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. Author manuscript; available in PMC: 2026 Jun 15.
Published in final edited form as: Behav Brain Res. 2026 Jun 1;513:116311. doi: 10.1016/j.bbr.2026.116311

Ethanol drinking involves astrocytes in male Wistar rats

Xiaoying Tan 1, Zheng-Ming Ding 1,2,*
PMCID: PMC13264698  NIHMSID: NIHMS2184197  PMID: 42229857

Abstract

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Studies have shown that ethanol altered expression of an astrocyte marker, i.e., glial fibrillary acidic protein (GFAP), in two key corticolimbic regions, the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). These regions comprise anatomically and functionally different subregions, i.e., the prelimbic (PL) and infralimbic (IL) cortex of the mPFC, the shell and core subregions of the NAc. However, ethanol effects on GFAP expression within these subregions remain largely unknown. In addition, effects of pharmacological manipulation of astrocytes on alcohol drinking have been understudied. Western blot was conducted to determine GFAP expression in subregions of the mPFC and NAc after chronic intermittent ethanol drinking. Fluorocitrate, a preferential metabolic inhibitor of astrocytes, was tested on intermittent ethanol drinking following administration into the lateral ventricle. Chronic intermittent ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell. Fluorocitrate reduced ethanol intake and preference without altering total fluid consumption. In addition, fluorocitrate did not affect water drinking when water was the only solution or basal locomotor activity. These results indicate that intermittent ethanol drinking induced GFAP elevation in a subregion-specific manner within the mPFC and NAc, and that a preferential metabolic inhibitor of astrocytes selectively attenuated ethanol drinking without non-specific inhibition of water drinking or general activity. Together, these results suggest that astrocytes may play an important role in chronic intermittent ethanol drinking.

Keywords: Astrocyte, ethanol, fluorocitrate, medial prefrontal cortex, nucleus accumbens

1. Introduction

Alcohol use and alcohol use disorder (AUD) remain serious public health concerns in the USA. The 2022 National Survey on Drug Use and Health reported that, among people aged 12 or older, 48.7% (137.4 million) were current alcohol users and 10.5% (29.5 million) had a past year AUD [1]. Excessive alcohol use takes a heavy toll on individuals and the society, causing ~178,000 deaths and ~4 million years of potential life loss, and costing over $250 billion yearly [2]. AUD involves profound dysregulation of neurobiological systems within the mesocorticolimbic circuitry that underlies the rewarding effects and incentive salience of alcohol, the increase in negative emotion and stress, craving and loss of control over alcohol seeking. Major neurobiological systems dysregulated by alcohol include neurotransmitters such as monoamines and amino acids, and neuropeptides such as opioids and corticotropin-releasing factor, etc. [3]. Two of the three FDA-approved medications, i.e., naltrexone and acamprosate, act on alcohol-affected neurobiological systems to exert their therapeutic effects. However, these medications are only moderately effective and their clinical use is limited [4, 5], highlighting a critical need for better understanding of the broad mechanisms underlying AUD in order to identify novel targets for development of more effective treatment strategy.

Astrocytes are the most abundant glial cells in the brain and an integrative component of the neural network. Astrocytes provide support to neurons, e.g., maintenance of fluid and ion homeostasis, formation of blood brain barrier, supply of energy substrates, and regulation of extracellular neurotransmission, etc. [6]. Emerging evidence suggests that astrocytes may contribute to mediating effects of commonly misused drugs, including alcohol [7–11]. Human alcoholics and ethanol-exposed animals showed morphological changes in astrocytes in various brain regions, e.g., enlargement of the cell soma, increase in the size of the cytoplasm, and enhancement in the number of the cellular processes and branching [12–15]. Glial fibrillary acidic protein (GFAP) is a major protein component of astrocyte intermediate filaments and is most widely used as a marker for reactive astrocytes. Human alcoholics showed elevated GFAP mRNA levels in the nucleus accumbens (NAc) [16]. Chronic ethanol exposure and withdrawal in rodents increased GFAP expression in various brain regions, including medial prefrontal cortex (mPFC) and NAc [13, 15, 17–20]. The mPFC and NAc are two key corticolimbic regions closely involved in the development of AUD [3]. The mPFC comprises mainly the prelimbic (PL) cortex and infralimbic (IL) cortex. The NAc contains the shell and core subregions. These subregions exhibit substantial differences in neurochemistry, anatomy, and connectivity, leading to their differential role in behavior and addiction [21–24]. It is noted that previous studies investigated these regions either as a whole [16, 17], or by only one subregion but not the other [19]. Given the profound differences among these subregions, it is important to delineate ethanol effects on GFAP expression within these subregions.

