Core temperature measures and summed catalepsy scores for rats receiving acute subcutaneous injections of 0. Core temperature and behavioral score were recorded at 0, 0. The left panel of Figure 2 shows that pretreatment with 1. Figure 2. Core temperature measures and summed catalepsy scores for rats receiving either subcutaneous sc vehicle VEH or 1. The left panel of Figure 3 depicts the effects of 1. Because vehicle administration did not affect body temperature over the course of repeated injections, we compared the effects of JWH treatments across days to those of vehicle treatment on day 1.

On day 1 of JWH exposure, temperature was significantly reduced from vehicle at the 1, 2, and 4 h timepoints. By day 3 of treatment, hypothermia was observed only at the 1 h timepoint, and on days 6 and 7, no reduction in temperature was observed. Figure 3. Core temperature measures and summed catalepsy scores for rats receiving either subcutaneous vehicle VEH or 1.

The right panel of Figure 3 depicts the effects of 1. Vehicle administration did not significantly alter summed catalepsy scores on day 1 of treatment, and there was no change in scores for vehicle-treated rats over the 7-day treatment regimen.

Since vehicle administration did not change catalepsy scores over the course of treatment, we compared the effects of JWH treatment across days to the effects of vehicle treatment on day 1.

Using this analysis, JWH increased catalepsy scores compared to vehicle Kruskal—Wallis statistic Rats received 0. Similar non-significant effects between pretreatment groups were observed at day 7.

DOI did not significantly affect core body temperature in rats pretreated with JWH or vehicle at either test day data not shown. Figure 4. Summed scores for wet dog shakes and back muscle crawls skin jerks induced by a subcutaneous challenge injection of 0. Behavioral scores were recorded at 0, 0. A separate cohort of rats was given 0. Figure 5.

Summed scores for serotonin syndrome behaviors and mean temperature recordings induced by a subcutaneous challenge injection of 0. Behavioral scores and core temperatures were recorded at 0, 0.

Post hoc tests revealed that temperature was significantly decreased in the JWH group compared to the vehicle group at 1. Figure 6. Time-course of core body temperature changes induced by a subcutaneous challenge injection of 0. Temperatures were recorded at 0, 0.

The psychiatric literature supports a strong relationship between heavy cannabis use and risk for subsequent psychosis and schizophrenia In addition, misuse of synthetic cannabinoids such as JWH and its analogs is associated with induction of more severe psychotic symptoms when compared to the effects of marijuana 26 , The aim of the present study was to use the popular synthetic cannabinoid JWH to further explore the relationship between repeated cannabinoid exposure and serotonergic dysregulation.

JWH 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. 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 Finally, our data show that rats receiving daily injections of JWH develop profound tolerance to its hypothermic and cataleptic effects, such that these effects are nearly absent after 7 days of treatment.

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 — 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. It is noteworthy that we observed trends for augmented wet dog shakes and attenuated skin jerks in rats exposed to JWH, but these effects did not reach significance, perhaps due to variability in the behavioral data. We also administered a submaximal dose of 0.

Hill et al. This hypothesis was later supported by the work of Franklin et al. It is well known that HU displays a much longer time course of action when compared to other synthetic cannabinoids, including JWH, and may bind pseudo-irreversibly to the CB 1 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, while such responses to THC and CP 55, ceased after 5 h.

Thus, the discrepancies between our results and those of Hill et al. 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 CB 1 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 treatments when compared to those receiving repeated vehicle treatments.

In a previous study, Hill et al. Both hypothermia and corticosterone release are presumably mediated by 5-HT 1A receptors in the brain 31 , thus Hill et al. It seems possible that discrepancies between our results and those of Hill et al. On the other hand, Zavitsanou et al. Our data demonstrating an increase in 5-HT 1A receptor sensitivity after exposure to JWH 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 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 injections. Rats in our study had implantable temperature transponders to facilitate the non-invasive measurement of body temperature. JWH 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 administration in rats are consistent with previous findings from our laboratory and others, which show dose-related hypothermic effects of JWH as assessed by radiotelemetry or rectal probes to measure core temperatures 33 — As the repeated injection procedure progressed in our study, rats began to develop tolerance to both the hypothermic and cataleptic effects produced by JWH By day 5 of repeated treatments, the effects of JWH 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 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 CB 1 receptors Likewise, acute JWH is reported to induce downregulation of CB 1 receptors in cultured neurons by a mechanism involving rapid receptor internalization The experiments of Tai et al. The apparently contradictory findings between our results and those of Tai et al.

