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. Author manuscript; available in PMC: 2018 Jan 1.
Published in final edited form as: Genes Brain Behav. 2017 Jan;16(1):139–148. doi: 10.1111/gbb.12363

Alcohol-dependent molecular adaptations of the NMDA receptor system

N Morisot 1, D Ron 1,*
PMCID: PMC5444330  NIHMSID: NIHMS860081  PMID: 27906494

Abstract

Phenotypes such as motivation to consume alcohol, goal-directed alcohol seeking and habit formation take part in mechanisms underlying heavy alcohol use. Learning and memory processes greatly contribute to the establishment and maintenance of these behavioral phenotypes. The N-methyl-d-aspartate receptor (NMDAR) is a driving force of synaptic plasticity, a key cellular hallmark of learning and memory. Here, we describe data in rodents and humans linking signaling molecules that center around the NMDARs, and behaviors associated with the development and/or maintenance of alcohol use disorder (AUD). Specifically, we show that enzymes that participate in the regulation of NMDAR function including Fyn kinase as well as signaling cascades downstream of NMDAR including calcium/calmodulin-dependent protein kinase II (CamKII), the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and the mammalian target of rapamycin complex 1 (mTORC1) play a major role in mechanisms underlying alcohol drinking behaviors. Finally, we emphasize the brain region specificity of alcohol's actions on the above-mentioned signaling pathways and attempt to bridge the gap between the molecular signaling that drive learning and memory processes and alcohol-dependent behavioral phenotypes. Finally, we present data to suggest that genes related to NMDAR signaling may be AUD risk factors.

Keywords: Addiction, alcohol, AMPA, amygdala, CaMKII, Fyn, kinase, mTOR, NMDA, nucleus accumbens, phosphatase, PTPalpha, signaling, STEP, striatum


A prominent theory of AUD is the alcohol-dependent ‘hijacking’ of learning and memory processes which leads to pathological alcohol seeking and taking (Hyman et al. 2006; Torregrossa et al. 2011). Repeated exposure to alcohol promotes synaptic plasticity in key brain regions contributing to the formation of strong memories related to the rewarding experience of alcohol consumption (Torregrossa et al. 2011). In parallel, learning of the adverse consequences associated with harmful alcohol use may be attenuated (Torregrossa et al. 2011). As a result, patients suffering from AUD exhibit excessive alcohol intake, escalation of consumption and uncontrolled seeking and taking behaviors, which are key characteristics of AUD (Apa 2013; Koob & Volkow 2010). Thus, a better understanding of the molecular events underlying the aberrant form of learning and memory associated with AUD will enhance the discovery of new potential therapeutic targets.

The NMDAR is a critical factor of learning and memory functions (Mayford et al. 2012), and a primary target of alcohol (Ron and Wang 2009). In recent years, the molecular mechanisms that drive (the GO pathways) or that prevent (the STOP pathways) the development of AUD have started to unravel (Ron & Barak 2016), and we focus here on the contribution of the NMDAR signaling to the GO pathways in studies that used chronic voluntary consumption of alcohol in rodents. Specifically, we provide a description of the normal function of the NMDAR in learning and memory processes. We then detail the molecular events that promotes NMDAR activity, in particular Fyn kinase-dependent mechanisms, as well as signaling downstream of the receptor, including CaMKII, which are both critically affected by excessive alcohol drinking. Next, we review the potential link between alcohol-induced NMDAR inhibition and downstream signaling involving mTORC1. Finally, we review evidence from human genetic studies suggesting that variants in NMDAR-related genes contribute to the risk of developing AUD.

The NMDAR system

Glutamate, the major excitatory neurotransmitter in the mammalian brain, contributes to various crucial brain functions, including learning and memory (Mayford et al. 2012). Glutamate signaling is mediated through the activation of two families of transmembrane receptors: the G protein-coupled metabotropic receptors (mGluRs) and the ionotropic receptors, NMDA, AMPA and Kainate receptors (Traynelis et al. 2010). The NMDAR is a tetramer comprised of an obligatory subunit GluN1, and the regulatory subunits GluN2A-D and GluN3A-B (Traynelis et al. 2010). The most common regulatory subunits are GluN2A and GluN2B that differentially control the electrophysiological properties of the channel such as conductance and open probability (Traynelis et al. 2010). Each GluN2 subunit is composed of a long intracellular C-terminal sequence which contains binding sites for scaffolding proteins as well as kinase phosphorylation sites that control channel trafficking, membranal localization and activity (Traynelis et al. 2010). The NMDARs are enriched in the postsynaptic density (PSD), which is a dense network of hundreds of proteins including kinases, phosphatases, small G proteins and scaffolding proteins that mediate functional and structural plasticity of the excitatory synapses (Kennedy 2000).

