Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2022 Apr 1.
Published in final edited form as: Psychopharmacology (Berl). 2021 Jan 8;238(4):1099–1109. doi: 10.1007/s00213-020-05757-9

Blockade of alcohol excessive and “relapse” drinking in male mice by pharmacological cryptochrome (CRY) activation

Yan Zhou 1,*, Mary Jeanne Kreek 1
PMCID: PMC7969462  NIHMSID: NIHMS1665282  PMID: 33420591

Abstract

Rationale:

Metabolic dysfunction, mood disorders, anxiety disorders, and substance abuse disorders are associated with disruptions in circadian rhythm and circadian clock gene machinery. While the effects of alcohol on several core components of the clock genes have been described in rodent models, pharmacological activation or inhibition of clock gene functions has not been studied on alcohol drinking behaviors.

Objectives:

We investigated whether cryptochrome (CRY1/2) activator KL001 altered alcohol intake in mice in excessive and relapse-like alcohol drinking models.

Methods:

Mice, subjected to 3 weeks of chronic intermittent alcohol drinking (two-bottle choice, 24-h access every other day) (IAD) developed excessive alcohol intake and high preference. We evaluated the pharmacological effects of KL001 after either 1-day acute withdrawal from IAD or 1-week chronic withdrawal using the alcohol deprivation effect (ADE) model.

Results:

Single pretreatment with KL001 at 1–4 mg/kg reduced alcohol intake and preference after acute withdrawal in a dose-related manner. The effect of KL001 on reducing excessive alcohol consumption seems alcohol-specific, as the compound does not alter sucrose (caloric reinforcer) or saccharin (non-caloric reinforcer) consumption in mice. Both single- and multiple- dosing regimens with an effective dose of KL001 (4 mg/kg) prevented the ADE after chronic withdrawal, with no tolerance development after the multi-dosing regimen.

Conclusions:

Pre-treatment with KL001 (a CRY1/2 activator) reduces excessive and “relapse” alcohol drinking in mice. Our in vivo results with a CRY activator suggest a possible novel target for alcohol treatment intervention.

Keywords: circadian clock gene, CRY activator, KL001, excessive alcohol drinking, alcohol deprivation effect

Introduction

Sleep is profoundly disrupted after excessive alcohol consumption, and disruption of circadian clock machinery may enhance the risk of alcohol disorder and use [Koob and Colrain 2020]. Circadian clocks depend on on a highly regulated system of transcriptional and translational feedback loops that coordinate clock-controlled gene expression, driving circadian patterns of behavior [Patke et al. 2020]. Among these clock genes are Cryptochrome (Cry) family members, including Cry1 and Cry2. CRY1 and CRY2, with Periods (encoded by Per1, Per2 and Per3), are part of a repressor complex that interacts with core transcriptional activators (CLOCK and BMAL1), and act as negative regulators of the circadian clock system to inhibit their own and other transcriptions. In mice, CRY1 with CRY2 in the suprachiasmatic nucleus (SCN) is involved in the generation of circadian rhythms, with minimal expression levels in the dark and with peak levels during the light phase [Miyamoto and Sancar 1998; Edwards et al 2016]. Further studies have found that CRY dysregulation is related to food intake, diabetes, obesity, anxiety-like and depression-like behaviors in rodents [Takahashi et al. 2008; Zhang et al. 2010; Hirota et al. 2012; Barclay et al. 2013; De Bundel et al. 2013; Griebel et al. 2014; Humphries et al. 2016; Hühne et al. 2020; Porcu et al. 2020], and probably to a variety of diseases in humans, including sleep phase disorder, major depressive disorder and anxiety [Patke et al. 2017; Onat et al. 2020].

In both humans and laboratory animals, genetic and neurobiological studies have provided supportive results, demonstrating that among multiple actions of alcohol in the CNS, excessive alcohol use can lead to long-lasting circadian desynchronization with altered expression of various core components of the clock genes, which could be involved in the positive or negative reinforcing aspects of alcohol addiction [Partonen 2015; Koob and Colrain 2020]. Early studies found that mice lacking clock gene Per2 or D-box binding protein show increased alcohol intake and preference [Spanagel et al. 2005; Le-Niculescu et al. 2008]. Alcohol exposure in mice during early postnatal days or in adult rats alters some core components of the clock genes (Cry1, Per2) in the SCN, cerebellum and liver [Farnell et al. 2008; Filiano et al. 2013]. Together, these findings have suggested that the clock genes are possibly involved in the developments of alcohol abuse and excessive consumption.

Withdrawal from alcohol is associated with increased stress responsivity and persistent negative affective symptoms, such as anxiety and depression, the severity of which is associated with excessive alcohol consumption or relapse susceptibility [Koob and Kreek 2007; Zhou and Kreek 2018]. Beyond the role in the control of circadian rhythm, Cry genes have a direct influence in anxiety-related behaviors and cognitive function in rodents. Specifically, the deletion of Cry1 or of both Cry1 and Cry2 is associated with anxiety-like behaviors in mice [De Bundel et al. 2013; Hühne et al. 2020]. As such, we hypothesized that desynchronized or impaired CRY activity may contribute to the negative affective states and increased stress responsivity that underlie negative reinforcement mechanisms driving alcohol drinking in withdrawal and that contribute to alcohol relapse following periods of abstinence.