Effects of pharmacological manipulation of astrocytes on alcohol drinking remain largely understudied. Among a limited selection of pharmacological tools selectively targeting astrocytes, fluorocitrate is an astrocyte-specific toxin widely used to probe astrocyte function in neurotransmission and behavior [25, 26]. Fluorocitrate is preferentially transported into astrocytes and compromises ATP production and astrocyte function [26, 27]. Studies have shown that fluorocitrate can inhibit reactive astrocytes and reduce GFAP expression and provide proactive effects in various behavioral models [28–30]. Our recent study demonstrates that nicotine self-administration enhanced GFAP expression in a key corticolimbic region and that administration of fluorocitrate into the lateral ventricle attenuated nicotine self-administration in rats [31]. These findings suggest that fluorocitrate can be a valid tool for probing the role of astrocytes on alcohol drinking that is associated with changes in GFAP expression.

The objective of the current study was to investigate the potential role of astrocytes in alcohol drinking. Experiments were performed to determine (a) effects of chronic ethanol drinking on GFAP expression in different subregions of the mPFC and NAc, and (b) effects of fluorocitrate on alcohol drinking.

2. Materials and Methods

2.1. Animals.

Young adult male Wistar outbred rats starting at ~8 weeks old were obtained from Inotiv (Indianapolis, IN USA) and housed in a vivarium controlled for constant temperature and humidity. The room was maintained on a reversed 12h light-dark cycle with light off at 9:30am and on at 9:30pm. Experimental procedures were performed during the dark phase. Acclimation period was approximately one week. Rats were housed in groups (2–4/cage) upon arrival and individually in separate cages during ethanol or water drinking. Cages were enriched with a polycarbonate play tunnel and nestlets. Food and water were available ad libitum. Protocols used were approved by the Institutional Animal Care and Use Committee at Pennsylvania State University College of Medicine. All experiments were performed in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals [32].

2.2. Chemical agents.

NaCl, KCl, CaCl2, MgCl2, fluorocitrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (190 proof) was obtained from Greenfield Global USA, Inc (Brookfield, CT, USA). Bupivacaine (0.5%) was purchased from Hospira, Inc. (Lake Forest, IL, USA). Carprofen (5 mg/kg) was acquired from Zoetis Inc. (Kalamazoo, MI, USA). All chemicals were dissolved in distilled water to desired concentrations.

2.3. Intermittent ethanol drinking.

Rats received access to ethanol with a two-bottle choice, intermittent access procedure as described previously [33]. Specifically, rats were given access to an ethanol bottle and a water bottle during three 24h sessions per week on Monday, Wednesday, and Friday, and were given access to only a water bottle during the rest of the week each week. No additional shaping procedure was used to induce ethanol preference. For the Western blot study, rats were divided into two groups with one group (n=9) receiving 20% ethanol vs water and the other (n=8) receiving water only for 11 weeks. For the fluorocitrate study, rats (n=9) received ethanol drinking with ethanol concentration ascending from 2% for week 1, 4% for week 2, 6% for week 3, 10% for weeks 4 and 5, to 20% for weeks 6 to 12. Access to ascending concentrations of ethanol has been shown in previous studies to be effective in inducing escalation of ethanol drinking in both rats and mice [34–37]. Positions of ethanol and water bottles were randomly alternated every week, and fluid intake was recorded to the nearest 0.1 g by weighing bottles 3 times a week, at the same time body weights were taken. Ethanol intake was converted to grams of ethanol per kilogram of body weight per day. Ethanol preference was calculated as the ratio of ethanol to total fluid intake in volume.

