MAPKs comprise three distinct signaling pathways, namely, the extracellular signal-regulated (ERK_1/2), the c-Jun N-terminal (JNK), and the p38 protein kinases (Boulton and Cobb 1991; Krishna and Narang 2008; Robinson and Cobb 1997). When phosphorylated, or “activated”, MAPK can translocate to the nucleus and facilitate gene transcription (Chen et al. 1992). Of the three subfamilies, the ERK_1/2/MAPK pathway has been the most implicated in addictive processes by a mounting body of research demonstrating sensitivity of this pathway in neural plasticity, learning and memory, and drug reinforcement (Adams and Sweatt 2002; Ferguson et al. 2006; Grewal et al. 1999; Mazzucchelli et al. 2002; Thomas and Huganir 2004). In addition, ERK_1/2 is abundantly expressed in brain regions that modulate addiction such as the prefrontal cortex, nucleus accumbens, bed nucleus of the stria terminalis and amygdala, and is generally increased by drugs of abuse, including cocaine, d-amphetamine, nicotine, morphine and Δ⁹-tetrahydrocannabinol (Choe et al. 2002; Lu et al. 2006; Ortiz et al. 1995; Valjent et al. 2004).

The effect of ethanol on ERK_1/2 activity has been less extensively studied. Consistent with other abused substances, acute ethanol increases ERK_1/2 phosphorylation in the prefrontal cortex, shell of the nucleus accumbens, central and basolateral nucleus of the amygdala, paraventricluar nucleus of the hypothalamus and in the Edinger-Westphal nucleus (Bachtell et al. 2002; Sharko and Hodge 2007). Similarly, ERK_1/2 phosphorylation is increased in the basolateral amygdala and nucleus accumbens shell by response contingent presentation of cues formerly paired with ethanol reinforcement (Schroeder et al. 2008). Conversely, other studies have shown have shown a decrease in the phosphorylation of ERK_1/2 by acute ethanol (Chandler and Sutton 2005; Hendrickson et al. 1998; Kalluri and Ticku 2002; Sanna et al. 2002; Tsuji et al. 2003). Together, these studies indicate that the relationship between acute ethanol and ERK_1/2 activity needs to be further elucidated, and it is possible that the ERK_1/2/MAPK pathway functionally regulates ethanol self-administration and other behavioral impairments associated with alcohol abuse and alcoholism.

ERK_1/2 is phosphorylated within the activation loop of the kinase on both a threonine and a tyrosine residue by the MAPK/ERK_1/2 kinase (MEK_1/2; Crews and Erikson 1992; Seger and Krebs 1995). This activated form of the protein (pERK_1/2) can induce expression of transcription factors including ELK, CREB, c-fos, and others (Adams et al. 2000; Sgambato et al. 1998; Treisman 1996; Vanhoutte et al. 1999). PD098059 (IC₅₀=2–7 μM), U0126 (IC₅₀=0.06–0.07 μM), and SL 327 (IC₅₀=0.18–0.22 μM) are ligands that inhibit the phosphorylation of MEK_1/2, which prevents the downstream phosphorylation of ERK_1/2 (Atkins et al. 1998; Favata et al. 1998). Systemic injection of SL 327 decreases levels of pERK_1/2 immunoreactivity by 62–91%, in the prefrontal cortex, nucleus accumbens, dorsal striatum, BNST, and amygdala, and a growing number of studies have shown that this class of drugs can significantly modulate behavior (Valjent et al. 2006b). For example, SL 327 blocks acute psychostimulant-induced locomotor activity, the induction of behavioral sensitization to cocaine and d-amphetamine, the expression of conditioned place preference to several drugs of abuse, the consolidation of fear memories, memory retention, long-term potentiation, the parental retrieving behavior of C57BL/6J mice, and cytokine-induced immobility in the forced swim test (Atkins et al. 1998; Cestari et al. 2006; Davis et al. 2000; Kuroda et al. 2007; Mazzucchelli and Brambilla 2000; Salzmann et al. 2003; Schafe et al. 2000; Shi and McGinty 2006; Valjent et al. 2006a, b; Wu and Lin 2008). Likewise, site-specific microinjections of U0126 or PD098059 into the amygdala significantly reduce immobility in the forced swim test, cocaine seeking after 30 days of withdrawal, and consolidation of pavlovian fear conditioning memories (Duvarci et al. 2005; Huang and Lin 2006; Lu et al. 2005; Schafe et al. 2000). Furthermore, intra-ventral tegmental area (VTA) microinjection of U1026 has recently been shown to prevent glial cell line-derived neurotrophic factor (GDNF)-induced decreases in ethanol self-administration (Carnicella et al. 2008). This study provides the most convincing evidence to date to support our central hypothesis that activity of the ERK_1/2/MAPK pathway might be involved in modulating the reinforcing properties of ethanol.

