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. Author manuscript; available in PMC: 2026 Feb 12.
Published in final edited form as: Addict Biol. 2022 Jul;27(4):e13178. doi: 10.1111/adb.13178

Role of suppressing GLT-1 and xCT in ceftriaxone-induced attenuation of relapse-like alcohol drinking in alcohol-preferring rats

Sujan C Das 1,2, Yusuf S Althobaiti 1,3, Alaa M Hammad 1,4, Fawaz Alasmari 1,5, Youssef Sari 1
PMCID: PMC12892963  NIHMSID: NIHMS2143916  PMID: 35754102

Abstract

Alcohol dependence results in long-lasting neuroadaptive changes in mesocorticolimbic system, especially in the nucleus accumbens (NAc), which drives relapse-like ethanol drinking upon abstinence or withdrawal. Within NAc, altered glutamate homeostasis is one of the neuroadaptive changes caused by alcohol dependence. Accumbal glutamate homeostasis is tightly maintained through glutamate transporter 1 (GLT-1) and cystine-glutamate antiporter (xCT). But the role of GLT-1 and xCT in relapse-like ethanol drinking is poorly understood. Here, we used alcohol-preferring (P) rats in relapse-like ethanol drinking paradigm to (a) determine the effect of relapse-like ethanol drinking on gene and protein expression of GLT-1 and xCT in NAc, measured by quantitative polymerase chain reaction (qPCR) and Western blot, respectively; (b) examine if glutamate uptake is affected by relapse-like ethanol drinking in NAc, measured by radioactive glutamate uptake assay; (c) elucidate if upregulation of either/both GLT-1 or/and xCT through ceftriaxone is/are required to attenuate relapse-like ethanol drinking. The GLT-1 or xCT protein expression was suppressed during ceftriaxone treatments through microinjection of GLT-1/xCT anti-sense vivo-morpholinos. We found that relapse-like ethanol drinking did not affect the gene and protein expression of GLT-1 and xCT in NAc. The glutamate uptake was also unaltered. Ceftriaxone (200 mg/kg body weight, i.p.) treatments during the last 5 days of abstinence attenuated relapse-like ethanol drinking. The suppression of GLT-1 or xCT expression prevented the ceftriaxone-induced attenuation of relapse-like ethanol drinking. These findings confirm that upregulation of both GLT-1 and xCT within NAc is crucial for ceftriaxone-mediated attenuation of relapse-like ethanol drinking.

Keywords: GLT-1, glutamate uptake, relapse-like ethanol drinking, xCT, vivo-morpholinos

1 |. INTRODUCTION

Alcohol dependence is a complex neuropsychiatric disorder causing long-lasting neuroadaptive changes in the meso-corticolimbic system of brain and thus triggers relapse if abstinence or withdrawal is achieved. To date, numerous preclinical studies have been conducted to elucidate druggable targets for preventing relapse-like ethanol drinking. Those druggable targets include opioid receptors (antagonist, naltrexone),1 α1-adrenergic receptors (antagonist, prazosin),2 glutamate receptors and ethanol-derived acetaldehyde (D-penicillamine).3 In targeting glutamatergic neurotransmission, the involvement of NMDA and AMPA receptors is considered as one of the mechanisms for inducing relapse-like ethanol drinking, and thus, blockers/modulators of these receptors have been shown to prevent relapse to ethanol.4,5 But very little is known about the involvement of glutamate transporters in relapse-like ethanol drinking.

Nucleus accumbens (NAc), a key brain region of mesocorticolimbic system, receives glutamatergic inputs from multiple brain regions (medial prefrontal cortex, hippocampus, and amygdala).6 The altered glutamate homeostasis within NAc has been reported to be associated with ethanol-seeking behaviour.7 Within NAc, glutamate homeostasis is tightly regulated through glutamate transporter 1 (GLT-1, one among five subtypes of excitatory amino acid transporters, that clears ~90% extracellular glutamate concentration) and cystine-glutamate antiporter (xCT, contributes to ~60% of extracellular glutamate concentration). To date, it is well-established that chronic ethanol exposure increases extracellular glutamate concentration in NAc, which persists even beyond acute withdrawal period.8,9 Thus, clearing off excess glutamate from extra-synaptic space through upregulation of GLT-1 is a possible way to restore the glutamate homeostasis and attenuate relapse-like ethanol drinking.