2.4. Western blot.

GFAP protein levels were determined following procedures previously described [31, 38]. Briefly, brain tissue was micro-punched from the PL cortex, the IL cortex, the NAc shell, and the NAc core. Total protein was extracted with a NucleoSpin® RNA/Protein purification kit (MACHEREY-NAGEL GmbH & Co., Duren, Germany) following the manufacturer’s instruction. Protein content was determined with the Qubit® Protein Assay on a Qubit 4 fluorometer (ThermoFisher Scientific, Waltham, MA, USA). Western blot was carried out on a ProteinSimple Wes automated western blot platform (Bio-techne, Minneapolis, MN, USA). A 12–230 kDa separation microplate kit was used with 0.6–1.5 μg protein loaded onto the plate. Primary antibodies included mouse anti-GFAP antibody MAB360 (1:500; MilliporeSigma, Burlington, MA, USA), and rabbit anti-GAPDH antibody ab9485 (1:100; Abcam, Cambridge, UK). Densitometric analysis of bands of interest were performed using the Compass analytical software from ProteinSimple ver. 5.0.1.

2.5. Stereotaxic surgery and microinjection.

Rats underwent stereotaxic surgery for guide cannula implantation for microinjection following procedures previously described [31, 39]. Briefly, rats were anesthetized with 2–3% isoflurane inhalation and then implanted with one 22-gauge guide cannula (P1 Technologies, Roanoke, VA, USA) aimed at the lateral ventricle (AP +1.3 mm, ML −0.9 mm, DV −3.0 mm). Stylets were inserted into cannula with a 0.5-mm extension beyond the guide cannula. Bupivacaine and carprofen were applied as analgesia during surgery. Following surgery, rats recovered for at least 5 days during which rats were handled regularly.

Rats were acclimated to the microinjection procedure through a mock injection session 1 day prior to the test. During the mock injection, rats were taken out of home cages and placed in the microinjection chambers. Then, stylets were removed and reinserted for at least 3 times. An infusion pump was turned on for 1 minute to produce the injection noise, but no solution was infused. After that, rats were returned to home cages. On the day of the microinjection, a 28-gauge microinjector (P1 Technologies, Roanoke, VA, USA) was inserted into the lateral ventricle with 1 mm extension beyond the guide cannula. The injector was connected via a PE50 tubing to a 25-μl Hamilton syringe mounted on a Harvard Apparatus PhD infusion pump. A ringer solution (147 mM NaCl, 3 mM KCl, 1.2 mM CaCl2, 1.2 mM MgCl2) or fluorocitrate (0.5 or 1.0 nmol in the ringer solution) was microinjected into the lateral ventricle in 1 μl over 2 minutes. After the microinjection, the injector remained in place for 2 more minutes before being removed. The microinjection was conducted ~15 min prior to the drinking session and rats returned to ethanol drinking with ethanol and water intake taken at 1, 4, and 24 hours into the drinking session. Fluorocitrate was administered in a within-subject design in which each rat received all doses of fluorocitrate and the vehicle in a random order with treatment sessions separated by non-treated sessions to allow drinking to return to baseline levels prior to the next treatment.

2.6. Open field test.

Locomotor activity was assessed in an open field test following procedures previously described [40]. Briefly, the apparatus comprised four opaque walls (L × H: 100 × 35 cm) that fit solidly in a nonreflective slotted base. Two opaque quad dividers split the apparatus into four 50 × 50 cm arenas with each arena housing one rat. Distance traveled was tracked with an ANY-maze video tracking system (Stoelting Co., Wood Dale, IL USA). A cross-over design was employed so that rats received the ringer solution in one session and 1.0 nmol fluorocitrate in another session, counterbalanced. Treatments were conducted 3 days apart. For each session, rats received the microinjection of fluorocitrate or the ringer solution into the lateral ventricle. Approximately 15min later, rats were placed in the arena and distance traveled was tracked for 60 min.

