Effects of alcohol on the membrane excitability and synaptic transmission of medium spiny neurons in the nucleus accumbens

Abstract

Chronic and excessive alcohol drinking lead to alcohol dependence and loss of control over alcohol consumption, with serious detrimental health consequences. Chronic alcohol exposure followed by protracted withdrawal causes profound alterations in the brain reward system that leads to marked changes in reinforcement mechanisms and motivational state. These long-lasting neuroadaptations are thought to contribute to the development of cravings and relapse. The nucleus accumbens (NAcc), a central component of the brain reward system, plays a critical role in alcohol-induced neuroadaptive changes underlying alcohol-seeking behaviors. Here we review the findings that chronic alcohol exposure produces long-lasting neuroadaptive changes in various ion channels that govern intrinsic membrane properties and neuronal excitability, as well as excitatory and inhibitory synaptic transmission in the NAcc that underlie alcohol-seeking behavior during protracted withdrawal.

Introduction

The mesolimbic dopamine system is part of the motivational system that regulates appropriate behavioral responses to natural reinforcers such as food, drink and sex. Natural rewards rarely produce long-lasting changes in the mesolimbic system (Hyman, Malenka, & Nestler, 2006). However, alcohol like all other drugs of abuse acutely activates the mesolimbic dopamine system and, upon chronic exposure, produces long-lasting functional alterations in the brain reward system that leads to marked changes in reinforcement mechanisms and motivational state (Koob & Nestler, 1997; Wise, 1998). This persistent functional reorganization of the reward system defines a new equilibrium, different from homeostasis, which allows an apparent stability around a new ‘set-point’, termed allostasis (Le Moal, 2009). Allostatic mechanisms are involved in maintaining a functioning brain reward system, but at a price. For instance, persistent neuroadaptations induced by chronic alcohol exposure produce a negative affect state which contributes to craving for alcohol thereby enhancing the risk of relapse (Breese et al., 2005; Koob, 2003; Sinha, 2001).

The nucleus accumbens (NAcc) is a key structure of the mesolimbic dopaminergic rewarding system involved in behavioral effects of alcohol associated with dependence. Two functionally and anatomically distinct sub-regions, the core and the shell compose the NAcc. These NAcc sub-regions are specifically involved in alcohol-seeking and drug-seeking behaviors (Chaudhri, Sahuque, Cone, & Janak, 2008; Fuchs, Evans, Parker, & See, 2004; Fuchs, Ramirez, & Bell, 2008). A recent study has demonstrated that pharmacological inactivation of the NAcc core reduces conditioned responding to discrete alcohol cues, supporting an important role for the NAcc core in cue-induced relapse to alcohol-seeking (Chaudhri, Sahuque, Schairer, & Janak, 2010). In contrast, pharmacological inactivation of the NAcc shell attenuates the renewal of conditioned responding triggered by the alcohol context suggesting a role for the NAcc shell in context-induced relapse to alcohol-seeking (Chaudhri et al., 2010). The NAcc is mainly (∼95%) composed of GABAergic medium spiny neurons (MSNs) which project massively to the ventral pallidum, substantia nigra and the ventral tegmental area (VTA) (Chang & Kitai, 1985; Kalivas, Churchill, Klitenick, 1993; Nauta, Smith, Faulla, Domesick, 1978; Usuda, Tanaka, & Chiba, 1998; Xia et al., 2011). MSNs also form functional GABAergic interconnections within the NAcc (Taverna, van Dongen, Groenewegen, & Pennartz, 2004). MSNs receive extensive glutamatergic inputs from limbic areas such as prefrontal cortex, hippocampus and basolateral amygdala (Brog, Salyapongse, Deutch, & Zahm, 1993; Sesack & Grace, 2010). The remaining neurons in the NAcc are subpopulations of local circuit cholinergic and GABAergic interneurons (Bennett & Bolam, 1994; de Quidt & Emson, 1986; Hussain, Johnson, & Totterdell, 1996; Vincent & Johansson, 1983; Vincent et al., 1983; Zhou, Vincent, & Dani, 2002). In addition to the well-described dopaminergic input from the VTA which is crucial in mediating reward-related information (Beckstead, Domesick, & Nauta, 1979; Dahlstrom & Fuxe, 1965; Fallon & Moore, 1978; Swanson, 1982), GABAergic neurons from the VTA also send projections to the NAcc (Van Bockstaele & Pickel, 1995), thus providing a reciprocal inhibitory loop to this mesolimbic circuit. Several studies have shown that MSNs play a central role in mediating the reinforcing properties of alcohol (Nie, Rewal, Gill, Ron, & Janak, 2011; Rewal et al., 2011; Rewal et al., 2009). However, we still have a limited understanding of the molecular and cellular mechanisms involved in the effect of alcohol on MSNs that lead to alcohol-induced behavior. The purpose of this article is to provide an overview of the findings showing that acute and chronic alcohol (ethanol) exposure can cause neuroadaptive changes in neuronal excitability, glutamatergic and GABAergic synaptic transmission in the NAcc that underlie important alcohol-induced behaviors.

