Cue-induced reinstatement of alcohol-seeking behavior is associated with increased ERK_1/2 phosphorylation in specific limbic brain regions: Blockade by the mGluR5 antagonist MPEP

Abstract

Relapse to alcohol use after periods of abstinence is a hallmark behavioral pathology of alcoholism and a major clinical problem. Emerging evidence indicates that metabotropic glutamate receptor 5 (mGluR5) antagonists attenuate relapse to alcohol-seeking behavior but the molecular mechanisms of this potential therapeutic effect remain unexplored. The extracellular signal-regulated kinase (ERK_1/2) pathway is downstream of mGluR5 and has been implicated in addiction. We sought to determine if cue-induced reinstatement of alcohol-seeking behavior, and its reduction by an mGluR5 antagonist, is associated with changes in ERK_1/2 activation in reward-related limbic brain regions. Selectively-bred alcohol-preferring (P) rats were trained to lever press on a concurrent schedule of alcohol (15% v/v) vs. water reinforcement. Following 9 days of extinction, rats were given an additional extinction trial or injected with the mGluR5 antagonist MPEP (0, 1, 3, or 10 mg/kg) and tested for cue-induced reinstatement. Brains were removed 90-min later from the rats in the extinction and MPEP (0 or 10 mg/kg) conditions for analysis of p-ERK_1/2, total ERK_1/2, and p-ERK₅ immunoreactivity (IR). Cue-induced reinstatement of alcohol-seeking behavior was associated with a three to five-fold increase in p-ERK_1/2 IR in the basolateral amygdala and nucleus accumbens shell. MPEP administration blocked both the relapse-like behavior and increase in p-ERK_1/2 IR. p-ERK_1/2 IR in the central amygdala and NAcb core was dissociated with the relapse-like behavior and the pharmacological effect of mGluR5 blockade. No changes in total ERK or p-ERK₅ were observed. These results suggest that exposure to cues previously associated with alcohol self-administration is sufficient to produce concomitant increases in relapse-like behavior and ERK_1/2 activation in specific limbic brain regions. Pharmacological compounds, such as mGluR5 antagonists, that reduce cue-induced ERK_1/2 activation may be useful for treatment of relapse in alcoholics that is triggered by exposure to environmental events.

Introduction

Alcoholism is a complex neuropsychiatric disorder that is characterized by recurring cycles of chronic drinking, abstinence and relapse. Emerging evidence suggests that alcohol and other drugs of abuse may produce maladaptive changes in a variety of molecular and cell signaling pathways that lead to enduring changes in brain structure and function (Crews et al., 1996, Fadda and Rossetti, 1998, Nestler and Aghajanian, 1997). These neuroadaptations are thought to regulate the progressive and recurring behavioral pathologies that occur in alcoholism (Breese et al., 2005, Pandey et al., 2003, Ron and Jurd, 2005). Understanding drug-induced plasticity in brain and behavioral functions is of potential importance for development of new pharmacotherapies for problems associated with alcoholism, such as relapse (Anton et al., 1995, Heinz et al., 2003, Mann, 2004).

A growing number of studies have shown that metabotropic glutamate receptor 5 (mGluR5) activity regulates alcohol-related behavioral processes in animal models. For example, the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) inhibits the reinforcing (Cowen et al., 2005, Hodge et al., 2006, Schroeder et al., 2005) and discriminative stimulus effects of investigator- (Besheer and Hodge, 2005) and self-administered (Besheer et al., 2006) alcohol in rodents. Similarly, pharmacological blockade of mGluR5 prevents relapse-like behavior as measured by cue-induced reinstatement (Backstrom et al., 2004) and alcohol deprivation procedures (Schroeder et al., 2005). Although these findings indicate mGluR5 blockade attenuates relapse to alcohol-seeking behavior, the molecular or cell signaling mechanisms associated with this potential therapeutic effect remain unexplored.

