
==== Front
0367050
6438
Pharmacol Biochem Behav
Pharmacol Biochem Behav
Pharmacology, biochemistry, and behavior
0091-3057
1873-5177

38679080
10.1016/j.pbb.2024.173776
nihpa2017097
Article
The CB1 negative allosteric modulator PSNCBAM-1 reduces ethanol self-administration via a nonspecific hypophagic effect
Buechler Harley M. a1
Sumi Mousumi a1
Madhuranthakam Indu Mithra a1
Donegan Christa a
DiGiorgio Frank Jr. a
Acosta Alisha A. a
Uribe Sarah a
Rahman Mohammad A. a
Sorbello Alison a
Fischer Bradford D. b
Keck Thomas M. a*
a Rowan University, Glassboro, NJ 08028, United States
b Cooper Medical School of Rowan University, Camden, NJ 08103, United States
1 These authors contributed equally to this work.

* Corresponding author at: Department of Biological & Biomedical Sciences and Department of Chemistry & Biochemistry, College of Science & Mathematics, Rowan University, Glassboro, NJ 08028, United States. keckt@rowan.edu (T.M. Keck).
26 8 2024
7 2024
27 4 2024
04 9 2024
240 173776173776
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Alcohol use disorder (AUD) affects >15 million people in the United States. Current pharmacotherapeutic treatments for AUD are only modestly effective, necessitating the identification of new targets for medications development. The cannabinoid receptor type 1 (CB1) has been a target of interest for the development of medications for substance use disorders and other compulsive disorders. However, CB1 antagonists/inverse agonists (e.g., rimonabant) have severe side effects that limit their clinical utility, including anxiety, depression, and suicide. Recent development of CB1 negative allosteric modulators (NAMs), including PSNCBAM-1, may provide an alternative mechanism of attenuating CB1 signaling with reduced side effects. PSNCBAM-1 has not yet been evaluated for effects in models of AUD. In this study, we investigated the effects of the CB1 NAM, PSNCBAM-1, in rodent models of AUD using adult male mice. PSNCBAM-1 dose-dependently attenuated oral ethanol self-administration (8 % w/v ethanol in water), significantly reducing ethanol rewards at a dose of 30 mg/kg, but not at 10 or 18 mg/kg. PSNCBAM-1 also dose-dependently attenuated palatable food self-administration (diluted vanilla Ensure), significantly reducing food rewards at 18 and 30 mg/kg PSNCBAM-1. PSNCBAM-1 did not affect conditioned place preference for 2 g/kg ethanol. These results suggest PSNCBAM-1 reduces ethanol-taking behavior via a nonspecific hypophagic effect and does not reduce the rewarding effects of ethanol.

PSNCBAM-1
Cannabinoid receptor
Alcohol self-administration
Alcohol use disorder
Conditioned place preference
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pmc1. Introduction

Alcohol use disorder (AUD) is a serious health condition with massive global impact, characterized by uncontrollable alcohol consumption due to physical and emotional dependence on alcohol and negative effects when alcohol is not consumed (Yang et al., 2022). The 2021 National Survey on Drug Use and Health (NSDUH) determined that 28.6 million adults in the United States aged 18 and above suffered from AUD in the prior year. Globally, in 2019, alcohol consumption was the cause of death for 2.07 million men and 374,000 women (Alcohol’s Effects on Health, n.d.). Current treatments for AUD have low rates of success, indicating a clear need for new pharmacotherapeutics.

The cannabinoid receptor type 1 (CB1), a G protein-coupled receptor (GPCR), expressed in the peripheral and central nervous system, is an intriguing target for development of medications for substance use disorders (Soler-Cedeno and Xi, 2022; Manzanares et al., 2018). The endogenous cannabinoid system signals primarily via CB1 and the cannabinoid receptor type 2 (CB2). CB1 is widely expressed throughout the brain, regulating learning, memory, decision-making, pain, and energy metabolism (Romero-Torres et al., 2023). CB1 signaling controls a wide variety of physiological functions, such as food intake, energy balance, cardiovascular functions, reproductive functions, immune modulation, and cell apoptosis (Janero, 2012).

