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. Author manuscript; available in PMC: 2021 Jul 1.
Published in final edited form as: Addict Biol. 2019 Jun 19;25(4):e12783. doi: 10.1111/adb.12783

Cannabidiol inhibits sucrose self-administration by CB1 and CB2 receptor mechanisms in rodents

Guo-Hua Bi 1,#, Ewa Galaj 1,#, Yi He 1, Zheng-Xiong Xi 1,*
PMCID: PMC6920611  NIHMSID: NIHMS1028474  PMID: 31215752

Abstract

A growing number of studies suggest therapeutic applications of cannabidiol (CBD), a recently FDA-approved medication for epilepsy, in treatment of many other neuropsychological disorders. However, pharmacological action and the mechanisms by which CBD exerts its effects are not fully understood. Here we systemically examined the effects of CBD on oral sucrose self-administration in rodents and explored the receptor mechanisms underlying CBD-induced behavioral effects using pharmacological and transgenic approaches. Systemic administration of CBD (10, 20, 40 mg/kg, i.p.) produced a significant reduction in sucrose self-administration in rats and in wild type (WT) and CB1−/− mice, but not in CB2−/− mice. CBD appeared to be more efficacious in CB1−/− mice than WT mice. Similarly, pretreatment with AM251, a CB1R antagonist, potentiated, while AM630, a selective CB2R antagonist, blocked CBD-induced reduction in sucrose self-administration, suggesting the involvement of CB1 and CB2 receptors. Furthermore, systemic administration of JWH133, a selective CB2R agonist, also produced a dose-dependent reduction in sucrose self-administration in WT and CB1−/− mice, but not in CB2−/− mice. Pretreatment with AM251 enhanced, while AM630 blocked JWH133-induced reduction in sucrose self-administration in WT mice, suggesting that CBD inhibits sucrose self-administration possibly by CB1 receptor antagonism and CB2 receptor agonism. Taken together, the present findings suggest that CBD may have therapeutic potential in reducing binge eating and the development of obesity.

Keywords: Cannabidiol, cannabinoid, sucrose self-administration, CB1 receptor, CB2 receptor, feeding behavior

Introduction

It is well known that excessive food intake and binge eating contribute to the national epidemic of obesity. The endocannabinoid system has been implicated in numerous aspects of eating-related behaviors and disorders (Jager and Witkamp, 2014; Lau et al., 2017). Previous studies have demonstrated that CB1R agonists can induce overeating (Williams and Kirkham, 1999), while CB1R antagonists suppress food intake and weight gain in rodents (Colombo et al., 1998; McLaughlin et al., 2003), suggesting potential use of CB1R antagonists in treatment of body overweight and obesity. However, the related clinical trials have been terminated worldwide due to the depressive and anxiogenic properties of CB1R antagonists (Le Foll et al., 2009; Patel and Hillard, 2006; Tambaro et al., 2013).

In recent years much attention has been given to cannabidiol (CBD), the second major component extracted from the Cannabis sativa plant (Mechoulam et al., 1970). Unlike Δ9-tetrahydrocannabinol (Δ9-THC), the major psychoactive component in cannabis, CBD is devoid of psychotropic effects (Martin-Santos et al., 2012; Viudez-Martínez et al., 2018). Recent research suggests that CBD may have a wide range of medical applications in treatment of epilepsy (Devinsky et al., 2016; Sands et al., 2018), anxiety (Crippa et al., 2011; Zuardi et al., 1993), schizophrenia (Morgan and Curran, 2008; Schubart et al., 2011), neurodegenerative disorders (Esposito et al., 2006; García-Arencibia et al., 2007) and even cancer (Kenyon et al., 2018). A significant progress in cannabinoid research has been made such as CBD was approved by the USA Food and Drug Administration (FDA) in June 2018 for treatment of epilepsy. Furthermore, it has been reported that CBD may have therapeutic potential in controlling food intake and preventing obesity. A number of studies indicate that CBD can inhibit food consumption (Farrimond et al., 2012; Ignatowska-Jankowska et al., 2011; Sofia and Knobloch, 1976) and responding for food or sweetened water in rats and monkeys (Brady and Balster, 1980; Hiltunen et al., 1989). In contrast, a lack of CBD effects on food-related behaviors in rats or mice has also been reported (Musty and Sands, 1978; Scopinho et al., 2011; Wiley et al., 2005). Thus, more research is required to determine the role of CBD in controlling body weight and obesity. Given recent findings that CBD has the ability to reduce alcohol (Viudez‐Martínez et al., 2018), cocaine (Luján et al., 2018) or methamphetamine (Hay et al., 2018) self-administration, in the present study, we systemically examined the pharmacological action of CBD on oral sucrose self-administration and explored the receptor mechanisms through which CBD alters sucrose-taking.

