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. 2023 Mar 27;58(3):280–288. doi: 10.1093/alcalc/agad019

Serotonin regulation of intermittent and continuous alcohol drinking in male and female C57BL/6J mice with systemic SB242084 and buspirone

Yeasong E Ko 1, Lara S Hwa 2,✉
PMCID: PMC10168709  PMID: 36973222

Abstract

This study aims to assess the therapeutic potentials of novel serotonergic compounds in treating alcohol use disorders by investigating the effects of SB242084 and buspirone on intermittent and continuous alcohol consumption in male and female mice. Adult male and female C57BL/6J mice were given two-bottle choice to 20% ethanol and water on an intermittent or continuous availability schedule. Drug testing consisted of intraperitoneal injections of 0.3, 1, 3 mg/kg SB242084 or 1, 3, 10 mg/kg buspirone, and subsequent alcohol and water consumption were measured. To monitor the drug effects on anxiety-like and locomotor behavior, the highest dose of each compound was administered before free activity in an open field. SB242084 dose-dependently attenuated alcohol drinking for intermittent alcohol drinking in male mice but did not significantly alter alcohol drinking in mice given continuous access. Two-hour and four-hour female drinking behavior was unaffected by SB242084. In comparison, buspirone not only suppressed intermittent and continuous alcohol drinking in both males and females but also reduced distance traveled in the open field test. Observed differences in responses to SB242084 between drinking groups may imply differing neural mechanisms between episodic and continuous drinking driven by serotonin. Reductions in drinking after buspirone treatment may be related to non-specific properties. These findings suggest the therapeutic potential of compounds blocking the 5-HT2C receptor for alcohol use disorders.

Keywords: alcohol drinking, intermittent, continuous, SB242084, buspirone

Short Summary: Systemic administration of SB242084, a serotonin 2C receptor antagonist, reduced intermittent alcohol drinking in male C57BL/6J mice. However, SB242084 did not reduce continuous alcohol drinking in male mice or drinking in female mice. Buspirone, a serotonin 1A partial agonist, reduced intermittent alcohol consumption in males and females and locomotor activity in the open field.

Introduction

Alcohol misuse continues to pose a great threat to society, and alcohol use disorder (AUD) is among the most widespread mental health disorders around the world (Rehm and Shield, 2019). Although the prevalence of AUDs in men remains greater than in women, the increases in alcohol use and high-risk drinking across genders are still significant public health concerns (Grant et al., 2017). AUDs can form through a long-term pattern of excessive drinking, and some have suggested that heavy use over time should be integral to defining AUDs (Rehm et al., 2013). We have a great need to continue developing pharmaceutical treatments for AUD.

Studies have long implicated the role of serotonin (5-HT) in mediating AUD and mental health disorders, like depression and anxiety, though their relationship is complex due to this co-morbidity (McBride et al., 1993; Marcinkiewcz et al., 2015). For example, buspirone, a partial 5-HT 1A receptor (5-HT1A) agonist and dopamine D2, D3, and D4 antagonist, has been historically used to reduce voluntary alcohol drinking in humans and animals (Bruno, 1989; Kranzler and Meyer, 1989; Hedlund and Wahlstrom, 1996). Compounds that are selective for 5-HT2C receptors are also known to modify alcohol self-administration behaviors (Tomkins et al., 2002; Yoshimoto et al., 2012; Tabbara et al., 2021). Foundational studies in rats demonstrated that buspirone and the 5-HT2C antagonist, SB242084, alleviate deficits in social interaction during withdrawal from ethanol liquid diet (Overstreet et al., 2003). It is thought that 5-HT drugs may have efficacy through anti-anxiety mechanisms rather than anti-drinking mechanisms (Malec et al., 1996).

