
==== Front
7605074
6087
Neuroscience
Neuroscience
Neuroscience
0306-4522
1873-7544

38909675
10.1016/j.neuroscience.2024.06.017
nihpa2018466
Article
Effects of MC-100093 on Ethanol Drinking and the Expression of Astrocytic Glutamate Transporters in the Mesocorticolimbic Brain Regions of Male and Female Alcohol-Preferring Rats
Alotaibi Ahmed a
Travaglianti Shelby a
Wong Woonyen a
Abou-Gharbia Magid b
Childers Wayne b
Sari Youssef a*
a University of Toledo, College of Pharmacy and Pharmaceutical Sciences, Department of Pharmacology and Experimental Therapeutics, Toledo, OH 43614, USA
b Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA 19140, USA
* Corresponding author. Address: University of Toledo, College of Pharmacy & Pharmaceutical Sciences Department of Pharmacology & Experimental Therapeutics Health Science Campus, 3000 Arlington Avenue, Toledo, OH, 43614, USA. youssef.sari@utoledo.edu (Y. Sari).
13 9 2024
06 8 2024
22 6 2024
17 9 2024
552 8999
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Chronic ethanol consumption increased extracellular glutamate concentrations in several reward brain regions. Glutamate homeostasis is regulated in majority by astrocytic glutamate transporter 1 (GLT-1) as well as the interactive role of cystine/glutamate antiporter (xCT). In this study, we aimed to determine the attenuating effects of a novel beta-lactam MC-100093, lacking the antibacterial properties, on ethanol consumption and GLT-1 and xCT expression in the subregions of nucleus accumbens (NAc core and NAc shell) and medial prefrontal cortex (Infralimbic, mPFC-IL and Prelimbic, mPFC-PL) in male and female alcohol-preferring (P) rats. Female and male rats were exposed to free access to ethanol (15% v/v) and (30% v/v) and water for five weeks, and on Week 6, rats were administered 100 mg/kg (i.p) of MC-100093 or saline for five days. MC-100093 reduced ethanol consumption in both male and female P rats from Day 1–5. Additionally, MC-100093 upregulated GLT-1 and xCT expression in the mPFC and NAc subregions as compared to ethanol-saline groups in female and male rats. Chronic ethanol intake reduced GLT-1 and xCT expression in the IL and PL in female and male rats, except there was no reduction in GLT-1 expression in the mPFC-PL in female rats. Although, MC-100093 upregulated GLT-1 and xCT expression in the subregions of NAc, we did not observe any reduction in GLT-1 and xCT expression with chronic ethanol intake in female rats. These findings strongly suggest that MC-100093 treatment effectively reduced ethanol intake and upregulated GLT-1 and xCT expression in the mPFC and NAc subregions in male and female P rats.

glutamate
nucleus accumbens
ethanol dependence
GLT-1
xCT
==== Body
pmcIntroduction

Changes in glutamate homeostasis is associated with the development of drug dependence (Kalivas et al., 2009). Glutamate projections in the mesocorticolimbic pathways are critical in mediating response to drug rewards and drugs of abuse. Among many projecting glutamatergic pathways, there are glutamatergic signals that project from the medial prefrontal cortex (mPFC), amygdala, and hippocampus to the nucleus accumbens (NAc) (McFarland et al., 2003; LaLumiere and Kalivas, 2008; Parent et al., 2009; Mitrano et al., 2010; Papp et al., 2012). The NAc is divided into two distinct sub-regions termed the nucleus accumbens shell (NAc-shell) and the nucleus accumbens core (NAc-core). Glutamatergic projections from the mPFC to the NAc are suggested to be involved in regulating reward-related behaviors and drug dependence (Torregrossa et al., 2008). The mPFC regulates reward and motivation, and its disruption triggers the loss of control over compulsive drug-seeking behaviors in drug dependence (Goldstein and Volkow, 2011). There are at least four subregions in the mPFC, two of which, mPFC-infralimbic (mPFC-IL) and mPFC-prelimbic (mPFC-PL), have been shown to regulate drug-seeking behavior differently. The mPFC-PL appears to drive drug-seeking behavior and reinstatement of drug-seeking after extinction or abstinence, while the mPFC-IL is involved in the inhibition of drug-seeking behavior and suppresses the reinstatement of drug-seeking (Zavala et al., 2003; Pelloux et al., 2013; West et al., 2014; Moorman et al., 2015; Gutman et al., 2017). It is important to note that disruption of mPFC-NAc glutamatergic pathways might contribute to the development of dependence to drugs of abuse, including ethanol (Kalivas and Volkow, 2005; Moussawi and Kalivas, 2010; Goodwani et al., 2017).