Alcohol exposure alters electrical membrane properties in NAcc MSNs

The regulation of the electrical membrane properties of MSNs is critical in the integration and processing of drug-related information converging on the NAcc. In vivo, MSNs display two membrane potential states: a hyperpolarized down-state and a more depolarized up-state during which action potential discharge occurs (O'Donnell & Grace, 1995; Wilson & Kawaguchi, 1996). Dysregulation of this two-state potential has been proposed as a major neuroadaptation underlying addiction (Kim, Park, Lee, Park, & Kim, 2011; Mu et al., 2010). Systemic administration of ethanol reduces glutamate-activated and spontaneously active MSNs in the NAcc of freely moving rats (Criado, Lee, Berg, & Henriksen, 1995, 1997). Although the action of acute ethanol has largely been studied, the mechanisms involved in this action of ethanol on MSN firing are still not yet fully understood.

Action of ethanol on voltage-gated ion channels

Activity of MSNs is regulated by the electrical membrane properties that depend on properties of voltage-gated ion channels involved in the generation of action potential (AP), including voltage-dependent Na⁺, Ca²⁺ and K⁺ channels (Nisenbaum, Xu, & Wilson, 1994; Steephen & Manchanda, 2009). Several studies have shown that brain expression of mRNAs coding for multiple voltage-gated ionic channels varies between lines of rodents known to differ markedly in voluntary alcohol consumption (Mulligan et al., 2006; Mulligan et al., 2011; Ponomarev et al., 2006; Tabakoff et al., 2008). For instance, it has been shown that the levels of alcohol consumption was positively correlated with the expression levels of the mRNA coding for the Na⁺ channel β4 subunit (scn4b), an auxiliary subunit involved in the regulation of neuronal activity (Grieco, Malhotra, Chen, Isom, & Raman, 2005), in several brain regions, including the NAcc (Mulligan et al., 2006; Tabakoff et al., 2008). This suggests that high voluntary alcohol consumption could be due at least in part to the reduction of the inhibitory effect of ethanol on Na⁺ channels produced by the high levels of expression of scn4b. Indeed, acute application of high concentration of ethanol (100–200 mM) inhibits voltage-gated Na⁺ channel functions expressed into Xenopus oocytes (Horishita & Harris, 2008; Shiraishi & Harris, 2004) and in dorsal root ganglion neurons (Wu & Kendig, 1998). In addition, the presynaptic effects of ethanol on glutamatergic and GABAergic synaptic transmission of dopamine neurons in the VTA have been shown to depend on tetrodotoxin-sensitive Na⁺ channels (Deng, Li, Zhou, & Ye, 2009; Xiao et al., 2009; Xiao & Ye, 2008). All together, these studies suggest that voltage-gated Na⁺ channels play an important role in the processing of alcohol-related information. Thus, further studies will be needed in order to investigate the mechanisms involved in the effects of ethanol on Na⁺ channels of MSNs in the NAcc.

The inhibition of L-type voltage-gated Ca²⁺ channel (VGCCs) by acute ethanol has been described in several preparations (Calton, Wilson, & Moore, 1999; Carlen et al., 1993; Hendricson, Thomas, Lippmann, & Morrisett, 2003; Walter & Messing, 1999; Zucca & Valenzuela, 2010). Alterations in L-type VGCC number and activity have been related tochronic ethanol effects and withdrawal hyperexcitability (Dolin, Little, Hudspith, Pagonis, & Littleton, 1987; Guppy, Crabbe, & Littleton, 1995; Guppy & Littleton, 1994; Perez-Velazquez, Valiante, & Carlen, 1994). Behavioral experiments have demonstrated that L-type VGCC inhibitors attenuate the signs of the alcohol withdrawal syndrome and reduce the neuronal hyper-excitability observed after withdrawal from chronic alcohol (Bailey, Molleman, & Little, 1998; Smith, Watson, Stephens, & Little, 1999; Veatch & Gonzalez, 2000; Watson & Little, 2002; Whittington & Little, 1991). In the NAcc, it has been shown that L-type VGCC activity is increased during acute ethanol withdrawal (Rossetti, Isola, De Vry, & Fadda, 1999). Interestingly, inhibition of L-type VGCCs attenuates certain behavioral/neurological signs associated with acute ethanol withdrawal by reversing the depletion of extracellular dopamine levels in the NAcc of rats withdrawn from ethanol (Rossetti et al., 1999). Further studies will be needed to characterize the long-lasting effects of ethanol on VGCCs in the NAcc to determine their involvement in alcohol relapse.

The current–voltage relationship of MSNs is characterized by an inward rectification detectable at hyperpolarized membrane potentials (Belleau & Warren, 2000; O'Donnell & Grace, 1993). This inward rectification has been attributed to the activation of voltage-dependent inwardly rectifying K⁺ (K_IR) channels (Belleau & Warren, 2000). K_IR channels are the major determinants of the input resistance and the hyperpolarized resting membrane potential of MSNs during the down-state (Nisenbaum & Wilson, 1995). Interestingly, acute ethanol drinking modulates the expression of mRNAs coding for the K_IR channel subunits in the NAcc of alcohol-preferring mice (Mulligan et al., 2011). In vitro, acute application of ethanol induces a hyperpolarization associated with a decrease in input resistance of MSNs in the striatum (Blomeley, Cains, Smith, & Bracci, 2011). This ethanol-induced hyperpolarization is abolished in the presence of barium suggesting that the effect of ethanol may be due to an increase in potassium conductance through barium-sensitive K_IR channels (Blomeley et al., 2011). We showed that chronic intermittent ethanol (CIE) exposure followed by 40 days of withdrawal produced an increase in the inward rectification of the current-voltage relationship associated with a decrease in the input resistance of MSNs in the NAcc core (Marty & Spigelman, 2011). Moreover, the resting membrane potential of MSNs was more hyperpolarized in the CIE-treated animals (Marty & Spigelman, 2011). These results suggest that CIE treatment could cause a long-lasting increase in K_IR channel activity which could be critical in the state transition and synaptic integration of MSNs in NAcc core (Steephen & Manchanda, 2009).

