Glutamate plasticity woven through the progression to alcohol use disorder: a multi-circuit perspective

Two-bottle choice

Two-bottle choice (2BC) involves offering the option to drink either a diluted ethanol-containing solution (concentrations range from 3 to 30%) or water for a fixed amount of time (Table 1). 2BC allows for the measurement of both voluntary consumption and ethanol preference over water and can be used as a single protocol or be combined with others to generate the desired level of drinking. In other words, first-day BACs may indicate low-level drinking, but weeks of 2BC could produce intoxicating BACs. This section focuses on 2BC studies that assess baseline ethanol preference, but daily limited-access studies that generate more binge-like drinking are discussed in the next section.

The studies are unequivocal that NMDA and AMPA regulate 2BC drinking, and both competitive and non-competitive GluN antagonists reduce 2BC intake^13–15. For example, NMDA and AMPA infused into the lateral hypothalamus can both increase 2BC consumption¹⁴. However, GluN antagonists and glycine B site blockade can importantly reduce motor coordination to achieve these effects^13–16. Similarly, GluN2A knockout mice show alcohol-induced impairments in motor coordination from wild-types (WTs) but do not show differences in consumption¹⁷. Other glutamate-related knockout lines also do not differ in 2BC drinking compared with WTs (for example, AMPA GluR1, GluN1 glycine, and mGluR5)^18–21. Pharmacological manipulations of mGluRs, specifically mGluR5 antagonists and mGluR7 agonists, are effective at reducing 2BC intake in rats^22–24. In complementary experiments, blockade with mGluR7 antagonist MMPIP or shRNA in the NAc can increase low-dose alcohol intake and preference^24,25. Since Homer2 knockout mice drink less than WTs in 2BC²⁶, it suggests that downstream signaling molecules are also important beyond glutamate receptor binding and clearance. It is worth mentioning that the US Food and Drug Administration (FDA)-approved medication for alcohol dependence, acamprosate, for which the glutamatergic mechanism of action is controversial, reduces 2BC drinking in rats²⁷. There is a glaring gap in the literature for which glutamatergic circuits in the brain may govern low-dose ethanol drinking. We need this critical information for insight into higher-dose plasticity.

Another important variable on the outcome of 2BC drinking and potential neuroadaptations is strain. Classic comparisons contrast between drinking behavior of C57BL/6J mice and DBA/2J mice, yet many inbred strains have been assessed for 2BC²⁸. Although specific sucrose-fading procedures can be used to induce ethanol drinking in DBA/2J mice (for example, 29) or bypassing ethanol taste altogether (for example, 30), this mouse strain drinks much less than C57BL/6J mice. 2BC preference may be related to strain differences in the effect of glutamate and NMDA on the brain in vitro^31,32 and differences in gene expression in response to acute ethanol³³. Also, alcohol-preferring (P) rats, genetically selected for high alcohol drinking, have a loss of the mGluR2 receptors that may contribute to escalated alcohol intake³⁴. Comparing high-drinking and low-drinking strains caused from trait selection or from inbred lines would increase the understanding of how glutamate-related genes influence drinking behavior.

Operant self-administration of alcohol

Operant self-administration is a powerful method for mice, rats, and monkeys to assess ethanol reinforcement. Via these methods, rodents will typically self-administer amounts ranging from 0.5 to 2 g/kg depending on factors such as session length, reinforcement schedule, and alcohol concentration by pressing a lever, spinning a wheel, or poking the nose into a receptacle (Table 1). Uncompetitive GluN antagonists ketamine and memantine reduce operant responding for ethanol with mechanistic target of rapamycin signaling, likely regulating the anti-alcohol effects of ketamine³⁵. Both mGluR5 and mGluR1 blockade and mGluR7-positive allosteric modulation decrease alcohol self-administration in rats and mice^36–40, particularly in the NAc^41,42. As in 2BC studies, some have seen ethanol-induced sedation and hypnosis with mGluR5 antagonist MPEP and mGluR2/3 antagonist LY341495⁴³ and non-specific reductions in sucrose self-administration^39,44. This may be due in part to mGluR5 influencing D1 receptors in seeking behavior⁴⁵.