Neuronal pathways that receive signals through CB1s in the CNS contribute to the rewarding effects of certain non-drug stimuli and many addictive drugs, leading to their excessive intake and pathological consequences. Recent studies have found that CB1 activity can cause a shift from normal behavior to impaired decision making and repetitive drug intake; pharmacologically targeting CB1 is one strategy to reverse these effects and ultimately treat substance use disorders (Manzanares et al., 2018; Henderson-Redmond et al., 2016).

Rimonabant (SR1417116A) was the first clinically available, potent, selective, orally active CB1 receptor antagonist (Soyka et al., 2008). Both in vitro and in vivo studies show that rimonabant antagonizes the behavioral and pharmacological effects induced by CB1 agonists (Elbatsh et al., 2012; Järbe and DiPatrizio, 2005; Järbe et al., 2010; Eckard et al., 2020). An appetite suppressant that was shown to cause weight loss and improve cardiovascular risk factors like dyslipidemia, waist circumference, and blood pressure, rimonabant was initially approved in Europe in 2006 for the treatment of obesity (Després et al., 2005; Sam et al., 2011). In 2008, the drug was withdrawn from the market due to serious psychiatric adverse effects, such as suicidal thoughts, anxiety, and depression (Sam et al., 2011).

In studies relevant to AUD, rimonabant reduced alcohol-taking and -seeking behaviors (Colombo et al., 2007; Arnone et al., 1997; Economidou et al., 2006). For instance, in studies involving selectively bred Sardinian alcohol-preferring (sP) rats, rimonabant reduced voluntary alcohol consumption (Colombo et al., 1998; Serra et al., 2001; Vinod et al., 2012). Furthermore, clinical studies of rimonabant revealed its effectiveness in smoking cessation and reducing alcohol dependence (Robinson et al., 2018; Cahill and Ussher, 2011). These findings suggest that pharmacotherapies that attenuate CB1 signaling hold promise as potential therapeutic agents in the treatment of AUD.

The clinical failure of rimonabant is typically attributed to its full antagonism, and likely inverse agonism, of CB1 signaling, producing severe and life-threatening mood disorders (Sam et al., 2011; Cahill and Ussher, 2011; Le Foll et al., 2009). To avoid serious adverse events, several research teams have focused on developing novel CB1 negative allosteric modulators (NAMs) with the idea that NAMs would attenuate CB1 signaling in an activity-dependent manner, providing some of the desired therapeutic effects with reduced side-effect profiles (Bertini et al., 2017; Nguyen et al., 2017a).

PSNCBAM-1 is classified as a NAM based on its effects in efficacy assays, although it enhances agonist binding (Nguyen et al., 2017a; Horswill et al., 2007; Cawston et al., 2013). PSNCBAM-1 reduces the overall activation of CB1 by agonists by modulating receptor internalization and thus its effects on cAMP accumulation, overall decreasing the cell’s response to CB1 activation. (Cawston et al., 2013; Gamage et al., 2017) Other CB1 NAMs share this set of pharmacological characteristics (Price et al., 2005). PSNCBAM-1 has not yet been evaluated for effects in rodent models of AUD. This study aimed to investigate the effects of PSNCBAM-1 in models of AUD using adult male mice, with the working hypothesis that PSNCBAM-1 would reduce alcohol reward and alcohol-taking and -seeking behavior. To test this hypothesis, we evaluated the effects of PSNCBAM-1 in ethanol conditioned place preference (CPP) and self-administration studies.

2. Materials and methods

2.1. Animals

All studies used drug-naïve male C57BL/6 mice, starting approximately 8 weeks of age. Mice were obtained from Charles River Laboratories (Wilmington, MA). All studies were carried out in the vivarium at Cooper Medical School of Rowan University (CMSRU). Mice were caged in polycarbonate cages (four animals per cage) in a temperature- and humidity-controlled vivarium, with ad libitum access to food and water, and enrichment provided by paper Bio-Huts and/or nestlets. All studies were conducted in procedure rooms separate from the housing facility during the light phase of the light/dark cycle (lights on at 7:00, lights off at 19:00). Animals were weighed every day and evaluated for general health and behavioral parameters. All the experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Rowan University. The CMSRU animal facility of Rowan University is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.

