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Published in final edited form as: Neuropharmacology. 2024 Apr 16;252:109947. doi: 10.1016/j.neuropharm.2024.109947

β-caryophyllene inhibits heroin self-administration, but does not alter opioid-induced antinociception in rodents

Ewa Galaj 1,*, Guo-Hua Bi 2, Zheng-Xiong Xi 2
PMCID: PMC11077898  NIHMSID: NIHMS1988882  PMID: 38631564

Abstract

A growing body of research indicates that β-caryophyllene (BCP), a constituent present in a large number of plants, possesses significant therapeutic properties against CNS disorders, including alcohol and psychostimulant use disorders. However, it is unknown whether BCP has similar therapeutic potential for opioid use disorders. In this study, we found that systemic administration of BCP dose-dependently reduced heroin self-administration in rats under an FR2 schedule of reinforcement and partially blocked heroin-enhanced brain stimulation reward in DAT-cre mice, maintained by optical stimulation of midbrain dopamine neurons at high frequencies. Acute administration of BCP failed to block heroin conditioned place preference (CPP) in male mice, but attenuated heroin-induced CPP in females. Furthermore, repeated dosing with BCP for 5 days facilitated the extinction of CPP in female but not male mice. In the hot plate assay, pretreatment with the same doses of BCP failed to enhance or prolong opioid antinociception. Lastly, in a substitution test, BCP replacement for heroin failed to maintain intravenous BCP self-administration, suggesting that BCP itself has no reinforcing properties. These findings suggest that BCP may have certain therapeutic effects against opioid use disorders with fewer unwanted side-effects by itself.

Key terms: endocannabinoid; BCP, β-caryophyllene; conditioned place preference; intracranial self-stimulation; heroin; drug self-administration, analgesia

Introduction

There is an interesting interaction between the endocannabinoid and opioid systems in a variety of biological functions, including intestinal motility (Basilico et al., 1999; Frederickson et al., 1976; Kulkarni-Narla and Brown, 2001), emesis (Simoneau et al., 2001), modulation of stress (Corchero et al., 1999; Valverde et al., 2000), anxiety (Berrendero and Maldonado, 2002; Marín et al., 2003) and the dopamine system (Tanda et al., 1997). Both cannabinoids and opioids produce antinociception (Megens et al., 1998; Vivian et al., 1998; Wang et al., 2020) and cannabinoid receptor agonists [e.g., delta-9-tetrahydrocannabinol (THC), CP55,940 and WIN 55,212] can enhance the analgesic effects of opioids (Cichewicz et al., 1999; Cichewicz and McCarthy, 2003; Maguire et al., 2013; Weed et al., 2018). There is also evidence indicating that the interaction of these two systems is critical for the development of addiction (Befort, 2015; Fattore et al., 2005). For example, genetic deletion of mu opioid receptors in MOR-knockout (MOR-KO) mice abolishes the development of conditioned place preference (CPP) produced by a low dose of THC or conditioned place aversion produced by a high dose of THC (Ghozland et al., 2002). Pharmacological blockade of CB1 receptors (CB1Rs) reduces heroin self-administration in rats (He et al., 2019) and the expression of morphine CPP in mice (Zhang et al., 2016). Likewise, CB2 receptor (CB2R) agonists such as M1710 and LY2828360 reduce the development of antinociceptive tolerance to morphine and attenuate morphine-induced physical dependence (Li et al., 2019; Lin et al., 2018). Our group has previously reported on the importance of cannabinoid CB1 (He et al., 2019; Xi et al., 2008), CB2 (Galaj et al., 2020a; Jordan et al., 2020; Xi et al., 2011), and peroxisome proliferator-activated receptors (PPARα and PPARγ receptors) (Galaj et al., 2021) in addiction [for comprehensive reviews see (Galaj and Xi, 2019; Jordan and Xi, 2019; Soler-Cedeno and Xi, 2022)].