The NMDAR contributes to the strengthening of synaptic connections (synaptic plasticity), that are essential to the formation of long-term memory (Mayford et al. 2012). Short-term synaptic plasticity is achieved through transient synaptic transmission changes (milliseconds to minutes), which then quickly return back to baseline levels, whereas long-term plasticity is achieved by repeated stimulation that causes persistent changes (hours to days) in the synaptic connections (Mayford et al. 2012). The NMDAR-dependent long-term potentiation (LTP) is the basis of synaptic plasticity (Malenka 2003; Mayford et al. 2012), and is associated with (1) the forward trafficking of AMPARs, (Malinow & Malenka 2002), (2) de novo protein synthesis (Kasai et al. 2010) and (3) enlargement of existing spines in addition to the formation of new spines (Matsuzaki et al. 2005). The NMDAR-dependent LTP was initially described in the hippocampus but was later observed in various brain regions important for the development of AUD, including the ventral tegmental area, the nucleus accumbens (NAc), the dorsal striatum, the prefrontal cortex (PFC) and amygdala (Bernier et al. 2011; Britt et al. 2012; Cho et al. 2012; Taylor et al. 2016; Wang et al. 2012).

Src family tyrosine kinases and the NMDAR

This section focuses on modulation of NMDAR activities through tyrosine phosphorylation and dephosphorylation. Kinase phosphorylation of the GluN2A and GluN2B subunits plays a major role in the localization, activation state and physiological properties of the NMDARs (Traynelis et al. 2010). Kinases including protein kinase C, CaMKII, and cyclin-dependent kinase 5 phosphorylate the GluN2 subunits on serine and threonine sites (Ron 2004), while the Src family of tyrosine kinases (PTKs), Fyn and Src phosphorylates the GluN2 subunits on tyrosine sites. (Ohnishi et al. 2011; Ron 2004; Trepanier et al. 2012). Fyn and Src are the best characterized kinases modulating the NMDAR function. As such, the kinases play an important role in synaptic plasticity, learning and memory (Ohnishi et al. 2011; Salter & Kalia, 2004; Trepanier et al. 2012).

Fyn and Src are composed of regulatory and catalytic domains (Engen et al. 2008). In their inactive conformation, Fyn and Src are phosphorylated on tyrosine 528 (mouse)/531 (rat/human) (PhosphoTyr528/531[Fyn/Src]) enabling the formation of an intramolecular bond with the SH2 domain that keeps the kinases in a closed inactive conformation (Engen et al. 2008). Dephosphorylation of this site results in a conformational change, allowing the kinases to undergo autophosphorylation at tyrosine 417 (mouse)/420 (rat and human) (PhosphoTyr417/420[Fyn/Src]), which is the hallmark of the active kinase (Engen et al. 2008). Conversely, dephosphorylation of PhosphoTyr417/420 inhibits Fyn and Src activity (Engen et al. 2008). The phosphatase responsible for dephosphorylating the inactive site PhosphoTyr527/530[Fyn/Src], and thus that enables the activation of the kinases is the protein tyrosine phosphatase alpha (PTPalpha) (Bhandari et al. 1998; Ponniah et al. 1999; Su et al. 1999; Vacaresse et al. 2008; Vacaru & den Hertog 2010; Zheng et al. 2000). The termination of Fyn activation in the brain is mediated via the dephosphorylation of the active phosphorylation site Tyr417/[Fyn] by the striatal-enriched tyrosine phosphatase (STEP) (Goebel-Goody et al. 2012). Although studies have mainly focused on STEP dephosphorylating Fyn thereby inhibiting kinase activity (Goebel-Goody et al. 2012), it is plausible that similar mechanisms hold true for Src. Finally, the activity of STEP is controlled in part, by protein kinase A (PKA) (Goebel-Goody et al. 2012). Specifically, PKA phosphorylates STEP resulting in an inhibition of the activity of the phosphatase thereby facilitating kinase activity (Goebel-Goody et al. 2012).

Once active, Fyn phosphorylates tyrosine residues within the cytoplasmic tail of GluN2B subunits (Ohnishi et al. 2011; Salter & Kalia 2004; Trepanier et al. 2012). The consequence of Fyn-mediated phosphorylation of GluN2B is the enhancement of channel function (Trepanier et al. 2012; Yaka et al. 2002, 2003a), which is due, at least in part, of increased membranal retention of GluN2B (Dunah et al. 2004; Nakazawa et al. 2001; Prybylowski et al. 2005). Both Fyn (Yaka et al. 2002) and PTPalpha (Lei et al. 2002) are part of the NMDAR complex, and the close proximity of Fyn to GluN2B allows the efficient phosphorylation of the subunit (Sato et al. 2008; Tezuka et al. 1999; Yaka et al. 2002; Yaka et al. 2003a). Finally, STEP was reported to dephosphorylate GluN2B leading to channel internalization (Snyder et al. 2005). How alcohol co-opts the Fyn signaling-dependent regulation of NMDAR to drive alcohol-related phenotypes is reviewed herein.