Glutamate homeostasis is disrupted with ethanol exposure and dependence, leading to increased extracellular glutamate concentrations in mesocorticolimbic brain regions (Das et al., 2015; Alasmari et al., 2018). Several glutamate transporters mediate the regulation of extracellular glutamate concentration. Among these glutamate transporters, glutamate transporter 1 (GLT-1, its human homolog is excitatory amino acid transporter 2, EAAT2) regulates the uptake of the majority of extracellular glutamate concentrations in the brain. Chronic exposure or self-administration to drugs of abuse can lead to downregulation of GLT-1, an effect that is associated with increased extracellular glutamate concentrations at the synaptic cleft (Rao and Sari, 2012; Das, et al., 2015; Roberts-Wolfe and Kalivas, 2015; Alasmari, et al., 2018; Kim et al., 2018; Abulseoud et al., 2022). Extensive studies from our laboratory demonstrated clearly that chronic ethanol consumption downregulated the expression of GLT-1 in mesocorticolimbic brain regions, and this effect was associated with increased extracellular glutamate concentration in the brain of alcohol-preferring (P) rats (Rao and Sari, 2012; Sari et al., 2013; Alhaddad et al., 2014; Aal-Aaboda et al., 2015; Rao et al., 2015; Rao et al., 2015). Alternatively, cystine-glutamate exchanger (xCT) is another glutamate transporter that is responsible for the release of astrocytic glutamate in exchange for cystine (Baker et al., 2002; Moran et al., 2005). Several studies from our lab showed that chronic ethanol exposure decreased xCT expression in several brain reward regions of P rats (Alhaddad, et al., 2014; Rao, et al., 2015; Hakami et al., 2016).

Our lab has been studying the effects of GLT-1 upregulators on attenuating the effects of chronic exposure to drugs of abuse, including ethanol. Thus, our studies have clearly demonstrated that ceftriaxone, a beta-lactam antibiotic known to upregulate GLT-1 (Rothstein et al., 2005), decreased ethanol consumption in rats, and this effect was associated with the attenuation of downregulation of GLT-1 and xCT expression and consequently normalizing the extracellular glutamate concentration in the brain reward regions such as the NAc (Sari et al., 2011; Sari et al., 2013; Alhaddad, et al., 2014; Rao and Sari, 2014; Das, et al., 2015). In addition to ceftriaxone, other beta-lactam antibiotics are effective on attenuating ethanol consumption and relapse behaviors, in part through upregulation of GLT-1 and xCT expression in brain reward regions such as NAc and mPFC (Alasmari et al., 2015; Alasmari et al., 2016; Hakami, et al., 2016). Importantly, a recent study from our lab revealed the efficacy of a novel beta-lactam, MC-100093, in reducing ethanol intake in P rats, and this effect was associated with upregulation of GLT-1 in NAc (Alhaddad et al., 2022). It is important to note that the pharmacokinetics of MC-100093 were performed in a previous study, which showed that MC-100093 displayed 28% oral bioavailability (F = 28%) in rats, and the drug was associated with a 14% brain/plasma ratio after intraperitoneal (i.p) injection (Knackstedt et al., 2021). This latter study revealed that MC-100093 was associated with a 23.5% enhancement of glutamate uptake with an IC50 of 0.1 μM in an astrocyte-neuron co-culture model. In addition, MC-100093 lacks antimicrobial activity against gram-positive and gram-negative bacteria (Knackstedt, et al., 2021). In this study, we examined the effects of MC-100093 on attenuating ethanol consumption in both male and female P rats. Additionally, this study explored the neurocircuits involving the mPFC subregions (mPFC-IL and mPFC-PL) and the NAc subregions (NAc-shell and NAc-core) to determine any changes in the expression of GLT-1 and xCT in P rats of both sexes exposed to ethanol and MC-100093.

Experimental procedures

Animals

Male and female alcohol-preferring (P) rats were acquired from Indiana University School of Medicine, Indianapolis, IN at the age of 80–85 days. All animals were single housed in a room with temperature at 22 °C and 50% humidity in a 12 h light/dark cycle. Rats had free access to food and water throughout the experimental procedures. All the experimental procedures were approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC) under IACUC protocol# 400160.

Ethanol intake measurements

At the age of 90–100 days, 5–7 male and female rats were assigned randomly to three groups: (1) Water-drinking (Water-Saline) group with no access to ethanol and served as a control group; (2) Ethanol-Saline group, which was exposed to continuous free-choice access to ethanol (15% and 30%, v/v, concurrently), and water for five weeks, and saline i.p. injections were performed from Days 1–5 on Week 6; and (3) Ethanol-MC-100093 group, which had continuous free-choice access to ethanol (15% and 30%, v/v), and water for five weeks, and MC-100093 i.p. injections were performed from Days 1–5 on Week 6. Ethanol and water intake were measured as g/kg/day. Rats were required to meet the criterion of an average ethanol consumption of 4 g/kg/day to be included in the study, for at least 2 weeks before saline vehicle or MC-100093 i.p. injections, as adopted in previous studies (Alhaddad, et al., 2022;Sari, et al., 2011;Sari and Sreemantula, 2012). Average ethanol and water consumptions throughout Week 5 served as a baseline. At Week 6 of the experiment, ethanol-MC-100093 group received MC-100093 at a dose of 100 mg/kg (i.p.) once daily for five days, and ethanol-saline and water-saline groups received saline vehicle as an i.p injection for five consecutive days. Water and ethanol intakes were measured daily 24 h after the first i.p. injections of saline or MC-100093 and 24 h after the last i.p. injections of saline or MC-100093.

Brain tissue harvesting

After 24 h of receiving the last i.p. injection of either saline or MC-100093, rats were euthanized by CO2 inhalation followed by decapitation with a guillotine. Brains were harvested and immediately frozen on dry ice and stored at −80 °C. Cryostat (Leica) was used to isolate the mPFC subregions (mPFC-IL and mPFC-PL), and subregions of the NAc (NAc-core and NAc-shell). Brains were micro-punctured using the stereotaxic coordinates following the Rat Brain Atlas (Paxinos and Watson, 2006). The isolated brain regions were stored at −80 °C for subsequent protein detection using Western Blot assay.

