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. Author manuscript; available in PMC: 2023 Jan 12.
Published in final edited form as: Alcohol. 2022 Oct 12;105:43–51. doi: 10.1016/j.alcohol.2022.09.005

Drinking history dependent functionality of the dorsolateral striatum on gating alcohol and quinine-adulterated alcohol front-loading and binge drinking

Meredith R Bauer 1, Megan M McVey 1, Stephen L Boehm II 1,*
PMCID: PMC9835618  NIHMSID: NIHMS1860465  PMID: 36240946

Abstract

After an extended alcohol-drinking history, alcohol use can transition from controlled to compulsive, causing deleterious consequences. Alcohol use can be segregated into two distinct behaviors, alcohol seeking and alcohol taking. Expression of habitual and compulsive alcohol seeking depends on the dorsolateral striatum (DLS), a brain region thought to engage after extended alcohol access. However, it is unknown whether the DLS is also involved in compulsive-like alcohol taking. The purpose of this experiment was to identify whether the DLS gates compulsive-like binge alcohol drinking. To ask this question, we gave adult male and female C57BL/6J mice a binge-like alcohol-drinking history, which we have previously demonstrated to produce compulsive-like alcohol drinking (Bauer, McVey, & Boehm, 2021), or a water-drinking history. We then tested the involvement of the DLS on gating binge-like alcohol drinking and compulsive-like quinine-adulterated alcohol drinking via intra-DLS AMPA receptor antagonism. We hypothesized that pharmacological lesioning of the DLS would reduce compulsive-like quinine-adulterated alcohol (QuA) drinking, but not non-adulterated alcohol drinking, in male and female C57BL/6J mice. Three important findings were made. First, compulsive-like alcohol drinking is significantly blunted in cannulated mice. Because of this, we conclude that we were not able to adequately assess the effect of intra-DLS lesioning on compulsive-like alcohol drinking. Second, we found that the DLS gates binge-like alcohol drinking initially, which replicates findings in our previous work (Bauer, McVey, Germano, Zhang, & Boehm, 2022). However, following an extended alcohol history, the DLS no longer drives this behavior. Finally, alcohol and QuA front-loading is DLS-dependent in alcohol-history mice. Intra-DLS NBQX altered these drinking behaviors without altering ambulatory locomotor activity. These data demonstrate the necessity of the DLS in binge-like alcohol drinking before, but not following, an extended binge-like alcohol-drinking history and in alcohol front-loading in alcohol-history mice.

Keywords: AMPA receptor, Binge drinking, Compulsive, Dorsolateral striatum, Front-loading

Background

Alcohol use disorder (AUD) is a chronic disease that results in compulsive alcohol use, defined as drinking despite clear evidence of harmful consequences (American Psychiatric Association, 2013). Additionally, binge alcohol drinking, defined as >4 drinks for females and >5 drinks for males within 2 h, is related to the development of AUD (Addolorato et al., 2018; NIAAA, 2019). While binge drinking can occur prior to development of AUD, understanding the neurobiology of binge-like alcohol drinking during the development and maintenance of compulsive-like alcohol drinking is important. The dorsolateral striatum (DLS) is a brain region most notably involved in inflexible associative behaviors such as habitual and compulsive reward seeking (Yin, Knowlton, & Balleine, 2004, Yin, Knowlton, & Balleine, 2006; Corbit, Nie, & Janak, 2012, Corbit, Nie, & Janak, 2014; Giuliano, Belin, & Everitt, 2019). The DLS is also involved in maintaining excessive alcohol drinking (Darcq et al., 2016) and binge-like alcohol drinking (Bauer et al., 2022). However, it is unknown how the DLS is involved in binge-like alcohol drinking prior to development of compulsive-like alcohol drinking or after compulsive-like alcohol drinking is established. The purpose of this experiment was to assess binge-like and compulsive-like alcohol drinking early (no alcohol binge-drinking history) and late (after a drinking history that has been shown to produce compulsive-like alcohol drinking) in the drinking history using male and female C57BL/6J mice.

Compulsive alcohol drinking in the human population is exemplified by a compulsive alcohol drinker who may choose to drink alcohol during work hours, being fully aware that this behavior may result in the loss of income or may compulsively drink a non-beverage alcohol solution like methanol being fully aware that this may result in an aversive taste and poisoning (Green et al., 2018; Lachenmeier, Rehm, & Gmel, 2007). Compulsive-like alcohol drinking has been modeled in rodents by adulterating the alcohol solution during free access with the bitter compound, quinine (Hopf & Lesscher, 2014; Wolffgramm & Heyne, 1991). Adulterating the alcohol solution with quinine results in a highly aversive flavor with no other known negative consequence. Animals who are compulsive will consume the quinine-adulterated alcohol (QuA) solution readily. We utilized the binge-like alcohol drinking mouse model, Drinking-in-the-Dark (Rhodes, Best, Belknap, Finn, & Crabbe, 2005; Thiele, Crabbe, & Boehm, 2014), to assess compulsive-like QuA drinking. Specifically, we found that male and female C57BL/6J mice with a 3-week binge-like alcohol-drinking history drink significantly more of a 500–μM QuA solution than water-history mice, and that these mice drink the same amount of QuA as alcohol on the day prior (Bauer, McVey, & Boehm, 2021). Importantly, for mice to be considered compulsive drinkers, QuA intake should differ from a control group but not from the last day of alcohol access. Thus, we have established a robust model of compulsive-like alcohol drinking, which can easily be used to test both compulsive-like and non-compulsive binge drinking.