This research aims to assess the effects of buspirone and the 5-HT2C antagonist SB242084 on the continuous and intermittent drinking behavior in both male and female mice. Every other day, intermittent access (IA) to alcohol has been a method of generating higher levels of voluntary drinking in rodents compared to levels of drinking observed on ad libitum, continuous access (CA) schedules (Spear, 2020). Some features of an IA schedule, such as the repeated opportunities to drink between periods of abstinence, may translate to heavy, episodic drinking in humans. This behavioral pharmacology report is unique in that male and female mice on different schedules of drinking are compared within 5-HTergic drug testing. We hypothesize that SB242084 and buspirone will significantly attenuate ethanol consumption in the IA drinking schedule, with potential differences in drug effects on the CA drinking schedule. General fluid consumption and locomotor activity in the open field will also be monitored for off-target, sedative properties of these 5-HTergic compounds. These behavioral studies targeting 5-HT1A and 5-HT2C receptors using different drinking schedules provide insight into the neuroadaptations underlying harmful drinking patterns in people.

Materials and Methods

Animals

The 6–8-week-old male and female C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME). Upon arrival, the mice were given at least 1 week of habituation to acclimate to the vivarium in a 24 h reversed light–dark cycle room (7 a.m. lights off, 7 p.m. lights on). Singly housed mice had ad libitum access to Rodent LabDiet 5001 (Lab Supply, Northlake, TX) and water. The Baylor University Institutional Animal Care and Use Committee approved these experiments.

Experimental design

C57BL/6J alcohol-drinking mice (n = 12/group) were given either IA or CA to two-bottle choice to represent long-term heavy drinking or frequent, lower drinking behavior, respectively. The IA schedule consisted of 24 h access to 20% ethanol on alternating days of the week, with water available at all times (Albrechet-Souza et al., 2015; Hwa et al., 2016). There was no ramp of increasing ethanol concentrations, which is a slight modification from the original Hwa et al. (2011) description. The CA schedule consisted of 24 h access to 20% ethanol every day of the week, with water available at all times. Fluid consumption was measured by collecting before drinking and after drinking weights of Drinko Measurer 10-ml glass tubes and 90-mm sipper tubes (Amuza, San Diego, CA). Fluid spillage was calculated by subtracting measurements collected from a drip cage with no animal present. Mice underwent total 12 drinking days, which spanned ~4 weeks for the IA condition and 12 consecutive days for the CA condition. Following the 12 drinking days, drug testing occurred across 4 test days with either SB242084 (0, 0.3, 1, and 3 mg/kg, i.p.) or buspirone (0, 1, 3, and 10 mg/kg, i.p.) in a Latin square design. Bottles were weighed ~3 h into the dark cycle according to consistent drinking schedules, which was during 24-h withdrawal for IA mice and no withdrawal for CA mice. Mice were habituated to three separate saline injections before drug testing.

Additional tests of the effects of SB242084 or buspirone on anxiety-like and locomotor behavior were performed using an open field during 24 h withdrawal from alcohol. Drinking groups were combined, and half of the mice received the highest drug dose, 3 mg/kg SB242084 or 10 mg/kg buspirone, and the other half received the vehicle 20 min before being placed into the corner of an open arena. Mouse activity was videorecorded in a 45 x 45 cm open arena for 10 min. Locomotor behavior was analyzed with the EthoVision 14 software (Noldus, Leesburg, VA) for behavioral tracking.

Drugs

SB242084 (Sigma-Aldrich, Atlanta, GA), a 5-HT2C antagonist, was suspended in a vehicle of 0.5% DMSO in saline in the doses of 0.3, 1, and 3 mg/kg (Yoshimoto et al., 2012). Buspirone (Sigma-Aldrich), a 5-HT1A partial agonist and dopamine D2, D3, D4 antagonist, was dissolved in saline in the doses of 1, 3, and 10 mg/kg (Martin et al., 1992; Rodgers et al., 1997). Drugs were administered intraperitoneally in a volume of 10 ml/kg 20 min before either testing for two-bottle choice consumption or locomotor activity in the open field.

