Synthetic cannabinoids are novel psychoactive substances with pharmacological similarity to the phytocannabinoid Δ⁹-tetrahydrocannabinol (THC), the main psychoactive ingredient in marijuana. Over the last decade, herbal smoking blends consisting of plant material laced with synthetic cannabinoids (i.e., “spice” products) have emerged in the recreational drug marketplace. Analytical investigations of the first spice products revealed that a primary psychoactive component was naphthalen-1-yl-(1-pentylindol-3-yl)methanone, also known as JWH-018 (1, 2). JWH-018 and many of its structural analogs were found in spice products during 2010 through 2013, and JWH-018 is still present on the street today (3, 4). JWH-018 is a potent agonist at cannabinoid type-1 (CB₁) and cannabinoid type-2 (CB₂) receptors, which displays at least threefold higher binding affinity than THC at both receptors (5, 6). When administered to mice, JWH-018 produces effects consistent with other CB₁ receptor agonists, including hypothermia, analgesia, reduced motor activity, and catalepsy (5, 6). In drug discrimination studies, JWH-018 fully substitutes for the THC stimulus cue in both mice and rats (7–9).

Several lines of clinical evidence support a relationship between heavy cannabis use and risk for development of psychosis and schizophrenia (10–13). Although the precise underpinnings of schizophrenia are not fully understood, dysregulation of brain serotonin (5-HT) systems has been implicated in certain psychotic symptoms, such as paranoia and hallucinations (14–16). Preclinical studies in rodents show that exposure to CB₁ receptor agonists can influence 5-HT receptor responsiveness in vivo. For example, Darmani reported that acute pretreatment with various cannabinoids, including THC and the potent cannabinoid receptor agonist (6aR,10aR)-9-(hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6H,6aH,7H,10H,10aH-benzo[c]isochromen-1-ol (HU-210), inhibits behavioral effects of the 5-HT_2A/2C agonist 2,5-dimethoxy-4-iodoamphetamine (DOI) in mice (17). By contrast, Hill et al. found that repeated treatment with HU-210 for 12 days enhances wet dog shakes induced by DOI in rats (18). Franklin et al. found that 7-day exposure to the cannabinoid agonist 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl) cyclohexyl]-5-(2-methyloctan-2-yl)phenol (CP 55,940) enhances cortisoterone release induced by DOI in rats, and this effect is accompanied by upregulation of 5-HT_2A receptors in the hypothalamus (19). Recent evidence suggests a direct interaction between CB₁ and 5-HT_2A receptors in rat brain. In particular, Viñals et al. showed that CB₁ and 5-HT_2A receptor heteromers are present in hippocampus and other brain regions related to memory formation, and these heteromers are necessary for the amnesic effects of THC, but not its analgesic effects (20).

Despite the continued misuse of synthetic cannabinoids by humans, little is known about the functional consequences of repeated administration of JWH-018 or related substances found in spice products. Given the emerging evidence for interactions between cannabinoid and 5-HT systems in the brain, we sought to determine the effects of repeated treatment with JWH-018 on the behavioral responsiveness to selective 5-HT receptor agonists. Specifically, male Sprague-Dawley rats were treated for seven consecutive days with JWH-018, then challenged with DOI or the 5-HT_1A receptor agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) at 1 day and 7 days after the last JWH-018 treatment. Body temperatures and catalepsy scores were determined during the repeated dosing regimen of JWH-018, while body temperatures and agonist-induced behaviors were measured following challenge doses of 5-HT drugs. We hypothesized that repeated exposure to JWH-018 would enhance subsequent behavioral responsiveness to DOI in rats [e.g., see Ref. (18)]. Such increases in 5-HT_2A/2C activity produced by cannabinoid exposure could contribute to adverse psychiatric symptoms associated with cannabinoid use.

