Effects of (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male, alcohol-preferring rats

Abstract

Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT-1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol-preferring (P) rats and that upregulation of the GLT-1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)-(-)-5-methyl-1-nicotinoyl-2-pyrazoline (MS-153), on ethanol drinking and expressions of GLT-1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free-choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS-153 or vehicle for 5 days. The results show that MS-153 treatment significantly reduces ethanol consumption. It was revealed that GLT-1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol-vehicle-treated rats (ethanol-vehicle group) compared with water-control animals. MS-153 treatment upregulated GLT-1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS-153 in these glutamate transporters for the attenuation of ethanol-drinking behavior.

Keywords: GLT-1; MS-153; amygdala; glutamate; hippocampus.

Figure 1

(A) Effects of MS-153 treatment on average daily ethanol intake (g/kg/day) in male P rats exposed to five weeks of continuous free choice of ethanol and water. Statistical analyses demonstrated a significant difference between MS-153 treated group (n=5) and ethanol vehicle group (n=10). Additionally, independent t-test revealed a significant decrease in ethanol intake with MS-153 (50 mg/kg, i.p.) treated group from Day 1 (24 hrs after the first i.p. injection) through Day 5 as compared to ethanol vehicle group. (B) Effects of MS-153 treatment on average daily water intake (ml/kg/day) in male P rats exposed to five weeks of continuous free choice of ethanol and water. Statistical analysis revealed a significant increase in water consumption in MS-153-treated P rats as compared to ethanol vehicle-treated P rats, on days 1, 3 and 4. (C) Effects of MS-153 treatment on body weight (grams) of male P rats exposed to five weeks of continuous free choice access to ethanol and water. Statistical analysis of animals’ body weight data revealed no significant difference in body weight between the ethanol MS-153-treated group and the ethanol vehicle-treated group during the entire period of the study. (D) Effect of MS-153 treatment on total fluid intake of male P rats exposed to continuous free choice access to ethanol and water. Statistical analysis showed significant increase in total fluid intake in MS-153 treated group, on days 3 and 4, as compared to vehicle treated group. Data are shown as mean ± SEM. (*p<0.05; **p<0.01; **p<0.001).

Figure 2

Effect of MS-153 on GLT-1 expression in the amygdala. Upper panel: Representative immunoblots of GLT-1 and β-tubulin, a loading control, in the amygdala. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of GLT-1 in the MS-153-treated group (50 mg/kg, i.p.; n=5) as compared to the ethanol vehicle-treated group (n=5). In addition, statistical analysis revealed a significant downregulation of GLT-1 in the ethanol vehicle group as compared to the ethanol naïve vehicle group. Data are shown as mean ± SEM. (**p<0.01).

Figure 3

Effect of MS-153 on xCT expression in the amygdala. Upper panel: Representative immunoblots of xCT and β-tubulin, a loading control, in the amygdala. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of xCT in the MS-153 treatment group (50 mg/kg, i.p.; n=5) as compared to the ethanol vehicle group (n=5). Alternatively, statistical analysis revealed a significant downregulation of xCT in the ethanol vehicle group as compared to the ethanol naïve vehicle group. Data are shown as mean ± SEM. (*p<0.05; **p<0.01).

Figure 4

Effect of MS-153 on GLT-1 expression in the hippocampus. Upper panel: Representative immunoblots of GLT-1 and β-tubulin, a loading control, in the Hipp. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of GLT-1 in the MS-153 treatment group (50 mg/kg, i.p.; n=5) as compared to the ethanol vehicle group (n=5). Alternatively, statistical analysis revealed a significant downregulation of GLT-1 in the ethanol vehicle group as compared to the ethanol naïve vehicle group. Data are shown as mean ± SEM. (*p<0.05; ***p<0.01).

Figure 5

Effect of MS-153 on xCT expression in hippocampus. Upper panel: Representative immunoblots of xCT and β-tubulin, a loading control, in the Hipp. Lower panel: Quantitative analysis of the immunoblots demonstrated significant upregulation of xCT in the MS-153 treatment group (50 mg/kg, i.p.; n=5) as compared to the ethanol vehicle group (n=5). Alternatively, statistical analysis showed a significant downregulation of xCT in the ethanol vehicle group as compared to the ethanol naïve vehicle group. Data are shown as mean ± SEM. (*p<0.05; **p<0.01).