulating alcohol seeking. However, the lack of selective pharmacological tools has made it difficult to differentiate between receptor subtypes within the D1 and the D2 families.

Extended alcohol intake with periods of withdrawal significantly affects extracellular levels of dopamine (Ericson et al., 2020; Thielen et al., 2004), and alters dopamine D1 and D2 receptor binding sites in brain-subregions such as NAc, dorsal striatum and amygdala (Kim et al., 1997; Sari et al., 2006). Reduced dopamine D2 receptor expression in PFC further parallels with alcohol-induced CPP (Rotter et al., 2012). These changes in dopaminergic neurotransmission may in turn contribute to an imbalance between excitation and inhibition via striatal medium spiny projection neurons (MSNs), which may further promote alcohol seeking (Cheng et al., 2017).

Compounds that increase or stabilize dopamine levels have been shown to prevent reinstatement and suppress relapse-like drinking in the alcohol deprivation effect model [ADE; (Fredriksson et al., 2019; Libarino-Santos et al., 2020; Soderpalm et al., 2020; Spanagel & Holter, 1999; Sutera et al., 2016)]. Inhibition of the dopamine degrading enzyme cathechol-O-methyltransferase (COMT) also reduces cue-induced reinstatement in male rats (McCane et al., 2018). Importantly, both activation and inhibition of dopamine receptor signaling may affect alcohol seeking in a similar manner, either by acting as a replacement treatment analogous to opioid maintenance therapy, or by blocking rewarding effects of alcohol and affecting goal-directed behavior. Alcohol induced changes in dopaminergic neurotransmission appear to be highly time-dependent in relation to alcohol use, complicating interpretation of findings (Hirth et al., 2016).

Dysfunction of D2-like receptor signaling in particular has been associated with alcohol seeking (Blum et al., 1995). Systemic administration or bilateral NAc injection of a dopamine D2 receptor antagonist robustly decreases alcohol seeking responses during extinction trials (Czachowski et al., 2002; Samson & Chappell, 2004), which may be linked to the role of accumbal dopamine D2 receptor for processing information related to stimulus control and goal-directed behavior. After longer exposure periods, the dopamine D2-like receptor dependency appears to shift towards the dorsal striatum (Corbit et al., 2014). Local administration of the combined D1/D2 dopamine receptor antagonist flupenthixol in the dorsolateral striatum (DLS) dose-dependently decreases seeking responses, and the sensitivity to the antagonist predicted vulnerability to subsequent development of compulsive alcohol seeking on a second order schedule (Giuliano et al., 2019). Dorsal striatal dopamine levels are linearly correlated with the persistence of compulsive alcohol seeking, which is seen in a subset of animals (Giuliano et al., 2019). In addition to its effects in the DLS, flupenthixol also suppresses alcohol seeking when administered locally in the amygdala, but not in the NAc core (Gremel & Cunningham, 2009). This is in line with a postulated role of amygdala in the progressive shift from ventral to dorsal striatum as drug seeking behavior becomes an incentive habit (Belin et al., 2009).

The dopamine D3 receptor may be of particular interest when assessing alcohol seeking and cue-induced reinstatement. Cue-induced alcohol-seeking is suppressed by D3 antagonists (Vengeliene et al., 2006). Furthermore, systemic administration of a D3 antagonist, or a partial agonist, suppresses relapse-like behavior following alcohol deprivation in long-term alcohol drinking Wistar rats. Alcohol-induced up-regulation of dopamine D3 receptors in this model is particularly prominent in the dorsal striatum, suggesting that it may contribute to alcohol-seeking and relapse (Vengeliene et al., 2006).

In cocaine self-administration, a seminal paper proposed a model for individual addiction vulnerability, based on three criteria thought to parallel key clinical phenomena of addiction: inability to abstain during a signaled period of reward unavailability, increased motivation assessed using a progressive ratio schedule, and persistent alcohol intake despite aversive foot shocks. In this model, the minority of rats that met all three criteria also showed increased cocaine-seeking, both when induced by priming and by drug-associated cues (Deroche-Gamonet et al., 2004). A similar model was recently applied to alcohol (Domi et al., 2019; Jadhav et al., 2017, 2018). Rats that reached all three criteria showed increased dopamine D1 and decreased dopamine D2 receptor mRNA expression in the DLS three months later. This supports a role for dopaminergic dysregulation in compulsive alcohol seeking and suggests that dopaminergic neuroadaptations may persist even after protracted abstinence. Interestingly, while cue-induced reinstatement is insensitive to D4 antagonists, blockade of D4 receptors suppresses stress-induced reinstatement (Kim et al., 2020).

Taken together, dopamine D1-like receptor signaling in the NAc appears to be important for regulating alcohol intake, while D2-like receptors in the dorsal striatum seem to be most important for alcohol seeking and reinstatement (Table 1). At the same time, dopamine D1 receptors in the dorsal mPFC play a key role in cocaine-induced reinstatement of cocaine seeking (Devoto et al., 2016), and stress-induced activation of VTA dopamine projection to the PFC has been proposed to induce reinstatement of cocaine-seeking behavior via a glutamatergic projection to the NAc core (McFarland et al., 2004). Since ablation of mPFC neurons projecting to the NAc has been shown to block cue-induced reinstatement of alcohol seeking (Keistler et al., 2017), it is possible that similar pathways are also recruited during cue-guided alcohol seeking.

2.4 Section IV: glutamatergic signaling in alcohol seeking behaviors

Alcohol seeking has been especially linked to glutamatergic changes in amygdalo-cortico-striatal circuits (Burnett et al., 2016), and involves both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. It has been proposed that, as alcohol use becomes more compulsive, a transition in neural control occurs from metabotropic to ionotropic receptors (Hwa et al., 2017). However, the role of glutamatergic receptors and their subunit composition in alcohol seeking differs depending on localization (Burnett et al., 2016). Potentiation of glutamatergic activity after prolonged heavy drinking results in long-lasting plasticity mainly in corticostriatal synapses that sustain alcohol seeking (Ma et al., 2017; Meinhardt et al., 2013). Impairments in neuroplasticity produced by chronic alcohol exposure in brain areas involved in cognitive processes may dampen behavioral flexibility and promote habitual seeking behaviors (Kroener et al., 2012; Renteria et al., 2018).

The ventral and dorsal striatum are differentially implicated in alcohol addiction. They receive glutamatergic inputs from both amygdalar and cortical projections that innervate MSNs, and interact with dopaminergic inputs to these cells (Lobo & Nestler, 2011). It has been reported that extracellular levels of glutamate are increased in the basolateral amygdala (BLA) and NAc core during cue-induced reinstatement of alcohol seeking (Gass et al., 2011). Furthermore, the mPFC-NAc pathway is necessary for cue-induced reinstatement of alcohol seeking. Selective ablation of glutamatergic mPFC neurons projecting to NAc but not BLA prevented cue-induced reinstatement, without influencing extinction. Reinstatement was also prevented by ablation of amygdalar projections to the NAc (Keistler et al., 2017).

It has been proposed that, similar to other addictive drugs, alcohol seeking becomes a maladaptive habit that relies on a shift from of control from the ventral and dorsomedial striatum (DMS) to the DLS (Belin & Everitt, 2008; Giuliano et al., 2019; Willuhn et al., 2012). While DMS receives glutamatergic inputs from associative cortices, DLS is innervated by sensorimotor cortex and the thalamus (Bolam et al., 2000; Reig & Silberberg, 2014). Potentiation of glutamatergic inputs to the DLS, together with dopamine-related changes, is one of the main mechanisms behind the emergence of habitual alcohol seeking (Barker et al., 2015; Corbit et al., 2014).

Thus, a plethora of glutamatergic neuroadaptations contribute to the emergence of alcohol addiction-like behaviors, including alcohol seeking. In the following, we describe some molecular mechanisms that involve glutamatergic neurotransmission, and have been shown to be important for alcohol seeking in rodent models (Table 3). We focus on the potential for targeting metabotropic and ionotropic glutamate receptors in order to rescue maladaptive changes that occur with chronic use.

TABLE 3 Compounds targeting glutamatergic neurotransmission in alcohol seeking behavior

2.4.1 Ionotropic glutamate receptors

Glutamate produces its direct effects on neuronal excitability and firing through iGluRs, N-methyl-d-aspartate receptors (NMDAR), α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPAR) and kainate receptors that act as ligand-gated ion channels (Traynelis et al., 2010). These receptors mediate fast excitatory neurotransmission and are critically important for synaptic plasticity in brain regions that mediate alcohol seeking and taking (Bell et al., 2016). Chronic alcohol exposure and withdrawal result in increased activity and expression of both NMDARs and AMPARs, resulting in neuroadaptations that reduce behavioral flexibility (Christian et al., 2012; Krystal et al., 2003; Wang et al., 2012). NMDARs have been extensively studied for their involvement in both cue-induced reinstatement of alcohol seeking and compulsive alcohol seeking, i.e. seeking (rather than taking) behavior that continues despite negative consequences.

As noted in Section I, compulsive alcohol seeking has recently been shown to emerge alongside compulsive drinking (Giuliano et al., 2018, 2019). It has previously been shown that alcohol-induced neuroadaptations of accumbal NMDARs promote alcohol taking that is punished with footshock or quinine adulteration (Seif et al., 2013, 2015). Also, punishment-resistant alcohol-seeking in mice increases following a history of alcohol dependence, and is accompanied by increased expression of NMDAR subunits GluN1 and GluN2A in the medial orbitofrontal cortex (OFC) (Radke et al., 2017). A recent study examined molecular pathways involved in habitual alcohol seeking. A random interval schedule of reinforcement was used to promote the emergence of habitual responding (Dickinson et al., 1983), and satiety-induced outcome devaluation was used to test habitual behavior. The GluN2B NMDAR subunit in the OFC, a brain region that projects to the dorsal striatum, was found to mediate habitual alcohol seeking through a mechanism involving mTORC1 signaling (Morisot et al., 2019). These finding are in agreement with previous reports indicating that activation of mTORC1 signaling is a key mechanism behind heavy alcohol use and relapse (Ben Hamida et al., 2019; Laguesse et al., 2017).

Up-regulation of NMDARs GluN2B subunit expression in corticostriatal circuits is critical for promoting reinstatement of alcohol-seeking (Wang et al., 2010). NMDAR antagonists have shown efficacy in blocking both priming-induced reinstatement, and relapse-like behavior after protracted abstinence in the ADE model (Spanagel, 2009; Vengeliene et al., 2005; Wang et al., 2010). By contrast, NMDARs do not seem to play a role in cue-induced reinstatement of alcohol seeking (Bäckström & Hyytiä, 2004; Eisenhardt et al., 2015). Suppressed cue-induced reinstatement of alcohol seeking was seen with the clinically approved medication acamprosate, and was attributed to NMDA-mediated effects (Bachteler et al., 2005), but it has since become clear that effects of acamprosate are complex, and not likely to be mediated through direct actions at NMDARs (Spanagel et al., 2014).

Similar to NMDARs, AMPAR function is also enhanced after chronic alcohol exposure, and NMDAR-dependent increase in AMPAR activity has been shown to trigger drug seeking (Christian et al., 2012; Gipson et al., 2013; Shen et al., 2011). Accordingly, the AMPA positive allosteric modulator (PAM) aniracetam potentiates cue-induced reinstatement of alcohol seeking in alcohol-preferring P rats (Cannady et al., 2013). Chronic alcohol also disrupts Ca²⁺ /calmodulin-dependent protein kinase II (CaMKII)-AMPA signaling in the PFC and amygdala, increasing the risk of relapse to alcohol seeking through CaMKII-dependent activation of AMPARs (Cannady, Fisher, et al., 2017; Salling et al., 2017). In agreement with these findings, the selective AMPAR receptor antagonist GYKI 52,466 blocks cue-induced reinstatement and ADE in rats (Sanchis-Segura et al., 2006). Moreover, AMPAR/kainate mixed antagonists (CNQX and NBQX) are able to reduce cue-induced reinstatement of alcohol seeking (Bäckström & Hyytiä, 2004; Czachowski et al., 2012; Sciascia et al., 2015). Selective kainate receptor antagonists such as LY466195 have mostly been studied for their ability to reduce alcohol intake (Van Nest et al., 2017), and their potential to influence alcohol seeking has to our knowledge not yet been studied.

2.4.2 Metabotropic glutamate receptors

A more fruitful category of potential therapeutic targets may be offered by mGluRs, which are widely expressed in both neurons and glial cells of the central nervous system in (Niswender & Conn, 2010)). mGluRs are GPCRs that mediate slow neurotransmission through modulation of second messenger levels and ion-channel activity (Conn & Pin, 1997). They are located in the proximity of the synaptic cleft in both pre-and postsynaptic neurons (Cartmell & Schoepp, 2000; Shigemoto et al., 1997). Briefly, eight metabotropic glutamate receptors have been identified, and categorized into three groups, based on sequence similarity, signal transduction pathways, and pharmacological properties. Group I consists of mGluR1 and 5; group II of mGluR2 and 3; while mGluR4, 6, 7, and 8 belong to Group III. Group I mGluRs are Gq-coupled, are mainly present in postsynaptic neurons, and when activated, trigger a cascade that ultimately results in increased intracellular Ca²⁺ levels. Activation of Group I mGluRs also triggers changes in transcriptional regulation and gene expression. Group II and III, which are Gi/o coupled, are mostly localized at presynaptic terminals and in astrocytes, where they modulate the release of glutamate and other neurotransmitters (Wang & Zhuo, 2012; Yin & Niswender, 2014). The involvement of mGluRs in alcohol seeking has been extensively studied, with most findings focusing on mGluR5 and 2, which have been considered potential medication targets.

Alcohol acutely dampens mGluR1/5 function, but protracted alcohol use potentiates both the expression and activity of these receptors (Zorumski et al., 2014). Using a drug discrimination procedure, it was shown that activation of accumbal mGluR5s is essential for interoceptive effects of alcohol (Besheer et al., 2009). Accordingly, competitive mGluR5 antagonists as well as mGluR5 negative allosteric modulators (NAMs) attenuate cue-induced reinstatement of alcohol seeking, both when administered systemically, and when microinjected into the NAc or the BLA (Bäckström et al., 2004; Caprioli et al., 2018; Sinclair et al., 2012). Using the selective mGluR5 NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), it was shown that suppressed reinstatement of alcohol seeking following down-regulated mGluR5 transmission involves the extracellular signal-regulated kinases 1/2 (ERK_1/2) signaling pathway (Schroeder et al., 2008). ERK_1/2 signaling is downstream of mGluR5, and is activated in amygdala inputs to the ventral striatum by contingent presentation of alcohol associated cues. Within this circuitry, ERK_1/2 phosphorylation was associated with increased cue-induced reinstatement of alcohol seeking, and this effect was counteracted by MPEP (Schroeder et al., 2008).

A potential interpretation of these findings is that mGluR5s are involved in associative learning that links alcohol-associated cues with alcohol effects, becomes progressively strengthened over the course of developing alcohol addiction, persists into protracted abstinence, and contributes to alcohol seeking under non-reinforced conditions. In addition to blocking the recall of these alcohol-memories as reviewed above, facilitating their extinction may also offer treatment opportunities. Exposure-based extinction of alcohol cue-reactivity is a clinical treatment of alcohol addiction, but its efficacy is limited (Mellentin et al., 2017), and could potentially be strengthened using medications. In that context, the mGluR5 PAM CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide) has been shown to facilitate extinction of cue-conditioned alcohol seeking (Gass et al., 2014). This effect was mediated through mGluR5 modulation of small-conductance calcium activated potassium (K_Ca²) channels, and was obtained both with systemic and intra-infralimbic/PFC activation of mGluR5s (Cannady, McGonigal, et al., 2017).

In contrast with mGluR5, mGluR1 effects on alcohol seeking have not been extensively studied, with most of the literature focusing on mGluR1 PAMs and NAMs effects on alcohol consumption (Besheer et al., 2008; Cozzoli et al., 2014; Lum et al., 2014).

mGluR2-mediated control of glutamatergic neurotransmission through presynaptic modulation of glutamate release has received considerable interest as a pharmacological target in several psychiatric disorders (Crupi et al., 2019). Prolonged alcohol exposure has been shown to disrupt mGluR2 function by down-regulating expression of Grm2, the gene encoding this receptor. Deficits in corticostriatal and cortico-amygdala mGluR2-mediated feedback inhibition of glutamate release have been shown to promote reinstatement of alcohol seeking (Lovinger & McCool, 1995; Meinhardt et al., 2013). High levels of glutamate in the BLA and NAc have been detected during cue-induced reinstatement of alcohol seeking, together with an alcohol-induced mGluR2 down-regulation in mPFC (Gass et al., 2011; Meinhardt et al., 2013). Genetically selected alcohol-preferring P rats lack mGluR2s, and show escalation of alcohol consumption and resistance to alcohol drinking devaluation (Timme et al., 2020; Zhou et al., 2013).

In agreement with the findings reviewed above, the mixed mGluR2/3 agonist LY379268 has been shown to reduce reinstatement of alcohol seeking, both when reinstatement was induced by cues and by footshock stress (Bäckström & Hyytiä, 2005; Kufahl et al., 2011; Sidhpura et al., 2010; Zhao et al., 2006). However, LY379268 and other orthosteric group II mGluR agonists are unable to discriminate between the respective contribution of mGluR2 and mGluR3. In a recent paper, we therefore tested the effect of the selective mGluR2 PAM AZD8529 on alcohol taking and seeking behaviors (Augier et al., 2016). Cue- but not stress-induced reinstatement of alcohol seeking was potently blocked by AZD8529 in Wistar rats. This effect was absent in alcohol preferring P rats lacking functional mGluR2s. Of note, AZD8529 also reduced 20% alcohol self-administration, but this effect was marginal compared to the potent and specific blockade of reinstatement induced by alcohol-associated stimuli. Together with previous findings obtained with mixed mGluR2/3 agonists, this indicates that mGluR2 are specifically involved in cue-induced alcohol seeking. A potential role of mGluR3 remains to be evaluated. Thus, mGluR2 agonists or PAMs may have therapeutic potential, and prevent relapse to alcohol use.

Results are generally less promising with manipulations targeting group III mGluR_S, mGluR7, and mGluR8. Upon intracerebroventricular administration, a mixed mGlu4/mGlu7 agonist was reported to reduce reacquisition of alcohol self-administration after a period of abstinence (Lebourgeois et al., 2018); while the GluR₈ receptor agonist (S)-3,4-DCPG attenuated cue-induced reinstatement of alcohol seeking, but only at doses with motor-suppressant effects (Bäckström & Hyytiä, 2005).

2.4.3 Targeting glutamatergic transmission for therapeutic purposes

The findings reviewed here clearly support the notion that glutamatergic transmission plays a critical role in alcohol seeking and relapse. The complexity of ionotropic glutamate receptors and their subunits may seem to offer a rich landscape for development of therapeutics for alcohol addiction. Decades of experience from attempts to develop medications targeting NMDA receptors for ischemic excitotoxicity, epilepsy and Parkinson's disease, to name a few indications that have been pursued, suggest otherwise (Low & Roland, 2004). One possible conclusion is that ionotropic effects of glutamate may simply be too fundamental for brain function to allow modulation for therapeutic purposes while maintaining adequate safety and tolerability. In contrast, preclinical findings indicate that presynaptic agonists of mGluR2/3 receptors and antagonists of postsynaptic mGluR5 may be effective pharmacotherapeutic treatments in alcohol seeking behavior and relapse.

2.5 Section V: GABAergic neurotransmission and alcohol seeking

GABAergic neurotransmission plays an important role in a wide range of alcohol effects, from initial intoxication to alcohol seeking and relapse (Enoch, 2008; Heilig et al., 2011). GABA is the principal inhibitory neurotransmitter of the brain, and inhibits the activity of mesolimbic dopamine neurons (Bowery & Smart, 2006). It acts on two classes of receptors: ionotropic GABA_A receptors, which are ligand-gated chloride channels that also include the GABA_A ρ subclass (previously known as GABA_C receptor), and the metabotropic GABA_B receptor, that is G protein coupled, and regulates the activity of potassium and calcium channels (Bormann, 2000; Kuriyama et al., 1993). Alcohol affects GABAergic transmission both via pre- and post-synaptic mechanisms, and has both acute and long-term effects on GABAergic transmission (Roberto & Varodayan, 2017). Increased propensity for relapse, driven by negative reinforcement, has been suggested to result from a dysregulation of peptidergic neuromodulatory systems that converge on GABAergic circuitry within the extended amygdala (George & Hope, 2017; Koob, 2008). The extended amygdala comprises the CeA, the NAc shell, and the lateral portion of the BNST (Heimer & Alheid, 1991).

CeA plays a role in both footshock- and yohimbine-induced alcohol seeking (Walker et al., 2017, 2020). In vivo studies and ex vivo slice electrophysiology studies show that chronic alcohol exposure, which promotes a range of alcohol addiction-like behaviors, increases GABA release in the CeA through both pre- and post-synaptic mechanisms (Roberto et al., 2003, 2004). In agreement with those observations, we recently found that impaired clearance of GABA within the CeA, caused by a low expression of the GABA transporter GAT-3, is associated with pathological high choice for alcohol over a natural reward, and continued self-administration of alcohol despite punishment and adulteration with quinine. Decreased expression of GAT-3 in the amygdala was accompanied by decreased expression of several GABA_A receptor subunit transcripts, presumably reflecting a compensatory down-regulation in response to the sustained increase in GABAergic tone (Augier et al., 2018). Of note, SP-mediated activation of NK1 receptors in CeA promotes GABAergic transmission in this structure, and this effect is potentiated in rats with a history of alcohol dependence (Khom et al., 2020). These finding suggest that pharmacological interventions with an ability to restore GABA homeostasis within the CeA may have therapeutic potential in alcohol addiction (Heilig et al., 2019; Spanagel, 2018).

