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Published in final edited form as: Psychopharmacology (Berl). 2022 Jan 31;239(3):887–895. doi: 10.1007/s00213-022-06079-8

N6-Substituated Adenosine Analog J4 Attenuates Anxiety-like Behaviors in Mice

Lee Peyton 1, Brandon Emanuel León 2, Hesham Essa 1, Yijuang Chern 3, Doo-Sup Choi 1,4
PMCID: PMC9063204  NIHMSID: NIHMS1795532  PMID: 35102423

Abstract

Rationale

Withdrawal from chronic alcohol exposure produces various physical and mental withdrawal symptoms. Activation of adenosine receptors is known to inhibit withdrawal-induced excitation. However, limited studies investigate how adenosine analogs may prove helpful tools to alleviate alcohol-withdrawal related affective behaviors.

Objectives

This study aimed to investigate the effects of J4 compared with saline using the mice vapor or voluntary ethanol drinking model on behavioral endpoints representing ethanol-withdrawal negative emotionality commonly observed during abstinence from chronic alcohol use.

Methods

We subjected C57BL/6J mice to chronic intermittent ethanol (CIE) exposure schedule to investigate how 72 h withdrawal from alcohol alters affective-like behavior. Next, we determined how treatment with J4, a second-generation adenosine analog, influenced affective behaviors produced by alcohol withdrawal. Finally, we determined how J4 treatment alters voluntary ethanol drinking using the two-bottle choice drinking paradigm.

Results:

Our results show that 72 h withdrawal from chronic intermittent ethanol exposure produces limited if any affective-like disturbances in male C57BL/6J mice exposed to 4 cycles ethanol vapor. Most importantly, J4 treatment irrespective of ethanol exposure decreases innate anxiety-like behavior in mice.

Conclusions:

Withdrawal from chronic intermittent ethanol exposure and subsequent behavioral testing 72 h later produces minimal affective-like behavior. J4 treatment did however reduce marble burying behavior and increased time spent in open arms of elevated plus maze, suggesting J4 may be useful as a general anxiolytic.

Keywords: adenosine, J4, two-bottle choice, marble burying, elevated-plus maze

INTRODUCTION

Drug addiction, including alcohol use disorder (AUD), has conventionally been thought of as a chronic, relapsing disorder-occurring in three stages: the compulsion to seek and consume alcohol, loss of control over its intake, and emergence of negative affective state upon alcohol withdrawal (Mason 2017). Importantly, negative affect (depressed mood, anxiety, irritability, and sleep disturbance) is hypothesized to play a central role in the chronic relapsing nature of AUD and the development of alcohol addiction, as the increase in alcohol consumption becomes reinforcing to alleviate the negative affect produced from its abstinence (Sidhu et al. 2018). More simply, alcohol is seen as a negative reinforcing agent (Koob 2017) driving increased drinking behavior. The role of this withdrawal-related phenomenon in the development and sustaining nature of alcohol addiction remains a topic of debate (Heilig et al. 2010; Sidhu et al. 2018). Both rat and mice models have been instrumental in furthering our understanding of this phenomena, with research suggesting that alcohol-induced affect disturbance arises partly due to long-term disruption in the balance between glutamate and γ-amino butyric acid (GABA) neurotransmission (Wang et al. 2021).

Adenosine acts as a neurotransmitter and exerts its function through interacting with four well-identified G protein coupled receptor (GPCR) as A1R, A2AR, A2BR, and A3R (Dunwiddie and Masino 2001; Fredholm et al. 2005). Over the years, adenosine receptors have come to be implicated in many neurological and psychiatric disorders (Fredholm et al. 2005). Especially, adenosine signaling is an important contributor in AUD, mood, and anxiety disorders (Asatryan et al. 2011; Dunwiddie and Masino 2001; Nam et al. 2013a; Nam et al. 2013b; Nam et al. 2012; van Calker et al. 2019). Moreover, neurophyschopharmacological studies have suggested a close harmony between psychiatric disorders (e.g., AUD and mood-related illness) and the modulation of the adenosinergic signaling system within the CNS (Yamada et al. 2014). Strikingly, both depression and anxiety are closely synced to perturbations in adenosine signaling. The comorbidity of anxiety disorders and substance use disorder including AUD is common (Turner et al. 2018) with anxiety causally thought to contribute to alcohol craving and consumption (McCaul et al. 2017).