CamKII and the NMDAR

At its resting state, the pore of the NMDAR is blocked by Mg2+ ions (Traynelis et al. 2010). Presynaptic stimulation causes a strong postsynaptic depolarization mediated by AMPAR which enables the removal of Mg2+ and, together with glutamate binding, leads to channel opening resulting in the entry of calcium (Ca2+) and sodium (Na+) ions (Traynelis et al. 2010). Ca2+ entering the NMDAR pore binds calmodulin, which associates with, and activates CaMKII (Irvine et al. 2006). CaMKII is a serine and threonine kinase composed of 12 independent subunits (Irvine et al. 2006). Once Ca2+ is bound, CaMKII is autophosphorylated by neighboring subunits of the holoenzyme, a mechanism that keeps the kinase active even after Ca2+ levels decrease and calmodulin dissociates from the kinase (Irvine et al. 2006). The autonomous activity of the holoenzyme is considered to be a molecular transducer of synaptic plasticity and long-term memory (Coultrap & Bayer 2012; Herring & Nicoll 2016; Muller et al. 2016). Specifically, NMDAR-dependent LTP is mediated by CaMKII phosphorylation and forward trafficking of AMPARs to the synaptic membrane (Herring & Nicoll 2016; Malinow & Malenka 2002). How alcohol promotes signaling activation downstream of the NMDAR is discussed herein.

Alcohol and NMDAR phosphorylation

Although the inhibition of NMDARs by acute alcohol exposure has been most extensively studied (Lovinger et al. 1989, Ron and Wang 2009), chronic alcohol increases NMDAR function in numerous brain regions (Abraham 2008; Floyd et al. 2003; Grover et al. 1998; Gulya et al. 1991; Iorio et al. 1992; Smothers et al. 1997; Wang et al. 2007, 2010). This section focuses on mechanisms underlying enhanced NMDAR signaling by repeated cycles of alcohol drinking and withdrawal.

Alcohol and Fyn signaling

Over the past decade, we have generated data to suggest that the Fyn signaling pathway is activated in the striatum of rodents in response to alcohol exposure. The striatum can be divided into ventral (NAc), dorsomedial (DMS) and dorsolateral (DLS) subregions based on neuroanatomical and functional divergences (Everitt & Robbins 2013). We found that the level of PhosphoTyr417[Fyn] and thus the activity of the kinase is increased in the dorsal striatum in response to alcohol exposure (Wang et al. 2007). Furthermore, we found that contingent and non-contingent activation of Fyn by alcohol is localized to the DMS and was not observed in the DLS or in the NAc (Darcq et al. 2014; Gibb et al. 2011; Wang et al. 2007, 2010). Although both Src and Fyn are expressed throughout the brain, we generated data to suggest that alcohol specifically activates Fyn in the dorsal striatum. Specifically, we used an immunoprecipitation assay to pull down the kinase and detected the specific Fyn phosphorylation by alcohol (Wang et al. 2007). In contrast, Src was not activated by alcohol in the dorsal striatum (Wang et al. 2007). Interestingly, the activation of Fyn by alcohol is long-lasting. Specifically, repeated daily systemic administration of a non-hypnotic dose of alcohol or a 7-week intermittent access (IA) to 20% alcohol in a two-bottle choice paradigm (IA20%-2BC) led to Fyn activation in the DMS of mice and rats that was maintained even after 16–24 h of withdrawal (Darcq et al. 2014; Gibb et al. 2011; Wang et al. 2010). We further showed that Fyn activation by alcohol is not due to alterations in the levels of the kinase (Wang et al. 2011) but instead resulted from the lateral movement of PTPalpha in close proximity to Fyn (Gibb et al. 2011), and through PKA-mediated phosphorylation of STEP, which in turn prevented the ability of STEP to dephosphorylate the kinase (Darcq et al. 2014). The PKA has been shown to be an upstream transducer of many of alcohol's actions in the brain (Ron & Barak 2016), and Xu et al. recently reported that the alcohol-mediated PKA-dependent phosphorylation and thus inhibition of STEP prevented the phosphatase from dephosphorylating and inactivating PTPalpha (Xu et al. 2015), thus enabling PTPalpha to translocate into lipid rafts and activate Fyn (Gibb et al. 2011; Xu et al. 2015). Curiously, similar to the activation profile of Fyn in response to alcohol exposure, PKA-dependent phosphorylation of STEP was localized to the DMS and was not detected in other striatal regions, (Darcq et al. 2014; Gibb et al. 2011; Wang et al. 2010; Xu et al. 2015). Finally, moderate consumption of alcohol modeled by a continuous access to 10% alcohol in a 2BC paradigm (CA10%-2BC) did not activate Fyn in the DMS of mice (Fig. 1, original data), suggesting that Fyn is activated only in response to heavy drinking of alcohol.