Western Blot analyses

Immunoblot assays were performed to determine the expression of GLT-1, xCT and β-tubulin in mPFC-IL, mPFC-PL, NAc-core, and NAc-shell of all groups as described previously (Rao and Sari, 2014). Briefly, brain samples were homogenized in lysis buffer supplemented with protease inhibitor and total protein was quantified using protein assay (BioRad, Hercules, CA, USA). Equal amount of the samples was loaded on polyacrylamide gel (10–20%). Subsequently, proteins were transferred on a PVDF membrane and blocked with 5% milk in Tris-buffered saline Tween-20 (1x TBST) for 30 min at room temperature. Membranes were then incubated overnight at 4 °C with one of the following antibodies: rabbit anti-GLT-1 (1:1000, Abcam, AB41621), and/or rabbit anti-xCT (1:1000, Abcam, AB175186). Mouse anti-β-tubulin (1:1000; Cell Signaling D71G5) was used as a loading control antibody. On the following day, membranes were washed five times with 1x TBST followed by incubation with appropriate secondary antibody (1:4000) for 60 min at room temperature. Chemiluminescent reagents (Super Signal West Pico, Pierce Inc.) were used to detect proteins using a ChemiDoc imaging system (BioRad, USA). GLT-1 and xCT expression were normalized against β-tubulin, a control loading protein. Imaging System and ImageJ software version 1.53a were used to quantify and analyze the expression of GLT-1 and xCT in the subregions of the mPFC and NAc. Data from water-control group were represented as 100% and all other values were expressed relative to this control to evaluate the changes in protein expression. The ratios for treated animals in the ethanol/saline and ethanol/MC-100093 groups were normalized to the mean ratios for the control group (water/saline group). The control ratio was set at 100, and the results from each of the treated groups were expressed as a percentage relative to the water/saline group value of 100%. We normalized the ethanol/saline and ethanol/MC-100093 groups data to water/saline group to reduce any differences of contrast with Western blots for each set of groups (control/water/saline, ethanol/saline, and ethanol/MC-100093 groups) as it was performed in previous studies (Hammad et al., 2021; Alhaddad, et al., 2022).

Statistical analyses

All statistical analyses were conducted using GraphPad Prism (10). Two-way (mixed) ANOVA followed by Bonferroni multiple comparison post-hoc test was performed to analyze daily ethanol intake, average daily water intake and body weight. One-way ANOVA followed by Newman-Keuls post hoc tests were used to analyze Western blot data. All statistical analyses data were reported as a p < 0.05 of significance.

Results

Effect of MC-100093 treatment on ethanol consumption, ethanol preference and body weight in male P rats

Statistical analysis using two-way ANOVA revealed significant main effects of Day [F(5, 61) = 10.04, p < 0.0001], and Treatment [F(1, 61) = 88.97, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 61)= 4.754, p = 0.0010]. Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 1 through Day 5 of treatment in the ethanol-MC-100093 group compared to the ethanol-saline group (Fig. 1A). Ethanol preference was calculated as total ethanol consumption/total fluid consumption × 100 from daily ethanol and water consumption (Goodwani et al., 2015). Two-way ANOVA revealed a significant main effect of Day [F(5, 71)= 9.958, p < 0.0001] and Treatment [F(1, 71)= 122.8, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 71)= 5.815, p = 0.0001]. Bonferroni multiple comparison test showed a significant decrease in ethanol preference (%) in ethanol-MC-100093 group as compared to the ethanol-saline group starting on Day 1 through Day 5 (Fig. 1B). Statistical analysis of water consumption data revealed a significant Treatment × Day interaction [F(10, 95)= 2.219, p = 0.0228] and Treatment [F(2, 95)= 70.46, p < 0.0001]. Bonferroni multiple comparison test showed a significant increase in water consumption from treatment Day 1 through Day 5 in the ethanol-MC-100093 group compared to the ethanol-saline group (Fig. 1C). Body weights (g) of rats were monitored throughout the study. MC-100093 treatment had no significant effect on body weights in male P rats (Fig. 1D).

Effect of MC-100093 treatment on ethanol consumption, ethanol preference and body weight in female P rats