2.2. Drugs

PSNCBAM-1 (1-(4-chlorophenyl)-3-(3-(6-(pyrrolidin-1-yl)pyridin-2-yl)phenyl)urea; Tocris) was dissolved in 10 % DMSO, 10 % Tween 80, and 80 % saline vehicle for all studies, using sonication to achieve a clear solution. PSNCBAM-1 was administered via i.p. injections in all experiments at a volume of 10 mL/kg.

190 proof ethanol was purchased from Koptec. For CPP experiments, ethanol was mixed with physiological saline to achieve the desired mass/volume dilution and administered via i.p. injections. For self-administration experiments, ethanol was diluted in Vanilla Ensure™ and water to achieve the desired mass/volume dilution and was administered orally via operant chamber delivery. All dilutions were made weekly and stored at 4 °C between experiments.

2.3. Behavioral procedures

2.3.1. Locomotor activity

This test was conducted to determine whether PSNCBAM-1 induced substantial locomotor disruptions in mice.

2.3.2. Apparatus

Four 40 × 40 cm2 modular open field instruments were organized on a vertical shelving unit, two per shelf, for locomotor experiments. A small fluorescent light and a USB camera connected to a PC running Any-maze software were placed above each chamber. Each test apparatus and floor insert were cleaned with 70 % isopropyl alcohol and allowed to dry completely before and after all training and testing processes including between each animal in serial testing.

2.3.3. Treatment groups

This study used 32 drug-naïve male C57BL/6 mice. An initial set of 8 vehicle animals were compared to 8 receiving 30 mg/kg PSNCBAM-1. A later study compared 8 vehicle animals to 8 receiving 18 mg/kg PSNCBAM-1. No differences were seen between the vehicle groups so all data were collapsed.

2.3.4. Testing

All animals were exposed to the open field twice over the course of two days prior to testing to minimize novelty-induced locomotor activity. Animals were given a saline injection after 15 min in the open field during these 55-min pre-exposures to acquaint them with the handling and injection technique. During these pre-exposures, behavior was not recorded. On the test day, animals were given i.p. injections of 10 mg/kg PSNCBAM-1 or 30 mg/kg PSNCBAM-1 or vehicle mixture and placed in the open field chambers to record their behavior and activity.

2.3.5. Analysis

Locomotor activity for each animal was recorded in 5 min intervals. Overall locomotor activity is represented as the total distance traveled following PSCNBAM injection.

2.3.6. Food and ethanol self-administration

Self-administration studies were conducted to examine the effects of PSNCBAM-1 on operant behavior reinforced by a palatable food reward or ethanol. Animals were trained to nose poke to obtain food or ethanol. After sufficient training, animals were injected with the drug under investigation and evaluated for its anti-addiction property in terms of reduced ethanol self-administration.

2.3.7. Apparatus

Self-administration training and testing used mouse operant chambers (Med Associates, Fairfax, VT) placed in sound-attenuating cabinets and connected to a computer running MED-PC software (version 4). Each operant chamber featured two nose poke holes equipped with infrared beams. A liquid dipper was located between the two nose poke holes, connected via tubing to a pump and syringe that delivered the programmed reward. For both food and ethanol self-administration studies, the left nose poke hole was paired with food or ethanol reinforcers, while the right nose poke hole had no programmed consequences. Upon earning a programmed reward, the syringe pump discharged the liquid reinforcer for 3 s (delivering an approximate 0.1 mL volume). All rewards were accompanied by light and tone stimuli during the duration of the 3 s reward delivery. There was no programmed timeout.

2.3.8. Treatment groups

Two separate groups of 8 mice were used for food and ethanol self-administration studies, respectively. Animals in the ethanol group received identical food self-administration training before a fading procedure was used to transition them to ethanol. All eight mice met the training criteria for the food self-administration. 7 of 8 mice met the training criteria for the ethanol self-administration and one mouse was excluded from PSNCBAM-1 testing and analysis.

