Effects of novel beta-lactam, MC-100093, and ceftriaxone on astrocytic glutamate transporters and neuroinflammatory factors in nucleus accumbens of C57BL/6 mice exposed to escalated doses of morphine

Abstract

Chronic exposure to opioids can lead to downregulation of astrocytic glutamate transporter 1 (GLT-1), which regulates the majority of glutamate uptake. Studies from our lab revealed that beta-lactam antibiotic, ceftriaxone, attenuated hydrocodone-induced downregulation of GLT-1 as well as cystine/glutamate antiporter (xCT) expression in central reward brain regions. In this study, we investigated the effects of escalating doses of morphine and tested the efficacy of novel synthetic non-antibiotic drug, MC-100093, and ceftriaxone in attenuating the effects of morphine exposure in the expression of GLT-1, xCT, and neuroinflammatory factors (IL-6 and TGF-β) in the nucleus accumbens (NAc). This study also investigated the effects of morphine and beta-lactams in locomotor activity, spontaneous alternation percentage (SAP) and number of entries in Y maze since opioids have effects in locomotor sensitization. Mice were exposed to moderate dose of morphine (20 mg/kg, i.p.) on days 1, 3, 5, 7, and a higher dose of morphine (150 mg/kg, i.p.) on day 9, and these mice were then behaviorally tested and euthanized on Day 10. Western blot analysis showed that exposure to morphine downregulated GLT-1 and xCT expression in the NAc, and both MC-100093 and ceftriaxone attenuated these effects. In addition, morphine exposure increased IL-6 mRNA and TGF-β mRNA expression, and MC-100093 and ceftriaxone attenuated only the effect on IL-6 mRNA expression in the NAc. Furthermore, morphine exposure induced an increase in distance travelled, and MC-100093 and ceftriaxone attenuated this effect. In addition, morphine exposure decreased the SAP and increased the number of arm entries in Y maze, however, neither MC-100093 nor ceftriaxone showed any attenuating effect. Our findings demonstrated for the first time that MC-100093 and ceftriaxone attenuated morphine-induced downregulation of GLT-1 and xCT expression, and morphine-induced increase in neuroinflammatory factor, IL-6, as well as hyperactivity. These findings revealed the beneficial therapeutic effects of MC-100093 and ceftriaxone against the effects of exposure to escalated doses of morphine.

Keywords: Drug dependence; GLT-1; Locomotor activity; Opioid; xCT.

Fig. 1

Experimental design and timeline of the study.

Fig. 2

Effects of repeated exposures to moderate dose and single higher dose of morphine and treatment with ceftriaxone and MC-100093 in body weight of male C57BL/6 mice. Statistical analyses didn’t show any significance difference between control and treatment groups in body weight. N = 8–9/group.

Fig. 3

Effects of ceftriaxone and MC-100093 in locomotor activity and short memory Y maze in mice exposed to repeated moderate dose and single higher dose of morphine. (A) Locomotor behavioral analysis showed that ceftriaxone and MC-100093 attenuated morphine-induced increases in total distance travelled. B) Short memory Y maze analysis did not show any significance difference in the spontaneous alternation percentage (SAP) between morphine, and ceftriaxone-morphine- and MC-100093-morphine-treated groups. C) Statistical analysis did not show any significance difference in the number of arms entries in Y Maze between morphine and ceftriaxone-morphine and MC-100093-moprhine treated groups. *p < 0.05; ***p < 0.001; N = 6–7 per group.

Fig. 4

Effects of ceftriaxone and MC-100093 in protein expression of GLT-1 and xCT in the NAc of mice exposed to repeated moderate dose and single higher dose of morphine. Quantitative analysis of Western blots showed that morphine exposure downregulated GLT-1 protein expression in the NAc as compared to control group, and ceftriaxone and MC-100093 increased GLT-1 protein expression as compared to morphine treated group. Similarly, morphine exposure downregulated xCT protein expression in the NAc, and ceftriaxone and MC-100093 increased xCT protein expression as compared to morphine treated group. Control group data are represented as 100 %, and data are represented as mean ± S.E.M. *p < 0.05; **p < 0.01. N = 5 per group.

Fig. 5

Effects of ceftriaxone and MC-100093 in IL-6 mRNA (C) and TGF mRNA (D) in the NAc of mice exposed to repeated moderate dose and single higher dose of morphine. RT_PCR quantitative analysis showed that morphine exposure increased IL-6 mRNA expression in the NAc as compared to control group. Importantly, ceftriaxone and MC-100093 attenuated morphine-induced upregulation of IL-6 mRNA expression in the NAc. Furthermore, morphine exposure increased TGF-β mRNA expression in the NAc as compared to control group. There was a significant difference between control and morphine-MC100093 groups. However, there were no significance differences in the expression of TGF-β mRNA between morphine, morphine-MC100093 and morphine-ceftriaxone in the NAc. Control group data are represented as 100 %, and data are represented as mean ± S.E.M. *p < 0.05; **p < 0.01; N = 5 per group.