Adenosine is an ever-present nucleoside within the central nervous system (CNS) where studies have highlighted its importance in the regulation of many physiological and pathological conditions (Burnstock 2008; Cheffer et al. 2018). Separate from canonical neurotransmission in which vesicles mediate neurotransmission, adenosine can arise intracellularly from the degradation of adenosine monophosphate (AMP) and released by bidirectional equilibrative nucleoside transporters (ENT) 1–4. Additionally, adenosine can form from the extracellular catabolism of released nucleotides by ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) or ecto-5’-nucleotidase (CD73) with CD73 being responsible for the preferential breakdown of AMP within the extracellular space and being the best-characterized enzymatic source of adenosine (Augusto et al. 2013; Latini and Pedata 2001). ENT1 shows the highest expression in the striatum and is one of the main transporters responsible for the regulation of adenosine concentration (Choi et al. 2004; Hong et al. 2019b; Nam et al. 2012). Interestingly, chronic alcohol exposure has been shown to downregulate ENT1 expression in cultured neuronal cells, with our previous work suggesting that downregulation of ENT1 decreases extracellular adenosine concentration (Chen et al. 2016; Jia et al. 2020a; Nam et al. 2012). In the striatum, pharmacologic activity of adenosine is mediated predominately by the Gsα-coupled adenosine A2AR receptor (A2AR) mainly expressed on striatopallidal neurons, allowing for inhibitory control through the indirect pathway of the basal ganglia circuitry (Hong et al. 2019b; Nam et al. 2013a). Relevant to AUD, A2AR has shown and important role in mediating alcohol consumption’s cellular and behavioral responses.

Pure adenosine and adenosine analogues are anxiolytic (Chen et al. 2007; Dunwiddie and Masino 2001). J4 is a synthetic second-generation BBB-penetrable N6-substituated adenosine analog derived from the rhizomes of Gastrodia elata, an herb medicinally used for over 1500 years in Asia (Chen et al. 2016). J4 dually targets A2AR (Ki, 1.7 μM) and ENT1 (Ki, 50 nM), thereby resulting mainly in the suppression of the transporter and probably a low-level activation of the receptor (Chang et al. 2021; Huang et al. 2011). Previous studies utilizing J4 have shown that its oral bioavailability reaches 48% with a brain-to-blood ratio approximately 16% (Chang et al. 2021). Recently, our work has indicated parent compound of J4, NHBA, dampens alcohol drinking and seeking behaviors (Hong et al. 2019b). However, exploration of these compounds in the regulation of negative emotionality induced from alcohol withdrawal remains. Therefore, we investigated whether J4 would attenuate alcohol-withdrawal potentiated negative affective behavior.

MATERIALS AND METHODS

Animals

Male C57BL/6J mice were purchased from Jackson Laboratories and were utilized for experiments when they reached between 10 and 12 weeks old. Animals were group-housed under 12:12 h lighting conditions with lights on at 0600 and lights off at 1800, and provided ad libitum access to food and water. All procedures used throughout the duration of this experiment were approved by the Mayo Clinic Institutional Animal Care and Use Committee and performed in accordance with NIH guidelines. A total of 84 male C57BL/6J mice were used for this study. A total of 3 cohorts of mice were used for this experiment: Cohort 1 (open-field testing), Cohort 2 (CIE, elevated plus maze, and marble burying task), Cohort 3 (2-bottle choice). For cohort 2, mice were tested in marble burying task during morning prior to testing in EPM which occurred during the afternoon.

Drugs

J4 was provided by Dr. Yijuang Chern (Institute of Biomedical sciences, Academia Sinica, Taipei, Taiwan). A stock solution of J4 was prepared in a 5% DMSO solution at a concentration of 1 mg/mL. A working solution of J4 was utilized for experiments by diluting with saline and injecting (i.p.) at a dose of 0.03 (mg/kg) in 0.03% DMSO.