2.3.9. Food restriction

Before each training or testing session, mice were food restricted for approximately 21 h, but had ad lib access to water. Every day, mice were weighed and held within 10 % of their free-feeding weight. Animals were allowed ad lib access to chow for 1 h after each training or testing session. In prior pilot studies, we were unsuccessful in achieving reliable ethanol self-administration in mice without maintaining food self-administration.

2.3.10. Training

All mice were trained initially to nose poke for a palatable food reward, diluted vanilla Ensure™ (50:50 water:Ensure), initially using a fixed-ratio 1 (FR1) in which a single nose poke into the reinforcer-paired nose poke hole resulted in one reward delivery. As each mouse earned >90 of 100 possible food rewards in a 2-hour period over three days on a given FR level, the FR was successively increased, from FR2 to FR3 and FR4. Once stable FR4 food self-administration was achieved, animals moved onto drug testing (food self-administration group) or an ethanol fading procedure.

2.3.11. Ethanol fading procedure

Ethanol was gradually introduced into the diluted vanilla Ensure™, incrementally increasing the percentage of ethanol from 2 %, to 4 %, to 6 %, then to 8 % w/v (equivalent to 10.13 % v/v), replacing the water in the baseline 50:50 water:Ensure mixture. Then, the Ensure was incrementally replaced with water until reaching a final 8 % w/v mixture of ethanol and water in each earned reward. Animals progressed to the next dilution after earning a consistent number of earned reinforcers (± 10 %) for at least 3 straight days. Baseline 8 % w/v ethanol self-administration averaged ~10 rewards over a 2-hour training session; mice appeared visibly intoxicated at the end of training sessions (altered gait, sluggish responses) due to the relatively high training dose (averaging consumption of ~0.8 g alcohol/2 h). This progressive exposure to ethanol, leading up to the final concentration used in our study, spanned a period of approximately 100 training sessions over four months, ensuring that animals had a substantial history of ethanol experience before testing was conducted.

2.3.12. Testing

After meeting FR4 training criteria for food or ethanol intake, mice were tested with varying doses of PSNCBAM-1 (10 mg/kg, 18 mg/kg, 30 mg/kg, or vehicle) using a Latin square design. PSNCBAM-1 was given i. p. 5 min before placing the animal inside the operant chamber and beginning a standard 2-hour procedure. Each drug testing day was followed by two intervening days of training. A 5-min drug pretreatment time was chosen on the basis of prior pharmacokinetics studies suggesting a relatively short drug half-life (~13 min in rat liver microsomes (Nguyen et al., 2017b)) and a desire to maximize plasma drug concentrations during the 2-hour self-administration session. Pilot studies confirmed robust behavioral effect with a 5-min pretreatment time.

2.3.13. Conditioned place preference (CPP)

CPP is a method for evaluating the subjective effects of drugs of abuse. The CPP test works on the idea that primary reinforcers like legal or illegal drugs, food, water, or sex are coupled with contextual stimuli that gain secondary reinforcing characteristics through Pavlovian contingency.

2.3.14. Apparatus

The three-chamber CPP apparatus (Med Associates, Fairfax, VT) placed in sound attenuating cabinets and connected to a computer running MED-PC software (version 5). Each apparatus has two larger compartments with white or black walls and different floor grates. These compartments are connected via a smaller central gray compartment with closable sliding doors which could be used to block or allow the free movement of the mice into and out of the white and black compartments.

2.3.15. Initial preference

Before conditioning/preference training, animals were tested for a predisposed side preference by placing mice in the chambers with full access to all the compartments for 30 min, to remove any bias from the experiment. The ratio of time spent in one test compartment over the total time spent in the central compartment was calculated as a measure of initial compartment preference. Time spent in the central compartment was recorded but not considered in the calculation. Twelve animals with an initial preference of >65 % for one test compartment were excluded from training or further testing. The drug-paired and vehicle-paired compartments for each animal were assigned using a Latin square design, which evenly distributed compartment pairing, box placement, and first-day treatment among all treatment groups, as per an unbiased experimental procedure.

An unbiased procedure was used to divide animals into separate treatment groups and plan each training regimen. While biased techniques can provide increased sensitivity for detecting medication effects dependent on the animal’s baseline motivational states, or for detecting anxiolytic and anti-aversive drug effects, they also have a larger false-positive rate, which we wanted to minimize.