Sucrose is a natural energy source and reward (sweet) substance that provides higher reward valence even than cocaine (Lenoir et al., 2007). The consumption of highly sweetened diets is now highly prevalent in developed countries and is thought to contribute to the current obesity epidemic (Malik et al., 2006). Therefore, oral sucrose self-administration is a commonly used animal model to study feeding, binge-eating and food-taking disorders. CBD has been shown to have multiple acting targets including CB1 and CB2 receptors (Pertwee, 2008), GPR55 receptor (Ryberg et al., 2007), 5-HT1A receptor (Russo et al., 2005), GPR3, GPR6, GPR12 (Laun et al., 2018), μ and δ opioid receptors (Kathmann et al., 2006), vallinoid receptor 1 (TRPV1) (Bisogno et al., 2001) and peroxisome proliferator‐activated receptor γ (PPAR γ) (Campos Alline Cristina et al., 2012). However, more recent studies suggest that CBD is a potent allosteric modulator of CB1 and CB2 receptors with nanomolar binding affinity (Martínez-Pinilla et al., 2017; Navarro et al., 2018; Tham et al., 2019; Thomas et al., 2007). These new findings suggest that CB1 and CB2 receptors could be important targets in the pharmacological action produced by CBD. To test this hypothesis, we used pharmacological and transgenic approaches to explore a possible involvement of CB1 and CB2 receptors in CBD-induced behavioral effects.

Methods

Animals

Adult male Long Evans rats and mice were used in this study. Male wild-type (WT) and CB1−/− mice with C57BL/6J genetic backgrounds were bred at the National Institute on Drug Abuse (NIDA) from CB1+/− breeding pairs that were generously donated by Dr. Andrea Zimmer. CB2−/− mice with C57BL/6J genetic backgrounds were bred from CB2+/− breeding pairs that were generously donated by Dr. George Kunos (National Institute on Alcohol Abuse and Alcoholism). All animals were matched for age (8–14 weeks) and weight (rats, 250–350 g; mice, 25–35 g). They were housed in a climate-controlled animal colony room on a reversed light-dark cycle (lights on at 7:00 p.m., lights off at 7:00 a.m.) with free access to food and water. One week prior to the start of experiments animals received daily rations of food in order to maintain their weights at 85% of free feeding values. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Research Council and were approved by the Animal Care and Use Committee of the NIDA of the US National Institutes of Health (NIH).

2.2. Drugs

Cannabidiol (CBD) was generously provided by the NIDA Drug Supply Program and was dissolved in the 5% cremophor. Sucrose (Sigma-Aldrich) was dissolved in 0.9% physiological saline to achieve 5% concentration that was delivered in a volume of 0.1 or 0.02 ml onto a food trough. JWH133, AM251, and AM630 were purchased from Tocris Bioscience. JWH133 was dissolved in soya oil-based Tocrisolve-100 (Tocris Bioscience, USA). AM251 and AM630 were dissolved in 5% cremophor and were administered intraperitoneally (i.p.).

Procedure

Operant conditioning chambers

Sucrose self-administration experiments were conducted in operant conditioning chambers (Med Associates, US), each placed in a ventilated, sound-attenuating cubicle. Each operant chamber was equipped with two levers located 2.5 cm above the floor, a cue light, a speaker located 5 cm above the active lever and a food trough onto which liquid sucrose was delivered upon a lever press.

Oral Sucrose Self-Administration

Procedures for oral sucrose self-administration in rats and mice were the same as we reported previously (You et al., 2018; Zhang et al., 2015). Briefly, animals were trained to self-administer sucrose under a fixed ratio 1 (FR1) schedule of reinforcement during daily 3-hour sessions. Responding on an active lever activated the syringe pump causing the delivery of 5% liquid sucrose onto a liquid food receptacle (0.1 ml per delivery in rats, 0.02 ml per delivery in mice) and the presentation of the light/tone cue above the active lever. Responses on an inactive lever were counted but had no consequences. During the 4.2-s infusion period, additional responses on the active lever were recorded, but did not lead to additional infusions. To prevent satiation of sucrose reward, we set a maximal number of 100 sucrose deliveries during each 3-h session. Animals were tested with different compounds once stable sucrose self-administration was achieved, defined as i) at least 20 sucrose rewards earned per 3-h session, ii) less than 20% variability in daily sucrose intakes across two consecutive sessions, and iii) an active/inactive lever press ratio exceeding 2:1. All animals met these criteria before being tested with one of the compounds.

