After 2 weeks of acclimation to the vivarium, rats were subjected to surgical procedures and subsequently used for experiments. Rats were rapidly anesthetized with isoflurane using a drop jar which contained a raised floor above a gauze pad saturated with 5 mL of isoflurane. Once fully anesthetized, each rat received a surgically implanted IPTT transponder Bio Medic Data Systems, Seaford, DE, USA to facilitate the non-invasive measurement of body temperature via a portable radio frequency reader system handheld reader.
Animals were individually housed postoperatively and allowed 7—10 days for recovery. As a first step in our study, we examined the dose—response effects of acute JWH administration in a cohort of 12 rats.
Rats were tested once per week for three consecutive weeks. On test day, rats were moved to the testing room in their home cages and given 1 h to acclimate. Feeding trays were removed, and wire lids were placed atop the cages. Rats received sc injections of JWH 0.
Immediately before injection, and at various times thereafter 0. Observers were not blind to the drug treatment condition. Rats were assigned a catalepsy score based on three behaviors: immobility absence of movement , flattened body posture, and splayed limbs limbs spread out away from the center of the body. Once dose—response experiments were completed, we next tested the effect of pretreatment with the CB 1 receptor antagonist rimonabant on the responses induced by JWH in a cohort of 12 rats.
Rats were pretreated with either 1. Body temperature measurements and behavior scoring were carried out as described previously for acute dose—response experiments. Results from the acute dose—response experiments demonstrated that 1. Thus, this dose was used for the repeated injection experiments carried out in a group of 32 rats.
The repeated dosing with JWH or its vehicle was carried out in the vivarium. Rats fitted with surgically implanted sc temperature transponders received a single sc injection of either 1.
Immediately before injection, and at 1, 2, and 4 h post-injection, body temperature was measured using the handheld reader, and animals were observed for 90 s. One day after the last repeated treatment with JWH or vehicle i. One cohort of 16 rats received 0. The doses of DOI and 8-OH-DPAT were based on preliminary dose—response experiments, which identified drug doses evoking robust behavioral changes that were less than maximal data not shown.
The specific non-contingent behaviors induced by DOI were wet dog shakes and back muscle contractions i. Both behaviors are known to be mediated by 5-HT 2A receptors in rats 21 — The numbers of wet dog shakes and skin jerks present during the observation period were tallied. Wet dog shakes were defined as a rapid and sudden rotation of the head, neck, and shoulders from one side to the other, analogous to the way a wet dog may shake to dry itself. Skin jerks were defined as brief paraspinal muscle contractions of the back muscles in a tail to head direction.
Possible scores for each behavior were 0 behavior absent , 1 behavior present , or 2 behavior intense or continuous. At the end of the observation period, the scores for the three behaviors were summed to produce a 5-HT syndrome score for each time point. After acute serotonergic drug challenge, body temperatures were measured using the handheld reader at 0. Data were tabulated, analyzed, and graphically depicted using GraphPad Prism version 5.
Statistical analyses were performed on data from all 7 days of the JWH repeated administration experiment, however, Figure 3 only shows data from selected days to make the graphs easier to interpret.
The left panel of Figure 1 illustrates the effect of acute JWH administration on core body temperature in male rats. It is worth noting that 0. As seen in the right panel of Figure 1 , JWH dose-dependently increased the summed catalepsy behavioral score Kruskal—Wallis statistic Figure 1.
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. Group comparison was done using the unpaired t -test. We also observed behavioral changes after administration of JWH, including suppression of locomotor activity, impaired walking, ataxia, extensor rigidity in both hind limbs, straub tail, muscular jerks, rearing, and low-intensity behavioral seizures Suppl. Video S1. The righting reflex was preserved, whereas the touch escape response was impaired.
Animals also showed dyspnea gasps and profound catatonia Suppl. Video S2. Video S3. Table 1. To clarify whether cannabinoids induce seizure activity in a dose-dependent manner, we quantified spike frequency after acute i. The result showed a clear dose-response relationship, with the number of spikes increasing with dose Pretreatment with AM resulted in the complete abolition of electrographic seizures: the EEG showed a normal pattern in all animals tested Fig.
Figure 2 , however no specific behavioral tests, beyond simple observation were conducted. To examine the pharmacokinetics of JWH, blood was collected at different time points after administration of the drug at a dose of 2.
After separation of the serum, JWH and 6 metabolites were measured. Serum JWH concentration reached JWH 5-OH indole metabolite increased in the serum at a much lower range of concentrations, with a peak at 0. Other metabolites of JWH were detected at low serum levels, with the highest value less than 0. On the other hand, a substantial body of literature on cannabinoids in animal models shows mostly anticonvulsive effects However, few of these used EEG recordings to assess epileptic events and many of them induced seizures either electrically or pharmacologically, changing signalling pathways and brain states prior to cannabinoid application.
Since the discovery of the cannabinoid system CB 1 R, CB 2 R and endogenous cannabinoids , their physiological properties have been extensively discussed and contradictory findings have been reported in humans as well. Despite such attempts to use medical marijuana in the treatment of intractable epilepsy, the medical evidence supporting its use for the treatment of neurological diseases is not sufficiently convincing 26 , and evidence for the safety and efficacy of cannabinoids that would allow them to be used clinically remains weak.
Because we intentionally used higher doses which produce toxicity and induce seizures, these doses might have effects not representative of those seen with typical medicinal or recreational human consumption.
It would be interesting in the future to also test lower doses, typically used medicinally or recreationally to determine whether the effect is lost or diminished. As we show here, seizures induced by cannabinoids are typically mild and almost asymptomatic to the untrained eye - therefore careful EEG evaluation would be needed.
However, it is important to note that dose conversion always requires some consideration, because human bioavailability and metabolism may differ significantly from that of animals. Furthermore, differences between human and animal receptor sensitivity or density have been shown to affect human pharmacologic or toxicologic outcomes In general, number of studies on JWH pharmacokinetics are limited and sample sizes with equal conditions are small, which makes the comparison of the different studies difficult.
Another point is the different route of administration, and it is possible that inhalation might have a faster pharmacokinetic effect than intraperitoneal injection.
The activity of metabolites of JWH is unknown, but may be important because such metabolites may exert synergistic effects and reinforce the SCs toxicity. This may reflect a species difference for JWH metabolism, and thus potentially indicate different toxicity. This increase of concentration could partially explain the long-term electrographic and behavioral changes after JWH administration in mice.
To answer these questions, further studies on JWH metabolites and their toxicity need to be conducted in mice. CB 1 Rs are expressed in both excitatory glutamatergic and inhibitory GABAergic terminals; therefore, the distribution of CB 1 R-expressing activated neurons may play a key role in generating different behavioral and neurophysiological effects Our results clearly show that the epileptogenic properties of cannabinoids are mediated via CB 1 receptors.
The greatest danger therefore for accidental overdoses seems to stem from SCs with high CB 1 R affinity. These substances are often not controlled, since they are newly synthesized and therefore not yet banned by regulatory agencies. 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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