Recently, we have been interested in therapeutic utility of beta-caryophyllene (BCP), an FDA-approved food additive that is naturally present in cannabis, black pepper, cloves and other herbs (Mediavilla and Steinemann, 1997; Sharma et al., 2016). BCP, was initially identified as a selective CB2R agonist with anti-inflammatory properties (Cho et al., 2007; Gertsch, 2008; Gertsch et al., 2008). Later studies showed that BCP reduces capsaicin-induced pain (Katsuyama et al., 2013), thermal hyperalgesia and mechanical allodynia (Klauke et al., 2014). BCP also exerts anxiolytic, anti-depressive effects (Bahi et al., 2014) and can reduce drug and alcohol-reinforced behaviors (Al Mansouri et al., 2014; Galaj et al., 2021; He et al., 2021, 2020). We have demonstrated that systemic administration of BCP dose-dependently reduces nicotine self-administration in rats and mice and attenuates nicotine-enhanced brain-stimulation reward (He et al., 2020). A reduction in nicotine taking after BCP administration can be blocked by AM630, a selective CB2R antagonist but is still evident in CB2-KO mice, suggesting that BCP effects are mediated by CB2 and non-CB2 receptor mechanisms (He et al., 2020). Indeed, we have shown that BCP attenuates cocaine self-administration via PPARα and PPARγ receptors (Galaj et al., 2021a). In addition, we reported that BCP reduces cocaine CPP, reinstatement of cocaine- (Galaj et al., 2021) and methamphetamine-seeking (He et al., 2021), indicating its efficacy against relapse. Pretreatment with BCP can attenuate the rewarding effects of psychostimulants, as evidenced in the electrical or optogenetic brain stimulation reward paradigms (Galaj et al., 2021; He et al., 2021, 2020) and in a microdialysis study showing a significant reduction in METH-induced increases in extracellular dopamine in the nucleus accumbens (NAc) (He et al., 2021).

However, it is still unknown whether BCP can attenuate opioid self-administration, CPP and antinociceptive effects, and whether BCP effects are sex-specific.

Thus, in the present study, we used male and female mice and rats in various behavioral paradigms to explore the efficacy of BCP on addiction-related behaviors. First, we investigated whether BCP treatment can reduce intravenous heroin self-administration and heroin-enhanced optogenetic brain-stimulation reward, indicative of its therapeutic utility in reducing opioid-reinforced behaviors. We also assessed whether BCP reduces heroin CPP and facilitates the extinction of CPP. Lastly, we investigated the effect of BCP on opioid-induced antinociception and potential reinforcing effects of BCP, two important issues to consider in the medication development for opioid use disorders and pain management. We used rats and mice in this study to increase reproducibility of the data. Accessibility to DAT-cre mice allowed us to test BCP impact on the reinforcing and rewarding effects of optical brain stimulation reward. Because it is difficult to train mice to self-administer heroin, we opted for a rat model that is highly reliable and has been used for decades. Overall, with this set of experiments we assessed the BCP therapeutic utility against addictive-like behaviors.

Methods

This study was carried out in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources on Life Sciences, National Research Council, 2011) and was approved by the National Institute on Drug Abuse Institutional Animal Care and Use Committee. This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

Animals:

Male Long-Evans rats (n=12) were purchased from Charles River Laboratories (Frederick, MD, USA) and used in Exp.1–2. DAT-IRES-cre knocked-in mice (n=6 males and n=6 females) with C57BL/6J genetic background were bred at the National Institute on Drug Abuse Intramural Research Program and used in Exp.3. Breeders were purchased from Jackson Laboratory (B6.SJL-Slc6a3tm1.1(Cre)Bkmn/J; stock # 006660). Male (n=26) and female (n=31) C57BL/6J wild type (WT) mice obtained from our in-house colony were used in Exp. 4–5. All animals were housed in climate-controlled animal colony rooms 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.

Surgery:

The procedures for jugular catheter surgery and drug self-administration were conducted as previously reported (Galaj et al., 2022, 2020a). Briefly, rats were anesthetized by an intraperitoneal (i.p.) injection of ketamine/xylazine (90/10 mg/kg, i.p.) and their scalp and the neck were shaved, and cleaned with iodine. An ophthalmic ointment (Paralube Vet ointment) was applied to the eyes to prevent corneal drying. During and after surgery the rats were placed on a heating pad to prevent hypothermia. For catherization, a small incision was made to the right of the midline of the neck. The jugular vein was exposed and the tip of a microrenathane catheter (Braintree Scientific Inc., MA, US) was inserted into the right atrium of the heart. The catheter was secured to the vein with silk sutures and its free end was fed subcutaneously around the back of the neck to exit through the scalp incision. The catheter was then connected to a connector (a modified 24-g cannula; Plastics One, Roanoke, VA, US). The connector was then mounted onto the skull using jeweler’s stainless-steel screws and dental acrylic. To maintain its patency, the catheter was filled with 0.05 ml of heparin (200 U/ml of saline) and gentamicin (40 mg/ml) immediately after surgery and daily thereafter. The rats were given five days of recovery before IVSA began.

For the optical intracranial self-stimulation (oICSS) experiment, as described previously (Galaj et al., 2020b; He et al., 2021), DAT-cre mice were anesthetized and microinjected with excitatory channelrhodopsin (AAV5-EF1α-DIO-ChR2-EYFP; UNC Vector Core, Chapel Hill, NC, US). Bilateral injections were made to the VTA (AP −3.1, ML ±0.8, DV −4.25), using a 10 μL Nanofil syringe and Micro 4 Pump (World Precision Instruments, FL, US) at a rate of 50 nL/min for a total volume of 150 nL per VTA side. Bilateral custom-made ferrule fibers were implanted (200 μm inner diameter, Doric Lenses) 0.5 mm above the injection site.