Two-way ANOVA revealed significant main effects of Day [F(5, 54)= 3.014, p = 0.0180], and Treatment [F(1, 54)= 49.92, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 54)= 2.477, p = 0.0431]. Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 1 through Day 5 in the ethanol-MC-100093 group as compared to the ethanol-saline group (Fig. 2A). Furthermore, we analyzed ethanol drinking between male and female groups. A Two-way ANOVA analysis showed a significant main effect of Day [F(5, 66)= 20.53, p < 0.0001], and Sex [F(1, 66)= 17.68, p < 0.0001] between male and female groups. A post hoc analysis using Bonferroni multiple comparison test showed a significant decrease in ethanol consumption from Day 3 through Day 5 in the ethanol-MC-100093 male group compared to the ethanol-MC-100093 female group (Fig S1.B). Two-way ANOVA revealed a significant main effect of Day [F(5, 55)= 5.999, p = 0.0002] and Treatment [F(1, 55)= 67.73, p < 0.0001] as well as a significant Treatment × Day interaction [F(5, 55)= 3.608, p = 0.0068]. Bonferroni multiple comparison test showed a significant decrease in ethanol preference (%) in ethanol-MC-100093 group as compared to the ethanol-saline group starting on Day 1 through Day 5 (Fig. 2B). Statistical analysis of water consumption data revealed a significant a significant main effect of Day [F(5, 72)= 2.725, p = 0.0260] and Treatment [F(2,72)= 128.3, p < 0.0001] and a significant Treatment × Day interaction [F(10, 72)= 3.709, p = 0.0005]. Bonferroni multiple comparison test showed a significant increase in water consumption from Day 1 through Day 5 in the ethanol-MC-100093 group as compared to the ethanol-ealine group (Fig. 2C). Body weights (g) of rats were monitored throughout the study. MC-100093 treatment had no significant effect on body weights in female P rats (Fig. 2D).

Effect of MC-100093 on the expression of GLT-1 in the mPFC-IL of male and female P rats

One-way ANOVA showed a significant difference in GLT-1 expression in the mPFC-IL [F(2, 12)= 34.30, p < 0.0001] in male P rats (Fig. 3A). Newman-Keuls post-hoc analysis revealed a significant decrease in GLT-1 expression in the mPFC-IL of the ethanol-saline group as compared to the water-saline group (p < 0.01), while MC-100093 attenuated ethanol-induced downregulation of GLT-1 expression (p < 0.0001, p < 0.001 respectively) (Fig. 3A). Alternatively, there was a significant difference in GLT-1 expression among the three groups in the mPFC-IL of female P rats [F(2, 12)= 12.47, p < 0.0012] (Fig. 4A). Newman-Keuls post-hoc analysis showed MC-100093 upregulated GLT-1 expression in the mPFC-IL of female rats as compared to ethanol-saline group (p < 0.001) and water-saline group (p < 0.05), whereas a significant reduction in the expression of GLT-1 was found in the ethanol-saline group as compared to the water-saline group (p < 0.05) (Fig. 4A).

Effect of MC-100093 on the expression of xCT in the mPFC-IL of male and female P rats

One-way ANOVA revealed a significant difference in the expression of xCT in the mPFC-IL of male P rats among all tested groups [F(2, 12)= 13.81, p < 0.0008] (n = 5/group) (Fig. 3B). Newman-Keuls revealed a significant decrease in the expression of xCT in the mPFC-IL of the ethanol-saline group as compared to the water-saline group (p < 0.05), while treatment with MC-100093 reversed the effect of ethanol and significantly increased the expression of xCT (p < 0.001) (Fig. 3B). In female P rats, there was a significant difference in xCT expression among the three groups in the mPFC-IL [F(2, 12)= 14.04, p < 0.0007] (Fig. 4B). Newman-Keuls post-hoc analysis showed a significant upregulation in the expression of xCT in MC-100093-treated female rats as compared to ethanol-saline and water-saline groups (p < 0.001 and p < 0.01 respectively), whereas a significant reduction in the expression of xCT was found in the ethanol-saline group as compared to the water-saline group (p < 0.05) (Fig. 4B).

Effect of MC-100093 on the expression of GLT-1 in the mPFC-PL of male and female P rats

In male P rats, one-way ANOVA showed a significant difference in GLT-1 expression in the mPFC-PL among all tested groups [F(2, 12)= 22.65, p < 0.0001] (Fig. 3C). Newman-Keuls post-hoc analysis revealed a significant decrease in GLT-1 expression in the mPFC-PL of the ethanol-saline group as compared to the water-saline group (p < 0.05), while its expression was significantly increased in group treated with MC-100093 (p < 0.001) as compared to the ethanol-saline group and water-saline group (p < 0.01) (Fig. 3C). Furthermore, there was a significant difference in GLT-1 expression among the three groups in the mPFC-PL of female P rats [F(2, 12)= 8.886, p = 0.0043] (Fig. 4C). Newman-Keuls post-hoc analysis showed a significant upregulation of GLT-1 expression in the mPFC-PL of MC-100093-treated group as compared to ethanol-saline and water-saline groups (p < 0.01 and p < 0.05 respectively) (Fig. 4C). No significant changes were detected between ethanol-saline and water-saline groups in the expression of GLT-1 in the mPFC-PL of female P rats.

Effect of MC-100093 on the expression of xCT in the mPFC-PL of male and female P rats

One-way ANOVA revealed a significant difference in the expression of xCT in the mPFC-PL of male P rats among all tested groups [F(2, 12)= 11.79, p = 0.0015] (n = 5/group) (Fig. 3D). Newman-Keuls test revealed a significant decrease in the expression of xCT in the mPFC-PL of the ethanol-saline group as compared to the water-saline group (p < 0.05), while treatment with MC-100093 reversed the effect of ethanol and significantly increased the expression of xCT (p < 0.01) (Fig. 3D). In female P rats, there was a significant difference in xCT expression among the three groups in the mPFC-PL [F(2, 11)= 13.31, p = 0.0009] (Fig. 4D). Newman-Keuls post-hoc analysis showed a significant upregulation of the expression of xCT in MC-100093-treated female rats as compared to ethanol-saline and water-saline groups (p < 0.001 and p < 0.05 respectively), whereas a significant reduction in the expression of xCT was found in the ethanol-saline group as compared to the water-saline group (p < 0.05) (Fig. 4D).