The behavioral implications of modulating MAPK activity are largely unknown. A handful of studies have shown that the MEK_1/2 inhibitors SL 327 and U0126 block cue-induced reinstatement, locomotor sensitization to psychostimulants, memory consolidation, LTP, and parental care (Atkins et al. 1998; Cestari et al. 2006; Davis et al. 2000; Kuroda et al. 2007; Lu et al. 2005; Valjent et al. 2006a, b). A recent study by Carnicella et al.( 2008) found that intra-VTA GDNF infusion inhibits ethanol, but not sucrose, self-administration and that this effect is mediated by MAPK signaling since pretreatment with U1026 prevented the GDNF-induced reduction in drinking. Together with our series of experiments, these studies are some of the first to demonstrate that ethanol self-administration is altered due to MEK_1/2 inhibition. Furthermore, the effect of SL 327 on ethanol self-administration is intriguing because it suggests that MEK_1/2 inhibition can functionally alter behavior by potentiating self-administration behavior. Increases in ethanol self-administration have been found after administration of a wide variety of compounds including allopregnanolone, estradiol, μ opioid agonists, κ opioid antagonists, A₂ antagonists and intra-raphe muscimol (Arolfo et al. 2004; Ford et al. 2004, 2005; Hodge et al. 1995; Janak et al. 1998; Mitchell et al. 2005; Quirarte et al. 2007; Reid et al. 2002; Sabino et al. 2007; Sinnott et al. 2002; Tomkins et al. 1994; Ulm et al. 1995; Vacca et al. 2002; Zhang and Kelley 2002). Despite the diverse nature of these ligands, they all have a biphasic effect on self-administration; low doses increase and high doses decrease drinking. This further suggests that there may be a singular common mechanism that underlies escalations in ethanol self-administration, perhaps via an intracellular mechanism such as pERK_1/2.

Conceptually, increases in drug-reinforced responding are difficult to interpret because they can result from either increased motivation to self-administer or blockade of the pharmacological effects of ethanol (i.e., requiring more reinforcements to achieve the same subjective effect). In this study, we characterized mouse performance on a variety of PR schedules of alcohol reinforcement as one index of motivation. PR responding was found to be stable across repeated testing and insensitive to a range of SL 327 doses. Importantly, mice were not consuming pharmacologically active amounts of ethanol under this schedule so it is unlikely that the subjective effects of the consumed ethanol or satiety were interacting with their performance. These results on PR responding for ethanol argue against the hypothesis that inhibition of pERK_1/2 increased the motivation to consume ethanol. Instead, it is possible that SL 327 (30 mg/kg) might have antagonized the acute pharmacological effects of ethanol to increase ERK_1/2 activity, leading to increased ethanol self-administration. The latter interpretation is consistent with a wide body of literature regarding mechanisms of drug self-administration. For instance, a primary pharmacological effect of cocaine is to block the dopamine transporter, which increases synaptic dopamine and activates dopamine receptors. Accordingly, administration of low doses of D1-like or D2-like receptor antagonists has been shown to increase cocaine self-administration (Ettenberg et al. 1982; Koob et al. 1987; Woolverton 1986). Analogous evidence shows that low to moderate doses of the opiate antagonist naloxone increase morphine (Weeks and Collins 1976; Woods et al. 1975) and heroin (Ettenberg et al. 1982; Koob et al. 1984) self-administration. Higher doses of antagonists generally reduce drug self-administration. Thus, one plausible interpretation of the increase in self-administration observed in this study following pretreatment with SL 327 is that ERK_1/2 activation is a primary pharmacological effect of ethanol that supports its reinforcing properties.

