Intra-amygdala inhibition of ERK(1/2) potentiates the discriminative stimulus effects of alcohol

Abstract

Extracellular signal-regulated kinase (ERK(1/2)) has been implicated in modulating drug seeking behavior and is a target of alcohol and other drugs of abuse. Given that the discriminative stimulus (subjective/interoceptive) effects of drugs are determinants of abuse liability and can influence drug seeking behavior, we examined the role of ERK(1/2) in modulating the discriminative stimulus effects of alcohol. Using drug discrimination procedures, rats were trained to discriminate a moderate intragastric (IG) alcohol dose (1g/kg) versus water (IG). Following an alcohol (1g/kg) discrimination session phosphorylated ERK(1/2) (pERK(1/2)) immunoreactivity (IR) was significantly elevated in the amygdala, but not the nucleus accumbens. Therefore, we hypothesized that intra-amygdala inhibition of ERK(1/2) would disrupt expression of the discriminative stimulus effects of alcohol. However, intra-amygdala or accumbens administration of the MEK/ERK(1/2) inhibitor U0126 (1 and 3μg) had no effect on the discriminative stimulus effects of the training dose of alcohol (1g/kg). Contrary to our hypothesis, intra-amygdala infusion of U0126 (3μg) potentiated the discriminative stimulus effects of a low alcohol dose (0.5g/kg) and had no effect following nucleus accumbens infusion. Importantly, site-specific inhibition of pERK(1/2) in each brain region was confirmed. Therefore, the increase in pERK(1/2) IR in the amygdala following systemic alcohol administration may be reflective of the widespread effects of alcohol on the brain (activation/inhibition of brain circuits), whereas the site specific microinjection studies confirmed functional involvement of intra-amygdala ERK(1/2). These findings show that activity of the ERK signaling pathway in the amygdala can influence the discriminative stimulus effects of alcohol.

Figure 1. Confirmation of discriminative stimulus control by alcohol

(A) Before testing, discriminative stimulus control was confirmed in all rats by testing a cumulative alcohol substitution curve (n=23). Dose-dependent substitution for the 1 g/kg alcohol dose was observed, demonstrating that the training procedures established reliable stimulus control. (B) Response rate was reduced by the highest alcohol dose (1.7 g/kg). Horizontal dashed lines (>80%) denote full substitution for the discriminative stimulus effects of alcohol. Graphed values are expressed as mean ± s.e.m. *p<0.05 vs. 0.1 g/kg alcohol (Student Newman Keuls post hoc).

Figure 2. Alcohol administration increases pERK_1/2 IR in specific nuclei of the amygdala

(A) Robust discrimination performance and similar response rates on the final test session for rats administered water or the alcohol training dose (1 g/kg; n=6/group). Horizontal dashed lines (>80%) denote full substitution for the discriminative stimulus effects of alcohol. (B) Animals were sacrificed approximately 90 min following alcohol/water administration. Significant elevations in pERK_1/2 positive IR were observed in the subnuclei of the amygdala (CeA, BLA, and LaDL) in the alcohol group relative to the Water group. In contrast, no changes in pERK_1/2 IR were observed in the nucleus accumbens core or shell. (C) Representative photomicrographs (20X) of pERK_1/2 IR in the CeA (central portion of the CeA shown in photomicrograph) and (D) nucleus accumbens core (medial to the anterior commissure shown in photomicrograph) after water and alcohol (1 g/kg, IG) administration. Scale bars, 100 μm. Graphed values are expressed as mean ± s.e.m. *p<0.05 vs. water (t-test).

Figure 3. MEK/ERK_1/2 inhibition in the amygdala or nucleus accumbens does not alter the discriminative stimulus effects of alcohol (1 g/kg, IG)

(A) Intra-amygdala infusion of the MEK/ERK_1/2 inhibitor U0126 (n=9) 30 min prior to alcohol (1 g/kg, IG) did not alter the discriminative stimulus effects of alcohol or (B) response rate. (C) Illustration depicting accurate amygdala injector placements aimed at the CeA and a corresponding photomicrograph showing an injector tract (arrow). (D) Intra-accumbens infusion of U0126 (n=9) also did not alter the discriminative stimulus effects of alcohol, but (E) produced significant response rate reductions. (F) Illustration depicting accurate nucleus accumbens injector placements aimed at the core and a corresponding photomicrograph showing an injector tract (arrow). Horizontal dashed lines (>80%) denote full substitution for the discriminative stimulus effects of alcohol. Graphed values are expressed as mean ± s.e.m. *p<0.05 vs. vehicle (Student Newman Keuls post hoc).

Figure 4. Intra-amygdala MEK/ERK_1/2 inhibition in the amygdala potentiates the discriminative stimulus effects of a low alcohol dose (0.5 g/kg, IG)

(A) In the same rats, amygdala infusion of U0126 30 min prior to alcohol (0.5 g/kg, IG; n=9) potentiated the discriminative stimulus effects of alcohol, without altering response rate. (B) In contrast, intra-accumbens infusion of U0126 did not alter the discriminative stimulus effects of the low alcohol dose (0.5 g/kg, IG; n=9), but significantly reduced response rate, indicating differential involvement of the two brain regions in regulating the discriminative stimulus effects of alcohol. To evaluate whether MEK/ERK_1/2 inhibition alone produced alcohol-like discriminative stimulus effects, the rats were administered U0126 30 min prior to water (IG). (C) Intra-amygdala U0126 did not produce alcohol-like effects (i.e., low alcohol-appropriate responses) and did not alter response rate. (D) Similarly, intra-accumbens U0126 did not produce alcohol-like effects, but significantly reduced response rate. Horizontal dashed lines (>80%) denote full substitution for the discriminative stimulus effects of alcohol. Graphed values are expressed as mean ± s.e.m. *p<0.05 vs. vehicle (paired t-test).

Figure 5. Confirmation of decreased pERK_1/2 IR following U0126 administration

Following the completion of testing, rats received a unilateral infusion of vehicle and U0126 and were sacrificed 30 min later. (A) Intra-amygdala U0126 (3 μg; n=4) significantly reduced pERK_1/2 IR in the CeA. Representative photomicrographs (20X) showing decreased pERK_1/2 IR following U0126 administration in the CeA. Pictures (central portion of the CeA) are taken proximal to the injector tracts to avoid artifacts associated with tissue penetration from the microinjections. (B) Similarly, intra-accumbens U0126 (3 μg; n=5) administration significantly reduced pERK_1/2 in the core. (D) Representative photomicrographs (20X) showing decreased pERK_1/2 IR following U0126 administration in the nucleus accumbens core (medial to the anterior commissure). Scale bars, 100 μm. Pictures are taken proximal to the injector tracts to avoid artifacts associated with tissue damage from the microinjections.