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. Author manuscript; available in PMC: 2023 Mar 1.
Published in final edited form as: Alcohol. 2021 Dec 1;99:1–8. doi: 10.1016/j.alcohol.2021.11.004

Orbitofrontal cortex subregion inhibition during binge-like and aversion-resistant alcohol drinking

Kristen M Schuh 1,†, Elizabeth A Sneddon 1,†, Austin M Nader 1, Marissa A Muench 1, Anna K Radke 1,*
PMCID: PMC8844094  NIHMSID: NIHMS1767306  PMID: 34863917

Abstract

Two important contributors to alcohol-related problems and alcohol use disorder (AUD) are binge- and compulsive-like drinking. The orbitofrontal cortex (OFC), a brain region implicated in outcome valuation and behavioral flexibility, is functionally altered by alcohol exposure. Data from animal models also suggests that both the medial (mOFC) and lateral (lOFC) subregions of the OFC regulate alcohol-related behaviors. The current study was designed to examine the contributions of mOFC and lOFC using a model of binge-like and aversion-resistant ethanol (EtOH) drinking in C57BL/6J male and female mice. The inhibitory Designer Receptor Exclusively Activated by Designer Drugs (DREADD) hM4Di were used to inhibit neurons in either the mOFC or the lOFC in mice drinking 15% EtOH in a two-bottle limited access modified drinking in the dark paradigm. The effects of chemogenetic inhibition on consumption of quinine-adulterated EtOH, water, and water + quinine was also assessed. Inhibiting the mOFC did not alter consumption of EtOH or aversion-resistant drinking of EtOH + quinine. In contrast, inhibition of neurons in the lOFC increased consumption, but not preference, of EtOH alone. mOFC and lOFC inhibition did not alter water or quinine adulterated water intake, indicating the effects shown here are specific to EtOH drinking. These data support the role of the lOFC in regulating alcohol consumption but fail to find a similar role for mOFC.

Keywords: orbitofrontal cortex, drinking in the dark, alcohol, binge drinking, compulsive, aversion-resistant

Graphical Abstract

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Introduction

Alcohol Use Disorder (AUD) continues to be a substantial health problem in the United States with an estimated 15 million adults that have been diagnosed (NIAAA, 2021). Binge drinking of alcohol is an important contributor to alcohol-related problems and costs (NIAAA, 2021). Another key marker of AUD is compulsive drinking, which can be defined as excessive alcohol drinking that persists despite negative consequences (Association and Others, 2013). Given the magnitude of AUD and other alcohol-related problems, it is vital to study the neural mechanisms underlying these types of problem drinking as this knowledge could lead to the development of new and more effective pharmacotherapies.

The orbitofrontal cortex (OFC) is an area of interest in alcohol dependence because its function is altered by alcohol exposure (Cazares et al., 2021; Moorman, 2018; Shields & Gremel, 2020). OFC volumes are lower in patients with AUD (Demirakca et al., 2011; Durazzo et al., 2011; Wang et al., 2016) and such volumetric differences are associated with both heavy drinking (O’Neill et al., 2001) and relapse (Durazzo and Meyerhoff, 2020). In rodents, dendritic spine density increased in the OFC following withdrawal from alcohol (McGuier et al., 2015). Beyond structural changes, alcohol exposure also increases (Radke et al., 2017; Nimitvilai et al., 2017, 2018; Mitchell et al., 2012) and decreases (Volkow & Fowler, 2000; Jin et al., 2012; Mitchell et al., 2012; Nimitvilai et al., 2018) various neurotransmitter systems in the OFC and both promotes and suppresses OFC neuron excitability (Renteria et al., 2018; Gioia and Woodward, 2021; Nimitvilai et al., 2015; Badanich et al., 2013; Cannady et al. 2020). Collectively, these neuroanatomical, neurochemical, and neurophysiological changes are thought to contribute to decision-making deficits (e.g., impaired flexibility and response inhibition) often observed in AUD patients (Lee et al., 2013; Fillmore & Rush, 2006; Vanes et al., 2014) and alcohol-exposed rodents (Badanich et al., 2011; McMurray et al., 2014, 2016; Gass et al., 2014; Rodberg et al., 2017).