A secondary behavioral consequence of MEK_1/2 inhibition was decreased locomotor behavior after high dose administration of SL 327. Consistent with the literature, open field activity was significantly reduced in ethanol self-administering mice by 50–100 mg/kg SL 327 (Valjent et al. 2006b). However, behaviors during the ethanol self-administration session were unaffected by 50 mg/kg indicating that the motor activation that occurs during a self-administration session is sufficient to overcome the mildly sedative effects of SL 327 at this dose. Interestingly, this context-dependent locomotor-suppressant effect of 50 mg/kg SL 327 was not observed in mice that had been trained to self-administer sucrose (i.e., 50 mg/kg SL 327 did not reduce the frequency of duration of any behavior). Importantly, both ethanol and sucrose mice showed a significantly greater degree of sedation after injection of 100 mg/kg SL 327 in both basal (open field) and stimulated (behavior in the operant conditioning chamber) motor activity. It is known that voluntary home-cage ethanol drinking (Spanos and Hodge 2008) and forced exposure to high doses of ethanol by vapor inhalation (Sanna et al. 2002) leads to a downregulation of pERK_1/2 expression. While the effect of daily ethanol intake on pERK_1/2 under operant self-administration conditions is not known, the differential response of the ethanol self-administering mice to SL 327 suggests that modest levels of ethanol self-administration might also reduce pERK_1/2, which could lead to heightened locomotor sensitivity to ERK_1/2 inhibition.

The role of a second MAPK subfamily, JNK, in the regulation of behavior has been less extensively studied but shares many common characteristics with ERK_1/2. JNK is widely recognized for its activation in response to neuronal cell death and oxidative stress, and it has recently been shown to be an important downstream effector of glutamate activity (Borsello et al. 2003; Carboni et al. 2008; Herdegen et al. 1998; Mukherjee et al. 1999; Schwarzschild et al. 1997). In striatal tissue, the group 1 metabotropic glutamate receptor (mGluR) agonist DHPG has been shown to cause a threefold increase in JNK and pERK_1/2 phosphorylation. This increase is blocked by the group 1 mGluR5 antagonist MPEP, but not the group 1 mGluR1 antagonist CPCCOEt, indicating that this effect is mGluR5 but not mGluR1 dependent (Choe and Wang 2001; Yang et al. 2006). This connection between mGluR5 and JNK activity is relevant because it (1) links JNK phosphorylation to a receptor system that is known to modulate ethanol drinking and (2) suggests that ERK_1/2 and JNK can be both activated by the same pharmacological ligands and thus may regulate common processes (Backstrom et al. 2004; Besheer et al. 2006; Hodge et al. 2006; Lominac et al. 2006; McMillen et al. 2005; Schroeder et al. 2005). Because of this relationship, we were interested in investigating whether inhibition of JNK activity by AS 601245 would produce a similar behavioral consequence as ERK_1/2 inhibition. At high doses (30 and 56 mg/kg), reductions in both ethanol self-administration and locomotor activity were observed after injection of AS 601245; low doses (3–10 mg/kg) were ineffective. Thus, the potentiation of ethanol self-administration appears to be ERK_1/2-specific. This finding is in accordance with an earlier study showing that intra-accumbal amphetamine-conditioned place preference can be blocked by a MEK but not JNK inhibitor (Gerdjikov et al. 2004). Together, these data support the hypothesis that ERK_1/2 signaling may be critical for mediating reward-related learning and reinforcement.

A major role of the MAPK pathways in the adult central nervous system is the regulation of neural and behavioral plasticity, including memory processes (Chandler 2003; Impey et al. 1999; Sweatt 2004). For example, SL 327 (100 mg/kg) has been shown to block cued and contextual fear conditioning in a manner that is dissociated from motor effects in rats (Atkins et al. 1998). Similarly, JNK inhibition blocks memory formation and retrieval of an inhibitory avoidance task (Bevilaqua et al. 2003). Thus, it is plausible that the non-specific reductions in ethanol and sucrose self-administration observed after the high doses of the MEK_1/2 and JNK inhibitors in this study reflect memory disruption. However, several factors seem to preclude this interpretation. First, it is important to reiterate that doses of the MAPK inhibitors that reduced operant responding also produced significant decreases in spontaneous locomotor activity in both ethanol- and sucrose-exposed mice, which indicates pleiotropic behavioral effects at high doses of these compounds and complicates interpretations related specifically to memory. Second, although SL 327 (30 mg/kg) inhibits spatial memory in mice (Selcher et al. 1999), this dose specifically enhanced ethanol self-administration without effect on sucrose self-administration in the present study, which is inconsistent with memory disruption. Thus, the apparent lack of effect of SL 327 on memory in this study may reflect differential functional regulation or procedural differences between tests of operant conditioned behavior as compared to tests that are commonly used to assess ERK_1/2 regulation of memory (e.g., Morris water maze, fear conditioning). Alternatively, the enhancement of ethanol-reinforced responding by SL 327 may reflect neuroplasticity in ERK_1/2 function recruited away from memory processes toward regulation of drug reinforcement, which is consistent with other evidence showing that a long history of ethanol self-administration can usurp the function of homeostatic regulatory systems (Kelley et al. 2001).