Effect of MC-100093 on the expression of GLT-1 in the NAc-shell of male and female P rats

In male P rats, one-way ANOVA showed a significant difference in GLT-1 expression in the NAc-shell among all tested groups [F(2, 12)= 16.68, p = 0.0003] (Fig. 5A). A significant decrease in GLT-1 expression in the NAc-shell of the ethanol-saline group as compared to the water-saline group was revealed by Newman-Keuls post-hoc analysis (p < 0.05), while its expression was significantly increased in group treated with MC-100093 as compared to the ethanol-saline and water-saline groups (p < 0.001, p < 0.01 respectively) (Fig. 5A). Moreover, there was a significant difference in GLT-1 expression among the three groups in the NAc-shell of female P rats [F(2, 12)= 10.41, p = 0.0024] (Fig. 6A). Newman-Keuls post-hoc analysis showed a significant upregulation of GLT-1 expression in the NAc-shell of MC-100093 treated female rats (p < 0.01), however, there was no significant reduction in the expression of GLT-1 in the ethanol-saline group compared to the water-saline group (Fig. 6A).

Effect of MC-100093 on the expression of xCT in the NAc-shell of male and female P rats

One-way ANOVA revealed a significant difference in the expression of xCT in the NAc-shell of male P rats among all tested groups [F(2, 12)= 40.16, p = 0.0006] (Fig. 5B). Newman-Keuls revealed a significant decrease in the expression of xCT in the NAc-shell of the ethanol-saline group as compared to the water-saline group (p < 0.05), while treatment with MC-100093 increased the expression of xCT (p < 0.001) (Fig. 5A). In female P rats, there was a significant difference in xCT expression among the three groups in the NAc-shell [F(2, 12)= 8.526, p = 0.0050] (Fig. 6B). Newman-Keuls post-hoc analysis showed a significant upregulation in the expression of xCT in the MC-100093 treated group as compared to ethanol-saline and water-saline groups (p < 0.01), whereas no significant changes in the expression of xCT was found in the ethanol-saline group as compared to the water-saline group (Fig. 6B).

Effect of MC-100093 on the expression of GLT-1 in the NAc-core of male and female P rats

In male P rats, one-way ANOVA showed a significant difference in GLT-1 expression in the NAc-core among all tested groups [F(2, 12)= 10.12, p = 0.0027] (Fig. 5C). Newman-Keuls post-hoc analysis revealed that GLT-1 expression was significantly increased in group treated with MC-100093 as compared to the ethanol-saline and water-saline groups (p < 0.01 and p < 0.05 respectively) (Fig. 5C). There was no difference in the expression of GLT-1 in ethanol-saline group compared to water group. Furthermore, there was a significant difference in GLT-1 expression among the three groups in the NAc-core of female P rats [F(2, 12)= 5.176, p = 0.0239] (Fig. 6C). Newman-Keuls post-hoc analysis showed a significant upregulation of GLT-1 expression in the NAc-core of MC-100093-treated female rats (p < 0.05) (Fig. 6C). No significant changes were detected between ethanol-saline and water-saline groups in the expression of GLT-1 in NAc-core of female P rats.

Effect of MC-100093 on the expression of xCT in the NAc-core of male and female P rats

One-way ANOVA revealed a significant difference in the expression of xCT in the NAc-core of male P rats among all tested groups [F(2, 12)= 14.52, p < 0.0006] (Fig. 5D). Newman-Keuls revealed a significant decrease in the expression of xCT in the NAc-core of the ethanol-saline group as compared to the water-control group (p < 0.05), while treatment with MC-100093 reversed the effect of ethanol, and significantly increased the expression of xCT (p < 0.001). In female P rats, there was a significant difference in xCT expression among the three groups in the NAc-core [F(2, 12)= 13.87, p < 0.0008] (Fig. 6D). Newman-Keuls post-hoc analysis showed a significant upregulation of xCT expression in ethanol-MC-100093 group as compared to ethanol-saline and water-saline groups (p < 0.001 and p < 0.01 respectively), whereas no significant changes in the expression of xCT was found in the ethanol-saline group as compared to the water-saline group.