Experiment 1. Effects of CBD on sucrose self-administration in rats

We first assessed the effects of CBD on sucrose self-administration in rats under a FR5 schedule of reinforcement. Animals (n=12) were first trained to self-administer sucrose under FR1 schedule of reinforcement and then on a FR5 schedule of reinforcement. After stable self-administration was achieved for at least 3 consecutive days, rats were injected with one of the CBD doses (vehicle, 20 or 40 mg/kg, i.p.) 30 min prior to a self-administration session. On following days rats were re-stabilized and later re-tested with a different dose of CBD. The order of CBD doses that the animals were tested with was counterbalanced. Only the animals (n=9) received all three doses of CBD treatment were included in the data analysis.

Experiment 2. Effects of CBD on sucrose self-administration in WT, CB1−/−, CB2−/− mice

To ensure that our results are reproducible across different species and to determine the potential involvement of CB1 and/or CB2R mechanisms in CBD action, we then assessed the effects of CBD on sucrose self-administration in WT (n=12), CB1−/− (n=6) and CB2−/− (n=7) mice. The experimental procedures for sucrose self-administration in mice were the same as those in Experiment 1, except the FR1 schedule of reinforcement was used throughout the experiment. The drug treatment was the same as described above. Only the animals (9 WT, 6 CB1-KO, 6 CB2-KO) received all three doses of CBD treatment were included in the data analysis.

Experiment 3. The effects of CBD treatment on sucrose self-administration in the presence of CB1 or CB2 receptor antagonism

To confirm the findings observed in the above transgenic mice, we further assessed the effects of CB1 or CB2R antagonists on CBD-induced reduction in sucrose self-administration. After stable responding was achieved, WT mice were divided into 4 groups – vehicle (5% cremophor) group (n=12), vehicle + CBD (20 mg/kg) group (n=12), AM251 (3 mg/kg) + CBD (20 mg/kg) group (n=12), and AM630 (3 mg/kg) + CBD (20 mg/kg) group (n=8). Thirty min prior to the test session mice were injected according to the assigned treatment and later were allowed to self-administer sucrose under a FR1 schedule of reinforcement, under the same conditioned as described above. We then observed the effects of vehicle (Trocrisolve-100, n=12), AM251 (3 mg/kg; n=11) or AM630 (3 mg/kg; n=9) alone on oral sucrose self-administration under a FR1 schedule of reinforcement in WT mice.

Experiment 4. The effect of CBD treatment on sucrose self-administration under a PR schedule of reinforcement in WT mice

To determine whether CBD treatment alters motivation for sucrose self-administration WT mice were trained to self-administration sucrose first under the FR1 and then PR schedules of reinforcement. Under a PR schedule, requirement of lever presses for a single sucrose delivery was progressively raised within each session according to the following PR series: 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492 and 603 so that the animal reached a break point (Li et al., 2017; Richardson and Roberts, 1996). Final-ratio (or break-point) was defined as the number of lever presses completed for the last sucrose delivery before a one-hour period during which no sucrose reward was obtained. Animals were allowed to continue daily sessions of sucrose self-administration until day-to-day variability in final ratio fell within 1–2 ratio increments for 3 consecutive days. On the testing day, 30 min prior to a test session, mice were injected with one of the CBD doses (0, 20 or 40 mg/kg) and then allowed to press the lever for sucrose under the PR schedule of reinforcement. The order of drug injections was counterbalanced.

Experiment 5. The effects of JWH133 on sucrose self-administration in mice

In this experiment, we further explored whether the selective CB2R agonist, JWH133, alters sucrose-self administration in WT (n=11), CB1−/− (n=6), and CB2−/− mice (n=6) in a way similar to CBD. The self-administration procedures were the same as in the Experiment 2. After stable responding was achieved for at least 3 consecutive days, each animal was injected with one of the JWH133 doses (vehicle, 10 or 20 mg/kg, i.p.) 30 min prior to self-administration test session. Each animal received 3 doses of drug injections 3–5 days apart.