2.3.16. Treatment groups

30 mice were ultimately used for this study. Animals were divided into three groups following initial preference testing. The groups received 18 mg/kg PSNCBAM-1 (n = 10), 30 mg/kg PSNCBAM-1 (n = 10), or vehicle (n = 10) 5 min prior to the reinforcer (2.0 g/kg ethanol) for training days on the ethanol-paired side. Animals received vehicle doses prior to training on the saline-paired side. 18 mg/kg and 30 mg/kg PSNCBAM-1 doses were tested because these doses showed clear behavioral effects in the food and ethanol self-administration studies detailed below. The 5-min pretreatment time was chosen to match the self-administration studies.

2.3.17. Drug conditioning

10 training sessions were conducted over a 10-day period for the primary acquisition or conditioning to take place. Animals were given drug or vehicle injections immediately before placing the animal inside the CPP apparatus and confined in the specified test chamber for 30 min during training. Drug and vehicle exposures alternated daily, with the pattern of exposures counterbalanced within groups across days and apparatuses.

2.3.18. Preference test

In a CPP expression trial experiment, trained mice were given no injection and were given free access to the complete apparatus (including both drug- and vehicle-paired compartments) for 30 min.

2.3.19. Analysis

For both the initial (Δpretest) and final preference (Δposttest) tests, a preference score was generated by subtracting the seconds spent in the vehicle-paired chamber from the seconds spent in the drug-paired compartment (time in the central compartment was ignored). The following formula was used to compute an overall preference score for a specific pharmacological dosage: Preference score (sec) = Δposttest – Δpretest.

2.3.20. Data analysis

Data from all studies was collected and analyzed (GraphPad Prism 6) using one-way or repeated-measures ANOVA, as appropriate, followed by pre-planned Bonferroni analyses.

3. Results

The effects of PSNCBAM-1 were tested in initial control studies to determine whether PSNCBAM-1 would produce general behavioral disruptions that might complicate interpretations of other planned behavioral tests. Initial control studies indicated that 10 and 30 mg/kg, i.p., PSNCBAM-1 did not significantly disrupt locomotor activity in the open field (Fig. 1). One-way ANOVA analysis of total post-injection locomotor activity revealed no significant effect of treatment (F(2,29) = 0.5594, p > 0.5).

3.1. CPP

Three different dosages, 10 mg/kg, 18 mg/kg, and 30 mg/kg, in addition to a vehicle mixture, were examined as a pretreatment to a 2.0 g/kg dose of ethanol or saline vehicle for the PSNCBAM-1 CPP tests (Fig. 2). PSNCBAM-1 administration during conditioned place preference training had no effect on the rewarding value of 2.0 g/kg ethanol. A one-way ANOVA study of preference for the ethanol-paired compartment indicated that PSNCBAM-1 administration had no significant impact (F (2,27) = 0.3603, p > 0.7). A paired two-tailed t-test of initial preference vs. final preference for the ethanol-paired compartment for the vehicle group indicates that ethanol induced a significant place preference in the vehicle-only treatment group (t(9) = 2.370, p = 0.0419).

3.2. Palatable food self-administration

All mice were trained initially to self-administer a 50 % Ensure/50 % water combination for food self-administration. The mice were evaluated in a Latin square design with two different dosages of PSNCBAM-1, 18 mg/kg and 30 mg/kg, and vehicle. Mice were given i.p. injections of PSNCBAM-1 or vehicle prior to a 2-hour self-administration session on test days. PSNCBAM-1 inhibited palatable food self-administration in a dose-dependent manner, considerably lowering food rewards at 18 mg/kg and 30 mg/kg (Fig. 3). One-way repeated-measures ANOVA revealed a significant effect of PSNCBAM-1 treatment (F (3,21) = 8.410, p = 0.0007). Pre-planned Bonferroni tests revealed a significant difference between vehicle treatment and 18 mg/kg (t = 3.426, p < 0.05) and 30 mg/kg PSNCBAM-1 (t = 4.298, p < 0.01).