Our lab and others have shown that upregulation of both GLT-1 and xCT (through ceftriaxone, CEF) within NAc can attenuate relapse-like ethanol drinking.10 Although upregulation of both GLT-1 and xCT was associated with CEF-induced attenuation of relapse-like ethanol drinking, it is unclear whether upregulation of only one of these two transporters was sufficient to impart similar results. To better understand the contributory role of GLT-1 and xCT in CEF’s anti-relapse-like effects on ethanol drinking, we used here relapse-like ethanol drinking model with male alcohol-preferring rats. Here, we investigated the following: First, we determined whether relapse-like ethanol drinking affects the gene and protein expression of GLT-1 and xCT in NAc. Second, we determined if relapse-like ethanol drinking imparts any change in glutamate uptake in NAc. Third, we reconfirmed the attenuation of relapse-like ethanol drinking through CEF treatment. Fourth, we determined the effect of suppression of GLT-1 on CEF-induced attenuation of relapse-like ethanol drinking. Finally, we investigated the effect of xCT suppression on CEF-induced attenuation of relapse-like ethanol drinking.

2 |. MATERIALS AND METHODS

2.1 |. Subjects

All animal housing and experimental procedures were performed in accordance with National Institutes of Health guidelines for laboratory animal use. Animal experimental protocols were approved by the Institutional Animal Care and Use Committee of The University of Toledo. Male alcohol-preferring (P) rats were obtained from Indiana University, Indianapolis, IN. P rats were singly housed with 21°C and 50% humidity in 12 h light/dark cycle. Food and water were available unrestricted throughout the experimental procedures. All experiments were performed in light cycle.

2.2 |. Voluntary ethanol drinking procedure

At the age of 90 days, male P rats were exposed to free choice of ethanol (15% and 30%, v/v, concurrently) and/or water for five (5) weeks. Then P rats went through ethanol abstinence for 2 weeks. After 2 weeks of ethanol abstinence, P rats were re-exposed to voluntary ethanol drinking for 1 week. Body weight, ethanol drinking (as gm/kg/day) and water drinking were measured three times a week during five weeks of voluntary drinking starting on third week (baseline drinking) and every day during relapse-like ethanol drinking (7 days). The following experimental groups have been included in this study: CON = ethanol-naïve group drinking water only (n = 6–7 for glutamate uptake assay; n = 6 for Western blot and quantitative polymerase chain reaction (qPCR); same group of P rats served as CON for Western blot and qPCR, whereas separate group of P rats served as CON for glutamate uptake assay), EtOH-Sal = ethanol drinking group treated with saline (n = 6–7 for glutamate uptake assay; n = 6 for Western blot and qPCR; same group of P rats served as EtOH-Sal for Western blot and qPCR, whereas separate group of P rats served as EtOH-Sal for glutamate uptake assay), EtOH-CEF = ethanol drinking group treated with CEF (n = 7), GLT-1 S-Sal = ethanol drinking group microinjected with GLT-1 sense vivo-morpholinos and treated with saline (n = 6), GLT-1 AS-Sal = ethanol drinking group microinjected with GLT-1 anti-sense vivo-morpholinos and treated with saline (n = 8), GLT-1 AS-CEF = ethanol drinking group microinjected with GLT-1 anti-sense vivo-morpholinos and treated with CEF (n = 7), xCT S-Sal = ethanol drinking group microinjected with xCT sense vivo-morpholinos and treated with saline (n = 6), xCT AS-Sal = ethanol drinking group microinjected with xCT anti-sense vivo-morpholinos and treated with saline (n = 7), xCT AS-CEF = ethanol drinking group microinjected with xCT anti-sense vivo-morpholinos and treated with CEF (n = 7).

2.3 |. Stereotaxic surgeries for guide cannulas implantation

On first day of ethanol abstinence, guide cannulas (Plastic One, 26G) were surgically implanted bilaterally (AP + 1.8, ML ± 1.5, DV – 5 mm) above the NAc (core and/or shell). Cocktail (70:30) of ketamine (75 mg/kg) and xylazine (5 mg/kg) were used for anaesthesia. Flunixin (1 mg/kg, i.m.) was injected subcutaneously to relieve post-operative pain. P rats had 7 days of recovery period before the first microinjections of vivo-morpholinos.