Measured variables and statistical analysis

Statistical analyses were performed on the collected data using GraphPad Prism 9 graphing and statistical software. A mixed model repeated measures analysis of variance (ANOVA) was used to compare two schedules of alcohol drinking, intermittent versus continuous, across time. Mixed model ANOVA was run to analyze the effects of drug dose and schedule on alcohol drinking behavior at 2, 4, and 24 h time points. Drug effects on alcohol consumption (g/kg) and alcohol preference (%) were measured. Alcohol preference is the ratio between 20% ethanol fluid consumed divided by the total fluid consumed. Total distance traveled (cm) and time spent in the center (s) were measured in the open field for 10 min. Holm-Sidak post hoc t-tests were performed to compare drug doses to the vehicle dose, or IA versus CA. Male and female mice were analyzed separately for most comparisons but were directly compared for activity in the open field.

Results

A mixed model repeated measures ANOVA revealed an interaction between schedule of access and time on daily alcohol drinking (g/kg) for adult male C57BL/6J mice, given either intermittent alcohol (IA) or continuous alcohol (CA) [F11, 500 = 3.801, P < 0.0001] in addition to a main effect of schedule [F1, 46 = 27.88, P < 0.0001; Fig. 1A]. Post hoc multiple comparisons indicated the IA males had greater daily g/kg drinking than CA males for Sessions 2, 5, 6, 8, 9, 11, and 12 [Session 2, t = 4.998, P = 0.0001; Session 5, t = 4.333, P = 0.0010; Session 6, t = 4.358, P = 0.0009; Session 8, t = 5.005, P = 0.0001; Session 9, t = 6.530, P < 0.0001; Session 11, t = 4.818, P = 0.0003; Session 12, t = 3.554, P = 0.0122]. Additionally, a mixed model repeated measures ANOVA on daily alcohol drinking showed an interaction between schedule and time in female mice [F11, 505 = 4.057, P < 0.0001] in addition to a main effect of schedule [F1, 46 = 5.116, P = 0.0285; Fig. 1B]. Specifically, Sessions 7, 8, and 10 were different between IA and CA females [Session 7, t = 3.184, P = 0.0294; Session 8, t = 4.746, P = 0.0005; Session 10, t = 3.891, P = 0.0053]. Daily alcohol preference was also different between IA male mice and CA male mice as analyzed by a mixed effects model [interaction schedule × time: F11, 498 = 2.806, P = 0.0015; schedule: F1, 46 = 21.70, P < 0.0001; Fig. 1C]. Sessions 2, 5, 6, 9, and 11 had greater alcohol preference in IA versus CA males [Session 2, t = 4.398, P = 0.0017; Session 5, t = 3.219, P = 0.0309; Session 6, t = 3.024, P = 0.0486; Session 9, t = 4.842, P = 0.0006; Session 11, t = 4.879, P = 0.0003]. In females, daily alcohol preference was also different between IA and CA groups [schedule: F1, 46 = 4.887, P = 0.0321; Fig. 1D]. There were no post hoc differences between schedules for female alcohol preference.

Figure 1.

Figure 1

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Daily alcohol drinking (grams/kilogram/24 h) of two-bottle choice to 20% ethanol or water for adult male (A) or female (B) C57BL/6J mice given either every-other-day intermittent alcohol (IA, filled symbols) or daily continuous alcohol (CA, open symbols); daily alcohol preference in 24 h was also measured over time for male (C) and female (D) mice given IA (filled symbols) or CA (open symbols); N = 24/group. ****P < 0.0001 versus CA; *P < 0.05 versus CA