Self-administration training techniques are also useful to investigate cue-induced reinstatement, or seeking behavior, following extinction of the alcohol-paired cues (Table 1). In operant self-administration protocols, cue-induced reinstatement or stress-induced reinstatement of alcohol seeking after a period of extinction training is also interpreted to be a form of relapse⁴⁶ (Table 1). We discuss the literature here instead of the relapse section, as no alcohol is consumed during reinstatement tests. There have been mixed reports for the ability of competitive GluN antagonists to affect reinstatement^47,48. For mGluRs, it is not surprising that mGluR5 antagonism and mGluR2/3 agonism reduce cue-induced reinstatement, alcohol seeking in Pavlovian spontaneous recovery, and enhanced sensitivity to the attenuation of conditioned reinstatement^49–52, but there are varying reports for whether these compounds affect baseline self-administration. Gass et al.⁵³ found evidence for increased glutamate transmission from the basolateral amygdala (BLA) to NAc core during cue-induced reinstatement of alcohol seeking. Glutamate transmission and transport may be mediated through adenosine ENT1⁵⁴ since N-acetylcysteine and ceftriaxone, which alter glial reuptake and release of glutamate, also alter alcohol self-administration⁵⁵. Downstream signaling molecules such as PKCε, ERK, and CaMKII/AMPA in the PFC and amygdala have been well established in alcohol self-administration and cue-induced reinstatement^56–59. Specifically, amygdalar CaMKII/AMPA activation promotes self-administration and drinking^60–62, whereas inhibition of CaMKII in the PFC increases the positive reinforcing effects of alcohol⁶³. Others have explored the activation of mGluR2 amygdala to hippocampus pathway in cue-induced alcohol seeking, where mGluR-mediated synaptic depression is impaired in the hippocampus³⁴. It seems that subregions of the amygdala and also the PFC are recruited during this low-level drinking.

Alcohol-discriminative stimulus effects

Alcohol discrimination tasks are useful to assess the neurobiological mechanisms underlying the discriminative stimulus effects (for example, interoceptive effects) of low and high alcohol doses (Table 1). However, it is important to note that these tasks do not involve alcohol drinking but rather experimenter-administered alcohol. We have known for decades that the discriminative stimulus properties of ethanol are mediated by GluNs and GABAA^64–66. Specifically, lower alcohol doses (for example, 0.5–1 g/kg) engage GABA receptor systems whereas higher doses (>2 g/kg) involve NMDA receptor systems. Rats and cynomolgus monkeys can discriminate alcohol from glutamate release inhibitors and NMDA ligands, showing that they have partial alcohol-like effects^67,68. This is different from the discrimination of acamprosate, where acamprosate fails to substitute for an alcohol cue, suggesting that it is not a substitution drug⁶⁹. Besheer et al. have shown that alcohol discrimination is co-regulated by mGluR5 in the NAc and the mGluR2/3 in the amygdala^70–73 and that inhibition of MEK/ERK(1/2) in the amygdala, but not NAc, potentiates the effects of a low alcohol dose⁷⁴. Recent work with stress hormone corticosterone links both mGluR5 and mGluR2/3 in the sensitivity to alcohol^75,76, suggesting a role for neuropeptide modulation of glutamatergic circuits. Furthermore, in addition to the NAc, a functional role for the medial PFC (mPFC) in modulating sensitivity to low alcohol doses has been shown^66,77. An interesting contribution from the Holmes lab shows that GluN2B in corticostriatal circuits governs choice learning and choice shifting⁷⁸. Although this learning is not in the presence of alcohol, they show a dissociation between OFC GluN2B in choice shifting and dorsal striatum GluN2B in choice learning. These findings suggest it is possible that learning about alcohol through discrimination tasks recruits distinct populations of both iGluR and mGluR in subcortical sites, although more research is required to confirm how this contrasts from habitual learning in the striatum.

Overall, there is ample evidence demonstrating PFC plasticity in alcohol-seeking behavior and low-dose alcohol drinking at a stage engaging positive reinforcement and the euphoric effects of the drug. Although 2BC studies have tested several facets of the glutamate system using knockout mice, there is a gap of knowledge in iGluRs in alcohol self-administration studies. This may be confounded by the fact that competitive GluRN antagonists mimic the interoceptive properties of alcohol. More recent studies have implicated GluRA in the rostromedial tegmental nucleus in alcohol seeking⁷⁹. Another behavioral outcome of low-dose acute, self-administered alcohol (1 g/kg) is an increase in inter-male aggression in a subset of mice⁸⁰. Memantine, neramexane, and mGluR5 antagonist MTEP interacted with alcohol to further increase alcohol-heightened aggression in mice, whereas mGluR2/3 agonist LY379268 did not⁸¹. CRF type-1 receptors regulate serotonin function from the dorsal raphe nuclei (DRN)- mPFC to alter alcohol-heightened aggression⁸², so glutamate may influence the mPFC for the expression of low-dose alcohol-related behavior.