Figure 1. Moderate consumption of alcohol does not trigger Fyn activation in the DMS.

Figure 1

Mice had CA10% (black) or water only (white) in a 2BC paradigm for 21 days, and the DMS was dissected immediately after the last 24-h alcohol drinking session. (a) pY417/420[Fyn/Src] as well as the total protein level of Fyn and actin, which was used as a loading control, were measured by western blot analysis. (b) Histogram shows the mean ratio of pY417/420[Fyn/Src] to total Fyn ± SEM, expressed as percentage of water controls. Data analysis indicates that CA10% does not affect Fyn activation in the DMS (t10 = 0.92, P = 0.38, unpaired Student's t-test). n = 6.

As described in above, one of the major substrates of Fyn in the brain is GluN2B, and as expected, the pattern of GluN2B phosphorylation in response to both contingent and non-contingent alcohol exposure resembles the pattern of Fyn activation by alcohol. Specifically, repeated daily systemic administration of alcohol for 7 days, or 7–8 weeks of IA20%-2BC resulted in a long-lasting phosphorylation of GluN2B at the synaptic membrane (Ben Hamida et al. 2013; Darcq et al. 2014; Wang et al. 2010), and similar to Fyn, the phosphorylation was localized to the DMS and was not detected in the DLS or NAc (Darcq et al. 2014; Gibb et al. 2011; Wang et al. 2010). Finally, GluN2B phosphorylation by alcohol led to increased membranal stabilization of GluN2B and a long-lasting facilitation of NMDAR activity (Wang et al. 2007, 2010, 2011). Together, these studies suggest that repeated cycles of alcohol exposure and withdrawal activate Fyn in the DMS, which in turn phosphorylates GluN2B resulting in the enhancement of channel activity (Fig. 2). This conclusion is supported by experiments in which lentivirus gene delivery was used to infect the DMS with short hairpin RNA (shRNA) sequence to knockdown PTPalpha or STEP mRNA. As predicted, knockdown of PTPalpha in the DMS prevented alcohol-dependent Fyn activation and GluN2B phosphorylation (Ben Hamida et al. 2013). Conversely, downregulation of STEP in the DMS potentiated the alcohol-mediated Fyn activation and GluN2B phosphorylation (Darcq et al. 2014). Curiously, Hicklin et al. reported that ex vivo exposure of hippocampal neurons to alcohol increased STEP-mediated dephosphorylation of GluN2B, which the authors suggested contributes to the inhibitory actions of alcohol of the NMDAR activity (Hicklin et al. 2011; Wu et al. 2011). These data are opposite to the finding that GluN2B is phosphorylated in response to ex vivo exposure of hippocampal neurons to alcohol (Yaka et al. 2003b). It is important to note however that the high basal level of GluN2B phosphorylation described by Hicklin et al. could be masking the actions of alcohol.

Figure 2. Molecular pathways transducing alcohol's signal in the DMS.

Figure 2

Alcohol activates PKA, which phosphorylates STEP inhibiting the activity of the phosphatase. Inhibition of STEP allows for the long-lasting activation of Fyn. Alcohol also enables the membranal colocalization of Fyn with its activator PTPalpha. Active Fyn phosphorylates GluN2B, which enhances the activity of the channel. Calcium entry through the GluN2B-containing NMDARs enables the activation of CaMKII. CaMKII activation promotes the forward trafficking of the AMPAR subunits, which in turn contributes to synaptic plasticity and alcohol dependent behavioral phenotypes.