Drugs:

Heroin hydrochloride (provided by NIDA IRP Pharmacy) was dissolved in 0.9% saline to achieve a final dose of 1 mg/kg for intraperitoneal injections or 0.05 mg/kg for intravenous deliveries. BCP was purchased from Millipore-Sigma and dissolved in saline containing 10% DMSO and 15% Tween to achieve doses of 25, 50 and 100 mg/kg and was administered 30 min prior to the test.

Apparatus:

Drug self-administration and substitution experiments were conducted in Med-Associates standard rat operant conditioning chambers equipped with 2 levers, a light and tone above the active lever, and a fluid line through which a rat was connected to a Razel 3.33 RPM pump filled with a 10 ml syringe. A heroin CPP experiment was conducted in Med-Associates standard three-compartment place preference chambers (17 l x13 w x13 h conditioning compartments and middle compartment 10 l × 13 w 13 h in cm). Each compartment had distinct walls (black or grey) and floors (grid or bars). An oICSS/brain stimulation reward experiment was conducted in Med-Associates standard mouse operant conditioning chambers equipped with 2 levers, a light above the active lever, patch cords (Doric Lenses INC, Quebec, Canada), Doric FC/FC swivel through which a mouse was connected to a 473 nm laser (OEM Laser Systems Inc, Draper, UT, US) and Master-9 interface. Antinociception was assessed using a hot plate device (Model 39, IITC Life Science Inc., CA, US).

Procedure:

Exp. 1. The impact of BCP on heroin self-administration under a FR2 reinforcement schedule

We first assessed whether BCP can reduce heroin self-administration. Rats (n=12) were trained to self-administer heroin (0.05 mg/kg/ infusion) under an FR2 schedule of reinforcement during daily 3-h sessions. Responding on the active lever activated a syringe pump for 4.65 s causing the delivery of heroin and the illumination of the light above the active lever. Responses on the inactive lever were counted but had no consequences. During the infusion period, additional responses on the active lever were recorded, but did not lead to additional infusions. Once animals demonstrated a pattern of stable responding (<20% variability in daily heroin intake across 3 consecutive sessions), they were tested with different doses of BCP (0, 50 and 100 mg/kg), randomly chosen. BCP was administered intraperitoneally 30 min prior to the test session. After establishing a new baseline, animals were tested with other doses of BCP, 3–5 days apart. We chose BCP doses that do not produce motor impairment, as we had demonstrated in previous studies

Exp. 2. Substitution test with BCP

In this experiment we examined potential reinforcing effects of BCP, one of the most important issues to consider in the medication development for substance use disorders. We trained 12 rats to self-administer heroin intravenously (0.05 mg/kg/injection) on an FR2 schedule of reinforcement, as described in Exp.1 and in our previous study (Galaj et al., 2021). Once animals showed a reliable pattern of IVSA, we replaced heroin with intravenous BCP at 0.5 and 1 mg/kg/injection doses and assessed whether animals would maintain IVSA, indicative of abusive liability of BCP. After 8 sessions of BCP substitution test, we reintroduced an intravenous heroin (0.05 mg/kg/injection) and measured the number of heroin infusions self-administered.

Exp. 3. The impact of BCP on heroin-enhanced brain stimulation reward maintained by optogenetic activation of VTA DA neurons.

To assess whether BCP can attenuate the rewarding effects of heroin, we used an optogenetic brain stimulation reward paradigm with DAT-cre mice, as reported previously (Galaj et al., 2020b; He et al., 2020). DAT-cre mice (n=6 males and 6 females), with ChR2 expression targeting the VTA DA neurons, were connected to a 473-nm wavelength laser (OEM Laser Systems, UT) by two optic patch cords (200-μm core diameter), and Doric FC/FC fiber rotary joint. During each 1-h session, each press on the active lever activated the light above the lever and delivered a 1-s pulse train of blue light (473 nm, 10 mW, 5 ms pulse duration, 50 Hz) depolarizing VTA DA cells. Pressing the inactive lever yielded no stimulation. Once animals’ responding stabilized (<20% variability in responding for at least 3 consecutive sessions), a multiple stimulation frequency schedule was introduced. Every 10 min stimulation frequency was decreased from 100 to 50, 25, 10, 5 and finally to 1 Hz, and lever presses at each frequency were counted. The testing phase began once stable brain stimulation reward responding was achieved with < 20% variation across 3 consecutive sessions. Mice received two injections prior to the test session: an injection of BCP (0, 25, 50 mg/kg; i.p.) and 15 min later an injection of heroin (1 mg/kg). 15 min later, mice were allowed to lever-press for brain-stimulation reward. After each test, mice received additional sessions until a new baseline was established and later were re-tested with a different dose of BCP + heroin. After completion of the above behavioral experiment, the immunohistochemistry assay was used to verify AAV-ChR2-EGFP expression in VTA DA neurons in DAT-cre mice, using the methods we reported previously (Galaj et al., 2020b; He et al., 2020).