Experiment 6. The effects of JWH133 on sucrose self-administration in the presence of CB1 or CB2 antagonism

Similarly as in Experiment 3, we used pharmacological approaches to observe whether pretreatment with CB1 or CB2R antagonist can block JWH133-induced reduction in sucrose self-administration. Again, the experimental procedures were the same as in Experiment 3. Additional four groups of WT mice – vehicle (Tocrisolve-100; n=12), vehicle (Tocrisolve-100) + JWH133 (20 mg/kg; n=11), AM251 (3 mg/kg) + JWH133 (20 mg/kg; n=9), and AM630 (3 mg/kg) + JWH133 (20 mg/kg; n=8), were used to assess the effects of AM251 or AM630 pretreatment on JWH133 action in sucrose self-administration.

Data analyses

All data were expressed as mean ± S.E.M. In Experiments 1 and 2 percentage of baseline sucrose intake for each animal was calculated by dividing the number of sucrose deliveries on the test day by the average number of sucrose deliveries earned in the last two baseline days (before the test day).

One-way ANOVAs for repeated measures (RM) over drug dose were used for statistical analysis followed with post-hoc Bonferroni tests with correction for multiple group comparisons. Statistical significance was defined as p<0.05.

Results

CBD inhibits sucrose self-administration in rats

Figure 1A shows the acquisition of responding for sucrose that reached the asymptote after 5 sessions of training. Figure 1B shows that systemic administration of CBD produced a trend but not a significant reduction in the total number of sucrose self-administration in rats (one-way RM ANOVA, F2, 16 = 3.316, p=0.062). However, when we analyzed percent baseline of sucrose intake, we found that CBD treatment produced a significant and dose-dependent reduction in sucrose self-administration (Fig. 1C, F2, 16 = 4.159, p=0.035). Post-hoc Bonferroni tests revealed that the 40 mg/kg, but not 20 mg/kg, dose of CBD significantly reduced sucrose intake (p<0.05). Figure 1D shows inactive lever responding, indicating that CBD had no effect on inactive lever presses (F2, 16 = 1.542, p=0.224), suggesting no significant sedative effects under the CBD treatment.

Figure 1.

Figure 1

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The effects of CBD on sucrose self-administration under a FR5 schedule of reinforcement in rats. A. Mean (± SEM) number of sucrose deliveries during the training B: Mean (± SEM) number of sucrose deliveries in the absence or presence of the CBD treatment. C: Percent baseline of sucrose intake. D. Mean (± SEM) number of inactive lever presses. *p < 0.05, compared to vehicle (0 mg/kg) control group.

CBD inhibits oral sucrose self-administration in mice

Figure 2A shows the acquisition of responding for sucrose during the training where CB1-KO mice took longer to stabilize and reach the asymptote. Figure 2B shows that systemic administration of CBD produced a significant and dose-dependent reduction in sucrose self-administration in WT (one-way RM ANOVA, F2, 16 = 3.938, p=0.04) and CB1−/− mice (F2, 10=2.97, p=0.01), but not in CB2−/− mice (F2, 10 = 0.892, p=0.44). Figure 2C shows that CBD treatment significantly inhibited percent baseline of sucrose intake in WT (one-way RM ANOVA, F2, 16 = 3.91, p=0.041) and CB1−/− (F2, 10 = 7.37, p=0.01) mice, but not in CB2−/− (F2, 10 = 1.546, p=0.26) mice. Post-hoc Bonferroni tests for multiple group comparisons revealed that CBD, at 20 mg/kg, significantly lowered sucrose intake in WT and CB1−/− mice (p<0.05). Figure 2D shows that systemic administration of CBD did not alter inactive lever presses in any strain of mice.

Figure 2.

Figure 2

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The effects of CBD on sucrose self-administration under a FR1 schedule of reinforcement in WT, CB1−/− and CB2−/− mice. A. Mean (± SEM) number of sucrose deliveries during the training. B: Mean (± SEM) number of sucrose deliveries in the absence or presence of the CBD treatment. C: Percent baseline of sucrose intake. D. Mean (± SEM) number of inactive lever presses. *p<0.05, compared to vehicle control group in each genotype of mice.