The psychiatric literature supports a strong relationship between heavy cannabis use and risk for subsequent psychosis and schizophrenia (12). In addition, misuse of synthetic cannabinoids such as JWH-018 and its analogs is associated with induction of more severe psychotic symptoms when compared to the effects of marijuana (26, 27). Previous studies in rats demonstrate that exposure to synthetic cannabinoids can induce enhanced sensitivity to 5-HT_2A/2C receptor activation (18) and upregulation of 5-HT_2A/2C receptors in specific brain regions (28, 29). The aim of the present study was to use the popular synthetic cannabinoid JWH-018 to further explore the relationship between repeated cannabinoid exposure and serotonergic dysregulation. JWH-018 is a potent non-selective cannabinoid receptor agonist that was found in the first generation of spice products (1, 2). The present experiments yielded three primary findings. First, in contrast to the results of others [e.g., see Ref. (18)], we detected no significant difference in responsiveness to the 5-HT_2A/2C receptor agonist DOI between rats pretreated with synthetic cannabinoids compared to those pretreated with vehicle. Second, we found a modest and significant enhancement of sensitivity to behavioral and hypothermic effects induced by 8-OH-DPAT in rats exposed to repeated injections of JWH-018. Finally, our data show that rats receiving daily injections of JWH-018 develop profound tolerance to its hypothermic and cataleptic effects, such that these effects are nearly absent after 7 days of treatment.

In our experiments, male rats were subjected to seven consecutive days of JWH-018 injections, then given a challenge dose of either the 5-HT_2A/2C agonist DOI or the 5-HT_1A agonist 8-OH-DPAT at 1 and 7 days after cessation of the repeated dosing regimen. Typical behavioral responses to DOI administration in rats are wet dog shakes (analogous to the head twitch response in mice) and back muscle contractions, also known as skin jerks (21–23). These responses are accepted as specific indicators of 5-HT_2A receptor activation since the effects are blocked by selective 5-HT_2A receptor antagonists. We found no significant difference in the number of wet dog shakes or skin jerks induced by DOI between the cannabinoid-treated and vehicle-treated groups at either time point. Our findings differ from those of Hill et al., who reported that 12 days of HU-210 administration in rats increases DOI-induced wet dog shakes but decreases skin jerks (18). It is noteworthy that we observed trends for augmented wet dog shakes and attenuated skin jerks in rats exposed to JWH-018, but these effects did not reach significance, perhaps due to variability in the behavioral data. We also administered a submaximal dose of 0.1 mg/kg DOI for our experiments, whereas Hill et al. administered a 10-fold higher dose. Hill et al. theorized that the differential effects of HU-210 on the two behaviors induced by DOI could be due to region-specific changes in 5-HT_2A receptors caused by the cannabinoids. This hypothesis was later supported by the work of Franklin et al., who found that 7-day administration of CP 55,940 increased DOI-induced prolactin release, while producing no change in brain levels of 5-HT_2A receptor mRNA (19). It is well known that HU-210 displays a much longer time course of action when compared to other synthetic cannabinoids, including JWH-018, and may bind pseudo-irreversibly to the CB₁ receptor. Hruba and McMahon found that rhesus monkeys trained to discriminate THC from vehicle continued to emit drug-appropriate responses for 48 h after administration of HU-210, while such responses to THC and CP 55,940 ceased after 5 h. The same study found that rimonabant treatment increased the ED₅₀ values of THC and CP 55,940 discrimination by 12.5 fold, while only causing a 3.8-fold increase for HU-210 (30). Thus, the discrepancies between our results and those of Hill et al. could be due to the use of different cannabinoid agonists for the repeated treatment regimen.

Darmani administered a range of doses of THC, HU-210 and CP 55,940 to mice, followed by an injection of DOI 20 min later, and found that the cannabinoids dose-dependently reduce DOI-induced behaviors (17). Our study used a repeated cannabinoid administration paradigm followed by the administration of DOI after 1 and 7 days of withdrawal, so this may help to explain the differences between our results and those of Darmani. The present findings in rats show that administration of CB₁ agonists causes considerable catalepsy (see Figures 1–3), so it seems possible that suppression of motor activity caused by acute cannabinoids could influence subsequent behavioral effects of 5-HT_2A receptor agonists. We purposefully designed our experiments to examine the responsiveness to 5-HT agonists at 1 and 7 days after the acute effects of cannabinoid administration had subsided.