Exp. 4. The impact of BCP on heroin conditioned place preference (CPP) and extinction of heroin CPP

Six groups of WT mice (n=8–11/group) were tested for the effects of BCP on the expression of heroin CPP. During two pre-exposure sessions, mice were placed in the middle compartment of the CPP apparatus and allowed to freely explore three compartments for 15 min. The pre-exposure sessions were then followed by a conditioning phase consisting of 4 heroin and 4 saline conditioning sessions. For all of these sessions the doorways between compartments were closed. Prior to each conditioning session each mouse was injected with heroin (1 mg/kg) or saline intraperitoneally and immediately placed in the heroin or saline compartments. Heroin was paired with either the preferred compartment (determined as the compartment in which a mouse spent the most time during preexposure) or the non-preferred compartment. On the day following the last conditioning session, all mice were tested for heroin CPP. Thirty min prior to the test, mice were injected with one dose of BCP [males: 0 (n= 8), 25 (n=8) and 50 mg/kg (n=10); females: 0 (n=10); 25 (n=11), and 50 mg/kg (n=11)]. Each mouse was then placed in the middle compartment of the CPP apparatus and allowed to freely explore all three compartments for 15 min. The time spent in the heroin and saline compartment was measured.

We then tested whether BCP can facilitate the extinction of already established CPP. For 5 consecutive days mice were injected with the same dose of BCP as previously. Thirty min post injection mice were placed in the CPP apparatus and allowed free exploration of all compartments for 15 min. The time spent in each chamber was measured.

Exp. 5. The effect of BCP on heroin-induced antinociception.

Male and female mice (n=13/group) were utilized in this experiment. Mice’ basal and heroin-induced response to heat was measured using a standard hot plate. Briefly, the plate was warmed up to a fixed temperature of 52.5°C. An animal was placed on the hotplate and the latency to the first nociceptive response (i.e., paw shaking, lifting or licking their paws, rearing, or jumping) was measured. After establishing the baseline, mice were injected with BCP (0, 25, 50 mg/kg; ip.). Fifteen min later, mice received an injection of heroin (3 mg/kg; ip). The latency to the first nociceptive response was measured at 0, 15, 30, 60, 90 and 120 post-heroin injection, 2 times at each time point. Three days later animals underwent another nociception test, also with heroin (3 mg/kg; ip) but this time, with another dose of BCP, randomly chosen. The cut-off time for the test was 60 s to avoid tissue damage.

Results

Exp. 1. BCP dose dependently reduced heroin self-administration in rats

Fig. 1A shows the effects of BCP on heroin IVSA under an FR2 reinforcement schedule. Treatment with BCP dose-dependently reduced heroin IVSA. This observation was confirmed by a one-way ANOVA revealing a significant BCP effect (F2,35 = 11.42, p<0.001). Post-hoc Dunnett’s tests revealed statistically significant reductions in heroin intake after 50 mg/kg (p<0.05), 100 mg/kg of BCP (p<0.001), when compared to vehicle. As shown in Fig. 1B, BCP at the 100 mg/kg, but not 50 mg/kg, dose caused a significant reduction in active lever presses (F2,35 = 9.71, p<0.01) but had no significant impact on inactive lever presses (F2,35 = 1.80, p=0.20; Fig. 1C), suggesting heroin-specific attenuating effects.

Figure 1.

Figure 1.

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Effects of BCP on heroin self-administration and in a substitution test A. BCP produced a dose-dependent reduction in the number of intravenous infusions self-administered by rats under an FR2 schedule of reinforcement. (* p<0.05, compared to vehicle) B. BCP produced a significant reduction in active lever presses in a dose-dependent manner. (* p<0.05, compared to vehicle) C. BCP had no effect on inactive lever presses. D. In substitution test, rats did not maintain drug IVSA when BCP was substituted for heroin, but regained responding once heroin was reintroduced.

Exp. 2. BCP failed to maintain self-administration in a substitution test

In Exp. 2, we substituted BCP for intravenous heroin to assess reinforcing effects of BCP. As shown in Fig. 1D, rats acquired heroin IVSA within the first few days and failed to maintain IVSA once heroin was replaced with intravenous BCP. During substitution tests, their responding significantly declined reaching 2–4 infusions per 3-hour session. This was true for 0.5 and 1 mg/kg/infusion dose of BCP. However, when heroin was reintroduced, animals slowly regained heroin IVSA, but they never reached the pre-BCP-substitution level. This observation was confirmed by a one-way ANOVA revealing a significant session effect (F18, 189 = 12.49, p<0.001) and by post hoc Dunnett’s tests revealing significant differences in drug IVSA with BCP substitution (0.5 and 1 mg/kg/infusion) as well post-BCP heroin (ps<0.05), as compared to Session 1. No significant differences in heroin IVSA were observed during the first 5 sessions (ps>0.05).