OFC activity is also implicated in alcohol-seeking behaviors in rodent self-administration models. This function is consistent with the known role of the OFC in encoding the sensory features and value of expected outcomes, including both rewards and punishments (O’Doherty et al., 2001; Schoenbaum et al., 2011; Becker et al., 2017; Moorman, 2018). For example, both increases and decreases in OFC neuronal activity have been associated with ethanol (EtOH) preference in rats (Hernandez and Moorman, 2020) and increases in neural activity in the OFC have been observed during reinstatement of EtOH seeking (Bianchi et al., 2018). Further the OFC has been shown to play a role in habitual alcohol-seeking (Morisot et al., 2019). Chemogenetic or pharmacological inactivation of the lateral OFC (lOFC) reduces cue-induced reinstatement of EtOH seeking (Hernandez et al., 2020; Arinze and Moorman, 2020) while neurotoxic lesions of the lOFC increases EtOH consumption in rats (Ray et al., 2018). Consistent with the findings regarding alcohol-induced deficits in OFC function discussed above, lOFC regulation of consumption may be specific to animals rendered dependent on EtOH (den Hartog et al., 2016). Further, EtOH-dependent mice with lOFC lesions exhibited less sensitivity to quinine added to an EtOH solution (den Hartog et al., 2016), suggesting this region plays a role in aversion-resistant drinking. In the medial OFC (mOFC), we previously demonstrated that chronic EtOH vapor-induced increases in NMDA-receptor expression and function were associated with greater resistance to footshock punishment during an operant EtOH-seeking task (Radke et al., 2017). Thus, it appears that alcohol-induced changes in both lOFC and mOFC may contribute to high levels of consumption and a tendency to drink despite negative consequences.

Based on these prior findings, we sought to determine whether selective chemogenetic inhibition of the mOFC and lOFC would influence binge-like and aversion-resistant EtOH drinking. We used a twobottle, limited access, “drinking in the dark” (DID) paradigm in C57BL/6J mice (after Sneddon et al., 2019). In this modified DID paradigm, mice are given access to both water and 15% EtOH in the home cage for a period of two hours, three hours into the dark cycle, as mice are most active and are most likely to consume EtOH during this time (Sprow and Thiele, 2012). To study aversion-resistant drinking, quinine, a bitter tastant, was added to the EtOH bottle (Sneddon et al., 2021; Radke et al., 2020; Darevsky et al., 2019; Seif et al., 2015; 2013; Lesscher et al., 2010). To inhibit the mOFC and lOFC, we used the Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) approach (Roth, 2016). Our results suggest that activity in lOFC, but not mOFC, regulates alcohol consumption but that neither subregions participates in aversion-resistant drinking.

Materials and Methods

Subjects

35 C57BL/6J male and female mice were bred from breeding pairs purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed at Miami University in standard shoe box udel polysufone rectangular mouse cages (18.4 cm × 29.2 cm × 12.7 cm). Post-weaning and prior to surgery, mice were group-housed (2–4 mice per cage). Throughout the study, animals resided in a temperature-controlled room and were on a 12:12 hour light/dark cycle (lights on at 7:00 AM) with access to LabDiet 5001 standard food and reverse-osmosis (RO) water provided ad libitum. Animal care followed the guidelines set by the National Institute of Health and the Institutional Animal Care and Use Committee at Miami University approved all procedures.

Drugs

EtOH (15%) was prepared volume/volume in RO water. Quinine hemisulfate (100 μM; Q1250-50G, Millipore-Sigma, St. Louis, MO) was prepared in either 15% EtOH or RO water. All solutions were made fresh daily. Water soluble clozapine-n-oxide (CNO) dihydrochloride (Tocris, Batch no: 2A/218143) was dissolved in 0.9% sterile saline (1.0 mg/kg; Sneddon et al., 2021) and saline served as the vehicle (VEH) solution for control injections. CNO was stored in 1.5 mL centrifuge tubes, in a dark container and kept frozen at −20°C until testing. Approximately one hour prior to testing, CNO was thawed for use. CNO and VEH were delivered in an intraperitoneal (i.p.) injection volume of 10 mL/kg.