Blockade of CB2, not CB1, receptors blocks CBD action

Figure 3A (left panel) shows that CBD, at 20 mg/kg, significantly inhibited sucrose self-administration in the absence of AM251 (a CB1R antagonist) or AM630 (a CB2R antagonist). This effect was enhanced in the presence of AM251 pretreatment but blocked by AM630 pretreatment. A one-way ANOVA revealed a significant treatment effect (F3, 44 = 20.388, p<0.001, Figure 3A). Post-hoc Bonferroni tests for multiple group comparisons indicated that CBD-induced reduction in self-administration and AM251-induced enhancement of CBD action were statistically significant. Figure 3A (right panel) shows neither AM251 nor AM630 alone altered sucrose intake in WT mice (one-way ANOVA, F2, 29 = 0.617, p=0.574). Figure 3B shows that CBD treatment significantly lowered break-point for sucrose self-administration in WT mice (one-way ANOVA, F2, 16 = 9.55, p=0.001). Post-hoc Bonferroni tests revealed that reductions produced by 10 and 20 mg/kg doses of CBD were statistically significant as compared to the vehicle control group (p<0.001).

Figure 3.

Figure 3

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The effects of CBD on sucrose self-administration in WT mice in the presence of AM251 (a CB1R antagonist, 3 mg/kg) or AM630 (3 mg/kg, a CB2R antagonist) or under PR schedule of reinforcement. A: Mean (± SEM) number of sucrose intake under a FR1 schedule of reinforcement. B: PR break-point in sucrose self-administration in the presence or absence of CBD. *p<0.05, **p<0.01, ***p<0.001, compared to (Veh + Veh) group (A) or in vehicle (0 mg/kg) control group. #p<0.05, compared to (Veh+CBD) group.

JWH133 inhibits sucrose self-administration in mice

Based on our findings that a CB2R mechanism underlies the pharmacological action of CBD, we assessed whether systemic administration of JWH133, a highly selective CB2R agonist, can produce a similar reduction in sucrose self-administration. Figure 4A shows that JWH133, at 10 and 20 mg/kg doses, significantly inhibited sucrose self-administration in WT (F2, 20 = 16.0, p<0.001, one-way RM ANOVA) and CB1−/− (F2, 10 = 8.184, p<0.01) mice, but not in CB2−/− mice (F2,10 = 0.455, p=0.647). Post-hoc Bonferroni tests indicated that reductions observed in WT or CB1−/− mice were statistically significant after 10 mg/kg or 20 mg/kg CBD treatment (p<0.05).

Figure 4.

Figure 4

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The effects of JWH133, a selective CB2R agonist, on sucrose self-administration in mice. A: Treatment with JWH133 (10, 20 mg/kg) dose-dependently inhibited sucrose self-administration in WT and CB1−/− mice, but not in CB2−/−mice. B: Pretreatment with AM630 (3 mg/kg, i.p.), but not AM251 (3 mg/kg, i.p.), blocked JWH133-induced reduction in sucrose self-administration **p < 0.01, **p<0.001, compared to vehicle (A) or (Veh + Veh) (B) control group.

AM630 blocks JWH133 action in sucrose self-administration

Figure 4B shows the effects of pretreatment with AM251 or AM630 on CBD action, indicating that AM251 (3 mg/kg, i.p.) failed, but AM630 blocked CBD-induced reduction in sucrose self-administration. A one-way ANOVA revealed a significant treatment effect (F3, 36 = 12.00, p<0.001). Post-hoc Bonferroni tests revealed that CBD-induced reduction was statistically significant after vehicle or AM251 pretreatment (p<0.05).

Discussion

The goals of this study were to investigate whether CBD can reduce sucrose intake in rodents and to explore the receptor mechanisms underlying CBD action. The major findings include: 1) systemic administration of CBD produced a significant and dose-dependent reduction in sucrose-self administration in rats and mice under FR1, FR5 and PR schedules of reinforcement, suggesting a reduction in sucrose reward; 2) pharmacological blockade or genetic deletion of CB1Rs failed to block CBD-induced reduction in sucrose self-administration. However, blockade of CB1Rs by AM251 produced an effect similar to CBD, while pretreatment with AM251 produced an enhancement in CBD action, suggesting possible involvement of CB1R antagonism in CBD action; 3) pharmacological blockade or genetic deletion of CB2Rs blocked the pharmacological action of CBD in sucrose self-administration, suggesting a CB2R mechanism involvement; and finally, 4) stimulation of CB2Rs by JWH133 produced a reduction in sucrose self-administration in a way similar to CBD. Taken together, these findings suggest that CBD-induced reduction in sucrose self-administration may be mediated by inhibition of CB1Rs and stimulation of CB2Rs.