GABAergic transmission in the BNST also contributes to stress-induced reinstatement of drug seeking and negative affect associated with addiction (Lebow & Chen, 2016; Lowery-Gionta & Kash, 2014). BNST is a major target of CeA projection neurons, about 80% of which are GABAergic (Gungor & Pare, 2016; Le Gal LaSalle et al., 1978; Weller & Smith, 1982). Pina and colleagues have shown that electrolytic ablation of the BNST both before and after conditioning reduced the magnitude of cue-induced alcohol seeking in mice (Pina et al., 2015).

BNST sends extensive projections to the VTA (Dong & Swanson, 2004, 2006; Kudo et al., 2012). These predominantly innervate non-dopaminergic VTA neurons, and are made up by distinct populations whose activity promotes divergent motivational states. Specifically, a majority of VTA-projecting BNST neurons are GABAergic, and their activation produces anxiolytic-like and rewarding effects. In contrast, activity of a smaller population of glutamatergic BNST inputs to the VTA is anxiogenic and aversive (Jennings et al., 2013; Kudo et al., 2012). A population of CRH-expressing GABA neurons intrinsic to the BNST controls the balance between these BNST outputs by inhibiting the activity of the anxiolytic output neurons, and is itself under influence of serotonergic inputs from the dorsal raphe nucleus (Marcinkiewcz et al., 2016). A recent report showed that chronic intermittent alcohol exposure results in dysregulation of this local GABAergic BNST microcircuit, an effect which may promote negatively reinforced alcohol seeking during withdrawal and protracted abstinence (Pati et al., 2020). In this study, withdrawal from alcohol exposure resulted in increased excitability of the CRH-expressing GABAergic interneurons, accompanied by decreased activity of putatively anxiolytic-acting non-CRH BNST neurons that project to both lateral hypothalamus (LH) and VTA.

In the striatum, chronic alcohol exposure reduces GABAergic transmission, and this effect may separately contribute to increased alcohol seeking and intake (Lovinger & Kash, 2015). After prolonged alcohol exposure, a decrease in GABAergic transmission has been shown in the DLS, DMS and NAc of both mice and monkeys (Cuzon Carlson et al., 2011; Liang et al., 2014; Wilcox et al., 2014). This is accompanied by decreases in both the amplitude and the frequency of miniature inhibitory postsynaptic currents (mIPSCs), suggesting that the underlying mechanism may be either a decrease of GABA release from existing synapses, or a decrease in the number of GABAergic synapses onto dorso-striatal MSNs (Lovinger & Kash, 2015). Alcohol-mediated disinhibition of the DLS may thus result from weakened GABAergic inhibition of this structure, providing a potential mechanistic basis for habitual alcohol seeking (Corbit et al., 2012).

Alcohol-induced changes in GABAergic transmission are in part attributable to changes in the function and sensitivity of GABA_A and GABA_B receptors (Davies, 2003; Gass & Olive, 2012; Grobin et al., 1998; Kumar et al., 2009). This makes it important to understand adaptations of GABAergic receptors in the different stages of alcohol addiction.

2.5.1 GABA_A receptors in relapse to alcohol seeking

GABA_A and GABA_B receptors both contribute to acute as well as chronic effects of alcohol, including sedation, tolerance, withdrawal, and motivational effects (Chester & Cunningham, 2002; Colombo et al., 2004; June et al., 2003; Lobo & Harris, 2008; Olsen & Liang, 2017).

GABA_A receptors are ligand-gated chloride channels composed of five subunits arranged around a central pore. They are assembled from α(1–6) and β(1–3) subunits which are obligate. Assembled receptors may also contain γ(1–3), δ, ε, π, or θ subunits. In addition, ρ(1–3) subunits exist, but do not co-assemble with classical GABA_A receptors; instead, they homooligomerize to form GABA_A ρ receptors. The composition from multiple subunits allows for an extensive heterogeneity of receptor formation, varies between brain regions, and determines the pharmacological profile of the receptor, including its responses to alcohol (Olsen & Sieghart, 2009). Acute and chronic alcohol exposure induces transient changes in GABA_A receptor subunit levels, composition, and regional and subcellular localization (Kumar et al., 2009).

Alcohol potentiates GABA_A receptor-mediated synaptic transmission via an increase in GABA release from presynaptic terminals in a multitude of brain regions (Spanagel, 2009), also including the cerebellar cortex (Valenzuela & Jotty, 2015). Prolonged exposure to chronic alcohol facilitates GABAergic transmission, and the rebound renders the brain hyperexcitable during alcohol withdrawal (Lovinger, 2008; Roberto et al., 2012; Steffensen et al., 2009). Chronic alcohol exposure can induce GABA_A receptor down-regulation, producing tolerance and withdrawal, and disrupting a wide range of behaviors (Forstera et al., 2016). GABA_A receptors in the CeA regulate alcohol-maintained responding in alcohol preferring rats (Foster et al., 2004), but there is little evidence for a role of GABA_A receptors in alcohol seeking behaviors.

A recent study showed that chronic administration of the GABA_A α1-preferring antagonist, 3-isopropoxy-β-carboline hydrochloride (5–20 mg/kg), selectively reduced alcohol self-administration but not alcohol seeking assessed under a chained schedule of reinforcement in baboons (Holtyn et al., 2017). By contrast, the same compound failed to attenuate drinking in rhesus macaques (Sawyer et al., 2014), while in alcohol preferring P rats, the analogue 3- β-carboline hydrochloride reduced alcohol maintained responding when injected in the ventral pallidum, a key node in neural circuits that control relapse to alcohol seeking (Harvey et al., 2002; Prasad & McNally, 2020). In mice lacking the GABA_A α1 receptor subunit, reduction of alcohol drinking was also accompanied by a reduction in saccharin and sucrose consumption (Blednov et al., 2003; June et al., 2007), suggesting that these effects are not specific for alcohol.

2.5.2 Role of GABA_B receptors in alcohol seeking

In contrast with the limited and contradictory evidence on the role of GABA_A receptors in alcohol seeking, extensive data have accumulated over recent years for a role of GABA_B receptors in this behavior (see Table 4).

TABLE 4. Compounds targeting GABAergic neurotransmission in alcohol seeking behavior

GABA_B receptors are G-protein coupled, and require dimerization of two subunits (GABA_B1 and GABA_B2) to be functional (Robbins et al., 2001). Both pre-and postsynaptic activation of GABA_B receptors inhibit neurotransmitter release through neuronal hyperpolarization, which results from increased potassium and decreased calcium permeability (Bettler & Tiao, 2006). Preclinical and clinical evidence suggests that activation of GABA_B receptors holds promise as a mechanism for treatment of alcohol addiction (Addolorato et al., 2009; Augier, 2021; Farokhnia et al., 2018; Heilig & Egli, 2006).

Results with GABA_B activation in animal models of alcohol addiction were recently reviewed (Holtyn & Weerts, 2020). In brief, much of the data have been obtained with baclofen, a selective GABA_B receptor agonist that for more than four decades has been clinically approved as a treatment for muscle spasticity. Baclofen has been shown to reduce the reinforcing properties of alcohol and the severity of alcohol withdrawal. It also prevents reinstatement of alcohol seeking [recently reviewed in (Colombo & Gessa, 2018)]. In the alcohol deprivation model of relapse-like drinking, baclofen (1, 1.7 and 3 mg/kg, i.p.) abolished alcohol intake of Sardinian alcohol preferring (sP) rats after 7 days of abstinence (Colombo et al., 2003). These findings were then replicated with baclofen (3 mg/kg i.p.) in sP rats using different alcohol concentrations (10%, 20%, and 30% (v/v) (Colombo et al., 2006). However, following long-term voluntary alcohol drinking with repeated deprivation cycles, chronic baclofen (1 and 3 mg/kg i.p.) reduced relapse-like drinking only at the highest dose. This effect was not specific, since this dose also induced sedative effects and altered food intake inducing a significant body loss (Vengeliene et al., 2018).

Thus, in rodent models, the separation between baclofen doses with specific effects on alcohol-related behaviors and doses that produce non-specific sedative effects or otherwise impair performance (i.e. the therapeutic index) is limited, and variable. The variable findings on baclofen doses required in different relapse studies may be explained by the use of different strains and lines of rats, because of innate differences in their GABAergic transmission. For instance, genetically selected msP rats show increased GABA levels in the CeA at baseline compared to Sprague Dawley and Wistar rats, and this may render them more sensitive to inhibition of GABA release by GABA_B activation (Herman et al., 2013). Two additional factors that may influence the dose–response relationship of baclofen are differences in alcohol drinking history, and the acute versus chronic nature of baclofen administration.

Cue-induced relapse to alcohol seeking is reduced by baclofen across species, including rats (both msP and Wistar), as well as baboons, a species in which baclofen also facilitated extinction of responding for alcohol (Duke et al., 2014; Maccioni et al., 2008; Vengeliene et al., 2018). Baclofen has also been reported to reduce yohimbine-induced relapse to alcohol seeking in rats, and to attenuate alcohol seeking in an odor recognition task in mice, in the latter model presumably by blunting the corticosterone response to the footshock (Rabat et al., 2019; Williams et al., 2016). The latter effect is in line with a well-documented clinical relationship between HPA axis activity, craving, and relapse to alcohol use (Blaine & Sinha, 2017; Sinha et al., 2011; Stephens & Wand, 2012). It is also in agreement with a recent clinical study that reported reduced cortisol levels in alcohol-dependent patients that received treatment with baclofen (Geisel et al., 2019).

Clinically, baclofen has shown promising, although conflicting, results for the treatment of alcohol addiction (Addolorato et al., 2007; Pierce et al., 2018; Agabio et al., 2018; see also Agabio & Leggio, 2018; Augier, 2021 for review on this topic), but safety and tolerability concerns have prevented its approval as a clinical alcohol addiction treatment (ANSM, 2017; Bowery, 2006; Garbutt, 2018). Some of these concerns are related to tolerance and dose-escalation, phenomena expected with chronic GPCR agonist treatment. PAMs of the GABA_B receptor have the potential to avoid these effects, by targeting a site that is topographically distinct from the orthosteric GABA binding sites, and instead potentiating the effect of GABA upon its binding to the receptor (Froestl, 2010; Perdona et al., 2011). GABA_B PAMs have received considerable interest as potential therapeutics for alcohol addiction in recent years (Augier, 2021; Holtyn & Weerts, 2020; Maccioni & Colombo, 2019). The majority of studies that have examined GABA_B PAMs support their improved selectively and wider therapeutic index in reducing alcohol seeking and taking.

For instance, we have recently found that the selective GABA_B PAM, ADX71441 (3, 10 mg/kg), potently suppressed both cue- and stress (footshock)-induced reinstatement of alcohol seeking in Wistar rats (Augier, Dulman, Damadzic, et al., 2017). Moreover, ADX71441 attenuated stress-induced neuronal activity, indexed by cFos activity, in an interconnected network of brain structures that included NAc shell, mPFC and the dorsal raphe nucleus. Surprisingly, although neuronal activity in the CeA was also reduced by ADX71441 (3 mg/kg), it did not correlate with relapse-like behavior. Based on these findings, ADX71441 may act on distinct but converging networks that mediate relapse, or, alternatively, on a common neuronal pathway that promotes alcohol seeking initiated by stress, drug cues or drug priming (Kalivas & Volkow, 2005).

In addition, the novel GABA_B PAM, CMPPE, (10, 30 mg/kg i.p), reduced relapse to alcohol drinking in a repeated alcohol deprivation model, and suppressed cue-induced reinstatement of alcohol seeking in Wistar rats (Vengeliene et al., 2018). It also showed efficacy in reducing cue-induced reinstatement of alcohol seeking when administered at lower doses (2.5, 5 mg/kg i.p) in alcohol preferring sP rats (Maccioni, Fara, et al., 2019). Another novel GABA_B PAM, COR659, suppressed cue-induced reinstatement of alcohol seeking when administered at the lowest dose tested (2.5 mg/kg), and reduced alcohol self-administration in a manner that was maintained when the compound was administered chronically (Maccioni, Colombo, et al., 2019).

The mechanism(s) through which GABA_B activation prevents relapse to alcohol seeking remain unclear. One candidate mechanism is through inhibition of mesolimbic dopaminergic neurons. Activation of GABA_B receptors in the VTA results in a decreased dopamine release both in the NAc and the mPFC. Moreover, a GABA_B PAM injected in the VTA enhanced GABA_B inhibition of dopaminergic neuronal firing (Chen et al., 2005). Presumably related to these effects, microinjections of baclofen into the VTA dose-dependently suppressed cue-induced alcohol seeking in rats (Leite-Morris et al., 2008). It was recently reported that dissociable mesolimbic dopamine pathways control responding triggered by discrete alcohol-associated cues and alcohol associated contexts, respectively. Inhibition of inputs from the VTA to the NAc core reduced alcohol seeking triggered by an alcohol associated cue, irrespective of context. In contrast, silencing terminals of VTA inputs to the NAc shell selectively reduced cue-induced alcohol seeking in an alcohol-associated context (Valyear et al., 2020). If activation of GABA_B receptors can attenuate activity of VTA neurons that belong to both these populations, it may therefore be able to broadly prevent relapse both when induced by discrete cues, and by contextual stimuli.

In summary, activation of GABA_B receptors appears to hold considerable promise as a treatment for prevention of alcohol seeking and relapse. GABA_B PAMs may be able to avoid the safety and tolerability issues that limit the use of the existing orthosteric GABA_B agonist baclofen. Developing GABA_B PAMs that are safe for human use, and evaluating their efficacy as alcohol addiction medications is currently one of the most promising avenues for bringing forward novel therapeutics for this indication.

3 CONCLUDING REMARKS

Since their introduction in the early 1980s, animal models that use reinstatement of drug seeking following extinction have been widely used to study mechanisms of relapse. The application of these models to alcohol seeking has generated a vast literature, most (but not all) of which we have reviewed here. This work has made major advances in the past decades, and has identified multiple biological systems that contribute to alcohol seeking.

The overarching question ahead of the field is whether these advances and the biological systems they have identified are able to predict activity to prevent craving and relapse in people with alcohol addiction. Because of multiple failures in clinical development, industry efforts to develop psychiatric medications have dramatically decreased, and the ability of animal models to predict clinical activity in psychiatric disorders in general has been broadly questioned (Hyman, 2014). The landscape in addictive disorders is no different (Venniro et al., 2020).

So, are studies of alcohol seeking in animals a worthwhile effort? Some insights into this critical question may be gained from the present review. Specifically, we do not believe that data support any blanket conclusions that animal models of alcohol-seeking and relapse are, or are not, able to predict clinical activity in a meaningful way. The landscape that has emerged is in fact much more complex. On one hand, human and animal findings obtained with naltrexone and other tools targeting MOP strongly support a predictive validity of both priming- and cue-induced reinstatement models. Although somewhat more complex, findings with baclofen and GABA_B activation also generally support a predictive validity of relapse models in animals. On the other hand, the lack of success with CRH1 antagonists in clinical development, together with other data, may seem to prompt just the opposite conclusion.

Our preliminary attempt to integrate these observations is that a more nuanced view of the animal findings is needed. Predictive validity is not an all or nothing phenomenon, and is not necessarily first and foremost a property of the model. Instead, it is likely to vary dramatically across biological systems studied. A key factor that determines this variation is probably the degree to which the respective system and its elements are conserved across species, something that has received far too little attention. One implication of this realization is that dedicating decades of preclinical studies to any particular biological system may not be the optimal strategy, at least not if neuroscience of alcohol addiction wants to contribute to better outcomes for patients. Instead, signals from animal models should as quickly as possible be put to test in human proof-of-principle, biomarker-based studies, as outlined and recently tested by the NIMH Fast-Fail initiative for KOP antagonism in depression (Pizzagalli et al., 2020). If human translation is not supported, continued preclinical work is associated with a high opportunity cost.

With this type of strategy in mind, we believe that a priority list of targets for clinical fast-fail evaluation is suggested by the literature reviewed here. At the top of this list, in our assessment, is development of safe and well-tolerated GABA_B PAMs, which should be evaluated for prevention of both stress- and cue-induced craving and relapse in patients. A priority that comes in a close second is evaluation of KOP antagonists, which based on the data reviewed here would be predicted to prevent stress-induced craving and relapse, potentially offering a useful combination with naltrexone. In third position is evaluation of mGluR2 PAMs. We are less optimistic about interventions targeting dopaminergic or ionotropic glutamate receptors, in part because of the fundamental role these have on brain function and behavior, which may make it difficult to develop medications that combine efficacy with acceptable safety and tolerability.

Summary

Treating addiction is difficult because many people relapse. Over half of patients with alcohol addiction start drinking again within three months, a rate that has not changed much. Stress, alcohol-related cues, or even a small amount of alcohol can trigger a chain of behaviors leading to relapse. A strong urge to drink, called "craving," often comes before relapse and can predict future drinking.

Animal Models for Studying Alcohol Seeking Behavior

Alcohol Seeking in Reinstatement Models to Study Craving and Relapse

Scientists often use animal models to study relapse and its brain mechanisms. In these "reinstatement" models, animals first learn to self-administer alcohol. However, getting rodents to drink alcohol can be challenging due to its taste. Researchers have developed methods to overcome this, such as pre-exposing animals to alcohol or extended training, but these can complicate studies. Newer methods allow animals to self-administer alcohol without these extra steps.

After animals learn to self-administer alcohol, they go through an "extinction" phase where the alcohol reward is removed, and their alcohol-seeking behavior decreases. Then, researchers can reintroduce triggers like cues or stress to see if alcohol-seeking behavior returns, which models craving in humans.

Mini-Dictionary of Terms

Operant Self-Administration: A method where an animal performs an action (like pressing a lever) to get a reward, such as food or a drug. This teaches the animal to associate the action with the reward.

Reinstatement: A standard animal model for studying drug relapse. After training, the animal's drug-seeking behavior is extinguished. Then, various triggers (stress, drug cues, or a small dose of the drug) are used to see if drug-seeking returns.

Stress-Induced Reinstatement: Animals are trained to self-administer a drug with associated cues. Their drug-seeking is then extinguished while the cues are still present. Stress is then used to trigger a return of drug-seeking.

Cue-Induced Reinstatement: Animals learn to self-administer a drug with associated cues. Their drug-seeking is then extinguished when the cues are absent. Reintroducing these cues later causes a return of drug-seeking.

Drug-Induced Reinstatement: Animals are trained to self-administer a drug with an associated cue. Drug-seeking is then extinguished while the cues are present. A small dose of the drug is then used to trigger a return of drug-seeking.

Alcohol injections can cause rats to resume alcohol-seeking, but cues or contexts linked to alcohol are more powerful triggers. For example, specific smells or visual cues can strongly prompt relapse-like behavior, especially in rats that prefer alcohol. Physical stressors like footshock or drugs that cause anxiety can also lead to a strong return of alcohol seeking. However, the stressors used in animal studies are often different from the psychosocial stressors that affect humans, which might limit how well these models translate to human addiction. Researchers are exploring new methods, such as social defeat stress, to create more relevant animal models.

Compulsive Alcohol Seeking

Compulsive alcohol use, where drinking continues despite negative consequences, is a key feature of alcohol addiction. Understanding how controlled alcohol use turns into compulsive use is a major goal for researchers. Many studies have looked at compulsive drinking, where animals continue to drink alcohol even if it's made bitter or causes a mild electric shock. More recently, studies have started to examine compulsive seeking of alcohol, which occurs before actual intake.

A recent study used a four-phase protocol to investigate compulsive alcohol seeking in rats. First, rats learned to associate a light with alcohol availability. Then, they learned to press a "taking lever" for alcohol. Next, they had to press a "seeking lever" to make the taking lever available. Finally, pressing the seeking lever was sometimes punished with a mild electric shock. This study found that about a third of the rats continued to seek alcohol despite the punishment, suggesting individual differences in vulnerability to compulsive seeking. Another approach, using multiple criteria from addiction diagnostic guidelines, also identified a subset of rats that showed persistent alcohol seeking even when it led to punishment. These findings highlight that some individuals are more prone to developing compulsive alcohol behaviors.

Neurobiological Mechanisms Mediating Alcohol Seeking

Section I: Opioid Systems and Alcohol Seeking

Opioid systems are an important area of study for alcohol addiction because opioid antagonists like naltrexone are approved treatments. Naltrexone reduces heavy drinking and prevents progression from a single drink to full relapse, similar to how it blocks alcohol-induced reinstatement in rats. It also lessens cue-induced craving in humans. Studies show higher levels of mu-opioid receptors (MOP) in the brains of abstinent alcohol-addicted individuals, which are linked to craving. Alcohol also releases natural opioids in areas of the brain involved in reward. These findings suggest that animal models of relapse have some relevance to human addiction.

Opioid systems are complex, involving different peptide families (endorphins, dynorphins, enkephalins, nociceptin) and their corresponding receptors (MOP, kappa-opioid (KOP), delta-opioid (DOP), and nociceptin (NOP)). These receptors influence both the pleasurable and unpleasant effects of alcohol. MOP activation generally promotes the rewarding effects of alcohol, while KOP activation is linked to its aversive or unpleasant aspects.