Action of ethanol on BK and SK channels

Large- (BK) and small-conductance (SK) Ca²⁺-activated K⁺ channels represent one of the several important pharmacological targets of ethanol (Brodie, Scholz, Weiger, & Dopico, 2007; Mulholland, Becker, Woodward, & Chandler, 2011; Mulholland et al., 2009; Treistman & Martin, 2009). Acute ethanol has been found to increase BK and decrease SK channel activity in several preparations (Brodie, McElvain, Bunney, & Appel,1999; Davies et al., 2003; Dopico, Lemos, & Treitman, 1996; Dreixler, Jenkins, Cao, Roizen, & Houamed, 2000).

BK channels are potent regulators of spike repolarization, firing frequency and fast afterhyperpolarization (fAHP) in MSNs of the NAcc (Ishikawa et al., 2009). By speeding up spike repolarization, BK channels can facilitate high-frequency action potential discharge in response to excitatory synaptic input (Gu, Vervaeke, & Storm, 2007). In genetically modified mice, BK subunit composition influences the response to ethanol and controls the development of ethanol tolerance at the molecular, cellular and behavioral levels (Martin et al., 2008; Treistman & Martin, 2009). In the striatum, acute ethanol exposure induces a rapid and selective degradation of BK mRNA, via an epigenetic mechanism involving microRNA miR-9, resulting in a reorganization of BK mRNA splice variants encoding for BK channel isoforms with different intrinsic properties and sensitivity to ethanol (Pietrzykowski et al., 2008). In the NAcc, acute application of ethanol potentiates the open probability of somatic BK channels (Martin et al., 2004). In a recent study, it has been shown that chronic ethanol exposure induces a persistent tolerance of BK channels to acute ethanol in MSNs which is dependent upon the duration of previous ethanol exposure (Velazquez-Marrero et al., 2011). This longer-persistence tolerance is thought to be due to a reorganization of BK channel subunits resulting in increased expression of an alcohol-resistant BK channel isoform called STREX (stress axis-regulated exon) (Velazquez-Marrero al., 2011). STREX channels are more sensitive to calcium indicating that these channels have higher levels of activity and larger BK current for a given calcium concentration. Interestingly, our findings have shown that CIE treatment induced a faster repolarization of the AP and a potentiation of the fAHP amplitude in NAcc MSNs (Marty & Spigelman, 2011). All together, these studies suggest that alcohol-induced reorganization of BK channel isoforms could be critical in the development of tolerance, dependency and addiction to alcohol.

It has been shown recently that chronic exposure of self-administrated ethanol followed by 3 weeks of withdrawal decreased SK channel function resulting in decreased slow AHP amplitude and increased excitability of MSNs in the NAcc core (Hopf et al., 2010). In addition, in vivo infusion of SK channel activator into the NAcc core selectively reduced motivation to obtain alcohol after withdrawal. Interestingly, chlorzoxazone, a US Food and Drug Administration (FDA)-approved positive modulator of Ca²⁺-activated K⁺ channels (Cao, Dreixler, Roizen, Roberts, & Houamed, 2001; Liu, Lo, & Wu, 2003), reduces alcohol-seeking behavior in a rat model of CIE exposure (Hopf et al., 2011). By contrast, CIE treatment administrated by oral intubation did not produce any modification of the slow AHP and firing rate of MSNs in the NAcc core (Marty & Spigelman, 2011).

The relative dose, duration and the mode of administration of ethanol represent important criteria in the interpretation and comparison of the results between studies, as this could explain the apparent discrepancies between findings. Furthermore, the composition of the intrapipette solutions used for electrophysiological recordings could also explain the differences in the experimental results of different studies. For instance, the addition of high concentration of EGTA (ethylene glycol tetraacetic acid), a calcium chelator, in the intrapipette solution could mask a possible effect of ethanol on ion channels that are activated by intracellular calcium. Therefore, all these different parameters should be carefully considered when comparing the effects of ethanol from different studies in order to avoid misleading interpretations.

All together, these results suggest that chronic alcohol exposure induces long-lasting alterations of voltage-gated ion channels which regulate the electrical membrane properties of MSNs. These persistent neuroadaptations could lead to the alteration of processing and integration of information converging on the NAcc which may have important implications for alcohol-related behaviors.

Alcohol exposure alters synaptic glutamatergic transmission in the NAcc

The MSNs receive major excitatory glutamatergic inputs from different part of the brain such as the hippocampus, prefrontal cortex, amygdala and thalamus (Brog et al., 1993; Sesack & Grace, 2010). The regulation of glutamatergic transmission is critical for the maintenance of homeostasis of the reward system. The neuroadaptive changes of synaptic glutamatergic transmission have been shown to play an important role in the development of addictive behaviors (Kalivas, Lalumiere, Knackstedt, & Shen, 2009). Indeed, pharmacological inhibition of AMPA receptor (AMPAR) and NMDA receptor (NMDAR) has been shown to attenuate reinstatement of alcohol-seeking behavior (Backstrom & Hyytia, 2004; Sanchis-Segura et al., 2006). Therefore, alteration of glutamatergic transmission into the NAcc could induce profound changes in the reward system leading to the expression of drug-related disorders characteristic of the allostatic state (David, Ansseau, & Abriani, 2005; Kalivas, 2009; Stuber et al., 2011).