Exp. 3. BCP partially blocked the rewarding effects of heroin in an optogenetic brainstimulation reward paradigm

Next, we used an optogenetic brain stimulation reward paradigm to assess whether BCP can reduce the rewarding effects of heroin. As shown in Fig. 2A, an excitatory ChR2 virus was injected bilaterally into the VTA and optic fiber ferrules were implanted 0.5 mm above it. Four weeks post-surgery, DAT-cre mice were trained to press a lever that activated ChR2-infected VTA DA cells, leading to membrane depolarization and rapid DA cell firing (Fig. 2B). As shown in Fig. 2C, excitatory ChR2 virus was robustly expressed in the VTA of DAT-cre mice. Mice acquired lever pressing for optogenetic brain stimulation reward maintained by different stimulation frequencies (1–100Hz), as shown in Fig. 2D and 2F. When we analyzed a total number of lever presses, we found that 1 mg/kg dose of heroin enhanced lever presses for brain stimulation reward. Pretreatment with 25 mg/kg of BCP failed to block this effect but 50 mg/kg of BCP dampened a heroin enhancing effect (Fig. 2E). A one-way ANOVA revealed no significant group effect (F3, 35 = 2.68, p=0.08), but individual t-tests revealed that the heroin and (heroin + BCP 25) groups were significantly different from the vehicle (t9=2.32; p=0.04 and t9=2.29; p=0.03, respectively). The (Heroin + BCP 50) group was indistinguishable from the vehicle group (t9=0.91; p=0.41).

Figure 2.

Figure 2.

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Effects of BCP on heroin-enhanced brain stimulation reward in DAT-cre mice. A. A diagram showing that an excitatory AAV-ChR2 virus was microinjected into the bilateral ventral tegmental area (VTA) of DAT-cre mice. B. A diagram illustrating that optical stimulation upon lever pressing activated midbrain dopamine cells leading to rapid firing of DA neurons. C. Representative images illustrating AAV-ChR2-eYFP co-expression with tyrosine hydroxylase (TH) in VTA DA neurons in DAT-Cre mice. D. Representative brain stimulation reward records from a single session of a mouse pressing a lever for different stimulation frequencies. E. Systemic administration of heroin (1 mg/kg) enhanced total lever presses maintained by 6 stimulation frequencies. BCP, at 50 mg/kg, attenuated this effect. (*p<0.05 as compared to the vehicle). F. BCP pretreatment attenuated heroin-enhanced brain stimulation reward in a dose-dependent manner. (*p<0.05, as compared to vehicle; #p<0.05, as compared to heroin group)

As shown in Fig. 2F, DAT-cre mice made more lever presses for higher (25, 50, 100 Hz) than lower stimulation frequencies (1, 5, 10 Hz). A dose of 1 mg/kg heroin increased lever presses for 5, 10, 25, 50 Hz, but not 1 or 100 Hz, stimulation frequencies. Pretreatment with BCP failed to block these effects, except at 100 Hz (BCP at 50 mg/kg). A two-way ANOVA revealed a significant dose × frequency interaction (F15, 216 = 3.39, p<0.001). Tukey’s multiple comparison tests revealed that the heroin group was significantly different from vehicle at 50, 25, 10 and 5 Hz (ps<0.05), but not significantly different from the BCP groups, except at 100Hz. 50 mg/kg of BCP significantly reduced heroin effects at 100 Hz (p<0.05). These findings suggest that heroin was able to enhance brain stimulation reward and BCP partially blocked this effect.

Exp. 4. BCP attenuated heroin conditioned place preference (CPP) in females but not males

We then tested whether BCP can attenuate the expression of heroin CPP. As shown in Fig. 3A and 3B, male and female mice in different groups showed similar initial preference. After conditioning, control male mice and those treated with BCP at 25 and 50 mg/kg showed significant preference for heroin-paired compartment (Fig. 3A). A two-way ANOVA revealed a main effect of phase (i.e., CPP) (F1, 23 = 34.68, p<0.001), but no effect of BCP (F2, 46 = 0.03, p>0.05) and no phase × BCP dose interaction (F2, 23 = 0.05, p>0.05) in males. In contrast, female control mice showed significant heroin CPP but those treated with 25 or 50 mg/kg dose of BCP did not, when compared to the pre-exposure data (Fig. 3B). A two-way ANOVA revealed a main effect of phase (F1, 29 = 14.39, p<0.001), but no effect of BCP (F2, 29 = 0.03, p>0.05) and no phase × BCP dose interaction (F2, 29 = 0.69, p>0.05) in females. However, a careful analysis with individual t-tests, revealed a significant phase effect for vehicle group (t9=4.28; p=0.02), but not for BCP at 25 mg/kg (t10=2.08; p=0.07) and BCP at 50 mg/kg (t10=1.23; p=0.24). These findings suggest that BCP at least in part attenuates heroin CPP in females, but not males.