Chronic Intermittent Ethanol Exposure (CIE)

As previously described, ethanol vapor was delivered in plexiglass inhalation chambers (Starski et al. 2020). Briefly, mice were immediately exposed to EtOH vapor for 16 h from 1700 to 0900, followed by an 8 h room air exposure (Fig. 1a). This process was repeated for 4 consecutive days, followed by 3 consecutive days of room air exposure. Before each 16 h EtOH vapor exposure, mice were intraperitoneally injected with 1.5 (g/kg) priming dose of 20% pure EtOH diluted in saline (Sigma Aldrich; Burlington, MA). After 4 weeks of CIE exposure, mice were allowed 72 h withdrawal and randomly distributed into groups: Air + Vehicle (n = 10), Air + J4 (n = 10), CIE + Vehicle (n = 10), CIE + J4 (n =10). For the air groups, mice were subjected to the exact same schedule of exposure, except they received intraperitoneal priming injections of saline, and were placed in plexiglass chambers delivering room air. J4 treatment was delivered (i.p.) to groups receiving drug treatment immediately following their last removal from the vapor chamber in week 4, for 3 consecutive days prior to experimentation (Fig. 1a).

Figure 1.

Figure 1.

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(a) Schematic representation of chronic intermittent ethanol (CIE) schedule. Mice were exposed to EtOH vapor for 16 h/day for 4 consecutive days followed by a 72-h withdrawal period. This 7-day schedule of EtOH vapor exposure was repeated for 4 weeks. Upon the final removal from the EtOH vapor chamber on weeks 4, J4 was injected once daily (i.p) for 3 consecutive days during the 72-h withdrawal period. (b) Schematic representation of two-bottle choice voluntary drinking paradigm. Briefly, mice were allowed 24 h access to two bottles (water and EtOH). Mice were gradually escalated to 10% EtOH drinking in a stepwise fashion (3% to 6% to 10%) after which, the behavioral effects of J4 treatment on voluntary EtOH drinking, preference, and behavior following 72 h withdrawal determined.

Two-Bottle Choice

Voluntary oral EtOH consumption and preference were determined using the two-bottle choice test in mouse home cage (Fig. 1b). Mice were allowed 24 h access to two bottles (one containing water and the other containing EtOH) and group housed in the BioDAQ liquid choice home cages (BioDAQ; Research Diets, New Brunswick, NJ). The concentration of EtOH was increased from 3% to 6% to 10% every fourth day. After reaching 10% EtOH concentration, mice were individually housed, and placed into home cages equipped and allowed 24 h access to two sipper bottles (one containing water and the other containing 10% EtOH). Mice were allowed 14 days habituation to their new drinking environment before any monitoring of ethanol consumption or preference. After habituation period, EtOH drinking behavior was determined.

To determine the effects of J4 treatment on EtOH drinking, EtOH consumption and preference were measured 4 h after either vehicle or J4 (0.03 mg/kg, i.p.) was administered once daily at 17:00 h. Bottle measurement occurred during the dark cycle (active cycle) of mice at 21:00 h.

Open-Field Test

To evaluate motor activity, several doses of J4 was i.p. injected (n = 4–5 per group) 30 min prior to mice being tested using the open-field test (OFT). The OFT was performed in sound attenuated chambers (Med-Associates, Inc., St. Albans City, VT) equipped with infrared beams and Activity Monitor (Med-Associates). Mice were placed in the center of a Plexiglass box (27 cm × 27 cm × 20.3 cm) and permitted to freely explore the chamber for 30 min (Jia et al. 2020b).