2.4 |. Microinjections of vivo-morpholinos

Vivo-morpholinos were purchased from Gene Tools, Inc. (Philomath, OR, USA). The sequences of vivo-morpholinos were as follows: anti-sense GLT-1, 5′-TGTTGGCACCCTCGGTTGATGCCAT-3′; sense GLT-1, 5′-TACCGTAGTTGGCTCCCACGGTTGT-3′; anti-sense xCT, 5′-TGGCCACAACTGGCTTTCTGACCAT-3′ and sense xCT, 5′-TACCAGTCTTTCGGTCAACACCGGT-3′. The base sequences for vivo-morpholinos were selected based on the previous literatures showing effective suppression of respective transporters.11,12 After 1 week of surgical recovery, vivo-morpholinos (30 pmol, 1 μl/hemisphere) were microinjected into NAc (2 mm below the guide cannula) through internal cannulas (33 G, Plastic One) connected to infusion pump (Harvard Apparatus) with infusion rate of 0.5 μl/min. The internal cannulas were left at injection site for additional 1 min for diffusion. Microinjections were carried out for three consecutive days (Day 8 through Day 10 of ethanol abstinence).

To investigate the individual upregulatory effects of GLT-1 and xCT on relapse-like ethanol drinking, CEF (a potent upregulator of GLT-1 and xCT) was i.p. injected in rats paired with anti-sense GLT-1 and anti-sense xCT vivo-morpholinos. CEF (200 mg/kg) was injected for five consecutive days starting on third day of microinjections (last 5 days of ethanol abstinence). The CEF 200 mg/kg dose was used because this dose has been reported to prevent ethanol withdrawal syndrome completely in P rats.13 The guide cannula sites were verified histologically. For validation of GLT-1 and xCT suppression, GLT-1/xCT sense and anti-sense vivo-morpholinos were microinjected contra-laterally into ethanol-naïve (CON) P rats for three consecutive days. Five days after third microinjection (start point of relapse to ethanol), rats were euthanized, brains were flash frozen and later, NAc tissues were micro-punched for western blot experiment.

2.5 |. Radioactive glutamate uptake assay

Na+-dependent/-independent glutamate uptakes in NAc were determined using crude membrane fractionation as described previously.14 Briefly, fresh NAc samples (left and right hemisphere NAc were pooled) were homogenized in 500 μl of cold 0.32 M sucrose buffer containing 10 mM HEPES and 1 mM EDTA (pH 7.4). The homogenized samples were centrifuged at 1000 × g for 10 min (4°C), and the resulting pellets were discarded. The resulting supernatants were centrifuged at 15 000 × g for 20 min (4°C) to pellet down the crude membrane fractions. Pellets were re-suspended in Krebs–Ringer’s phosphate (KRP) buffer containing 140 NaCl, 1.2 CaCl2, 1.2 KH2PO4, 5 HEPES, 1 MgCl2 and 10 glucose (in mM) (pH 7.4). For Na+-independent glutamate uptake, NaCl in KRP buffer was replaced with 140 mM choline chloride. The glutamate uptake was initiated with the addition of 3H-glutamate (2 μCi/ml) in the presence of non-radioactive glutamate (1 μM) in final volume of 250 μl. The incubation time at 37°C was 15 min, and then glutamate uptake was stopped by putting samples on ice. Then samples were centrifuged at 1000 × g for 10 min (4°C), and supernatants were discarded. The pellets were further washed with ice-cold choline-containing KRP buffer to remove excess 3H-glutamate. The pellets were lysed with 1% SDS and radioactivity was counted in liquid scintillation counter. The protein concentration in each sample was determined by Lowry method.15 The glutamate uptake was measured as disintegrations per minute (dpm/mg of protein/min) and later converted into arbitrary units.

2.6 |. Western blot and qPCR

Gene expression of GLT-1 and xCT within NAc following relapse-like ethanol drinking was determined through quantitative PCR as described previously.16 The same CON group and the same EtOH-Sal group were used for both the Western blot and qPCR experiments. The right hemisphere NAc was used for Western blot, and the left hemisphere was used for qPCR. Briefly, the real-time PCR was carried out in iCycler (Bio-Rad Laboratories, Germany) with reaction mixture containing reverse-transcribed cDNA as template, SYBR green as fluorescent dye and primers of the respective genes (GLT-1, xCT and GAPDH). The relative mRNA expression of the respective genes was quantified using 2–ΔΔCT method.

The protein expression of GLT-1, xCT and GAPDH or beta-Tubulin within NAc was determined through Western blot analysis as described preciously.17 Very briefly, extracted proteins were transferred onto PVDF membrane and incubated with appropriate primary (@4°C, overnight) and secondary antibodies (@RT, 1 h). The blots were developed into HyBlot CL film and quantified through MCID system. The data were represented as ratio of GLT-1/and xCT/GAPDH or beta-Tubulin.