Surgery

Surgical procedures were the same as those described in Sneddon et al. (2021). Using isoflurane, mice were anesthetized then were placed into a stereotaxic instrument (model SAS75, Kopf Instruments Inc., Tujunga, CA). To ensure the head was flat, the nose bar was set to 0 mm and then was adjusted accordingly. A midline incision was made over the skull and then bilateral holes were drilled into the skull using the following coordinates: mOFC (n = 20, male = 11, female = 9): AP: +2.35, ML: +/−0.3, DV: −2.5 and lOFC (n = 15, male =10, female = 5): AP: +2.6, ML: +/− 1.2, DV: −2.5, in respect to Bregma. All coordinates were identified using the Franklin & Paxinos Mouse Brain Atlas (Franklin & Paxinos, 2008). The viral vector AAV-hSyn-hM4DGi-mCherry (ID: 50475-AAV8, Addgene, generously provided by Dr. Bryan Roth) was injected using a 32-gauge Hamilton syringe in a volume 0.3 μL per side over the course of 7 min (titer ≥ 7×1012 vg/mL). The virus was allowed 3 min to diffuse before the needle was removed from the surgical site. The needle was sanitized with a sterile EtOH wipe and was allowed to dry before its next use. After the final injection, VetBond tissue adhesive (Santa Cruz Biotechnology, Inc. Dallas, TX) was used to close to surgical site. The home cage was then placed on a heating pad for at least 30 min before it was transported to the colony room. For three days, mice were given standard post-operative care and had access to 50–80 mg/kg/day of ibuprofen available in the drinking water. Recovery time lasted a period of at least one week and mice had at least three weeks between the surgery and the first CNO injection.

EtOH drinking in the dark

EtOH was self-administered in a two-bottle limited access model for two hours a day, five days each week (Monday-Friday) over the course of three weeks (Fig. 1A). One bottle contained 15% EtOH, while the other bottle contained RO water. Bottles were alternated each day to prevent a side bias. Bottles were weighed before and after each two-hour drinking session. Two “dummy” cages were placed on the same rack as the mice to account for spillage or evaporation. To assess if inhibition altered baseline EtOH consumption, mice received a CNO injection 10 min prior to the onset of drinking session 11. Quinine hemisulfate (100 μM) was added to the 15% EtOH on drinking sessions 13–15. A subset of the mice in the mOFC experiment (n = 11, male = 6, female = 5) did not receive a CNO injection on session 11. Mice were randomly assigned to receive an injection of CNO (1.0 mg/kg) or VEH (0.9% saline) in a crossover design on drinking sessions 13 and 15, 10 min before the start of the two-hour drinking session. To habituate subjects to injections, saline injections (0.2 cc/subject) were given to all mice one week prior to drinking. Additional habituation injections were given immediately following the two-hour drinking sessions on Tuesdays and Thursdays during the first two weeks of drinking to ensure that stress effects of the injection would not alter EtOH drinking.

Figure 1. Timelines of the 15% ethanol and water DID paradigms.

Figure 1.

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A) Timeline of the drinking in the dark task. Mice were presented with water or 15% EtOH in their home cage and consumption was measured over a 2-h period for 12 drinking sessions. On session 11, mice received a CNO injection to assess the effects of chemogenetic inhibition on baseline EtOH drinking. On sessions 13 – 15, quinine (100 μM) was added to the EtOH solution. On sessions 13 and 15 mice received either a CNO or VEH injection 10 min prior to the test period. B) Approximately two weeks following the last quinine + EtOH session, mice were given access to two water bottles over a 2-h period for 5 drinking sessions. On sessions 6–10, quinine (100 μM) was added to one of the water bottles. On sessions 2, 4, 7, and 9 mice received either a CNO or VEH injection 10 min prior to testing.

Water drinking in the dark

Next, the effects of chemogenetic inhibition on water consumption and quinine sensitivity were assessed. Two weeks following EtOH drinking, mice were given access to two bottles of RO water for two hours a day for a total of 5 drinking sessions (Fig. 1B). The following week, mice had access to RO water and quinine adulterated (100 μM) water for two hours a day for a total of 5 drinking sessions. During both weeks, on sessions 2 and 4, mice were randomly assigned to receive an injection of CNO or VEH in a crossover design.