MOP Receptors and Alcohol Seeking

Studies in animals mirror human findings, showing that naltrexone, which primarily acts on MOP, reduces alcohol seeking triggered by a small dose of alcohol or by alcohol-related cues. Naltrexone consistently inhibits cue-induced alcohol seeking but has less effect on stress-induced seeking. A selective MOP antagonist, GSK1521498, also reduced alcohol drinking and cue-induced alcohol seeking in rats. In a model of compulsive alcohol seeking, GSK1521498 was more effective at suppressing seeking behavior in rats that were identified as compulsive drinkers, suggesting it might be particularly useful for individuals with severe addiction.

Overall, preclinical data strongly support that blocking MOP can help prevent alcohol craving and relapse, aligning with clinical results. However, the benefits seen clinically with current MOP blockers are modest. It is unclear if more selective MOP antagonists would provide greater benefits.

KOP Receptors and Alcohol Seeking

KOP receptors and their natural ligand, dynorphin (DYN), are important for stress responses and negative emotions in addiction. Long-term alcohol use can alter the KOP/DYN system, contributing to negative emotional states that encourage excessive drinking as a way to self-medicate. Increased KOP activity during prolonged abstinence may worsen negative emotions, driving alcohol seeking. Therefore, KOP blockers might be useful treatments for alcohol addiction.

In rats, KOP antagonists like nor-binaltorphimine (nor-BNI) suppress alcohol seeking caused by both physical (footshock) and pharmacological (yohimbine) stress. Activating KOP receptors can also trigger alcohol seeking, an effect blocked by nor-BNI. This stress-induced reinstatement involves the CRH1 receptor, indicating a pathway where DYN acts before CRH. KOP antagonists have also been shown to block cue-induced alcohol seeking.

While KOP blockade shows promise, developing suitable drugs for clinical use has been challenging due to complex pharmacology and safety concerns with older compounds. A newer generation of KOP antagonists, such as CERC-501, is being evaluated. CERC-501 has shown promise in preclinical tests, reversing anxiety during alcohol withdrawal and blocking stress-induced alcohol seeking without affecting cue-induced seeking or other behaviors. This suggests that combining KOP and MOP antagonists could be a good treatment strategy, with CERC-501 potentially addressing the negative emotional states that naltrexone does not.

DOP Receptors and Alcohol Seeking

Fewer studies have focused on the role of DOP receptors in alcohol seeking. However, some evidence suggests that DOP receptors might be involved in cue-induced alcohol seeking. Selective DOP antagonists have been shown to reduce alcohol seeking triggered by environmental cues and stress.

NOP Receptors in Alcohol Seeking

The nociceptin/orphanin FQ (N/OFQ)-NOP system influences pain, emotions, and addiction. Initially, it was thought that activating NOP receptors would reduce drug motivation. However, similar effects have also been seen with NOP antagonists, making it unclear which approach is best for addiction treatment.

Activating NOP receptors, whether with N/OFQ itself or drug mimics, has been shown to lessen alcohol withdrawal symptoms, reduce relapse after not drinking, and decrease stress-induced alcohol seeking. This is observed in both non-dependent rats and those with a history of dependence. Nociceptin can also suppress cue-induced alcohol seeking. Brain studies show that alcohol causes more significant changes in the N/OFQ-NOP system in alcohol-dependent rats. Interestingly, a potent NOP agonist (SR-8993) reduced alcohol withdrawal anxiety and attenuated both stress- and cue-induced alcohol seeking. Paradoxically, an NOP antagonist (LY2940094) also prevented alcohol taking and stress-induced alcohol seeking, and blocked alcohol-induced dopamine release.

One theory to explain these conflicting findings is that NOP receptors might quickly become less responsive after activation by agonists. This could mean that giving NOP agonists might effectively lead to an "antagonist-like" effect over time. More human research is needed to determine if targeting the NOP system is a viable treatment strategy for alcohol addiction and whether agonists or antagonists would be more effective. Early human studies with an NOP antagonist have been inconclusive.

Section II: Other Peptides Involved in Alcohol Seeking

Corticotropin-Releasing Hormone (CRH)

CRH is a peptide known for its role in stress responses, and it is found throughout the brain, including areas involved in alcohol seeking like the CeA and BNST. CRH acts through two receptor subtypes, CRH1 and CRH2. Stress-induced alcohol seeking is primarily linked to CRH1 receptors. CRH1 signaling acts as an "alarm system" activated by uncontrollable stress.

Blocking CRH signaling consistently prevents stress-induced alcohol seeking without affecting cue-induced seeking. This effect has been shown with both direct brain administration and systemic administration of CRH1 antagonists. In alcohol-dependent animals, CRH and CRH1 receptors are increased in the CeA, making these animals more sensitive to CRH1 blockers in preventing stress-induced reinstatement.

Despite promising preclinical data, human studies using CRH1 antagonists have not supported these findings, with trials failing to reduce stress-induced craving or improve other psychiatric conditions. This suggests that CRH1 antagonists are unlikely to be effective for treating alcohol addiction.

Substance P (SP) and Its Neurokinin 1 (NK1) Receptor

SP is a peptide that belongs to the tachykinin family, and it primarily binds to the NK1 receptor. NK1 receptors are found in brain regions involved in both pleasurable and unpleasant behaviors, modulate stress responses, and regulate alcohol-related behaviors. Developing NK1 antagonists for animal studies was challenging due to differences between human and rodent receptors, but the development of specific compounds like L822429 overcame this.

Studies with L822429 showed that blocking NK1 receptors systemically prevents stress-induced reinstatement of alcohol seeking, but not cue-induced seeking. In alcohol-preferring rats, higher NK1 expression in the CeA is linked to increased sensitivity to stress-induced alcohol seeking, and blocking NK1 in this area reduces this effect. Over-expressing NK1 receptors in the CeA makes rats more vulnerable to stress-induced alcohol seeking. Activation of NK1 receptors by SP increases GABA release in the CeA, and this effect is enhanced in alcohol-dependent rats. These findings suggest that NK1 receptors in the CeA promote sensitivity to stress-induced relapse.

Human studies with NK1 antagonists showed some initial promise in reducing stress-induced craving and brain responses to negative stimuli in patients with alcohol addiction. However, a larger study with unselected patients did not meet its primary goal, though some secondary analyses suggested a benefit. The inconsistent results in clinical trials for depression, where NK1 antagonists were originally explored, were later attributed to the need for nearly complete receptor blockade for therapeutic effects. Therefore, NK1 antagonism might still be a viable treatment if highly brain-penetrant drugs are given at adequate doses to anxious alcohol-addicted patients, but further evaluation is unlikely.

Section III: The Role of Dopaminergic Neurotransmission in Alcohol Seeking

Alcohol activates dopamine neurons in the brain, leading to increased dopamine release in reward-related areas like the NAc and mPFC. This dopamine release contributes to alcohol's rewarding effects, and blocking dopamine receptors can reduce alcohol self-administration and seeking. Alcohol-induced dopamine release also causes brain changes that may promote addiction, including a less active mPFC, leading to poorer impulse control and increased vulnerability to relapse. The role of dopamine depends on the specific brain region, duration of alcohol use, and type of alcohol-seeking behavior being studied.

Dopamine acts through five types of G protein-coupled receptors (D1-D5), divided into D1-like (D1 and D5) and D2-like (D2, D3, D4) families. D1 and D2 receptors are the most common and are highly expressed in reward-related brain areas. Extended alcohol use and withdrawal affect dopamine levels and D1/D2 receptor binding in various brain regions. Reduced D2 receptor expression in the PFC is linked to alcohol-related conditioned place preference. These changes can create an imbalance in striatal neurons, promoting alcohol seeking.

Drugs that increase or stabilize dopamine levels can prevent reinstatement and suppress relapse-like drinking. Blocking the dopamine-degrading enzyme COMT also reduces cue-induced reinstatement. Both activating and inhibiting dopamine signaling can affect alcohol seeking, possibly by replacing alcohol's effects or by blocking its rewarding properties. Changes in dopamine transmission related to alcohol use are highly dependent on timing, making interpretation complex.

Dysfunction of D2-like receptor signaling is particularly associated with alcohol seeking. Blocking D2 receptors, especially in the NAc, reduces alcohol seeking during extinction. After longer alcohol exposure, D2-like receptor dependency may shift to the dorsal striatum. Blocking dopamine D1/D2 receptors in the dorsolateral striatum (DLS) reduces seeking, and this sensitivity can predict compulsive alcohol seeking. High dopamine levels in the dorsal striatum are linked to persistent compulsive alcohol seeking. Blocking these receptors in the amygdala also suppresses alcohol seeking, supporting the idea that the amygdala plays a role in the shift from goal-directed to habitual drug seeking.

The dopamine D3 receptor is also relevant for alcohol seeking and cue-induced reinstatement. D3 antagonists or partial agonists can suppress cue-induced alcohol seeking and relapse-like behavior after alcohol deprivation. Increased D3 receptors in the dorsal striatum after long-term alcohol drinking may contribute to seeking and relapse. A model of addiction vulnerability showed that rats meeting all criteria for addiction had increased D1 and decreased D2 receptor expression in the DLS, suggesting long-lasting dopamine system changes. While D4 antagonists do not affect cue-induced reinstatement, they can suppress stress-induced reinstatement.

Overall, D1-like receptors in the NAc seem important for regulating alcohol intake, while D2-like receptors in the dorsal striatum are crucial for alcohol seeking and reinstatement. D1 receptors in the dorsal mPFC are also key for cocaine-induced reinstatement, and similar pathways might be involved in alcohol seeking.

Section IV: Glutamatergic Signaling in Alcohol Seeking Behaviors

Alcohol seeking is strongly linked to changes in glutamate signaling within brain circuits that connect the amygdala, cortex, and striatum. This involves both ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). As alcohol use becomes more compulsive, control may shift from mGluRs to iGluRs. The specific roles of these receptors vary by brain region and their subunit composition. Prolonged heavy drinking can lead to lasting changes in glutamate activity, particularly in corticostriatal connections, which support alcohol seeking. These changes in brain plasticity can reduce behavioral flexibility and promote habitual seeking.

The ventral and dorsal striatum are involved in different aspects of alcohol addiction. Both receive glutamate signals from the amygdala and cortex, which interact with dopamine inputs to striatal neurons. During cue-induced alcohol seeking, glutamate levels increase in the basolateral amygdala (BLA) and NAc core. The mPFC-NAc pathway is necessary for cue-induced alcohol seeking; blocking glutamate neurons from the mPFC to the NAc prevents it. Amygdala projections to the NAc also play a role.

Similar to other addictions, alcohol seeking may become a maladaptive habit, shifting control from the ventral and dorsomedial striatum (DMS) to the dorsolateral striatum (DLS). The DLS receives glutamate inputs from motor and sensory areas, and enhanced glutamate signals there, along with dopamine changes, are key drivers of habitual alcohol seeking.

Many glutamate-related brain changes contribute to alcohol addiction-like behaviors. Researchers are exploring ways to target mGluRs and iGluRs to reverse these harmful changes.

Ionotropic Glutamate Receptors

iGluRs, including NMDARs, AMPARs, and kainate receptors, directly affect how neurons fire and are crucial for brain plasticity in regions involved in alcohol seeking. Chronic alcohol exposure and withdrawal increase the activity and number of both NMDARs and AMPARs, leading to brain changes that reduce behavioral flexibility. NMDARs have been widely studied for their role in cue-induced and compulsive alcohol seeking (seeking despite negative consequences).

Compulsive alcohol seeking, like compulsive drinking, involves NMDARs. Alcohol-induced changes in NMDARs in the NAc promote alcohol taking even when it's punished. Punishment-resistant alcohol seeking in mice with alcohol dependence is linked to increased NMDAR subunits (GluN1 and GluN2A) in the medial orbitofrontal cortex (OFC). In habitual alcohol seeking, the GluN2B NMDAR subunit in the OFC, which connects to the dorsal striatum, is involved through a signaling pathway called mTORC1. This aligns with previous findings linking mTORC1 to heavy alcohol use and relapse.

Increased GluN2B NMDARs in corticostriatal circuits are crucial for promoting alcohol seeking reinstatement. NMDAR antagonists can block both priming-induced reinstatement and relapse-like behavior after long abstinence. However, NMDARs do not seem to play a role in cue-induced reinstatement. While acamprosate was thought to have NMDA-mediated effects on cue-induced reinstatement, its actions are now understood to be more complex.

Like NMDARs, AMPAR function is enhanced after chronic alcohol exposure, and this increase can trigger drug seeking. Activating AMPARs with drugs can increase cue-induced alcohol seeking. Chronic alcohol also disrupts a signaling pathway involving CaMKII and AMPARs in the PFC and amygdala, increasing relapse risk. Conversely, AMPAR antagonists can block cue-induced reinstatement and relapse after alcohol deprivation. Mixed AMPAR/kainate antagonists also reduce cue-induced alcohol seeking. The potential of selective kainate receptor antagonists for influencing alcohol seeking is not yet well-studied.

Metabotropic Glutamate Receptors

mGluRs are promising therapeutic targets, widely found in brain cells. They are G protein-coupled receptors that slowly regulate brain signals by affecting messenger levels and ion channels. There are eight types of mGluRs, divided into three groups. Group I (mGluR1 and 5) are mainly postsynaptic, increasing intracellular calcium and affecting gene expression. Groups II (mGluR2 and 3) and III (mGluR4, 6, 7, 8) are usually presynaptic, modulating the release of glutamate and other neurotransmitters. mGluR5 and mGluR2 have been the main focus for alcohol seeking research.

Alcohol initially reduces mGluR1/5 function, but prolonged use increases their expression and activity. Activating mGluR5s in the NAc is important for the internal effects of alcohol. Blocking mGluR5s, either with antagonists or negative allosteric modulators (NAMs), reduces cue-induced alcohol seeking when given systemically or injected into the NAc or BLA. The mGluR5 NAM MPEP reduces alcohol seeking reinstatement by affecting the ERK1/2 signaling pathway, which is activated in amygdala-to-striatum connections by alcohol cues.

These findings suggest that mGluR5s are involved in the associative learning that links alcohol cues to its effects, which strengthens with addiction and contributes to seeking. Besides blocking the recall of these memories, facilitating their extinction could also be a treatment strategy. The mGluR5 positive allosteric modulator (PAM) CDPPB has been shown to improve the extinction of cue-conditioned alcohol seeking.

mGluR1's effects on alcohol seeking have not been as extensively studied as mGluR5.

mGluR2, which controls glutamate release, is a promising target for psychiatric disorders. Long-term alcohol exposure disrupts mGluR2 function by reducing its gene expression. Deficiencies in mGluR2-mediated feedback inhibition of glutamate release in corticostriatal and cortico-amygdala pathways promote alcohol seeking reinstatement. High glutamate levels are seen in the BLA and NAc during cue-induced alcohol seeking, along with mGluR2 downregulation in the mPFC. Rats that prefer alcohol often lack functional mGluR2s and show increased alcohol consumption and resistance to stopping drinking.

Consistent with these findings, the mGluR2/3 agonist LY379268 reduces reinstatement of alcohol seeking caused by cues and stress. However, this drug does not differentiate between mGluR2 and mGluR3. A selective mGluR2 PAM, AZD8529, powerfully and specifically blocked cue-induced alcohol seeking, but not stress-induced seeking, in normal rats; this effect was absent in alcohol-preferring rats lacking functional mGluR2s. This suggests that mGluR2s are specifically involved in cue-induced alcohol seeking. Therefore, mGluR2 agonists or PAMs could be potential treatments to prevent alcohol relapse.

Manipulating group III mGluRs (mGluR7 and mGluR8) has shown less promising results. Some agonists have reduced alcohol self-administration or cue-induced seeking, but often with motor-suppressant side effects.

Targeting Glutamatergic Transmission for Therapeutic Purposes

Research clearly shows that glutamate signaling is vital for alcohol seeking and relapse. While the complexity of iGluRs might seem to offer many targets, decades of failed drug development for conditions like epilepsy suggest that modulating iGluRs for therapeutic purposes without significant side effects is challenging because they are so fundamental to brain function. In contrast, preclinical findings suggest that mGluR2/3 presynaptic agonists and mGluR5 postsynaptic antagonists may be effective treatments for alcohol seeking and relapse.

Section V: GABAergic Neurotransmission and Alcohol Seeking

GABAergic neurotransmission is crucial for many effects of alcohol, from intoxication to seeking and relapse. GABA is the brain's main inhibitory neurotransmitter, reducing the activity of dopamine neurons. It acts on two types of receptors: ionotropic GABAA receptors (ligand-gated chloride channels) and metabotropic GABAB receptors (G protein-coupled, affecting potassium and calcium channels). Alcohol impacts GABAergic transmission both acutely and long-term. Increased relapse risk, driven by negative reinforcement, is thought to stem from dysregulation of peptide systems that affect GABA circuits in the extended amygdala (CeA, NAc shell, BNST).

The CeA is involved in stress-induced alcohol seeking. Chronic alcohol exposure increases GABA release in the CeA through both pre- and post-synaptic mechanisms. Impaired GABA clearance in the CeA, due to low levels of the GABA transporter GAT-3, is linked to a stronger preference for alcohol over natural rewards and continued alcohol self-administration despite punishment. This reduced GAT-3 expression is accompanied by decreased GABAA receptor subunits, possibly a compensatory response to higher GABA levels. SP-mediated activation of NK1 receptors in the CeA boosts GABAergic transmission, and this effect is enhanced in alcohol-dependent rats. These findings suggest that drugs that restore GABA balance in the CeA could be therapeutic.

GABAergic transmission in the BNST also contributes to stress-induced drug seeking and negative emotions associated with addiction. The BNST projects widely, including to the VTA. Most VTA-projecting BNST neurons are GABAergic and produce calming, rewarding effects, while a smaller group of glutamatergic BNST neurons cause anxiety and aversion. A population of CRH-expressing GABA neurons within the BNST controls this balance. Chronic alcohol exposure disrupts this local GABAergic circuit in the BNST, which may promote alcohol seeking during withdrawal.

In the striatum, chronic alcohol reduces GABAergic transmission, which may contribute to increased alcohol seeking and intake. This reduction, seen in the DLS, DMS, and NAc, involves decreases in GABA release or fewer GABAergic synapses. Weakened GABAergic inhibition in the DLS may contribute to habitual alcohol seeking.

Alcohol-induced changes in GABAergic transmission are partly due to changes in GABAA and GABAB receptor function and sensitivity, highlighting the need to understand these adaptations throughout addiction stages.

GABAA Receptors in Relapse to Alcohol Seeking

GABAA and GABAB receptors contribute to both acute and chronic effects of alcohol, including sedation, tolerance, withdrawal, and motivation. GABAA receptors are chloride channels made of five subunits, allowing for diverse receptor formations and varied responses to alcohol. Acute and chronic alcohol exposure transiently alters GABAA receptor subunit levels and locations.

Alcohol enhances GABAA receptor-mediated transmission by increasing GABA release in many brain regions. Prolonged alcohol exposure facilitates GABAergic transmission, leading to hyperexcitability during withdrawal. Chronic alcohol can also cause GABAA receptor downregulation, leading to tolerance and withdrawal and disrupting various behaviors. GABAA receptors in the CeA regulate alcohol consumption in alcohol-preferring rats, but there is limited evidence for their role in alcohol seeking behaviors.

A recent study showed that a GABAA α1-preferring antagonist reduced alcohol self-administration but not alcohol seeking in baboons. However, similar compounds have inconsistent effects in other animal models, and effects may not be specific to alcohol.

Role of GABAB Receptors in Alcohol Seeking

In contrast to the limited evidence for GABAA receptors, substantial data support a role for GABAB receptors in alcohol seeking. GABAB receptors are G protein-coupled and, when activated, inhibit neurotransmitter release by affecting potassium and calcium channels. Both preclinical and clinical evidence suggest that activating GABAB receptors is a promising approach for treating alcohol addiction.

Baclofen, a selective GABAB receptor agonist, reduces alcohol's reinforcing properties, eases withdrawal, and prevents reinstatement of alcohol seeking. In models of relapse, baclofen abolished alcohol intake after abstinence, though the specific dose needed varied and sometimes caused non-specific sedative effects or reduced food intake, limiting its "therapeutic index." This variability may be due to differences in rat strains, drinking history, and acute vs. chronic administration.

Baclofen consistently reduces cue-induced relapse to alcohol seeking across species, including rats and baboons. It also reduces yohimbine-induced relapse and alcohol seeking in an odor recognition task, possibly by blunting stress responses. This aligns with clinical findings linking HPA axis activity, craving, and relapse, and studies showing baclofen reduces cortisol levels in alcohol-dependent patients.

Clinically, baclofen has shown promising but mixed results for alcohol addiction treatment. Concerns about safety, tolerability, tolerance, and dose-escalation have hindered its approval. GABAB receptor positive allosteric modulators (PAMs) are being explored to potentially avoid these issues, as they enhance the effect of natural GABA without directly activating the receptor, offering a wider therapeutic window.

GABAB PAMs have shown promise in preclinical studies. ADX71441, a selective GABAB PAM, powerfully suppressed both cue- and stress-induced alcohol seeking in rats. It also reduced stress-induced brain activity in areas involved in relapse. Other novel GABAB PAMs, CMPPE and COR659, have also reduced relapse-like drinking and cue-induced alcohol seeking.