CBD inhibits oral sucrose intake

Cannabis contains more than 100 chemical components that share a similar chemical cannabinoid structure. CBD was isolated from cannabis in 1940 (Adams et al., 1940) and then identified as a non-psychotropic cannabinoid in 1970 (Mechoulam et al., 1970). Since then, a growing body of research has shown that CBD could be a useful and promising medication for the treatment of epilepsy, substance abuse and dependence, schizophrenia, pain, anxiety, depression, sleep disorders, and Parkinson’s disease (Crippa et al., 2018; Hermann and Schneider, 2012). In June 2018, the FDA approved Epidiolex (CBD) for treatment of seizures associated with two rare and severe forms of epilepsy.

An important finding in the present study is that CBD is also effective in attenuation of sucrose intake and motivation for sucrose, as assessed by reductions in sucrose self-administration and break-points for sucrose reward. CBD appears more potent and effective in mice than in rats since higher doses of CBD are required in rats to produce a significant reduction in sucrose self-administration. This may be related to the different pharmacokinetic profiles in rats versus mice. The present finding is consistent with previous reports that CBD is effective and useful at controlling food intake, body weight and preventing the development of obesity (Farrimond et al., 2012; Ignatowska-Jankowska et al., 2011; Sofia and Knobloch, 1976; Wierucka-Rybak et al., 2014). It is also consistent with previous reports that CBD may have a therapeutic value to treat substance use disorders and obesity as it has the ability to reduce rewarding effects of alcohol (Viudez‐Martínez et al., 2018), cocaine and methamphetamine (Hay et al., 2018; Katsidoni et al., 2013; Luján et al., 2018). Although CBD failed to alter heroin self-administration, it reduced cue-induced reinstatement of drug seeking (Ren et al., 2009) and facilitated the extinction of psychostimulant-induced conditioned place preference (CPP) (Parker et al., 2004). Studies with human subjects revealed that CBD reduced cigarette consumption in smokers (Morgan et al., 2013) and counteracted euphoric as well as negative effects of Δ9-THC in cannabis users (Dalton et al., 1976; Karniol et al., 1974; Zuardi et al., 1982). Interestingly, CBD itself lacks rewarding properties as it failed to induce CPP, withdrawal symptoms or altered motor behavior (Parker et al., 2004; Viudez‐Martínez et al., 2018). It also failed to affect brain stimulation reward in rodents (Katsidoni et al., 2013). These findings suggest that CBD has no abuse potential by itself (Martin-Santos et al., 2012).

Role of CB1 receptor in CBD action

However, a challenge in CBD research is that the molecular targets underlying CBD’s action are ambiguous. Based on the structural similarity to Δ9-THC, it was initially believed that similar receptor mechanisms underlie CBD’s action. However, experimental evidence suggests that CBD has a very low affinity (Ki =10~30 μM) and shows little agonist activity (EC50 >10 μM) at the CB1 and CB2Rs, as compared to Δ9-THC (Ki = 3~80 nM for CB1/CB2Rs) (Pertwee, 2008), suggesting non-CB1 and non-CB2 mechanism involvement. Alternatively, a growing body of literature indicates that CBD has higher affinity at many other molecular targets, including GPR55, TRPV1, PPAR γ, opioid and serotonin receptors (5-HT1A) (Ibeas Bih et al., 2015; Morales and Reggio, 2017). However, a majority of the above findings derives from in vitro cell lines and currently, there is a lack of convincing evidence indicating that any of these targets directly contribute to therapeutic effects produced by CBD in vivo or in human clinical trials.

The second important finding in the present study is that CBD-induced reduction in sucrose self-administration may be related to CB1R inactivation. This is based on the findings that pharmacological blockade of CB1Rs by AM251 produced a reduction in sucrose self-administration in a way similar to CBD, and pretreatment with AM251 (30 min prior to CBD) produced an augmented reduction in sucrose self-administration. This finding is consistent with previous observation of food intake and body weight in rats fed with a high-fat or high sugar diet (Wierucka-Rybak et al., 2014). In addition, growing evidence demonstrates that CBD may act as allosteric CB1R antagonist (see discussion below). These findings suggest that AM251 and CBD may act on CB1Rs, producing an additive or synergistic effect although their exact binding sites on CB1Rs are unclear. We note that genetic deletion of CB1Rs failed to alter CBD action. This should not be used to exclude CB1R involvement since in the absence of CB1Rs, CBD may act on other targets (such as CB2Rs, discussed below) producing pharmacological effects. Overall, the hypothesis about the role of CB1Rs in CBD action are consistent with previous reports that Δ9-THC and the endocannabinoid anandamide increase food consumption by stimulation of CB1Rs (Farrimond et al., 2011; Jamshidi and Taylor, 2001; Williams and Kirkham, 2002a, 2002b). Accordingly, blockade of CB1Rs produces an inhibitory effect on food-taking behavior.