Alcohol drinking behaviors were monitored after systemic administration of the 5-HT2C receptor antagonist SB242084. Drinking behavior was monitored during the first 2 h and 4 h of two-bottle choice exposure. A two-way repeated measures ANOVA showed an effect of schedule during this initial 2 h period [F1, 22 = 8.306, P = 0.0087; Fig. 2A]. Holm-Sidak multiple comparisons showed that the IA alcohol drinking (g/kg/2 h) was greater than CA at the 0.3 mg/kg dose of SB242084 [t = 4.213, P = 0.0018]. After 4 h two-bottle choice, a mixed-effects analysis showed an interaction between schedule and SB242084 treatment [F3,65 = 3.022, P = 0.0359; Fig. 2B] and an effect of schedule [F1,22 = 16.27, P = 0.0006]. Post hoc comparisons confirmed 3 mg/kg SB242084 specifically reduced alcohol drinking (g/kg/4 h) compared to vehicle in the IA male drinkers [t = 2.990, P = 0.0118]. IA drinking (g/kg/4 h) was greater than CA drinking at the vehicle, 0.3, and 1 mg/kg SB242084 doses [vehicle t = 2.860, P = 0.0158; 0.3 mg/kg t = 4.225, P = 0.0002; 1 mg/kg t = 2.557, P = 0.0244] but not at 3 mg/kg SB242084. Male 24 h alcohol drinking (g/kg) was also affected by SB242084 [F2.641, 57.23 = 3.348, P = 0.0301] and schedule [F1, 22 = 31.94, P < 0.0001; Fig. 2C]. IA males had greater daily consumption than CA males at all doses of SB242084 [vehicle t = 7.581, P < 0.0001; 0.3 mg/kg t = 5.335, P = 0.0001; 1 mg/kg t = 4.103, P = 0.0011; 3 mg/kg t = 2.457, P = 0.0224]. Two-hour alcohol preference was higher for IA males compared to CA males [schedule: F1,22 = 23.81, P < 0.0001; Fig. 2D], specifically at the vehicle and 0.3 mg/kg doses [vehicle t = 3.053, P = 0.0236; 0.3 mg/kg t = 4.603, P = 0008]. Alcohol preference at the 4-h point trended toward an interaction between schedule and drug [F3, 65 = 2.483, P = 0.0686; Fig. 2E] but only an effect of schedule was found [F1, 22 = 26.43, P < 0.0001]. Four-hour alcohol preference was greater in IA versus CA males at the vehicle, 0.3, and 1 doses of SB242084 [vehicle t = 3.078, P = 0.0205; 0.3 mg/kg t = 6.806, P < 0.0001; 1 mg/kg t = 3.037, P = 0.0205]. 24-hour preference was also affected by schedule [F1, 22 = 23.41, P < 0.0001], specifically at the vehicle, 0.3, and 1 mg/kg doses of SB242084 [vehicle t = 4.396, P = 0.0020; 0.3 mg/kg t = 5.036, P = 0.0005; 1 mg/kg t = 3.153, P = 0.0123; Fig. 2F].

Figure 2.

Figure 2

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Alcohol (EtOH) drinking (g/kg) and EtOH preference after systemic administration of SB242084, a serotonin 2C antagonist, across 2-, 4-, and 24-h two-bottle choice offered on an IA (filled symbols) or CA schedule (open symbols); drinking (g/kg) panels are A–C, G–I and accompanying preference panels are D–F, J–L; male behavior is shown in A–F (n = 12/group), and female behavior is G–L (n = 12/group); ##P < 0.01 versus CA; #P < 0.05 versus CA; *P < 0.05 versus vehicle

Female drinking behaviors were also analyzed after systemic administration of SB242084. A two-way repeated measures ANOVA with schedule and drug as factors indicated there was an effect of schedule on 2 h alcohol g/kg consumption [F1,22 = 7.989, P = 0.0098; Fig. 2G]. IA and CA g/kg/2 h were different at the 0.3 mg/kg dose of SB242084 [t = 3.322, P = 0.0134]. Neither SB242084 nor schedule affected 4 h alcohol drinking g/kg in females [Fig. 2H]. SB242084 generally affected female 24 h alcohol drinking (g/kg) with a main effect of drug [F2.815, 61.93 = 4.037, P = 0.0124] and schedule [F1, 22 = 4.375, P = 0.0482; Fig. 2I]. The highest dose of SB242084 reduced female 24 h drinking (g/kg) compared to vehicle [t = 2.981, P = 0.0370] in IA females. An effect of schedule was also present for 2 h alcohol preference, but no drug effects were observed [F1,22 = 6.650, P = 0.0171; Fig. 2J]. Alcohol preference at 4 h was affected by SB242084 [F2.147, 47.22 = 4.075, P = 0.0210; Fig. 2K] and schedule [F1, 22 = 6.524, P = 0.0181]. Specifically, 1 mg/kg SB242084 decreased 4 h alcohol preference compared to vehicle in CA females [t = 3.524, P = 0.0142]. The 24-h preference was also generally affected by drug [F2.568, 56.50 = 3.753, P = 0.0205; Fig. 2L] and schedule [F1, 22 = 5.114, P = 0.0340] with no significant post hoc comparisons.