Elevated Plus Maze

To assess anxiety-like behavior following withdrawal from ethanol vapor, mice (n= 10 per group) were tested using the Elevated plus maze (EPM) (Med-Associates). EPM was performed using a plus-shaped maze consisting of two open arms (35 cm × 6 cm) and two closed arms (5 cm × 6 cm × 22 cm) with a connecting center zone (6 cm × 6 cm) elevated 74 cm above the floor. Mice with i.p. injected with either drug (0.03 mg/kg) or vehicle for 3 consecutive days at 17:00 h following the final ethanol vapor exposure. On the day of testing, mice were treated with either drug or vehicle 30 min prior to being placed in the center of the EPM facing an open arm. Mice were allowed to explore freely within the maze for a 10 min duration. The amount of time spent in the open arms, frequency to enter open arms, and latency to enter open arms were recorded and analyzed using Ethovision-XT video tracking software (Noldus Inc, Netherlands).

Marble Burying Task

To assess anxiety-like behavior after ethanol withdrawal, we used the marble burying test as described (Angoa-Pérez et al. 2013; Ruby et al. 2011). Mice with i.p. injected with either drug (0.03 mg/kg) or vehicle for 3 consecutive days at 17:00 h following the final ethanol vapor exposure. On the day of testing, mice were treated with either drug or vehicle 30 min prior to testing in the marble burying task. Each mouse was placed in a cage containing 20 marbles, spaced 4 cm from one another on top of compressed cedar chip rodent bedding to a depth of 5 cm. Each mouse was allowed 30 minutes to bury marbles. Afterwards, the mouse was carefully removed, and the number of marbles buried up to 2/3 their depth in bedding was recorded for each mouse (Deacon 2006).

Statistical Analysis

All data are expressed as mean ± SEM (standard error of the mean). One-Way or Two-way ANOVA was used to compare the difference between groups where appropriate with Bonferroni post hoc analysis. Statistical significance was set at P<0.05 and all statistical calculations performed using GraphPad Prism 9 (La Jolla, California).

RESULTS

High dose J4 treatment negatively regulates locomotor activity

Activation of adenosine receptors is known to cause ataxia and sedation. To determine the appropriate dose of J4 for experimentation, we performed an open-field test to monitor locomotor activity. Systemic i.p. injection of J4 significantly altered total distance travelled (Fig. 2a, F (3,15) = 12.74, P = 0.01). Bonferroni’s post hoc analysis for vehicle vs. J4 (0.1 mg/kg), P = 0.006. Stereotypic behavior was also recorded during the open-field testing session with no effects of drug treatment regarding vertical counts (Fig. 2b, F (3,15) =0.83, P = 0.27) or jump counts (Fig. 2c, F (3,15) = 3.04, P = 0.07). These results indicate that 0.1 mg/kg dose of J4 negatively regulates locomotion

Figure 2.

Figure 2.

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The effects of J4 treatment on locomotor activity were determined using open-field test (OFT). J4 treatment (0.1 mg/kg) significantly decreased total distance traveled during a 30 min open field session (a). Additionally, J4 treatment had no effect on vertical counts (b) or jump counts (c). n = 4/5 per group.

J4 treatment suppresses innate anxiety-like behavior during EPM

To assess the anxiolytic effects J4 treatment during EtOH withdrawal, mice were treated with chronic intermittent ethanol vapor sessions according to the experiment 1 schedule (Fig 1a) using a separate group of mice from the OFT. In the EPM, we found that four cycles of chronic intermittent EtOH exposure followed by 72 h withdrawal and drug treatment had no effects within the first 5 min on frequency to enter open arms (Fig. 3a, exposure F(1,36) = 0.01, P = 0.89, drug treatment F (1,36) = 0.74, P = 0.39, interaction F(1,36) = 0.62, P = 0.43), or latency to enter open arms (Fig. 3b, exposure F (1,36) = 0.22, P = 0.63, drug treatment F (1,36) = 0.31, P = 0.57, interaction F (1,36) = 0.17, P = 0.67). Interestingly, J4 treatment was shown to increase time spend in the open arms of EPM irrespective of ethanol exposure (exposure F (1,36) = 0.19, P = 0.65, drug treatment F(1,36) = 4.29, P = 0.04, interaction F (1,36) = 1.33, P = 0.25) (Fig. 3c). When looking at the resulting 10 min of the EPM, frequency to enter open arms (Fig. 3d, exposure F(1,36) = 1.27, P = 0.26, drug treatment F(1,36) = 0.07, P = 0.77, interaction F (1,36) = 0.71, P = 0.40), latency to enter open arms (Fig. 3e, exposure F(1,36) = 0.07, P = 0.79, drug treatment F(1,36) = 1.35, P = 0.25, interaction F (1,36) = 0.15, P = 0.69), or time spent in the open arms (Fig. 3F, exposure F(1,36) = 0.04, P = 0.82, drug treatment F (1,36) = 1.65, P = 0.20, interaction F (1,36) = 1.39, P = 0.24) were significantly altered. These results indicate that CIE combined with 72 h withdrawal is not anxiogenic, however, J4 treatment at 0.03 mg/kg reduces innate anxiety-like behavior of mice.