2.7. Histology.

Microinjector placements were verified as previously described [39]. At the end of microinjection study, rats were euthanized with CO2 overdose and bromophenol blue was microinjected into the lateral ventricle. Brains were quickly removed and frozen at −80°C. Brain sections (40 μm thick) were sliced on a cryostat microtome and stained with cresyl violet for the determination of placements with the reference to the rat brain atlas of Paxinos & Watson [41].

2.8. Statistical analysis.

Data are presented as mean ± SEM and analyzed in SPSS ver. 31.0. Time course data on drinking and locomotor activity were analyzed with linear mixed modeling for repeated measures followed by Bonferroni multiple comparisons. For Western blot, densitometric data of the protein of interest were first normalized against the loading control GAPDH. Then, values from the Water control group were averaged and were used to normalize other values. Data were analyzed with t tests. The significant level was set at p < 0.05.

3. Results

3.1. Effects of intermittent ethanol drinking on GFAP expression in key corticolimbic regions

During intermittent ethanol drinking, rats gradually escalated ethanol intake from ~ 2 g/kg/d during week 1 to ~ 5 g/kg/d at the end of week 5 and maintained intake at this level thereafter (Fig. 1A; linear mixed modeling: F31, 248 = 8.6, p < 0.001). In addition, rats gradually increased ethanol preference from ~20% during the first week to more than 50% over time (Fig. 1B; linear mixed modeling: F31, 248 = 10.2, p < 0.001). During the last week of drinking, 6 out of 9 rats reached more than 50% ethanol preference with the average preference at 61 ± 2%.

Figure 1.

Figure 1.

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Effects of chronic intermittent ethanol (E; n=9) or water (W; n=8) drinking on protein levels of glial fibrillary acidic protein (GFAP), a molecular marker for astrocytes, in key corticolimbic subregions. A. Escalation of ethanol intake during intermittent access to 20% ethanol (linear mixed modeling: F31, 248 = 8.6, p < 0.001). B. Ethanol preference during intermittent access to 20% ethanol (linear mixed modeling: F31, 248 = 10.2, p < 0.001). C. GFAP protein levels in the prelimbic cortex (PL; t test: t15 = 2.7, p = 0.016), infralimbic cortex (IL; t test: t15 = 0.7, p = 0.5), nucleus accumbens shell (t test: t15 = 0.531, p = 0.603), and nucleus accumbens core (t test: t15 = 2.591, p = 0.02) subregions. * p < 0.05, significantly different from the W group. D. Representative Wes images of GFAP and GAPDH in each region.

Effects of ethanol drinking on GFAP expression were shown in Fig. 1C & 1D. GFAP levels were significantly higher in the ethanol group than those in the water group (Fig. 1C) in the PL cortex (t15 = 2.7, p = 0.016) and NAc core (t15 = 2.591, p = 0.02), but not in the IL cortex (t15 = 0.7, p = 0.5) or NAc shell (t15 = 0.531, p = 0.603).

3.2. Effects of fluorocitrate on ethanol drinking during intermittent access

During ethanol drinking prior to the fluorocitrate treatment, rats gradually escalated ethanol intake from ~ 1 g/kg/d during week 1 to ~ 4 g/kg/d toward the end of week 12 (Fig. 2A; linear mixed modeling: F35, 280 = 6.1, p < 0.001). Ethanol preference gradually decreased from ~60% during week 1 to ~20% at the beginning of week 6 when ethanol concentrations increased from 2% to 20%, and then slowly increased to ~40% with additional 7 weeks of drinking on 20% ethanol (Fig. 2B; linear mixed modeling: F35, 280 = 4.2, p < 0.001). During the last week of drinking, 3 out of 9 rats reached more than 50% ethanol preference with the average ethanol preference at 53 ± 1%.

Figure 2.

Figure 2.