Discussion

The present findings demonstrate that treatment with MC-100093 at a dose of 100 mg/kg (i.p.) reduced ethanol intake in male P rats, and for the first time we report that MC-100093 is effective in reducing ethanol consumption in female P rats. These results are in parallel with a previous study from our laboratory that showed MC-100093 reduced ethanol intake in male P rats with a dose of 50 mg/kg (i.p.) (Alhaddad, et al., 2022). The study by Alhaddad and colleagues compared the effect of MC-100093 at a dose of 50 mg/kg in attenuating ethanol drinking to ceftriaxone. The study also compared the effect of MC-100093 and ceftriaxone on GLT-1 and xCT expression in the subregion of NAc shell in male P rats. MC-100093 (50 mg/kg, i.p.) attenuated moderately ethanol intake as compared to ceftriaxone at a dose of 200 mg /kg, i.p. In this study, we focused on testing higher dose of MC-100093 (100 mg/kg, i.p.) as well as we focused on the neurocircuits of PFC and NAc subregions regarding the expression of GLT-1 and xCT. Importantly, we investigated the effects of ethanol consumption and MC-100093 at dose of 100 mg/kg in these neurocircuits to determine for any potential sex difference. Thus, this study included female P rats for the first time and examined the effects of ethanol and MC-100093 in the expression of GLT-1 and xCT in the neurocircuits involving the subregions of mPFC and NAc for comparison with male P rats. MC-100093-induced reduction in ethanol intake was associated with upregulation of GLT-1 expression in the subregions of the mPFC and NAc. The reduction of ethanol drinking was associated with a decrease in ethanol preference starting from Day 1 throughout Day 5 in the ethanol-MC-100093 group as compared to the ethanol-saline group. It is important to note that the reduction of ethanol consumption was associated with a significant increase in water intake in the ethanol-MC-100093 group. This suggests that ethanol-MC-100093 group tend to have less preference to ethanol (Fig. 1B and Fig. 2B), and rats tend to consume more water as compared to ethanol-saline group (Fig. 1C and Fig. 2C). Moreover, MC-100093 treatment showed no effect on the body weight of male and female P rats, which indicates its specificity in reducing ethanol consumption. This is in line with a previous study, showing that MC-100093 has no effect on body weight and sucrose intake (Knackstedt, et al., 2021). In addition, other beta lactam antibiotic, ceftriaxone was proven not to influence body weight or sucrose intake (Sari, et al., 2011; Qrunfleh et al., 2013). This indicates the MC-100093 specificity in reducing ethanol consumption without affecting body weight. Nevertheless, it is noteworthy that MC-100093 was associated with a higher attenuation effect on ethanol consumption in male P rats exceeding female P rats. This effect was apparent and continued to increase daily in reducing ethanol intake through the end of the experiment. Whereas MC-100093 attenuation effect in female remained by some means equal across the last three days. Furthermore, a previous study investigated ethanol consumption between male and female P rats, showed that female P rats tend to consume more ethanol than male P rats (Bell et al., 2011).

The mesocorticolimbic pathway plays an important role in mediating the rewarding effects of drugs of abuse, including ethanol. The mPFC sends and receives glutamatergic projections into the NAc and other brain regions (Mcdonald et al., 1996). Therefore, consumption of ethanol is linked to the dysregulation of the glutamatergic pathways in the mesocorticolimbic system, specifically in the mPFC and NAc. (Kapasova and Szumlinski, 2008; Ding et al., 2012; Ding et al., 2013). Microdialysis studies in rats revealed increased of extracellular glutamate concentrations following chronic ethanol consumption in the NAc (Das, et al., 2015). Furthermore, magnetic resonance spectroscopy (MRS) studies in rats showed an increase in glutamate levels in the mPFC (Hermann et al., 2012). MRS studies in clinical settings also showed that ethanol dependent individuals had an increased glutamate levels in the mPFC (Hermann, et al., 2012; Frye et al., 2016) and NAc (Bauer et al., 2013) compared to healthy individuals. Thus, we focused on NAc and mPFC as target brain reward regions. Several astroglial glutamate transporters, GLT-1 and xCT, which regulate extracellular glutamate concentrations, are downregulated in the mesocorticolimbic reward pathways following ethanol consumption (Melendez et al., 2005; Szumlinski et al., 2007; Sari, 2013; Alhaddad, et al., 2014; Rao and Sari, 2014; Das, et al., 2015; Alasmari, et al., 2016) (Fig. 7). Decreased expression of GLT-1 transporter has been reported following exposure to other drugs of abuse such as cocaine (Knackstedt et al., 2010; Reissner et al., 2015; Kim, et al., 2018) and nicotine (Knackstedt et al., 2009) (Fig. 7). Ceftriaxone and other β-lactam antibiotics exhibited a reduction in ethanol consumption in male P rats and attenuated relapse-like ethanol intake behavior, which was in part associated with an upregulation of GLT-1 and xCT in the NAc and mPFC (Qrunfleh, et al., 2013; Rao and Sari, 2014; Rao, et al., 2015). In this study, we examined the expression of GLT-1 in two of the mPFC subregions, specifically, mPFC-IL and mPFC-PL. Results from the present study, for the first time, revealed that five weeks of chronic ethanol consumption was associated with a significant downregulation of GLT-1 expression in the mPFC subregions, mPFC-IL and mPFC-PL in male P rats, and MC-100093 was effective in upregulating GLT-1 expression in these brain subregions. Nonetheless, in female P rats, GLT-1 was downregulated in the mPFC-IL subregions only. However, MC-100093 was associated with the upregulation of GLT-1 expression in both the subregions of the mPFC. It is important to note that rats administered ethanol for four weeks showed a reduction in the expression of GLT-1 in other regions of the PFC (Villavicencio-Tejo et al., 2021). Note, our study is the first to show that ethanol intake for five weeks reduced GLT-1 expression in the subregions of the mPFC such as mPFC-PL and mPFC-IL in male rats, however, downregulation of GLT-1 was observed only in the mPFC-IL in female rats. This indicates possible sex difference in the mPFC-PL with GLT-1 expression. Importantly, xCT expression was downregulated in the subregions of mPFC in both female and male rats. In any cases, MC-100093 upregulated GLT-1 expression in both subregions of the mPFC in either female or male rats.