KL001 is a cell-permeable carbazolic compound that directly interacts with and stabilizes CRY1 and CRY2, preventing ubiquitin-dependent degradation and functioning as a CRY activator [Hirota et al. 2012]. However, there is no study using CRY activators in rodent models on alcohol consumption. In this study, therefore, we investigated whether the CRY activator KL001 could alter excessive alcohol intake in mice during acute (1 day) withdrawal from chronic (3-week) intermittent alcohol drinking (IAD), to explore its potential for development as a therapeutic agent for alcohol abuse. In the IAD model, after the mice had access to alcohol drinking for 3 weeks [two-bottle choice, alcohol drinking every other day], they displayed excessive alcohol consumption (~ 15–20 g/kg/day). Also, the IAD mice constituted an appropriate mouse model for studying alcohol “relapse”, as after chronic (1-week) withdrawal, they developed the alcohol deprivation effect (ADE, modeling the relapse episodes that occur in human alcoholics) [Vengeliene et al. 2014; Zhou et al. 2017]. Consequently, we further determined the pharmacological effects of KL001 in this ADE model, to evaluate the therapeutic potential of CRY activators in treating alcoholism.

MATERIAL AND METHODS

1. Animals.

Male adult C57BL/6J (B6) mice (8 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and group housed in ventilated cages fitted with steel lids and filter tops in a temperature-controlled room (21 °C). Mice were placed on a 12-hour reverse light-dark cycle with lights off at 7:00 am (zeitgeber time [ZT]12) upon arrival, acclimated for one week prior to experiments, and given ad libitum access to food and water. Animal care and experimental procedures were conducted according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences 1996) and were approved by the Institutional Animal Care and Use Committee of the Rockefeller University.

2. Materials.

KL001 (Tocris Inc., Bristol, UK) were dissolved in 1% DMSO plus physiological saline. Alcohol solutions (15% or 30% v/v) were prepared from 190 proof absolute ethyl alcohol (Pharmco-AAPER, Brookfield, CT, USA) and dissolved in tap water. Sucrose and saccharin (Sigma-Aldrich Inc., St. Louis, MO, USA) were diluted in tap water.

3. Procedures.

3.1. Chronic Intermittent Alcohol Drinking (IAD).

In C57BL/6J mice, this model has been used for many years in our and other laboratories [Hwa et al. 2011; Zhou et al. 2017]. Mice had access to alcohol drinking in their home cages for 3 weeks in this two-bottle choice paradigm with alcohol drinking every other day. At the time when the mice started individual housing (1 week before the experiments), water bottles were exchanged for those with sipper tubes to acclimate the mice to the sipper tubes (without ball bearings). Starting at 3 hours after lights off (10:00 am, ZT15), both water and alcohol (15%) sipper tubes were placed on their home cages. These sipper tubes were 10-ml pipettes that were cut at both ends, sealed with a rubber stopper, and fitted with a stainless-steel straight sipper tubing with a ball bearing at the end to prevent alcohol leakage. The left / right position of the water and alcohol tubes was randomly set to avoid any development of side preference. The alcohol tubes were refilled with fresh alcohol solution and left for 24 hours before replaced with the water tubes. By mixing alcohol with tap water to reach the appropriate (v /v) alcohol concentration (15 or 30%), alcohol solutions were prepared fresh every 48 hours. Alcohol and water intake values (to the nearest 0.1 ml) were recorded after 0, 4, 8 and 24 hours of alcohol access (ZT15, ZT19, ZT23 and ZT15, respectively) on the drinking days, and used to calculate consumed alcohol intake (i.e., g / kg) and relative preference for alcohol (alcohol intake /alcohol intake + water intake). After 3 weeks of IAD with 15% alcohol, mice rapidly acquired alcohol drinking behaviour and subsequently daily alcohol intake reached to about 15 g/kg/day (Table S2), with a high preference ratio above 0.8 at zeitgeber time [ZT]15–19 (Figure 2). As reported before, this 3-week voluntary IAD paradigm was found to give rise to blood alcohol concentrations (BAC) around 0.50 – 0.63 mg/ml, which were within the range required to produce specific psycho-pharmacological effects of alcohol [Zhou et al. 2017].

Figure 2. Effects of single administration of KL001 (4 mg/kg) at zeitgeber time [ZT]15 on alcohol intake (A), water (B), preference ratio (C) and total fluid (D) in mice after 1 day of withdrawal from 3-week chronic intermittent alcohol drinking.

Figure 2

Open in a new tab

(1) Control group: mice (n=10) received one vehicle injection (i.p.) before the drinking test; and (2) KL001 group: mice (n=9) received one KL001 injection (4 mg/kg, i.p.) 30 min before the drinking session. Alcohol (15%) and water intake values were recorded after 0, 4, 8 and 24 hours of alcohol access (Starting at 3 hours after lights off) (zeitgeber time [ZT]15). *p<0.05 or **p<0.01 vs. control at the same time point. Individual values are presented as dot plots with mean ± range.

In the following experiments, mice were randomized as the vehicle (1% DMSO plus physiological saline)-treated and the drug (KL001)-treated groups had matched body weight and similar alcohol intake 1 day before the test day. The drug dissolved in the vehicle was administered by an experimenter, blinded to the treatments given to the experimental groups.

3.1.1. Single administration after 0 hour of alcohol access at ZT15.

After 1 day of withdrawal, at ZT15, alcohol (15% concentration) was presented 30 min after a single injection of KL001 (1, 2 or 4 mg/kg in 1% DMSO, i.p.) or vehicle, and then alcohol and water intake values were recorded after alcohol access at 0, 4, 8 and 24 hours (ZT15, ZT19, ZT23 and ZT15, respectively). The KL001 doses chosen were based on our pilot study.

3.1.2. Single administration after 4 hours of alcohol access at ZT19.

Similar study was conducted, except that single injection of KL001 with 4 mg/kg (the most effective dose at ZT15) or vehicle was given at ZT19 (4 hours after alcohol access). Alcohol and water intake values were recorded after KL001 injection at 0, 4 and 20 hours (ZT19, ZT23 and ZT15, respectively).

