We report here that all doses of CEF tested attenuated ethanol consumption in P rats, but only the highest doses tested (100 and 200 mg/kg) were associated with an up-regulation of GLT1 expression in the PFC and NAc. Increases in the expression of GLT1 appear to be inversely associated with a post treatment attenuation of ethanol intake. It is noteworthy that the levels of ethanol intake (between 6 and 7 g/kg/day) exhibited by the saline-treated P rats result in repeated, pharmacologically relevant (at least 40–50 mg%) blood alcohol levels (Bell et al., 2006a
; Murphy et al., 1986
). Although there were significant differences in the water intake between saline- and CEF-treated groups, there were no significant differences in the body weight between all the groups. The increase in water intake could be due to the fact that decreases in ethanol intake in the CEF-treated groups were compensated, in part, by the increases in water intake.
Glutamate transmission in key brain regions of the reward circuit including PFC and NAc plays a critical role in dependence-related behaviors, including locomotor sensitization and drug-seeking behavior (Kalivas et al., 2009
; Sari et al., 2009
). There is a relatively high concentration of glutamate in the PFC and NAc, which is associated with addiction-related changes in cognition, emotion, sensory input and subsequent motor output (McFarland and Kalivas, 2001
). The importance of glutamate projections from the PFC, particularly to the NAc and the VTA, has been confirmed by clinical neuroimaging studies during craving for commonly abused drugs such as ethanol, cocaine, methamphetamine, heroin and nicotine (Childress et al., 1999
; Dom et al., 2005
; Garavan et al., 2000
; Goldstein and Volkow, 2002
; Wexler et al., 2001
; Xiao et al., 2006
). We tested for changes in GLT1 protein expression levels within the PFC and NAc regions because the interactions between these two regions mediate, at least in part, drug reward (Kalivas et al., 2009
). Our interest in these regions also stems from their glutamatergic input from the amygdala and hippocampus, key players in initiating drug-seeking behavior as well (Kalivas et al., 2009
Ethanol exposure has been demonstrated to alter glutamatergic activity in the mesocorticolimbic circuit. Previous studies, using Cologne ‘ALKO’ Alcohol-Accepting (cAA) rats, investigating the effects of 20 months of ethanol exposure on glutamatergic function in the cerebral cortex (Schreiber and Freund, 2000
) found that ethanol-exposed cAA rats displayed decreased glutamate transporter activity compared with naïve cAA rats (Schreiber and Freund, 2000
). It is noteworthy that both the Alko Alcohol (AA) (the foundation stock for cAA rats) and P rats were selectively bred for ethanol preference, using similar criteria, and both used Wistar rats, albeit from different colonies and progenitors (Bell et al., 2005
; Sommer et al., 2006
). The specific involvement of GLT1 in addiction has been tested in drug abuse models as well. For example, activation of GLT1 by MS-153 effectively attenuated morphine, methamphetamine and cocaine conditioned place preference in mice (Nakagawa et al., 2005
). Additionally, our laboratory has reported that CEF attenuates cue-induced cocaine relapse in a dose-dependent manner (Sari et al., 2009
). In accordance, Kalivas et al. (2009
) found similar effects on cocaine relapse with CEF (Knackstedt et al., 2010
). This relapse was accompanied by an increase in GLT1 expression in the PFC and NAc. Additionally, CEF was found to increase accumbal cysteine/glutamate exchanger (xCT) expression in a rat model of cocaine relapse-like behavior (Knackstedt et al., 2010
). This later study demonstrated that CEF-induced increase in the xCT level was correlated with down-regulation of extracellular levels of glutamate.
In the brain, CEF is the most potent β-lactam antibiotic in inducing up-regulation or activation of GLT1 (Miller et al., 2008
; Rothstein et al., 2005
; Sari et al., 2010
). Furthermore, single daily injections of 200 mg/kg CEF for five consecutive days in mice increased glutamate uptake in the striatum, a primary target of cortical glutamate input (Miller et al., 2008
). Thus, CEF appears to have a direct central effect on glutamate transporter function.