Following these findings, we also found a downregulation of GLT-1 expression in the NAc-shell but not in the NAc-core following five weeks of chronic ethanol consumption in male P rats, and MC-100093 treatment resulted in attenuating ethanol consumption by upregulating GLT-1. These findings are in line with previous studies from our laboratory demonstrated that ethanol drinking for five weeks resulted in the downregulation of GLT-1 expression in the NAc-shell but not in the NAc-core in male P rats (Alhaddad, et al., 2022). MC-100093 treatment has been effective in upregulating GLT-1 expression in these subregions of the NAc in male P rats. In female P rats, chronic ethanol drinking did not show a significant change in GLT-1 expression in the NAc-shell and NAc-core. Importantly, MC-100093 upregulated GLT-1 in both subregions of the NAc, and this may have attenuated any dysfunctional effect in glutamate homeostasis that might be caused by chronic exposure to ethanol in these subregions of the NAc. Studies are warranted to determine changes in glutamate homeostasis in female and male P rats exposed chronically to ethanol and treated with MC-100093.

Changes in the expression of the xCT system, which is involved in exchanging intracellular glutamate with extracellular cystine, have been implicated in the development of drug dependence (Kalivas, 2009; Knackstedt, et al., 2010; Alhaddad, et al., 2014). This system can impact mGluR2/3, a presynaptic receptor that regulates the negative feedback for synaptic glutamate release (Kalivas, 2009). Hence, we explored the expression of xCT in the subregions of the NAc and mPFC in this study. Thus, chronic ethanol consumption downregulated xCT expression in both the mPFC subregions in male and female P rats. However, chronic ethanol consumption downregulated xCT expression only in the subregions of the NAc in male P rats but not female P rats, which indicate possible sex difference. It is important to note that MC-100093 treatment upregulated xCT expression in both the NAc and the mPFC subregions in male and female P rats.

This study focused on the glutamatergic projections involving the mPFC-NAc neurocircuit. As these projections are implicated in different behavioral effects among other classes of drugs of abuse. It is important to note that glutamatergic projections from the mPFC-PL to the NAc-core may regulate drug seeking, while glutamatergic projections from the mPFC-IL to the NAc-shell are suggested to mediate extinction to drug seeking behavior (LaLumiere and Kalivas, 2008; Peters et al., 2008; Peters et al., 2009; LaLumiere et al., 2010). The projections from mPFC to the NAc are involved in the generation of ethanol-cue association and induction of reinstatement to ethanol, which was significantly decreased by the ablation of mPFC neurons projecting to the NAc (Klenowski, 2018). Furthermore, a study showed that extinction learning was associated with changes in the glutamatergic neuronal plasticity in the mPFC-IL and PL (Gass et al., 2014). Other studies also indicated that mPFC-PL and mPFC-IL were found to play a role in inhibiting drug seeking behavior and reinstatement (Peters, et al., 2008; Pelloux, et al., 2013; Gutman, et al., 2017). Therefore, it is critical to investigate these glutamatergic projections between male and female rats exposed to chronic ethanol.

In this study, the effect of chronic ethanol intake on these projections was distinct between the two sex groups within the two different brain subregions. For instance, in male P rats, a significant decrease in the expression of GLT-1 was found in the mPFC-IL and mPFC-PL, and a significant decrease in the expression of GLT-1 was found in the NAc-shell but not the NAc-core. Alternatively, female P rats showed a differential effect of chronic ethanol consumption within the same subregions. The effect of ethanol in the mPFC showed a significant downregulation of GLT-1 expression in the mPFC-IL but not in the mPFC-PL. There were no changes in the expression of GLT-1 in the NAc-shell or NAc-core. To the best to our knowledge, there is less known about the effects of exposure to ethanol and other drugs of abuse on the expression of GLT-1 and xCT in the neurocircuits involving the subregions of NAc and mPFC in females. We suggest that the lack of effects of ethanol intake on the expression of GLT-1 and xCT in the NAc subregions may be associated with changes in sex hormones. Future studies are warranted to use ovariectomized female rats to determine whether hormonal changes can have an effect in the modulation of GLT-1 and xCT expression in model of chronic ethanol exposure in the subregions of NAc.

In conclusion, the results from this study indicate that treatment with MC-100093 at a dose of 100 mg/kg (i.p.) decreased ethanol consumption in male and female P rats, partly by increasing GLT-1 and xCT expression in the mPFC and NAc subregions. The present study supports the rationale that MC-100093 treatment reduced ethanol intake by restoring basal extracellular glutamate concentrations in the mesocorticolimbic neuronal circuits partly through upregulation of GLT-1 and xCT expression. Further studies are needed to determine the molecular mechanisms of MC-100093 and investigate dose-dependent effects in ethanol intake and the expression of these astrocytic glutamate transporters in brains of both male and female rats.

Supplementary Material

Figure S1

Acknowledgments

The research was supported by the National Institutes of Health, NIAAA, grant# AA029674.