2.7 |. Statistical analyses

The amount of ethanol and water drinking during the relapse period were analysed using two-way analysis of varaince (ANOVA) followed by Bonferroni’s multiple comparison tests. Two group comparisons in gene expression, glutamate uptake and protein expression data were performed through unpaired t test. All data are presented as mean ± SEM. The level of significance was set to p < 0.05.

3 |. RESULTS

3.1 |. CEF attenuates relapse-like ethanol drinking

The relapse-like ethanol drinking paradigm has been depicted in Figure 1A. The amounts of ethanol and water drinking between EtOH-Sal and EtOH-CEF were analysed through two-way ANOVA followed by Bonferroni’s multiple comparison test. Two-way ANOVA, followed by Bonferroni’s multiple comparison test, revealed that there is a significant effect of treatment in ethanol drinking (F (1,6) = 14.65, p = 0.0087) (Figure 1B). Bonferroni’s multiple comparison test further revealed that the amount of ethanol drinking in EtOH-CEF group was significantly decreased (vs. EtOH-Sal) during relapse Day 17 through Day 21 (p = 0.027 for Day 17, p = 0.042 for Day 18, p = 0.0017 for Day 19, p = 0.0014 for Day 20 and p = 0.0039 for Day 21) (Figure 1B).

FIGURE 1.

FIGURE 1

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Effect of ceftriaxone (CEF) treatments on relapse-like ethanol drinking. The relapse-like ethanol drinking paradigm has been depicted in (A). CEF (200 mg/kg/day, i.p.) treatments during last 5 days of ethanol abstinence significantly lowered the ethanol drinking during relapse Day 17 through Day 21 (p = 0.0087) (B). CEF treatments also increased the amount of water drinking during the relapse Day 15 through Day 21 (p = 0.0036). All data are expressed as mean ± SEM. n = 7/group. *, p < 0.05; **, p < 0.01; @, p < 0.001; &, p < 0.0001. EtOH-Sal, ethanol drinking rats treated with saline; EtOH-CEF, ethanol drinking rats treated with ceftriaxone

Similarly, two-way ANOVA showed that there is a significant effect of treatment in water drinking during relapse-period (F (1,6) = 21.29, p = 0.0036) (Figure 1C). Bonferroni’s multiple comparison test depicts that the amount of water drinking in EtOH-CEF group was significantly increased (vs. EtOH-Sal) during the Day 15 through Day 21 (p = 0.036 for Day 15, p = 0.029 for Day 16, p = 0.002 for Day 17, p = 0.0001 for Day 18, p < 0.0001 for Day 19, p = 0.0001 for day 20 and p = 0.044 for Day 21) (Figure 1C).

3.2 |. Relapse-like ethanol drinking did not affect gene and protein expression of GLT-1 and xCT

The effect of relapse-like ethanol drinking on gene expression of GLT-1 and xCT in NAc was determined through real-time, quantitative PCR. Two-tailed unpaired t test revealed that relative mRNA expression of GLT-1 in NAc was not significantly different between control (CON) and ethanol-drinking (EtOH-Sal) rats (t(10) = 0.566, p = 0.58) (Figure 2A). Similarly, relapse-like ethanol drinking did not significantly affect the accumbal relative mRNA expression of xCT in ethanol-drinking (EtOH-Sal) rats compared with control (CON) rats (t(10) = 2.019, p = 0.071) (Figure 2B).

FIGURE 2.

FIGURE 2

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Effect of relapse-like ethanol drinking on relative gene and protein expression of GLT-1 and xCT in nucleus accumbens (NAc) of P rats. Relapse-like ethanol drinking for 7 days did not affect the gene expression (A) (p = 0.58) and protein expression (C) (p = 0.96) of GLT-1 in nucleus accumbens of P rats. Similarly, relative gene (B) (p = 0.07) and protein expression (D) (p = 0.68) of xCT in NAc was not significantly affected by chronic ethanol drinking. All data are expressed as mean ± SEM. n = 6/group. CON, control group (ethanol-naïve); EtOH-Sal, ethanol-drinking group injected with saline; C, CON; E, EtOH-Sal

The effect of relapse-like ethanol drinking on protein expression of GLT-1 and xCT in NAc was determined through Western blot analysis. Two-tailed unpaired t test revealed that relative protein expression of GLT-1 was not significantly different between control (CON) and ethanol-drinking (EtOH-Sal) rats (t(10) = 0.048, p = 0.963) (Figure 2C). Similarly, relapse-like ethanol drinking did not affect relative protein expression of xCT in ethanol-drinking (EtOH-Sal) rats compared to control (CON) rats (t(10) = 0.4166, p = 0.686) (Figure 2D).