Figure 3.

Figure 3.

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Effects of BCP on heroin conditioned place preference in male and female WT mice. A. Systemic administration of BCP failed to attenuate heroin CPP in males. B. BCP dose-dependently attenuated heroin CPP in females (*p<0.05, as compared to pre-exposure). C. Repeated administration of BCP for 5 days had no impact on CPP extinction in males. D. Repeated administration of BCP facilitated CPP extinction in females on Sessions 4 and 5. (*p<0.05, as compared to vehicle; S=session).

We continued the BCP treatment for the next 4 days to assess whether BCP can facilitate extinction of heroin-induced CPP. As shown in Fig. 3C, with repeated testing, control and BCP-treated male mice showed similar reductions in heroin CPP (from Session 1 to Session 5). Repeated treatment with BCP had no impact on heroin CPP extinction in males. A two-way ANOVA confirmed this observation and revealed a significant session effect (F4,92 = 3.46, p<0.01) but no significant BCP effect (F2,23 = 0.28, p>0.05) and no session × BCP dose interaction (F8, 92 = 0.52., p>0.05). Interestingly, control female mice persistently showed heroin CPP, even by session 5 (Fig. 3D). A two-way ANOVA revealed no significant session effect (F4,116 = 0.66, p>0.05), no significant BCP effect (F2,29 = 2.03, p>0.05) and no session × BCP dose interaction (F8, 116 = 0.81., p>0.05) in females. On sessions 4 and 5 significant differences emerged when comparing 50 mg/kg group to the vehicle group using, individual t-tests (Session 4: t19 = 2.1; p<0.05 and Session 5: t19 = 2.45; p<0.05). Thus, this data suggests that repeated dosing with BCP has no impact on CPP extinction in males, but significantly facilitates the extinction of CPP in females.

Exp. 5. BCP did not alter heroin-induced antinociception in male and female mice.

In Exp. 5, we assessed whether BCP can alter heroin analgesic effects. As shown in Fig. 4A and 4B, mice in different groups showed a similar basal level of nociception. On average, first responses to pain were observed within 7.0–7.5 sec. However, when heroin (3 mg/kg) was administered, the latency to the first response reached on average 37–38 sec., indicating heroin produced significant analgesic effects with a peak effect at 15 min post injection. These effects were short lasting and a steady decline in analgesic effects was already evident 30 min post-injection. Unexpectedly, BCP did not produce a significant enhancement in heroin analgesic effects nor did it extend the duration of antinociception (Fig. 4A and 4B). A two-way ANOVA revealed a significant time effect (F5,60 = 107.8, p<0.001), but no BCP effect (F2,60 = 0.94, p>0.05) and no time × BCP dose interaction (F10, 60 = 1.33, p>0.05) in males. Similarly, in females, a two-way ANOVA revealed a significant time effect (F5,60 = 111.1, p<0.001), but no BCP effect (F2,60 = 0.32, p>0.05) and no time × BCP dose interaction (F10, 60 = 1.55, p>0.05).

Figure 4.

Figure 4.

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Effects of BCP on heroin-produced antinociception in male (A) and female (B) mice. Systemic administration of BCP failed to alter heroin-induced antinociception.

Discussion

In this study, we evaluated the potential utility of BCP against heroin-related behaviors using gold-standard animal models of addiction. We found that: 1) BCP significantly and dose-dependently reduced heroin self-administration in rats under an FR2 schedule of reinforcement. 2) In the substitution test, rats did not maintain drug IVSA once BCP was substituted for intravenous heroin, suggesting that BCP produces low rewarding and reinforcing effects. 3) Heroin reliably enhanced optogenetic brain-stimulation reward maintained by activation of VTA DA neurons and BCP partially blocked this effect in male and female DAT-cre mice. 4) BCP attenuated the expression of heroin CPP in female but not male WT mice. 5) Repeating treatment with BCP facilitated the extinction of heroin CPP in females but not males. 6) BCP, at the same doses that inhibited heroin self-administration, failed to alter a heroin analgesic effect. Lastly, 7) sex differences in the BCP effects emerged in the CPP and CPP extinction paradigms. Together, these findings suggest that BCP produces mild but significant therapeutic effects on heroin-related behaviors without unwanted effects on its own. The observed attenuating effects cannot be explained by sedation or motoric effects as BCP at the selected doses does not reduce locomotor activity in rats and mice, nor does it affect rotarod performance in mice (Galaj et al., 2021; He et al., 2021, 2020).