The DLS receives glutamatergic input, which is gated by post-synaptic AMPARs on medium spiny neurons (MSNs). AMPA receptors are ionotropic glutamate receptors that are involved in fast-acting neurotransmission in the CNS and are vital for long-term potentiation (LTP). LTP is dependent on the membrane insertion of post-synaptic AMPA receptors, where glutamate binding to AMPA receptors is necessary to produce sufficient post-synaptic depolarization. The development of compulsive-like alcohol drinking is likely mediated by the region-specific glutamatergic input into the AMPA-gated MSNs in the dorsal striatum, potentially resulting in the shift from goal-directed to compulsive alcohol drinking behavior. In support of this idea, Corbit et al. (2014) found that antagonism of DLS AMPA receptors returned habitual alcohol-responding rats to goal-directed and Haggerty, Muñoz, Pennington, Grecco, and Atwood (2022) found that altering cortical input into the DLS reduces binge alcohol drinking in binge-experienced (but not binge-inexperienced) rats. Finally, work from our lab found that AMPAR antagonism in the DLS significantly reduces binge-like alcohol drinking, without altering locomotor activity, following short drinking history (1 week; Bauer et al., 2022). Thus, AMPAR gating in the DLS may be key to plasticity changes resulting in development and maintenance of compulsive-like binge alcohol drinking.

Previously, researchers have used the competitive AMPA/kainate receptor antagonist, NBQX, to investigate the role of AMPA receptors in alcohol drinking. NBQX has a relatively high binding affinity for AMPA receptors (KD = 47 nM; Dev, Petersen, Honoré, & Henley, 1996) and is more selective for AMPA receptors by 30-fold than it is for kainate receptors, making it a useful pharmacological agent to assess AMPA receptors (Sheardown, Nielsen, Hansen, Jacobsen, & Honoré, 1990). Previous work investigating the role of AMPA receptors and alcohol consumption have demonstrated efficacy of NBQX, both systemically to reduce alcohol consumption (Bauer, Garcy, & Boehm; 2020; Ruda-Kucerova, Babinska, Luptak, Getachew, & Tizabi, 2018; Stephens & Brown, 1999) and site-specifically in the DMS (Wang et al., 2012) and DLS to reduce alcohol seeking (Corbit et al., 2014) and taking (Bauer et al., 2022). While the previously mentioned research has demonstrated the role of AMPA receptors in alcohol consumption, no work to our knowledge has demonstrated the role of DLS AMPA receptors in a limited-daily access alcohol consumption model of compulsive binge-like drinking and non-compulsive binge-like alcohol drinking across the drinking history. Additionally, female mice are rarely used in research and sex differences are almost never assessed. To expand on this literature, we sought to test whether the DLS gates compulsive, QuA, and non-compulsive alcohol drinking in male and female C57BL/6J mice.

Materials and methods

Animals

Experimentally naïve adult male and female C57BL/6J mice (PND [postnatal day] 62–90 at test start date; n = 7–11 per group, N = 120 mice total) were acquired from Jackson Laboratories (Bar Harbor, Maine, United States). Animals were individually housed in a vivarium with a 12:12-h reverse lighte–dark cycle for at least one week prior to the start of experiments. Throughout the experiment, animals received food (Lab Diet 5001, Rodent Diet) and water ad libitum with the exception of water bottle removal during the 2-h drinking sessions. Procedures were approved by the IUPUI School of Science Institutional Animal Care and Use Committee and conformed to the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).

Drinking-in-the-dark (DID)

DID is a limited-access model of binge-like alcohol consumption (Thiele et al., 2014). Mice receive one 10-mL ball-bearing sipper tube of 20% v/v alcohol in tap water into their home cages in place of the regular water bottle for 2 h, 3 h into the dark cycle, each day. C57BL/6J mice demonstrate binge-like alcohol consumption during DID, achieving 2-h intakes approaching 3.0 g/kg and blood alcohol levels surpassing 0.08 g/dL (Bauer et al., 2021; Kasten & Boehm, 2014). DID of either alcohol or water (control mice) occurred for a total of 24 days. On days 22 and 24, mice were given either alcohol or QuA DID, counterbalanced across day, and intakes were recorded at the 20- and 120-min timepoints. For a timeline of procedures, see Figure 1. Consumption was measured by reading the sipper tubes to the nearest 0.025 mL. Volumes were adjusted for leak based on the volume leaked from a tube in an empty cage on the same rack.

Fig. 1. Timeline of experimental procedures and cannula placements.

Fig. 1.

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All mice underwent bilateral cannulation surgery. Following recovery, alcohol-history (top) or water-history (bottom) mice underwent DID for a total of 24 days. On days 22 and 24, mice were microinjected with one of three concentrations of the AMPAR antagonist, NBQX, immediately before DID. The concentration of drug infused on day 22 was the same as for day 24. The solution presented during DID on days 22 and 24 was either alcohol or QuA. These were presented in a counterbalanced order, such that the effect of NBQX was tested for alcohol and QuA. Retro-orbital sinus bloods were collected immediately following DID on day 24. Placements from DLS surgeries are shown. Placements were compared with Paxinos and Watson’s Mouse Brain Atlas.

Solutions

190 proof alcohol was purchased from Pharmco, Inc. (Brookfield, Connecticut, United States) and was added to tap water to create a 20% v/v alcohol solution for use in DID. On test days (either day 22 or 24), the 20% ethanol solution was adulterated with quinine hemi-sulfate for a QuA concentration of 0.5 mM (0.1957 g/L) as we have previously done (Bauer et al., 2021). Quinine hemi-sulfate was purchased from Millipore Sigma (St. Louis, Missouri, United States).

Locomotor monitoring

Home cage locomotor activity was monitored during the drinking sessions on infusion days. Data were collected using the Opto M-3 system (Columbus Instruments), which collects locomotor activity by summating beam breaks over a set time interval from infrared beams that surround the perimeter of the home cage. This has been previously described in depth elsewhere (Bauer et al., 2020).