Nevertheless, compelling evidence has been generated to indicate that Fyn plays a critical role in alcohol-related behaviors. Pioneer studies showed that genetic deletion of Fyn (Fyn−/−) resulted in higher sensitivity of mice to the acute sedative hypnotic actions of alcohol, which required GluN2B phosphorylation (Boehm et al. 2003; Miyakawa et al. 1997; Yagi 1999; Yaka et al. 2003c). The early data regarding the role of Fyn in alcohol drinking in the 2BC paradigm have been controversial. For instance, we did not find differences in alcohol intake between wild-type (WT) and Fyn−/− mice even when mice were bred on two different backgrounds; C57BL/6J or 129SVJ (Yaka et al. 2003c), whereas Boehm et al. (2003) observed a reduction of alcohol drinking in Fyn−/− mice compared to WT mice (Boehm et al. 2003). More intriguing was the observation that alcohol consumption and preference were reduced in Fyn overexpression transgenic mice (Boehm et al. 2004). Fyn−/− mice display abnormal arrangements of cells in specific brain regions as evident by undulations in granule cell layer in the hippocampus (Grant et al. 1992). Thus, possible compensatory mechanisms during development may be the explanation of these discrepancies. Moreover, it is important to note that these early studies used the CA10%-2BC paradigm, which, as we show herein, does not produce an activation of Fyn in the DMS (Fig. 1), and possibly in other brain regions, thus providing an explanation to the lack of changes in the consumption of 10% alcohol intake in the Fyn−/− mice vs. WT littermates (Yaka et al. 2003c).

Numerous studies link the Fyn signaling pathway to the scaffolding protein postsynaptic density protein (PSD-95). PSD-95 associates with Src PTKs (Kalia and Salter, 2003), and the phosphorylation of GluN2B enhances the binding of PSD-95 to the subunit (Rong et al., 2001). PSD-95 also associates with STEP and the association destabilizes STEP leading to its degradation (Won et al., 2016). Interestingly, PSD-95 KO consume less alcohol compared to WT (Camp et al., 2011), however, PSD-95 KO mice consume more alcohol compared to baseline after deprivation (Camp et al., 2011).

More recently, models of operant self-administration of 20% alcohol have been proven useful to study multiple aspects of AUD including heavy alcohol intake, alcohol seeking and relapse (Carnicella et al. 2014). In this paradigm, rats undergo a period of 7 weeks of IA20%-2BC followed by an operant self-administration training period during which rats press on a designated ‘active’ lever to obtain a drink of alcohol (Carnicella et al. 2014). After acquisition of self-administration of 20% alcohol, cannula is be implanted in specific brain regions to pharmacologically manipulate the activity of an enzyme within a signaling pathway. Infusion of the Fyn inhibitor, PP2, in the dorsal striatum (Wang et al. 2007) and specifically into the DMS of rats (Wang et al. 2010) prior to a self-administration session reduced the number of presses on the active lever and the amount of alcohol consumed (Wang et al. 2007, 2010). In contrast, PP2 infusion into the NAc or DLS did not alter alcohol self-administration, indicating that the role of Fyn on alcohol intake is brain region-specific (Wang et al. 2007, 2010). PP2 infusion in the dorsal striatum did not affect the inter-response interval distribution, thus ruling out general locomotor alterations (Wang et al. 2007). To test whether the contribution of Fyn in the DMS to self-administration is generalized to other reinforcing substances, Wang et al. trained rats to self-administer sucrose, a natural reward, and showed that Fyn inhibition in the DMS did not affect sucrose self-administration, suggesting that Fyn specifically drives alcohol drinking behavior (Wang et al. 2010). Furthermore, Wang et al. also tested the contribution of Fyn in the DMS to relapse to alcohol seeking. To model relapse, rats trained to self-administer alcohol undergo an extinction training in which the active lever is not reinforced (i.e. no alcohol is delivered upon pressing the active lever) (Carnicella et al. 2014; Epstein et al. 2006). Once the operant behavior is extinguished, the reinstatement test is carried out in which a small drop of alcohol is placed in the reward port and lever presses previously associated with alcohol are recorded (Carnicella et al. 2014; Epstein et al. 2006). The sensory cues (i.e. odor + taste) of the alcohol prime trigger a robust reinstatement of alcohol seeking (Carnicella et al. 2014). Using this model, Wang et al. found that Fyn inhibition in the DMS reduces the reinstatement of alcohol seeking, showing a key role for Fyn in mechanisms underlying relapse to alcohol use (Wang et al. 2010). Together, these data strongly suggest that Fyn in the DMS plays a role in intake of alcohol as well as relapse to alcohol seeking.