Figure 3.

Figure 3.

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Elevated plus maze assessment of J4 treatment following 72 h EtOH vapor withdrawal. i.p. administration of J4 (0.03 mg/kg) had no effect on frequency to enter open arms (a), latency to enter open arms (b), nor time spent in open arms (c) after a 5-minute assessment. Likewise, a 10-minute assessment of EPM activity provided similar results for frequency to enter open arms (d), Latency to enter open arms (e) and time spent in open arms (f). n = 10 per group.

Marble burying behavior is reduced by J4 treatment

The marble burying assay is a common behavioral test to measure anxiety or compulsive-like constructs. To assess the potential anxiolytic or anti-compulsive activity of J4 produced from 72 h EtOH vapor withdrawal, mice performed the marble bury test. Results of the marble burying test revealed J4 treatment significantly alters burying behavior (Fig. 4a and b, exposure F (1,36) = 0.002, P = 0.95, drug treatment F (1,36) = 12.17, P = 0.001, interaction F (1,36) = 0.32, P = 0.57). Bonferonni’s post hoc analysis for CIE+Vehicle vs. CIE + J4, P = 0.04. These results suggest minimal effects of CIE exposure on burying behavior however systemic injection of J4 suppresses the anxiogenic or compulsive response to glass marbles.

Figure 4.

Figure 4.

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Marble burying task assessment following 72 h withdrawal from chronic intermittent EtOH vapor exposure (CIE). (a) Administration (i.p.) J4 (0.03 mg/kg) in CIE treated mice reduced the number of marbles buried after withdrawal. (b) Representative image depicting marble burying behavior (right panel). n = 10 per group

J4 treatment minimally effects voluntary ethanol drinking or preference

Previously, our group found that the first generation N6-substituated adenosine analog from which J4 is derived, reduced ethanol drinking behaviors (Hong et al. 2019b). Thus, we examined whether J4 would similarly reduce voluntary ethanol consumption in mice using the two-bottle choice assay. After acclimation to 10% ethanol drinking, we investigated whether J4 contributes to ethanol consummatory behaviors. Once per day i.p. treatment of J4 (0.03 mg/kg) showed a limited change in grams of ethanol consumed (Fig. 5a, treatment F (1, 23) = 1.65, P = 0.21, time F (2, 46) = 3.02, P = 0.05, interaction F (2, 46) = 0.14, P = 0.86) or preference for ethanol (Fig. 5b, treatment F (1, 23) = 0.71, P = 0.40, time F (2, 46) = 2.65, P = 0.08, interaction F (2, 46) = 1.39, P = 0.25) which suggests J4 treatment does not alter ethanol drinking behavior.

Figure 5.

Figure 5.

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Effects of J4 treatment on 10% ethanol drinking. Three consecutive days J4 treatment showed no differences in the amount of ethanol consumed (a) or the preference (b) for ethanol versus vehicle treated counterparts. n =11–13 per group.