Histology

Following the completion of behavioral experiments, mice underwent an intracardiac perfusion. Mice were first anesthetized with ketamine-xylazine (100 mg/kg ketamine + 10 mg/kg xylazine). After anesthetization, animals were intracardially perfused with 0.9% saline followed by a 30% formalin solution (weight/volume). Brains were stored in 30% sucrose formalin in 4°C for a least 48 h before being sectioned. Brains were sectioned with a cryostat into 40 μM sections at −20 °C. VECTASHIELD mounting medium with DAPI counterstain (Vector Labs, Burlingame, CA), was used to coverslip slides. An Olympus AX-70 Wide-field Multi-mode microscope was used to image sections at 4X magnification. Any subjects were removed from analysis if there was no viral expression or if the regions of interest were missed. Adobe Photoshop, GNU Image Manipulation Program (GIMP) 2.10, BioRender, and GraphPad Prism software were used to create images.

Data Analysis

Bottle weights were expressed in grams of EtOH or mL of water consumed per kilogram of body weight for each mouse and was then averaged across groups. Consumption was calculated as =(Initial Bottle Weight − Final Bottle Weight) − Average Dummy Bottle Weights. Preference was calculated as =(Volume of EtOH/(Volume of EtOH + Water Consumption))*100.

A priori, sex differences were not expected, thus groups were not powered (n = 14; Sneddon et al., 2019) to assess sex differences. Data were first analyzed with sex as a factor, but no sex differences emerged. Therefore, data were collapsed and analyses between sex were not reported. Any data from bottles spilling or measurement error were excluded from all analyses. In these cases, data from drinking session 1–10 were analyzed using a Mixed Effects analysis of variance (ANOVA). Analyses can be interpreted similar to an RM ANOVA in the presence of random missing values. In cases where the assumption of sphericity was violated (ε < 0.75), the Greenhouse-Geisser correction was applied. Post-hoc Dunnett’s tests were used to assess changes in consumption or preference compared to the first drinking session. Outliers were identified by using Grubbs’ test with α = 0.05 as the criteria.

To analyze consumption and preference on baseline drinking sessions (i.e., sessions 10, 11, and 12) and aversion-resistant drinking sessions (i.e., sessions 13 and 15) RM ANOVAs were used. Post hoc Dunnett’s test comparing daily consumption to the first drinking session were used to assess escalation of intake and preference. For aversion resistant sessions, a RM ANOVA was also used to assess whether the order of VEH/CNO presentation affected drinking. Because the order of VEH/CNO presentation did not affect the results, data were collapsed for visualization and analysis by paired t-test. To demonstrate aversion-resistance, consumption on session 10 was compared to the quinine session on which VEH was injected using a paired t-test. The threshold of significance for all statistical test was (α = 0.05). All analyses were conducted using GraphPad Prism 9.0.2 (La Jolla, CA).

Results

Inhibition of the mOFC does not influence consumption of EtOH or EtOH + quinine

To assess the contribution of neurons in the mOFC to binge-like and aversion-resistant EtOH intake, chemogenetic inhibition with the designer receptor hM4Di was utilized. All mice were injected with AAV-hSyn-hM4DGi-mCherry as we have previously found that CNO alone does not influence the behavior of C57BL/6J mice in the modified DID task (Sneddon et al., 2021). mCherry viral expression was observed in cell bodies in the mOFC. The average range of expression along the anterior-posterior axis was 1.07 mm (Fig. 2A). All mice (n = 20, male = 11, female = 9) showed consistent consumption of 15% EtOH throughout drinking sessions 1–10. A Mixed Effects ANOVA revealed no significant effect of session (F(3.668, 68.871) = 1.880, p = 0.129) (Fig. 2B). Mice also consistently preferred 15% EtOH vs. water. A Mixed Effects ANOVA uncovered a significant main effect of session (F(5.881, 104.795) = 4.346, p < 0.001). A post hoc Dunnett’s test showed that sessions 6 (p = 0.047), 7 (p = 0.001), 8 (p = 0.009), 11 (p = 0.007), and 12 (p = 0.025) were significantly higher vs. session 1, suggesting that preference for EtOH escalated over the course of the experiment (data not shown).