3.2. Sucrose (caloric reinforcer) and saccharin (non-caloric reinforcer) drinking.

With 4 mg/kg KL001 (the most effective dose tested for reducing alcohol intake) at ZT15, we further tested the specificity of the action of KL001 on alcohol using sucrose or saccharin drinking behavior following the IAD or after single administration in alcohol naïve mice. In the following experiments, 15% alcohol IAD exposure was identical to those in the above experiment as described in section 3.1. After 3 weeks of IAD, the alcohol tube was switched to sucrose for 3 sessions with stable intakes. The mice assigned to the vehicle-treated or KL001-treated groups had similar sucrose intake 24 hours before the test day. On the test day, sucrose (2%) and water intake values were recorded after 0, 4, 8 and 24 hours of sucrose access (ZT15, ZT19, ZT23 and ZT15, respectively). In parallel separate experiments, saccharin drinking (0.1%) was tested after 3 weeks of IAD with an identical procedure.

3.2.1. Single administration of KL001 at ZT15 on sucrose or saccharin drinking after 3-week IAD.

A single i.p. injection of KL001 (4 mg/kg) in 1% DMSO or vehicle was given 30 min before the sucrose or saccharin were presented at ZT15. Mice were assigned to one of two treatment groups: vehicle as control and KL001.

3.2.2. Single administration of KL001 at ZT15 on sucrose or saccharin drinking in alcohol-naïve mice.

The procedures were identical to the above, except the mice were exposed to sucrose or saccharin only (after 3 training sessions).

3.3. The alcohol deprivation effect (ADE) after 1-week withdrawal from 3-week IAD.

In C57BL/6J mice, this model has been used for many years in our laboratory [Zhou et al. 2017] (Table S1). Mice had 24-h access to alcohol vs water on alternating days for 3 weeks. In the baseline session, 15% alcohol and water intake values were recorded at 0, 4, 8 and 24 hours (ZT15, ZT19, ZT23 and ZT15, respectively) during the 3rd week of IAD. The alcohol bottles were then removed from the cages, and after 7 days of withdrawal, the alcohol (30%) bottles were presented 3 hours after the dark cycle at ZT15 and the alcohol and water intake values were recorded at 0, 4, 8 and 24 hours (ZT15, ZT19, ZT23 and ZT15, respectively) in the ADE session.

3.3.1. Single administration of KL001 in the ADE model.

The mice assigned to the vehicle-treated or KL001-treated groups had similar alcohol intake in the baseline session. Control group: mice received one vehicle injection (1% DMSO, i.p.) before the ADE test; and KL001 group: mice received one KL001 injection (2 or 4 mg/kg in 1% DMSO, i.p.) before the ADE test at ZT15. Then, alcohol was presented 30 min after KL001 or vehicle injection, and alcohol and water intake values were recorded after 0, 4, 8 and 24 hours of alcohol access (ZT15, ZT19, ZT23 and ZT15, respectively). The KL001 doses chosen (2–4 mg/kg) were based on the above IAD experiments.

3.3.2. Repeated administration of KL001 in the ADE model.

In the following experiments, alcohol exposure procedure was identical to the above experiment in section 3.3.1, with the following exceptions: mice received 5 consecutive daily administrations of vehicle or KL001 (4 mg/kg) at ZT15 during the 1-week withdrawal. On the ADE test day, alcohol was presented at ZT15 1 day after the last KL001 injection, and then alcohol and water intake values were recorded after 0, 4, 8 and 24 hours of alcohol access (ZT15, ZT19, ZT23 and ZT15, respectively).

3.4. Single administration of KL001 on locomotor activity.

Male alcohol-naïve mice were injected with KL001 (4 mg/kg, i.p.) or vehicle. At 10:00 am (ZT15) 30 mins after the injection, mice were placed into the appropriate chamber in a place conditioning apparatus for 30 min, and locomotor activity was assessed as the number of “crossings”, defined as breaking the light beams at either end of the conditioning chamber as described before in details [Zhou and Kreek 2019]. The KL001 dose and injection schedule (30 min before the test) at ZT15 were based on the above IAD experiments.

3.5. Single administration of KL001 on anxiety-like behavior.

Male alcohol-naïve mice were injected with KL001 (4 mg/kg, i.p.) or vehicle. At 10:00 am (ZT15) 30 mins after the injection, mice were placed on central platform of the elevated plus maze facing the open arm and allowed to explore the maze for 5 min. The number of closed arm entries, open arm entries and time spent in open arms were recorded manually as described before in details [Maiya et al. 2009; Zhou and Kreek 2019].

4. Data analysis.

We performed power analyses to determine the number of mice required to provide statistically significant results, based on the levels of differences seen previously [Zhou et al. 2017], and predicted that these studies require (>6–8) animals per group. For dose response analysis on KL001, group differences for alcohol intake and preference ratios at the 4-hour recording time were analyzed using one-way ANOVA for treatments with different doses (Figure 1). In the experiments with KL001, alcohol (or sucrose or saccharin) intake, water intake, total fluid and preference ratio differences across the different groups were analyzed using two-way ANOVA with repeated measures for time (0–4h, 4–8h vs 8–24h interval) (Figures 2–5). In the ADE experiments, alcohol intake differences across the different groups were analyzed using two-way ANOVA for treatment (vehicle vs drug) and for session (baseline vs ADE), with testing our a priori hypothesis that there was an ADE based on the published findings [Zhou et al. 2017] (Figure 6). All the ANOVA analysis was followed by Tukey Honest Significance Difference (HSD) post hoc tests or planned comparison. For other behavioral measures, the differences between groups were analyzed using Student’s t-test. The accepted level of significance for all tests was p < 0.05. All statistical analyses were performed using Statistica (version 5.5, StatSoft Inc, Tulsa, OK).