In separate groups of mice, alcohol drinking behavior was recorded after systemic administration of buspirone, a partial 5-HT1A agonist. A mixed effects analysis revealed that there was an effect of buspirone on initial 2 h alcohol drinking (g/kg) in males [F2.375, 51.45 = 8.143, P = 0.0004; Fig. 3A]. Specifically, alcohol drinking (g/kg/2 h) was suppressed by the 10 mg/kg buspirone dose versus vehicle in the IA males [t = 4.700, P = 0.0019] but not CA males. Results after 4 h were similar to those found at 2 h, where there was a drug effect of buspirone [F2.423, 52.51 = 5.467, P = 0.0044; Fig. 3B]. Post hoc comparisons show that 10 mg/kg buspirone reduced alcohol drinking (g/kg/4 h) compared to vehicle in IA males [t = 3.341, P = 0.0196]. The 24-h alcohol drinking (g/kg) in males was also affected by buspirone [F2.484, 53.82 = 3.229, P = 0.0375] and schedule [F1, 22 = 11.76, P = 0.0024; Fig. 3C]. IA males consumed more daily alcohol (g/kg) than CA males at the 3 and 10 mg/kg doses of buspirone [3 mg/kg t = 3.916, P = 0.0022; 10 mg/kg t = 4.277, P = 0.0012]. Similar to results seen in the SB242084 experiments, there was an effect of schedule for 2 h alcohol preference after buspirone [F1, 22 = 22.68, P < 0.0001; Fig. 3D]. Alcohol preference was greater in IA males compared to CA males at all buspirone doses [vehicle t = 3.618, P = 0.0058; 1 mg/kg t = 2.590, P = 0.0175; 3 mg/kg t = 3.836, P = 0.0058; 10 mg/kg t = 4.156, P = 0.0027]. Alcohol preference after 4 h was not altered by buspirone, but there was an effect of schedule [F1, 22 = 26.09, P < 0.0001; Fig. 3E]. Four-hour alcohol preference was greater in IA males compared to CA males at all doses of buspirone [vehicle t = 4.522, P = 0.0006; 1 mg/kg t = 3.970, P = 0.0017; 3 mg/kg t = 3.862, P = 0.0017; 10 mg/kg t = 5.067, P = 0.0002]. The 24-h preference was also affected by schedule [F1, 22 = 36.43, P < 0.0001; Fig. 3F], but not drug, at all doses of buspirone [vehicle t = 3.950, P = 0.0008; 1 mg/kg t = 4.337, P = 0.0006; 3 mg/kg t = 5.987, P < 0.0001; 10 mg/kg t = 6.702, P < 0.0001].

Figure 3.

Figure 3

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Alcohol (EtOH) drinking (g/kg) and EtOH preference after systemic administration of buspirone, a serotonin 1A agonist, across 2-, 4-, and 24-h two-bottle choice offered on an IA (filled symbols) or CA schedule (open symbols); drinking (g/kg) panels are A–C, G–I, and accompanying preference panels are D–F, J–L; male behavior is shown in A–F (n = 12/group), and female behavior is G–L (n = 12/group); ##P < 0.01 versus CA; #P < 0.05 versus CA; **P < 0.01 versus vehicle