Extracellular signal-regulated kinase (ERK) is one potential downstream mediator of mGluR5 activity (Yang et al., 2004) that is also known to modulate adaptive processes such as learning, memory, and synaptic plasticity (Sweatt, 2004, Thomas and Huganir, 2004) as well as maladaptive forms of plasticity associated with drug use (Girault et al., 2007, Valjent et al., 2006). The two closely related isoforms of ERK (ERK₁ and ERK₂, or ERK_1/2) are activated by dual phosphorylation on both threonine and tyrosine residue by MEK_1/2 (Anderson et al., 1990); reviewed by Cobb (1999). Activated ERK_1/2 phosphorylates cellular targets or translocates to the nucleus where it activates specific gene transcription factors (Grewal et al., 1999). By regulating cellular activities and gene transcription, the ERK_1/2 cascade transduces the activity of a variety of extracellular and intracellular signals into enduring changes in CNS structure and function (Pearson et al., 2001, Qi and Elion, 2005, Treisman, 1996, Valjent et al., 2000, Valjent et al., 2006).

Alcohol exposure produces changes in the active (i.e., phosphorylated) form of ERK_1/2 (p-ERK_1/2) in brain regions that are of significance to behavioral pathologies associated with addiction (Chandler and Sutton, 2005, Davis et al., 1999, Kalluri and Ticku, 2002a, Sanna et al., 2002, Tsuji et al., 2003). For example, acute ethanol has been shown to produce a dose- and time-dependent decrease in p-ERK_1/2 levels in mouse and rat cerebral cortex (Chandler and Sutton, 2005, Kalluri and Ticku, 2002b, Tsuji et al., 2003); but other studies have shown that acute ethanol increases ERK_1/2 phosphorylation in the Edinger–Westphal nucleus (Bachtell et al., 2002) and amygdala (Pandey et al., 2008). Forced exposure to chronic ethanol and withdrawal alter ERK_1/2 phosphorylation in brain regions that regulate alcohol self-administration and relapse-like behavior, including the amygdala (Pandey et al., 2008, Sanna et al., 2002).

Although the mechanisms by which alcohol alters ERK_1/2 phosphorylation are not fully characterized, converging evidence indicates that mGluR5 may regulate both ERK_1/2 activation and the neural effects of alcohol. For example, pharmacological activation of Group I metabotropic glutamate receptors (mGluR1 and mGluR5), via site-specific infusion of the agonist DHPG, increases ERK_1/2 phosphorylation in rat striatum (Choe and Wang, 2001). This effect of the nonselective agonist is fully blocked by the mGluR5 antagonist MPEP in cultured striatal neurons but not altered by an mGluR1 antagonist (Yang et al., 2004), which suggests mGluR5 specificity in ERK_1/2 activation. Other data show that ethanol inhibits mGluR5 activity in vitro (Minami et al., 1998), which could lead to reduced ERK_1/2 activation as noted above. When taken together with evidence that cue-induced reinstatement of alcohol-seeking behavior has recently been linked to increase ERK_1/2 phosphorylation in the amygdala (Radwanska et al., 2008), these findings suggest that mGluR5 antagonist-induced effects on relapse-like behavior may be associated inhibition of ERK_1/2 activation in specific brain regions.

To address this hypothesis, the present study was designed to first determine if the mGluR5 antagonist MPEP would inhibit cue-induced reinstatement of alcohol-seeking behavior in selectively-bred alcohol-preferring (P) rats (Li et al., 1979). Second, we examined p-ERK_1/2 immunoreactivity (IR) in the nucleus accumbens and amygdala following extinction, reinstatement, and MPEP treatment to determine if the behavioral effects of MPEP are associated with altered ERK_1/2 activation. These brain regions were chosen for study because they are key elements in the neurobiological regulation of associative learning processes in drug addiction and reward (Everitt et al., 1999), are known to mediate the reinforcing and subjective effects of alcohol (Besheer et al., 2003, Hodge et al., 1995, Hodge and Cox, 1998, Schroeder et al., 2003), and have recently been linked to alcohol relapse-like behavior in rodents (Dayas et al., 2007, Zhao et al., 2006) and cue-induced craving in abstinent human alcoholics (Schneider et al., 2001). Third, we examined total ERK_1/2 IR to establish if changes in p-ERK_1/2 IR were associated with altered abundance of the kinase. Finally, adjacent brain sections were processed for p-ERK₅ IR in an effort to address potential specificity of ERK_1/2 activation in cue-induced reinstatement of alcohol-seeking behavior and its blockade by MPEP.