Bilateral cannulation

The DLS was targeted for bilateral cannulations using a Kopf stereotaxic alignment system at predetermined coordinates (M/L: ±2.5 mm, A/P: +0.38 mm, and D/V: −3.0 mm). This has been described in depth elsewhere (Bauer et al., 2022). Following surgery, mice received a single subcutaneous injection of 5 mg/mL carprofen (a post-operative pain treatment) at 10 mL/kg and were placed on a heating pad until recovery from anesthesia (approximately 30 min). Mice were given ad libitum food and water and at least 48 h of recovery prior to DID. Following the completion of the behavioral data collection, brains were taken for verification of cannula placement. Mice were humanely euthanized, brains were extracted, and flash-frozen in 2-methylbutane at −20 to −40 °C. Brain slices were stained with cresyl violet and placements were determined using the Paxinos and Franklin brain atlas (Paxinos & Franklin, 2001) as a reference.

Stylet changing and habituation

Stylets were changed daily for all of the mice. Mice were habituated to the microinjection procedure over the five days leading up to the procedure. This resulted in an incrementally increased length of restraint each day. The first restraint length was 30 s, and the length increased by 30 s each day until the restraint time reached that of the length of an infusion for the mock injection (2 min and 30 s).

Microinjections

Mock injections were given on day 20 immediately prior to DID by using microinjectors that extended 0.5 mm past the cannula and by restraining the mouse for the duration of a microinjection (2.5 min). Mice were microinjected on day 22 and 24 with one of three drug concentrations [saline (control), 0.15 μg/side, or 0.5 μg/side NBQX] into the DLS. On days 21 and 23, mice were returned to their original drinking solution and were not infused. Mice were injected with the same drug concentration on both infusion days. The drug concentration groups were assigned (counterbalanced) based on 2-h consumption on the mock injection day. Microinjection procedures have been described in depth elsewhere (Bauer et al., 2022).

Blood alcohol concentrations (BACs)

On day 24, immediately following DID, mice had retro-orbital sinus bloods taken. Blood plasma was spun down on a centrifuge at 13,805 g for 5 min, plasma was pipetted out, and blood samples were stored at −20 °C. BACs were determined using an Analox EtOH Analyzer (Analox Instruments; Lunenburg, Massachusetts, United States). The Analox was calibrated with a 5-μL injection of 100-mg/dL ethanol standard. Following calibration, blood plasma was then briefly vortexed and approximately 5 μL were pipetted into the analyzer. BACs were immediately displayed and cataloged. The Analox was recalibrated every 5–10 samples to ensure accurate readouts.

Experimental design and statistical analyses

Analysis of Variance (ANOVA) was used to determine differences in drinking, locomotor activity, and BACs. Sex was included as a factor in all initial analyses. If sex differences were not found, we collapsed on sex as a factor for subsequent analyses and graphing. Baseline alcohol drinking was assessed with RM one-way ANOVA. The effect of NBQX concentration on mock injection was tested with one-way ANOVA. Test of compulsive-like drinking was done with a paired t test for alcohol-history animals and unpaired t test when comparing water- vs. alcohol-history animals. Order effects were tested with RM two-way ANOVA. Twenty-minute and 2-h intakes were assessed with one-way ANOVAs. Normality and sphericity were assessed in each test and were corrected for if violated. Differences were considered significant at p < 0.05. Figures were made using GraphPad Prism 8, and data were analyzed using R Studio (www.r-project.org).

Results

Baseline alcohol or water drinking (days 1–21) for male and female mice is shown in Figure 2. Alcohol consumption is displayed in grams consumed per kilogram of body weight, and water consumption is displayed in milliliters consumed per kilogram of body weight. All analyses initially included sex as a factor. Sex did not have a significant main effect nor interaction (p’s > 0.05), so we collapsed on sex as a factor. RM one-way ANOVA of day and NBQX concentration on water intake revealed a main effect of day (F(20, 1080) = 3.84, p < 0.0001, η2G = 0.05) (Fig. 2A). One-way ANOVA of drug concentration on mock injection day in water-history mice revealed no significant differences (p > 0.05) (Fig. 2B). RM one-way ANOVA of day and NBQX concentration on alcohol intake revealed a main effect of day (F(20, 980) = 3.19, p < 0.001, η2G 0.04) (Fig. 2C). One-way ANOVA of drug concentration on mock injection day in alcohol-history mice revealed no significant differences (p > 0.05) (Fig. 2D). Drug group assignments were chosen based on day 20 (mock injection) alcohol consumption so that baseline drinking was similar across drug groups.

Fig. 2. Baseline drinking.

Fig. 2.

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Average daily water consumption per concentration of NBQX across the 21 days prior to intra-DLS NBQX in male and female (A) mice (n = 7–11/sex/concentration). RM one-way ANOVA revealed a main effect of day (***p < 0.001) for male and female mice. Water intake across drug concentrations on mock injection day in male and female (B) mice. One-way ANOVA revealed no significant effects. Average daily alcohol consumption per concentration of NBQX across the 21 days prior to intra-DLS NBQX in male and female (C) mice (n = 7–11/sex/concentration). RM one-way ANOVA revealed a main effect of day (***p < 0.001) for male and female mice. Alcohol intake across drug concentrations on mock injection day in male and female (D) mice. Data are displayed as mean ± standard error of the mean.

To determine whether alcohol-history mice drank QuA compulsively, we assessed drinking as previously done (Bauer et al., 2021). All analyses initially included sex as a factor. There were no other main effects or interactions including sex (p’s > 0.05), so we collapsed on sex as a factor. First, we assessed whether QuA and alcohol drinking were at the same level in alcohol-history mice. Paired t test of solution in alcohol-history mice revealed a main effect of drinking solution (t(18) = 4.7, p < 0.001) (Fig. 3A). We next assessed whether alcohol-history mice drank significantly more QuA than water-history mice. Unpaired t test of solution in male and female mice revealed no significant differences in QuA intake (p’s > 0.05) (Fig. 3B). These data do not support the development of compulsive-like QuA drinking in alcohol-history mice. In other words, alcohol-history mice in the current study did not develop quinine-resistant drinking after three weeks of DID as we had previously observed, published, and replicated (Bauer et al., 2021).