Acute effects of ethanol on synaptic glutamatergic transmission in the NAcc

Acutely, ethanol has been shown to alter glutamate transmission within the NAcc. Alcohol reduces presynaptic release of glutamate within the NAcc and inhibits glutamate postsynaptic receptor function (Nie, Madmba, & Siggins, 1994; Zhang, Hendricson, & Morrisett, 2005). NMDAR-mediated currents are more sensitive to the inhibitory effect of low concentrations of ethanol, whereas AMPAR-mediated currents are only affected by high concentration of ethanol (Nie et al., 1994). Acute ethanol vapor exposure can alter the expression of glutamatergic synaptic plasticity by blocking the induction of NMDAR-dependent long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) elicited in MSNs from the NAcc shell (Jeanes, Buske, & Morrisett, 2011). This acute effect of ethanol is thought to be due to the potent inhibitory action of ethanol on GluN2B-containing NMDAR function in the NAcc (Maldve et al., 2002; Zhang et al., 2005). In addition, this inhibition of NMDAR function by acute ethanol has been shown to be responsible for the ethanol-induced attenuation of the NMDAR-dependent long-term potentiation (LTP) of EPSCs in several brain structures including the striatum (Izumi, Nagashima, Murayama, & Zorumski, 2005; Morrisett & Swartzwelder, 1993; Schummers & Browning, 2001; Tokuda, Izumi, & Zorumski, 2011; Weitlauf, Egli, Grueter, & Winder, 2004; Xie et al., 2009; Yin, Park, Adermark, & Lovinger, 2007). By contrast, chronic alcohol exposure produces an upregulation of NMDAR function and subunit expression in the mesolimbic system during withdrawal from alcohol (Lack, Diaz, Chappell, DuBois, McCool, 2007; Szumlinski, Ary, Lominac, Klugmann, & Kippin, 2008; Wang et al., 2007; Wang, Lanfranco, Gibb, & Ron, 2011). Indeed, CIE treatment has recently been shown to facilitate the LTP induction of NMDAR-mediated EPSCs in VTA dopaminergic neurons during short-term withdrawal (Bernier, Whitaker, & Morikawa, 2011). In addition, chronic ethanol exposure has been reported to facilitate the induction of NMDAR-dependent LTP in hippocampal CA1 neurons, demonstrating that acute and chronic ethanol exposure differentially affect the induction of LTP (Fujii, Yamazaki, Sugihara, & Wakabayashi, 2008). These results suggest that increased NMDAR function may be, at least in part, responsible for the neuronal hyperexcitability observed during withdrawal from chronic ethanol treatment (Follesa & Ticku, 1995; Hendricson et al., 2007; Kumari & Ticku, 2000).

Ethanol affects the regulation of extracellular glutamate homeostasis in the NAcc

NAcc extracellular glutamate is an important determinant of ethanol drinking. Acute and chronic ethanol administration has been shown to increase extracellular levels of glutamate in the NAcc (Dahchour, Hoffmanm, Deitrich, & de Witte, 2000; Lallemand, Ward, De Witte, & Verbanck, 2011; Melendez, Hicks, Cagle, & Kalivas, 2005; Szumlinski et al., 2007) (Table 1). Experimental manipulations that elevate or lower NAcc extracellular glutamate levels in the NAcc can modulate ethanol drinking behavior by increasing or decreasing ethanol consumption, respectively (Kapasova & Szumlinski, 2008; Szumlinski et al., 2008). In addition, chronic alcohol administration sensitizes the response to an acute alcohol challenge to elevate extracellular glutamate levels in the NAcc (Szumlinski et al., 2007). Consistently, a recent study has shown that an increase in NAcc core extracellular glutamate levels is associated with cue-induced reinstatement of alcohol-seeking behavior (Gass, Sinclair, Cleva, Widholm, & Olive, 2011). This alcohol-induced dysregulation of extracellular glutamate levels could be due to an alteration in the mechanism of release and re-uptake of glutamate as shown in cocaine studies (Baker et al., 2003; Pierce, Bell, Duffy, & Kalivas, 1996). In the NAcc core, basal extracellular glutamate is mainly derived from constitutive cystine/glutamate (cys/glu) exchange (Baker, Xi, Shen, Swanson, & Kalivas, 2002). The release of cytoplasmic glutamate is linked to cystine uptake and occurs more in glial cells than neurons. Extracellular glutamate levels are also regulated by glutamate transporters (Danbolt, 2001), in particular the glial transporter GLT1 located at the vicinity of the synaptic cleft. GLT1 allows to buffer glutamate released from presynaptic vesicle and via cys/glu exchanger (Pendyam, Mohan, Kalivas, & Nayar, 2009). In addition, the deletion of the Homer2 gene, a member of the Homer family involved in the regulation of glutamate signaling in the postsynaptic density, has been shown to reduce the levels of glutamate in the NAcc (Szumlinski et al., 2005). This regulation of extracellular glutamate levels allows for tonic stimulation of metabotropic glutamate receptors which, in turn, control synaptic glutamatergic transmission. Indeed, it has been shown that activation of cys/glu exchanger in the NAcc stimulates presynaptic metabotropic glutamate receptor 2/3 (mGlu_2/3) activation, thereby decreasing the synaptic glutamate release probability (Moran, McFarland, Melendez, Kalivas, & Seamans, 2005). Self-administration of cocaine reduces the expression of both cys/glu exchanger and GLT1 in the NAcc core (Knackstedt, Melendez, & Kalivas, 2010; Madayag et al., 2007). In addition, repeated cocaine administration reduces the expression of Homer1 in the NAcc during protracted withdrawal (Swanson, Baker, Carson, Worley, & Kalivas, 2001). Consequently, repeated cocaine exposure strongly reduces the basal levels of extracellular glutamate leading to an increase in synaptic release of glutamate via decreased tonic activation of release-regulating mGlu_2/3 and mGlu_1/5 in the NAcc core (Madayag et al., 2007; Miguens et al., 2008; Pierce et al., 1996; Swanson et al., 2001; Xi, Baker, Shen, Carson, & Kalivas, 2002). Activation of cys/glu exchanger and GLT1 has been shown to restore basal extracellular glutamate levels in the NAcc core and prevent cue-induced reinstatement of cocaine seeking (Baker et al., 2003; Knackstedt et al., 2010; Moussawi et al., 2009; Sondheimer & Knackstedt, 2011). Consistently, stimulation of mGlu_2/3 to reduce glutamate release has been shown to attenuate cocaine seeking (Baptista, Martin-Fardon, & Weiss, 2004; Peters & Kalivas, 2006). Interestingly, reduction in glutamatergic transmission via activation of mGlu_2/3 or inhibition of postsynaptic mGlu₅ attenuates alcohol-seeking behavior (Adams, Short, & Lawrence, 2010; Backstrom & Hyytia, 2005; Johnson et al., 2007). These findings suggest that like cocaine, alcohol produces similar neuroadaptive changes of synaptic glutamatergic transmission in the NAcc which lead to the development of alcohol-seeking behavior.