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Effects of intraventricular administration of fluorocitrate (Fluo; in nmol) on ethanol intake in g/kg/day and ethanol preference during intermittent ethanol drinking (n=9). A. Escalation of ethanol intake prior to the fluorocitrate treatment (linear mixed modeling: F35, 280 = 6.1, p < 0.001). B. Ethanol preference prior to the fluorocitrate treatment (linear mixed modeling: F35, 280 = 4.2, p < 0.001). C. Ethanol intake in g/kg/day (n=9/treatment condition) with the fluorocitrate treatment. Linear mixed modeling followed by Bonferroni multiple comparisons. 0–1h: F2,24 = 3.582, p = 0.043; 1–4h: F2,24 = 0.921, p = 0.412; 4–24h: F2,24 = 6.712, p = 0.005; total: F2,24 = 5.567, p = 0.01. D. Ethanol preference (n=9/treatment condition) with the fluorocitrate treatment. 0–1h: F2,24 = 0.274, p = 0.763; 1–4h: F2,24 = 0.695, p = 0.509; 4–24h: F2,24 = 7.769, p = 0.003; total: F2,24 = 8.983, p = 0.001. * p < 0.05, significantly lower than the ringer treatment.

Shortly after ethanol acquisition, rats received surgery for implantation of a guide cannula into the lateral ventricle and recovered for approximately 7 days prior to the fluorocitrate treatment. Effects of fluorocitrate on ethanol intake in g/kg/day and ethanol preference were shown in Fig. 2C & D. Fluorocitrate reduced total ethanol intake during the 24h session (Fig. 2C; linear mixed modeling: F2, 24 = 5.567, p = 0.01). The higher concentration of fluorocitrate significantly reduced ethanol intake (p = 0.003), whereas the lower concentration of fluorocitrate induced a trend toward significant reduction in ethanol intake (p = 0.053). The reduction mainly occurred during the first hour with the lower concentration of fluorocitrate (linear mixed modeling: F2, 24 = 3.582, p = 0.043) and during hours 4–24 with the higher concentration of fluorocitrate (linear mixed modeling: F2, 24 = 6.712, p = 0.005). Similarly, fluorocitrate decreased ethanol preference during the 24h session (Fig. 2D; Linear mixed modeling: F2, 24 = 8.983, p = 0.001) with the decrease mainly during hours 4–24 with both concentrations of fluorocitrate (linear mixed modeling: F2, 24 = 7.769, p = 0.003). Following the fluorocitrate treatment, ethanol intake returned to control levels typically within 2 additional drinking sessions. For example, ethanol intake was 4.3 ± 0.6 g/kg/d during the 2nd drinking session after the 1 nmol fluorocitrate treatment session and was not different from the level after the control treatment (4.4 ± 0.6 g/kg/d). These results suggest that fluorocitrate produced reversible effects on ethanol drinking.

Effects of fluorocitrate on the volumes of water, ethanol and total fluid during the two-bottle choice, intermittent access were shown in Fig. 3. Fluorocitrate increased the volume of water consumed during the 24h session (Fig. 3A; linear mixed modeling: F2, 24 = 6.540, p = 0.005) with the significant increase during hours 4–24 with both concentrations (linear mixed modeling: F2, 24 = 5.838, p = 0.009). In contrast, fluorocitrate at the higher concentration significantly reduced the volume of ethanol consumed during the 24h session (Fig. 3B; linear mixed modeling: F2, 24 = 5.765, p = 0.009). The reduction mainly occurred during hours 4–24 (linear mixed modeling: F2, 24 = 6.734, p = 0.005). As a result, the volume of total fluid consumed was not significantly altered over the 24h session (Fig. 3C; linear mixed modeling: F2, 24 = 1.835, p = 0.181).

Figure 3.

Figure 3.

