In order to evaluate the effects of acute exposure to JWH-018, mice were injected once with either a vehicle or JWH-018 (1 mg/kg, i.p.). We commonly observed induction of abnormal symptoms, such as rotational and seizure-like behavior, by JWH-018 at least one hour after administration. Behavioral analyses were conducted two hours after administration of JWH-018. Locomotor and anxiety-like behavior were evaluated in the open-field test, and novel object recognition (NOR) as well as tail suspension tests were conducted to compare amnesic effects of JWH-018 to those of cannabicyclohexanol (CCH) and Δ⁹-THC. The results of the open-field test suggested that JWH-018 increases locomotor exploration while decreasing anxiety-like behavior in a novel environment (Fig. 1A,B). In addition, NOR test revealed a decrease in the recognition index of the JWH-018 group when compared with the control group (Fig. 1C). On the basis of this observation, it was strongly suggested that JWH-018 produces an impairment of memory retention in mice, which is in concordance with previous reports of memory impairments resulting from acute injections of JWH-018²⁷. Along these lines, tail suspension tests revealed a higher immobile time for the JWH-018 group, whereas it was shortened in the CCH group compared with vehicle-injected controls (Fig. 1D). These results could help to identify a behavioral pharmacological marker for synthetic cannabinoid abuse in the future.

Hippocampal extracts were subjected to metabolomic analysis using UPLC-TOF/MS. The principal component analyses of the metabolomic data obtained from UPLC-TOF/MS in positive-ion modes (Fig. 2A,B) revealed that the clusters of plots in the two groups were distinctly separated from each other. This observation suggests that the hippocampal metabolome varies markedly between JWH-018 treatment and JWH-018/AM251 co-administration. The S-plot was used to select potential biomarkers and visualize metabolomic changes. The farther red dots were the more likely biomarker candidates because of their high contributions and correlations (Fig. 2C,D).

By aligning mass and retention times of ions to a previous database, we observed that many metabolites were significantly altered, including as amino acids, endocannabinoids, and tricarboxylic acid (TCA)-cycle intermediates (see the Supplemental Table 1 for the detailed conditions). Due to the nature of these observations, some ingredients were further investigated. This reflects the metabolic response of mice to JWH-018 exposure revealing potential markers of metabolomic perturbation. Furthermore, providing sufficient data to foray on the research and exploration of its underlying mechanisms. For example, we observed a reduction of N-acetyl-aspartate (NAA) in the brain following JWH-018 exposure. NAA is typically present at a high concentration in the brain and is a neuron-specific marker for monitoring neuronal damage associated with central nervous system (CNS) diseases although it lacks specificity for any particular disease^28,29. Therefore, reduction of NAA suggested neuronal damage following JWH-018 exposure. Additionally, glutamic acid, a major excitatory neurotransmitter in the brain, was increased in the JWH-018-treated group compared to the control group. Furthermore, administration of JWH-018 resulted in increases of phenylalanine and tyrosine levels, and succinic acid, which is an intermediate of the TCA-cycle indicating the influence of the TCA cycle for energy metabolism and supply. Although we did not address the functional meaning of these significantly varied metabolites in depth in this study, understanding the contribution of these metabolite alterations may potentially aid clarifying the effects of synthetic cannabinoids on the CNS in the future.

Importantly, we observed for the first time that JWH-018 treatment resulted in a significant elevation of AEA and 2-AG levels, which may indicate alterations in fatty acid metabolism. AEA and 2-AG are endogenous ligands of the CB1 receptor, serving as mediators for the retrograde suppression of synaptic transmission and modulate synaptic plasticity^27,30,31, which plays a pivotal role in learning and memory. An accumulation of endocannabinoids suppressing synaptic long-term potentiation could elicit an aggravation of memory impairment. Therefore, it was important to confirm whether JWH-018 induces the accumulation of endocannabinoids. Furthermore, application of a specific CB1 receptor antagonist could help to elucidate the mechanism underlying an increase in endocannabinoids by JWH-018.