Fig. 3. Failure to demonstrate compulsive-like alcohol drinking.

Fig. 3.

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To determine whether we observed compulsive-like alcohol drinking, we ran the following analyses in saline-infused animals. A. Paired t test determined a main effect of solution (****p < 0.0001). B. Two-way ANOVA revealed no significant differences (p’s > 0.05). Data are displayed as mean ± SEM.

Despite the lack of quinine-resistant drinking, we nevertheless went on to assess the effect of intra-DLS AMPAR antagonism on 20-min and 2-h alcohol and QuA drinking in water-history mice (Fig. 4), and 20-min front-loading and 2-h drinking in alcohol-history mice (Fig. 5). All analyses initially included sex as a factor initially. No main effects or interactions involved sex (p’s > 0.05), so we collapsed on sex as a factor. Twenty-minute alcohol and QuA drinking are not considered front-loading, as front-loading in C57BL/6J mice develops as a consequence of alcohol-drinking history. However, we include these analyzed data and refer to them as 20-min for the water-history mice, as opposed to front-loading as we do in the alcohol-history mice. One-way ANOVA of NBQX concentration on 20-min alcohol or QuA intake in water-history mice revealed no significant differences (p’s > 0.05) (Fig. 4A and B). One-way ANOVA of NBQX concentration on 2-h alcohol intake in water-history mice revealed a main effect of NBQX (F(2,53) 3.45, p < 0.05, η2G = 0.12) (Fig. 4C). RM two-way ANOVA in of order of solution presented (Alcohol first vs. QuA first) and concentration of NBQX on 2-h QuA intake in water-history mice revealed a main effect of order (F(1, 49) = 5.4, p < 0.05, η2G = 0.1) (Fig. 4D). Thus, we assessed QuA intake in water-history mice separately by order. Subsequent analysis indicated a significant interaction of solution order and drug concentration (F(2, 49.48) = 3.44, p < 0.05, η2G = 0.13). Post hoc Bonferroni-corrected t test comparison determined that saline-infused mice drank significantly more QuA if they were given QuA the second time compared with the first time (t(49) = 3.38, p < 0.01) (Fig. 4D). Additionally, post hoc Bonferroni-corrected t test determined that 0.15-mg/side NBQX significantly reduced QuA intake compared with saline when the QuA was offered the second time (t(49) = 2.97, p < 0.01) (Fig. 4D).

Fig. 4. Intra-DLS AMPAR antagonism on alcohol and QuA intake in water-history mice.

Fig. 4.

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One-way ANOVA on 20-min alcohol (A) or QuA (B) intake revealed no significant effects of drug concentration (p > 0.05). One-way ANOVA on 2-h alcohol (C) intake revealed a main effect of NBQX (*p < 0.05). A significant order effect was observed in water-history animals given QuA (*p < 0.05); therefore, data were separated by solution order. NBQX significantly reduced QuA drinking in water-history animals given QuA second (**p < 0.05), but not first (p > 0.05). Intake was also significantly higher in saline animals given QuA second (*p < 0.05) (D). Data are displayed as mean ± standard error of the mean.

Fig. 5. Intra-DLS AMPAR antagonism on alcohol and QuA intake in alcohol-history mice.

Fig. 5.

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Two-way ANOVA on 20-min alcohol (A) intake revealed a significant main effect of sex (*p < 0.05) and NBQX concentration (*p < 0.05). One-way ANOVA on 2-min QuA (B) intake revealed a main effect of drug concentration (*p < 0.05). One-way ANOVA on 2-h alcohol (C) or QuA (D) intake revealed no significant differences (p > 0.05). Data are displayed as mean ± standard error of the mean.

Intra-DLS AMPAR antagonism on 20-min and 2-h alcohol and QuA drinking in alcohol-history mice is shown in Figure 5. RM two-way ANOVA of order of solution presented (Alcohol first vs. QuA first) and concentration of NBQX on 2-h QuA intake in alcohol-history mice did not reveal a main effect of order or interaction of order and NBQX concentration (p’s > 0.05). Thus, we collapsed on order as a factor. Two-way ANOVA of NBQX concentration on 20-min alcohol front-loading in alcohol-history mice revealed a significant main effect of sex, (F(2, 47) = 2.8, p < 0.05, η2G = 0.1), a main effect of NBQX concentration (F(2, 47) = 3.1, p < 0.05, η2G = 0.12), but no interaction of sex or concentration (Fig. 4A). Kruskal–Wallis test of NBQX concentration on 20-min QuA front-loading in alcohol-history mice revealed a significant main effect of concentration (H(3) = 6.41, p < 0.05) (Fig. 5B). Post hoc Dunn’s multiple-comparison test determined that 0.5 μg/mL was significantly lower than the saline-infused group (p < 0.05) (Fig. 5B). One-way ANOVA of NBQX concentration on 2-h alcohol or QuA intake in alcohol-history mice revealed no significant differences (p’s > 0.05) (Fig. 5C and D).

Blood alcohol concentration (BAC) correlations with alcohol and QuA are shown in Figure 6. Pearson’s correlation in water-history mice determined that alcohol (R2 = 0.54, p < 0.0001) and QuA (R2 = 0.605, p < 0.0001) blood alcohol concentrations significantly and positively predicted intake (Fig. 6A). Average BAC for water-history mice given alcohol was 73.81 ± 14.25 mg/dL and for QuA was 31.66 ± 6.19 mg/dL; data not shown. Average Pearson’s correlation in alcohol-history mice also determined that alcohol (R2 = 0.65, p < 0.0001) and QuA (R2 = 0.4, p < 0.001) BACs significantly and positively predicted intake (Fig. 6B). Average BAC for alcohol-history mice given alcohol was 89.14 ± 17.41 mg/dL and for QuA was 23.09 ± 5.3 mg/dL; data not shown. Thus, while both alcohol- and water-history animals binge drank alcohol as demonstrated by BACs of approximately 80 mg/dL, QuA-drinking animals did not reach BAC levels near a binge.