3.3 |. Glutamate uptake in NAc in rats undergoing relapse-like ethanol drinking

Glutamate uptake was determined through radio-active glutamate uptake assay in crude membrane fractionation of NAc. Two-tailed unpaired t test revealed that Na+-dependent glutamate uptake in NAc of ethanol-drinking (EtOH-Sal) group was not significantly different compared to control (CON) group (t(12) = 1.387, p = 0.1907) (Figure 3A). Similarly, Na+-independent glutamate uptake was unaffected in NAc EtOH-Sal group compared with control group (CON) (t(10) = 0.360, p = 0.726) (Figure 3B).

FIGURE 3.

FIGURE 3

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Effect of relapse-like ethanol drinking on glutamate uptake and effectiveness of vivo-morpholinos on suppression of GLT-1 and xCT in nucleus accumbens (NAc) of P rats. Relapse-like ethanol drinking did not affect either Na+-dependent (A) (p = 0.19) or Na+-independent (B) (p = 0.72) glutamate uptake in NAc of P rats. n = 6–7/groups. The microinjections timeline has been depicted in (C). Three-day microinjections of GLT-1and xCT antisense vivo-morpholinos (30 pmol) significantly suppressed the GLT-1 expression (D) (p = 0.038) and xCT expression (E) (p < 0.0001), respectively, in NAc after 5 days of last microinjection compared to GLT-1 and xCT sense vivo-morpholinos (30 pmol) microinjections, respectively. n = 5/group. All data are expressed as mean ± SEM. CON, control group (ethanol-naïve); EtOH-Sal, ethanol-drinking group injected with saline; GLT-1 S, GLT-1 sense group; GLT-1 AS, GLT-1 antisense group; xCT S, xCT sense group; xCT AS, xCT antisense group; S, sense; AS, anti-sense

3.4 |. Verification of vivo-morpholinos to suppress GLT-1 and xCT

The timeline for verification of GLT-1 and xCT AS vivo-morpholinos in suppressing GLT-1 and xCT, respectively, has been depicted in Figure 3C. The protein expression of GLT-1 and xCT was determined through Western blot analysis in sense and anti-sense hemisphere of NAc after 5 days of last microinjection. Unpaired t test revealed that the anti-sense GLT-1 vivo-morpholinos significantly suppressed GLT-1 protein expression after 5 days of last microinjection compared with sense GLT-1 vivo-morpholinos (t(8) = 2.485, p = 0.03) (Figure 3D). Similarly, unpaired t test revealed that the anti-sense xCT vivo-morpholinos significantly suppressed xCT protein expression after 5 days of last microinjection compared to sense xCT vivo-morpholinos (t(8) = 10.56, p < 0.0001) (Figure 3E).

3.5 |. Effects of GLT-1 suppression on CEF-induced relapse-like ethanol and water drinking

The timeline for GLT-1 S and AS vivo-morpholinos microinjections has been depicted in Figure 4A. GLT-1 sense and GLT-1 anti-sense vivo-morpholinos were microinjected into NAc of ethanol drinking rats for 3 days starting on Day 8 of ethanol abstinence. Following microinjections, GLT-1 sense microinjected rats were treated with saline, and GLT-1 anti-sense microinjected rats were treated with CEF (200 mg/kg/day) for five consecutive days. Two-way repeated measure ANOVA, followed by Bonferroni’s multiple comparison tests, revealed that there is no significant effect of treatment (F (2, 18) = 1.58, p = 0.2329) and no significant day × treatment interaction (F (14, 126) = 1.068, p = 0.3927) in ethanol drinking (Figure 4B).

FIGURE 4.

FIGURE 4

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Effect of GLT-1 suppression in nucleus accumbens (NAc) on ceftriaxone-induced reversal of relapse-like ethanol drinking in P rats. The timeline for guide cannula implantation, GLT-1 S and AS vivo-morpholinos microinjections, ceftriaxone (CEF) treatments and relapse-like ethanol drinking has been depicted in (A) (inset: representative image of a guide cannula track). Suppression of GLT-1 through GLT-1 antisense vivo-morpholinos reversed the ceftriaxone (200 mg/kg, i.p.)-induced attenuation of relapse-like ethanol drinking (B) in relapse Day 15 through Day 21. In contrast, ceftriaxone treatments increased water drinking in Day 15 through Day 20 (C). n = 6–8/groups. @, p < 0.01; &, p < 0.001; *, p < 0.05. GLT-1 S-Sal, GLT-1 sense group treated with saline; GLT-1 AS-Sal, GLT-1 antisense group treated with saline; GLT-1 AS-CEF, GLT-1 antisense group treated with ceftriaxone. All data are represented as mean ± SEM