Table 1.

Effects of ethanol on the extracellular levels of glutamate in the NAcc.

References	Animal model	Ethanol treatment	Measurement period	Levels of extracellular glutamate in the NAcc
Dahchour et al., 2000	Male HAS (High-alcohol sensitivity) and LAS (Low-alcohol sensitivity) rats	Acute i.p. injection of EtOH 2 g/kg	Up to 3 h after EtOH injection	↗ from 100 to 180 min after injection
Melendez et al., 2005	Male Sprague- Dawley rats	Chronic EtOH exposure: EtOH l g/kg i.p. daily for 7 days	24 h and 14 days after the last EtOH injection	↗ at 24 h. = at 14 days. Note: No change in GLT1 protein expression at 24h
Sziimlinski et al., 2007	Male alcohol- preferring C57BL/6J (B6) mice	Scheduled High Alcohol Consumption (SHAC): 6 doses of 5% EtOH (v/v) solution every other day (SHAC6)	Day 1 for SHAC1 (1st dose of EtOH), and day 18 for SHAC6 (6th dose of EtOH)	Basal levels SHAC1 = SHAC6. ↗ during EtOH consumption only in SHAC6. ↗ after acute EtOH challenge (2 g/kg, i.p) only in SHAC6
Kapasova & Szumlinski, 2008	Male alcohol- preferring C57BL/6J (B6) mice Male alcohol- avoiding DBA2/J (D2) mice	Chronic intermittent EtOH exposure: 1–8 doses of EtOH 2 g/kg i.p. every other day	1st and 8th EtOH injection	Basal levels = prior the 1st and 8th EtOH injection in both B6 and D2 mice. ↗ after the 1st EtOH injection only in D2 mice. ↗ after the 8th EtOH injection only in B6 mice. Notes: APDC (mGlu2/3 agonist) ↘ EtOH intake in B6 and D2; TBOA (glutamate reuptake inhibitor) ↗ EtOH intake only in B6 mice
Szumlinski et al., 2008	Male alcohol- preferring C57BL/6J (B6) mice	Chronic intermittent EtOH exposure: 8 doses of EtOH 2 g/kg i.p. every other day	After acute i.p. injection of EtOH 2 g/kg	↗ in control mice. ↗↗ in mice overexpressing Homer2 in the NAcc. Note: overexpression of Homer2 in the NAcc ↗ EtOH intake
Lallemand et al., 2011	Male Wistar rats	Chronic intermittent ethanol exposure: EtOH 2 or 3 g/kg administrated by gavage	24 h after the last dose of EtOH	Basal levels ↘ only in EtOH 2 g/kg group. ↗ after acute EtOH challenge in both groups
Gass et al., 2011	Male Wistar rats	EtOH self- administration exposure: EtOH l mg/kg i.v. on a FR1 schedule	During reinstatment testing session	↗ during cue-induced reinstatement of EtOH-seeking behavior.