Figure 1. Dose responses of single administration of KL001 (0, 1–4 mg/kg) on reducing alcohol intake (A) and preference ratio (B) in mice (n=8) after 1 day of withdrawal from 3-week chronic intermittent alcohol drinking.

Figure 1

Open in a new tab

Data were collected at the 4-hour time point on the testing day (zeitgeber time [ZT]15–19). * p<0.05 vs. control (KL001 at 0 mg/kg). Individual values are presented as dot plots with mean ± range.

Figure 5. No effect of single administration of KL001 (4 mg/kg) at zeitgeber time [ZT]15 on saccharin intake (A), water intake (B) or preference ratio (C) in mice.

Figure 5

Open in a new tab

(1) Control group: mice (n=7) received one vehicle injection (i.p.) before the drinking test; and (2) KL001 group: mice (n=7) received one KL001 injection (4 mg/kg, i.p.) 30 min before the drinking session. Saccharin (0.1%) and water intake values were recorded after 0, 4, 8 and 24 hours of saccharin access (Starting at 3 hours after lights off) (zeitgeber time [ZT]15). Individual values are presented as dot plots with mean ± range.

Figure 6. Effects of single administration (A) or five repeated administration (B) of KL001 (4 mg/kg) at zeitgeber time [ZT]15 on alcohol intake in an alcohol deprivation effect model after 4 hours of alcohol access (zeitgeber time [ZT]15–19) in mice after 1 week of withdrawal from 3-week chronic intermittent alcohol drinking.

Figure 6

Open in a new tab

A. (1) Control group: mice (n=6) received one vehicle injection (i.p.); and (2) KL001 group: mice (n=6) received one KL001 injection (4 mg/kg, i.p.) 30 min before the drinking session. B. (1) Control group: mice (n=7) received five vehicle injections (i.p.); and (2) KL001 group: mice (n=7) received five KL001 injections (4 mg/kg, i.p.) during the 1-week withdrawal with the last one 1 day before alcohol were presented again. Alcohol (30%) and water intake values were recorded after 0 and 4 hours of alcohol access (Starting at 3 hours after lights off) (zeitgeber time [ZT]15). * p<0.05 vs. control baseline, and + p<0.05 vs. ADE. Individual values are presented as dot plots with mean ± range.

RESULTS

1. Single administration of KL001 reduced alcohol (but not sucrose or saccharin) intake and preference after 1 day of withdrawal from chronic IAD.

1.1. Dose-response of KL001 on IAD.

At the 4-hour time point between ZT15–19, the full-dose response of single KL001 administration (0, 1, 2 and 4 mg/kg) in terms of 15% alcohol intake and preference is presented in Figure 1. For intake (Figure 1A), one-way ANOVA revealed a significant effect of KL001 [F (3, 28) = 3.7, p<0.05], and Tukey HSD post hoc analysis showed that in comparison with the vehicle group, the KL001-treated mice at 4 mg/kg had less intake than the vehicle-treated ones [p<0.05]. For preference ratio (Figure 1B), there was a significant effect of KL001 [one-way ANOVA, F (3, 28) = 3.4, p<0.05], and the KL001 treatment at 4 mg/kg significantly decreased the preference ratio [Tukey HSD test, p<0.05].

1.2. Single KL001 at 4 mg/kg reduced 15% alcohol intake and preference.

For intake, two-way ANOVA with repeated measures revealed a significant effect of KL001 treatment [F (1, 17) = 5.2, p<0.05], and planned comparison analysis showed that the KL001-treated mice had less intake than the vehicle-treated ones at 4 hours (ZT15–19) [p<0.01] (Figure 2A). In comparison to the vehicle controls, 4 mg/kg KL001 reduced mean alcohol intake by approximately 50% after 4 hours, associated with a compensatory increase in water intake [p<0.05] (Figure 2B) (resulting in virtually unchanged total fluid intake [Figure 2D] at 4 hours [ZT15–19]). For preference ratio, there was a significant interaction between KL001 treatment and time interval [two-way ANOVA, F (1, 17) = 4.8, p<0.05], and the KL001-treated mice had less preference than the controls at 4 hours (ZT15–19) [planned comparison, p<0.01] (Figure 2C).

Furthermore, we evaluated the KL001 effect on the entire 24-hour consumption and found that the KL001-treated group had less intake (10 ± 1.1 g/kg/day) than the control group (15 ± 0.6 g/kg/day) [Student’s t-test (1, 17) = 5.3, p<0.05], with no significant difference in preference ratios (control: 0.65 ± 0.08; KL001: 0.60 ± 0.11). Also, during the entire 24-hour drinking session, the KL001 treated mice drunk more water (1.4 ± 0.31 ml/day) than the controls (0.69 ± 0.13 ml/day) [Student’s t-test (1, 17) = 4.9, p<0.05].

In another experiment, we tested whether the effect of KL001 was time-dependent during the day, and single injection of KL001 at 4 mg/kg was thus given at ZT19 (instead of ZT15). As shown in Figure 3, KL001 had no effect on either intake or preference after 4 hours (ZT19–23) or 20 hours (ZT23–15) of the KL001 injection.

Figure 3. No effect of single administration of KL001 (4 mg/kg) at zeitgeber time [ZT]19 on alcohol intake or preference ratio in mice.

Figure 3

Open in a new tab

(1) Control group: mice (n=6) received one vehicle injection (i.p.); and (2) KL001 group: mice (n=6) received one KL001 injection (4 mg/kg, i.p.) at ZT19, 4 hours after alcohol access. Alcohol (15%) and water intake values were recorded after 4 and 20 hours of alcohol access. Individual values are presented as dot plots with mean ± range.