Bonferroni’s multiple comparison tests showed no significant difference in ethanol drinking between GLT-1 sense-saline (GLT-1 S-Sal) and GLT-1 anti-sense-saline (GLT-1 AS-Sal) groups from Day 15 through Day 21 (p < 0.99 for Days 15, 17, 18, 19 and 20; p = 0.96 for Day 16; and p = 0.703 for Day 21). Bonferroni’s multiple comparison tests also revealed that the relapse-like ethanol drinking was not significantly different between GLT-1 AS-Sal and GLT-1 AS-CEF groups from Day 15 through Day 21 (p = 0.184 for Day 15; p > 0.99 for Days 16, 17 and 21; p = 0.07 for Day 18; p = 0.186 for Day 19 and p = 0.518 for Day 20). The baseline ethanol drinking is not significantly different among GLT-1 S-Sal, GLT-1 AS-Sal and GLT-1 AS-CEF groups (Figure 4B).

We also determined the effect of GLT-1 suppression on water drinking during relapse period. Two-way repeated measure ANOVA, followed by Bonferroni’s multiple comparison test, revealed that there was a significant effect of treatment (F (2, 19) = 14.32, p = 0.0002) and a significant day × treatment interaction (F (14, 133) = 2.721, p = 0.0015) in water drinking. Bonferroni’s multiple comparison test showed that water drinking between GLT-1 S-Sal and GLT-1 AS-Sal groups was not significantly different from relapse Day 15 through Day 21 (p = 0.245 for Day 16 and p < 0.99 for Days 15, 17, 18, 19, 20, 21). In contrast, CEF (200 mg/kg/day) treatments significantly increased water drinking in GLT-1 AS-CEF group compared with GLT-1 AS-Sal group on relapse Day 15 through 20 (p = 0.0018 for Day 15; p = 0.0012 for Day 16; p = 0.001 for Day 17; p = 0.0001 for Day 18; p = 0.0003 for Day 19 and p = 0.037 for Day 20). The baseline water drinking was not significantly different among GLT-1 S-Sal, GLT-1 AS-Sal and GLT-1 AS-CEF groups (Figure 4C).

3.6 |. Effects of suppression of xCT on CEF-induced relapse-like ethanol and water drinking

The timeline for xCT S and AS vivo-morpholinos microinjections has been depicted in Figure 5A. xCT sense and xCT anti-sense vivo-morpholinos were microinjected into NAc of ethanol drinking rats for 3 days starting on Day 8 of ethanol abstinence. Following microinjections, xCT sense microinjected rats were treated with saline, and xCT anti-sense microinjected rats were treated with CEF (200 mg/kg/day) for five consecutive days. Two-way ANOVA, followed by Bonferroni’s multiple comparison tests, revealed that there is no significant day × treatment interaction (F (14, 119) = 1.086, p = 0.378) in ethanol drinking.

FIGURE 5.

FIGURE 5

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Effect of xCT suppression in nucleus accumbens (NAc) on ceftriaxone-induced reversal of relapse-like ethanol drinking in P rats. The timeline for guide cannula implantation, xCT S and AS vivo-morpholinos microinjections, ceftriaxone (CEF) treatments and relapse-like ethanol drinking has been depicted in (A) (inset: representative image of guide cannula track). Suppression of xCT through xCT antisense vivo-morpholinos reversed the ceftriaxone (200 mg/kg, i.p.)-induced attenuation of relapse-like ethanol drinking (B) in relapse Day 15 through Day 21. In contrast, ceftriaxone treatments increased water drinking in relapse Day 15 through Day 21 (B). n = 6–7/groups. @, p < 0.001; &, p < 0.0001. xCT S-Sal, xCT sense group treated with saline; xCT AS-Sal, xCT antisense group treated with saline; xCT AS-CEF, xCT antisense group treated with ceftriaxone. All data are represented as mean ± SEM

Bonferroni’s multiple comparison tests showed no significant difference in ethanol drinking between xCT sense-saline (xCT S-Sal) and xCT anti-sense-saline (xCT AS-Sal) groups from relapse Day 15 through Day 21 (p < 0.99 for Days 15, 16, 17, 18, 20 and 21; p = 0.136 for Day 19). Bonferroni’s multiple comparison tests also revealed that the relapse-like ethanol drinking was not significantly different between xCT AS-Sal and xCT AS-CEF groups from relapse Day 15 through Day 21 (p = 0.239 for Day 15; p = 0.206 for Day 16; p < 0.99 for Days 17 and 18; p = 0.082 for Day 19; p = 0.776 for Day 20 and p = 0.062 for Day 21). The baseline ethanol drinking was not significantly different among xCT S-Sal, xCT AS-Sal and xCT AS-CEF groups (Figure 5B).