The exact mechanisms by which GABAB activation prevents relapse are still being investigated. One possibility is through inhibiting dopamine neurons in the VTA, which reduces dopamine release in the NAc and mPFC. Activating GABAB receptors in the VTA has been shown to suppress cue-induced alcohol seeking. Since separable dopamine pathways control responses to discrete cues and contexts, GABAB activation might broadly prevent relapse triggered by both.

In summary, activating GABAB receptors appears very promising for preventing alcohol seeking and relapse. GABAB PAMs could overcome the safety issues of existing drugs. Developing safe GABAB PAMs for human use and testing their effectiveness in alcohol addiction is a high priority for new treatments.

Concluding Remarks

Animal models of drug seeking, especially reinstatement models, have greatly advanced our understanding of alcohol relapse mechanisms over the past decades, identifying many biological systems involved.

A crucial question is whether these findings can predict how well treatments will prevent craving and relapse in humans. There is debate about the predictive power of animal models in psychiatric disorders generally. For addictive disorders, the situation is similarly complex.

The data do not support a simple "yes" or "no" answer to whether animal models of alcohol seeking reliably predict clinical activity. The reality is more nuanced. On one hand, findings with naltrexone and other MOP-targeting tools strongly suggest that both priming- and cue-induced reinstatement models have predictive validity. Baclofen and GABAB activation also generally support the predictive validity of these relapse models. On the other hand, the failure of CRH1 antagonists in clinical trials points to the opposite conclusion.

A more refined view is needed: predictive validity is not all-or-nothing and likely varies greatly across the biological systems studied. A key factor is probably how well a system is conserved across species, which has received too little attention. This means that focusing decades of preclinical study on a single biological system might not be the best strategy for advancing alcohol addiction treatment. Instead, signals from animal models should be tested quickly in human proof-of-principle studies, using biomarkers, as exemplified by the NIMH Fast-Fail initiative for KOP antagonism in depression. If human translation is not supported, continuing preclinical work in that area becomes less efficient.

With this strategy in mind, a priority list for rapid clinical evaluation emerges from this review. At the top are safe and well-tolerated GABAB PAMs, which should be tested for preventing stress- and cue-induced craving and relapse. Second, KOP antagonists should be evaluated for preventing stress-induced craving and relapse, potentially in combination with naltrexone. Third, mGluR2 PAMs warrant consideration. Less optimism surrounds interventions targeting dopaminergic or ionotropic glutamate receptors due to their fundamental roles in brain function, which make it difficult to develop effective and safe medications.

Summary

Treating addiction is challenging because many patients relapse after they stop using alcohol. Even after many years, about half of people with alcohol addiction relapse within three months. Stress, reminders of alcohol, or even a small amount of alcohol can trigger a strong urge to drink, known as craving, which often leads to relapse. This urge to drink can predict how likely someone is to relapse in the future.

Animal Models for Studying Alcohol Seeking Behavior

Alcohol Seeking in Relapse Models to Study Craving and Relapse

Researchers often use animal models to study relapse and its brain mechanisms. In these models, animals first learn to self-administer alcohol. However, alcohol's bitter taste can make this difficult for rodents, similar to humans who are not used to alcohol. Various methods, such as water deprivation or sugar solutions, have been used to overcome this, but some recent studies have shown that animals can learn to self-administer alcohol without these additional steps.

Once animals learn to self-administer alcohol, the next step is extinction training, where the alcohol is no longer available. This causes the animals to stop seeking alcohol. Then, "reinstatement" occurs when certain triggers, such as cues or stress, cause the animals to start seeking alcohol again, even though it's not available. This seeking behavior is considered a model of craving in humans.

Different types of triggers can cause reinstatement. Alcohol-associated cues (like smells or visual signals) and stress are particularly effective. For example, specific smells or combining a smell with a visual cue can strongly trigger alcohol seeking, especially in rats bred to prefer alcohol. Physical stressors, like mild electric shocks, or pharmacological stressors, like the anxiety-inducing drug yohimbine, can also lead to robust alcohol seeking. However, since human stressors are often social or psychological, there might be differences in the brain mechanisms involved compared to the physical stressors used in animal studies. More research is needed to see if findings from social stress models in animals apply to alcohol seeking.

Compulsive Alcohol Seeking

A key feature of alcohol addiction is continuing to seek and use alcohol despite negative consequences. Understanding how alcohol use becomes compulsive is a major area of research. Most animal studies have looked at compulsive drinking, where animals continue to drink alcohol even if it's made bitter or causes a mild electric shock. More recently, researchers have started to study compulsive seeking of alcohol, which occurs before actual drinking.

One method involves a four-phase protocol where rats first learn to associate a light cue with alcohol. Then they learn to press a lever to take alcohol, followed by a phase where they press a different "seeking" lever to make the "taking" lever appear. Finally, the seeking lever is associated with mild electric shocks. Using this protocol, researchers identified different groups of rats: some showed compulsive seeking, continuing to press the seeking lever despite shocks, while others reduced their seeking. This suggests individual differences in vulnerability to compulsive alcohol seeking.

Another approach uses a multi-criteria model to identify rats prone to addiction. In this model, alcohol seeking is measured during periods when no alcohol is available. Some rats show persistent alcohol seeking, and a subset of these also continue to seek alcohol despite electric shocks. These findings confirm that individual rats have different vulnerabilities to developing addiction-like behaviors.

Neurobiological Mechanisms Mediating Alcohol Seeking

Opioid Systems and Alcohol Seeking

Opioid systems are an important starting point for understanding alcohol seeking because drugs like naltrexone, which target these systems, are approved treatments for alcohol addiction. Naltrexone helps prevent heavy drinking after a person has a "slip" and drinks a small amount of alcohol, similar to how it blocks alcohol-induced relapse in rats. It also appears to reduce craving caused by alcohol-related cues. Studies show that people with alcohol addiction have more mu-opioid receptors (MOP) in certain brain areas, which correlates with cravings. Also, alcohol can release natural opioids in the brain, suggesting MOP activation plays a role in alcohol's rewarding effects.

Opioid systems are complex, involving different peptide families and receptor types (MOP, kappa-opioid (KOP), delta-opioid (DOP), and nociceptin (NOP)). These receptors are involved in both the positive and negative effects of alcohol. For example, MOP activation generally increases alcohol's rewarding effects, while KOP activation is linked to unpleasant effects.

MOP Receptors and Alcohol Seeking

Blocking MOPs with naltrexone reduces both alcohol seeking and the motivational responses to alcohol-related cues in animals, similar to human observations. Naltrexone primarily inhibits cue-induced alcohol seeking rather than stress-induced seeking. Selective MOP antagonists also reduce alcohol seeking in various animal models, including those studying compulsive seeking. These preclinical findings strongly support MOP blockade as a treatment for alcohol craving and relapse, consistent with clinical results. However, the overall clinical effectiveness of these medications is modest, and it's unclear if more selective MOP antagonists would offer significant improvements over existing treatments.

KOP Receptors and Alcohol Seeking

KOPs and their natural ligand, dynorphin (DYN), are important for stress responses and negative emotions in addiction. Long-term alcohol exposure can alter the KOP/DYN system, leading to negative emotional states that encourage excessive drinking to feel better. Increased KOP activity during withdrawal may contribute to these negative feelings and promote alcohol seeking. Therefore, KOP antagonists are considered potential treatments for alcohol addiction.

In rats, KOP antagonists like nor-BNI reduce alcohol seeking triggered by stress. Activating KOPs, on the other hand, can trigger alcohol seeking, which can then be blocked by KOP antagonists. This suggests that DYN acts upstream of other stress-related systems to produce stress-induced relapse. KOP antagonists also block cue-induced alcohol seeking. However, the complex actions of older KOP antagonists have made it difficult to develop them for clinical use. A new generation of KOP antagonists, such as CERC-501, show promise. CERC-501 has been shown to reverse anxiety during alcohol withdrawal and block stress-induced alcohol seeking in rats, without affecting cue-induced seeking. This suggests that KOP antagonists primarily target negative emotional states that drive alcohol seeking, and could be combined with MOP antagonists (like naltrexone) for a more comprehensive treatment strategy.

DOP Receptors and Alcohol Seeking

Fewer studies have focused on the role of DOP receptors in alcohol seeking. However, some evidence suggests that DOPs may be involved in cue-induced alcohol seeking. For example, a selective DOP antagonist has been shown to inhibit alcohol seeking triggered by environmental cues and stress.

NOP Receptors in Alcohol Seeking

The NOP system, involving the peptide nociceptin/orphanin FQ (N/OFQ) and its receptor, has been widely studied in addiction. Initially, it was thought that activating NOPs would reduce motivation for drugs, but both NOP agonists and antagonists have shown similar effects, making it unclear which approach is better for treatment.

Activating NOPs with agonists has been shown to reduce alcohol withdrawal symptoms, relapse after alcohol deprivation, and stress-induced alcohol seeking in rats, especially in alcohol-dependent animals. Central administration of nociceptin also suppresses cue-induced alcohol seeking. Studies have shown that alcohol-dependent rats have more pronounced changes in the N/OFQ-NOP system in certain brain regions, linking this system to excessive drinking.

However, NOP antagonists have also shown similar positive effects, such as preventing alcohol taking and stress-induced relapse, and blocking alcohol-induced dopamine release. One explanation for these seemingly contradictory results is that NOP receptors might undergo rapid desensitization when activated by agonists, essentially leading to an antagonist-like effect over time. More human data is needed to determine if targeting the NOP system is a viable treatment strategy and whether agonists or antagonists would be more effective.

Other Peptides Involved in Alcohol Seeking

Corticotropin-releasing Hormone (CRH)

CRH is a peptide known for its role in the stress response and is found in brain areas important for alcohol seeking. Its actions are primarily mediated by CRH1 receptors. CRH1 receptors act as an "alarm system," becoming active during uncontrollable stress.

Blocking CRH signaling consistently blocks stress-induced alcohol seeking in animals, without affecting cue-induced seeking. This was shown with both brain injections of peptide antagonists and systemic administration of selective CRH1 antagonists. In alcohol-dependent rats, CRH and CRH1 receptor expression are increased in certain brain regions, making these animals more sensitive to CRH1 blockade for stress-induced relapse. These findings suggested that CRH1 antagonists could suppress stress-induced craving in humans, a predictor of relapse.

However, clinical trials with CRH1 antagonists in humans for alcohol addiction have unfortunately not shown positive results, despite efforts to ensure the drugs reached their target. Combined with their failures in other stress-related psychiatric conditions, it is currently considered unlikely that this mechanism will be useful for treating alcohol addiction.

Substance P (SP) and its Neurokinin 1 (NK1) Receptor

SP is a peptide that primarily binds to the NK1 receptor, which is involved in stress responses and several alcohol-related behaviors. Developing NK1 antagonists for rodents has been challenging due to species differences in the receptors. However, with the development of specific rat NK1 antagonists, studies have shown that blocking NK1 receptors can prevent stress-induced relapse to alcohol seeking, with high behavioral specificity (not affecting cue-induced relapse). In alcohol-preferring rats, increased NK1 expression in a specific brain region (CeA) is linked to increased sensitivity to stress-induced relapse, and blocking NK1 receptors in this area reduces it. Activation of NK1 receptors in the CeA also increases GABA release, and this effect is stronger in alcohol-dependent rats. These findings suggest that NK1 receptors in the CeA promote sensitivity to stress-induced relapse.

While early human studies with an NK1 antagonist showed some promise in reducing stress-induced craving and brain responses to negative emotions, later clinical trials did not confirm these findings. The inconsistent results in depression trials and the subsequent discontinuation of NK1 antagonist development were later attributed to the need for nearly complete receptor blockade for therapeutic effects, which may not have been achieved in all studies. It is possible that NK1 antagonism could still be a viable treatment for anxious alcohol-addicted patients if a highly brain-penetrant drug is administered at adequate doses, but this may never be tested.

The Role of Dopaminergic Neurotransmission in Alcohol Seeking

Alcohol activates dopamine neurons in the brain, leading to increased dopamine release in reward-related areas like the nucleus accumbens (NAc) and prefrontal cortex. This dopamine release contributes to alcohol's reinforcing effects, and blocking dopamine receptors can reduce both alcohol self-administration and relapse. Alcohol-induced dopamine changes also contribute to brain plasticity, potentially leading to impaired impulse control and increased relapse risk. The role of dopamine depends on the specific brain region, duration of alcohol use, and type of alcohol seeking behavior being studied.

Dopamine effects are mediated by five receptor types, divided into D1-like and D2-like families. D1 and D2 receptors are the most common and are highly expressed in reward circuits. Prolonged alcohol use and withdrawal affect dopamine levels and D1/D2 receptor binding in various brain regions. These changes in dopamine signaling can affect the balance of excitation and inhibition in brain cells, promoting alcohol seeking.

Drugs that increase or stabilize dopamine levels can prevent relapse-like drinking. However, both activating and inhibiting dopamine receptors can affect alcohol seeking in similar ways, either by replacing alcohol's rewarding effects or by blocking them. D2-like receptor signaling, particularly in the dorsal striatum, is associated with alcohol seeking, especially after longer periods of alcohol exposure. Blocking D2-like receptors can reduce alcohol seeking, and sensitivity to this blockade may predict vulnerability to compulsive alcohol seeking. The D3 receptor may also be important, as antagonists and partial agonists of D3 receptors suppress cue-induced alcohol seeking and relapse-like behavior.

Studies in rats have shown that those exhibiting addiction-like behaviors (such as inability to abstain, increased motivation, and persistent intake despite consequences) also show changes in D1 and D2 receptor expression, suggesting long-lasting dopamine dysregulation. While D4 antagonists do not affect cue-induced relapse, they can suppress stress-induced relapse. Overall, D1-like receptors in the NAc seem important for regulating alcohol intake, while D2-like receptors in the dorsal striatum are crucial for alcohol seeking and relapse. Dopamine pathways involving the prefrontal cortex and NAc also play a role in cue-guided alcohol seeking.

Glutamatergic Signaling in Alcohol Seeking Behaviors

Alcohol seeking is strongly linked to changes in glutamate signaling within brain circuits involving the amygdala, cortex, and striatum. Both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors are involved, and it's thought that as alcohol use becomes more compulsive, control shifts from mGluRs to iGluRs. The specific role of these receptors can vary depending on their location in the brain. Long-term heavy drinking strengthens glutamatergic activity in certain brain synapses, sustaining alcohol seeking. Chronic alcohol exposure can also impair brain plasticity, reducing behavioral flexibility and promoting habitual seeking.

The ventral and dorsal striatum, which receive glutamate inputs from the amygdala and cortex, are differentially involved in alcohol addiction. Increased glutamate levels are observed in the basolateral amygdala and NAc during cue-induced alcohol seeking. Pathways connecting the prefrontal cortex to the NAc are essential for cue-induced relapse. Alcohol seeking is also thought to become a maladaptive habit, shifting control from the ventral and dorsomedial striatum to the dorsolateral striatum (DLS). Increased glutamate inputs to the DLS, along with dopamine changes, are key mechanisms behind habitual alcohol seeking.

Researchers are focusing on targeting mGluRs and iGluRs to reverse these maladaptive changes caused by chronic alcohol use.

Ionotropic Glutamate Receptors

iGluRs, including NMDARs, AMPARs, and kainate receptors, are crucial for fast excitatory neurotransmission and synaptic plasticity in brain regions involved in alcohol seeking. Chronic alcohol exposure and withdrawal increase the activity and expression of NMDARs and AMPARs, leading to brain changes that reduce behavioral flexibility. NMDARs have been extensively studied for their role in both cue-induced and compulsive alcohol seeking (seeking despite negative consequences).

Compulsive alcohol seeking is linked to neuroadaptations in NMDARs, with increased expression of certain NMDAR subunits (GluN1 and GluN2A) in the orbitofrontal cortex (OFC) in mice that seek alcohol despite punishment. The GluN2B NMDAR subunit in the OFC also mediates habitual alcohol seeking through a specific signaling pathway (mTORC1). Upregulation of the GluN2B subunit in certain brain circuits is critical for promoting alcohol seeking relapse. NMDAR antagonists can block priming-induced relapse and relapse-like behavior after long abstinence, but they do not seem to play a role in cue-induced relapse.

AMPAR function is also enhanced after chronic alcohol exposure, and this can trigger drug seeking. AMPAR positive modulators can increase cue-induced alcohol seeking, while AMPAR antagonists can block cue-induced relapse and relapse-like drinking after abstinence. Selective kainate receptor antagonists have mainly been studied for reducing alcohol intake, and their effect on alcohol seeking is less known.

Metabotropic Glutamate Receptors

mGluRs are a promising group of therapeutic targets. They are G protein-coupled receptors that mediate slow neurotransmission and are found in both neurons and glial cells throughout the central nervous system. There are eight types of mGluRs, divided into three groups based on their signaling pathways. Group I mGluRs (mGluR1 and 5) are mainly postsynaptic and increase intracellular calcium levels, influencing gene expression. Group II and III mGluRs are mostly presynaptic and modulate the release of glutamate and other neurotransmitters. mGluR5 and mGluR2 have been particular focuses for medication development.

While alcohol initially dampens mGluR1/5 function, prolonged alcohol use increases their expression and activity. Activating mGluR5s in the NAc is essential for alcohol's internal effects. Competitive mGluR5 antagonists and negative modulators reduce cue-induced alcohol seeking, both when given systemically or injected directly into brain regions like the NAc or BLA. Blocking mGluR5s reduces alcohol seeking by affecting specific signaling pathways (ERK1/2). These findings suggest mGluR5s are involved in the associative learning that links alcohol cues to its effects, which strengthens with addiction and contributes to seeking. Facilitating the extinction of these alcohol memories, for example, by using mGluR5 positive modulators, could also be a treatment strategy.

In contrast, mGluR1's effects on alcohol seeking have not been as extensively studied.

mGluR2-mediated control of glutamate release is another area of interest. Prolonged alcohol exposure can disrupt mGluR2 function by reducing its expression. Deficits in mGluR2-mediated feedback inhibition of glutamate release in certain brain circuits promote alcohol seeking. High glutamate levels in the BLA and NAc during cue-induced relapse are accompanied by reduced mGluR2 in the mPFC. Alcohol-preferring rats, which lack functional mGluR2s, show increased alcohol consumption and resistance to devaluation. Mixed mGluR2/3 agonists have been shown to reduce both cue- and stress-induced alcohol seeking. More specifically, a selective mGluR2 positive modulator potently blocked cue-induced, but not stress-induced, alcohol seeking in rats, further supporting the specific involvement of mGluR2 in cue-induced alcohol seeking. Thus, mGluR2 agonists or modulators may have therapeutic potential to prevent relapse.

Manipulations targeting Group III mGluRs (mGluR7 and mGluR8) have shown less promising results in alcohol seeking studies.

Targeting Glutamatergic Transmission for Therapeutic Purposes

Research clearly shows that glutamate signaling is critical for alcohol seeking and relapse. While the complexity of iGluRs might seem to offer many drug targets, past attempts to develop NMDAR medications for other conditions have often failed due to fundamental roles in brain function, suggesting it may be hard to modulate them safely. In contrast, preclinical findings suggest that presynaptic agonists of mGluR2/3 receptors and antagonists of postsynaptic mGluR5 may be effective and safer pharmacological treatments for alcohol seeking and relapse.

GABAergic Neurotransmission and Alcohol Seeking

GABA, the brain's main inhibitory neurotransmitter, plays a significant role in many alcohol effects, from intoxication to seeking and relapse. It acts on two types of receptors: ionotropic GABAA receptors and metabotropic GABAB receptors. Alcohol affects GABAergic transmission both acutely and long-term. Dysregulation of peptidergic systems converging on GABAergic circuits in the extended amygdala (including CeA, NAc shell, and BNST) is thought to drive relapse through negative reinforcement.

The CeA is involved in stress-induced alcohol seeking. Chronic alcohol exposure increases GABA release in the CeA. Impaired GABA clearance in the CeA, due to low levels of the GAT-3 transporter, is associated with a preference for alcohol over natural rewards and continued alcohol use despite negative consequences. The substance P system also promotes GABAergic transmission in the CeA, and this effect is stronger in alcohol-dependent rats. These findings suggest that restoring GABA balance in the CeA could be a therapeutic strategy.

GABAergic transmission in the BNST also contributes to stress-induced drug seeking and negative emotions. The BNST projects to the VTA, with different populations of BNST neurons affecting motivation in opposing ways. Chronic alcohol exposure can disrupt a local GABAergic circuit in the BNST, promoting alcohol seeking during withdrawal.

In the striatum, chronic alcohol reduces GABAergic transmission, which may contribute to increased alcohol seeking. This reduction in GABAergic inhibition, particularly in the DLS, could be a mechanism behind habitual alcohol seeking. Alcohol-induced changes in GABAergic transmission are partly due to altered GABAA and GABAB receptor function and sensitivity.

GABAA Receptors in Relapse to Alcohol Seeking

Both GABAA and GABAB receptors are involved in alcohol's acute and chronic effects. GABAA receptors are chloride channels made of multiple subunits, which vary in the brain and determine their response to alcohol. Chronic alcohol exposure can lead to GABAA receptor downregulation, causing tolerance and withdrawal. While GABAA receptors in the CeA regulate alcohol-maintained responding, there is limited evidence for their direct role in alcohol seeking. Some studies suggest GABAA α1-preferring antagonists can reduce alcohol self-administration but not necessarily alcohol seeking, and these effects may not be specific to alcohol.

Role of GABAB Receptors in Alcohol Seeking

In contrast to GABAA receptors, there is extensive evidence for the role of GABAB receptors in alcohol seeking. GABAB receptors are G protein-coupled and inhibit neurotransmitter release. Activating GABAB receptors is a promising mechanism for treating alcohol addiction.

