Effects of ampicillin, cefazolin and cefoperazone treatments on GLT-1 expressions in the mesocorticolimbic system and ethanol intake in alcohol-preferring rats
Introduction
Chronic exposure to drugs of abuse causes dysregulation of glutamatergic neurotransmission, with alterations to glutamatergic projections from the prefrontal cortex (PFC) to the nucleus accumbens (NAc) (Kalivas, 2009, Kalivas and Volkow, 2011, Quintero, 2013). These projections are part of the mesocorticolimbic reward pathway and has been shown to mediate drug-seeking and relapse behaviors (Kalivas, 2009, Kalivas and Volkow, 2011, Quintero, 2013, Wise and Koob, 2014). Furthermore, recent studies have demonstrated that substance abuse is associated with impaired glutamatergic release, leading to an imbalance in glutamatergic homeostasis within the mesocorticolimbic pathway (Parsegian and See, 2014, Shen et al., 2014).
Ethanol, similar to other drugs of abuse, is known to significantly alter extracellular glutamate concentrations in the mesocorticolimbic pathway, which is due, at least partially, to impaired clearance of glutamate from the synapse (Melendez et al., 2005, Ding et al., 2013). These elevated NAc glutamate concentrations following ethanol intake, in turn, appear to promote continued excessive ethanol consumption (Griffin et al., 2014). Consistent with these findings, we have demonstrated a significant downregulation of the major glutamate transporter, glutamate transporter 1 (GLT-1, its human homolog is excitatory amino acid transporter 2, EAAT2), in the NAc of alcohol-preferring (P) male rats after five weeks of free-choice ethanol exposure compared to their ethanol-naïve counterparts (Sari and Sreemantula, 2012, Sari et al., 2013).
Importantly, upregulation of GLT-1 expression in the mesocorticolimbic pathway is associated with restored glutamate homeostasis and attenuated drug-seeking behavior (Knackstedt et al., 2010, Rasmussen et al., 2011). While ceftriaxone treatment has yielded promising results in reducing drug abuse in cocaine, ethanol, and methamphetamine exposure animal models (Sari et al., 2009, Sari et al., 2011, Abulseoud et al., 2012, Rao and Sari, 2014), other β-lactam antibiotics, identified earlier as GLT-1 upregulators (Rothstein et al., 2005), have not been evaluated for their in vivo efficacy. Therefore, the aim of this study is to evaluate the effect of other FDA approved β-lactam antibiotics – ampicillin (AMP), cefazolin (CZN) or cefoperazone (CPZ) treatments – on daily ethanol intake in male P rats following five weeks of free-choice ethanol exposure. Since 5 weeks of chronic ethanol exposure results in a consistent reduction of GLT-1 expression in the NAc and PFC (Sari and Sreemantula, 2012, Sari et al., 2013), ethanol-naïve animals were not included in this study.
In order to associate the changes in ethanol consumption following these treatments with changes in glutamatergic activity, GLT-1 expressions in the NAc and PFC were compared between the β-lactam-treated and saline-treated groups. To confirm the previously established pharmacological mechanism of GLT-1 upregulation in these brain regions (Wu et al., 2010), phosphorylation of signaling molecule AKT was also measured in the NAc and PFC of treated vs. control groups. Finally, to determine the CNS bioavailability of drug treatments, the cerebrospinal fluids (CSF) from AMP-, CZN-, and CPZ-treated P rats were analyzed by high-performance liquid chromatography (HPLC). Furthermore, we determined the effects of these β-lactam antibiotics on sucrose intake, a consummatory control for ethanol-drinking behavior. In addition, the N-methyltetrazolethiol side chain present in β-lactams is known to exhibit disulfiram-like effects on ethanol metabolism via inhibition of the enzyme, aldehyde dehydrogenase-2 (ALDH2) (Matsubara et al., 1987). Therefore, liver samples collected from AMP-, CZN-, and CPZ-treated P rats were analyzed for ALDH2 activity, an enzyme accountable for 60% of hepatic acetaldehyde metabolism (Weiner, 1987).
Section snippets
Animals
Adult male P rats were obtained from the Indiana University School of Medicine, Indianapolis, IN and housed in standard plastic tubs with corn-cob bedding in the Department of Laboratory Animal Resources vivarium at The University of Toledo. All animals had ad lib access to food and water during the study, and the animal vivaria were maintained at a temperature of 21 °C on a 12-h light/dark cycle (0600 h/1800 h). All of the animal experimental protocols were approved by the Institutional Animal
Effects of AMP, CZN, or CPZ treatment on ethanol consumption by male P rats
Fig. 1A illustrates the effects of AMP, CZN, or CPZ treatment on daily ethanol consumption (g/kg/day) in male P rats. Data analysis using GLM repeated measures revealed a significant main effect of Day [F(1,5) = 61.9, p < 0.001] and a significant Treatment × Day interaction [F(3,15) = 2.36, p < 0.01]. A one-way ANOVA followed by two-tailed Dunnett’s t-tests demonstrated a significant reduction (p ⩽ 0.01) in ethanol consumption in animals treated with AMP, CZN, or CPZ, starting on Day 2 and lasting through
Discussion
We report in this study that AMP, CZN, and CPZ treatments attenuated ethanol drinking compared to the saline-treated control group. Importantly, we have demonstrated that the reduction in ethanol intake following treatments with AMP, CZN, and CPZ was associated in part with significant upregulation of the major glutamate transporter, GLT-1, in regions of the mesocorticolimbic reward pathway (the NAc and the PFC). Moreover, AMP, CZN, and CPZ treatment were found to significantly increase the
Conflict of interest
The authors declare no conflict of interest. The views expressed in this manuscript are strictly those of the authors and do not necessarily represent those held by the National Institutes of Health (NIH) or NIAAA.
Acknowledgments
This work was supported by Award Number R01AA019458 (Y.S.) and in part by Award Number AA13522 (R.L.B.) from the National Institute on Alcohol Abuse and Alcoholism (NIAAA). The authors would also like to thank Dr. Nicolas L. Chiaia for providing training for CSF collection.
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