1.3. No effect of single KL001 (4 mg/kg) on sucrose intake or preference.

As alcohol is a caloric reinforcer, the specificity of the action of KL001 on alcohol intake was tested after single administration of 4 mg/kg KL001 on 2% sucrose (caloric reinforcer) drinking in mice after chronic IAD. As tested at the same time point between ZT15–19 in the above IAD experiments, KL001 had no significant effect on either intake or preference on sucrose consumption (Figure 4). Further, in alcohol-naïve mice (n = 6), there was no effect of 4 mg/kg KL001 on 2% sucrose intake (control mice: 1.7 ± 0.15 g/kg; KL001 mice: 2.0 ± 0.26 g/kg) or preference ratio (control: 0.90 ± 0.04; KL001: 0.92 ± 0.02) between ZT15–19.

Figure 4. No effect of single administration of KL001 (4 mg/kg) at zeitgeber time [ZT]15 on sucrose intake (A), water intake (B) or preference ratio (C) in mice.

Figure 4

Open in a new tab

(1) Control group: mice (n=9) received one vehicle injection (i.p.) before the drinking test; and (2) KL001 group: mice (n=6) received one KL001 injection (4 mg/kg, i.p.) 30 min before the drinking session. Sucrose (2%) and water intake values were recorded after 0, 4, 8 and 24 hours of sucrose access (Starting at 3 hours after lights off) (zeitgeber time [ZT]15). Individual values are presented as dot plots with mean ± range.

1.4. No effect of single KL001 (4 mg/kg) on saccharin intake or preference.

The specificity of the KL001 action was further tested on saccharin (non-caloric reinforcer) intake. After stable intake observed, the mice assigned to the vehicle-treated or KL001-treated groups showed similar saccharin intake 24 hours before the test day. Similar to the above sucrose experiment, the KL001 at 4 mg/kg had no significant effect on either intake or preference on 0.1% saccharin consumption (Figure 5). Furthermore, in alcohol-naïve mice (n=6), there was no significant effect on saccharin drinking (control mice: 0.17 ± 0.03 g/kg; KL001 mice: 0.14 ± 0.04 g/kg) or preference ratio (control: 0.96 ± 0.03; KL001: 0.95 ± 0.03).

2. Effect of single or repeated administration of KL001 on ADE.

In the single-dose KL001 experiment, we first tested KL001 at 2 mg/kg and found that there was no significant effect at this low dose (Figure S1). Then we tested 4 mg/kg dose and the data are presented in Figure 6A. On alcohol intake between ZT15–19, two-way ANOVA revealed a significant effect of KL001 treatment [F (1, 20) = 5.9, p<0.05] and a significant interaction between treatment and session [F (1, 20) = 5.7 p<0.05]. Further Tukey HSD analysis showed that (1) in the control group (vehicle-treated mice), there was significant effect of ADE [p<0.05]; and (2) the KL001-ADE mice had less intake than the control-ADE ones in the ADE session [p<0.05] (Figure 6A). There was no difference in preference ratio between the control and KL001 groups in the ADE session (control mice: 0.49 ± 0.08; KL001 mice: 0.46 ± 0.11). There were no significant effects of single KL001 treatment after 8 or 24 hours (between ZT19–23 and ZT23–15) in the ADE session (Table S3A).

In the repeated KL001 experiment, we tested the effect of KL001 at 4 mg/kg and found a similar reducing effect on the ADE as the above single-dose experiment. On alcohol intake between ZT15–19 (Figure 6B), two-way ANOVA revealed a significant effect of KL001 treatment [F (1, 24) = 4.8, p<0.05], and a significant interaction between session and treatment [F (1, 24) = 4.9, p<0.05]. Tukey HSD analysis showed that the KL001-ADE mice had less intake than the control-ADE ones [p<0.05]. To test our a priori hypothesis that there was an ADE, we included the Tukey HSD test result showing that in the control group, the ADE was significant [p<0.05] (Figure 6B), though 2-way ANOVA did not reveal any significant effect of session. For preference ratio, there was no difference between the vehicle- and KL001-treated groups in the ADE session (control mice: 0.58 ± 0.06; KL001 mice: 0.56 ± 0.07). There were no significant effects of the repeated KL001 treatment after 8 or 24 hours (between ZT19–23 and ZT23–15) in the ADE session (Table S3B).

3. No effect of single KL001 on locomotor activity.

We tested whether KL001 could induce sedation in mice at 4 mg/kg required to decrease alcohol drinking and found no difference on the number of 30-min crossings in alcohol-naïve mice at 10:00 am (ZT15) 30 min after the KL001 injection (control mice 233 ± 57, n=6; KL001 mice 261 ± 47, n=8).

4. No effect of single KL001 on anxiety-like activity.

The number of closed arm entries, open arm entries and time spent in open arms were scored after KL001 at 4 mg/kg, and there was no significant effect of KL001 (Table 1). Of note, due to a small sample size (n=7–8), there were relatively large variations in the percentage entries into open arms.

Table 1.

No effect of single administration of KL001 (4 mg/kg) at Zeitgeber time [ZT]15 on anxiety-like activity in alcohol-naïve mice at 10:00 am (ZT15) 30 min after the injection. Data in table are presented as mean ± SEM.