3.3. Ethanol self-administration

To determine whether PSNCBAM-1 could reduce ethanol self-administration behavior, mice trained to self-administer 8 % (w/v) ethanol were tested with various doses of PSNCBAM-1 in a Latin square design (Fig. 4). On test days, mice were given i.p. injections of PSNCBAM-1 or vehicle prior to a 2-hour self-administration session.

One-way repeated-measures ANOVA revealed a significant effect of PSNCBAM-1 treatment (F (3,18) = 4.264, p = 0.0193). Pre-planned Bonferroni tests revealed a significant difference between vehicle and 30 mg/kg PSNCBAM-1 treatment (t = 3.016, p < 0.05) but not between vehicle and 18 mg/kg PSNCBAM-1 treatment (t = 2.537, p > 0.05).

4. Discussion

Studies have consistently shown that attenuation of CB1 signaling can suppress appetitive behaviors for food as well as drugs of abuse (Manzanares et al., 2018; Ward et al., 2009; Gobira et al., 2019; Maccioni et al., 2010; Luján et al., 2022; Yu et al., 2009; Li et al., 2008; Biala et al., 2009). PSNCBAM-1 is a CB1 NAM that acts centrally and peripherally. In the CNS, PSNCBAM-1 exerts effects opposite to that of CB1 agonists like THC and CP-55,940. Prior to this study, PSNCBAM-1 had never been evaluated for effects in rodent models of AUD.

In these studies, PSNCBAM-1 did not significantly affect the acquisition of place preference to 2.0 g/kg ethanol, suggesting that the doses tested (18 and 30 mg/kg) did not alter the rewarding value of 2 g/kg ethanol. In operant studies, PSNCBAM-1 attenuated food and ethanol (8 % w/v) self-administration at doses that did not affect CPP. Control experiments indicate that PSNCBAM-1 did not significantly affect locomotor activity in an open-field at doses that altered self-administration behavior. Taken together, these results suggest that PSNCBAM-1-mediated attenuation of ethanol self-administration was not driven by any reduction in the rewarding value of alcohol but rather a general hypophagic response. The results of this study are fully consistent with a prior investigation of PSNCBAM-1 that reported hypophagic effects of PSNCBAM-1 in rodents, reducing food intake and body weight (Horswill et al., 2007). Overall, these results do not support the utility of PSNCBAM-1 for AUD treatment.

Our reported outcome is seemingly at odds with results reported by Lovelock et al. (2022), in which the newer CB1 NAM RTICBM-74 (Nguyen et al., 2017b) reduced alcohol intake in rats trained to self-administer ethanol. However, substantial differences in methods employed may explain this divergence. Our experiments used mice rather than rats, used a lower ethanol concentration (10.13 % v/v compared to 15 % v/v) but longer self-administration sessions (2 h compared to 30 min) with a higher fixed-ratio (FR4 compared to FR2). PSNCBAM-1 and RTICBM-74 have substantially different pharmacokinetics (Nguyen et al., 2017b; Lovelock et al., 2022). Each of these procedural and pharmacological differences could have contributed to differences in our reported outcomes. Importantly, this study only evaluated male mice of a single strain, and all tests were performed within the light part of the light:dark cycle. Male and female mice have different sensitivities to the locomotor effects induced by ethanol (Dudek et al., 1991; Middaugh et al., 1992) and patterns of ethanol self-administration can vary between male and female mice (Middaugh and Kelley, 1999). Rats and mice also have different capacities for operant learning. Finally, we only tested a single dose of ethanol in our CPP studies—a dose that is at the peak of our ethanol dose-effect curve in pilot studies—and it is possible that PSNCBAM-1 may have influenced the rewarding value of smaller or larger ethanol doses.

The distinct pharmacological profiles and behavioral impacts of CB1 NAMs across different species underscore the complexity of cannabinoid signaling in substance use disorders. In the study by Gamage et al. (2017), CB1 NAM Org27569 was evaluated in mice using behavioral assays sensitive to CB1 orthosteric agonists and antagonists. Interestingly, unlike rimonabant, an orthosteric CB1 antagonist/inverse agonist, Org27569’s anorectic effects were independent of the CB1 receptor, indicating a different mechanism of action. The study found that Org27569 did not produce CB1-mediated effects alone and did not affect the actions of orthosteric agonists, challenging its utility as a ‘gold standard’ CB1 AM in in vivo settings. This highlights the complexity of CB1 NAM pharmacology and underscores the need for CB1 AMs with effects that translate from the molecular level to the whole animal.