Lasting effects of chronic ethanol exposure on synaptic glutamatergic transmission in the NAcc

Extensive studies using different ethanol exposure models have examined the effect of chronic ethanol exposure during withdrawal on glutamatergic receptor function in several brain regions including the mesolimbic dopamine system (Stuber, Hopf, Tye, Chen, & Bonci, 2010). In the NAcc of rats, it has been shown recently that CIE exposure induces the activation of AKT kinase and its intracellular target named mammalian target of rapamycin complex 1 (mTORC1) kinase (Neasta, Ben Hamida, Yowell, Carnicella, & Ron, 2010). mTORC1-related signaling plays an important role in the translocation of synaptic proteins (Hoeffer & Klann, 2010). Activation of the mTORC1-related pathway is thought to be responsible for the increased expression level of AMPAR subunit GluA1 and Homer in the NAcc observed at 24 h withdrawal after 3 months of CIE exposure (Neasta et al., 2010). Interestingly, inhibition of AKT or mTORC1 reduces alcohol-seeking behavior (Neasta et al., 2010; Neasta, Ben Hamida, Yowell, Carnicella, & Ron, 2011). Further, it has been shown that the deletion of the Homer2 gene impairs the development of behavioral and neurochemical plasticity after repeated ethanol administration (Szumlinski et al., 2005). In agreement, over-expression of Homer2 protein in the NAcc facilitates ethanol reinforcement and increases ethanol intake (Szumlinski et al., 2008). These studies suggest that mTORC1 and Homer2 activation may be involved in the altered expression of proteins involved in glutamatergic transmission after CIE treatment. For instance, Obara et al. (2009) have found that NMDAR subunits GluN2A and GluN2B, mGlu₅ and mGlu₁ protein expression increase in the NAcc core of rats after 24 h withdrawal following 6 months of CIE exposure. However, after 4 weeks of withdrawal only mGlu₅ expression remains increased (Obara et al., 2009). Interestingly, the therapeutic efficacy of acamprosate, a US FDA-approved drug used for treating human alcoholism, is thought to relate to the inhibition of mGlu₅ function (Blednov & Harris, 2008). These findings suggest that mGlu₅ could be involved in mechanisms mediating alcohol-seeking behavior.

There is additional evidence that chronic ethanol exposure and withdrawal produces a time-dependent modulation of glutamatergic receptor function in the NAcc. Indeed, it has been recently demonstrated that the NMDAR-dependent LTD of EPSCs induced in MSNs from the NAcc shell is abolished after 24 h withdrawal of chronic ethanol vapor exposure, instead resulting in LTP of EPSCs. However, after 72 h of withdrawal when the NMDAR- dependent LTD was still strongly reduced, the LTP was no longer observed (Jeanes et al., 2011). All together, these studies suggest that the neuroadaptive changes observed during early withdrawal may lead to distinct long-term neuroadaptations involving different neuronal mechanisms that are more likely to underlie alcohol-seeking behaviors.

Increasing evidence suggests similarities between mechanisms induced by drugs of abuse (Hyman et al., 2006; Kalivas et al., 2009; Nestler, 2001). Cocaine, alcohol and other drugs of abuse cause long-lasting neuroadaptations of glutamatergic system in the brain including the NAcc (Gass & Olive, 2008). Protracted withdrawal from cocaine induces a long-lasting potentiation of AMPAR EPSC amplitude in the NAcc core that plays an important role in cue-induced cocaine-seeking behavior (Conrad et al., 2008; Mameli et al., 2009). This potentiation of AMPAR synaptic transmission is due to an increase in the number of postsynaptic Ca²⁺-permeable GluA1 homomeric (GluA2-lacking) AMPARs which is characterized by an increase in single channel conductance (Conrad et al., 2008; Guire, Oh, Soderling, & Derkach, 2008; Mameli et al., 2009). Our recent findings show that CIE exposure followed by prolonged withdrawal (>40 days) induced a potentiation of AMPAR synaptic transmission associated with an increase in AMPAR unitary conductance in the NAcc core (Marty & Spigelman, 2011). These results suggest that the potentiation of glutamate transmission induced during protracted withdrawal from CIE treatment could be due to a redistribution of AMPAR subunit composition increasing the number of functional postsynaptic GluA2-lacking AMPARs as previously observed in cocaine studies (Conrad et al., 2008; Mameli, Balland, Lujan, & Luscher, 2007; Mameli et al., 2009). Thus, increased NAcc core glutamatergic transmission may be a critical neuroadaptation that could be responsible for cue-induced reinstatement of alcohol-seeking behavior, which in abstaining human alcoholics would promote alcohol craving and increase the risk of relapse.

Alcohol exposure alters synaptic GABAergic transmission in the NAcc

The MSNs receive strong inhibitory GABAergic inputs from the VTA (Sesack & Grace, 2010; Van Bockstaele & Pickel, 1995; Voorn, Jorritsma-Byham, Van Dijk, & Bujis, 1986). Moreover, MSNs from the NAcc send reciprocal GABAergic projections back to non-dopaminergic neurons in the VTA (Kalivas et al., 1993; Usuda et al., 1998; Xia et al., 2011). This reciprocal connection between the NAcc and the VTA is thought to mediate an inhibitory feedback to regulate dopamine release in the NAcc (Rahman & McBride, 2000, 2002). In addition, the GABAergic interneurons within the NAcc regulate the excitability of the MSNs (Taverna, Canciani, & Pennartz, 2007). Therefore, modulation of GABAergic transmission in the NAcc is important in the regulation of the reward system homeostasis.