In the present study, a lower dose of CEF (50 mg/kg) did not appear to increase GLT1 expression 3 days post treatment, but were effective in reducing ethanol intake. At the time point when GLT1 expression was determined, drinking levels in the 25 mg/kg dose group did not differ from control. Therefore, the lower doses may not have had a direct effect on GLT1 expression, at least not detectable by the methods used in the present study. This suggests that CEF may have additional pharmacological effects, or that its effect on GLT1 activity is secondary to an unknown primary effect. CEF has been shown to increase glutamate uptake in the rat hippocampus without increasing GLT1 expression (Lipski et al., 2007
). In addition, a previous study using Wistar rats tested a single CEF (200 mg/kg, i.p.) injection 90 min after middle cerebral artery occlusion. Although CEF did not increase GLT1 expression, the activity of GLT1 was increased in several brain regions including the hippocampus, striatum and frontal cortex (Thone-Reineke et al., 2008
One possible mechanism in which CEF acts indirectly on GLT1 may involve central glutathione (GSH) activity. An in vitro
study has shown that ceftrixaone treatment increased GSH and xCT levels (Lewerenz et al., 2009
). The CEF-induced increases in xCT and subsequent increases in GSH level may be one mechanism for reversing the glutamate transporter deficits caused by free radical oxidation. Ethanol withdrawal is associated with increases in oxygen-derived free radicals (Vallett et al., 1997
), which have been shown to inhibit glutamate uptake by oxidation of thiol groups (Volterra et al., 1994
). These authors reported that this effect was reversed by GSH administration. Additionally, in vitro
studies have shown that GSH prevents ethanol-induced gastric mucosal damage (Loguercio et al., 1993
; Mutoh et al., 1990
). A number of studies implicate high alcohol intake with abnormal, relative to low ethanol-drinking rodents, levels of GSH and/or enzymes associated with GSH in high ethanol-consuming rodent lines. Naïve high alcohol-preferring mice have greater gene expression for the GSH S-transferase, mu type 1 gene than their low alcohol-preferring counterparts, suggesting that this is a candidate gene for ethanol preference (Saba et al., 2006
). Naïve inbred P (iP) rats have greater GSH S-transferase, mu type 2 and GSH S-transferase gene expression in the hippocampus than their inbred alcohol non-preferring (iNP) counterparts (Edenberg et al., 2005
). A subsequent study found that iP rats have lower levels of GSH S-transferase, alpha 4 gene expression in the PFC, NAc, hippocampus, amygdala and caudate-putamen as well as lower levels of GSH S-transferase omega 1, and GSH S-transferase, mu type 3 (when expression levels across all five brain regions were averaged) than iNP rats (Kimpel et al., 2007
). Work with the AA and its Alko alcohol non-accepting (ANA) counterpart found that AA rats had higher GSH S-transferase alpha 4, mu 1 and mu 3, as well as GSH peroxidase 3 gene expression in the PFC than ANA rats (Sommer et al., 2006
). Again, these findings suggest that this family of genes modulates ethanol preference.
Regarding ethanol exposure, five consecutive daily injections of ethanol increased GSH S-transferase-alpha protein expression in the NAc of alcohol non-preferring (NP) rats compared with naïve NP rats (McBride et al., 2009
). Under operant conditions, ethanol self-administration by P rats increased GSH peroxidase 4 gene expression in the NAc relative to rats self-administering saccharin (Rodd et al., 2008
). Also, chronic ethanol consumption by P rats increases hydroxyacyl glutathione hydrolase gene expression in the NAc relative to naïve P rats (Bell et al., 2009
). However, it must be noted that these studies examined gene and/or protein expression levels, thus absolute levels of GSH activity were not determined. Thus, future studies addressing this important research question, ethanol-associated changes in GSH activity in vivo
, are needed.
In addition, activation of protein kinase C (PKC) induces a rapid down-regulation in the cell surface expression of several neurotransmitter transporters (Beckman et al., 1999
; Daniels and Amara, 1999
; Melikian and Buckley, 1999
; Qian et al., 1997
). In particular, activation of PKC caused a rapid decrease in the cell surface expression of GLT1 (Kalandadze et al., 2002
). Taken together, these findings suggest that CEF may act via a presently unidentified mechanism independent of the activation and/or up-regulation of GLT1. Further studies are warranted to investigate the full pharmacological activity of CEF in P rats.
Regarding GLT1 up-regulation, the precise cellular mechanism underlying this effect remains unknown. At least two pathways have been suggested, and they may have direct or indirect interactions with each other. First, Lee et al. (2008
) demonstrated that the canonical nuclear factor kB (NF-kB) signaling pathway is necessary for the CEF-induced increase in GLT1 in human primary fetal astrocytes. While NF-kB activity itself was not measured, ethanol consumption by P rats has been shown to increase, within the NAc shell, the expression of genes associated with this signaling pathway (McBride et al., 2010
). In addition, a previous study reported operant ethanol self-administration by inbred P rats reduced gene expression levels for the NF-kB-activating protein in the NAc (Rodd et al., 2008
), which may or may not correspond with decreased levels of the protein itself. Secondly, it has been shown that the mammalian target of rapamycin (mTOR) pathway is also involved in regulating GLT1 expression and subsequent glutamate uptake in vitro
, such that phosphorylation of mTOR by Akt appears to alter GLT1 expression levels (Wu et al., 2010
). As with the NF-kB signaling pathway, McBride et al. (2010
) have also reported that ethanol drinking by P rats increased gene expression for the Akt, a constituent of the Wnt/beta-catenin signaling pathway, and Akt1 proteins in the NAc shell. Again, this study was on gene expression levels. Thus, future in vivo
studies examining phosphorylation of mTOR, concomitant with altered GLT1 expression levels, following ethanol self-administration are needed.
In conclusion, we report here that CEF reduced ethanol intake in an animal model of alcohol abuse. In addition, the post treatment reduction in ethanol intake was dose dependent in nature, such that higher doses had a stronger effect. However, post treatment alterations in GLT1 expression levels within the PFC and NAc occurred only in the highest dose groups. Therefore, a direct action on GLT1 levels may be limited to our high CEF doses, while lower doses may act via other mechanisms. Given previous work indicating that up-regulation of GLT1 attenuates cue-induced reinstatement of cocaine-seeking behavior in rats (Knackstedt et al., 2010
; Sari et al., 2009
), the present findings indicate that CEF, as well as possibly other manipulators of GLT1 expression, is a potential therapeutic compound targeting ethanol and drug abuse/dependence.
This work was supported in part by R21AA016115 (Y.S.), DA 02451 (G.V.R.), U01AA13522 (R.L.B.) and R24AA015512 (Lawrence Lumeng).