The GABAB receptor agonist baclofen reduces alcohol's reinforcing properties, withdrawal severity, and prevents relapse to alcohol seeking in animal models. It has been shown to abolish alcohol intake after abstinence and reduce cue-induced alcohol seeking across species. Baclofen also reduces stress-induced alcohol seeking and can blunt stress hormone responses. Clinically, baclofen has shown mixed but promising results for alcohol addiction treatment, but safety and tolerability concerns (like sedation and tolerance) have hindered its approval.

GABAB positive allosteric modulators (PAMs) are being investigated to overcome these issues. PAMs enhance the effect of natural GABA without directly activating the receptor, potentially leading to better selectivity and a wider therapeutic window. Studies with GABAB PAMs have shown they can powerfully suppress both cue- and stress-induced alcohol seeking in rats, and also reduce stress-induced neuronal activity in related brain areas. Other novel GABAB PAMs have also shown efficacy in reducing relapse to alcohol drinking and cue-induced alcohol seeking, often at lower doses and with less side effects than baclofen.

The mechanisms by which GABAB activation prevents alcohol seeking are still being investigated, but one candidate is through inhibiting dopamine neurons in the VTA, which then reduces dopamine release in the NAc and mPFC. GABAB activation may broadly prevent relapse by attenuating VTA neuron activity involved in both cue- and context-induced alcohol seeking. In summary, GABAB receptor activation, particularly through PAMs, shows significant promise for preventing alcohol seeking and relapse, and developing safe and effective GABAB PAMs is a high priority for new addiction treatments.

Concluding Remarks

Animal models of alcohol seeking and relapse, developed since the 1980s, have significantly advanced our understanding of the biological systems involved. The key question now is whether these animal findings can predict effectiveness in preventing craving and relapse in humans. Given the challenges in psychiatric drug development, the predictive power of animal models for addiction is often questioned.

However, a nuanced view is needed. The ability of animal models to predict clinical outcomes is not a universal "all or nothing" phenomenon; it likely varies significantly across different biological systems. The degree to which a system is conserved across species is probably a crucial factor that has not received enough attention. This implies that focusing decades of preclinical study on a single biological system might not be the best strategy. Instead, promising signals from animal models should be tested quickly in human proof-of-principle studies using biomarkers. If human translation is not supported, continuing preclinical work on that target might be unproductive.

Based on this approach, a priority list for clinical evaluation emerges from the reviewed literature. At the top are safe and well-tolerated GABAB PAMs, which should be tested for preventing both stress- and cue-induced craving and relapse. Second, KOP antagonists are promising for preventing stress-induced craving and relapse, and could be combined with naltrexone. Third, mGluR2 PAMs warrant evaluation. Less optimism is held for targeting dopaminergic or ionotropic glutamate receptors, largely due to their fundamental roles in brain function, which could make it difficult to develop effective and safe medications.

Abbreviations

• P rats: Indiana alcohol Preferring rats

• CPP: Conditioned place preference

• MOP: Mu-opioid receptor

• NAc: Nucleus accumbens

• OFC: Orbitofrontal cortex

• KOP: Kappa-opioid receptor

• DOP: Delta-opioid receptor

• NOP: Nociceptin receptor

• DYN: Dynorphin

• nor-BNI: Nor-binaltorphimine

• CRH: Corticotropin-releasing hormone

• BNST: Bed nucleus of the stria terminalis

• JNK: c-Jun N-terminal kinase

• N/OFQ: Nociceptin/orphanin FQ

• CeA: Central nucleus of the amygdala

• GPCRs: G protein-coupled receptors

• HPA: Hypothalamic-pituitary-adrenal

• SP: Substance P

• NK: Neurokinin

• VTA: Ventral tegmental area

• (m)PFC: (Medial) prefrontal cortex

• MSNs: Medium spiny projection neurons

• ADE: Alcohol deprivation effect

• COMT: Catechol-O-Methyltransferase

• DLS: Dorsolateral striatum

• iGluR: Ionotropic glutamate receptors

• mGluR: Metabotropic glutamate receptors

• BLA: Basolateral amygdala

• DMS: Dorsomedial striatum

• NMDAR: N-methyl-d-aspartate receptors

• AMPAR: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors

• PAM: Positive allosteric modulator

• CaMKII: Ca2+/calmodulin-dependent protein kinase II

• NAM: Negative allosteric modulator

• MPEP: 2-Methyl-6-(phenylethynyl)pyridine

• ERK1/2: Extracellular signal-regulated kinases ½

• CDPPB: 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide

• LH: Lateral hypothalamus

• mIPSC: Miniature inhibitory postsynaptic current

• sP: Sardinian alcohol preferring

Introduction

A major challenge in treating addiction is preventing relapse after a person stops using alcohol. Research shows that more than half of those who have recently stopped drinking alcohol relapse within three months, a statistic that has not changed significantly over time. For individuals with alcohol addiction, certain events often lead to relapse, such as stressful situations, reminders of alcohol, or even a small amount of alcohol. A strong urge to drink, known as "craving," often precedes relapse. Studies have confirmed that the intensity of craving in response to triggers predicts the risk of future relapse.

Animal Models for Studying Alcohol Seeking Behavior

Alcohol Seeking in Reinstatement Models

Animal models are widely used to study relapse and its underlying brain mechanisms. These "reinstatement" models involve training animals to self-administer alcohol, then stopping the alcohol supply, and finally observing if certain triggers cause them to seek alcohol again. Getting animals to reliably self-administer alcohol can be challenging due to the taste of high alcohol concentrations. Researchers have developed methods to overcome this, such as water deprivation or prolonged training, though these can complicate study results. More recent methods have achieved stable alcohol self-administration without these extra steps.

Mini-Dictionary of Terms

Operant self-administration

This is a method where an animal learns to perform a specific action, like pressing a lever, to receive a reward, such as food or a drug solution. Typically, two levers are present: one that provides the reward ("active") and one that does nothing ("inactive"). If the animal repeatedly performs the action to get the reward, the drug is considered to be a "reinforcer" because it strengthens the animal's behavior.

Reinstatement

This is a standard animal model used to study drug relapse. After animals learn to self-administer a drug and then stop responding to it (extinction), they are tested to see if certain triggers cause them to seek the drug again. These triggers can include stress, a small dose of the drug itself (drug priming), or cues and contexts previously associated with the drug.

Stress-induced reinstatement

In this model, laboratory animals first learn to self-administer a drug, with the drug delivery being linked to specific cues like a tone or light. The animals then learn to stop responding for the drug while these drug-associated cues are still present.

Cue-induced reinstatement

Similar to stress-induced reinstatement, animals are initially trained to self-administer a drug in the presence of specific cues. Their drug-seeking responses are then reduced by removing the drug and the associated cues. During the reinstatement test, reintroducing these specific cues causes an increase in drug-seeking behavior.

Drug-induced reinstatement

Animals are trained to self-administer a drug, and drug delivery is paired with a specific cue. The animals then stop responding for the drug while the discrete cues are still present. Once drug-seeking behavior is low and stable, a small dose of the drug previously self-administered is given, which causes the animals to start seeking the drug again.

After animals learn to self-administer alcohol, the next step in reinstatement procedures is an "extinction" phase. During this phase, the action that previously led to alcohol delivery no longer provides the reward. As a result, the animals' responses on the alcohol-associated lever decrease or stop. When triggers are then introduced, such as specific cues or stress, the animals will resume their alcohol-seeking behavior even without the reward. The rate of this behavior is measured as an indicator of the animal's urge to obtain alcohol, similar to craving in humans.

Alcohol injections can cause rats to seek alcohol again, but cues linked to alcohol or stressful situations are more effective triggers. The type of cue matters; for example, olfactory cues can trigger relapse in rodents. Combining an olfactory cue with a visual cue can make the effect even stronger, especially in genetically alcohol-preferring rats. Physical stressors, such as foot shocks, or pharmacological stressors, such as the anxiety-inducing drug yohimbine, also effectively trigger alcohol seeking. However, the psychosocial stressors experienced by humans with alcohol addiction differ from the physical stressors used in animal studies. This difference might mean that the biological mechanisms found in animal studies are not always the same as those in humans. Researchers are exploring new animal models, such as using social defeat stress, to better mimic human experiences.

Compulsive Alcohol Seeking

A key characteristic of alcohol addiction is "compulsive" alcohol seeking and drinking, meaning it continues despite negative consequences. Understanding how alcohol use shifts from controlled to compulsive is crucial for addiction research. Most studies have focused on compulsive drinking, where animals continue to drink alcohol even if it is made bitter or if they receive an electric shock. Compulsive alcohol seeking—the act of seeking alcohol without actually consuming it, despite negative consequences—has only recently begun to be studied.

Researchers have developed models to separate alcohol seeking from alcohol taking. One such model involves four phases for rats. First, rats learn to associate a light cue with alcohol availability. Next, they learn to press a "taking lever" for alcohol delivery, accompanied by the cue light. In the third phase, a "seeking lever" is introduced; pressing this lever makes the taking lever available. Finally, in the fourth phase, the seeking-taking process is punished with mild foot shocks. Using this method, researchers found that some rats (34%) continued to seek alcohol despite the punishment, indicating compulsive seeking, while others significantly reduced their seeking behavior.

Another approach uses a "multicriteria paradigm" to identify rats prone to addiction, based on human diagnostic criteria. This model measures alcohol seeking during "no-drug" periods and also assesses alcohol seeking despite punishment. In these studies, only a subset of rats developed persistent and punishment-resistant alcohol seeking, confirming that individuals vary in their vulnerability to addictive behaviors.

Neurobiological Mechanisms Mediating Alcohol Seeking

Opioid Systems and Alcohol Seeking

Opioid systems are a good starting point for understanding the brain mechanisms behind alcohol seeking because medications like naltrexone, which block opioid receptors, are approved to treat alcohol addiction. Naltrexone reduces heavy drinking and prevents relapse from a small slip to severe drinking. It also helps suppress craving caused by alcohol cues. In people with alcohol addiction, higher levels of mu-opioid receptors (MOP) in the brain are linked to cravings during abstinence, and alcohol consumption in social drinkers causes the release of natural opioids in certain brain areas. This suggests that MOP activation influences the brain's reward system, which supports the idea that animal models of relapse can predict human responses.

Opioid systems are complex, involving different peptide families (endorphins, dynorphins, enkephalins, and nociceptin) and their corresponding receptors (MOP, kappa-opioid (KOP), delta-opioid (DOP), and nociceptin (NOP) receptors). These systems play many roles in addiction and are crucial for the reinforcing effects of drugs. For instance, MOP activation enhances the positive, rewarding effects of alcohol, while KOP activation is linked to the negative, unpleasant aspects of alcohol. The following sections describe how different opioid receptor subtypes affect alcohol seeking in animals.

MOP Receptors and Alcohol Seeking

Studies in animals show that naltrexone, a drug that blocks opioid receptors, reduces alcohol seeking triggered by a small dose of alcohol, similar to its effects in humans. Blocking MOP also reduces the desire for alcohol and alcohol seeking driven by alcohol-related cues. Naltrexone primarily targets MOP and has been shown to stop cue-induced alcohol seeking but not stress-induced seeking in rats. Other MOP blockers have also been found to reduce cue-induced alcohol seeking in rats and baboons. However, giving naltrexone during the extinction phase of alcohol seeking (when animals learn to stop seeking alcohol) did not significantly affect their later sensitivity to alcohol cues or their alcohol consumption.

A specific MOP blocker, GSK1521498, has been shown to reduce both alcohol drinking and cue-induced alcohol seeking in alcohol-preferring rats. In a model of compulsive alcohol seeking, GSK1521498 reduced alcohol seeking in both compulsive and non-compulsive rats, but the effect was stronger in compulsive rats, suggesting it might be more effective for individuals with more severe addiction. Unlike naltrexone, GSK1521498 only targets MOP and does not have partial agonist activity. Overall, preclinical research strongly supports blocking MOP as a way to prevent alcohol craving and relapse, which aligns with human study findings. However, the effectiveness in clinical trials has been modest, and it's unclear if more specific MOP blockers would offer better results than existing medications.

KOP Receptors and Alcohol Seeking

KOP receptors and their natural ligand, dynorphin (DYN), are important for stress responses and negative emotions in addiction, including alcohol addiction. Long-term alcohol use can change the KOP/DYN system, leading to negative emotional states that encourage heavy drinking to alleviate discomfort. Increased KOP sensitivity and reduced dopamine in a brain region called the NAc are key factors in the negative effects of alcohol withdrawal. Since increased DYN/KOP activity during prolonged abstinence can contribute to negative emotions that promote alcohol seeking, KOP blockers could potentially be useful treatments for alcohol addiction.

In rats, nor-binaltorphimine (nor-BNI), a KOP blocker, has been shown to stop alcohol seeking caused by both physical stress (footshock) and pharmacological stress (yohimbine). Conversely, activating KOP receptors with a drug called U50,488 causes alcohol seeking to return, and nor-BNI blocks this effect. Research also suggests that DYN acts before corticotropin-releasing hormone (CRH) in causing stress-induced alcohol seeking. Nor-BNI also reduces alcohol seeking when injected into the bed nucleus of the stria terminalis (BNST), indicating this area is important for DYN/KOP mechanisms in stress-induced alcohol seeking.

Nor-BNI has also been shown to block alcohol seeking triggered by cues. Another KOP blocker, JDTic, reduced anxiety-like behavior during withdrawal and decreased cue-induced alcohol seeking in rats. It also reduced relapse-like behavior in female alcohol-preferring rats. However, the exact mechanisms of these effects are complex due to nor-BNI and JDTic's complicated actions. KOP blockade has been considered a potential treatment for alcohol addiction, but effective KOP blockers suitable for clinical use have been scarce. A new generation of KOP blockers, like CERC-501, shows promise. CERC-501 reversed anxiety caused by alcohol withdrawal and blocked stress-induced alcohol seeking in rats, without affecting cue-induced seeking or other behaviors. This suggests that KOP activation is mainly linked to negative emotions that promote alcohol seeking. CERC-501's effects complement those of naltrexone, which specifically blocks cue-induced but not stress-induced alcohol seeking. Therefore, combining KOP and MOP blockade in treatment appears to be a good strategy.

DOP Receptors and Alcohol Seeking

Compared to MOP and KOP receptors, fewer studies have focused on the role of DOP receptors in alcohol seeking. Some evidence suggests that DOP receptors might be involved in cue-induced alcohol seeking. For example, naltrindole, a selective DOP blocker, reduced alcohol seeking triggered by environmental cues related to alcohol. This drug also suppressed alcohol seeking induced by both cues and contexts. Additionally, another DOP blocker, SoRI-9409, effectively reduced alcohol seeking caused by yohimbine-induced stress in rats.

NOP Receptors in Alcohol Seeking

The Nociceptin/orphanin FQ (N/OFQ)-NOP system is involved in pain, emotions, and other bodily functions. It has been extensively studied in addiction models, including those for alcohol. Initially, it was thought that activating NOP receptors would reduce motivation for addictive drugs. However, later observations showed that both NOP activators and blockers can produce similar effects, making it unclear which approach is better for developing addiction medications.

Activating NOP receptors, whether with nociceptin, related peptides, or small-molecule drugs, has been shown to reduce alcohol withdrawal symptoms, relapse after stopping alcohol, and stress-induced alcohol seeking. These effects have been observed in both non-dependent and alcohol-dependent rats. Administering nociceptin directly into the brain also reduced cue-induced alcohol seeking in alcohol-preferring rats. Electrophysiological studies have shown that alcohol causes greater changes in the N/OFQ-NOP system in alcohol-dependent rats compared to non-dependent rats. Also, alcohol-preferring rats have naturally higher levels of CRH1 receptors, which links CRH dysregulation with the N/OFQ-NOP system and excessive drinking.

A potent NOP activator, SR-8993, was found to reverse alcohol withdrawal-induced anxiety and reduce both stress- and cue-induced alcohol relapse in rats. Surprisingly, similar results were found with an orally available NOP blocker, LY2940094, which prevented alcohol consumption and stress-induced alcohol seeking in alcohol-preferring rats. LY2940094 also blocked alcohol-induced dopamine release in the NAc. To explain these conflicting findings, it has been suggested that NOP receptors rapidly become less sensitive when activated by drugs. This means that NOP activators might eventually cause a blocking effect.

Currently, it is not clear whether targeting the NOP system is a good strategy for developing alcohol addiction medications, or whether activators or blockers would be more effective. Human studies are needed to answer these questions. So far, a small human study with LY2940094 showed mixed results, not meeting its primary goal but showing some positive effects in secondary analyses.

Other Peptides Involved in Alcohol Seeking

Corticotropin-releasing Hormone (CRH)

CRH is a 41-amino acid peptide best known for its role in regulating stress hormones, but it is also found throughout the brain. Its role in alcohol-related behaviors has been extensively reviewed. In addition to the hypothalamus, CRH neurons are present in brain structures important for alcohol seeking, like the CeA and BNST. CRH acts through two types of G protein-coupled receptors (CRH1 and CRH2). Stress responses, including stress-induced alcohol seeking, are primarily mediated by CRH1 receptors in the CeA and BNST. CRH2 activation often has opposite effects to CRH1. CRH1 signaling acts as an "alarm system" that is usually quiet but becomes active during uncontrollable stress.

Blocking CRH signaling consistently stops stress-induced alcohol seeking, but does not affect cue-induced relapse-like behavior. This was first shown by administering a non-selective CRH antagonist directly into the brain and a selective CRH1 antagonist systemically; both blocked stress-induced alcohol seeking. These studies also indicated that CRH1 antagonism blocks stress-induced reinstatement independently of the HPA axis. Another study in alcohol-dependent rats showed that CRH antagonism selectively blocked stress-induced, but not cue-induced, reinstatement. After prolonged alcohol dependence, the expression of CRH and its CRH1 receptor increases in the CeA, making animals more sensitive to the effects of CRH1 blockade on stress-induced reinstatement.

These findings consistently suggest that in rodents, CRH1 receptors specifically mediate stress-induced, but not cue-induced, alcohol seeking. Also, a history of alcohol dependence makes animals more sensitive to CRH1 blockade in preventing relapse. This research predicted that CRH1 antagonists would reduce stress-induced craving in people with alcohol addiction, which is a known predictor of relapse. However, human studies have not supported these preclinical findings, despite efforts to ensure the drugs were active in the brain. Given the failures of CRH1 antagonists in treating other stress-related psychiatric conditions and their discontinuation in clinical development, it is unlikely that this mechanism will be used for alcohol addiction treatment.

Substance P (SP) and its Neurokinin 1 (NK1) Receptor

SP is an 11-amino acid peptide that is part of the tachykinin family, which also includes NKA and NKB. Tachykinins act through three receptor subtypes: NK1, NK2, and NK3. SP preferentially binds to the NK1 receptor. NK1 receptors are Gs/q-coupled G protein-coupled receptors found in various brain regions involved in both rewarding and unpleasant behaviors. They regulate stress responses and several behaviors related to alcohol.

Studying NK1 receptors in animals has been challenging due to differences in receptor structure and drug affinity between humans and rodents. This was overcome by developing L822429, an NK1 antagonist specifically designed to work well in rats. Using this drug, researchers found that blocking NK1 receptors throughout the body stopped stress-induced alcohol seeking, without affecting cue-induced seeking. In alcohol-preferring rats, NK1 expression in the CeA is higher due to a genetic variation. These rats are more sensitive to alcohol seeking triggered by the pharmacological stressor yohimbine, and infusing L822429 into the CeA suppresses this effect. Conversely, increasing NK1 receptors in the CeA of other rats makes them more sensitive to yohimbine-induced reinstatement. Recent findings also suggest that NK1 receptor activation by SP increases GABA release in the CeA, and this effect is stronger after alcohol dependence. These findings collectively show that NK1 receptors in the CeA increase sensitivity to stress-induced relapse and other alcohol-related behaviors.

Based on preclinical findings, the NK1 antagonist LY686017 was tested in a human study with recently detoxified alcohol-addicted patients. The study found that LY686017 reduced stress-induced craving and brain responses to negative emotional stimuli. A subsequent larger study, however, did not meet its primary goal, possibly because the patients were not highly anxious.

The development of NK1 antagonists was initially driven by their potential as antidepressants. However, inconsistent results in depression trials led to their discontinuation for stress-related psychiatric disorders. It was later discovered that NK1 antagonists require nearly complete receptor blockade to be effective, unlike most other GPCR antagonists. Therefore, it is possible that NK1 antagonism could still be a viable treatment for alcohol addiction if a highly brain-penetrant medication is administered at adequate doses to anxious alcohol-addicted patients. However, this possibility may never be explored.

The Role of Dopaminergic Neurotransmission in Alcohol Seeking

Alcohol activates dopamine neurons in the ventral tegmental area (VTA), leading to increased dopamine release in reward-related brain regions like the NAc and medial prefrontal cortex (mPFC). The rewarding effects of alcohol are partly due to this dopamine release, and blocking dopamine receptors reduces both alcohol self-administration and drug-seeking behaviors. Alcohol-induced dopamine release also causes brain changes that can promote addiction. These changes in brain circuits related to reward and memory can lead to reduced impulse control and increased vulnerability to drug relapse. Overall, the role of dopamine in alcohol seeking depends on the specific brain region, the duration of alcohol use, and the type of alcohol-seeking behavior being studied.