We also determined the effect of xCT suppression on water drinking during relapse period. Two-way ANOVA, followed by Bonferroni’s multiple comparison test, revealed that there was a significant day × treatment interaction (F (14, 126) = 3.049, p = 0.0004) in water drinking. Bonferroni’s multiple comparison test showed that water drinking between xCT S-Sal and xCT AS-Sal groups was not significantly different from relapse Day 15 through Day 21 (p > 0.99 for Day 15 through Day 21). In contrast, CEF (200 mg/kg/day) treatments significantly increased water drinking in xCT AS-CEF group compared with xCT AS-Sal group on relapse Day 15 through 21 (p = 0.0002 for Days 15 and 18; p < 0.0001 for Days 16, 17, 19, 20 and 21). The baseline water drinking was not significantly different among xCT S-Sal, xCT AS-Sal and xCT AS-CEF groups (Figure 5C).

4 |. DISCUSSION

In this study, we used relapse-like ethanol drinking paradigm (Figure 1A) with P rats. Hyper-glutamatergic state within meso-corticolimbic system, particularly NAc, promotes excessive ethanol drinking. That imbalanced glutamatergic state within NAc can stem from numerous factors including altered glutamate release/clearance in/out of the extra-synaptic space on which the current study based. Here, we addressed some fundamental questions to elucidate the underlying cause of altered glutamate homeostasis in relapse-like ethanol drinking within NAc in light of GLT-1 and xCT.

First, we determined whether relapse-like ethanol drinking affect the gene and protein expression of GLT-1 and xCT. GLT-1 is the major excitatory amino acid transporter that accounts for >90% of extracellular glutamate uptake.18 Alternatively, xCT releases glutamate into extrasynaptic space and accounts for ~60% of that extracellular glutamate.19 Glutamate uptake by GLT-1 and glutamate release through xCT should be in equilibrium to maintain physiological extracellular glutamate level. Altered protein expression of GLT-1 and/or xCT is one of the possibilities contributing to imbalanced glutamate homeostasis within NAc. Decreased GLT-1 expression within NAc would result in decreased glutamate clearance (assuming GLT-1 activity remain unchanged) leading to accumbal hyper-glutamatergic state and vice versa. In contrast, increased expression of xCT will lead to excess glutamate release into extrasynaptic space and vice versa. In this study, we found that 7 days of relapse-like ethanol drinking did not affect the gene and protein expression of GLT-1 and xCT (Figure 2). These findings rule out the above-mentioned possibilities of altered expression of GLT-1 and xCT in relapse-like ethanol drinking. Very interestingly, these findings mark that relapse-like ethanol drinking is pharmacologically different from chronic ethanol drinking as latter is associated with decreased GLT-1 expression leading to increased extracellular glutamate in NAc.20 These data are consistent with our previous findings that expression of xCT and GLT-1 was unaltered following relapse-like-ethanol drinking.10,21 Unaltered protein expression of GLT-1 and xCT following relapse-like ethanol consumption reported here is similar to the previous reports observed in intermittent ethanol consumption paradigm.22–24

Second, we determined if relapse-like ethanol drinking affect the glutamate uptake within NAc. Because relapse-like ethanol drinking did not change GLT-1 and xCT protein expression, there is a possibility of change in their activity. GLT-1 transports glutamate in Na+-dependent manner, whereas xCT (catalytic subunit of Xc– system) releases glutamate into extra-synaptic space in Na+-independent manner. Here, we reveal that relapse-like ethanol drinking for 7 days did not alter Na+-dependent (accounts for GLT-1) or Na+-independent (accounts for xCT) glutamate uptake in NAc (Figure 3A, B). These findings signify that glutamate uptake through GLT-1 or glutamate release through xCT does not contribute to hyper-neuroexcitation within NAc following relapse-like ethanol drinking. Similar results have been reported in recent years showing that repeated ethanol administration or chronic intermittent ethanol did not alter glutamate transport.25,26 Collectively, relapse-like ethanol drinking might arise through other pathways rather than contribution of deficit in GLT-1 and xCT. Possible pathways involving glutamatergic neurotransmission within NAc might be (a) involvement of GluN1 (NMDA receptor subunit) and GluA1 (AMPA receptor subunit) within medium-spiny neurons of NAc27; (b) dysregulation in synaptic glutamate release modulation through metabotropic glutamate receptors.28