Fig. 6. Blood alcohol concentrations (BACs).

Fig. 6.

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Immediately following DID on the final day of infusion, blood samples were taken and BACs were determined. Alcohol and QuA intakes significantly and positively predict BACs in A) water-history and B) alcohol-history mice. ***p < 0.001, ****p < 0.0001.

Lastly, during all infusion days, ambulatory home cage locomotor activity was recorded. We did not observe any significant effects of sex or drug concentration on locomotor activity (p’s > 0.05; data not shown).

Discussion

The primary goal of this experiment was to identify the role of the DLS in modulating binge-like and compulsive-like alcohol drinking early and late (after development of compulsive-like alcohol drinking) after an alcohol-drinking history using male and female C57BL/6J mice. We found that compulsive-like alcohol drinking was largely blunted in this experiment, even though we used a model of compulsive-like alcohol drinking we have previously demonstrated behaviorally and replicated (Bauer et al., 2021). While we were unable to test our original compulsive-like alcohol drinking hypothesis due to a failure of the model, we nevertheless have identified important functionality of the DLS for alcohol drinking. Specifically, we found that the DLS initially drives binge-like alcohol drinking. However, following an extended binge-drinking history, one in which we have previously demonstrated to cause compulsive-like alcohol drinking, the DLS no longer drives binge drinking. We also assessed alcohol and QuA front-loading in alcohol-history mice finding intra-DLS NBQX reduced front-loading of alcohol and QuA. Importantly, intra-DLS NBQX did not alter ambulatory locomotor activity during any of the drinking sessions. We assessed this in male and female mice, with adequate power to detect sex differences, finding no sex differences.

Our main finding that intra-DLS NBQX reduces alcohol drinking initially, but not after extended alcohol access, is indicative of binge drinking-induced plasticity in the DLS. These data suggest that over extended alcohol access, the DLS becomes less and less involved in gating binge-like alcohol drinking. This finding is in agreement with recent work investigating cortical inputs into the DLS (Haggerty et al., 2022). Haggerty et al. (2022) found that stimulating inputs into the DLS reduces binge-like alcohol drinking after several weeks of binge access but had no effect on initial binge-like alcohol drinking. We have previously shown that following a single week of binge-drinking history, intra-DLS NBQX reduces binge-like alcohol drinking in male and female mice (Bauer et al., 2022). This occurs without altering locomotor activity or saccharin drinking (though strong trends suggest intra-DLS NBQX may attenuate saccharin drinking). This, and the findings in this manuscript suggest that the plasticity occurring in the DLS is altered after binge-drinking histories of greater than a week in male and female C57BL/6J mice. These results and ours corroborate with the idea that the DLS functionality for driving binge-like alcohol drinking differs as a function of alcohol-drinking history.

Since the DLS is involved in compulsive alcohol seeking (Giuliano et al., 2019), we aimed to identify whether compulsive alcohol taking is also DLS-dependent. To do this we used a drinking history and QuA concentration in which we had previously shown and replicated robust compulsive-like QuA drinking (Bauer et al., 2021). Specifically, we considered drinking to be compulsive-like if 1) alcohol-history mice drink significantly more QuA than water-history mice, and 2) alcohol-history mice drink the same amount of alcohol at baseline than QuA. Thus, the alcohol-drinking history must cause specific behavioral changes relative to the water-history mice to be considered compulsive drinking. As laid out in Figure 3, we were not able to demonstrate compulsive drinking, by our measures, in our alcohol-history group. However, if one were to consider QuA drinking alone as a measure of compulsive-like alcohol drinking, then our results suggest the DLS is not involved in compulsive-like QuA drinking. This interpretation is in agreement with Giuliano et al. (2019), who assessed the DLS for both compulsive taking and seeking. Giuliano et al. (2019) found that intra-DLS DA antagonism reduces compulsive seeking, but not compulsive taking in an operant seek-take chain procedure in rats. Thus, in agreement with others, our data suggest the DLS does not drive compulsive-like alcohol taking. The DLS is involved in habitual alcohol seeking, where over extended alcohol seeking and taking, the DLS engages to drive habitual alcohol seeking (Corbit et al., 2012, 2014), but not alcohol taking (Corbit et al., 2014). Therefore, over extended alcohol access, it appears that while the DLS is engaging for habitual alcohol seeking, it is disengaging to promote continued alcohol drinking. This finding and interpretation have important implications for the field because it provides support for different mechanisms of alcohol seeking versus taking and identifies a novel functional role of the DLS across different alcohol drinking histories.

In addition to assessing binge-like drinking, we assessed front-loading of both alcohol and QuA in alcohol-history mice. Alcohol front-loading is observed when over an extended drinking history, animals increase the rate of intake early in the drinking session (Ardinger, Lapish, Czachowski, & Grahame, 2022). We have previously found that alcohol front-loading occurs for alcohol and QuA in alcohol-history mice under this timeline (Bauer et al., 2021). We found that intra-DLS NBQX reduced front-loading of alcohol and QuA. It has previously been suggested that reducing alcohol front-loading may have important translational aspects, as reduced front-loading has led to reduced 2-h intakes (Linsenbardt & Boehm; 2014). Our results differ from this finding as intra-DLS NBQX reduced front-loading, but not overall 2-h intakes in alcohol-history mice. However, the Linsenbardt and Boehm paper (2014) had a shorter DID history than ours. It is possible that the extra week of drinking history engaged other brain regions, which overwrote the DLS’ role in gating binge-like alcohol drinking. Our findings differ from another project (Haggerty et al., 2022) that found stimulating inputs into the DLS after a DID history decreased alcohol front-loading, which is essentially the opposite of what we found. However, Haggerty et al. (2022) used circuit manipulations, whereas we utilized pharmacological lesioning. Thus, the two projects are not directly comparable. Since alcohol front-loading is thought to be due to increased motivation for the reward, our data suggest that the DLS may be involved in motivation for alcohol and QuA.