Treatment Vehicle (n=7) KL001 (n=8)
Number of closed arm entries 19 ± 5 20 ± 6
% open arm entries 22 ± 9 24 ± 8
Time spent in open arms (%) 15 ± 3 17 ± 3
Open in a new tab

DISCUSSION

The first objective in our present study was to investigate the potential of the CRY activator KL001 in reducing excessive alcohol consumption in mice after 1-day acute withdrawal from chronic excessive alcohol drinking. Single administration of KL001 significantly reduced alcohol intake in a dose-related manner (1–4 mg/kg) (Figure 1A). The KL001-induced reduction of alcohol intake at the first 4-hour time point (ZT15–19) and during the entire 24-hour drinking session (Figure 2A) was coupled with a significant compensatory increase in water intake (Figure 2B), resulting in no change in total fluid intake (Figure 2D), suggesting the absence of a nonspecific sedative effect by KL001. Consistently, KL001 (1–4 mg/kg) produced a dose-related decrease in alcohol preference (Figure 2C). Further, the present study confirmed that it is unlikely that the effect of KL001 on alcohol intake was secondary to a general suppression of appetitive or consumption behaviors, since KL001 at 4 mg/kg (the effective dose on alcohol intake) did not decrease sucrose (caloric reinforcer) (Figure 4) or saccharin (non-caloric reinforcer) (Figure 5) intake. Our results also suggest that the enhanced CRY activity by KL001 does not modulate sucrose or saccharin reward, at least at the doses and time tested. Further, KL001 at 4 mg/kg did not produce any sedation in the spontaneous locomotor activity test or anxiety-like behavior in the elevated plus maze test with a small sample size (Table 1). Together, these results clearly demonstrate that the KL001-induced CRY activation plays a specific role in modulating excessive alcohol drinking.

Compared with the mice between ZT15–19, which had alcohol intake at the levels of around 5 g/kg/4 hours, the mice between ZT19–23 showed lower alcohol intake with the levels of 3–4 g/kg/4 hours (Figure 2), similar to those observed in our previous studies [Zhou et al. 2017, Zhou and Kreek 2019]. Here we purposely compared the effects of KL001 at different time points during the day between ZT15–19 and ZT19–23, and found that KL001 at 4 mg/kg altered alcohol drinking between ZT15–19 (Figure 2), but not between ZT19–23 (Figure 3). It suggests that the effect of KL001 on reducing alcohol intake is probably related to the circadian pattern of Cry gene expression and CRY activity after chronic alcohol exposure [Farnell et al. 2008; Filiano et al. 2013; Patke et al. 2017, 2020], which warrants the need for further analysis. Alternatively, the lack of significant effect of KL001 (at least at the dose tested here) may be a floor effect due to the lower basal alcohol intake between ZT19–23 than the one between ZT15–19.

Our second main objective was to investigate the potential of KL001 in preventing relapse-like drinking in mice after chronic withdrawal. The occurrence of an increase in mouse alcohol consumption after a certain period of imposed abstinence has been named as the alcohol deprivation effect (ADE), which has been considered as a rodent model of alcohol “relapse” behavior and alcohol craving with good predictive validity [Burattini et al. 2006; Vengeliene et al. 2014]. As shown in Figure 6A and 6B, mice displayed the ADE with increased alcohol intake after 1-week chronic withdrawal as observed in our previous studies [Zhou et al. 2017; Zhou and Kreek 2019]. In contrast, the mice pretreated with single KL001 administration at 4 mg/kg had a blunted ADE (Figure 6A), suggesting the involvement of impaired CRY activity in the “relapse” drinking. One concern about the use of CRY activators like KL001 is that the CRY activation after repeated KL001 administration could result in the development of tolerance. Therefore, we tested the effectiveness of the multiple-dosing regimen (5 daily injections before the ADE during the 1-week chronic withdrawal) to mimic the multiple-dosing treatment in the clinic. Similar to the effect of single KL001 on reducing the ADE (Figure 6A), the repeated pretreatments of KL001 during the alcohol withdrawal week at the same dose (4 mg/kg) efficiently prevented the ADE (Figure 6B), with no tolerance development after this multiple-dosing regimen. It is worth mentioning that the prevention of the ADE by this multiple-dosing regimen occurred with the last KL001 administration 1 day before alcohol re-exposure ADE. Our new results constitute an interesting finding of the anti-relapse properties of KL001 observed in “relapse” ADE drinking behaviour, and our promising in vivo data may provide new information about the therapeutic potential of CRY activators in the treatment of alcoholism.

Previous lines of experimental evidence have suggested chronic alcohol-exposed rodents as an animal model of alcohol withdrawal-induced “anxiety” or “depression” [Colombo et al. 1995; Zhou et al. 2011; Zhou and Kreek 2018]. These emotional states, and the search for the anxiolytic effects of alcohol, might in turn contribute to promoting the high levels of alcohol intake and preference that characterize the excessive drinking observed in mice. The CRY system is involved in “anxiety-” and “depression-” related behaviors [De Bundel et al. 2013; Schnell et al. 2015; Hühne et al. 2020; Porcu et al. 2020]. Of interest, it was recently found that CRY1/2 inhibits activation of D1 dopamine receptor neurons in the nucleus accumbens (probably through interacting with Gs protein) in a mouse “depression” model [Porcu et al. 2020]. Our hypothesis is that enhanced CRY activity by its stabilizers reverses the effect of alcohol withdrawal driving excessive alcohol intake or relapse-like drinking. Thus, as observed after acute withdrawal in mice (Figure 2), the increased CRY activity after KL001 administration (inhibiting CRY degradation) could be partially responsible for the subsequent reduction in alcohol intake. Of interest, behavioral responses to single KL001 (inhibition of alcohol drinking) were apparent not only at 1-day acute withdrawal time point, but also after 1-week chronic withdrawal in the ADE model (Figure 6), suggesting that CRY dysfunction (deficiency or desynchronized activity) may occur in early withdrawal and persist into chronic withdrawal. Because it is unknown how chronic excessive alcohol or “relapse” drinking regulated the Cry genes with other clock genes in a region-specific manner in mouse brains, other possible explanations should be considered. In Cry1 and Cry2 knockout mice, there is an increased level of circulating corticosterone, suggesting their roles on tonic inhibition of the hypothalamic–pituitary–adrenal axis [Lamia et al 2011], and an altered response of cocaine-induced neuronal activation in the striatum [De Bundel et al 2013]. In addition, it has been found that Per2 interplays with several key regulators of the stress systems, like glucocorticoids and its receptors [Yang et al. 2009], proopiomelanocortin [Agapito et al. 2010] and glutamate transporters [Spanagel et al. 2005]. Therefore, further experiments are necessary to fill important knowledge gaps such as the particular target genes or neuronal pathways regulated by CRY after chronic excessive alcohol consumption, withdrawal and “relapse” in rodent models.