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Effects of intraventricular administration of fluorocitrate (Fluo; in nmol) on the volumes of water, ethanol, and total fluid during intermittent ethanol drinking (n=9). A. The volume of water consumption (n=9/treatment condition). Linear mixed modeling followed by Bonferroni multiple comparisons. 0–1h: F2,24 = 0.239, p = 0.789; 1–4h: F2,24 = 0.922, p = 0.412; 4–24h: F2,24 = 5.838, p = 0.009; total: F2,24 = 6.540, p = 0.005. B. The volume of ethanol consumption (n=9/treatment condition). Linear mixed modeling followed by Bonferroni multiple comparisons. 0–1h: F2,24 = 3.289, p = 0.055; 1–4h: F2,24 = 0.982, p = 0.389; 4–24h: F2,24 = 6.734, p = 0.005; total: F2,24 = 5.765, p = 0.009. C. The volume of total fluid consumption (n=9/treatment condition). Linear mixed modeling followed by Bonferroni multiple comparisons. 0–1h: F2,24 = 2.373, p = 0.115; 1–4h: F2,24 = 0.768, p = 0.475; 4–24h: F2,24 = 2.24, p = 0.128; total: F2,24 = 1.835, p = 0.181. * p < 0.05, significantly different from the ringer treatment.

3.3. Effects of fluorocitrate on water consumption and basal locomotor activity

After the two-bottle choice ethanol drinking, rats proceeded to drink water only followed by open field test, fluorocitrate was tested on water alone consumption and basal locomotor activity for potential non-specific effects. Effects of fluorocitrate on water only consumption were presented in Fig. 4A. Fluorocitrate did not alter water consumption over a 24h period when water was the only available solution (Fig. 4A: linear mixed modeling: F1, 16 = 0.982, p = 0.336). Effects of fluorocitrate on locomotor activity were shown in Fig. 4B. Fluorocitrate failed to alter distance traveled in the open field (Fig. 4B: linear mixed modeling: F1, 16 = 0.131, p = 0.722).

Figure 4.

Figure 4.

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Effects of intraventricular administration of fluorocitrate (Fluo; in nmol) on consumption of water as the sole drinking fluid and basal locomotor activity in an open field test. A. The volume of water consumed over a 24h period (n=9/treatment condition). Linear mixed modeling: F1, 16 = 0.982, p = 0.336. B. Distance traveled over 60 min in the open field (n=9/treatment condition). Linear mixed modeling: F1, 16 = 0.131, p = 0.722.

4. Discussion

Our results demonstrate that chronic intermittent ethanol drinking enhanced GFAP protein expression in the PL cortex and NAc core, but not in the IL cortex or NAc shell, compared with water drinking. Microinjection of fluorocitrate into the lateral ventricle decreased ethanol intake and preference but did not alter total fluid consumption. In addition, fluorocitrate did not affect water consumption when water was the only drinking solution, nor did it change distance travelled in the open field test. These results indicate that fluorocitrate attenuated ethanol drinking without altering water drinking or general activity. Taken together, these results suggest that astrocytes may play an important role in ethanol drinking.

Ethanol drinking enhanced GFAP expression in the PL cortex of the mPFC and the core subregion of the NAc (Fig. 1B). These results are consistent with previous findings demonstrating higher GFAP levels in the NAc in human alcoholics [16] and in the mPFC in mice with chronic ethanol exposure [17] when these regions were studied as a whole. In contrast to our results, a previous study found that chronic ethanol drinking did not alter the number of GFAP+ cells in the PL cortex without examining the IL cortex [19]. Another study found that chronic ethanol drinking increased the number of GFAP+ cells in the shell but not core subregion of the NAc shortly after ethanol drinking [18]. Exact explanations for the difference between these previous studies and our study remain unknown. It is noted that different strains of rats were used: Wistar outbred rats in our study, alcohol preferring P rats in the Miguel-Hidalgo study, and Wistar Han® in the Bull et al study. P rats are selectively-bred for high alcohol preference and drinking, whereas Wistar and Wistar Han® rats are not [42]. Wistar and Wistar Han® rats are different in allelic frequency of Grm2cys407* allele that encodes a stop codon resulting in the loss of functional metabotropic glutamate 2 receptor [43]. This mutation has been linked to altered alcohol intake, impulsivity, risk taking and emotional behavior [43]. Another difference is the drinking procedures employed: intermittent access to 20% ethanol for 11 weeks in our study, continuous access to 10% ethanol for 2 or 6 weeks in the Miguel-Hidalgo study, and intermittent access to 20% ethanol for 12 weeks followed by a 24h withdrawal in the Bull et al study. It is possible that differences in rat strain and ethanol procedure may contribute to different results. Interestingly, number of GFAP+ cells was found to increase following a 3-day withdrawal in the PL cortex in the Miguel-Hidalgo study and following a 3-week withdrawal in both NAc shell and core subregions in the Bull et al study, suggesting that withdrawal may be an important factor affecting ethanol effects on GFAP expression in these subregions.