Dopamine produces its effects through five G protein-coupled receptors, divided into two families: D1-like (D1 and D5) and D2-like (D2, D3, and D4). D1 and D2 receptors are the most common and are highly expressed in reward-related brain areas. While most studies focus on D1 and D2, D3, D4, and D5 subtypes may also play specific roles in regulating alcohol seeking, but selective drugs to differentiate them are lacking.

Prolonged alcohol intake and withdrawal significantly affect dopamine levels outside cells and alter D1 and D2 receptor binding in regions like the NAc, dorsal striatum, and amygdala. Reduced D2 receptor expression in the PFC is also linked to alcohol-induced conditioned place preference. These changes in dopamine signaling can create an imbalance in striatal neurons, which may further promote alcohol seeking.

Drugs that increase or stabilize dopamine levels have been shown to prevent reinstatement and reduce relapse-like drinking in models of alcohol deprivation. Inhibiting an enzyme that degrades dopamine, COMT, also reduces cue-induced alcohol seeking in male rats. Interestingly, both increasing and inhibiting dopamine receptor signaling can affect alcohol seeking similarly, either by acting as a replacement treatment or by blocking the rewarding effects of alcohol and influencing goal-directed behavior. Alcohol's effects on dopamine are highly time-dependent, making interpretation of findings complex.

Dysfunction of D2-like receptor signaling, in particular, has been linked to alcohol seeking. Blocking D2 receptors systemically or in the NAc reduces alcohol seeking during extinction, possibly due to the NAc D2 receptor's role in processing cues and goal-directed behavior. After longer alcohol exposure, the dependence on D2-like receptors seems to shift towards the dorsal striatum. Administering a D1/D2 antagonist, flupenthixol, into the dorsolateral striatum (DLS) reduces alcohol seeking, and sensitivity to this antagonist predicted vulnerability to compulsive alcohol seeking. Dopamine levels in the dorsal striatum are directly related to the persistence of compulsive alcohol seeking in some animals. Flupenthixol also suppresses alcohol seeking when given in the amygdala, but not in the NAc core, supporting the idea that the amygdala plays a role in the shift from ventral to dorsal striatum as drug seeking becomes a habit.

The dopamine D3 receptor may be particularly relevant for alcohol seeking and cue-induced reinstatement. D3 antagonists suppress cue-induced alcohol seeking. Additionally, administering a D3 antagonist or partial agonist reduces relapse-like behavior after alcohol deprivation in rats that have consumed alcohol for a long time. Alcohol-induced increase of D3 receptors in the dorsal striatum in this model suggests it may contribute to alcohol seeking and relapse.

In a model of cocaine addiction, a seminal study proposed that individual vulnerability to addiction could be predicted by three criteria: inability to stop drug use during unavailability, increased motivation for the drug, and continued use despite negative consequences. Rats meeting all three criteria showed increased cocaine seeking. A similar model applied to alcohol found that rats meeting all three criteria had increased D1 and decreased D2 receptor mRNA expression in the DLS three months later, suggesting that dopamine system changes persist after abstinence and contribute to compulsive alcohol seeking. Interestingly, while D4 antagonists do not affect cue-induced reinstatement, they do suppress stress-induced reinstatement.

In summary, D1-like receptor signaling in the NAc seems important for regulating alcohol intake, while D2-like receptors in the dorsal striatum appear most crucial for alcohol seeking and reinstatement. At the same time, D1 receptors in the dorsal mPFC are key for cocaine-induced reinstatement of cocaine seeking, and stress-induced activation of VTA dopamine pathways to the PFC may trigger cocaine seeking through glutamate projections to the NAc. Since removing mPFC neurons that project to the NAc has been shown to block cue-induced alcohol seeking, similar pathways may also be involved in cue-guided alcohol seeking.

Glutamatergic Signaling in Alcohol Seeking Behaviors

Alcohol seeking has been particularly linked to changes in glutamate signaling within brain circuits involving the amygdala, cortex, and striatum. This includes both ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors. It has been suggested that as alcohol use becomes more compulsive, control shifts from metabotropic to ionotropic receptors. However, the role of different glutamate receptors varies depending on their location in the brain. Increased glutamate activity after long-term heavy drinking leads to lasting brain changes, especially in connections between the cortex and striatum, that maintain alcohol seeking. Impairments in brain flexibility caused by chronic alcohol exposure can reduce behavioral adaptability and promote habitual seeking behaviors.

The ventral and dorsal striatum play different roles in alcohol addiction. They receive glutamate signals from both the amygdala and the cortex, which interact with dopamine signals to their neurons. Studies have reported increased glutamate levels in the basolateral amygdala (BLA) and NAc core during cue-induced alcohol seeking. Furthermore, the pathway from the mPFC to the NAc is necessary for cue-induced alcohol seeking. Selectively removing glutamate neurons from the mPFC that project to the NAc (but not the BLA) prevented cue-induced reinstatement without affecting extinction. Removing amygdalar projections to the NAc also prevented reinstatement.

It has been proposed that, like with other addictive drugs, alcohol seeking becomes a maladaptive habit, with control shifting from the ventral and dorsomedial striatum (DMS) to the DLS. While the DMS receives glutamate signals from associative brain regions, the DLS is connected to sensorimotor areas and the thalamus. Increased glutamate signals to the DLS, along with dopamine-related changes, are considered a main mechanism behind the development of habitual alcohol seeking.

Many glutamate-related brain changes contribute to the emergence of alcohol addiction-like behaviors, including alcohol seeking. The following sections describe some molecular mechanisms involving glutamate signaling that are important for alcohol seeking in animal models. The focus is on the potential to target metabotropic and ionotropic glutamate receptors to correct maladaptive changes caused by chronic alcohol use.

Ionotropic Glutamate Receptors

Glutamate directly affects nerve cell activity through iGluRs, including NMDAR, AMPAR, and kainate receptors, which act as channels that open when glutamate binds to them. These receptors are crucial for rapid excitatory signals and brain plasticity in regions involved in alcohol seeking and taking. Chronic alcohol exposure and withdrawal increase the activity and expression of both NMDARs and AMPARs, leading to brain changes that reduce behavioral flexibility. NMDARs have been extensively studied for their role in both cue-induced alcohol seeking and compulsive alcohol seeking (seeking despite negative consequences).

Compulsive alcohol seeking has been shown to develop alongside compulsive drinking. Alcohol-induced changes in NMDARs in the NAc have been linked to alcohol consumption that is punished with footshock or bitter quinine. Also, alcohol-seeking that resists punishment increases in mice after a history of alcohol dependence, and this is associated with increased expression of NMDAR subunits GluN1 and GluN2A in the medial orbitofrontal cortex (OFC). A recent study on habitual alcohol seeking found that the GluN2B NMDAR subunit in the OFC, a brain region that connects to the dorsal striatum, mediates habitual alcohol seeking through a mechanism involving mTORC1 signaling. These findings align with previous reports that mTORC1 signaling is a key mechanism in heavy alcohol use and relapse.

Increased expression of the NMDAR GluN2B subunit in circuits connecting the cortex and striatum is critical for promoting alcohol seeking. NMDAR blockers have been effective in stopping both priming-induced reinstatement and relapse-like behavior after prolonged abstinence in the alcohol deprivation effect model. In contrast, NMDARs do not seem to play a role in cue-induced alcohol seeking. The medication acamprosate, which suppresses cue-induced alcohol seeking, was initially thought to act through NMDARs, but it is now understood that its effects are more complex and likely not directly mediated by NMDARs.

Similar to NMDARs, AMPAR function is enhanced after chronic alcohol exposure, and an NMDAR-dependent increase in AMPAR activity has been shown to trigger drug seeking. Accordingly, an AMPA positive allosteric modulator (PAM), aniracetam, strengthens cue-induced alcohol seeking in alcohol-preferring rats. Chronic alcohol also disrupts CaMKII-AMPA signaling in the PFC and amygdala, increasing the risk of alcohol relapse through CaMKII-dependent activation of AMPARs. In line with these findings, the selective AMPAR antagonist GYKI 52,466 blocks cue-induced reinstatement and the alcohol deprivation effect in rats. Furthermore, mixed AMPAR/kainate antagonists reduce cue-induced alcohol seeking. Selective kainate receptor antagonists have mostly been studied for their ability to reduce alcohol intake, and their potential effect on alcohol seeking has not yet been explored.

Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) may offer more promising therapeutic targets. These receptors are widely present in nerve cells and glial cells throughout the central nervous system. mGluRs are G protein-coupled receptors that mediate slower nerve signaling by controlling secondary messenger levels and ion channel activity. They are located near the synaptic cleft in both pre- and postsynaptic neurons. There are eight identified mGluRs, divided into three groups based on their similarities, signaling pathways, and drug properties. Group I (mGluR1 and 5) are mainly found in postsynaptic neurons and increase intracellular calcium levels when activated, leading to changes in gene expression. Groups II (mGluR2 and 3) and III (mGluR4, 6, 7, and 8) are mostly located at presynaptic terminals and in astrocytes, where they control the release of glutamate and other neurotransmitters. The involvement of mGluRs in alcohol seeking has been extensively studied, with most research focusing on mGluR5 and mGluR2 as potential drug targets.

Alcohol temporarily reduces mGluR1/5 function, but prolonged alcohol use increases both the expression and activity of these receptors. Studies have shown that activating mGluR5s in the NAc is essential for the internal effects of alcohol. Accordingly, mGluR5 antagonists and negative allosteric modulators (NAMs) reduce cue-induced alcohol seeking, whether given systemically or injected into the NAc or BLA. Research with the selective mGluR5 NAM MPEP showed that reduced mGluR5 signaling suppresses alcohol seeking by affecting the ERK1/2 signaling pathway. This pathway is activated in the amygdala's connections to the ventral striatum by alcohol-associated cues, and its activation is linked to increased cue-induced alcohol seeking, which MPEP counteracts.

These findings suggest that mGluR5s are involved in the learned association between alcohol cues and alcohol effects, an association that strengthens during addiction, persists into abstinence, and contributes to alcohol seeking. Besides blocking the recall of these alcohol memories, strengthening their extinction might also offer treatment opportunities. Exposure-based extinction therapy for alcohol cue-reactivity is a clinical treatment for alcohol addiction, but its effectiveness is limited and could potentially be enhanced by medications. In this context, the mGluR5 PAM CDPPB has been shown to facilitate the extinction of cue-conditioned alcohol seeking. This effect was mediated by mGluR5 modulation of certain potassium channels and was observed with both systemic and localized activation of mGluR5s.

In contrast to mGluR5, mGluR1's effects on alcohol seeking have not been widely studied, with most research focusing on how mGluR1 PAMs and NAMs affect alcohol consumption.

mGluR2-mediated control of glutamate signaling, through presynaptic modulation of glutamate release, has drawn significant interest as a drug target for several psychiatric disorders. Prolonged alcohol exposure has been shown to disrupt mGluR2 function by reducing the expression of the gene encoding this receptor. Deficiencies in mGluR2-mediated feedback inhibition of glutamate release in the cortex and amygdala have been shown to promote alcohol seeking. High levels of glutamate in the BLA and NAc have been detected during cue-induced alcohol seeking, along with an alcohol-induced decrease in mGluR2 in the mPFC. Genetically selected alcohol-preferring rats lack functional mGluR2s and show increased alcohol consumption and resistance to alcohol devaluation.

These findings are supported by studies showing that the mixed mGluR2/3 agonist LY379268 reduces alcohol seeking triggered by both cues and footshock stress. However, LY379268 and other similar drugs cannot differentiate between the specific contributions of mGluR2 and mGluR3. Therefore, researchers tested the selective mGluR2 PAM AZD8529 on alcohol consumption and seeking. AZD8529 strongly blocked cue-induced, but not stress-induced, alcohol seeking in rats. This effect was absent in alcohol-preferring rats lacking functional mGluR2s. While AZD8529 also reduced alcohol self-administration, its effect was much more pronounced and specific in blocking reinstatement caused by alcohol-associated cues. Together with previous findings, this indicates that mGluR2s are specifically involved in cue-induced alcohol seeking. The potential role of mGluR3 still needs to be explored. Thus, mGluR2 agonists or PAMs may have therapeutic potential in preventing relapse to alcohol use.

Results are generally less promising with drugs targeting group III mGluRs, mGluR7, and mGluR8. A mixed mGlu4/mGlu7 agonist was reported to reduce alcohol self-administration after abstinence when administered into the brain, while a mGluR8 agonist reduced cue-induced alcohol seeking, but only at doses that also caused motor suppression.

Targeting Glutamatergic Transmission for Therapeutic Purposes

The research clearly shows that glutamate signaling plays a critical role in alcohol seeking and relapse. The complex nature of ionotropic glutamate receptors and their subunits might seem to offer many opportunities for developing treatments for alcohol addiction. However, decades of attempts to develop drugs targeting NMDA receptors for conditions like stroke, epilepsy, and Parkinson's disease suggest otherwise. One possible conclusion is that the ionotropic effects of glutamate are too fundamental for brain function to allow modulation for therapeutic purposes while maintaining adequate safety and tolerability. In contrast, preclinical findings suggest that presynaptic agonists of mGluR2/3 receptors and antagonists of postsynaptic mGluR5 may be effective drug treatments for alcohol seeking behavior and relapse.

GABAergic Neurotransmission and Alcohol Seeking

GABAergic neurotransmission is crucial for a wide range of alcohol effects, from intoxication to alcohol seeking and relapse. GABA is the brain's main inhibitory neurotransmitter and reduces the activity of dopamine neurons in the reward system. It acts on two types of receptors: GABAA receptors (ligand-gated chloride channels) and GABAB receptors (G protein-coupled receptors that regulate potassium and calcium channels). Alcohol affects GABA signaling both before and after the synapse, with both immediate and long-term consequences. It has been suggested that an increased risk of relapse, driven by negative reinforcement, results from an imbalance in peptide-based signaling systems that affect GABA circuits within the extended amygdala, a brain region involved in fear and stress responses.

The CeA is involved in alcohol seeking induced by both footshock and yohimbine. Studies show that chronic alcohol exposure, which promotes various addiction-like behaviors, increases GABA release in the CeA through both pre- and post-synaptic mechanisms. Researchers recently found that impaired removal of GABA within the CeA, due to low expression of the GABA transporter GAT-3, is linked to a strong preference for alcohol over natural rewards and continued alcohol self-administration despite punishment. This decreased GAT-3 expression in the amygdala was accompanied by reduced expression of several GABAA receptor subunits, possibly as a compensatory response to the sustained increase in GABA activity. Notably, SP-mediated activation of NK1 receptors in the CeA promotes GABAergic transmission in this structure, and this effect is stronger in rats with a history of alcohol dependence. These findings suggest that drugs that can restore GABA balance within the CeA may have therapeutic potential for alcohol addiction.

GABAergic transmission in the BNST also contributes to stress-induced drug seeking and the negative emotions associated with addiction. The BNST receives many projections from the CeA, most of which are GABAergic. Studies have shown that surgically removing the BNST reduces cue-induced alcohol seeking in mice.

The BNST also sends extensive projections to the VTA, mainly to non-dopaminergic VTA neurons. These projections are made up of different populations of neurons that promote contrasting motivational states. Most VTA-projecting BNST neurons are GABAergic, and their activation produces anxiety-reducing and rewarding effects. In contrast, a smaller group of glutamate-releasing BNST inputs to the VTA causes anxiety and aversion. A population of CRH-expressing GABA neurons within the BNST controls the balance of these BNST outputs by inhibiting the anxiety-reducing neurons, and these neurons are themselves influenced by serotonin signals from the dorsal raphe nucleus. A recent study showed that chronic intermittent alcohol exposure disrupts this local GABAergic circuit in the BNST, which may promote alcohol seeking driven by negative reinforcement during withdrawal and prolonged abstinence. In this study, alcohol withdrawal led to increased activity of the CRH-expressing GABA neurons and decreased activity of the potentially anxiety-reducing non-CRH BNST neurons that project to the lateral hypothalamus (LH) and VTA.

In the striatum, chronic alcohol exposure reduces GABAergic transmission, which may separately contribute to increased alcohol seeking and intake. After prolonged alcohol exposure, a decrease in GABAergic transmission has been observed in the DLS, DMS, and NAc of mice and monkeys. This is accompanied by reductions in both the strength and frequency of inhibitory signals, suggesting that the underlying mechanism could be a decrease in GABA release or a reduction in the number of GABAergic connections to striatal neurons. Alcohol-induced weakening of inhibition in the DLS may therefore be a mechanism for habitual alcohol seeking.

Alcohol-induced changes in GABAergic transmission are partly due to changes in the function and sensitivity of GABAA and GABAB receptors. This makes it important to understand how GABAergic receptors adapt during the different stages of alcohol addiction.

GABAA Receptors in Relapse to Alcohol Seeking

Both GABAA and GABAB receptors contribute to the immediate and long-term effects of alcohol, including sedation, tolerance, withdrawal, and motivational effects.

GABAA receptors are ligand-gated chloride channels made up of five subunits arranged around a central pore. They include α(1–6) and β(1–3) subunits, which are always present, and can also contain γ(1–3), δ, ε, π, or θ subunits. Additionally, ρ(1–3) subunits exist but form a separate type of GABAA receptor. The combination of multiple subunits leads to a wide variety of receptors that differ between brain regions and determine how the receptor responds to alcohol. Acute and chronic alcohol exposure causes temporary changes in GABAA receptor subunit levels, composition, and location in the brain.

Alcohol enhances GABAA receptor-mediated signals by increasing GABA release from presynaptic terminals in many brain regions. Prolonged chronic alcohol exposure strengthens GABAergic transmission, and when alcohol is withdrawn, the brain becomes overexcited. Chronic alcohol exposure can also lead to GABAA receptor down-regulation, causing tolerance and withdrawal, and disrupting various behaviors. GABAA receptors in the CeA regulate alcohol consumption in alcohol-preferring rats, but there is little evidence that GABAA receptors play a significant role in alcohol seeking behaviors.

A recent study showed that long-term administration of a GABAA α1-preferring antagonist reduced alcohol self-administration but not alcohol seeking in baboons. In contrast, the same compound did not reduce drinking in rhesus macaques. In alcohol-preferring rats, a similar compound reduced alcohol-maintained responding when injected into the ventral pallidum, a key area for relapse to alcohol seeking. In mice lacking the GABAA α1 receptor subunit, reduced alcohol drinking was also accompanied by reduced consumption of saccharin and sucrose, suggesting that these effects are not specific to alcohol.

Role of GABAB Receptors in Alcohol Seeking

In contrast to the limited and conflicting evidence regarding GABAA receptors in alcohol seeking, extensive data have accumulated over recent years supporting a role for GABAB receptors in this behavior.

GABAB receptors are G-protein coupled and require two subunits (GABAB1 and GABAB2) to be functional. Activating GABAB receptors, both pre- and postsynaptically, inhibits neurotransmitter release by making neurons more negatively charged, which results from increased potassium and decreased calcium permeability. Preclinical and clinical evidence suggests that activating GABAB receptors holds promise as a treatment for alcohol addiction.

Results from GABAB activation in animal models of alcohol addiction have been reviewed recently. Much of the data comes from baclofen, a selective GABAB receptor agonist that has been approved for treating muscle spasms for over four decades. Baclofen has been shown to reduce the rewarding properties of alcohol and the severity of alcohol withdrawal. It also prevents the return of alcohol seeking. In the alcohol deprivation model of relapse-like drinking, baclofen eliminated alcohol intake in Sardinian alcohol-preferring rats after 7 days of abstinence. These findings were replicated with different alcohol concentrations. However, after long-term voluntary alcohol drinking with repeated periods of abstinence, chronic baclofen reduced relapse-like drinking only at the highest dose, and this effect was not specific, as it also caused sedation and weight loss.

Thus, in rodent models, the difference between baclofen doses that specifically affect alcohol-related behaviors and doses that cause non-specific sedative effects or impair performance (the "therapeutic index") is limited and varies. The inconsistent findings on baclofen doses in different relapse studies may be due to the use of different rat strains with innate differences in their GABAergic transmission. Two other factors that might influence baclofen's dose-response relationship are differences in alcohol drinking history and whether baclofen is given acutely or chronically.

Cue-induced relapse to alcohol seeking is reduced by baclofen across various species, including rats and baboons. In baboons, baclofen also helped animals stop responding for alcohol. Baclofen has also been reported to reduce yohimbine-induced alcohol seeking in rats and to lessen alcohol seeking in a fear memory task in mice, possibly by reducing the stress hormone corticosterone. This latter effect aligns with a well-documented clinical link between HPA axis activity, craving, and relapse to alcohol use. It also matches a recent clinical study that reported lower cortisol levels in alcohol-dependent patients treated with baclofen.

Clinically, baclofen has shown promising, though sometimes conflicting, results for treating alcohol addiction, but safety and tolerability concerns have prevented its approval as a clinical treatment. Some concerns relate to tolerance and dose escalation, which are expected with long-term use of G protein-coupled receptor agonists. GABAB receptor positive allosteric modulators (PAMs) have the potential to avoid these issues by targeting a different site on the receptor and enhancing the effect of GABA when it binds. GABAB PAMs have garnered significant interest as potential treatments for alcohol addiction in recent years. Most studies on GABAB PAMs support their improved selectivity and wider therapeutic window in reducing alcohol seeking and consumption.

