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Article by Fawaz Alasmaria,#, Laura E. Crotty Alexanderb,c,#, Jessica A. Nelsond, Isaac T. Schieferd, Ellen Breene, Christopher A. Drummondf , and Youssef Saria,*

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

Introduction

Electronic (e)-cigarettes are battery operated nicotine delivery devices that heat and aerosolize e-liquid, creating vapor [For review (Hahn et al., 2014)]. Most e-liquid on the market contains nicotine, thus users who are inhaling e-cigarette vapor are also inhaling nicotine (Crotty Alexander et al., 2015). Recently, e-cigarette use has increased globally as an alternative or supplement to conventional tobacco cigarette use (Yamin et al., 2010, Crotty Alexander et al., 2015, Schoenborn and Gindi, 2015). Emerging evidence demonstrates that exposure to e-cigarettes may induce several toxicological effects, including inflammation, decreased host defenses and DNA damage that is a precursor to neoplastic transformation (Vardavas et al., 2012, Hwang et al., 2016, Yu et al., 2016).

Although e-cigarettes were invented as a smoking cessation tool, they have been demonstrated to confer similar urges to smoke, as compared to conventional smoking (King et al., 2014). Withdrawal symptoms and high desire to smoke were associated with withholding of e-cigarettes (Dawkins et al., 2012). E-cigarettes deliver smaller amounts of nicotine per puff as compared to tobacco cigarettes, however, similar systemic nicotine and cotinine concentrations to combustible cigarettes have been achieved after exposure (Schroeder and Hoffman, 2014). It has been reported that some brands of e-cigarettes can deliver higher nicotine levels as compared to combustible cigarettes (Ramôa et al., 2015). It has been found that exposure to nicotine containing e-cigarette vapor during late prenatal and early postnatal life induced significant persistent behavioral alterations during adulthood compared to vehicle control (e-cigarettes without nicotine) and air-control mice (Smith et al., 2015).

In the present study, we investigated the effects of chronic exposure to e-cigarette vapor containing nicotine, using a well-established, clinically relevant mouse exposure system, on the glutamatergic system in female CD-1 mice. The rationale for using female, not male, mice in this study is that previous studies found that female animal models showed higher nicotine seeking behavior compared to male models (Donny et al., 2000, Torres et al., 2009).

In addition, a prior study found that female rats exhibited higher motivation to consume nicotine as compared to male and female ovariectomized rats (Donny et al., 2000). This suggests that hormones, including estradiol, play a crucial role in nicotine seeking behavior. Moreover, nicotine rewarding effects have been enhanced in female rats as compared to male rats (Torres et al., 2009).

Nicotinic acetylcholine receptors (nAChRs), including alpha-7 nAChR (α-7 nAChR), have been found to be upregulated or stimulated following exposure to nicotine (Auta et al., 2000, Buisson and Bertrand, 2001, Konradsson-Geuken et al., 2009, Alsharari et al., 2015). Importantly, α-7 nAChR is localized mainly in pre-synaptic glutamatergic neurons in the mesocorticolimbic areas (Marchi et al., 2002, Jones and Wonnacott, 2004, Feduccia et al., 2012) and this receptor has been found to modulate the majority of glutamate release in prefrontal cortex (PFC) and other brain regions following nicotine exposure (KonradssonGeuken et al., 2009, Feduccia et al., 2012, Bortz et al., 2013). However, there is little known about the effects of chronic nicotine exposure on α-7 nAChR expression. We here, for the first time, determined the expression of α-7 nAChR in frontal cortex (FC), striatum (STR) and hippocampus following six months of inhalation of e-cigarette vapor containing nicotine in female CD-1 mice.

Additionally, we and others have shown that chronic exposure to drugs of abuse reduced the expression of glutamate transporter-1 (GLT-1,) as well as cystine/glutamate antiporter (xCT) in the nucleus accumbens (NAc), HIP and amygdala (Knackstedt et al., 2009, Knackstedt et al., 2010, Alhaddad et al., 2014a, Alhaddad et al., 2014b, Aal-Aaboda et al., 2015). It is important to note that GLT-1 is responsible for clearance of a majority of the extracellular glutamate concentration into astrocytes (Tanaka et al., 1997, Danbolt, 2001). Decreased GLT-1expression was associated with significantly increased extracellular glutamate concentrations in the NAc in animals exposed to alcohol or heroin (Melendez et al., 2005, LaLumiere and Kalivas, 2008, Shen et al., 2014, Das et al., 2015). In addition, xCT plays a crucial role in glutamate homeostasis by exchanging extracellular cystine for intracellular glutamate (Baker et al., 2002, Shih et al., 2006). Moreover, the xCT system was found to be involved in attenuation of nicotine seeking (Knackstedt et al., 2009). Glutamate/aspartate transporter (GLAST) is another glutamate transporter, co-localized with GLT-1 in astrocytes, and is mainly expressed in the cerebellum and retina (Lehre and Danbolt, 1998, Danbolt, 2001). Although we did not observe any downregulation in GLAST expression following alcohol drinking for five weeks (Alhaddad et al., 2014b, Hakami et al., 2016), we aimed in this study to determine whether chronic nicotine exposure may affect the expression of this glia l glutamate transporter in the central reward brain regions. In the present study, the expression of GLT-1, xCT, and GLAST after six months of exposure to e-cigarette vapor containing nicotine was investigated in the FC, STR and HIP.

Several studies have detected nicotine and cotinine (the major metabolite and biomarker of nicotine) in plasma and urine (Hengen and Hengen, 1978, Curvall et al., 1982, Horstmann, 1985, Degen and Schneider, 1991, Mercelina-Roumans et al., 1996, Acosta et al., 2004, Chang et al., 2005, Levine et al., 2013). Few studies have assessed the concentration of nicotine and cotinine in specific brain regions, such as NAc and STR (Chang et al., 2005, Katner et al., 2015). However, there is little known about the bioavailability of nicotine in the brain following six months of inhalation. As compared to other routes of administration, nicotine inhalation has a fast and high rate of absorption as well as high rate of brain distribution [For review see (Le Houezec, 2003)]. Detection of high concentrations of nicotine and cotinine in the brain during chronic nicotine inhalation might suggest the degree of nicotine exposure associated with any changes in the glutamatergic system. In this study, Western blotting was used to quantify changes in the amount of proteins that are expressed in neurons and glia in different brain regions for air control and e-cigarette vapor containing nicotine groups. In addition, we determined nicotine and cotinine concentrations in the FC following six months of exposure to e-cigarette vapor.

Conclusion

We conclude here that nicotine delivery via e-cigarette vapor inhalation, using a physiological and clinically relevant exposure method, induced changes in the expression of glial glutamate transporters and nicotinic receptors. Chronic exposure to nicotine through inhalation of e-cigarette vapor containing nicotine increased α-7 nAChR expression in the mesocorticolimbic area. Moreover, e-cigarette exposure also reduced the expression of GLT-1 and xCT, which may lead to high extracellular glutamate concentrations in central reward brain regions. These data demonstrated that nicotine exposure alters glial glutamate transporters as well as nicotinic receptors, which might be key proteins in the development of nicotine dependence.