In summary, the clock genes are not only key components of the molecular circadian clock but also function as major regulators of metabolism and mood [Patke et al. 2020]. Though there are complicated interactions between the circadian clock genes and basic physiological and behavioral processes, circadian gene dysregulation has been found to associate with a variety of diseases, including alcohol use disorder. The present study for the first time shows initial and promising in vivo data that CRY activator decreases alcohol excessive and “relapse” drinking in mice, and suggests that pharmacological CRY activation may offer a novel strategy to treat alcohol use disorder and/or alcoholism.

Supplementary Material

1665282_Sup_Materials

Acknowledgement:

The authors thank Professor Michael W. Young and Dr. Alina Patke in Laboratory of Genetics at the Rockefeller University for providing their comments and discussions on the study. This work was supported by NIH AA021970 (YZ), Robertson Therapeutic Development Fund at the Rockefeller University (YZ) and Miriam and Sheldon G. Adelson Medical Research Foundation (MJK). Special thanks to Angelique Baehr for providing her editing corrections on the manuscript.

Footnotes

Conflict of interest: All authors declare that they have no conflicts of interest.

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of a an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

References

  1. Agapito M, Mian N, Boyadjieva NI, Sarkar DK (2010) Period 2 gene deletion abolishes beta-endorphin neuronal response to ethanol. Alcohol Clin Exp Res 34: 1613–1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barclay JL, Shostak A, Leliavski A, Tsang AH, Johren O, Muller-Fielitz H, Landgraf D, Naujokat N, van der Horst GT, Oster H (2013) High-fat diet-induced hyperinsulinemia and tissue-specific insulin resistance in Cry-deficient mice. Am J Physiol Endocrinol Metab 304: E1053–1063. [DOI] [PubMed] [Google Scholar]
  3. Burattini C, Gill TM, Aicardi G, Janak PH (2006) The ethanol self-administration context as a reinstatement cue: acute effects of naltrexone. Neuroscience 139:877–887. [DOI] [PubMed] [Google Scholar]
  4. Colombo G, Agabio R, Lobina C, Reali R, Zocchi A, Fadda F, Gessa GL (1995) Sardinian alcohol-preferring rats: A genetic animal model of anxiety. Physiol Behav 57: 1181–1185. [DOI] [PubMed] [Google Scholar]
  5. De Bundel D, Gangarossa G, Biever A, Bonnefont X, Valjent E (2013) Cognitive dysfunction, elevated anxiety, and reduced cocaine response in circadian clock-deficient cryptochrome knockout mice. Front Behav Neurosci 7: 152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Edwards MD, Brancaccio M, Chesham JE, Maywood ES, Hastings MH (2016) Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling. Proc Natl Acad Sci U S A 113:2732–2737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farnell YZ, Allen GC, Nahm SS, Neuendorff N, West JR, Chen WJ, Earnest DJ (2008) Neonatal alcohol exposure differentially alters clock gene oscillations within the suprachiasmatic nucleus, cerebellum, and liver of adult rats. Alcohol Clin Exp Res 32: 544–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Filiano AN, Millender-Swain T, Johnson R Jr, Young ME, Gamble KL, Bailey SM (2013) Chronic ethanol consumption disrupts the core molecular clock and diurnal rhythms of metabolic genes in the liver without affecting the suprachiasmatic nucleus. PLoS One 8: e71684. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Griebel G, Ravinet-Trillou C, Beeské S, Avenet P, Pichat P (2014) Mice deficient in cryptochrome 1 (cry1 (−/−)) exhibit resistance to obesity induced by a high-fat diet. Front Endocrinol (Lausanne) 5: 49. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hirota T, Lee JW, St John PC, Sawa M, Iwaisako K, Noguchi T, Pongsawakul PY, Sonntag T, Welsh DK, Brenner DA, Doyle FJ 3rd, Schultz PG, Kay SA (2012) Identification of small molecule activators of cryptochrome. Science 337: 1094–1097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hühne A, Volkmann P, Stephan M, Rossner M, Landgraf D (2020) An in-depth neurobehavioral characterization shows anxiety-like traits, impaired habituation behavior, and restlessness in male Cryptochrome-deficient mice. Genes Brain Behav 19: e12661. [DOI] [PubMed] [Google Scholar]
  12. Humphries PS, Bersot R, Kincaid J, Mabery E, McCluskie K, Park T, Renner T, Riegler E, Steinfeld T, Turtle ED, Wei ZL, Willis E (2016) Carbazole-containing sulfonamides and sulfamides: Discovery of cryptochrome modulators as antidiabetic agents. Bioorg Med Chem Lett 26: 757–760. [DOI] [PubMed] [Google Scholar]