The content of hippocampal endocannabinoids was determined using methods reported previously³² with slight modifications. Identification and quantification of AEA and 2-AG were performed using an UPLC-TOF/MS (LCT-Premier XE, Waters, Milford, MA) with TargetLynx^XS v4.1 software (Waters Co., Milford, MA). A five point calibration curve with concentration ranges of 0.01–1 μM (0.01, 0.05, 0.1, 0.5, 1) of AEA-d₄ and 3.125–50 μM (3.125, 6.25, 12.5, 25,50) of 2-AG-d₈, and blanks with and without the internal standards were prepared. A linear regression of the ratio of the peak area counts of AEA, 2-AG to their corresponding internal standards (anandamide-d₄ and 2-AG-d₈) vs. concentration was used to construct the calibration curves (Supplemental Table 2).

As shown in Figs 3–5, UPLC-TOF/MS analysis was performed in a selected ion-monitoring mode. The mean AEA and 2-AG tissue levels in the control group were determined to be 11.6 pmol/g and 26.5 nmol/g of tissue, respectively, which is in concordance with previous reports^33,34. Furthermore, this measurement confirmed that JWH-018 caused a 10.6- and 2.7-fold increase of these components in the hippocampus, respectively. This elevation was blocked by co-administration of AM251. Taken together, our results demonstrate that JWH-018 facilitates endocannabinoids accumulation in a CB1 receptor-dependent fashion in brain hippocampus.

Total protein and RNA extraction were carried out after behavioral tests. Mice were euthanized and hippocampi were rapidly collected, snap-frozen in liquid nitrogen, and stored at −80 °C until use. To investigate how JWH-018 treatment elicits accumulation of endocannabinoids in the hippocampus, we examined the expression of mRNAs coding for endocannabinoid synthesizing and metabolizing enzymes. Biosynthesis of AEA is primarily achieved by the enzyme N-acyl-phosphatidylethanolamines-hydrolyzing phospholipase D (PD)³⁵. Fatty acid amide hydrolase (FAAH) is an intracellular membrane-bound enzyme that degrades fatty acid amides and it is responsible for inactivation of AEA by catalyzing its breakdown to arachidonic acid (AA) and ethanolamine^36,37. The primary synthetic and metabolic enzymes for 2-AG have been identified as diacylglycerol lipase (DGL) and monoacylglycerol lipase (MAGL), respectively^38,39. JWH-018 treatment decreased the mRNA level of FAAH and MAGL. Conversely, co-treatment with AM251 completely restored the mRNA levels of these enzymes (Fig. 6).

By evaluating the mRNA and protein levels of BDNF, a marker of synaptic plasticity in the hippocampus, we aimed to investigate molecular changes that can be linked to behavioral effects caused by JWH-018. BDNF is known to modulate neuroplasticity and adaptive processes underlying learning and memory⁴⁰. BDNF was significantly decreased following exposure to JWH-018 both at mRNA and protein levels (Fig. 7). This effect was prevented by co-administration of AM251.

JWH-018 and CCH were provided from the National Institute of Health Sciences of Japan. 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide (AM251), (5Z,8Z,11Z,14Z)-N-(2-hydroxy-[1,1,2,2-D4]ethyl)icosa-5,8,11,14-tetraenamide (AEA-d₄), and 5Z,8Z,11Z,14Z-eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid 2-glyceryl ester (2-AG-d₈) were purchased from Abcam (Cambridge, UK). Δ⁹-THC was prepared from Cannabis by Professor S. Morimoto. All other reagents were of the highest grade commercially available. All working solutions were stored at −80 °C until use.

All procedures for the animal experiments in this study were reviewed and approved by the Ethics Committee of Animal Experiments, Kyushu University. All methods were performed in accordance with the relevant guidelines and regulations. Every effort was made to minimize the number of animals used and their suffering. C57BL/6J-Bom mice were purchased at the age of seven weeks. Animals were housed in a cage for one week after their arrival, and were provided with food and water ad libitum. The rearing room was controlled on a 12 h light-dark cycle (lights off at 7:00 pm), and the room temperature was maintained at 22 °C. We divided animals in three groups for investigation: JWH-018 treatment, co-treatment with JWH-018 and AM251, and control (vehicle) treatment. In the present study, JWH-018 dose was chosen based on previous studies^41,50. JWH-018 (1.0 mg/kg injected group) was initially dissolved in absolute ethanol (final concentration was 2%) and Tween-80 (2%) and brought to the final volume with saline (0.9% NaCl). AM251 (1.0 mg/kg) was administered 30 min prior to JWH-018 injection in the co-administration group. The vehicle solution contained 2% ethanol, 2% Tween-80, and saline. Similarly, ∆⁹-THC (3.0 mg/kg) and CCH (1.0 mg/kg) were prepared. Their dose were chosen based on previous studies^51,52. All drugs were administered intraperitoneally (i.p.), and injected at a volume of 0.1 ml per 10 g of body weight. Behavioral analyses occurred after administration of appropriate drug or vehicle treatment. After the behavioral tests, all the mice were sacrificed for brain sampling collection. The whole brain was placed in liquid nitrogen immediately and frozen at −80 °C until biochemical analysis, along with metabolomics, real-time polymerase chain reaction (RT-PCR), and western blotting.