For example, researchers recently found that the selective GABAB PAM, ADX71441, strongly suppressed both cue- and stress-induced alcohol seeking in rats. Additionally, ADX71441 reduced stress-induced brain activity in a network of interconnected brain structures, including the NAc shell, mPFC, and the dorsal raphe nucleus. Although neuronal activity in the CeA was also reduced by ADX71441, this reduction did not correlate with relapse-like behavior. These findings suggest that ADX71441 may act on distinct but converging brain networks that mediate relapse, or on a common pathway that promotes alcohol seeking triggered by stress, cues, or alcohol priming.

Furthermore, a new GABAB PAM, CMPPE, reduced relapse to alcohol drinking in a repeated alcohol deprivation model and suppressed cue-induced alcohol seeking in rats. It also showed effectiveness in reducing cue-induced alcohol seeking at lower doses in alcohol-preferring rats. Another novel GABAB PAM, COR659, suppressed cue-induced alcohol seeking at the lowest dose tested and reduced alcohol self-administration in a way that was maintained with chronic administration.

The specific mechanisms by which GABAB activation prevents relapse to alcohol seeking remain unclear. One possible mechanism is through inhibiting dopamine neurons in the reward system. Activating GABAB receptors in the VTA leads to decreased dopamine release in both the NAc and the mPPC. Moreover, a GABAB PAM injected into the VTA enhanced GABAB inhibition of dopamine neuron firing. Possibly related to these effects, injecting baclofen into the VTA dose-dependently suppressed cue-induced alcohol seeking in rats. It was recently reported that different dopamine pathways in the reward system control responses triggered by specific alcohol-associated cues versus alcohol-associated contexts. Inhibiting VTA inputs to the NAc core reduced alcohol seeking triggered by an alcohol-associated cue, regardless of the context. In contrast, silencing VTA inputs to the NAc shell selectively reduced cue-induced alcohol seeking only in an alcohol-associated context. If activating GABAB receptors can reduce the activity of VTA neurons in both these populations, it may be able to broadly prevent relapse triggered by both specific cues and contextual stimuli.

In summary, activating GABAB receptors appears to hold considerable promise as a treatment for preventing alcohol seeking and relapse. GABAB PAMs may be able to avoid the safety and tolerability issues that limit the use of the existing GABAB agonist, baclofen. Developing GABAB PAMs that are safe for human use and evaluating their effectiveness as alcohol addiction medications is currently one of the most promising avenues for developing new treatments for this condition.

Concluding Remarks

Animal models of drug seeking and extinction, introduced in the early 1980s, have been extensively used to study relapse mechanisms. Applying these models to alcohol seeking has generated a vast amount of research, much of which has been reviewed here. This work has led to significant progress in identifying multiple biological systems that contribute to alcohol seeking.

A major question facing the field is whether these advances and the identified biological systems can predict effective treatments for craving and relapse in people with alcohol addiction. Due to numerous failures in clinical development, pharmaceutical industry efforts to develop psychiatric medications have significantly decreased, and the ability of animal models to predict clinical activity in psychiatric disorders generally has been widely questioned. The situation is similar for addictive disorders.

So, are studies of alcohol seeking in animals a worthwhile effort? This review offers some insights into this critical question. It is not possible to draw broad conclusions that animal models of alcohol seeking and relapse either definitively do or do not predict clinical activity. The situation is much more complex. On one hand, human and animal findings with naltrexone and other drugs targeting MOP strongly support the predictive validity of models for both priming- and cue-induced reinstatement. Although somewhat more complex, findings with baclofen and GABAB activation also generally support the predictive validity of animal relapse models. On the other hand, the lack of success with CRH1 antagonists in clinical development, among other data, might suggest the opposite conclusion.

A preliminary interpretation of these observations is that a more nuanced view of animal findings is needed. Predictive validity is not an all-or-nothing concept and may not primarily be a property of the model itself. Instead, it likely varies significantly across the specific biological systems being studied. A key factor in this variation is probably the degree to which a system and its components are similar across species, a factor that has received too little attention. One implication of this understanding is that dedicating decades of preclinical studies to a single biological system may not be the best strategy, especially if alcohol addiction neuroscience aims to improve patient outcomes. Instead, signals from animal models should be tested as quickly as possible in human proof-of-principle, biomarker-based studies, as recently outlined and tested by initiatives for KOP antagonism in depression. If human translation is not supported, continuing preclinical work in that area comes with a high opportunity cost.

With this strategy in mind, a priority list of targets for rapid clinical evaluation is suggested by the reviewed literature. At the top of this list is the development of safe and well-tolerated GABAB PAMs, which should be evaluated for preventing both stress- and cue-induced craving and relapse in patients. A close second priority is the evaluation of KOP antagonists, which, based on the data, are predicted to prevent stress-induced craving and relapse, potentially offering a useful combination with naltrexone. In third position is the evaluation of mGluR2 PAMs. Researchers are less optimistic about interventions targeting dopamine or ionotropic glutamate receptors, partly due to their fundamental roles in brain function and behavior, which may make it difficult to develop medications that are both effective and have acceptable safety and tolerability.

Abbreviations

• P rats: Indiana alcohol Preferring rats

• CPP: conditioned place preference

• MOP: Mu-opioid receptor

• NAc: nucleus accumbens

• OFC: orbitofrontal cortex

• KOP: kappa-opioid receptor

• DOP: delta-opioid receptor

• NOP: nociceptin receptor

• DYN: dynorphin

• nor-BNI: nor-binaltorphimine

• CRH: corticotropin-releasing hormone

• BNST: bed nucleus of the stria terminalis

• JNK: c-Jun N-terminal kinase

• N/OFQ: nociceptin/orphanin FQ

• CeA: central nucleus of the amygdala

• GPCRs: G protein-coupled receptors

• HPA: hypothalamic-pituitary-adrenal

• SP: substance P

• NK: neurokinin

• VTA: ventral tegmental area

• (m)PFC: (medial) prefrontal cortex

• MSNs: medium spiny projection neurons

• ADE: alcohol deprivation effect

• COMT: Cathechol-O-Methyltransferase

• DLS: dorsolateral striatum

• iGluR: ionotropic glutamate receptors

• mGluR: metabotropic glutamate receptors

• BLA: basolateral amygdala

• DMS: dorsomedial striatum

• NMDAR: N-methyl-d-aspartate receptors

• AMPAR: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors

• OFC: orbitofrontal cortex

• PAM: positive allosteric modulator

• CaMKII: Ca2+/calmodulin-dependent protein kinase II

• NAM: negative allosteric modulator

• MPEP: 2-Methyl-6-(phenylethynyl)pyridine

• ERK1/2: extracellular signal-regulated kinases ½

• CDPPB: 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide)

• LH: lateral hypothalamus

• mIPSC: miniature inhibitory postsynaptic current

• sP: Sardinian alcohol preferring

Introduction

A major challenge in helping people with alcohol problems is stopping them from going back to drinking after they have stopped. Studies have shown that many people who stop drinking start again within three months. This number has not changed much over time. For people with alcohol problems, stress, things that remind them of alcohol, or even a small drink can make them want to drink heavily again. This strong urge to drink is often, but not always, what leads to a relapse.

Animal Models for Studying Alcohol Seeking

Studying Relapse in Animals

Scientists use animal models to study why people relapse. In these models, animals learn to get alcohol. Then, they are stopped from getting it, which makes them stop seeking it. After this, different things are used to see if the animals start seeking alcohol again. This helps scientists understand what makes someone relapse.

It can be hard to get animals to drink a lot of alcohol because they often don't like the taste of strong alcohol. To get them to drink, scientists sometimes make the animals thirsty or mix alcohol with sweet things. However, newer methods allow animals to drink alcohol without these extra steps.

Different Ways to Make Animals Seek Alcohol Again

After animals learn to drink alcohol, they go through a phase where they can no longer get alcohol by doing a certain action, like pressing a lever. They eventually stop trying. Then, different triggers are used to see if they start pressing the lever again. These triggers are meant to be like things that make people want to drink.

• Operant self-administration: Animals are taught to do something (like press a lever) to get a reward, such as food or a drug like alcohol. They learn to repeat the action if the reward makes them feel good.

• Reinstatement: After animals stop seeking a drug, scientists test if different triggers make them start seeking it again. This helps study relapse.

• Stress-induced reinstatement: Animals are given a drug along with certain cues (like a sound or light). They then stop seeking the drug. If they are exposed to stress, they might start seeking the drug again, even without the cues.

• Cue-induced reinstatement: Animals get a drug with certain cues. Then, they stop seeking the drug. If those cues are shown again, they might start seeking the drug.

• Drug-induced reinstatement: Animals get a drug with a cue. They stop seeking the drug. Then, a small amount of the drug itself is given, which can make them start seeking the drug again.

These methods show that cues related to alcohol or stressful situations can strongly make animals seek alcohol again. For example, some studies found that mild foot shocks made rats seek alcohol, but a small dose of alcohol did not.

However, stress in animals, like foot shocks, is different from the kind of stress people experience. This might mean that what we learn from animal studies about how stress causes relapse might not be exactly the same for humans. Scientists are trying to use animal models that are more like human social stress to see if the results are similar.

Other less common ways to study alcohol seeking in animals include models where they learn to prefer certain places associated with alcohol, or models where they run down a path to get alcohol.

Compulsive Alcohol Seeking

A key sign of alcohol addiction is when someone keeps drinking even when bad things happen because of it. Understanding how drinking goes from being controlled to being compulsive is a big goal in addiction research. Most animal studies have looked at compulsive drinking, such as when animals keep drinking alcohol even if it tastes bad or if they get a small electric shock with it. But studying compulsive alcohol seeking, where animals keep trying to get alcohol even when it's not available, is a newer area of study.

In one study, researchers trained rats to get alcohol in a specific way. First, rats learned that a light meant alcohol was coming. Then, they learned to press a "taking lever" to get alcohol when the light was on. Next, they had a "seeking lever" that, when pressed, made the "taking lever" appear. If they pressed the taking lever, they got alcohol and the light came on. Finally, they made the "seeking lever" sometimes give a small electric shock.

Using this method, they found that about one-third of the rats kept pressing the seeking lever even with the shocks. Another third greatly reduced their seeking, and the rest showed some reduction. This shows that some animals become compulsive seekers more easily than others.

Another way to study compulsive alcohol seeking uses different signs of addiction, similar to what doctors use to diagnose addiction in people. In these studies, some rats kept seeking alcohol even when it was not given, and some kept seeking it despite getting a shock. This confirmed that some animals are more likely to develop addictive behaviors.

Neurobiological Mechanisms of Alcohol Seeking

Opioid Systems and Alcohol Seeking

Opioid systems are a good place to start when looking at how the brain causes alcohol seeking. Medicines like naltrexone, which block opioids, are approved to help people with alcohol addiction. Naltrexone helps reduce heavy drinking, but it does not necessarily stop someone from drinking completely. This means it helps prevent a small slip from becoming a full relapse. This is similar to how naltrexone stops animals from seeking alcohol again after a small dose of alcohol. It also seems to help reduce the urge to drink when people see things related to alcohol.

In people with alcohol problems, certain opioid receptors in the brain are more active, which is linked to a stronger urge to drink. Also, when social drinkers have alcohol, their brains release natural opioids. These findings suggest that blocking opioid receptors can help reduce alcohol craving and relapse, supporting what we see in animal models.

Opioid systems are complex, involving different brain chemicals (like endorphins and dynorphins) and their receptors (like MOP, KOP, DOP, and NOP). These systems play many roles in addiction. Opioid receptors are found in important brain areas for drug rewards. Activating MOP receptors seems to increase the pleasant effects of alcohol, while activating KOP receptors causes unpleasant feelings.

MOP Receptors and Alcohol Seeking

Like in humans, a drug that blocks many opioid receptors, naltrexone, stopped animals from seeking alcohol when given a small dose of alcohol. Blocking MOP receptors also reduced animals' desire for alcohol and stopped them from seeking it when they saw alcohol-related things. Naltrexone seemed to stop seeking caused by cues, but not seeking caused by stress. Other studies also found that naltrexone and a more specific MOP blocker stopped cue-induced alcohol seeking.

However, giving naltrexone while animals were learning to ignore alcohol cues did not change how much they wanted alcohol later, or help them forget the cues faster. A newer, more specific MOP blocker, GSK1521498, reduced alcohol drinking and cue-induced alcohol seeking in rats. It also reduced compulsive alcohol seeking, especially in rats that were already compulsive. This suggests that this drug might be very helpful for people with severe alcohol addiction.

Overall, animal studies strongly suggest that blocking MOP receptors can help prevent alcohol craving and relapse, matching what is seen in people. However, the effects in humans are only moderate. It's not clear if newer, more specific MOP blockers will work better than current medicines.

KOP Receptors and Alcohol Seeking

KOP receptors and a natural brain chemical called dynorphin (DYN) are very important in how stress and bad feelings affect addiction, including alcohol addiction. Drinking a lot of alcohol for a long time can change the KOP/DYN system, leading to bad moods that make people drink more to feel better. Increased KOP activity during the time someone is not drinking might contribute to these bad feelings, which then make them seek alcohol. So, blocking KOP receptors might be a good way to treat alcohol addiction.

In rats, a KOP blocker called nor-BNI stopped alcohol seeking caused by stress, whether it was physical stress (foot shock) or a stress-inducing drug. Also, turning on KOP receptors made animals seek alcohol again, and nor-BNI stopped this. This stress-induced seeking was also stopped by another drug that blocks CRH1 receptors, suggesting that DYN acts before CRH in causing stress-induced relapse. Nor-BNI has also been shown to reduce cue-induced alcohol seeking. Other KOP blockers have also been found to reduce anxiety and cue-induced relapse in rats.

Because of these findings, blocking KOP receptors is seen as a possible treatment for alcohol addiction. However, old KOP blockers had problems, like long-lasting effects or side effects like heart problems. Newer KOP blockers are now being developed and tested.

One new KOP blocker, CERC-501, was tested in animals. It stopped anxiety from alcohol withdrawal and blocked stress-induced alcohol seeking. These effects were specific to stress, meaning it did not affect cue-induced seeking or increased nicotine drinking. This supports the idea that KOP activation is mainly linked to bad moods that encourage alcohol seeking. CERC-501's effects are different from naltrexone, which mainly stops cue-induced relapse. This suggests that combining KOP and MOP blockers could be a good treatment strategy. CERC-501 also seemed safe in animals, as it did not affect alcohol's calming effects, how the body processes alcohol, or general movement.

DOP Receptors and Alcohol Seeking

Not many studies have looked at the role of DOP receptors in alcohol seeking, but some suggest they might be involved in cue-induced seeking. For example, a specific DOP blocker reduced alcohol seeking caused by alcohol-related cues and environments. Another DOP blocker also reduced alcohol seeking caused by a stress-inducing drug.

NOP Receptors in Alcohol Seeking

Nociceptin/orphanin FQ (N/OFQ) is a brain chemical that works with NOP receptors. This system is involved in pain, emotions, and other body functions. It has also been studied a lot in addiction models, including alcohol. At first, scientists thought that turning on NOP receptors would reduce the desire for addictive drugs. However, blocking NOP receptors also showed similar effects, making it unclear whether turning them on or off would be better for treating addiction.

Turning on NOP receptors, with nociceptin or similar drugs, has been shown to reduce alcohol withdrawal symptoms, relapse after stopping alcohol, and stress-induced alcohol seeking. This was seen in both normal rats and rats that were more dependent on alcohol. Giving nociceptin into the brain also stopped cue-induced alcohol seeking in rats. Studies show that alcohol causes bigger changes in the N/OFQ-NOP system in alcohol-dependent rats.

Researchers also found that a strong NOP-activating drug, SR-8993, reduced anxiety from alcohol withdrawal and lessened both stress- and cue-induced relapse in rats. But, surprisingly, a NOP-blocking drug, LY2940094, also had similar effects, preventing alcohol drinking and stress-induced alcohol seeking. It also stopped alcohol from causing dopamine release in the brain.

To understand these confusing results, some scientists think that NOP receptors might quickly become less sensitive when activated by drugs. This could mean that giving NOP-activating drugs might actually lead to effects similar to blocking the receptors.

Right now, it's not clear if targeting the NOP system is a good way to treat alcohol addiction, or if activating or blocking the receptors would be best. More studies in humans are needed to figure this out. Early human studies with a NOP blocker were not clear, as the main goal was not met, but some other positive effects were seen.

Other Peptides Involved in Alcohol Seeking

Corticotropin-releasing Hormone (CRH)

CRH is a brain chemical known for its role in stress. It is found in many brain areas important for alcohol seeking. CRH works through two main types of receptors, CRH1 and CRH2. Stress-induced alcohol seeking is mainly controlled by CRH1 receptors in certain brain areas. CRH1 signaling acts like an "alarm system" that turns on during difficult stress.

Blocking CRH signaling strongly stops stress-induced alcohol seeking but does not affect seeking caused by cues. This was first shown with drugs given directly into the brain or taken systemically. Studies also showed that CRH1 blockers work even if the stress response system in the body (HPA axis) is not active. In rats with a history of alcohol dependence, CRH and CRH1 receptors are more active in certain brain areas, making them more sensitive to CRH1 blockers for stress-induced relapse.

All these findings in animals suggest that CRH1 receptors mainly control stress-induced relapse, not cue-induced relapse. This led to the idea that CRH1 blockers could stop stress-induced craving in people with alcohol problems, which is a known sign that predicts relapse.

However, studies in humans have not supported these animal findings. Even with drugs that were thought to be very effective at blocking CRH1 receptors, the results were not positive. Given that CRH1 blockers have also failed in other stress-related mental health conditions and their development has stopped, it is unlikely this approach will be used to treat alcohol addiction.

Substance P (SP) and its Neurokinin 1 (NK1) Receptor

SP is a brain chemical that works with NK1 receptors. These receptors are found in many brain regions involved in both wanting good things and feeling bad, and they affect stress responses and alcohol-related behaviors.

It has been hard to study NK1 receptors in animals using drugs made for humans, because the receptors are different between species. However, a special NK1 blocker for rats was developed. Using this drug, scientists found that blocking NK1 receptors throughout the body stopped stress-induced relapse but did not affect cue-induced relapse. In rats that prefer alcohol, NK1 receptors in a specific brain area are more active, making them more sensitive to stress-induced relapse. Blocking these receptors in that area reduced the stress-induced relapse. Also, increasing NK1 receptors in that brain area made rats more sensitive to stress-induced relapse. Recent studies also suggest that NK1 receptor activation by SP increases another brain chemical, GABA, in this area, and this effect is stronger after a history of alcohol dependence. These findings show that NK1 receptors in this brain area make animals more likely to relapse due to stress.

Based on animal findings, an NK1 blocker was tested in people with alcohol addiction. It reduced stress-induced craving and brain responses to negative emotions. However, a later larger study did not show strong positive results in all patients.

The development of NK1 blockers was partly driven by the hope that they could treat depression. But due to inconsistent results in depression studies, drug companies stopped developing them for stress-related mental health problems. It was later found that NK1 blockers need to block almost all of the receptors to be effective, which might not have been achieved in earlier studies. It is still possible that NK1 blockers could be a useful treatment for anxious people with alcohol addiction, if the drugs are strong enough and given in the right way. But it is unlikely this will be tested.

Role of Dopamine in Alcohol Seeking

Alcohol makes certain brain cells release more dopamine in areas related to reward and thinking, like the NAc and mPFC. The rewarding effects of alcohol partly depend on this dopamine release. Blocking dopamine receptors reduces both how much alcohol animals drink and how much they seek it. Alcohol-induced dopamine release also causes brain changes that can lead to addiction. These changes in brain areas for reward and memory can weaken self-control and make people more likely to relapse. The role of dopamine depends on the brain area, how long alcohol has been used, and the type of alcohol-seeking behavior being studied.

Dopamine works through five types of receptors, divided into two groups: D1-like (D1 and D5) and D2-like (D2, D3, D4). D1 and D2 receptors are the most common and are found in brain areas related to reward. While most studies focus on D1 and D2, D3, D4, and D5 might also have specific roles in alcohol seeking. However, it's hard to tell them apart without specific drugs for each type.

Long-term alcohol use and withdrawal affect dopamine levels and change D1 and D2 receptor sites in brain areas like the NAc and amygdala. Lower D2 receptor activity in a part of the brain is also linked to alcohol making animals prefer certain places. These changes in dopamine might cause an imbalance in brain cells that then encourages alcohol seeking.

Drugs that increase or stabilize dopamine levels have been shown to stop relapse-like drinking after animals have been without alcohol. Blocking an enzyme that breaks down dopamine also reduces cue-induced relapse in male rats. Interestingly, both increasing and decreasing dopamine signals can affect alcohol seeking in similar ways, either by acting like a replacement medicine or by stopping the rewarding effects of alcohol. How alcohol changes dopamine signals depends heavily on when the alcohol was used, which makes understanding the findings tricky.

Problems with D2-like receptor signaling, in particular, are linked to alcohol seeking. Blocking D2 receptors in the NAc reduces alcohol-seeking actions, which might be due to the D2 receptor's role in how the brain processes cues and goal-directed behavior. After longer periods of alcohol use, the importance of D2-like receptors seems to move to another brain area, the dorsal striatum. Blocking dopamine D1/D2 receptors in this area reduced seeking, and how sensitive an animal was to this blocker predicted how likely they were to become compulsive alcohol seekers. This blocker also stopped alcohol seeking when given in the amygdala, but not in the NAc. This supports the idea that the amygdala plays a role in shifting drug seeking from being controlled by one brain area to becoming a habit controlled by another.

The dopamine D3 receptor might be especially important for alcohol seeking triggered by cues. D3 blockers stop cue-induced alcohol seeking. Also, giving a D3 blocker or a partial activator stops relapse-like behavior after animals have been without alcohol for a long time. Alcohol increases D3 receptors in the dorsal striatum, which suggests they might contribute to alcohol seeking and relapse.