Figure 2. Inhibition of the mOFC did not influence binge-like or aversion-resistant ethanol consumption.

Figure 2.

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A) Visual representation of hM4Di DREADD expression and spread in neurons of the mOFC. B) Mice consistently consumed 15% EtOH across drinking sessions. No differences in C) 15% EtOH consumption or D) preference were observed when the mOFC was inhibited. No differences were observed when the mOFC was inhibited on E) 15% EtOH + quinine consumption, F) preference, G) water consumption, or H) quinine + water consumption.

To assess the effects of mOFC inhibition on EtOH intake, CNO was injected on session 11 in a subset of mice (n = 8, male = 4, female = 4). One subject was identified as an outlier (Grubbs’ test, α = 0.05) and was thus removed from the analyses. RM ANOVAs revealed no significant differences in consumption (F(1.531,10.720) = 0.571, p = 0.537) (Fig. 2C) or preference (F(1.423,9.963) = 2.100, p = 0.178) (Fig. 2D) when analyzing consumption across sessions 10, 11, and 12 (no injection on session 10 and 12). A RM ANOVA also showed that water consumption was not altered when the mOFC was inhibited on session 11 compared to sessions 10 and 12 (F(1.080,8.643) = 2.143, p = 0.179) (data not shown). Additional analyses were conducted to assess if there were differences between mice receiving CNO on session 11 (n = 8, male = 4, female = 4) vs. those that did not (n = 11, male = 6, female = 5). Unpaired t-tests found no significant differences in EtOH consumption (t(18) = 0.810, p = 0.429), preference (t(18) = 1.091, p = 0.290), or water consumption (t(18) = 1.557, p = 0.137) between these subsets of mice (data not shown).

Addition of 100-μM quinine to the EtOH bottle did not significantly change consumption, as evidence by a paired t-test between session 10 and the VEH session (t(20) = 1.480, p = 0.155), suggesting drinking was aversion-resistant. Because consumption on session 10 was unusually high in one mouse (~20 g/kg), this analysis was also performed with that animal removed (session 10 = 1.69 ± 0.54 g/kg; VEH session = 1.03 ± 0.28 g/kg) (t(19) = 1.167, p = 0.258). Further, a paired t-test between VEH and CNO sessions found that mOFC inhibition did not change consumption of quinine-adulterated EtOH (t(19) = 0.100, p = 0.921) (Fig. 2E), preference (t(19) = 0.668, p = 0.512) (Fig. 2F), or water consumption (t(19) = 0.442, p = 0.663) (data not shown).

Two weeks following EtOH exposure, mice underwent a quinine sensitivity test to assess the effects of mOFC inhibition on water and quinine + water intake. A paired t-test showed that mOFC inhibition in a subset of mice (n = 9, male = 5, female = 4) did not alter water intake alone (t(8) = 0.915, p = 0.387) (Fig. 2G) or intake of quinine-adulterated water (t(8) = 0.985, p = 0.354) (Fig. 2H).

Inhibition of the lOFC increases EtOH consumption

To investigate the contribution of neurons in the lOFC to binge-like and aversion-resistant EtOH intake, chemogenetic inhibition with the designer receptor hM4Di was utilized. mCherry viral expression was observed in cell bodies in the lOFC. The average range of expression along the anterior-posterior axis was 0.57 mm (Fig. 3A). All mice (n = 15, male = 10, female = 5) consistently consumed 15% EtOH across drinking sessions 1–10. A Mixed Effects ANOVA revealed no significant effect of session (F(4.135, 57.425) = 2.247, p = 0.073) (Fig. 3B). Mice consistently preferred 15% EtOH vs. water. A Mixed Effects ANOVA discovered no significant differences between session (F(3.945, 54.867) = 2.159, p = 0.087).

Figure 3. Inhibition of the lOFC increased binge-like ethanol consumption.

Figure 3.