We found a modest yet significant increase in the behavioral and hypothermic effects induced by 8-OH-DPAT in rats receiving repeated JWH-018 treatments when compared to those receiving repeated vehicle treatments. The augmented sensitivity to 8-OH-DPAT resolved by 7 days after cannabinoid exposure. In a previous study, Hill et al. found that repeated injections of HU-210 for 12 consecutive days reduce the hypothermic and corticosterone responses produced by 8-OH-DPAT in vehicle-treated animals (18). Both hypothermia and corticosterone release are presumably mediated by 5-HT_1A receptors in the brain (31), thus Hill et al. found that repeated administration of HU-210 decreases 5-HT_1A activity in response to agonism, whereas we found the exact opposite in rats exposed to JWH-018. It seems possible that discrepancies between our results and those of Hill et al. could be due to the use of different cannabinoid agonists, as noted above. On the other hand, Zavitsanou et al. demonstrated that repeated injections of HU-210 increase 5-HT_1A receptor density and mRNA levels in the hippocampus and amygdala of male rats (32), a finding consistent with the possibility of enhanced 5-HT_1A receptor responsiveness after cannabinoid exposure. Our data demonstrating an increase in 5-HT_1A receptor sensitivity after exposure to JWH-018 is a unique finding, and its relationship to the development of psychiatric symptoms following cannabinoid exposure warrants further study. Future research should determine whether 5-HT_1A upregulation occurs after repeated exposure to other synthetic cannabinoids. Importantly, and in contrast to existing findings using other cannabinoid compounds, our data show that repeated exposure to JWH-018 does not induce robust alterations in 5-HT_2A receptor responsiveness, but increases 5-HT_1A responsiveness.

In addition to assessing changes in serotonergic activity after cannabinoid exposure, one of the secondary aims of our study was to examine pharmacological responses to repeated JWH-018 injections. Rats in our study had implantable temperature transponders to facilitate the non-invasive measurement of body temperature. JWH-018 was shown to dose-dependently cause hypothermia and catalepsy, both of which were reversed by rimonabant (see Figure 2). The present data showing acute decreases in body temperature after JWH-018 administration in rats are consistent with previous findings from our laboratory and others, which show dose-related hypothermic effects of JWH-018 as assessed by radiotelemetry or rectal probes to measure core temperatures (33–36). As the repeated injection procedure progressed in our study, rats began to develop tolerance to both the hypothermic and cataleptic effects produced by JWH-018. By day 5 of repeated treatments, the effects of JWH-018 became submaximal at all time points, and continued to decrease in the two remaining days. By day 7 of repeated treatments, the temperature and cataleptic effects JWH-018 were not significantly different from vehicle-treated animals. Previous studies in mice have shown that repeated daily injections of THC or synthetic cannabinoids produce behavioral tolerance due to downregulation and desensitization of CB₁ receptors (37). Likewise, acute JWH-018 is reported to induce downregulation of CB₁ receptors in cultured neurons by a mechanism involving rapid receptor internalization (38).

The experiments of Tai et al. showed that mice develop tolerance to the hypothermic effects of JWH-018, but not the locomotor suppressing effects (39). The apparently contradictory findings between our results and those of Tai et al. may be due to species-specific differences between rats and mice. Tai et al. also showed that mice repeatedly exposed to THC develop a cross tolerance to the effects of JWH-018. The development of tolerance to cannabis is well documented, and the demonstration of tolerance to JWH-018 could have important clinical implications (40, 41). Dose escalation in human THC users is often observed as a means to overcome cannabis tolerance, but this phenomenon likely will not cause acute bodily harm. By contrast, dose escalation with JWH-018 or other potent synthetic cannabinoids could be more dangerous. Typical adverse effects arising from synthetic cannabinoid use are tachycardia, agitation, and nausea; more serious adverse events include seizures, acute kidney injury, new onset psychosis, severe cardiac crisis, and death (27, 42). Further research is required to determine if such dose escalation occurs in humans who use synthetic cannabinoids.

To summarize, we found that repeated treatment with the synthetic cannabinoid JWH-018 does not lead to significant changes in 5-HT_2A receptor responsiveness in rats, but produces transient increases in 5-HT_1A receptor responsiveness. These findings, unlike data generated using other synthetic cannabinoids, do not support the contention that exposure to cannabinoid receptor agonists universally leads to an increase in 5-HT_2A receptor responsiveness, suggesting that alteration of 5-HT_2A neurotransmission may not be responsible for the link between cannabinoid exposure and the subsequent development of psychotic symptoms. On the other hand, rats in our experiments developed tolerance to both hypothermia and catalepsy produced by JWH-018 after several consecutive days of treatment, findings which differ from prior work in mice suggesting that tolerance only develops to hypothermic effects. Synthetic cannabinoid tolerance in humans could potentially lead to dose escalation, which could be more dangerous with synthetic cannabinoids when compared to marijuana.

Experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Vivarium facilities were fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, and study procedures were approved by the NIDA Intramural Research Program Animal Care and Use Committee.

JE and MB were responsible for experiment design, statistical analysis, and manuscript writing. JE collected the data.

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