  13. Hwa LS, Chu A, Levinson SA, Kayyali TM, DeBold JF, Miczek KA (2011) Persistent escalation of alcohol drinking in C57BL/6J mice with intermittent access to 20% alcohol. Alcohol Clin Exp Res 35: 1938–1947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koob GF, Kreek MJ (2007) Stress, dysregulation of drug reward pathways, and the transition to drug dependence. Am J Psychiatry 164: 1149–1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Koob GF, Colrain IM (2020) Alcohol use disorder and sleep disturbances: a feed-forward allostatic framework. Neuropsychopharmacology 45: 141–165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lamia KA, Papp SJ, Yu RT, Barish GD, Uhlenhaut NH, Jonker JW, Downes M, Evans RM (2011) Cryptochromes mediate rhythmic repression of the glucocorticoid receptor. Nature 480: 552–556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Le-Niculescu H, McFarland MJ, Ogden CA, Balaraman Y, Patel S, Tan J, Rodd ZA, Paulus M, Geyer MA, Edenberg HJ, Glatt SJ, Faraone SV, Nurnberger JI, Kuczenski R, Tsuang MT, Niculescu AB (2008) Phenomic, convergent functional genomic, and biomarker studies in a stress-reactive genetic animal model of bipolar disorder and co-morbid alcoholism. Am J Med Genet B Neuropsychiatr Genet 147B: 134–66. [DOI] [PubMed] [Google Scholar]
  18. Maiya R, Zhou Y, Norris EH, Kreek MJ, Strickland S (2009) Tissue plasminogen activator modulates the cellular and behavioral response to cocaine. Proc Natl Acad Sci U S A. 106: 1983–1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miyamoto Y, Sancar A (1998) Vitamin B2-based blue-light photoreceptors in the retinohypothalamic tract as the photoactive pigments for setting the circadian clock in mammals. Proc Natl Acad Sci U S A 95: 6097–6102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Onat OE, Kars ME, Gül Ş, Bilguvar K, Wu Y, Özhan A, Aydın C, Başak AN, Trusso MA, Goracci A, Fallerini C, Renieri A, Casanova JL, Itan Y, Atbaşoğlu CE, Saka MC, Kavaklı İH, Özçelik T (2020) Human CRY1 variants associate with attention deficit/hyperactivity disorder. J Clin Invest 130: 3885–3900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Partonen T (2015) Clock genes in human alcohol abuse and comorbid conditions. Alcohol 49: 359–365. [DOI] [PubMed] [Google Scholar]
  22. Patke A, Murphy PJ, Onat OE, Krieger AC, Özçelik T, Campbell SS, Young MW (2017) Mutation of the Human Circadian Clock Gene CRY1 in Familial Delayed Sleep Phase Disorder. Cell 169: 203–215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Patke A, Young MW, Axelrod S (2020) Molecular mechanisms and physiological importance of circadian rhythms. Nat Rev Mol Cell Biol 21: 67–84. [DOI] [PubMed] [Google Scholar]
  24. Porcu A, Vaughan M, Nilsson A, Arimoto N, Lamia K, Welsh DK (2020) Vulnerability to helpless behavior is regulated by the circadian clock component CRYPTOCHROME in the mouse nucleus accumbens. Proc Natl Acad Sci U S A 117: 13771–13782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schnell A, Sandrelli F, Ranc V, Ripperger JA, Brai E, Alberi L, Rainer G, Albrecht U (2015) Mice lacking circadian clock components display different mood-related behaviors and do not respond uniformly to chronic lithium treatment. Chronobiol Int 32: 1075–89. [DOI] [PubMed] [Google Scholar]
  26. Spanagel R, Pendyala G, Abarca C, Zghoul T, Sanchis-Segura C, Magnone MC, Lascorz J, Depner M, Holzberg D, Soyka M, Schreiber S, Matsude F, Lanthrop M, Schuman G, Albrecht U (2005) The clock gene Per2 influences the glutamatergic system and modulates alcohol consumption. Nat Med 11: 35–42. [DOI] [PubMed] [Google Scholar]
  27. Takahashi JS, Hong HK, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 9: 764–775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Vengeliene V, Bilbao A, Spanagel R (2014) The alcohol deprivation effect model for studying relapse behavior: a comparison between rats and mice. Alcohol 48: 313–320. [DOI] [PubMed] [Google Scholar]
  29. Yang S, Liu A, Weidenhammer A, Cooksey RC, McClain D, Kim MK, Aguilera G, Abel ED, Chung JH (2009) The role of mPer2 clock gene in glucocorticoid and feeding rhythms. Endocrinology 150: 2153–2160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Zhang EE, Liu Y, Dentin R, Pongsawakul PY, Liu AC, Hirota T, Nusinow DA, Sun X, Landais S, Kodama Y, Brenner DA, Montminy M, Kay SA (2010) Cryptochrome mediates circadian regulation of cAMP signaling and hepatic gluconeogenesis. Nat Med 16: 1152–1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zhou Y, Colombo G, Carai MAM, Ho A, Gessa GL, Kreek MJ (2011) Involvement of arginine vasopressin and V1b receptor in alcohol drinking in Sardinian alcohol-preferring rats. Alcohol Clin Exp Res 35: 1876–1883 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zhou Y, Rubinstein M, Low MJ, Kreek MJ (2017) Hypothalamic-specific proopiomelanocortin-deficiency reduces alcohol drinking in male and female mice. Genes Brain and Behavior 16: 449–461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zhou Y, Kreek MJ (2018) Involvement of activated brain stress responsive systems in excessive and “relapse” alcohol drinking in rodent models: implications for therapeutics. J Pharmacol Exp Ther 366: 9–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Zhou Y, Kreek MJ (2019) Combination of Clinically Utilized Kappa-Opioid Receptor Agonist Nalfurafine With Low-Dose Naltrexone Reduces Excessive Alcohol Drinking in Male and Female Mice. Alcohol Clin Exp Res 43: 1077–1090. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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

Supplementary Materials

1665282_Sup_Materials
NIHMS1665282-supplement-1665282_Sup_Materials.doc (243.5KB, doc)

RESOURCES