Drinking in the dark

The prototypical procedure in mice to induce binge-like drinking is giving one bottle of alcohol, offered 3 hours into the active dark photoperiod for 2–4 hours, termed drinking in the dark (DID) (Table 1)^90,91. C57BL/6J mice typically drink 2–5 g/kg in a session. Even two alcohol “binges” in adolescent rats are sufficient to abolish long-term synaptic depression in hippocampal slices and to evoke cognitive deficits via a short-lasting, repeated blockade of GluN, inducing a change in the receptor subunit composition⁹². An earlier DID study showed that both acamprosate and MPEP decreased DID intake without affecting sugar or water drinking⁹³. Others have gone on to show that mGluR5 signaling affects PKCε in the NAc or central amgydala (CeA) to regulate DID^94,95. Specifically, repeated DID for 30 days elevates CeA levels of glutamate-associated proteins of Homer2a/b, mGluR1a, GluN2B, and PLCε 24 hours after withdrawal from binge drinking⁹⁵. Intra-CeA and intra-NAc mGluR1 negative allosteric modulator JNJ-16259685 also reduces DID intake^96,97. More recent studies have isolated downstream factors after DID such as mGluRs affecting AMPA receptor trafficking proteins like eukaryotic elongation factor 2 or decreased amygdalar CaMKIIαT286 phosphorylation^98,99. Importantly, this effect was isolated to the amygdala but not NAc or dorsal striatum. This may be related to the lack of difference in frequency and amplitude of spontaneous excitatory post-synaptic current (sEPSC) in dorsolateral striatum and dorsomedial striatum medium spiny neurons between 6 weeks’ DID and water-drinking mice¹⁰⁰. Also, moving away from the classic mesolimbic pathway, others have identified a novel ventral tegmental area (VTA)–bed nucleus of the stria terminalis (BNST) CRF circuit in DID¹⁰¹. CRF-R1 antagonists can reduce DID through intact CRF-R2 signaling, and inhibiting VTA-projecting BNST CRF neurons reduces DID¹⁰¹. Repeated 2 g/kg alcohol injections result in enhanced GluN-mediated LTP in VTA dopamine neurons¹⁰², so it is likely that this VTA-BNST glutamate pathway is altered during binge drinking in DID in a similar fashion.

Beyond DID, there are other daily limited-access procedures that lead to binge drinking in rodents. Permutations of DID exist, such as 2-hour daily access for 14 days in C57BL/6J mice, to study other facets of binge-like drinking, such as tolerance¹⁰³. The scheduled high alcohol consumption (SHAC) protocol involves water restriction for all but 90 minutes of water access, and every fourth day alcohol replaces water for 10–30 minutes¹⁰⁴. Systemic administration of mGluR5 antagonist MPEP decreases SHAC intake but also sucrose self-administration⁴⁴. Further studies have found a role for mGluR5-Homer2-PI3K signaling in the NAc in SHAC intake¹⁰⁵, which can be replicated in the DID protocol⁹⁴. Another limited-access protocol is multiple scheduled access (MSA), in which P rats are offered four 1-hour 2BC sessions separated by 2 hours across the dark cycle 5 days per week¹⁰⁶. Changes in gene expression in the NAc and amygdala after weeks of MSA drinking in P rats have been extensively studied^107–109, so what is needed is targeting how glutamate interacts between the sites through mGluRs and iGluRs¹¹⁰. MSA can lead to a transient increase in alcohol drinking after a weekend of deprivation¹⁰⁷, an alcohol deprivation effect (ADE), so it incorporates episodic drinking in both limited-access binge drinking and relapse-like drinking.

Alcohol deprivation effect

Relapse is also episodic in nature, both in the clinic and modeled with animals. Relapse, a hallmark of alcohol use disorders, is the resumption of drinking following a prolonged period of abstinence. With animals, experimenters can model relapse through the expression of the ADE. In this method, alcohol-drinking animals are deprived of alcohol for a period of time (for example, days to weeks), and then following this deprivation period, an escalation in alcohol drinking is observed following re-exposure to alcohol (Table 1). Intra-PFC glutamate and acamprosate separately reduce the ADE^111,112. However, many other glutamatergic compounds—GluN/glycine receptor antagonist L-701,324, GluN2B selective antagonist ifenprodil, GluN channel blocker neramexane, GluA/GluK antagonist CNQX, and Na⁺ channel blocker lamotrigine—attenuate the ADE similar to alcohol seeking during cue-induced reinstatement^47,48. To the best of our knowledge, there are no reports for the involvement of iGluR or mGluR circuitry in the ADE, but we hypothesize that it would be similar to plastic changes in DID or operant self-administration circuitry.