We previously reported that BCP exerts promising therapeutic potential for psychostimulant use disorders. In a series of studies, we demonstrated that BCP attenuates cocaine-, nicotine- and methamphetamine-IVSA under fixed-ratio and progressive ratio schedules of reinforcement, as indicative of BCP’s ability to reduce psychostimulant reward and intake (Galaj et al., 2021; He et al., 2021, 2020). In an electrical or optogenetic brain stimulation reward paradigm, cocaine and opioids boost the proclivity of rats and mice to work for rewarding brain self-stimulation (Humburg et al., 2021; Wise, 1980). We have shown that BCP can attenuate electrical and optogenetic brain-stimulation reward and nicotine- and methamphetamine-enhanced brain-stimulation reward (Galaj et al., 2021; He et al., 2020). In addition, BCP dose-dependently attenuates cocaine-enhanced brain stimulation reward maintained by optical stimulation of midbrain DA neurons in DAT-cre mice, but it does not produce a statistically significant reduction in electrical brain-stimulation reward in rats (Galaj et al., 2020a). In the present study, we demonstrated that BCP only partially blocked heroin-enhanced optical brain stimulation reward, which is consistent with our previous in vivo microdialysis study indicating that BCP alone produces mild but long-lasting decreases in extracellular DA in the NAc and that BCP pretreatment only partially attenuates methamphetamine-induced increases in extracellular NAc DA (He et al., 2020). These findings suggest that BCP may directly modulate the mesolimbic DA reward circuitry. Furthermore, BCP reduces drug-primed reinstatement of psychostimulant seeking and cocaine CPP in rats, indicative of its preventative effects against relapse (Galaj et al., 2020a; He et al., 2021). BCP has been shown to reduce alcohol consumption and alcohol CPP (Al Mansouri et al., 2014). These results are encouraging, especially given the fact that BCP has good blood brain barrier penetration (Varga et al., 2018) and a low toxicity profile (Oliveira et al., 2018; Schmitt et al., 2016). Importantly, BCP can be effective when administered systemically or orally (Galaj et al., 2020a; Gertsch, 2008) without producing motoric impairments, as demonstrated in locomotor activity and rotarod paradigms (Galaj et al., 2020a; He et al., 2021).

The endocannabinoid system has been found to play a critical role in the neurobiological substrate underlying drug addiction, including opioid addiction (Galaj and Xi, 2019; He et al., 2019; Jordan et al., 2020; Soler-Cedeno and Xi, 2022; Spanagel, 2020). CB1R antagonists reduce heroin IVSA (He et al., 2019) and morphine CPP (Zhang et al., 2016), while genetic deletion of CB2 receptors exacerbates morphine withdrawal in CB2 KO mice (Iyer et al., 2020), suggesting that stimulation of CB2 receptors might diminish opioid effects. Indeed, stimulation of CB2 receptors by CB2 agonists has been shown to attenuate morphine CPP in wild type mice but not in CB2 KO mice (Iyer et al., 2020); it reduces the development of antinociceptive tolerance to morphine and attenuates morphine-induced physical dependence (Li et al., 2019; Lin et al., 2018). A recent report confirmed these findings, demonstrating that LY2828360 (a CB2 agonist) suppressed the development of morphine tolerance and reversed the established morphine tolerance to greater extent in male mice than female mice (Carey et al., 2023). Likewise, SA-57 (an inhibitor of the primary endocannabinoid degrading catabolic enzymes such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)) has the ability to augment the antinociceptive effects of morphine and reduce heroin-reinforced nose pokes behavior under a fixed ratio 1 and progressive ratio schedules of reinforcement in mice (Wilkerson et al., 2017). These findings indicate that there is a unique interaction between the opioid and endocannabinoid system, and CB1 and CB2 receptors are important players mediating opioid-driven behaviors.

