Effect of Modulation of the Astrocytic Glutamate Transporters' Expression on Cocaine-Induced Reinstatement in Male P Rats Exposed to Ethanol

Abstract

Aim: Reinforcing properties of ethanol and cocaine are mediated in part through the glutamatergic system. Extracellular glutamate concentration is strictly maintained through several glutamate transporters, such as glutamate transporter 1 (GLT-1), cystine/glutamate transporter (xCT) and glutamate aspartate transporter (GLAST). Previous findings revealed that cocaine and ethanol exposure downregulated GLT-1 and xCT, and that β-lactam antibiotics restored their expression.

Methods: In this study, we investigated the effect of ampicillin/sulbactam (AMP/SUL) (200 mg/kg, i.p.), a β-lactam antibiotic, on cocaine-induced reinstatement and locomotor activity in male alcohol preferring (P) rats using free choice ethanol (15 and 30%, v/v) and water. We also investigated the effect of co-exposure to ethanol and cocaine (20 mg/kg, i.p.) on GLT-1, xCT and GLAST expression in the nucleus accumbens (NAc) core, NAc shell and dorsomedial prefrontal cortex (dmPFC).

Results: Cocaine exposure decreased ethanol intake and preference. Cocaine and ethanol co-exposure acquired place preference and increased locomotor activity compared to ethanol-exposed rats. GLT-1 and xCT expression were downregulated after cocaine and ethanol co-exposure in the NAc core and shell, but not in dmPFC. AMP/SUL attenuated reinstatement to cocaine as well attenuated the decrease in locomotor activity and ethanol intake and preference. These effects were associated with upregulation of GLT-1 and xCT expression in the NAc core/shell and dmPFC. GLAST expression was not affected after ethanol and cocaine co-exposure or AMP/SUL treatment.

Conclusion: Our findings demonstrate that astrocytic glutamate transporters within the mesocorticolimbic area are critical targets in modulating cocaine-seeking behavior while being consuming ethanol.

Fig. 1

Effect of cocaine and AMP/SUL treatment on water intake and body weight during conditioning and extinction phases. Rats had free access to three bottles of water and ethanol (15%) and (30%) for five weeks. Water intake and body weight were measured during the last two weeks and served as baseline. Rats were then administered cocaine (20 mg/kg/day, i.p.) or saline alternatively for 8 days, composing the conditioning phase, and water intake (A) and body weight (C) were assessed daily. Cocaine did not significantly alter water intake as compared to saline during conditioning. Body weight was not significantly changed following cocaine exposure during the conditioning phase. Rats were then received either AMP/SUL (200 mg/kg/day) or saline for 8 days, the extinction phase. Water intake (B) and body weight (D) were measured daily during the extinction phase. AMP/SUL significantly increased water intake during Day 2 through Day 8. Body weight was not altered during the extinction phase. ^****P < 0.0001 (n = 6–7 for each final groups) (^##P < 0.01, ^###P < 0.001 and ^####P < 0.0001 as compared to baseline within cocaine group).

Fig. 2

Time spent in each conditioning chamber and locomotor activity, measured as distance traveled, during different CPP phases. (A) Repeated measures one-way ANOVA followed by Bonferroni post-tests for preference ratio revealed a significant increase in time spent in cocaine-paired chamber in postconditioning and reinstatement compared to preconditioning and extinction in ethanol-cocaine-saline group. (B) Repeated measures one-way ANOVA followed by Bonferroni post-tests for preference ratio revealed a significant increase in time spent in cocaine-paired chamber in postconditioning in ethanol-cocaine-AMP/SUL group. (C) Repeated measures one-way ANOVA followed by Bonferroni post-tests for preference ratio revealed a significant decrease in time spent in cocaine-paired chamber in reinstatement compared to extinction in ethanol-cocaine-AMP/SUL group. (D) Two-way ANOVA repeated measure followed by Bonferroni post-tests showed a decrease in distance traveled in ethanol-saline-saline group compared to control W group in the preconditioning, conditioning, extinction and reinstatement phase, while ethanol-cocaine-saline and ethanol-cocaine-AMP/SUL groups showed decrease in distance traveled compared to water group in preconditioning phase only (Values shown as means ± S.E.M. ^**P < 0.01, ^***P < 0.001 as compared to water group [@@P < 0.01, @@@P < 0.001 and @@@@P < 0.0001 as compared to ESS group], [^###P < 0.001 as compared to ECS group] (n = 6–7 for each group).)

Fig. 3

Effects of ethanol, cocaine (20 mg/kg) and AMP/SUL (200 mg/kg) on the ratio of GLT-1/β-tubulin (A), xCT/β-tubulin (B) and GLAST/β-tubulin (C) in the NAc core. Quantitative analysis revealed a significant increase in the ratio of GLT-1/β-tubulin in ECA/S group compared to the ECS group. Significant downregulation of GLT-1 and xCT expression was revealed in the ECS group compared to the control W and ESS groups. Significant increase in the ratio of xCT/β-tubulin was revealed in ECA/S group compared to the ECS group. No significant difference in GLAST expression was revealed among all tested groups in the NAc core. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Values shown as means ± S.E.M. n = 6 for each group. Abbreviations: ECA/S, ethanol-cocaine-AMP/SUL; ECS, ethanol-cocaine-saline; W, water; ESS, ethanol-saline-saline.

Fig. 4

Effects of ethanol, cocaine (20 mg/kg) and AMP/SUL (200 mg/kg) on the ratio of GLT-1/β-tubulin (A), xCT/β-tubulin (B) and GLAST/β-tubulin (C) in the NAc shell. Quantitative analysis revealed a significant increase in the ratio of GLT-1/β-tubulin in ECA/S group compared to the ECS group. Significant downregulation of GLT-1 expression was revealed in the ECS and ESS groups compared to the control W group. Significant increase in the ratio of xCT/β-tubulin was revealed in ECA/S group compared to the ECS group. Significant downregulation of xCT expression was revealed in ECS and ESS groups compared to the control W group. No significant difference in GLAST expression was revealed among all tested groups in the NAc shell. ^*P < 0.05, ^**P < 0.01. Values shown as means ± S.E.M. n = 6 for each group. Abbreviations: ECA/S, ethanol-cocaine-AMP/SUL; ECS, ethanol-cocaine-saline; W, water; ESS, ethanol-saline-saline.

Fig. 5

Effects of ethanol, cocaine (20 mg/kg) and AMP/SUL (200 mg/kg) on the expression of GLT-1/β-tubulin (A), xCT/β-tubulin (B) and GLAST/β-tubulin (C) in the dmPFC. Quantitative analysis revealed a significant increase in the ratio of GLT-1/β-tubulin and xCT/β-tubulin in ECA/S group compared to control W group. No significant difference in GLAST expression was revealed among all tested groups in the dmPFC. ^*P < 0.05. Values shown as means ± S.E.M. n = 6 for each group. Abbreviations: ECA/S, ethanol-cocaine-AMP/SUL; ECS, ethanol-cocaine-saline; W, water; ESS, ethanol-saline-saline.