The molecular mechanisms through which CBD modulates CB1R function are unclear. As stated above, CBD has very low affinity at the orthosteric binding site of CB1 Rs (Bisogno et al., 2001; Pertwee, 2008), suggesting that CBD may not alter endocannabinoid binding to the orthosteric site of the CB1Rs. However, more recent studies suggest that CBD is a potent non-competitive negative allosteric modulator (or antagonist) of CB1Rs (Laprairie et al., 2015; Straiker et al., 2015; Tham et al., 2019.; Thomas et al., 2007). CBD reduces both G protein-dependent signaling and β-arrestin 2 recruitment using Δ9-THC and 2-AG as orthosteric probes at CB1Rs. These findings suggest that CBD-induced reduction in sucrose self-administration may be mediated by functional inhibition of CB1Rs at its allosteric binding site.

We note that in addition to binding to CB1Rs, CBD and AM251 also have nanomolar range binding affinities at other targets, such as GPR55 receptors (Ryberg et al., 2007) and μ-opioid receptors (Kathmann et al., 2006; Seely et al., 2012). AM251 has been shown to exert ‘off-target’ effects in vitro and in CB1−/− mice (Seely et al., 2012). Thus, it is conceivable that the robust reduction in sucrose intake observed with the combined treatment of AM251 and CBD might involve GPR55 and μ opioid receptor sites. In fact, others have shown that opioid receptors play an important role in food-related behaviors (Gosnell and Levine, 2009; Viudez-Martinez et al., 2018).

Role of CB2 receptor in CBD action

Perhaps the most important finding in the present study is that a CB2R mechanism may also underlie CBD action in sucrose self-administration. This is based on the several lines of evidence. First, deletion of CB2Rs abolished CBD action; second, pretreatment with AM630, a selective CB2R antagonist, prevented CBD action; third, JWH133, a selective CB2R agonist, produced a similar effect as CBD; and fourth, pretreatment with AM630 blocked JWH133 action on sucrose self-administration. These findings are consistent with previous reports indicating that a CB2R-dependent mechanism is closely associated with CBD-induced reduction in food intake, body weight and obesity (Deveaux et al., 2009; Ignatowska-Jankowska et al., 2011; Ishiguro et al., 2010) and with CBD-produced neuroprotection (Castillo et al., 2010; Hermann and Schneider, 2012).

The molecular mechanisms through which CBD modulates CB2R function are still not fully understood. As stated above, CBD has low affinity for either CB1 or CB2 orthosteric binding site, and therefore, diverse non-cannabinoid molecular targets have been proposed. Using [35S]-GTPγS binding assays, Pertwee and co-workers provided the first evidence for CBD to antagonize the effects of CP-55,940 at hCB2 and to display CB2R inverse agonism (Pertwee, 2008; Thomas et al., 2007). A decade later, CBD has been proposed as a potential allosteric ligand of CB2Rs with a high affinity at the allosteric binding site of CB2Rs even at nanomolar concentrations and the ability to significantly decrease affinity (KD) of the orthosteric agonist (red CM-157) (Martínez-Pinilla et al., 2017, 2016). In similar conditions, CBD by itself did not significantly affect CB2R-coupled cAMP and ERK½ signaling, but blocked the action of the selective CB2R agonist JWH133 in a dose-dependent manner at nanomolar concentrations. Furthermore, a very recent paper reported that CBD is a partial CB2R agonist in the absence of orthosteric ligands (Laprairie et al., 2015). Taken together, these data suggest that CBD acts as an allosteric CB2R inverse agonist and functionally antagonizes action produced by orthosteric CB2R agonists in a non-competitive manner.