Furthermore, the findings in our study resonate with the broader narrative of CB1 receptor modulation in addiction. The pharmacological actions of CB1 NAMs, as illustrated in Jing et al. (2014) and Gamage et al. (2017), demonstrate a nuanced and species-specific influence on addiction-related behaviors. While Org27569 showed efficacy in rat models of drug relapse, its effects in mouse behavioral assays were notably different, highlighting the challenges in translating CB1 receptor modulation across species. These studies collectively emphasize the importance of considering species differences when interpreting the effects of CB1 NAMs and their potential in treating substance use disorders.

In conclusion, we examined the effects of the CB1 NAM PSNCBAM-1 in mouse models relevant to AUD. Our results showed that PSNCBAM-1 pretreatment did not significantly disrupt locomotor activity or affect the rewarding value of 2.0 g/kg ethanol. PSNCBAM-1 dose-dependently attenuated ethanol self-administration, reducing ethanol rewards at the 30 mg/kg dose, but also dose-dependently attenuated palatable food self-administration, significantly reducing food rewards at the 18 mg/kg and 30 mg/kg doses. These results are most parsimoniously explained by a PSNCBAM-1-mediated effect on general appetitive behavior and not on the rewarding value of ethanol.

Acknowledgments

This work was supported by internal funds from Rowan University and Cooper Medical School of Rowan University. Equipment used for these studies were purchased with support from DA041560.

Dedication

This manuscript is dedicated to the memory of Sonnie Sheahan (Rowan B.S. in Biological Sciences, 2020) who contributed to initial alcohol conditioned place preference studies that formed a foundation for this work. His light was extinguished far too soon.

Data availability

Data will be made available on request.

Fig. 1. PSNCBAM-1 did not significantly alter locomotor activity in the open field. (A) 20 min after introduction into the open field, mice were injected with 10 or 30 mg/kg PSNCBAM-1 or vehicle and locomotor activity was recorded for an additional 40 min. (B) Overall post-injection distance traveled was not significantly different across treatments. All data are presented as means ± SEM.

Fig. 2. PSNCBAM-1 did not significantly alter conditioned place preference for 2 g/kg ethanol. Pretreatment with 18 or 30 mg/kg PSNCBAM-1 prior to ethanol treatment did not attenuate acquisition of ethanol place preference compared to vehicle control. All data are presented as means ± SEM.

Fig. 3. 18 and 30 mg/kg, but not 10 mg/kg, PSNCBAM-1 significantly decreased self-administration of a palatable food reward. Pretreatment with 18 or 30 mg/kg PSNCBAM-1 prior to a food self-administration session reduced the number of palatable food rewards received compared to vehicle control. All data are presented as means ± SEM. * p < 0.05, ** p < 0.01, compared to vehicle control.

Fig. 4. 30 mg/kg, but not 10 or 18 mg/kg, PSNCBAM-1 significantly decreased ethanol self-administration. Pretreatment with 30 mg/kg PSNCBAM-1 prior to an ethanol self-administration session reduced the number of ethanol rewards received compared to vehicle control. All data are presented as means ± SEM. * p < 0.05, compared to vehicle control.

Declaration of competing interest

On behalf of all authors, the corresponding author states that there are no conflicts of interest.

CRediT authorship contribution statement

Harley M. Buechler: Conceptualization, Data curation, Formal analysis, Writing – original draft. Mousumi Sumi: Conceptualization, Data curation, Formal analysis, Writing – original draft. Indu Mithra Madhuranthakam: Investigation, Writing – review & editing. Christa Donegan: Investigation. Frank DiGiorgio: Investigation. Alisha A. Acosta: Investigation. Sarah Uribe: Investigation. Mohammad A. Rahman: Investigation. Alison Sorbello: Investigation. Bradford D. Fischer: Methodology, Resources, Software, Writing – review & editing. Thomas M. Keck: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing.
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