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A) Visual representation of hM4Di DREADD expression and spread in neurons of the lOFC. B) Mice consistently consumed 15% EtOH across drinking sessions. When the lOFC was inhibited, there was an increase in C) consumption of 15% EtOH, but not D) preference. No differences were observed when the lOFC was inhibited on E) 15% EtOH + quinine consumption, F) preference, G) water consumption, or H) quinine + water consumption. * p = 0.012 (Paired t-test).

To assess the effects of lOFC inhibition on EtOH intake, CNO was injected on session 11. RM ANOVAs identified a significant difference in consumption when mice received a CNO injection on session 11 vs. sessions 10 and 12 (F(1.717,23.900) = 5.968, p = 0.010) (Fig. 3C) but no difference in preference (F(1.948,27.278) = 2.676, p = 0.089) (Fig. 3D). Post hoc Dunnett’s tests found that consumption on session 11 was significantly increased compared to session 12 (p = 0.022) but did not reach significance compared to session 10 (p = 0.075) (Fig. 3C). A RM ANOVA also revealed that water consumption was not altered when the lOFC was inhibited on session 11 compared to sessions 10 and 12 (F(1.810, 25.346) = 1.099, p = 0.343) (data not shown).

Evidence for aversion-resistance was found with a paired t-test demonstrating no significant differences when comparing session 10 to the VEH session (t(14) = 1.526, p = 0.149) (data not shown). Further, a paired t-test found that lOFC inhibition did not change consumption of quinine-adulterated EtOH (t(14) = 1.536, p = 0.147) (Fig. 3E), preference (t(14) = 0.993, p = 0.338) (Fig. 3F), or water consumption (t(14) = 0.949, p = 0.358) (data not shown).

Two weeks following EtOH exposure, mice underwent a quinine sensitivity test. A paired t-test showed that lOFC inhibition did not alter water intake alone (t(14) = 0.091, p = 0.929) (Fig. 3G) or intake of quinine-adulterated water (t(14) = 1.585, p = 0.132) (Fig. 3H).

Discussion

Here, we investigated the effects of mOFC and lOFC inhibition in a model of binge-like and aversion-resistant EtOH drinking. First, we found that mOFC inhibition did not alter consumption of 15% EtOH or quinine-adulterated 15% EtOH. We also verified that mOFC inhibition did not alter water consumption or quinine sensitivity. Interestingly, we discovered that inhibition of the lOFC increased consumption of 15% EtOH but not aversion-resistant intake of quinine-adulterated EtOH. The observed effect was specific to EtOH as inhibition of neither the lOFC or mOFC altered water consumption or sensitivity to quinine, as previously shown (Ramírez-Lugo et al., 2016). This effect is unlikely to be due to the effects of CNO alone, as we have previously reported no effect of CNO in the modified DID task (Sneddon et al., 2021). We did not find any differences in preference for 15% EtOH or quinine-adulterated EtOH when the mOFC and lOFC were inhibited (although preference for 15% EtOH almost met the threshold of significance in the mice with lOFC inhibition). These results show that the mOFC and lOFC function differently in regards to EtOH intake, which has been shown with other alcohol-related behaviors including cue-induced reinstatement of EtOH-seeking (Arinze and Moorman, 2020).

In a previous study, we uncovered an increase in NMDAR subunit expression and enhanced NMDAR-mediated currents in the mOFC of mice exposed to chronic EtOH vapor (Radke et al., 2017). This effect was associated with increases in resistance to an aversive stimulus (footshock) in an operant EtOH-seeking task in male mice (Radke et al., 2017). Although we observed an increase in NMDAR expression and function and aversion resistance in the same animals, a causal effect was not established in that prior study. As such, a primary motivation for the current work was to test whether manipulation of mOFC produces changes in aversion resistance during EtOH drinking. Surprisingly, we found that mOFC inhibition did not alter consumption of 15% EtOH at baseline or when quinine was added to the EtOH solution, with the latter finding suggesting that mOFC does not participate in aversion-resistant drinking. There are a few notable differences between our previous work and the current study, including the EtOH drinking paradigm (operant responding vs. home cage DID) and the punishment (footshock with lever press vs. quinine in EtOH) used, that could explain the lack of mOFC involvement seen here. Further, our prior study observed increases in aversion-resistance and NMDAR function two weeks following exposure to chronic intermittent EtOH vapor (Radke et al., 2017), raising the possibility that mOFC involvement in aversion-resistant drinking emerges only following dependent-inducing levels of EtOH exposure or after an extended withdrawal period. It is important to note that mice in the current study drank less EtOH (~1–2 g/kg) than is often observed using the DID model (e.g., Rhodes et al., 2005; Sneddon et al., 2019; Bauer et al., 2021) and we did not confirm blood EtOH levels. Additional studies are needed, perhaps using chronic vapor exposure, to confirm a role of the mOFC in compulsive-like alcohol drinking behaviors.