It is unknown how CBD inhibits sucrose self-administration via CB2Rs at neural circuit level. We and others have reported that CB2Rs are expressed in the brain and are functionally involved in drug reward and addiction (Jordan and Xi, 2019; Manzanares et al., 2018). CB2Rs have been identified on the cell bodies of dopaminergic (DA) neurons in the ventral tegmental area (VTA) (Zhang et al., 2017, 2014, 2015) as well as on the terminals of these neurons in the nucleus accumbens (NAc) (Aracil-Fernández et al., 2012; Foster et al., 2016), two brain regions critical for reward and addiction (Wise and Bozarth, 1985). We previously reported that stimulation of VTA or NAc CB2Rs inhibits neuronal firing of DA neurons and decreases DA release in the NAc (Zhang et al., 2017, 2014). Stimulation of CB2Rs by JWH133 inhibits cocaine self-administration, cocaine-induced locomotor hyperactivity and conditioned place preference (CPP), and electrical brain-stimulation reward (Delis et al., 2017; Spiller et al., 2019; Xi et al., 2011). Correspondingly, deletion of CB2Rs leads to enhanced CPP or locomotor response to cocaine or ethanol in mice (Canseco-Alba et al., 2018; Ortega-Álvaro et al., 2015; Powers et al., 2015; Xi et al., 2011), while overexpression of CB2Rs decreases cocaine sensitization and cocaine self-administration (Aracil-Fernández et al., 2012). These findings suggest that CBD-induced reduction in sucrose self-administration may be mediated by a CB2-dependent mechanism in the mesolimbic DA reward system.

Given that stimulation of CB2Rs by JWH133 (an orthosteric CB2R agonist) produces an inhibitory effect on VTA DA neuronal activity and NAc DA release (Xi et al., 2011; Zhang et al., 2014; Zhang et al., 2017; Ma et al., 2019), one may expect that CBD, an allosteric inverse agonist of CB2Rs, would produce an increase in VTA DA neuronal activity and NAc DA release. However, by directly measuring extracellular DA level in the NAc using in vivo brain microdialysis, we found that CBD alone produced a 20% reduction (non-significant) in extracellular NAc DA, and pretreatment with CBD significantly reduced cocaine-enhanced DA (Galaj et al., 2019), suggesting that a DA-dependent mechanism may underlie CBD action. We cannot use this data to support or against whether CBD is an allosteric CB2R inverse agonist since CBD has multiple acting targets, and the observed DA effect could be a final net effect of multiple actions after CBD administration. In addition, the effects of CBD on NAc DA are conflicting. For example, microinjections of CBD into a lateral ventricle (i.c.v.) or lateral hypothalamus produced significant increases in the extracellular DA in the NAc (Murillo-Rodriguez et al., 2011, 2006). However, intra-NAc microinjection of CBD produced an inhibitory effect on VTA DA neuronal activity by itself (Renard et al., 2016) and attenuated amphetamine-induced locomotor sensitization and VTA DA neuronal sensitization (Renard et al., 2017, 2016). Although the authors proposed that 5-HT1A mechanisms may underlie intra-NAc CBD-induced reduction in NAc DA (Hudson et al., 2018; Renard et al., 2017), there is no direct evidence to support it. Furthermore, CBD has been reported to inhibit synaptic uptakes of DA in vitro synaptosome preparations (Pandolfo et al., 2011). Clearly, more studies are required to determine whether and how CBD alters the mesolimbic DA system activity.

In addition to the above DA mechanism, CBD was reported to inhibit the degradation of the endocannabinoid anandamide by fatty acid amide hydrolase (Watanabe et al., 1996) and inhibit the cellular uptake of anandamide (Bisogno et al., 2001). Given that anandamide a weak partial CB2R agonist, it is unsure whether such an enhanced anandamide-CB2R mechanism is involved in CBD action observed in the present study. In contrast, the endocannabinoid 2-arachidonoylglycerol (2-AG) is a full CB1 and CB2 receptor agonist (Mackie et al., 1993). If CBD similarly elevates brain 2-AG levels, it may well explain the present finding. More studies are required to test this hypothesis.

Lastly, sucrose intake may also alter neuronal activity and hormonal release in the hypothalamus and mesolimbic regions, leading to the development of preference for palatable sweetened food and the alterations in metabolism and body weight (Mitra et al. 2011, 2016). Thus, it is conceivable that CBD, in addition to reducing sucrose reward acutely, may also reverse hypothalamic and mesolimbic dysfunctions, leading to persistent preference for healthy diet.

In summary, the present findings suggest that CBD may have certain therapeutic potential in controlling binge-eating and treating obesity. Our findings are of particular importance given prevalence of obesity in the United States and successful pharmacotherapies to treat obesity are yet to be developed. In the series of experiments, we have demonstrated that CBD and JWH133 have the ability to reduce food reward and motivation to seek sweetened food. Our data pave the way toward better understanding the role of CB1 and CB2 receptors in food reward and promoting CBD as potential therapeutics for the treatment and prevention of obesity.

Acknowledgments

Funding and Disclosure

This research was supported by NIDA-IRP (DA000620–02). None of the authors has any disclosure.

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