Acute effects of ethanol on synaptic GABAergic transmission in the NAcc

GABA_A receptors (GABA_ARs) represent one of the several important pharmacological targets of ethanol (Kumar et al., 2009; Lovinger, 1997; Weiner & Valenzuela, 2006). Modulation of GABAergic transmission in the central nervous system underlies many of the behavioral effects of ethanol, including sedative-hypnotic, anticonvulsant, cognitive-impairing and anxiolytic actions (Kumar et al., 2009). Alcohol tolerance and dependence also appear to be attributable, at least in part, to changes in the function of GABA_AR subunit composition and trafficking (Kumar, Fleming, & Morrow, 2004; Liang et al., 2007; Liang et al., 2006). Numerous studies have demonstrated that acute ethanol potentiates GABA_AR function (Weiner & Valenzuela, 2006). In the NAcc core, ethanol (44–100 mM) increases GABA currents in about 50% of MSNs recorded (Nie, Madamba, & Siggins, 2000). This ethanol-induced enhancement of GABA currents is blocked by the non-selective mGlu receptor antagonist MCPG (Nie et al., 2000). In addition, it has been shown that the inhibitory effect of ethanol on NMDAR-mediated synaptic transmission in the NAcc core depends in part on the activation of metabotropic GABA_B receptors (GABA_BRs) likely via a presynaptic mechanism (Steffensen, Nie, Criado, & Siggins, 2000). Interestingly, acamprosate, used therapeutically to reduce relapse, has the opposite effect of ethanol in the NAcc core. Acamprosate enhances NMDAR-mediated synaptic transmission and blocks presynaptic GABA_BRs (Berton, Franscesconi, Madamba, Zieglgansberger, & Siggins, 1998). Yet, baclofen, a muscle relaxant and agonist at GABA_BRs, is also very effective to suppress the alcohol withdrawal syndrome severity in human alcoholics with minimal side effects (Addolorato et al., 2006; Liu & Wang, 2011). Together these studies suggest that ethanol can modulate the intricate and reciprocal regulation of the GABAergic and glutamatergic transmission in the NAcc core that can lead to long-lasting neuroadaptations responsible for alcohol dependence and alcohol-seeking behavior.

Role of GABAergic receptor subunits of the NAcc in alcohol drinking behavior

Alcohol drinking behavior has been related to GABAergic transmissionin the NAcc. Morphometric studies have shown that alcohol preference is associated with increased density of GABAergic terminals in the NAcc (Hwang, Lumeng, Wu, & Li, 1990). Down-regulation of the α4-subunit of GABA_AR has been observed in the NAcc during early withdrawal from chronic ethanol exposure (Papadeas, Grobin, & Morrow, 2001). Such early decreases in GABA_AR α4-subunit expression are thought to underlie the early to lerance for alcohol (Liang et al., 2007). Recent studies have provided the first compelling evidence for a specific role of GABA_ARα4- and δ-subunits in mediating alcohol-drinking behavior (Nie et al., 2011; Rewal et al., 2011; Rewal et al., 2009). In an elegant series of studies, Janak and colleagues have demonstrated that selective reduction of the GABA_AR α4-subunit expressions specifically in the NAcc shell, using viral-mediated RNAi techniques, reduces ethanol self-administration and preference, which is accompanied by a reduction of the reinforcing effects of ethanol (Rewal et al., 2011; Rewal et al., 2009). In addition, Nie et al. (2011) have shown that selective reduction of the GABA_AR δ -subunit expression specifically in the medial NAcc shell decreases ethanol intake. Interestingly, GABA_AR δ-subunits can associate with α4- and β-subunits to form a functional α4 βδ GABA_AR isoform predominantly located at perisynaptic or extrasynaptic sites (Nusser, Sieghart, & Somogyi, 1998; Wei, Zhang, Peng, Houser, & Mody, 2003). The α4βδ GABA_ARs are highly sensitive to GABA and are responsible for the tonic inhibition in the presence of low extracellular GABA that regulates neuronal excitability (Farrant & Nusser, 2005). Low concentrations of ethanol selectively increase the tonic current mediated by δ subunit-containing GABA_ARs (Sundstrom-Poromaa et al., 2002; Wallner, Hanchar, & Olsen, 2003; Wei, Faria, & Mody, 2004). Tonic currents recorded in NAcc MSNs are also potentiated by low concentrations of acute ethanol (Liang, Suryanarayanan, Olsen, & Spigelman, 2009). Taken together, these results suggest that α4βδ GABA_ARs in the medial NAcc shell play an important role in alcohol drinking behavior. Another recent study demonstrated that inactivation of the NAcc shell by infusion of muscimol produces differential effects on the intake of ethanol, sucrose or ethanol/sucrose solutions (Stratford &Wirtshafter, 2011). This finding confirms the importance of the NAcc shell in mediating voluntary ethanol intake. All together, these studies show that the alterations of GABAergic receptors producedin the NAcc shellin responseto alcohol are determinant in the reinforcing properties of alcohol.