Our major finding regarding the lOFC is that chemogenetic inhibition increased 15% EtOH consumption, which agrees with some prior studies in rodents. Chemogenetic inhibition or neurotoxic lesions of the lOFC increased EtOH consumption after chronic vapor exposure in mice (den Hartog et al., 2016) and lOFC lesions significantly increased EtOH consumption using a voluntary, intermittent access paradigm in rats (Ray et al., 2018). Others have found that chemogenetic inhibition of lOFC did not alter 10% and 20% EtOH or sucrose consumption in a home-cage paradigm and had no effect on responding for EtOH in a fixed-ratio (FR) 1 operant paradigm (Hernandez et al., 2020). Another study found that pharmacological inactivation of the mOFC and lOFC with baclofen/muscimol also did not alter EtOH seeking in a FR3 operant paradigm (Arinze and Moorman, 2020). Hernandez and colleagues (2020) have recently speculated that these discrepancies may reflect whether animals are drinking in a goal-directed manner, noting that exposure to chronic vapor (den Hartog et al., 2016) or footshock (Ray et al., 2018) may promote EtOH consumption that is more habitual than intermittent access or operant response paradigms do alone. Considering that the DID paradigm promotes high levels of EtOH consumption and the development of aversion-resistant drinking in the alcohol-preferring C57BL/6J line (Lesscher et al., 2010; Lei et al., 2016; Sneddon et al., 2019; Bauer et al., 2021), our results appear to also support this idea. Under this framework, lOFC control of EtOH drinking would be weaker in paradigms that promote dependence and habitual consumption, making behavior more susceptible to neuronal inhibition in this region. The mechanisms underlying these effects may involve EtOH-induced activation of mTORC1 via GluN2bcontaining NMDA receptors, blockade of which has been shown to reduce responding for EtOH and restore goal-directed behavior in rats responding habitually for EtOH (Morisot et al., 2019). Others have shown that alcohol-related behaviors are modulated by monoamines (Nimitvilai et al., 2017b) and glycine receptor dependent processes (Badanich et al., 2013) in the lOFC. An interesting future avenue for this research would be to see if these mechanisms also contribute to escalations in EtOH drinking.

Our findings add to the literature by demonstrating that lOFC inhibition increases EtOH intake and may increase preference for EtOH in a DID model. We further found that lOFC inhibition did not increase consumption during EtOH drinking sessions when quinine was added to the solution. This finding contradicts one prior study, which found that lesions of the lOFC produced an increase in quinine-resistant EtOH drinking using a 24-h intermittent access model following chronic vapor EtOH exposure (den Hartog et al., 2016). Here again, one possibility is that mice in the current study did not reach a sufficient level of dependence to recruit OFC involvement in aversion-resistant drinking. Another important consideration is that mice in our lOFC experiment were already resistant to quinine aversion at the time of inhibition (i.e., quinine did not reduce consumption compared to baseline). Whereas, in the study by den Hartog and colleagues (2016), the control mice remained sensitive to quinine, reducing consumption on quinine sessions by ~40%. Thus, lOFC inhibition may be able to induce resistance to punishment but not increase it further in mice that are already aversion resistant. It would be useful for future studies to test whether lOFC and mOFC inhibition increases aversion-resistant drinking using higher quinine concentrations that are known to reduce EtOH consumption in the DID paradigm (Sneddon et al., 2019).