The brain was homogenized in four volumes of cold MeOH/CH₃CN/H₂O (2:2:1, v/v), vortexed for 30 s and incubated in liquid nitrogen for 1 min. Samples were allowed to thaw and subsequently sonicated for 10 min. Sample lysis in liquid nitrogen combined with sonication was repeated three times. To precipitate proteins, samples were incubated for 1 h at −20 °C, followed by 15 min centrifugation at 13,000 rpm and 4 °C. The resulting supernatant was removed and evaporated to dryness. The dry extracts were then reconstituted in 200 μL CH₃CN/H₂O (1:1, v/v). A portion (10 μL) of these samples was subjected to UPLC-TOF/MS (LCT-Premier XE; Waters, Milford, MA) for metabolomic analysis. The operating conditions for UPLC and TOF/MS, and the procedures for multivariate analysis were selected according to the methods described previously⁵⁵. Chromatographic separation was performed on a BEH column (100 × 2.1 mm, 1.7μm i.d.; Waters Corporation, Milford, MA) using a Waters’ ACQUITY UPLC System (Waters Corporation). The duration of the gradient program of 0.1% aqueous formic acid (solvent A) and acetonitrile (solvent B) was 0–2 min, 0.1% B; −6 min, 0.1–25% B; 6–10 min, 25–80% B; 10–12 min, 80–90% B; 12–21 min, 0–99.9% B; 21–23 min, 99.9% B; 23–24 min, 0.1% B; 24–26 min, 0.1% B. The flow rate was 0.3 ml/min.

For MS analysis, scan range was from m/z 50 to 1000 with a scan time of 0.3 s. Source temperature was set to 120 °C with a cone gas flow of 50 L/h. Desolvation gas flow was set to 600 L/h at a temperature of 350 °C. Capillary voltage was set at 2.4 kV for ESI⁺ mode. Mass spectrometry was operated in W optics mode using dynamic range extension. For accurate mass acquisition, a lock mass of leucine-enkephalin was used via a lock spray interface to ensure accuracy during the MS analysis.

The content of brain endocannabinoids was determined using UPLC-TOF/MS by the methods reported previously with some modifications³². Fifty mg of frozen hippocampal tissue were transferred to a borosilicate glass tube containing 1 mL ice-cold acetonitrile spiked with 10 μL of internal standards mixture (1 pmol anandamide-d₄ and 2 nmol 2-AG-d₈). The hippocampus was homogenized manually with a glass pestle, followed by sonication in an ice-water bath for 30 min. The homogenates were kept at −20 °C overnight to precipitate proteins. Samples were then centrifuged at 3000 rpm for 3 min. The supernatant was removed with a glass pipette to a new tube and the process repeated one additional time. The supernatant was dried under a stream of N₂ gas, resuspended in 300 μL methanol, then evaporated again. The residue obtained was reconstituted with 300 μL methanol/acetonitrile (2:1, v/v), and an aliquot (10 μL) was subjected to UPLC-TOF/MS analysis. The operating conditions for the LC were as follows: column, ACQUITY UPLC^® HSS T3 column (1.8 μm particle size, 2.1 × 100 mm, Waters); column temperature, 40 °C; sample room temperature, 4 °C; The mobile phase was water with 10 mM ammonium acetate and 0.1% formic acid (solvent A), and acetonitrile (solvent B); elution program [% of B in A (min)]: 2% (0–3), 2 to 60% (3–10), 60 to 100% (10–20), 100% (20–22), 100 to 2% (22–22.5) and 2% (22.5–25); and flow rate, 0.3 mL/min. Quantitative analyses were carried out in the selected ion monitoring (SIM) mode. Under these conditions, AEA and 2-AG were monitored in their protonated form (m/z 348.2895 (AEA + H) and 396.3117 (2-AG + NH₄), respectively. As 2-AG undergoes isomerization to 1-AG arising from acyl group migration during sample workup⁵⁶, the peak areas of both isomers were combined for 2-AG quantitation. 2-AG and 1-AG had retention times 15.44 and 15.74 min, respectively.