Our results suggest that the development and maintenance of ongoing ethanol drinking may involve astrocyte alterations within the PL cortex and NAc core. The involvement of the PL cortex is consistent with recent studies demonstrating that ethanol drinking induced astrocyte-specific transcriptome responses in the mPFC and that bi-directional regulation of astrocyte calcium signaling in the mPFC altered ethanol drinking and ethanol-induced intoxication in mice [44, 45]. In addition, our results indicate that chronic ethanol drinking induced region-selective GFAP alterations. These region-specific effects are consistent with the notion that astrocytes are heterogenous between and within brain regions [46, 47]. For example, astrocytes were found to differ profoundly in electrophysiological characteristics, Ca2+ signaling, morphology, transcriptomic and proteomic signatures, and astrocyte-synapse proximity between striatum and hippocampus, suggesting neural circuit-specific properties of astrocytes [48]. Astrocyte metabolism was also found to be different between cortex and corpus callosum [49]. Although regions examined in these studies are not the same as the ones investigated in our study, it is possible that astrocytes in these subregions may exhibit different properties, leading to distinct GFAP response to chronic ethanol drinking.

Fluorocitrate reduced ethanol drinking without altering total fluid consumption (Fig. 2 & 3). On the other hand, fluorocitrate did not alter water drinking when water was the only drinking solution, or distance travelled in the open field test (Fig. 4). In addition, a previous study demonstrated that intraventricular administration of fluorocitrate enhanced sucrose preference that was impaired by a lipopolysaccharide (LPS) treatment [30]. These findings suggest that fluorocitrate may selectively reduce ethanol drinking without non-specific inhibition of general reward processing or general activity. These results are consistent with our recent findings that fluorocitrate inhibited nicotine reinforcement [31]. Together, these findings add to the growing evidence implicating astrocytes in drug addiction [7, 9, 50].

Fluorocitrate displays relative specificity toward astrocytes [25]. Following uptake into astrocytes via astroglia-specific acetate transporters, fluorocitrate acts as a reversable inhibitor of aconitase to disrupt the tricarboxylic acid cycle, resulting in blockade of ATP production and inhibition of astrocyte function [25, 26]. Microinjection of fluorocitrate at 1 nmol into striatum selectively inhibited astrocytic metabolism and disrupted astrocyte ultrastructure and appearance without impacting neurons over at least 4 hours [51–53]. Fluorocitrate administered in our microinjection study (0.5 & 1 nmol) is within the range used in previous studies, suggesting that fluorocitrate may selectively alter astrocytes globally in the brain following intra-ventricular administration. It is noted in a recent study that fluorocitrate at concentrations at or greater than 250 μM could directly alter neuronal function and morphology, including decrease in ATP levels and calcium activity and increase in the size of dendritic spins [54]. Given these, a direct contribution from neurons may not be excluded in the current study.

