Alcohol alters the activation of ERK1/2, a functional regulator of binge alcohol drinking in adult C57BL/6J mice

Abstract

Background: Binge alcohol drinking is a particularly risky pattern of alcohol consumption that often precedes alcohol dependence and addiction. The transition from binge alcohol drinking to alcohol addiction likely involves mechanisms of synaptic plasticity and learning in the brain. The mitogen-activated protein kinase (MAPK) signaling cascades have been shown to be involved in learning and memory, as well as the response to drugs of abuse, but their role in binge alcohol drinking remains unclear. The present experiments were designed to determine the effects of acute alcohol on extracellular signaling-related kinases (ERK1/2) expression and activity and to determine whether ERK1/2 activity functionally regulates binge-like alcohol drinking.

Methods: Adult male C57BL/6J mice were injected with ethanol (EtOH) (3.0 mg/kg, intraperitoneally) 10, 30, or 90 minutes prior to brain tissue collection. Next, mice that were brought to freely consume unsweetened EtOH in a binge-like access procedure were pretreated with the MEK1/2 inhibitor SL327 or the p38 MAPK inhibitor SB239063.

Results: Acute EtOH increased pERK1/2 immunoreactivity relative to vehicle in brain regions known to be involved in drug reward and addiction, including the central amygdala and prefrontal cortex. However, EtOH decreased pERK1/2 immunoreactivity relative to vehicle in the nucleus accumbens core. SB239063 pretreatment significantly decreased EtOH consumption only at doses that also produced nonspecific locomotor effects. SL327 pretreatment significantly increased EtOH, but not sucrose, consumption without inducing generalized locomotor effects.

Conclusions: These findings indicate that ERK1/2 MAPK signaling regulates binge-like alcohol drinking. As alcohol increased pERK1/2 immunoreactivity relative to vehicle in brain regions known to regulate drug self-administration, SL327 may have blocked this direct pharmacological effect of alcohol and thereby inhibited the termination of binge-like drinking.

Keywords: Alcohol; Amygdala; Binge Drinking; ERK1/2; MAP Kinase; Nucleus Accumbens.

Figure 1. tERK1/2 and pERK1/2 immunoreactivity

Representative photomicrographs (100X) of the cytological patterns of ERK1/2 immunoreactivity in the nucleus accumbens (a) core and (b) shell, (c) central amygdala, and (d) basolateral amygdala (scale bar, 10 microns).

Figure 2. Effects of acute ethanol on ERK1/2 phosphorylation in the nucleus accumbens as a function of time

(A) Mean (± SEM) immunoreactivity of the pERK1/2 positive area in the nucleus accumbens core following 3.0 g/kg ethanol treatment expressed as relative change versus saline control. (B) Representative photomicrographs (20X) of the cytological pattern of pERK1/2 immunoreactivity in the core. (C) Mean (± SEM) immunoreactivity of the pERK1/2 positive area in the nucleus accumbens shell following 3.0 g/kg ethanol treatment expressed as relative change versus saline control. (D) Representative photomicrographs (20X) of the cytological pattern of pERK1/2 immunoreactivity in the shell (scale bar, 50 microns). *Significantly different from saline control at the given time point (p<0.05; Student’s t-test).

Figure 3. Effects of acute ethanol on ERK1/2 phosphorylation in the amygdala as a function of time

(A) Mean (± SEM) immunoreactivity of the pERK1/2 positive area in the central amygdala following 3.0 g/kg ethanol treatment expressed as relative change versus saline control. (B) Representative photomicrographs of the cytological pattern of pERK1/2 immunoreactivity in the central amygdala. (C) Mean (± SEM) immunoreactivity of the pERK1/2 positive area in the basolateral amygdala following 3.0 g/kg ethanol treatment expressed as relative change versus saline control. (D) Representative photomicrographs of the cytological pattern of pERK1/2 immunoreactivity in the basolateral amygdala (scale bar, 50 microns). *Significantly different from saline control at the given time point (p<0.05; Student’s t-test).

Figure 4. Systemic ERK1/2 inhibition increases binge-like ethanol intake

(A) No effect of pretreatment with the MEK1/2 inhibitor SL327 on ethanol intake emerged during the first hour of drinking. (B) Over the total four-hour drinking session, SL327 significantly increased ethanol drinking at the 10 and 30 mg/kg doses. (C) Mice achieved a binge-level of alcohol consumption during the 4-hour testing period. (D) SL327 pretreatment did not affect total distance traveled in a four-hour locomotor activity test. (E) SL327 pretreatment did not alter blood ethanol metabolism following an acute 2g/kg i.p. ethanol injection. *Significantly different than vehicle at the given dose (p<0.05; Student-Newman-Keuls)

Figure 5. Systemic p38 inhibition non-selectively decreases binge ethanol intake

(A) Pretreatment with the p38 inhibitor SB239063 (30 mg/kg) reduced ethanol intake during the first hour of ethanol drinking. (B) The effect was no longer present by the end of the four-hour drinking session. (C) Mice achieved a binge-level of alcohol consumption during the 4-hour testing period. (D) SB239063 pretreatment reduced total distance traveled in a one-hour locomotor activity test. (E) SB239063 pretreatment did not alter blood ethanol metabolism following an acute 2g/kg i.p. ethanol injection. *Significantly different than vehicle at the given dose (p<0.05; Student-Newman-Keuls)

Figure 6. Effects of SL327 are selective for ethanol reinforcement

Mice were given access to a 1% sucrose solution on the same schedule as the ethanol consumption. No effect of SL327 on sucrose consumption emerged over the (A) one-hour or (B) four-hour access period. (C) SL327 pretreatment did not alter time spent in the center vs. perimeter of the open field during locomotor activity testing. *Significantly different than vehicle at the given dose (p<0.05; Student-Newman-Keuls)