Third, we have reconfirmed that CEF treatments attenuate relapse-like ethanol drinking (Figure 1B). The CEF-induced ethanol attenuation was associated with significantly increased water drinking by CEF-treated P rats during the relapse period. We have consistently observed that CEF treatments increase water intake in P rats.10,29 The increased water drinking by CEF-treated P rats might be, at least in part, due to (a) compensatory drinking of more water to maintain the total fluid intake as ethanol intake was reduced; and (b) gastrointestinal side effects of CEF such as diarrhoea.30,31 P rats might have consumed more water to compensate the body fluid loss due to CEF-induced gastrointestinal adverse effects. Previously, we have demonstrated that CEF-induced attenuation of ethanol drinking is associated with upregulation of both GLT-1 and xCT.13,29 Thus, there is a possibility that upregulation of only GLT-1 or xCT is playing key role in CEF-induced attenuation of relapse-like ethanol drinking. To address this, we suppressed the expression of GLT-1 through microinjections of vivo-morpholinos during CEF treatments. To verify the effectiveness of vivo-morpholinos, we microinjected sense and anti-sense vivo-morpholinos contra-laterally for three consecutive days into NAc of naïve P rats. We found that anti-sense vivo-morpholinos were effective to suppress corresponding proteins after 5 days of last microinjection (Figure 3D,E).

We report here that GLT-1 suppression with anti-sense vivo-morpholinos did not affect the relapse-like ethanol drinking compared with control vivo-morpholinos (sense GLT-1) injected rats. This finding might be due to the fact that (a) downregulation of GLT-1 protein expression does not contribute to the relapse-like ethanol drinking and/or (b) downregulation of GLT-1 expression cannot add additional drinking amount on relapse-drinkers due to limit of maximum ethanol consumable by P rats. Interestingly, suppression of GLT-1 in CEF-treated rats prevented the CEF-induced attenuation of relapse-like ethanol drinking with increased water drinking (Figure 4). CEF is a potent upregulator of both GLT-1 and xCT.32,33 The present finding assures that GLT-1 upregulation is mandatory in mechanism of CEF to prevent relapse-like ethanol drinking although GLT-1 expression is unaffected by relapse-like ethanol drinking.

Fourth, we determined if upregulation of only xCT expression through CEF was sufficient enough to attenuate relapse-like ethanol drinking. Surprisingly, xCT suppression with anti-sense vivo-morpholinos in CEF-treated rats also prevented the CEF-induced attenuation of relapse-like ethanol drinking (Figure 5). These finding (GLT-1 or xCT suppression) confirms that CEF prevents relapse-like ethanol drinking in a mechanism that involves both GLT-1 and xCT. There are evidences that GLT-1 and xCT are functionally co-regulated as decreased extra-synaptic glutamate through uptake by GLT-1 activates glutamate release through xCT and vice versa.34,35 It has also recently been shown that CEF-mediated attenuation of cue-induced cocaine restatement requires upregulation of both GLT-1 and xCT.36 Similarly, N-acetyl cysteine (NAC), known to upregulate both GLT-1 and xCT, has recently been shown to decrease ethanol-taking/seeking in operant self-administration paradigm.37

In conclusion, relapse-like ethanol drinking did not affect the relative gene and protein expression of GLT-1 and xCT in NAc of P rats. There was also no change in accumbal glutamate uptake following relapse-like ethanol drinking. The CEF-mediated attenuation of relapse-like ethanol drinking required upregulation of both GLT-1 and xCT. Thus, upregulation of both GLT-1 and xCT can serve as a potential therapeutic intervention to attenuate relapse-like ethanol drinking.

ACKNOWLEDGEMENTS

This research study was conducted with the support of Award Number R01AA019458 (Y.S.) from National Institutes on Alcohol Abuse and Alcoholism (NIAAA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Alcohol Abuse and Alcoholism or the National Institutes of Health (NIH). The authors extend their appreciation to the research supporting project number (RSP2022R235), King Saud University, Riyadh, Saudi Arabia for the support. The authors have nothing to disclose.

Funding information

King Saud University, Grant/Award Number: RSP2022R235; National Institutes on Alcohol Abuse and Alcoholism (NIAAA), Grant/Award Number: R01AA019458

Footnotes

CONFLICT OF INTEREST

The authors declare that they have no conflicts of interest.

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