Mechanisms underlying fluorocitrate inhibition of ethanol drinking remain unknown. Intraventricular administration of fluorocitrate at 1 nmol has been shown to inhibit activated astrocytes indicated by decreasing GFAP expression in various behavioral models. For example, in an LPS-induced depressive model, fluorocitrate reversed LPS-induced increase in GFAP levels in hippocampus and cortex [30]. In a hyperalgesia model, fluorocitrate blocked trauma-enhanced GFAP expression in the parabrachial nucleus [29]. In an ischemic stroke model, fluorocitrate prevented ischemia-induced GFAP increase in hippocampus [28]. The reduction of GFAP levels has been associated with fluorocitrate-induced amelioration of depressive-like behaviors, pain hypersensitivity, or impairment of learning and memory in these animal models. Given these, it is possible that fluorocitrate may normalize ethanol-increased GFAP expression to reduce ethanol drinking. Unfortunately, the current study was unable to compare GFAP levels in the PL cortex and NAc core between the vehicle and fluorocitrate treatments because the within-subject design of the study resulted in the same animals receiving both vehicle and fluorocitrate at different time points. Such limitation prevents the establishment of a direct link between astrocytes in these two regions and the effects of fluorocitrate. Therefore, future studies are warranted to further test this hypothesis by examining effects of fluorocitrate administration into the PL cortex and NAc core on GFAP expression and ethanol drinking.

Glutamate transmission may be involved in fluorocitrate effects. Fluorocitrate has been shown to inhibit glutamine synthesis and release from neurons [52, 55]. Fluorocitrate inhibition of astrocyte ATP production may also reduce the energy-dependent glutamate gliotransmission [56]. Microdialysis studies demonstrated that perfusion of the NAc core and mPFC with fluorocitrate reduced local extracellular glutamate levels [31, 57]. Given the significance of glutamate transmission within these regions in alcohol drinking [58], it is highly possible that inhibition of astrocytes in the NAc core and mPFC may contribute, at least in part, to fluorocitrate inhibition of ethanol drinking. This is consistent with the selective GFAP response in the NAc core and PL cortex to ethanol drinking (Fig. 1B). In addition, fluorocitrate was found to decrease extracellular glutamate levels in other brain regions, such as striatum and hippocampus [52, 55]. Therefore, contribution from these brain regions may not be excluded.

Fluorocitrate can also alter extracellular dopamine transmission, including elevation of extracellular dopamine levels in the NAc core and striatum [31, 59]. Mixed findings have been reported regarding the effects of enhancing dopamine transmission in the NAc on ethanol drinking. Increase in extracellular dopamine transmission in the NAc core via the indirect agonist amphetamine facilitated oral ethanol self-administration [60, 61]. Elevation of extracellular dopamine levels in the NAc with a dopamine transporter inhibitor had no effect on ongoing ethanol drinking [62]. A recent study reported that enhancement of extracellular dopamine levels via shRNA knockdown of dopamine transporters in NAc reduced ethanol drinking in mice [63]. Taking together, these studies suggest that there is a possibility that elevation of extracellular dopamine levels in the NAc core may contribute to fluorocitrate inhibition of ethanol drinking. It will be interesting to test this hypothesis in future studies.

One limitation of the current study is that only male rats were included. The lack of inclusion of female rats may prevent the generalization of current findings to female rats. Nonetheless, our studies indicate that chronic ethanol drinking enhanced GFAP expression in the PL cortex and the NAc core, and that global inhibition of astrocyte energy metabolism inhibited ethanol drinking in male rats. These findings suggest that astrocytes may play an important role in the development and maintenance of ethanol drinking. Together, these findings enhance our understanding of astrocyte mechanisms involved in drug addiction.

Highlights.

  • Intermittent ethanol drinking enhanced GFAP levels in the PL cortex and NAc core.

  • Fluorocitrate inhibition of astrocytes reduced intermittent ethanol drinking.

  • Fluorocitrate did not alter total fluid consumption or basal locomotor activity.

Funding:

This study was supported by the U.S. National Institute on Drug Abuse grant DA044242 (ZMD). The content of this manuscript is solely the responsibility of the authors and does not necessarily represent the official views of the U.S. National institute on Drug Abuse.

Footnotes

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Declaration of Interests Statement: The authors declare no conflict of interest.

Declaration of Generative AI Use: The authors declare no generative AI use.

Data Availability:

The data that supports the findings of this study are available from the corresponding author upon reasonable request.

References

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Data Availability Statement

The data that supports the findings of this study are available from the corresponding author upon reasonable request.

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