Female drinking behaviors were recorded after systemic buspirone administration. A two-way repeated measures ANOVA with schedule and drug as factors showed an effect of buspirone [F3, 66 = 3.632, P = 0.0172] and schedule on 2-h alcohol g/kg consumption [F1, 22 = 13.37, P = 0.0014; Fig. 3G]. Holm-Sidak comparisons showed no specific doses contributing to these main effects. Neither buspirone nor schedule affected female 4-h alcohol drinking g/kg (Fig. 3H). The 24-h daily drinking g/kg was affected by buspirone [F2.499, 54.99 = 3.284, P = 0.0350; Fig. 3I] and schedule [F1,22 = 12.00, P = 0.0022]. The 24-h drinking was higher in IA than CA mice at the 1 mg/kg buspirone dose [t = 3.148, P = 0.0188]. Schedule also impacted 2-h alcohol preference in the absence of drug effects [F1, 22 = 5.840, P = 0.0244; Fig. 3J]. Alcohol preference at 4 h was affected by buspirone [F2.629, 57.84 = 2.993, P = 0.0444; Fig. 3K] and schedule [F1, 22 = 8.380, P = 0.0084]. Four-hour IA female alcohol preference was greater than CA preference at the 1 mg/kg buspirone dose [t = 3.505, P = 0.0100]. Lastly, there was an effect of schedule, but not drug, on 24-h alcohol preference in females [F1, 22 = 11.83, P = 0.0023; Fig. 3L]. The 24-h IA alcohol preference was higher than CA preference at the vehicle, 1, and 10 mg/kg buspirone doses [vehicle t = 2.562, P = 0.0386; 1 mg/kg t = 3.661, P = 0.0098; 10 mg/kg t = 3.102, P = 0.0195].

After testing for drug effects on alcohol drinking, mice were assessed for drug effects in the open field. A two-way ANOVA revealed an effect of SB242084 [F1, 44 = 8.374, P = 0.0059; Fig. 4A] and an effect of sex on distance traveled in the open field [F1, 44 = 9.040, P = 0.0044]. No significant differences were found within-sex or within-drug comparisons. As a proxy for anxiety-like behavior, time spent in the center zone (s) was also measured, revealing no sex or drug effects after SB242084 (Fig. 4B). In contrast to SB242084, a two-way ANOVA showed a large effect of buspirone on distance traveled [F1, 44 = 548.4, P < 0.0001; Fig. 4C]. Buspirone impaired distance traveled in both male and female mice compared to vehicle [male t = 15.15, P < 0.0001; female t = 17.97, P < 0.0001]. For time spent in the center zone of the open field (s), there was an effect of buspirone treatment [F1, 44 = 8.882, P = 0.0047; Fig. 4D] and sex [F1, 44 = 4.312, P = 0.0437]. Specifically, males spent less time in the center zone after buspirone administration compared to vehicle [t = 3.456, P = 0.0024].

Figure 4.

Figure 4

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Distance traveled (centimeters, A, C) and time spent in the center zone (seconds, B, D) by male and female C57BL/6J mice in an open field for 10 min after systemic administration of either vehicle (open bars) or 3 mg/kg SB242084 (filled bars, A and B) or 10 mg/kg buspirone (filled bars, C and D); N = 12/group; **P < 0.01 versus vehicle; ****P < 0.0001 versus vehicle

Discussion

Male C57BL/6J mice given IA to 20% ethanol showed high drinking behavior and alcohol preference, whereas those given CA to alcohol reduced their consumption. Female C57BL/6J mice given IA also exhibited high levels of alcohol drinking and preference, but the magnitude of difference from continuous availability was less than the difference observed in males. Systemic injections of SB242084 and buspirone reduced 2- and 4-h alcohol drinking in male mice drinking on an intermittent, but not continuous, access schedule. Alcohol drinking in female mice was not largely altered by either 5-HTergic drug. The highest dose of buspirone, however, also impaired locomotor behavior, clouding the interpretation of its alcohol-suppressing effects.

