Effects of chronic inhalation of electronic cigarettes containing nicotine on glial glutamate transporters and α-7 nicotinic acetylcholine receptor in female CD-1 mice

Abstract

Alteration in glutamate neurotransmission has been found to mediate the development of drug dependence, including nicotine. We and others, through using western blotting, have reported that exposure to drugs of abuse reduced the expression of glutamate transporter-1 (GLT-1) as well as cystine/glutamate antiporter (xCT), which consequently increased extracellular glutamate concentrations in the mesocorticolimbic area. However, our previous studies did not reveal any changes in glutamate/aspartate transporter (GLAST) following exposure to drugs of abuse. In the present study, for the first time, we investigated the effect of chronic exposure to electronic (e)-cigarette vapor containing nicotine, for one hour daily for six months, on GLT-1, xCT, and GLAST expression in frontal cortex (FC), striatum (STR), and hippocampus (HIP) in outbred female CD1 mice. In this study, we also investigated the expression of alpha-7 nicotinic acetylcholine receptor (α-7 nAChR), a major pre-synaptic nicotinic receptor in the glutamatergic neurons, which regulates glutamate release. We found that inhalation of e-cigarette vapor for six months increased α-7 nAChR expression in both FC and STR, but not in the HIP. In addition, chronic e-cigarette exposure reduced GLT-1 expression only in STR. Moreover, e-cigarette vapor inhalation induced downregulation of xCT in both the STR and HIP. We did not find any significant changes in GLAST expression in any brain region. Finally, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques, we detected high concentrations of nicotine and cotinine, a major metabolite of nicotine, in the FC tissues of e-cigarette exposed mice. These data provide novel evidence about the effects of chronic nicotine inhalation on the expression of key glial glutamate transporters as well as α-7 nAChR. Our work may suggest that nicotine exposure via chronic inhalation of e-cigarette vapor may be mediated in part by alterations in the glutamatergic system.

Keywords: Cotinine; E-cigarettes; GLT-1; xCT; α-7 nAChR.

Fig. 1

Effects of six months exposure to e-cigarette vapor (e-Cig) containing nicotine on α-7 nAChR expression in the FC, STR and HIP in female CD-1 mice. A) Immunoblot bands for α-7 nAChR and GAPDH (loading control) expression in the FC, STR and HIP. B) Unpaired t-test analysis of immunoblots showed a significant increase in the ratio of α-7 nAChR / GAPDH in FC and STR, but not in HIP, in e-cigarette exposed mice as compared with air controls (control value set to 100%). Data are shown as mean ± SEM (**p<0.01; *p<0.05), (n=4-5 for each group).

Fig. 2

Effects of six months of inhalation of e-cigarette vapor containing nicotine (e-Cig) on GLT-1 expression in the FC, STR and HIP in female CD-1 mice. A) Immunoblot bands for GLT-1 and GAPDH (loading control) expression in the FC, STR and HIP. B) Unpaired t-test analysis of immunoblots showed a significant decrease in the ratio of GLT-1 / GAPDH in STR, and not significant in FC and HIP, in e-cigarette exposed mice compared with air controls (control value set to 100%). Data are shown as mean ± SEM (*p<0.05), (n=4-5 for each group).

Fig. 3

Effects of six months e-cigarette vapor containing nicotine inhalation (e-Cig) on xCT expression in the FC, STR and HIP of female CD-1 mice. A) Immunoblot bands for xCT and GAPDH (loading control) expression in FC, STR and HIP tissues. B) Unpaired t-test analysis of immunoblots showed a significant decrease in the ratio of xCT / GAPDH in STR and HIP, but not significant in FC, in e-cigarette mice as compared with air controls (control value set to 100%). Data are shown as mean ± SEM (**p<0.01; *p<0.05), (n=4-5 for each group).

Fig. 4

Effects of six months e-cigarette vapor containing nicotine inhalation (e-Cig) on GLAST expression in the FC, STR and HIP. A) Immunoblot bands for GLAST and GAPDH (loading control) expression in FC, STR and HIP tissues. B) Unpaired t-test analysis of immunoblots showed non-significant changes in the ratio of GLAST / GAPDH in FC, STR and HIP in e-cigarette exposed mice as compared with air controls (control value set to 100%). Data are shown as mean ± SEM, (n=4-5 for each group).

Fig. 5

LC-MS/MS was used to quantify levels of nicotine and cotinine based on the ratio of the area under the curve of the MRM transitions for each analyte and a corresponding isotope labeled internal standard (cotinine-d³ and nicotine-d⁴). A) calibration curve for extracted blank brain samples spiked with varying concentrations of nicotine and cotinine with fixed concentration of nicotine-d⁴ and cotinine-d³. LC-MS/MS total ion chromatograms (TIC) for each analyte within each sample of B) Air-control group and C) e-cigarettes (e-Cig) group. D) calculated concentrations of each analyte based on TIC shown in A-C. Data analyzed using oneway ANOVA and shown as mean ± SEM (***= p<0.001), (n=5 for each group).