DISCUSSION

Here, we demonstrate the effects of J4, a second-generation adenosine analog derived from the active compound within the rhizome of Gastrodia elata, on ethanol-withdrawal related behaviors. Our findings highlight the role of purinergic adenosine modulation in Mus musculus EPM and marble burying behavior. We found, that dual ENT1 blockade combined with modest A2AR activation with J4 increased time spent in open arms of EPM and reduced the number of marbles buried in CIE vapor treated and ethanol naïve mice. Moreover, J4 was able to produce these effects without deficits in locomotion that are known to concomitantly occur with adenosine A2AR activation or elevated adenosine concentration. It should be made clear that the single concentration of J4 used in all experiments were determined from preliminary open-field testing, indicating 0.03 mg/kg J4 had no antagonistic effects on locomotion. Additionally, previous studies utilizing in-vivo microdialysis has shown J4 elevates extracellular adenosine levels within the hippocampus 30 minutes post-infusion with adenosine concentrations reaching its peak 1.5 hr after infusion (Chang et al. 2021).

A2AR are highly expressed within the striatum (Borroto-Escuela et al. 2018; Ferré et al. 2002; Fredholm et al. 2007; Fredholm et al. 2003; Fredholm and Svenningsson 2020; Sebastião and Ribeiro 2000), where they are found on GABAergic medium spiny neurons (MSN) (Kang et al. 2020; Shen et al. 2013; Shen et al. 2008). Activation of A2AR containing MSN neurons releases GABA (Asatryan et al. 2011; Nam et al. 2013b), an inhibitory neurotransmitter. Due to the unique pharmacological properties of J4 in the targeting of both ENT1 and A2AR thereby modulating adenosine tone, we sought to investigate whether J4 may produce calming effects in response to ethanol withdrawal-related behaviors. Based on literature(Lowery-Gionta et al. 2015; McCool and Chappell 2015), we hypothesized that mice exposed to numerous cycles of ethanol vapor for 16 hours each day for 4 consecutive days proceeded by a 3-day withdrawal period, a 7-day cycle repeated for 4 weeks, would produce lasting increase in negative affective behaviors (Angoa-Pérez et al. 2013; Karadayian et al. 2013; Rose et al. 2016; Sidhu et al. 2018). Contrary to our hypothesis, our results show limited changes to withdrawal-related behaviors. It should be noted however that during our CIE exposure paradigm, we did not use the alcohol dehydrogenase inhibitor pyrazole, which may have attributed to our minute effects of chronic ethanol exposure.

The EPM is an extensively used assay to measure anxiety-like behavior and evaluate potential use of suspected anxiolytic agents (Shoji and Miyakawa 2021). In our study, J4 treated animals displayed and increased in time spent in the open arms within the first 5 minutes of testing of the EPM, suggesting J4 treatment may dampen anxiety-like behavior (Jia et al. 2020b; Overstreet et al. 2004; Towner and Varlinskaya 2020). Interestingly, J4 treatment was able to produce these effects despite the limited anxiogenic effects of ethanol withdrawal.

J4 failed to curtail voluntary ethanol drinking in mice unlike its preceding compound, NHBA (Hong et al., 2019). Of note, J4 has higher binding affinity toward ENT1, and lower affinity toward the A2AR, compared to the first-generation molecule NHBA (Ho et al. 2020; Hsu et al. 2020). Thus, J4 mainly inhibits the ENT1, but not effectively activate the A2AR (Chang et al. 2021; Hsu et al. 2020; Huang et al. 2011). Interestingly, while activation of A2AR decreases ethanol drinking, inhibition of ENT1 increases ethanol drinking (Choi et al. 2004; Hong et al. 2019a; Nam et al. 2013b; Nam et al. 2011). It may be that the strong activity of J4 as an ENT1 blocker outweighs any potential effects of A2AR activation, explaining why J4 treated mice failed to reduced ethanol drinking in our present study, whereas NHBA was successful in reducing drinking in our previous published work (Hong et al., 2019). We would be remiss without mention of other preclinical investigations that have found major therapeutic benefit of J4 when mixed in the drinking water, allowing ad libitum access for several months (Chang et al. 2021), suggesting the effects of J4 may only be effective following long-term chronic treatment. Indeed, J4 and like compounds have beneficial effects in preclinical treatments of neurodegenerative diseases (including Huntington’s disease, Alzheimer’s disease, and aging) without detrimental side effects (Chang et al. 2021; Hsu et al. 2020; Huang et al. 2011; Kao et al. 2017).