Upstream of Fyn is PTPalpha, which is required for the activation of the kinase (Bhandari et al. 1998). In line with the molecular findings described in this section, PTPalpha in the DMS plays an important role in mechanisms underlying excessive alcohol drinking. Specifically, PTPalpha knockdown in the DMS reduced alcohol intake and preference in an IA20%-2BC paradigm in rats without affecting water or sucrose consumption (Ben Hamida et al. 2013). Furthermore, to test whether PTPalpha contributes to the development of excessive alcohol drinking, the DMS of naïve mice was infected with a lentivirus expressing an shRNA sequence targeting PTPalpha prior to the beginning of the IA20%-2BC paradigm. Downregulation of PTPalpha reduced the consumption of, and preference for, 6%, 10% and 20% alcohol solution (Ben Hamida et al. 2013), suggesting that PTPalpha in the DMS is required for the developmental phase of excessive alcohol drinking. Conversely, infection of DMS neurons with a lentivirus expressing an shRNA sequence targeting STEP enhanced alcohol drinking and preference (Darcq et al. 2014). STEP was shown to contribute to NMDAR-mediated fear memory in the amygdala (Paul et al. 2007) and Hicklin et al. found that STEP is required for alcohol attenuation of fear conditioning (Hicklin et al. 2011). Furthermore, STEP−/− mice displayed increased level of alcohol intake in the 2BC paradigm as compared with WT mice (Legastelois et al. 2015). While WT and STEP−/− mice drink similar amounts of the sweet rewarding solution, saccharin, STEP−/− mice consume more of bitter tasting solutions of quinine or denatonium, that typically trigger aversion in WT mice (Legastelois et al. 2015). To test whether genetic deletion of STEP abolished the sensitivity to aversive stimulus, Legastelois et al. used a conditioned place aversion paradigm in which mice are conditioned to receive an injection of lithium chloride, an malaise-inducing agent, in a specific compartment. Following conditioning, WT but not STEP−/− mice avoided spending time in the lithium chloride-paired compartment (Legastelois et al. 2015). These results suggest that STEP controls the consumption of alcohol in part by attenuating its aversive bitter taste.

As described above, the PTPalpha activation and STEP inhibition by excessive alcohol drinking converge to promote Fyn activation, which in turn, phosphorylates GluN2B resulting in the enhancement of NMDAR activity. Similar to the reduction of alcohol consumption in response to Fyn inhibition in the DMS, GluN2B-containing NMDAR blockade by infusion of ifenprodil in the dorsal striatum (Wang et al. 2007) and more specifically in the DMS (Wang et al. 2010) reduced alcohol operant self-administration. The latter effect was brain region-specific as intra-NAc (Wang et al. 2007) or intra-DLS (Wang et al. 2010) infusion of ifenprodil was ineffective at reducing alcohol self-administration. Similar to PP2, intra-DMS administration of ifenprodil did not alter sucrose self-administration (Wang et al. 2010). Moreover, intra-DMS infusion of ifenprodil was sufficient to reduce alcohol-priming-induced reinstatement of alcohol seeking similar to Fyn inhibition (Wang et al. 2010). Thus, NMDAR activity in the DMS is crucial to alcohol intake and relapse.

Alcohol and CaMKII signaling

The molecular transducer of NMDAR activation is CaMKII (Irvine et al. 2006), and a direct link between CaMKII and alcohol-dependent behavioral phenotypes has been established by two studies (Easton et al. 2013; Salling et al. 2014). Specifically, Salling et al. reported that moderate consumption of alcohol increases the protein level of CaMKIIalpha in the amygdala, and that the administration of the CaMKII inhibitors KN-93, or a CamKII inhibitory peptide into the mouse Central Amygdala (CeA) reduces self-administration of a sweetened solution of 10% alcohol (Salling et al. 2014). A second study by Easton et al. used transgenic mice expressing a mutant form of the kinase in which threonine at position 286 in the alpha subunit of the kinase has been mutated to alanine (αCaMKIIT286A) thereby preventing the autophosphorylation and thus the autonomous activation of the kinase (Easton et al. 2013). The authors showed that αCaMKIIT286A mice exhibited a delay in the onset of alcohol consumption as compared with WT mice (Easton et al. 2013), indicating that the CaMKII mediates the development of alcohol drinking behaviors.

Downstream of NMDAR-dependent activation of CaMKII is the phosphorylation and forward trafficking of the AMPAR. Salling et al. further reported that moderate consumption of alcohol increases the protein levels as well as the phosphorylation of the GluA1 subunit of AMPARs in the CeA (Salling et al. 2014). In addition, we showed that excessive alcohol intake initiates the forward trafficking of AMPAR subunits in the DMS suggesting that CaMKII may be activated in the DMS in response to consumption of excessive amounts of alcohol (Wang et al. 2012). Furthermore, the contribution of AMPAR to the behavioral effects of alcohol was shown by Corbit et al. (2014), Salling et al. (2014) and Wang et al. (2012). Specifically, intra-DMS infusion of the AMPAR antagonist, NBQX into the DMS of rat, reduced self-administration of a 20% alcohol solution (Wang et al. 2012), whereas intra-amygdala infusion of NBQX reduced self-administration of a sweetened solution of 10% alcohol in mice (Salling et al. 2014). Finally, intra-DLS infusion of NBQX in the DLS blocked the expression of alcohol habits (Corbit et al. 2014).