In previous experiments we have demonstrated robust compulsive-like alcohol drinking under the same drinking history (Bauer et al., 2021). The mice used in this experiment had several additional manipulations that may have interfered with compulsive-like drinking. First, these animals received bilateral cannulations, had stylets changed daily, and underwent a procedure to habituate the animals to stress prior to infusion, and then also received infusions. Immediately after infusion, animals were placed in their home cage and expected to drink the QuA. It is possible that the stress of stylet changing and infusions reduced the animals’ motivation to drink. Alternatively, the habituation procedure we employed may have sensitized the animals to stress. While we did not directly test such hypotheses, it is plausible that these moving parts all contributed to reduced motivation to drink the QuA solution. It is also worth noting that mice who receive bilateral cannulation surgery have head caps made of cement to hold the cannula in place. It is possible that the weight of the head cap in addition to the aversive solution caused a compound aversion that the mice could not overcome. Finally, it is worth noting that even drinking of the regular alcohol solution may be impacted in mice that are cannulated and injected. Indeed, the male and female mice in the present study consumed an average of about 3 g/kg in 2 h following the 3-week drinking history. In mice who did not receive bilateral cannulation surgery (Bauer et al., 2021) and under the same drinking history, male mice drank 4 g/kg and female mice drank around 6 g/kg in 2 h. Thus, while data vary from experiment to experiment, our findings suggest that the procedures associated with bilateral cannulation and subsequent microinjection may reduce overall willingness to drink.

We also observed a bizarre order effect in QuA drinking in water-history mice. Specifically, we observed that mice given QuA following alcohol exposure drank significantly more QuA than mice given QuA before alcohol exposure. We also found that in mice given QuA second, NBQX reduced drinking. This could suggest that a single alcohol exposure alters the propensity to drink QuA and that the DLS is involved in aversion drinking. However, it is also possible that the effect is spurious or that the initial QuA intake was too low, creating a floor effect. Since we were not able to collapse on order, this analysis was not powered adequately; therefore, it may or may not be relevant to the broader findings. Additionally, since both alcohol and QuA drinking were reduced by intra-DLS NBQX in water-history mice, and previous research (Bauer et al., 2022) found that intra-DLS NBQX trended toward reducing saccharin drinking, the DLS might be involved in overall consummatory behavior, regardless of reward value.

A novel and important aspect of our research experiment was the inclusion of and adequate power to assess sex differences. We did not find any significant interactions of sex as a factor and therefore conclude sex differences were not present. In our model of compulsive-like alcohol drinking and in previous research assessing sex differences within the DLS on binge-like alcohol drinking, interactions between sex also did not occur (Bauer et al., 2021, 2022). In our hands (Bauer et al., 2020, 2021) and others (Sneddon, White, & Radke, 2019), female mice binge-drink significantly more alcohol and QuA than male mice. Interestingly, we did not observe this effect in even basal drinking. We have observed a blunted sex difference in binge-like alcohol drinking previously (Bauer et al., 2022) in mice who underwent bilateral cannulation surgery. Since evidence exists that female mice may be more sensitive to stress (Peltier et al., 2019), it is possible that the increased stress that occurs with cannula, stylet changing, and handling could have caused a reduction in drinking. Additionally, female mice are smaller than male mice, but the headcaps are roughly the same size regardless of sex. Because of this it is also possible that the sheer weight of headcaps had a greater impact on female mice’ ability to drink, compared to male mice. Thus, while we did not find sex differences in the DLS’ ability to gate binge-like alcohol drinking, we did observe sex differences relative to what we typically see in animals who did not receive surgery.

Finally, we assessed locomotor activity during all infusions and did not find a significant effect on locomotion. This is important because it demonstrates our ability to reduce alcohol drinking without altering overall movement. Therefore, intra-DLS reductions on front-loading and binge-like alcohol drinking were alcohol-specific effects. QuA drinking has been modeled using mice and rats in the preclinical literature. Since we are making inferences on the compulsive-like alcohol drinking behavior in mice, it may be useful to consider behavioral similarities in compulsive-like drinking between the two species. For example, genetic selection for high alcohol drinking in both mice and rats demonstrates an innate bias toward compulsive drinking. For example, selectively bred high alcohol-preferring mice (HAP) demonstrate innate compulsive-like QuA drinking (Houck, Carron, Millie, & Grahame, 2019; Sneddon, Schuh, Fennell, Grahame, & Radke, 2022) and selectively bred alcohol-preferring rats (P rats) demonstrate greater compulsive-like drinking compared with Wistar rats following the same alcohol-drinking history (Timme et al., 2020). This demonstrates that the genetic selection for high alcohol drinking in mice and rats results in greater susceptibility to compulsive-like alcohol drinking. Previous research on compulsive-like alcohol drinking in mice has mixed findings on whether sex differences occur (for review see Radke, Sneddon, Frasier, & Hopf, 2021). For example, Sneddon et al. (2019) and Bauer et al. (2020) found no effect of sex on QuA drinking using C57BL/6J mice, while Fulenwider, Nennig, Price, Hafeez, and Schank (2019), Shaw et al. (2020), and Sneddon, Ramsey, Thomas, and Radke (2020) found that females demonstrate greater compulsive-like drinking than male mice. In the rat literature, female Long Evans rats drink more QuA than males (Randall, Stewart, & Besheer, 2017), suggesting a sex difference in the same direction as the reported sex differences in mice. However, a recent report using P rats found that male rats demonstrate greater compulsive-like drinking than female rats (Katner et al., 2022), suggesting a sex difference in the opposite direction as mice. These, and many other things, may be important considerations when comparing findings across mice and rats.