Alcohol dependence characterized by habitual/compulsive drug-seeking leading to drug-taking (consumption), inability to limit intake, and emergence of withdrawal symptoms when abstinent from alcohol is a major contributing factor to the chronic relapsing nature of AUD mainly due to the negative reinforcing nature of alcohol (Perez and De Biasi 2015; Roberto et al. 2021). Previous studies have pointed to the increase in marble burying behavior within C57BL/6J mice following withdrawal from CIE (Kimbrough et al. 2020; Okhuarobo et al. 2020; Rose et al. 2016; Sidhu et al. 2018). Similarly, our experiment showed a slight increase in the numbers of marbles buried after 72 h CIE withdrawal. Perhaps most intriguing, once per day treatment of J4 (0.03 mg/kg) for 3 days abrogated increased marble burying in mice irrespective of chronic ethanol vapor exposure, suggesting a specific role for adenosine signaling during this behavior. There is some debate about the interpretation of marble burying behavior. Digging, burrowing, and burying behaviors are fundamental components of normal rodent behavior (de Brouwer et al. 2019). Indeed, burying responses towards nonharmful and harmful objects have been historically noted, with behavior toward the latter objects more notable (de Brouwer et al. 2019). To oppose, several studies have suggested the etiology of marble burying behavior to reflect a repetitive, perseverative, somewhat obsessive-compulsive like behavior (Angoa-Pérez et al. 2013; Eissa et al. 2018; Thomas et al. 2009). However, within the context of chronic alcohol withdrawal, perhaps the best interpretation of marble burying within mice is a symptom similar to psychomotor agitation which has been characterized during alcohol withdrawal in humans (Association 2013; Okhuarobo et al. 2020). In agreement with our study, other investigators have keenly highlighted that pharmacological co-activation of both the A1R and A2AR with respective N6-cyclopentyladenosine (CPA) and 2-p-(2-carboxyethyl)phenethylamino-50-N-ethylcarboxamidoadenosine (CGS21680) dose dependently reduced repetitive behaviors further providing support for adenosine signaling in repetitive or compulsion (Lewis et al. 2019; Tanimura et al. 2010). Interestingly, it has been suggested that dysregulation of the cortico-striato-thalamo-cortical circuitry by decreased activity within the indirect basal ganglia pathway is linked to compulsive/repetitive behaviors (Tanimura et al. 2010). Despite limited activity of J4 on A2AR, future studies should investigate the dynamic expression of adenosine receptors (A1R, A2AR, A2BR, and A3R) and important enzymatic regulators of adenosine production following CIE exposure and subsequent treatment with J4 within the indirect basal ganglia pathway under the context of marble burying since J4 has previously been shown to increase extracellular adenosine levels within the hippocampus (Chang et al. 2021). Further studies should dive deeply into investigating the molecular and behavioral mechanisms governing augmentation in marble burying behavior. More specifically, the role of adenosine signaling during marble burying behavior should be further scrutinized as a potential treatment for preservative/compulsive or anxiety related behaviors. Considering the limited number of therapeutic treatments for these disorders, adenosine modulation by J4 may represent a potential alternative to suppress compulsive/anxiety-like actions.

ACKNOWLEDGEMENTS

We thank all members of the DSC.’s laboratory for interest, help and comments.

FUNDING

This work was supported by the Samuel C. Johnson Genomics of Addiction Program at Mayo Clinic, the Ulm Foundation, and the National Institute on Alcohol Abuse and Alcoholism (AA018779, AA029258, AG072898).

Footnotes

CONFLICT OF INTEREST

D-S Choi is a scientific advisory board member to Peptron Inc. Peptron had no role in preparation, review, or approval of the manuscript. Y Chern holds patents on treating neurodegenerative diseases and pain by J4. All the other authors declare no biomedical financial interests or potential conflicts of interest.

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