Alcohol and mTORC1 signaling

As detailed above, the NMDAR is an essential mediator of synaptic plasticity and learning and memory (Malenka 2003), and alcohol addiction is thought to be a maladaptive form of learning and memory (Hyman 2005; Nestler 2001). It is well established that ex vivo acute application of alcohol in various types of neurons inhibits the activity of NMDAR (Lovinger et al. 1989; Ron & Wang 2009). These findings raise an interesting question: how can alcohol on one hand inhibit the activity of the channel and on the other hand also contribute to alcohol-dependent learning and memory phenotypes? A clue for solving this puzzle stemmed from studies reporting that the inhibition of the NMDAR function activates mTORC1 (Dwyer & Duman 2013), a kinase responsible for the initiation of the translational machinery at dendrites (Buffington et al. 2014), that plays an essential role in synaptic plasticity and learning and memory (Hoeffer & Klann 2010; Santini et al. 2014). Specifically, a single systemic administration the NMDAR inhibitors ketamine or Ro25-6981 was shown to produce a rapid activation of mTORC1 in the medial PFC (mPFC), which, in turn, produced an increase in the protein levels of the synaptic proteins PSD-95 and the GluA1 subunit of AMPAR, as well as to an increase in the protein levels of the presynaptic protein, synapsin I (Li et al. 2010). These molecular changes lead to increased number of spines and to an increase in synaptic strength of mPFC neurons (Li et al. 2010). Thus, it is plausible that the acute inhibition of the NMDAR by alcohol initiates mTORC1-mediated synaptic and structural plasticity that in turn drives initial alcohol associated learning. In fact, we obtained data to suggest that this mechanism may be responsible, at least in part to alcohol-associated reward learning. The NAc is a key component of the brain reward system (Sesack & Grace 2010), which orchestrates the acquisition (learning) and expression (memory retrieval) of the rewarding and reinforcing properties of alcohol (Koob 2003). We previously reported that unlike in the DMS in which alcohol both inhibits and enhances NMDAR function, alcohol's actions in the NAc are exclusively inhibitory (Wang et al. 2007, 2010, 2011). Interestingly, we found that alcohol also activates mTORC1 in the NAc (Neasta et al. 2010). Specifically, we showed that acute systemic administration of alcohol as well as episodes of heavy alcohol drinking activates mTORC1 in the NAc shell of rodents (Laguesse et al. 2016; Neasta et al. 2010). We further showed that mTORC1 activation is detected in dopamine D1 receptor (D1R) expressing NAc neurons during the first session of alcohol drinking in naïve mice resulting in increases in synaptic strength of NAc D1R neurons (Beckley et al. 2016). Furthermore, similar to the actions of ketamine and Ro25-6981 (Li et al. 2010), excessive alcohol consumption initiated the activation of the translational machinery leading to the translation of synaptic proteins including the collapsin response mediator protein 2 (CRMP-2), HOMER, PSD-95, Arc and GluA1 (Beckley et al. 2016; Liu et al. 2016; Neasta et al. 2010). Furthermore, the increase in CRMP-2 levels in the NAc in response to long-term heavy alcohol drinking promotes microtubules assembly (Liu et al. 2016). Importantly, we showed that systemic administration of the selective mTORC1 inhibitor, rapamycin, decreased rodents ' alcohol seeking and drinking as well as alcohol place preference (Beckley et al. 2016; Neasta et al. 2010). Together, these data suggest an intriguing possibility that the inhibition of the activity of NMDAR by alcohol activates the mTORC1-dependent signaling pathway (Fig.3), which in turn drives the memory of alcohol reward. Interestingly, we recently found that long-term excessive drinking of alcohol activates the mTORC1 signaling pathway in the orbitofrontal cortex (Laguesse et al. 2016). It would therefore be of interest to determine whether the NMDAR is inhibited by alcohol in this brain region as well and if so, whether it is linked to the activation of mTORC1 and/or to increases in synaptic plasticity and alcohol-dependent behavioral phenotypes.

Figure 3. Molecular pathways transducing alcohol's signal in the NAc.

Figure 3

Alcohol inhibits the activity of the NMDARs, which may contribute to the activation of mTORC1. mTORC1 phosphorylates its downstream substrates 4-eukaryote binding protein (4-EBP) and the ribosomal protein S6 kinase (S6K) resulting in the induction of the translational machinery and in the translation of the microtubule-binding protein (CRMP-2), the scaffolding proteins HOMER and PSD-95 as well as the GluA1 subunit of AMPAR, all of which play a major role in synaptic plasticity and alcohol-related phenotypes.