In a study of cocaine use, a model was proposed for how likely an animal is to become addicted. It looked at three things: not being able to stop during a signaled no-drug period, how much effort an animal would put in to get the drug, and continuing to take the drug despite shocks. Rats that met all three criteria also sought more cocaine. A similar model was used for alcohol. Rats that met all three criteria showed changes in D1 and D2 dopamine receptors in the DLS months later, suggesting that dopamine problems can last a long time and are involved in compulsive alcohol seeking. Interestingly, while D4 blockers don't affect cue-induced relapse, they do stop stress-induced relapse.

In summary, D1-like dopamine signals in the NAc seem important for controlling how much alcohol is consumed. D2-like receptors in the dorsal striatum appear most important for alcohol seeking and relapse. At the same time, D1 receptors in another part of the brain (mPFC) are key for cocaine seeking caused by cocaine itself. And stress-induced activation of dopamine pathways from the VTA to the mPFC might cause cocaine seeking through a different pathway to the NAc. Since removing certain mPFC neurons that go to the NAc has been shown to stop cue-induced alcohol seeking, it's possible similar pathways are involved in alcohol seeking guided by cues.

Glutamate Signaling in Alcohol Seeking Behaviors

Alcohol seeking is strongly linked to changes in glutamate, a brain chemical, in parts of the brain that form the amygdala-cortex-striatum circuits. This involves two main types of glutamate receptors: ionotropic (iGluR) and metabotropic (mGluR). It is thought that as alcohol use becomes more compulsive, control shifts from metabotropic to ionotropic receptors. However, the role of these receptors can differ based on where they are in the brain. Stronger glutamate activity after long-term heavy drinking leads to lasting changes in brain connections that keep alcohol seeking going. Problems with brain flexibility caused by long-term alcohol use can also make habitual seeking behaviors stronger.

The ventral and dorsal parts of the striatum are involved in different ways in alcohol addiction. They receive glutamate signals from the amygdala and cortex, which also interact with dopamine signals. Studies have shown that glutamate levels increase in the amygdala and NAc core when animals are seeking alcohol due to cues. Also, the pathway from the mPFC to the NAc is needed for cue-induced alcohol seeking. Removing specific glutamate neurons in this pathway stopped cue-induced relapse without affecting how animals stopped seeking alcohol. Removing connections from the amygdala to the NAc also stopped relapse.

It is thought that, like with other addictive drugs, alcohol seeking becomes a bad habit that shifts from being controlled by the ventral and dorsomedial striatum (DMS) to the dorsolateral striatum (DLS). While DMS gets glutamate signals from brain areas related to thought, DLS gets signals from areas related to movement and senses. Stronger glutamate signals to the DLS, along with changes in dopamine, are key reasons why habitual alcohol seeking develops.

So, many glutamate-related changes in the brain lead to behaviors similar to alcohol addiction, including alcohol seeking. The next sections will look at specific ways that glutamate systems are involved and how targeting them might help.

Ionotropic Glutamate Receptors

Glutamate directly affects brain cell activity through iGluRs, which include NMDAR, AMPAR, and kainate receptors. These receptors are like gates that open to let charged particles into brain cells, and they are crucial for how brain connections change in areas important for alcohol seeking and taking. Long-term alcohol use and withdrawal increase the activity and number of both NMDARs and AMPARs, leading to brain changes that reduce behavioral flexibility. NMDARs have been studied a lot for their role in both cue-induced alcohol seeking and compulsive alcohol seeking (seeking despite negative effects).

As mentioned earlier, compulsive alcohol seeking has been found to develop along with compulsive drinking. Studies have shown that changes in NMDARs in the NAc due to alcohol make animals continue drinking alcohol even when punished with shocks or bad taste. Also, mice that kept seeking alcohol despite punishment after being alcohol-dependent showed more NMDAR subunits (GluN1 and GluN2A) in a brain area called the medial orbitofrontal cortex (OFC). A recent study looked at how habitual alcohol seeking develops. They found that a specific NMDAR subunit, GluN2B, in the OFC (a brain area that connects to the dorsal striatum) was involved in habitual alcohol seeking through a mechanism called mTORC1 signaling. These findings fit with earlier reports that mTORC1 signaling is a key part of heavy alcohol use and relapse.

An increase in the GluN2B NMDAR subunit in certain brain circuits is important for promoting alcohol seeking relapse. Drugs that block NMDARs have been effective in stopping relapse caused by a small dose of alcohol or after a long time without alcohol. However, NMDARs do not seem to play a role in cue-induced alcohol seeking. A medicine called acamprosate, which is approved for clinical use, did reduce cue-induced alcohol seeking, and this was thought to be due to its effects on NMDA, but it's now clear that acamprosate works in complex ways, not directly on NMDARs.

Like NMDARs, AMPAR function also increases after long-term alcohol exposure, and this increase has been shown to trigger drug seeking. So, a drug that makes AMPARs more active, aniracetam, makes cue-induced alcohol seeking stronger in rats that prefer alcohol. Long-term alcohol use also disrupts a system involving CaMKII and AMPA in the PFC and amygdala, which increases the risk of relapse through CaMKII-driven activation of AMPARs. In line with this, a selective AMPAR blocker stopped cue-induced relapse and craving after being without alcohol in rats. Also, drugs that block both AMPAR and kainate receptors can reduce cue-induced alcohol seeking. Selective kainate receptor blockers have mainly been studied for reducing alcohol intake, and their effect on alcohol seeking has not been widely explored.

Metabotropic Glutamate Receptors

Metabotropic glutamate receptors (mGluRs) might be more promising targets for treatment. They are found widely in brain cells and control slow brain signals by affecting other molecules inside the cells. mGluRs are located near the connections between brain cells. Eight types of mGluRs have been found, grouped into three categories based on how they work. Group I (mGluR1 and 5) are mainly on the receiving end of brain cells and increase calcium levels inside the cell, which can change gene activity. Group II and III (mGluR2, 3, 4, 6, 7, 8) are mainly on the sending end of brain cells and in support cells, where they control the release of glutamate and other brain chemicals. Scientists have studied mGluRs a lot for their role in alcohol seeking, especially mGluR5 and 2, as possible targets for medicines.

Alcohol first weakens mGluR1/5 function, but long-term alcohol use then makes these receptors more active. Studies showed that activating mGluR5 in a certain brain area (NAc) is crucial for how alcohol makes animals feel internally. So, drugs that block mGluR5, or drugs that make them less active, reduce cue-induced alcohol seeking, both when given systemically and when injected directly into the NAc or BLA. A specific mGluR5 blocker, MPEP, was shown to reduce alcohol seeking relapse by affecting a signaling pathway in the brain called ERK1/2. This pathway is activated by alcohol cues in the amygdala and contributes to increased cue-induced alcohol seeking, which MPEP then stops.

One way to understand these findings is that mGluR5s are involved in learning to connect alcohol cues with alcohol effects. This learning gets stronger with addiction, lasts even after someone stops drinking, and contributes to alcohol seeking when there is no alcohol reward. Besides blocking these alcohol memories, helping to "unlearn" them might also be a treatment. Therapy that exposes people to alcohol cues is used to treat alcohol addiction, but it's not always very effective. Medicines might make it better. In this regard, a drug that makes mGluR5 more active, CDPPB, has been shown to help animals "unlearn" cue-conditioned alcohol seeking. This effect was linked to mGluR5 affecting certain potassium channels, and was seen when the drug was given systemically or in a specific brain area.

The effects of mGluR1 on alcohol seeking have not been studied much, with most research focusing on how drugs affecting mGluR1 change alcohol consumption.

mGluR2's control over glutamate release has gained a lot of interest as a drug target for mental health problems. Long-term alcohol use has been shown to disrupt mGluR2 function by reducing the gene that makes this receptor. Problems with mGluR2's ability to reduce glutamate release in certain brain areas have been shown to promote alcohol seeking relapse. High levels of glutamate in the BLA and NAc have been found during cue-induced alcohol seeking relapse, along with less mGluR2 activity in the mPFC due to alcohol. Rats that prefer alcohol naturally have fewer mGluR2s and show increased alcohol consumption and are less affected by efforts to reduce alcohol's value.

In line with these findings, a drug that activates both mGluR2/3, LY379268, has been shown to reduce alcohol seeking relapse caused by cues and by footshock stress. However, this drug cannot tell apart the roles of mGluR2 and mGluR3. So, researchers tested a specific mGluR2-activating drug, AZD8529, on alcohol drinking and seeking. It strongly blocked cue-induced, but not stress-induced, alcohol seeking relapse in rats. This effect was not seen in rats that prefer alcohol and lack working mGluR2s. While AZD8529 also reduced alcohol drinking, its effect was much stronger and more specific in blocking relapse caused by alcohol-related cues. Along with earlier findings, this suggests that mGluR2s are specifically involved in cue-induced alcohol seeking. The role of mGluR3 still needs to be explored. Thus, drugs that activate mGluR2 might be good treatments to prevent alcohol relapse.

Results are generally less promising for drugs targeting group III mGluRs, mGluR7, and mGluR8. A drug that activates both mGlu4/mGlu7 was reported to reduce animals starting to self-administer alcohol again after stopping. And a drug that activates GluR8 reduced cue-induced alcohol seeking relapse, but only at doses that also made animals move less.

Targeting Glutamatergic Transmission for Treatment

The findings clearly show that glutamate signals are very important for alcohol seeking and relapse. The many types of ionotropic glutamate receptors and their parts might seem like many opportunities to make medicines for alcohol addiction. However, decades of trying to make drugs for NMDA receptors for conditions like stroke, epilepsy, and Parkinson's disease suggest otherwise. One reason might be that the immediate effects of glutamate are too basic for brain function, making it hard to change them for treatment without causing too many side effects. In contrast, animal studies suggest that drugs that activate mGluR2/3 receptors (on the sending side of brain cells) and drugs that block mGluR5 receptors (on the receiving side of brain cells) might be effective treatments for alcohol seeking and relapse.

GABAergic Neurotransmission and Alcohol Seeking

GABA, the main brain chemical that slows down brain activity, plays a big role in many alcohol effects, from feeling drunk to seeking alcohol and relapsing. It slows down the activity of dopamine neurons in the reward system of the brain. GABA works on two types of receptors: GABAA receptors (which are like gates for chloride) and GABAB receptors (which are linked to G proteins and control potassium and calcium channels). Alcohol affects GABA signals in different ways, both immediately and over a long time. It is thought that a problem with certain brain chemical systems that affect GABA in the extended amygdala (which includes the CeA, NAc shell, and BNST) leads to a greater chance of relapse due to feeling bad.

The CeA is involved in alcohol seeking caused by foot shocks and a stress-inducing drug. Studies show that long-term alcohol exposure, which leads to many alcohol addiction-like behaviors, increases GABA release in the CeA. This is due to effects both before and after the connection between brain cells. In line with this, scientists recently found that poor removal of GABA in the CeA, due to low levels of a GABA transporter (GAT-3), was linked to choosing alcohol over a natural reward and continuing to drink alcohol despite punishment or bad taste. Less GAT-3 in the amygdala was also linked to less GABAA receptor subunits, possibly because the brain was trying to adjust to the constant high levels of GABA. Importantly, SP activating NK1 receptors in the CeA boosts GABA activity there, and this effect is stronger in rats with a history of alcohol dependence. These findings suggest that medicines that can restore normal GABA levels in the CeA might help treat alcohol addiction.

GABA signals in the BNST also contribute to stress-induced drug seeking and the bad feelings linked to addiction. The BNST is a main target for CeA neurons, most of which use GABA. Studies have shown that damaging the BNST reduces cue-induced alcohol seeking in mice.

The BNST also sends many signals to the VTA. Most of these signals go to non-dopamine neurons in the VTA and create different motivations. Specifically, most BNST neurons that go to the VTA use GABA, and activating them reduces anxiety and feels rewarding. In contrast, a smaller group of BNST neurons that use glutamate and go to the VTA increase anxiety and cause bad feelings. A group of GABA neurons in the BNST that produce CRH controls the balance between these outputs. Recent research showed that repeated alcohol exposure causes problems in this local GABA circuit in the BNST, which might promote alcohol seeking due to feeling bad during withdrawal and long-term abstinence. In this study, alcohol withdrawal led to increased activity of the CRH-producing GABA neurons and decreased activity of other BNST neurons that reduce anxiety.

In the striatum, long-term alcohol exposure reduces GABA activity, and this might contribute to increased alcohol seeking and drinking. After long-term alcohol exposure, GABA activity has been shown to decrease in the DLS, DMS, and NAc of mice and monkeys. This comes with a decrease in the strength and frequency of small inhibitory signals between brain cells, suggesting that there might be less GABA released or fewer GABA connections. So, alcohol weakening GABA's inhibitory effect in the DLS might be a reason for habitual alcohol seeking.

Changes in GABA activity due to alcohol are partly because of changes in how GABAA and GABAB receptors work and how sensitive they are. This makes it important to understand how GABA receptors adapt during different stages of alcohol addiction.

GABAA Receptors in Relapse to Alcohol Seeking

Both GABAA and GABAB receptors play a role in both immediate and long-term effects of alcohol, including feeling drunk, getting used to alcohol, withdrawal, and motivation.

GABAA receptors are like gates that let chloride into brain cells and are made of five parts that surround a central opening. These parts can be different types, which leads to many different kinds of GABAA receptors in various brain areas. This variety affects how the receptors respond to alcohol. Alcohol makes GABAA receptor signals stronger by increasing GABA release from brain cells in many brain regions. Long-term alcohol use makes GABA signals stronger, and when alcohol is stopped, the brain becomes overactive during withdrawal. Long-term alcohol exposure can also reduce GABAA receptors, leading to tolerance and withdrawal, and messing up many behaviors. GABAA receptors in the CeA control alcohol drinking in rats that prefer alcohol, but there is not much evidence for their role in alcohol seeking behaviors.

A recent study showed that taking a GABAA alpha1-preferring blocker for a long time reduced alcohol drinking but not alcohol seeking in baboons. However, the same drug did not reduce drinking in other monkeys. In rats that prefer alcohol, a similar drug reduced alcohol drinking when injected into a key brain area involved in relapse. In mice without the GABAA alpha1 receptor part, reduced alcohol drinking also came with reduced sugar drinking, suggesting these effects are not only for alcohol.

Role of GABAB Receptors in Alcohol Seeking

Unlike the confusing evidence for GABAA receptors in alcohol seeking, a lot of data has shown that GABAB receptors are important for this behavior.

GABAB receptors are G-protein coupled receptors and need two parts to work. Activating GABAB receptors, both before and after the connection between brain cells, stops the release of brain chemicals by making brain cells less active. Animal and human studies suggest that activating GABAB receptors shows promise as a treatment for alcohol addiction.

Studies on GABAB activation in animal models of alcohol addiction were recently reviewed. Much of the data comes from baclofen, a drug that activates GABAB receptors and has been approved for muscle spasms for decades. Baclofen has been shown to reduce the rewarding effects of alcohol and the severity of alcohol withdrawal. It also prevents animals from seeking alcohol again. In a model of relapse-like drinking after being without alcohol, baclofen stopped alcohol intake in rats that prefer alcohol. These findings were repeated with different alcohol strengths. However, after long-term voluntary alcohol drinking with repeated periods of stopping, baclofen only reduced relapse-like drinking at the highest dose given for a long time. This effect was not specific, as this dose also caused calming effects and reduced food intake, leading to weight loss.

So, in animal models, the difference between baclofen doses that specifically affect alcohol behaviors and those that cause calming or other problems is small and varies. The different baclofen doses needed in various relapse studies might be due to using different types of rats, as they might have natural differences in their GABA systems. Two other factors that might affect baclofen's effects are how long animals have been drinking alcohol and whether baclofen is given for a short or long time.

Cue-induced alcohol seeking relapse is reduced by baclofen in many animals, including rats and baboons. In baboons, baclofen also helped them "unlearn" to seek alcohol. Baclofen has also been reported to reduce alcohol seeking relapse caused by a stress-inducing drug in rats, and to reduce alcohol seeking in mice during a smell test, likely by reducing the stress hormone response. This fits with what is known in humans about the link between stress system activity, craving, and alcohol relapse. It also matches a recent human study that found lower stress hormone levels in alcohol-dependent patients treated with baclofen.

In people, baclofen has shown promising, but mixed, results for treating alcohol addiction. However, concerns about safety and side effects have kept it from being approved as a treatment. Some concerns are about tolerance and needing higher doses over time, which is expected with long-term use of drugs that activate GPCRs. Drugs that make GABAB receptors more active (called PAMs) might avoid these issues. They work by making GABA's effect stronger when it binds to the receptor, rather than directly activating the receptor. GABAB PAMs have attracted a lot of interest as potential treatments for alcohol addiction recently. Most studies on GABAB PAMs support their better specificity and wider safety margin in reducing alcohol seeking and drinking.

For example, researchers recently found that a specific GABAB PAM, ADX71441, strongly stopped both cue- and stress-induced alcohol seeking relapse in rats. ADX71441 also reduced stress-induced brain activity in a network of brain areas. Surprisingly, while brain activity in the CeA was also reduced by ADX71441, it did not seem to be directly linked to the relapse-like behavior. Based on these findings, ADX71441 might work on different brain networks that lead to relapse, or on a common pathway that causes alcohol seeking from stress, cues, or a small drink.

Another new GABAB PAM, CMPPE, reduced alcohol relapse in a repeated alcohol deprivation model and stopped cue-induced alcohol seeking relapse in rats. It also worked at lower doses to reduce cue-induced alcohol seeking relapse in rats that prefer alcohol. A third new GABAB PAM, COR659, stopped cue-induced alcohol seeking relapse at the lowest dose tested and reduced alcohol self-administration, an effect that lasted with long-term use.

How GABAB activation stops alcohol seeking relapse is still unclear. One idea is that it works by slowing down dopamine neurons in the reward system. Activating GABAB receptors in the VTA reduces dopamine release in the NAc and mPFC. Also, a GABAB PAM injected in the VTA made GABAB's slowing effect on dopamine neurons stronger. It is thought that baclofen injected into the VTA reduced cue-induced alcohol seeking in rats. Recent research showed that different dopamine pathways control responses to specific alcohol cues versus general alcohol-related environments. Blocking signals from the VTA to the NAc core reduced alcohol seeking caused by a specific cue, regardless of the environment. In contrast, silencing signals from the VTA to the NAc shell specifically reduced cue-induced alcohol seeking in an alcohol-related environment. If activating GABAB receptors can reduce the activity of VTA neurons in both these groups, it might be able to prevent relapse broadly, whether caused by specific cues or general environments.

In short, activating GABAB receptors seems very promising as a way to prevent alcohol seeking and relapse. GABAB PAMs might avoid the safety and tolerability problems that limit the use of baclofen. Developing safe GABAB PAMs for human use and testing how well they work as alcohol addiction medicines is currently one of the most promising paths to finding new treatments for this problem.

Conclusion

Since the early 1980s, animal models that involve making animals seek drugs again after stopping have been widely used to study how relapse happens. Using these models for alcohol seeking has produced a lot of research, much of which has been reviewed here. This work has made great strides in recent decades and has found many biological systems that contribute to alcohol seeking.

The main question for the future is whether these discoveries and the biological systems they have found can predict how well treatments will prevent craving and relapse in people with alcohol addiction. Because of many failures in testing new drugs, the drug industry has greatly reduced its efforts to develop mental health medicines. The ability of animal models to predict how well drugs work for mental health problems, in general, has been questioned. The situation for addiction is similar.

So, are studies of alcohol seeking in animals worthwhile? This review offers some answers to this important question. We do not believe that the data support a general conclusion that animal models of alcohol seeking and relapse either can or cannot predict how well drugs work in humans in a meaningful way. The situation is actually much more complicated. On one hand, findings in both humans and animals with naltrexone and other tools that target MOP strongly suggest that models of relapse caused by a small drink or by cues are good predictors. While a bit more complex, findings with baclofen and GABAB activation also generally support that animal relapse models can predict human effects. On the other hand, the failure of CRH1 blockers in human studies, along with other data, might suggest the opposite conclusion.

Our first attempt to put these observations together is that we need a more careful look at the animal findings. Predicting how a drug will work is not an all-or-nothing thing, and it might not mainly depend on the model itself. Instead, it is likely to change a lot depending on the specific biological system being studied. A key factor that causes this difference is probably how similar the system and its parts are across different species, something that has not been given enough attention. One important point from this idea is that spending decades on studying any single biological system in animals might not be the best approach, at least if alcohol addiction science wants to help patients get better. Instead, clues from animal models should be tested as quickly as possible in early human studies that look for specific signs in the body, as recently tested by a special project for KOP blockers in depression. If human studies do not support the animal findings, continuing animal research on that system wastes a lot of time and resources.

With this strategy in mind, we believe that the research reviewed here suggests a list of targets for quick human testing. At the top of this list, in our opinion, is the development of safe and well-tolerated GABAB PAMs. These should be tested in patients to prevent both stress- and cue-induced craving and relapse. A close second priority is testing KOP blockers, which, based on the data, are predicted to prevent stress-induced craving and relapse, possibly making a good combination with naltrexone. In third place is testing mGluR2 PAMs. We are less hopeful about treatments that target dopamine or ionotropic glutamate receptors, partly because these systems play such a basic role in brain function and behavior. This might make it hard to develop medicines that work well without causing too many side effects.