Elsevier

Neuroscience

Volume 295, 4 June 2015, Pages 164-174
Neuroscience

Effects of ampicillin, cefazolin and cefoperazone treatments on GLT-1 expressions in the mesocorticolimbic system and ethanol intake in alcohol-preferring rats

https://doi.org/10.1016/j.neuroscience.2015.03.038Get rights and content

Highlights

  • Ampicillin, cefazolin and cefoperazone treatments reduced alcohol intake.

  • These drugs upregulated both GLT-1 and pAKT levels in the NAc and PFC.

  • Cefoperazone significantly inhibited hepatic ALDH2.

  • Ampicillin, cefazolin and cefoperazone have been found in CSF.

Abstract

Chronic ethanol consumption is known to downregulate expression of the major glutamate transporter 1 (GLT-1), which increases extracellular glutamate concentrations in subregions of the mesocorticolimbic reward pathway. While β-lactam antibiotics were initially identified as potent upregulators of GLT-1 expression, only ceftriaxone has been extensively studied in various drug addiction models. Therefore, in this study, adult male alcohol-preferring (P) rats exposed chronically to ethanol were treated with other β-lactam antibiotics, ampicillin, cefazolin or cefoperazone (100 mg/kg) once daily for five consecutive days to assess their effects on ethanol consumption. The results demonstrated that each compound significantly reduced ethanol intake compared to the saline-treated control group. Importantly, each compound significantly upregulated both GLT-1 and pAKT expressions in the nucleus accumbens and prefrontal cortex compared to saline-treated control group. In addition, only cefoperazone significantly inhibited hepatic aldehyde dehydrogenase-2 enzyme activity. Moreover, these β-lactams exerted only a transient effect on sucrose drinking, suggesting specificity for chronically inhibiting ethanol reward in adult male P rats. Cerebrospinal fluid concentrations of ampicillin, cefazolin or cefoperazone have been confirmed using high-performance liquid chromatography. These findings demonstrate that multiple β-lactam antibiotics demonstrate efficacy in reducing alcohol consumption and appear to be potential therapeutic compounds for treating alcohol abuse and/or dependence. In addition, these results suggest that pAKT may be an important player in this effect, possibly through increased transcription of GLT-1.

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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