The NMDAR signaling and AUD risk factors

The preclinical studies described above point to the critical role of NMDAR signaling in the development of AUD. Accordingly, several components of the NMDAR signaling pathway have been identified in human studies to be associated with AUD phenotypes. Specifically, single nucleotide polymorphisms (SNPs) within the Fyn gene were shown to be associated with increased risk of developing AUD and in increased severity of the disorder (Ishiguro et al. 2000; Pastor et al. 2009; Schumann et al. 2003). Furthermore, Gelernter et al. utilized data from the genome-wide association studies together with data from the Study of Addiction: Genetics and Environment and the Collaborative Study on the Genetics of Alcoholism and found that Fyn is localized within a gene network that was enriched for genes associated with alcohol dependence in both European Americans and African Americans (Han et al. 2013). Furthermore, several SNPs within the CaMKII gene including one located in the autophosphorylation site of the kinase were also found to be associated with the severity of AUD (Easton et al. 2013), and with increased frequency of alcohol consumption (Meyers et al. 2013). Finally, mutations within the AMPAR subunits as well as mTOR, and HOMER were also shown to be associated with increased alcohol use (Meyers et al. 2015). Although more studies are needed, these studies strongly suggest that the NMDAR network could be viewed as a potential AUD risk factor.

Summary and future directions

In this review, we summarized preclinical data showing that excessive alcohol drinking alters NMDAR signaling, which in turn contributes to the expression of alcohol-related behaviors such as alcohol reward, intake, seeking and relapse. In addition, human studies identified genetic variants within the NMDAR signaling pathway as risk factors for AUD. Together, findings in humans and rodents converge to support the important role of NMDAR signaling in the disease. Thus, targeting the NMDAR-associated molecular pathways affected by alcohol may represent a novel strategy to prevent and treat AUD in humans.

We described signaling mechanisms which center on the NMDAR, that may account, in part, for both the molecular (Figs. 2 3) and behavioral adaptations that ultimately drive alcohol-related phenotypes. We focused on alterations of NMDAR signaling in three brain regions, e.g. the CeA, the DMS and the NAc. However, such signaling mechanisms may occur in other brain regions. For instance, enhanced GluN2B-containing NMDAR currents has been observed in the basolateral amygdala (BLA) and the ventral bed nucleus of the stria terminalis (vBNST) following early cessation from chronic passive alcohol exposure using alcohol liquid diet or vapor administration, respectively (Floyd et al. 2003; Kash et al. 2009). Thus, it would be of interest to determine whether voluntary consumption of alcohol enhances the activity of the NMDARs in the BLA and vBNST, and if so, whether alcohol does so via the activation of the above-mentioned signaling pathways. However, it is plausible that non-physiological long-term exposure of rodents to alcohol (e.g. liquid diet and vapor exposure) produces different molecular outcomes compared with a voluntary drinking paradigm.

An intriguing question is the rather striking brain region selectivity of alcohol's effects. For example, the Fyn signaling pathway is activated by alcohol in the DMS but not in the DLS or NAc, whereas mTORC1 is activated in the NAc shell and the OFC, but not in other striatal or cortical regions. An intriguing possibility is that alcohol, by altering the lipid membrane fluidity (Chin et al. 1978, 1979), changes the composition of signaling proteins within lipid rafts. Lipid rafts are detergent-insoluble membrane microdomains which are enriched in glycosphingolipids, glycophosphatidylinositol-anchored proteins and cholesterol that localize signaling molecules into one membranal site (Allen et al. 2007). Signaling molecules are known to move in or out of lipid rafts depending on the activation signal (Allen et al. 2007). PTPalpha was shown to regulate the activity of Fyn in rafts (Maksumova et al. 2005; Vacaresse et al. 2008) and we previously showed that alcohol induces the association of PTPalpha and Fyn in lipid rafts in the DMS (Gibb et al. 2011). Interestingly, cholesterol content varies across brain regions (Svenningsson et al. 2004), and thus it is plausible that differences in cholesterol content between brain regions of similar neuronal composition such as the DMS and the NAc determine which signaling cascades will be activated in response to alcohol exposure.

Finally, we focused herein on NMDAR-dependent signaling molecules in the DMS, NAc and CeA. The 3 brain regions play a different role in learning and memory-dependent mechanisms such as goal-directed (Yin & Knowlton 2006), reward learning (Sesack & Grace 2010) and anxiety-related behaviors (Janak & Tye 2015), thus, future directions will be required to link these signaling cascades to specific circuitries that contribute to the behavior.

Acknowledgments

The authors would like to thank Khanhky Phamluong for technical assistance and past and present members of the Ron Laboratory for their scientific contribution. This work was supported by NIAAA P50 AA017072 (D.R.) and U01AA023489 (D.R.).

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