In conclusion, we demonstrate that the DLS has a different function for alcohol drinking depending on drinking history. Specifically, front-loading of alcohol and QuA, a behavior that develops as a consequence of a drinking history, is DLS-dependent. However, overall, 2-h alcohol and QuA intakes are only DLS-dependent in animals who do not have an alcohol-drinking history, but not in animals with a 3-week DID history. These effects were not sex-specific and occurred in the absence of altered locomotion. Additionally, compulsive-like alcohol drinking is disrupted in cannulated mice. This is an important consideration in methodologies for future experimental designs. The broad interpretation from these findings is that alcohol-experience-dependent plasticity in the DLS is differentially involved in binge-like and front-loaded drinking, key behaviors in the development of AUD. Future work should focus on better understanding the nature of this plasticity to advance novel prevention and treatment strategies for AUD.

Acknowledgments

This work was supported by the Indiana Alcohol Research Center and NIH/NIAAA grants AA07611, T32AA07462, and 1F31AA029273-01A1.

Footnotes

Declaration of competing interest

The authors declare no conflicts of interest.

References

  1. Addolorato G, Vassallo GA, Antonelli G, Antonelli M, Tarli C, Mirijello A, et al. (2018). Binge drinking among adolescents is related to the development of alcohol use disorders: Results from a cross-sectional study. Scientific Reports, 8, Article 12624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. American Psychiatric Association. (2013). Diagnostic and statistical manual of mental disorders (5th ed.). [Google Scholar]
  3. Ardinger CE, Lapish CC, Czachowski CL, & Grahame NJ (2022. Oct). A critical review of front-loading: A maladaptive drinking pattern driven by alcohol’s rewarding effects (10th, (10th,. 46 pp. 1772–1782). Alcohol Clin Exp Res. doi: 10.1111/acer.14924. Epub 2022 O10.1111/acer.14924ct 14. Review. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bauer MR, Garcy DP, & Boehm SL (2020). Systemic administration of the AMPA receptor antagonist, NBQX, reduces alcohol drinking in male C57BL/6J, but not female C57BL/6J or high-alcohol-preferring, mice. Alcoholism: Clinical and Experimental Research, 44(11), 2316–2325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bauer MR, McVey MM, & Boehm SL (2021). Three weeks of binge alcohol drinking generates increased alcohol front-loading and robust compulsive-like alcohol drinking in male and female C57BL/6J mice. Alcoholism: Clinical and Experimental Research, 45(3), 650–660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bauer MR, McVey MM, Germano DM, Zhang Y, & Boehm SL II (2022). Intra-dorsolateral striatal AMPA receptor antagonism reduces binge-like alcohol drinking in male and female C57BL/6J mice. Behavioural Brain Research, 418, Article 113631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Corbit LH, Nie H, & Janak PH (2012). Habitual alcohol seeking: Time course and the contribution of subregions of the dorsal striatum. Biological Psychiatry, 72(5), 389–395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Corbit LH, Nie H, & Janak PH (2014). Habitual responding for alcohol depends upon both AMPA and D2 receptor signaling in the dorsolateral striatum. Frontiers in Behavioral Neuroscience, 8, 301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Darcq E, Morisot N, Phamluong K, Warnault V, Jeanblanc J, Longo FM, et al. (2016). The neurotrophic factor receptor p75 in the rat dorsolateral striatum drives excessive alcohol drinking. Journal of Neuroscience, 36(39), 10116–10127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dev KK, Petersen V, Honoré T, & Henley JM (1996). Pharmacology and regional distribution of the binding of 6-[3H] nitro-7-sulphamoylbenzo [f]-quinoxaline-2, 3-dione to rat brain. Journal of Neurochemistry, 67(6), 2609–2612. [DOI] [PubMed] [Google Scholar]
  11. Fulenwider HD, Nennig SE, Price ME, Hafeez H, & Schank JR (2019). Sex differences in aversion-resistant ethanol intake in mice. Alcohol and Alcoholism, 54(4), 345–352. [DOI] [PubMed] [Google Scholar]
  12. Giuliano C, Belin D, & Everitt BJ (2019). Compulsive alcohol seeking results from a failure to disengage dorsolateral striatal control over behavior. Journal of Neuroscience, 39(9), 1744–1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Green A, Neff D, Giuliano G, Lee N, Turchin R, & Kunkel EJ (2018). Surrogate alcohol or nonbeverage alcohol consumption: The Surrogate Alcohol Questionnaire (SAQ). Psychosomatics, 59(4), 349–357. [DOI] [PubMed] [Google Scholar]
  14. Haggerty DL, Muñoz B, Pennington T, Grecco GG, & Atwood B (2022). Anterior insular cortex inputs to the dorsolateral striatum govern the maintenance of binge alcohol drinking. bioRxiv. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hopf FW, & Lesscher HMB (2014). Rodent models for compulsive alcohol intake. Alcohol, 48(3), 253–264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Houck CA, Carron CR, Millie LA, & Grahame NJ (2019). Innate and acquired quinine-resistant alcohol, but not saccharin, drinking in crossed high-alcohol-preferring mice. Alcoholism: Clinical and Experimental Research, 43(11), 2421–2430. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kasten CR, & Boehm SL II (2014). Intra-nucleus accumbens shell injections of R (+)-and S (−)-baclofen bidirectionally alter binge-like ethanol, but not saccharin, intake in C57Bl/6J mice. Behavioural Brain Research, 272, 238–247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Katner SN, Sentir AM, Steagall KB, Ding ZM, Wetherill L, Hopf FW, et al. (2022). Modeling aversion resistant alcohol intake in Indiana alcohol-preferring (P) rats. Brain Sciences, 12(8), 1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lachenmeier DW, Rehm J, & Gmel G (2007). Surrogate alcohol: What do we know and where do we go? Alcoholism: Clinical and Experimental Research, 31(10), 1613–1624. [DOI] [PubMed] [Google Scholar]