Abbreviations:

mPFC medial prefrontal cortex

NAc nucleus accumbens

NAc-shell nucleus accumbens shell

NAc-core nucleus accumbens core

Fig. 1. Effects of MC-100093 treatment (100 mg/kg, i.p.) on ethanol intake, ethanol preference, water intake, and body weight in Male P rats for five consecutive days. (A) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average ethanol intake was significantly reduced in ethanol-MC-100093 group as compared to the ethanol-saline group. (B) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that ethanol preference was significantly decreased in the ethanol-MC-100093 group as compared to the ethanol-saline group. (C) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons showed that the average water intake was significantly increased in ethanol-MC-100093 group as compared to the ethanol-saline group. (D) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that there was no significant difference in body weight among all tested groups. Values are expressed as mean ± SEM (n = 5–7/group), (* p < 0.05 and ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Fig. 2. Effects of MC-100093 treatment (100 mg/kg, i.p.) on ethanol intake, ethanol preference, water intake, and body weight in Female P rats for five consecutive days. (A) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average ethanol intake was significantly decreased in the ethanol-MC-100093 group as compared to the ethanol-saline group. (B) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that ethanol preference was significantly reduced in the ethanol-MC-100093 group as compared to the ethanol-saline group. (C) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that the average water intake was significantly increased in ethanol-MC-100093 group as compared to the ethanol-saline group. (D) Statistical analysis using two-way ANOVA followed by Bonferroni multiple comparisons test showed that there was no significant difference in body weight among all tested groups. Values are expressed as mean ± SEM (n = 5–7/group), (* p < 0.05 and ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Fig. 3. Effects of MC-100093 treatment (100 mg/kg, i.p.) for five days on the expression of GLT-1 and xCT in the mPFC-IL and mPFC-PL in Male P rats. (A) Immunoblot of GLT-1 and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. In addition, a significant downregulation in the expression of GLT-1 was found in the mPFC-IL in the ethanol-saline group as compared to water-saline group. (B) Immunoblot of xCT and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test revealed that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the mPFC-IL. (C) Immunoblot of GLT-1 and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in GLT-1 expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the mPFC-PL. (D) Immunoblot of xCT and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group in the mPFC-PL. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).

Fig. 4. Effects of MC-100093 treatment (100 mg/kg, i.p.) for five days on the expression of GLT-1 and xCT in the mPFC-IL and mPFC-PL in Female P rats. (A) Immunoblot of GLT-1 and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in GLT-1 expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the mPFC-IL. (B) Immunoblot of xCT and β-tubulin in the mPFC-IL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the mPFC-IL. (C) Immunoblot of GLT-1 and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. (D) Immunoblot of xCT and β-tubulin in the mPFC-PL. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the mPFC-PL. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Fig. 5. Effects of MC-100093 treatment (100 mg/kg, i.p.) for five days on the expression of GLT-1 and xCT in the NAc-shell and NAc-core in Male P rats. (A) Immunoblot of GLT-1 and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline and water-saline groups. In addition, a significant downregulation in the expression of GLT-1 in the ethanol-saline group as compared to water-saline group in the NAc-shell. (B) Immunoblot of xCT and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 significantly upregulated xCT expression as compared to ethanol-saline group. In addition, a significant downregulation in the expression of xCT in the ethanol-saline group as compared to water-saline group in the NAc-shell. (C) Immunoblot of GLT-1 and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed that post-treatment with MC-100093 significantly upregulated GLT-1 expression as compared to ethanol-saline group in the NAc-core. (D) Immunoblot of xCT and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that there was a significant decrease in xCT expression in the ethanol-saline group as compared to water-saline group, while post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the NAc-core. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Fig. 6. Effects of MC-100093 treatment (100 mg/kg, i.p.) for five days on the expression of GLT-1 and xCT in the NAc-shell and NAc-core in Female P rats. (A) Immunoblot of GLT-1 and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test revealed that MC-100093 treatment significantly upregulated GLT-1 expression as compared to the ethanol-saline group. (B) Immunoblot of xCT and β-tubulin in the NAc-shell. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed a significant upregulation of xCT expression as compared to ethanol-saline group following treatment with MC-100093. (C) Immunoblot of GLT-1 and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test indicated that MC-100093 treatment significantly upregulated GLT-1 expression as compared to the ethanol-saline and water-saline groups. (D) Immunoblot of xCT and β-tubulin in the NAc-core. Quantitative analysis using one-way ANOVA followed by Newman-Keuls test showed that post-treatment with MC-100093 significantly upregulated xCT expression as compared to the ethanol-saline group in the NAc-core. Water-saline group data were represented as 100% (relative to water-saline). Values are expressed as mean ± SEM (n = 5/group), (* p < 0.05, ** p < 0.01, and *** p < 0.001).

Fig. 7. Schematic diagram displaying the glutamatergic projections from the mPFC to the NAc, and the effects of chronic ethanol consumption on GLT-1 and xCT in the subregions of mPFC and NAc. Downregulation of the expression of GLT-1 and xCT after chronic ethanol consumption leads to the increase of synaptic glutamate concentration. MC-100093 attenuated ethanol consumption through the upregulation of these transporters.

CRediT authorship contribution statement

Ahmed Alotaibi: Writing – review & editing, Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Shelby Travaglianti: Investigation, Data curation. Woonyen Wong: Methodology, Investigation, Data curation. Magid Abou-Gharbia: Resources, Funding acquisition. Wayne Childers: Resources, Methodology. Youssef Sari: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

Declaration of interest statement

The authors declare that they have no financial interests or personal relationships in the work reported in this research article.

Ethical statement

We have read and have abided by the statement of ethical standards for manuscripts submitted to Neuroscience.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuroscience.2024.06.017.
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