Tai et al. The development of tolerance to cannabis is well documented, and the demonstration of tolerance to JWH could have important clinical implications 40 , 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 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 , 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 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 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.

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. J Mass Spectrom —7.

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This may lead users to falsely believe that they are safe for consumption. Combined with unknown doses and affinities, users may easily subject themselves to accidental overdose.

Patients that present to emergency rooms with a history of SC abuse should be carefully monitored for epileptic events using EEG recordings. Particularly since their symptoms might be misinterpreted as simple sedation, stupor or catatonia 33 , Use of plant-derived cannabinoids seems less risky, since their potency might be lower and anti-epileptogenic compounds may counterbalance the epilepsy risk posed by CB 1 R agonism. Nevertheless these cannabinoids, if overdosed, can lead to epileptic seizures, as we show here.

Thus, the same vigilance as for SCs seems prudent, if overdose is suspected. Controlled medical use of cannabinoids seems to carry the smallest risk. Doses are typically significantly lower than used in our studies to elicit seizures.

It should be noted, however that epileptic seizures as rare adverse side effects of medical marijuana use have been reported The CB 1 R agonists we have studied revealed strong proconvulsive properties, implying that any newly synthesized CB 1 R agonists may also exert similar behavioral effects and trigger seizures.

No specific medication is currently available to alleviate cannabinoid intoxication. The mechanism of toxicity to explain the seizures induced by cannabinoids remains unclear. Coupled with increased public use, constant production of new analogues and our lack of knowledge as to their long-term effects on human health show the need for further research and proper regulation. Finally, the potential introduction of CB 1 R antagonists, as a treatment for cannabinoid-induced seizures or other life-threatening conditions in the case of overdose, requires further investigation in the clinical settings.

All described experiments were replicated at least twice. Mice were housed in a temperature- and humidity-controlled environment and maintained on a h light: h dark cycle lights on at Food and water were available ad libitum. Experiments were performed in compliance with relevant Japanese and institutional laws and guidelines and approved by the Animal Ethics Committee of the University of Tsukuba, number 16— Efforts were made to reduce the number of animals used and to minimize any pain or discomfort they might have felt.

EMG signals were recorded from two insulated Teflon-coated electrodes that had been inserted into the bilateral neck muscles. Finally, the electrode assembly was fixed to the skull with self-curing dental cement, and the wound was then sutured. After surgery, the animals were administered an i.

Each cable was flexible so that the mice could move freely about their cages. A video of each animal was recorded using an infrared camera. Seizure spikes were detected and counted using peak analysis function of OriginLab v8. The doses of cannabinoids presently used here were selected based on the CB 1 R affinity of the ligands and the electrographic representation of mild seizure events. In fact, we intentionally selected relatively high doses that we knew to have an effect on EEG power spectra The conversion of an animal to human dose cannot be done directly, as in the few available studies on human volunteers most utilize the inhalation route, while we administered drugs intraperitoneally.

However, after applying the human estimation dose conversion 40 , 2. Blood was collected in Terumo Capiject Capillary Blood Collection Tubes Tokyo, Japan and, after centrifugation serum was transferred into plastic tubes.

A Coretecs C18 column 2. Quantification of JWH and six metabolites was performed using multiple reaction monitoring MRM of the transitions of precursor ions to product ions with each cone voltage and collision energy as shown in Table 1. During the analyses, we confirmed two transitions precursor ions and two product ions of each compound and their ratio. The drug concentrations in the samples were calculated using the peak—area ratios of the product ions for quantitative monitored for the target compounds versus IS.

The calibration curves for the determination were constructed by analysing extracted drug-free control serum spiked with the standard solution. Calibration curves of JWH and the metabolites were linear over the concentration range 0.

The limit of detection of each drug was 0. All animals were littermates and were assigned randomly to different groups. EEG analysis and spike quantification were performed by an experimenter who was unaware of the group to which the animal belonged.

Power analysis was used to determine the ideal sample size for behavioral experiments. In some cases, mice were excluded from analysis due to damage to or loss of the EEG electrode.

The data sets generated and analysed during the current study are available from the corresponding author on reasonable request. National Drug Threat Assessment Summary.

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