  20. Linsenbardt DN, & Boehm SL 2nd (2014). Alterations in the rate of binge ethanol consumption: Implications for preclinical studies in mice. Addiction Biology, 19(5), 812–825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. National Academies Press. (2011). Guide for the care and use of laboratory animals (8th ed.). [PubMed] [Google Scholar]
  22. National Institute of Alcohol Abuse and Alcoholism (NIAAA) (2019). (2019). Drinking levels defined. Available at: https://www.niaaa.nih.gov/alcohol-health/overview-alcohol-consumption/moderate-binge-drinking. (Accessed July 2022).
  23. Paxinos G, & Franklin KBJ (2001). The mouse brain in stereotaxic coordinates (2nd ed.). San Diego: Academic Press. [Google Scholar]
  24. Peltier MR, Verplaetse TL, Mineur YS, Petrakis IL, Cosgrove KP, Picciotto MR, et al. (2019). Sex differences in stress-related alcohol use. Neurobiology of Stress, 10, Article 100149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Radke AK, Sneddon EA, Frasier RM, & Hopf FW (2021). Recent perspectives on sex differences in compulsion-like and binge alcohol drinking. International Journal of Molecular Sciences, 22(7), 3788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Randall PA, Stewart RT, & Besheer J (2017). Sex differences in alcohol self-administration and relapse-like behavior in Long-Evans rats. Pharmacology, Biochemistry, and Behavior, 156, 1–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rhodes JS, Best K, Belknap JK, Finn DA, & Crabbe JC (2005). Evaluation of a simple model of ethanol drinking to intoxication in C57BL/6J mice. Physiology & Behavior, 84, 53–63. [DOI] [PubMed] [Google Scholar]
  28. Ruda-Kucerova J, Babinska Z, Luptak M, Getachew B, & Tizabi Y (2018). Both ketamine and NBQX attenuate alcohol drinking in male Wistar rats. Neuroscience Letters, 14(666), 175–180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shaw GA, Bent MAM, Council KR, Pais AC, Amstadter A, Wolstenholme JT, et al. (2020). Chronic repeated predatory stress induces resistance to quinine adulteration of ethanol in male mice. Behavioural Brain Research, 382, Article 112500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sheardown MJ, Nielsen EØ, Hansen AJ, Jacobsen P, & Honore T (1990). 2, 3-Dihydroxy-6-nitro-7-sulfamoyl-benzo (F) quinoxaline: A neuroprotectant for cerebral ischemia. Science, 247(4942), 571–574. [DOI] [PubMed] [Google Scholar]
  31. Sneddon EA, Ramsey OR, Thomas A, & Radke AK (2020). Increased responding for alcohol and resistance to aversion in female mice. Alcoholism: Clinical and Experimental Research, 44(7), 1400–1409. [DOI] [PubMed] [Google Scholar]
  32. Sneddon EA, Schuh KM, Fennell KA, Grahame NJ, & Radke AK (2022. Oct 20). Crossed high-alcohol-preferring mice exhibit aversion-resistant responding for alcohol with quinine but not foot shock punishment. Alcohol, 105, 35–42. 10.1016/j.alcohol.2022.09.006. Online ahead of print. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sneddon EA, White RD, & Radke AK (2019). Sex differences in binge-like and aversion-resistant alcohol drinking in C57BL/6J mice. Alcoholism: Clinical and Experimental Research, 43(2), 243–249. [DOI] [PubMed] [Google Scholar]
  34. Stephens DN, & Brown G (1999). Disruption of operant oral self-administration of ethanol, sucrose, and saccharin by the AMPA/kainite antagonist, NBQX, but not the AMPA antagonist, GYKI 52466. Alcoholism: Clinical and Experimental Research, 23(12), 1914–1920. [PubMed] [Google Scholar]
  35. Thiele TE, Crabbe JC, & Boehm SL 2nd (2014). “Drinking in the dark” (DID): A simple mouse model of binge-like alcohol intake. Current Protocols in Neuroscience, 68, 9–49, 1–9.49.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Timme NM, Linsenbardt D, Timm M, Galbari T, Cornwell E, & Lapish C (2020). Alcohol-preferring P rats exhibit aversion-resistant drinking of alcohol adulterated with quinine. Alcohol, 83, 47–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wang J, Hamida SB, Darcq E, Zhu W, Gibb SL, Fe Lanfranco M, et al. (2012). Ethanol-mediated facilitation of AMPA receptor function in the dorsomedial striatum: Implications for alcohol drinking behavior. Journal of Neuroscience, 32(43), 15124–15132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wolffgramm J, & Heyne A (1991). Social behavior, dominance, and social deprivation of rats determine drug choice. Pharmacology, Biochemistry, and Behavior, 38, 389–399. [DOI] [PubMed] [Google Scholar]
  39. Yin HH, Knowlton BJ, & Balleine BW (2004). Lesions of dorsolateral striatum preserve outcome expectancy but disrupt habit formation in instrumental learning. European Journal of Neuroscience, 19(1), 181–189. [DOI] [PubMed] [Google Scholar]
  40. Yin HH, Knowlton BJ, & Balleine BW (2006). Inactivation of dorsolateral striatum enhances sensitivity to changes in the action–outcome contingency in instrumental conditioning. Behavioural Brain Research, 166(2), 189–196. [DOI] [PubMed] [Google Scholar]

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