It is important to note that sex differences have been observed in lOFC activity in dependent mice following chronic vapor exposure (Nimitvilai et al., 2020; Nimitvilai et al., 2018). Further, female rodents are known to consume higher levels of EtOH compared to males across a range of drinking paradigms (Radke et al., 2021a; Radke et al., 2021b; Finn, 2020). Although we and others have demonstrated that males and females do not differ in aversion-resistant EtOH intake in the DID paradigm (Bauer et al., 2021; Sneddon et al., 2019), females do show greater vulnerability to aversion-resistant drinking using other voluntary drinking paradigms (Radke et al., 2020; Sneddon et al., 2020; Fulenwider et al., 2019; Xie et al., 2019). In the current study, both male and female mice drank robustly but no differences were observed between the sexes at any point during the study. Thus, while it is possible that sex differences in EtOH consumption and aversion-resistant drinking would emerge following chronic EtOH vapor exposure or in a different EtOH drinking paradigm, our current results suggest that OFC subregions do not contribute to female vulnerability to EtOH consumption or aversion-resistant drinking.

There are some notable limitations to the current study. First, although we did assess the effect of inhibition of the mOFC and lOFC on baseline EtOH drinking, we did not investigate if the injection of the vehicle solution (0.9% saline) altered EtOH consumption at baseline. To avoid effects of injection stress, all mice were given habituation injections. In both the current study and our prior work using the DID paradigm (Sneddon et al., 2021), we have failed to find an influence of injections on consumption. For example, vehicle injection did not influence aversion-resistant drinking in either experiment. Still, we cannot rule out the possibility that the observed effect of lOFC inhibition was influenced by comparison to a session on which no injection was given. This study also did not assess how OFC involvement in drinking behaviors is influenced by alcohol exposure, a variable that is known to influence aversion-resistant drinking (Radke et al., 2017; Radke et al., 2020; Hopf et al., 2010) and OFC function (den Hartog et al., 2016). In addition, the OFC consists of a diverse population of neurons that are GABAergic and glutamatergic (Jin et al., 2014; 2012), both of which were inhibited non-specifically using the current chemogenetic approach. It is quite possible the inhibition of GABA- or glutamatergic neurons specifically may induce changes in alcohol intake as decreases in GABA receptors (Jin et al., 2012) and increases in glutamate receptors (Jin et al., 2014) have been observed following alcohol exposure. Future studies may be able to work out the conditions required for OFC control of alcohol drinking behaviors more clearly by varying factors, such as exposure history and specific neuron type, that contribute to the development of dependence.

In conclusion, inhibition of the lOFC but not the mOFC increased EtOH drinking while neither region altered aversion-resistant EtOH intake. These results confirm the role of lOFC in alcohol-drinking behaviors and raise important questions for future work examining the role of the OFC in compulsive-like drinking despite negative consequences.

Highlights.

  • mOFC and lOFC chemogenetically inhibited during binge- and quinine-resistant drinking

  • Chemogenetic inhibition of the mOFC did not alter consumption of 15% EtOH

  • Chemogenetic inhibition of the mOFC did not alter quinine-resistant EtOH intake

  • Chemogenetic inhibition of the lOFC increased consumption of 15% EtOH

  • Chemogenetic inhibition of the lOFC did not alter quinine-resistant EtOH intake

Acknowledgements:

The authors would like to thank Brandon Arnold, Bridget Dames, Kaila Fennell, and Natalie Shand for assistance with behavioral experiments.

Funding Sources:

This work was supported by the National Institutes of Health (AA027915 to AKR and NS118727 to EAS) and awards from the Office of Research for Undergraduates (KMS and AMN) at Miami University.

Abbreviations:

AUD

Alcohol use disorder

EtOH

ethanol

OFC

orbitofrontal cortex

mOFC

medial orbitofrontal cortex

lOFC

lateral orbitofrontal cortex

DREADDs

Designer Receptors Exclusively Activated by Designer Drugs

CNO

clozapine-N-oxide

VEH

vehicle

DID

drinking in the dark

Footnotes

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Declaration of interest: The authors declare no conflicting interests in this work.

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