The current studies found that systemic administration of a 5-HT2C antagonist reduced two-bottle choice drinking behavior in male mice that drank alcohol on an intermittent schedule but not continuous schedule. Previous findings suggesting that heavy exposures to alcohol may increase 5-HT1C or 5-HT2C receptor expression in limbic areas, and SB242084 reducing vapor-enhanced drinking or social anxiety-like behavior after alcohol vapor exposure agree with the current findings in male mice (Fu et al., 2020; Marcinkiewcz et al., 2015; Yoshimoto et al., 2012). Expression of ethanol-induced locomotor sensitization can be blocked by intra-nucleus accumbens injection of SB242084 in male Swiss mice (Andrade et al., 2011). This may give insight into how 5-HT2C receptor blockade in episodic presentations of alcohol in males is more effective in reducing drinking compared to CA drinking. It is possible that multiple withdrawal episodes, which may occur during the restricted, IA, protocol, may induce 5-HT2C plasticity to a greater extent than drinking in the CA condition. Indeed, a recent report confirms that intra-lateral habenula infusion of SB242084 suppresses operant self-administration of alcohol after IA in male Long-Evans rats (Fu et al., 2020). Follow up experiments should assess 5-HT2C receptor expression in these limbic areas after various protocols of voluntary drinking in males and females.

Additionally, we found that the largest dose of buspirone reduced intermittent, two-bottle choice drinking behavior in male mice; however, this dose greatly suppressed locomotor behavior in an open arena. Buspirone and other partial 5-HT1A agonists, like tandospirone, have been shown to alleviate anxiety-like behavior during withdrawal from alcohol liquid diet, chronic intermittent ethanol vapor, and 12-week repeated drinking in the dark binge drinking (Overstreet et al., 2003; Lowery-Gionta et al., 2015; Belmer et al., 2018). These reports suggest that these drugs dampen the adaptive changes associated with repeated withdrawal and heavy exposure. Our results imply that 2 weeks of CA drinking in mice may not recruit 5-HT1A neuroplasticity to a similar extent as the intermittent drinking condition. Studies have found increased 5-HT1A receptor binding throughout the brains of rhesus monkeys that consumed long-term alcohol (Hillmer et al., 2014), suggesting that exposure time may be a crucial variable. In addition, drinking after 8 weeks of CA to alcohol and water can be reduced by 20 mg/kg buspirone in male Sprague–Dawley rats but not after fewer weeks (Hedlund and Wahlstrom, 1996). With high doses of buspirone, it is unsurprising that we observed reduced locomotor behavior, as acute treatment of buspirone has prominent anxiolytic effects from 2.5 to 5.0 mg/kg range that are accompanied by general behavioral suppression (Martin et al., 1992; Rodgers et al., 1997). These off-target effects confound the drinking suppression in the male IA mice, although buspirone did not affect drinking in CA male mice. In females, distance traveled was suppressed, but center zone duration in the open field and drinking behavior in both IA and CA mice remained largely unaffected by buspirone.