In a series of studies, we have demonstrated that BCP produces its effects via CB2R and non-CB2R mechanism. For example, we found that pretreatment with AM630, a selective CB2R antagonist, but not AM251, a CB1R antagonist, can block the attenuating effects of BCP on nicotine IVSA in WT mice and rats. Genetic deletion of CB2Rs in CB2-KO mice significantly reduces basal levels of nicotine SA, as compared to WT mice and it also blocks a low dose, but not high dose, BCP-induced reduction in nicotine IVSA, suggesting the involvement of both CB2R and non-CB2R mechanisms (He et al., 2020). Similar effects have been observed with methamphetamine (He et al., 2021) and cocaine (Galaj et al., 2021). Although the literature indicates that some therapeutic effects of BCP are mediated by multiple receptors (Bento et al., 2011; Cho et al., 2015; Hernandez-Leon et al., 2020; Irrera et al., 2019; Katsuyama et al., 2013; Paula-Freire et al., 2014; Tian et al., 2019; Wu et al., 2014; Yang et al., 2017), we found no direct evidence of the involvement of CB1Rs, CB2Rs, GPR55, MORs or TLR-4 in BCP action in cocaine self-administration (Galaj et al., 2021), as pretreatments with respective antagonists or selective deletion of CB1Rs, CB2Rs or GPR55 failed to block BCP-induced reduction in cocaine self-administration. In contrast, GW6471 (a PPARα antagonist) and GW9662 (a PPARγ antagonist) reversed BCP-induced reductions in cocaine IVSA, while PPARα and PPARγ agonists mimicked BCP effects, suggesting that BCP therapeutic effects against cocaine are most likely mediated by activation of PPARα and/or PPARγ receptors directly or indirectly (Galaj et al., 2021). PPARα and PPARγ receptors are nuclear receptors that regulate gene expression and are expressed within the mesolimbic system (Hempel et al., 2023). Our group has shown that PPARγ, but not PPARα, agonists inhibit brain stimulation reward maintained by optical activation of VTA DA neurons and PPARα or PPARγ antagonists reverse the Δ9-THC-induced effects (Hempel et al., 2023).

Although the present study does not provide direct evidence, the interaction between opioids and PPAR have been reported by others (Ghavimi et al., 2015; Kashiwagi et al., 2021; Royal et al., 2004; Zhao et al., 2022). Pioglitazone, a PPARγ agonist, has been shown to reduce heroin IVSA under FR1 and progressive ratio schedules of reinforcement in rats and the reduction in responding for heroin can be blocked by a PPARγ antagonist (de Guglielmo et al., 2015). Similarly, pharmacological stimulation of PPARγ attenuates morphine withdrawal and abolishes stress- and heroin-induced reinstatement of drug seeking (de Guglielmo et al., 2017). Pioglitazone can also attenuate the development of tolerance to the analgesic effects of morphine but this effect is blocked by a PPARγ antagonist and is absent in the PPARγ-KO mice (de Guglielmo et al., 2014). Human laboratory studies report promising results of PPARγ agonists in reducing heroin cravings (Jones et al., 2018) but no effect on abuse liability of oxycodone (Jones et al., 2016), reinforcing effects of heroin (Jones et al., 2018) or opioid withdrawal (Schroeder et al., 2018). Thus, future studies should further investigate the role of these receptors, and other endocannabinoid receptors, in opioid-driven behaviors.

A rising interest in medical marijuana has prompted researchers to investigate potential sex differences in phytocannabinoid’s effectiveness, safety, tolerance and pharmacokinetics (Aviram et al., 2023; Hempel et al., 2018; Nguyen et al., 2020; Salviato et al., 2021; Wiley et al., 2021). Studies show that female, but not male, adolescent rats develop tolerance to the hypothermic effects of THC after repeated exposure, despite similar plasma THC levels (Aviram et al., 2023). Cannabidiol, another phytocannabinoid, produces anti-depressant-like effects in male mice in the tail suspension test (Silote et al., 2021) but anxiogenic effects in females in the Y-maze test (Wanner et al., 2021). While a number of findings suggest that there are no sex differences in cannabinoids’ effect on addiction-related behaviors (Hempel et al., 2018; Wakeford et al., 2017), some have shown that THC treatment impacts fentanyl self-administration in female but not male rats (Aviram et al., 2023). Only two studies have identified sex-differences in the BCP effects. While BCP produces more pronounced analgesic responses in male than in female mice (Ceccarelli et al., 2020), it promotes more rapid wound healing in females (Koyama et al., 2019). In the present study, we found that BCP produces more potent effects in females than males in reducing heroin-induced CPP and the extinction of CPP. However, it is possible that males require more extensive treatment or a higher dose of BCP to facilitate CPP extinction. We found no sex-differences in BCP action in the brain stimulation reward or antinociception paradigms.

In conclusion, in the present study we demonstrated and BCP can attenuate heroin IVSA in rats and in part, heroin-enhanced brain stimulation reward in DAT-cre mice. BCP also attenuates heroin-induced CPP in female but not male mice and fails to alter heroin analgesic effects. These results, in conjunction with previous reports, suggest that BCP is a promising ligand for treating psychostimulant use disorders and has certain therapeutic effects against opioid use disorders.

Highlights:

  • β-caryophyllene (BCP) reduces heroin self-administration in rats

  • BCP attenuates the expression and extinction of heroin conditioned place preference in female but not male mice

  • BCP fails to alter heroin antinociceptive effects in mice

Acknowledgments:

This work was supported by the National Institute on Drug Abuse Intramural Research Program funding to ZXZ and the National Institute on Drug Abuse under award number 1R15DA057501 - 01A1 to EG, Colgate University

Footnotes

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Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflict of interest.

Data Availability:

We are happy to share any protocols or programs that we used to collect these data. The data will be made available upon direct requests to the corresponding author.

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