It appears that episodic drinking, the amorphous transition between low-dose and high-dose intake, engages both reward-related and stress-related glutamate brain processes. A single DID protocol is mGluR5 antagonist-responsive, whereas repeated DID for a month alters changes in downstream glutamate proteins. Multiple glutamatergic compounds reduce the ADE and cue-induced reinstatement, so perhaps these protocols in combination with others would be more apt for screening medications for the clinic.

Intermittent access to alcohol

Cycles of binging and withdrawal occur in the transition to developing an alcohol use disorder. We can model voluntary alcohol drinking in between periods of abstinence, or alcohol deprivation, with 24-hour intermittent access to 2BC alcohol^131–133. Weeks of intermittent alcohol access can lead to drinking despite adverse consequences¹³⁴ and signs of withdrawal such as handling-induced convulsions and decreased social interactions^135,136. Giving access to alcohol for a 24-hour period may cause variability in when animals choose to drink, so researchers can also measure fluid consumption during the initial 2-, 4-, and/or 6-hour access within the 24-hour period. With this, front-loading behavior may be observed accompanied by high BACs after 2-hour access (Table 1)¹³⁵. Additionally, smaller segments within 24-hour access allow drug manipulations to be assessed^137,138.

Acamprosate reduces intermittent alcohol drinking in rats but not continuous-access alcohol drinking¹³². Confirming clinical reports, outbred mice drinking on intermittent access to alcohol for 8 weeks show increased extracellular glutamate in the mPFC during withdrawal compared with 1 week of drinking and compared with water drinkers¹³⁶. Early reports with intermittent-access drinking in rats, drinking 7 g/kg per day, have enhanced post-synaptic GluA function in VTA neurons in the absence of any change in pre-synaptic glutamate release¹³⁹. Similarly, glutamatergic and GABAergic synaptic transmission are altered in the striatum of non-human primates with extended access for 3 years¹⁴⁰. Six months of continuous access and intermittent access to alcohol consumption in P rats produce selective increases in group 1 mGluR/Homer2/GluN2 expression within the NAc core and CeA^141,142. Intermittent alcohol can produce short-term increases in Homer/glutamate receptor expression within both the NAc core and the CeA, which may increase the aversion of early alcohol withdrawal and consequently augment the negative reinforcing properties of alcohol. Modulators of the glutamate transporters reduce heavy drinking on a continuous-access schedule (15% and 30% ethanol) in P rats and the increased extracellular glutamate compared with water drinkers¹⁴³. These P rats also have enhanced expression of glutamate transporters EAAT2/GLT1 and xCT in the NAc and PFC, suggesting a role for targeting glutamate uptake in heavy drinking¹⁴⁴. Long-term intermittent alcohol recruits GABA and CRF neurons in the mPFC during withdrawal and disconnects the PFC–CeA pathway, suggesting that dysregulation of mPFC interneurons may be an early index of glutamate/GABA neuroadaptation in alcohol dependence¹⁴⁵. Impaired executive control over motivated behavior accompanies negative reinforcement during withdrawal. Seif et al.¹⁴⁶ show that cortical activation of NAc hyperpolarization-active GluN mediates aversion-resistant intermittent alcohol intake. Both the mPFC to NAc core and insula to NAc core mediate both quinine- and footshock-resistant alcohol drinking on an intermittent-access schedule. It appears that corticolimbic sites are integral to glutamate plasticity caused by chronic intermittent drinking.

Withdrawal from chronic intermittent ethanol vapor and other forced alcohol methods

There are several other protocols that forcibly induce a post-dependent state in animals, such as repeated high-dose alcohol injections, alcohol liquid diet, and chronic intermittent ethanol (CIE) vapor exposure (Table 1). Studies on brain glutamate during alcohol withdrawal have been most extensively explored using these methods, since they surpass the aversive taste of alcohol drinking solutions to induce heavy BACs. However, it is important to note that CIE is used to render rodents ethanol-dependent to subsequently increase voluntary ethanol intake, not only to maintain high BACs^147–149. Microdialysis studies have shown increased glutamate in the striatum¹⁵⁰, NAc^87,151–153, and hippocampus^1,154 during withdrawal in alcohol-exposed rats and mice. These results are similar to 5 g/kg alcohol gavage injections for 2–4 weeks causing increased glutamate in striatum, hippocampus, and substantia nigra 8–12 hours after the last ingestion¹⁵⁵. To counteract excitotoxicity, acamprosate and GluN antagonists have been used to decrease alcohol drinking and to alleviate symptoms of alcohol withdrawal, including increased glutamate tone and convulsive events^{112,151,156–158}. It is important to note that pharmacologically increasing glutamate transmission in the NAc with TBOA, a glutamate reuptake inhibitor, can increase drinking in both non-dependent and CIE-dependent mice¹⁵³. Alternatively, decreasing glutamate transmission in the NAc by activating group II mGluRs reduces drinking, although the effect was stronger in dependent mice. These results comparing glutamate in non-dependent and dependent animals have similar directionality with different magnitudes, so there may be separate but overlapping actions in the NAc for treating drinking versus withdrawal symptoms with glutamatergic compounds.