A more surprising finding in the current studies was the lack of acute treatment effects of both SB242084 and buspirone on female C57BL/6J mice. However, this study is among the first to use both female and male mice, so more research is needed to understand the effects of systemic injections of SB242084 and buspirone on two-bottle choice drinking behaviors of female mice. There are baseline sex differences in 5-HT1 receptor binding in rodent and human brains (Fischette et al., 1983; Jovanovic et al., 2008), which therefore may contribute to sex-linked emotional disturbances. Stress-related sex differences in 5-HT regulation has been characterized (Jones and Lucki, 2005; Goel and Bale, 2010), which may be involved after drinking in females. Since periods of heavy substance misuse and withdrawal are theorized to engage stress neurocircuitry (Koob and Le Moal, 2008), a possible underlying mechanism behind the observed sex differences may be corticotropin-releasing factor and its influence over 5-HT signaling in drinking females (Valentino et al., 2013). For example, intra-dorsal raphe nucleus microinjections of a corticotropin-releasing factor type-1 receptor antagonist reduces IA drinking in male mice (Hwa et al., 2016), but this may be distinct for female mice. Our experiments found that administration of the 5-HT2C antagonist slightly increased intermittent two-bottle choice drinking in female mice but suppressed it in male mice. There is precedent for SB242084 increasing ethanol-motivated responding in both high- and low-drinking male rats (Tomkins et al., 2002). Agonism of the 5-HT2C receptor using lorcaserin has been shown to reduce two-bottle choice alcohol consumption in female Long-Evans rats, IA drinking in male and female Long-Evans rats, and drinking in the dark in C57BL/6J male mice (Rezvani et al., 2014; Tabbara et al., 2021; Fletcher et al., 2022). Since intermittent exposure to ethanol vapor can lead to increased 5-HT2C receptor mRNA editing and expression in the accumbens and dorsal raphe of C57BL/6J male mice (Wantanabe et al., 2014), SB242084 may act by binding to 5-HT2C receptors that have been upregulated following IA drinking. It is also possible that 5-HT2C receptors may play an indirect role in regulating dopamine release in females, as SB242084 has been reported to increase striatal dopamine associated with response vigor of motivated behavior (Bailey et al., 2018). Since the females on both access schedules also show higher levels of drinking than the males, a general downregulation in 5-HT2C receptors could develop in females divergently from the males. Further avenues of investigation could compare lorcaserin to SB242084, as it may be predicted to alter drinking in female mice differently from males.

A limitation of the experimental design is that 12 consecutive drinking days is shorter time compared to 12 sessions across 4 weeks. To further parse out if these neuroadaptations are affected by duration versus schedule, other conditions such as 4 weeks of CA or 2 weeks of IA would be additionally needed. Speculating on length of time for detecting differences between the two models may not be as crucial as the repeated nature of intermittent exposure since as few as 5 days of intermittent ethanol vapor can produce changes in brain 5-HT2C signaling (Marcinkiewcz et al., 2015). Another strategy to differentiate the effects of schedule could be equating approximate cumulative drinking—e.g. administer drug treatment after a goal of 200 g/kg accumulated across either continuous or intermittent drinking sessions. And, as previously discussed, it is likely that these mechanisms underlying schedules of access may be sex dependent.

Conclusion

Ultimately, this research points to differences in treatment efficacy of two 5-HTergic compounds using two different patterns of alcohol drinking in male and female mice. Modeling different patterns of drinking may be useful for distinguishing if an episodic schedule of availability produces 5-HTergic circuitry distinct from everyday drinking, therefore yielding insight into more efficacious medications for treating AUD. Here, we confirm that 5-HT receptors may be promising targets for treating AUD, yet this study only scratches the surface given the wide variety of 5-HT receptor subtypes and the need to distinguish between the functional roles of autoreceptors versus postsynaptic receptors on chronic alcohol consumption and withdrawal-related pathology (Kenna, 2010; Müller et al., 2020). As the prevalence of AUD in females is increasing over time (Grant et al., 2017), it is imperative to understand preventative behavioral strategies in combination with efficacious pharmacology for sex-specific treatment of AUD.

Acknowledgements

The authors would like to thank Anna Lodge and Mariana Dejeux for their assistance in this research and writing.

Contributor Information

Yeasong E Ko, Department of Psychology and Neuroscience, Baylor University, Waco, TX 76798, United States.

Lara S Hwa, Department of Psychology and Neuroscience, Baylor University, Waco, TX 76798, United States.

Conflict of interest

None declared.

Funding

This work was supported by the National Institutes of Health (grant number R00 AA027576), an Honors College Student Research Materials Grant from the Baylor University Honors College, and an Undergraduate Research and Scholarly Achievement Research Grant from the Baylor University Office of Engaged Learning.

Data availability

The data underlying this article will be deposited into an open-source public data repository, BEARdata (http://beardata.baylor.edu), which is part of the Texas Data Repository.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data underlying this article will be deposited into an open-source public data repository, BEARdata (http://beardata.baylor.edu), which is part of the Texas Data Repository.

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