Chronic alcohol effects on integration of reward information processing in mesolimbic circuits

Alcohol-induced modulation of neuronal intrinsic membrane properties and synaptic transmission in NAcc microcircuits could alter the occurrence of suprathreshold postsynaptic depolarizations triggering APs. The AP constitutes the pertinent physiological coding event required for the induction of spike-time dependent plasticity (STDP), an activity-dependent bi-directional long-term synaptic plasticity, that can modulate the strength of synaptic inputs in MSNs and interneurons (Fino, Deniau, & Venance, 2008, 2009; Fino, Glowinski, & Venance, 2005; Fino & Venance, 2010; Pawlak & Kerr, 2008; Shen, Flajolet, Greengard, & Surmeier, 2008). The potentiation of glutamatergic inputs could enhance the integration of dendritic information and increase the likelihood of AP initiation at the soma (Xu, Ye, Poo, & Zhang, 2006). Alcohol-induced increase in excitatory postsynaptic inputs could promote the occurrence and the magnitude of STDP in the NAcc neurons. In contrast, inhibitory GABAergic inputs can modulate the degree of depolarizations elicited by excitatory inputs (Fitzpatrick, Akopian, & Walsh, 2001), thereby influencing the firing of MSNs and the occurrence of STDP. The modulation of the NAcc neuronal network plasticity by alcohol could lead to important alterations in the integration and transmission of pertinent information to output structures, such as the VTA which plays a major role in the behavioral effects of alcohol (Hyman et al., 2006; Nestler, 2001).

It is essential to take into account the possible effects of ethanol on the electrical properties and synaptic transmission of NAcc interneurons since these interneurons are directly involved in the regulation of NAcc microcircuits and control information processing of MSNs. Although cholinergic interneurons constitute less than 1% of the local neural population within the NAcc (Rymar, Sasseville, Luk, & Sadikot, 2004), a recent study has demonstrated their importance in reward-related responses to cocaine (Witten et al., 2010). Optogenetic inhibition of cholinergic interneurons increases MSN electrical activity in the NAcc and suppresses cocaine conditioning in freely moving rats (Witten et al., 2010). Such studies reinforce the importance of NAcc microcircuits in mediating drug-related behaviors.

In vivo studies have shown that alcohol modulates the release of dopamine in the NAcc. Acute ethanol self-administration increases the activity of VTA dopaminergic neurons producing an increase in dopamine release within the NAcc involved in the rewarding effect of ethanol (Brodie, Shefner, & Dunwiddie, 1990; Melendez et al., 2002; Weiss, Lorang, Bloom, & Koob, 1993; Yim & Gonzales, 2000). In contrast to acute ethanol, withdrawal from repeated ethanol administration is characterized by decreased levels of dopamine in the NAcc associated with a decrease in the activity of VTA dopaminergic neurons (Bailey, O'Callaghan, Croft, Manley, & Little, 2001; Diana, Pistis, Carboni, Gessa, & Rosseti, 1993; Shen, 2003; Shen & Chiodo, 1993). This depletion of dopamine in the mesolimbic system is believed to be responsible, at least in part, for dysphoria and anhedonia associated with alcohol protracted withdrawal which may trigger the drug-seeking behavior characteristic of addicts (Weiss & Porrino, 2002). Indeed, it has been shown that alcohol-dependent rats are more motivated during withdrawal to obtain ethanol in an operant self-administration task than non-dependents, and that ethanol consumption reverses the withdrawal-associated decreased dopamine levels in the NAcc (Weiss et al., 1996). The reciprocal connections between the NAcc and the VTA are critical in the regulation of dopamine release (Rahman & McBride, 2000, 2002). Thus, decreases in dopamine levels during withdrawal may result at least in part from the long-term effects of ethanol on the NAcc microcircuitry presented above. Further, ethanol-induced modifications of the VTA microcircuitry will likely affect both dopaminergic and GABAergic neurotrans-mission in the NAcc (Hopf et al., 2007; Hyman et al., 2006; Ortiz et al., 1995; Stuber et al., 2008). Similarly, excitatory inputs into the NAcc from the prelimbic cortex, hippocampus and amygdala will differentially affect reward information processing in the NAcc. Therefore, it is essential to consider the multiple effects of alcohol on the different brain structures in order to have a better understanding of the mechanisms responsible for the alcohol-induced neuroadaptations in the mesolimbic dopamine system.

While it may be tempting to speculate on what the demonstrated alcohol-induced alterations in membrane properties and synaptic transmission of MSNs might do to affect reward information processing in the NAcc circuitry, such speculation is premature. Hypothesis-driven experiments which utilize selective activation (or suppression) of various neuronal pathways of mesolimbic circuitry should be able to provide answers to these questions in the near future.

Conclusions

Collectively, the results from the studies described above provide compelling evidence that MSNs in the NAcc play a key role in mediating behavioral effects of alcohol. These studies have demonstrated that acute and chronic ethanol exposure alters electrical membrane properties of MSNs, and produces a dysregulation of glutamatergic and GABAergic transmission in the NAcc via intricate pre- and postsynaptic mechanisms. In addition, several studies have been able to relate the neuroadaptive changes produced by ethanol in the NAcc to several important behavioral symptoms of alcohol dependence such as alcohol consumption and alcohol-seeking behaviors.

Although the mechanisms involved in short-term effects of alcohol have been largely described, the long-lasting neuroadaptive changes induced by alcohol in the NAcc during protracted withdrawal from chronic alcohol exposure are still unclear. These long-lasting neuroadaptations are more likely to contribute to the development of the allostatic state mediating susceptibility to cravings and relapse. A better understanding of alcohol-induced long-lasting neuroadaptive changes in the different sub-regions of the NAcc should be useful in the development of specific pharmacological compounds to reduce alcohol-seeking behavior during protracted withdrawal.