In accordance with clinical studies, the PFC is a large target of glutamate plasticity in alcohol dependence. CIE results in increased GluN-mediated activity in the mPFC and increased GluN1 and GluN2B subunit expression^159,160. Mice that show “compulsive-like” behaviors after CIE exhibit increased NMDA currents in the orbitofrontal cortex compared with air-exposed controls¹⁶⁰. Rescue of infralimbic PFC mGluR2 deficit restores control over alcohol-seeking behavior¹⁶¹. It appears that mGluR2 and mGluR5 can target symptoms of withdrawal (but see¹⁶²). Acamprosate improved attention set-shifting of alcohol-exposed animals but did not alter the concurrent changes in synaptic transmission or membrane excitability of mPFC neurons, indicating that the changes are not the pharmacological targets of acamprosate in the recovery of mPFC functions¹⁶³. Abulseoud et al.¹⁶⁴ showed that attenuation of alcohol withdrawal by ceftriaxone induced upregulation of glutamate transporter EAAT2. Reduction of EAAT2 likely contributes to a hyperglutamatergic state in the ENT1 knockout mice^54,165,166. Some have suggested that increasing glutamate uptake through transporters has a potential therapeutic role in the treatment of alcohol dependence¹⁶⁷ (but see¹⁶⁸). Aberrations in PFC function entangle reduced executive control and poor decision making in alcoholics¹⁶⁹.

The extended amygdala—composed of the BNST, BLA, and CeA—is particularly vulnerable to glutamate plasticity caused by CIE treatment. Chronic alcohol exposure produces neuroadaptations in glutamatergic transmission in the CeA^170,171, and GluN2B-containing GluNs are most sensitive to CIE^170,172,173. CIE, but not continuous vapor exposure, increases BNST GluN-mediated EPSCs, not from altered glutamate release but from an increase in GluN2-containing GluN¹⁷⁴, suggesting that repeated cycles of exposure and withdrawal are necessary for these adaptations to occur. CIE enhances long-term potentiation formation in the BNST in GluN2B knockout mice through extrasynaptic GluN¹⁷⁵. Stress-induced alterations in anxiety-like behavior were absent following bilateral infusion of GluK1 agonist ATPA into the BLA, which augmented BLA GABAergic neurotransmission, and stress increased the amplitude of sEPSC and miniature inhibitory post-synaptic current¹⁷⁶. A regulatory stress neuropeptide could be nociceptin, since nociceptin application decreases glutamate transmission and blocks alcohol-induced effects in the CeA of naïve and CIE rats, but nociceptin antagonist revealed tonic inhibitory activity of nociceptin on evoked CeA glutamatergic transmission only in alcohol-dependent rats¹⁷⁷. Changes in the extended amygdala indicate a transition from positive reinforcement to negative reinforcement as stress neuropeptides like nociceptin, CRF, and dynorphin are more engaged¹⁷⁸.

Together, chronic forced or voluntary access to alcohol affects glutamate in multiple subcortical sites like the PFC and extended amygdala, and this agrees with the clinical literature. In addition to these sites, many others have examined the hippocampus as a crux of CIE-induced glutamatergic changes. Group I mGluRs and GluN2B-containing GluNs in CA1 and cortex impair LTD, reduce spine density, and disrupt learning^179,180 (but see 138). This may be related to the enhanced stress systems recruited during repeated exposure to and withdrawal from alcohol. In line with this hypothesis, corticohippocampal GluN2B is engaged during repeated swim stress¹⁸¹. This circuitry is also recruited in other addictive disorders. Glutamate homeostasis is a mediator of long-term drug-seeking behavior, especially through disruptions of the cysteine/glutamate exchanger and EAAT2/GLT1¹⁸². Alterations in glutamate transmission after chronic alcohol exposure and withdrawal are evident, but some effects are also likely to be unique to withdrawal alone. Future research can tease apart these dynamic distinctions or suggest that they are interconnected.