For the simultaneous quantitation of AEA and 2-AG, a linear regression of the ratio of the peak area counts of AEA and 2-AG to their corresponding internal standards (AEA-d₄ and 2-AG-d₈) versus concentration was used to construct the calibration curves. Calibration curves in clean extraction solvent were used, as the sample matrix always contained 2-AG and AEA. Calibration standard solutions were prepared using stock solutions of AEA-d₄, 2-AG-d₈. To accommodate the two endocannabinoids in the individualized quantitation method, the calibration set consisted of five dilutions, containing AEA-d₄, from 0.01–1 μM (0.01, 0.05, 0.1, 0.5, 1) and containing 2-AG-d₈ from 3.125–50 μM (3.125, 6.25, 12.5, 25, 50). Product ions were monitored in selected ion monitoring (SIM) mode. Injection volumes for samples and standards were 10 μL with needle overfill. Five-point calibration curves were performed in triplicate and 2-AG-d₈ peaks or AEA-d₄ were integrated using Masslynx software version 4.1(Waters Corp., Milford, MA). A linear regression of the ratio of the peak area counts of AEA or 2-AG to their corresponding internal standards (AEA-d₄ and 2-AG-d₈) versus concentration was used to construct the calibration curves. The linear regression correlation coefficients (r²) for the AEA and 2-AG calibration curves were 0.999 or higher for all calibration curves.

The expression of mRNAs was quantified by real-time RT-PCR as previously described. Primer sequences are shown in Supplemental Table 3. Briefly, total RNA was extracted from hippocampal tissue using the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). The obtained RNA (250 ng) was treated with genomic DNA (gDNA) Eraser for removal gDNA contamination, and reverse-transcribed to cDNA using the PrimeScript RT reagent kit (TaKaRa Bio Inc., Shiga, Japan). The cDNA was amplified with Fast SYBR Green Master Mix (Life Technologies, Carlsbad, CA, U.S.A.), using the StepOnePlus Real-time PCR system (LifeTechnologies). PCR conditions were as follows: 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. The relative levels of mRNA were determined using the 2^−ΔCT method. The amount of target mRNA was normalized to β-actin mRNA, and is shown as a ratio to the control).

Total protein was extracted from the remaining hippocampal tissue. The hippocampus was homogenized in four volumes (relative to the tissue weight) of 25 mM Tris-HCl (pH 7.4) containing 1 mM EGTA, 1 mM EDTA, 1 mM sodium fluoride, and a protease inhibitor cocktail (Roche Diagnostics, Tokyo, Japan), and centrifuged at 9,000 × g for 20 min. Protein concentration was measured by the Lowry assay⁵⁷ . Protein samples (10 µg) were separated on 12 or 15% SDS-polyacrylamide gels and transferred to polyvinylidenedifluoride membrane (PVDF, Millipore, MA) in an ATTO electrophoresis system (ATTO, Tokyo, Japan). After blocking for 1 h at room temperature with TBS-T (Tris-buffered saline with 0.1% Tween) containing 5% skimmed milk and 0.3% BSA, the membranes were at 4 °C overnight with primary antibodies against specific proteins. For the chemiluminescent visualization, membranes were incubated with secondary antibody conjugated with HRP at room temperature for 1 h. After washing six times with TBS containing 0.05% Triton X-100, the membrane was immersed in a Clarity Western ECL substrate (Bio-Rad) for 5 min at room temperature. The relative levels of the target proteins were determined using a ChemiDoc™ MP system operated by Image Lab™ software (Bio-Rad) in the auto-exposure mode.

One-way analysis of variance (ANOVA) was used for performing differences among different groups followed by the Student Newman-Keuls test. For all comparisons, p < 0.05 was considered to indicate statistical significance. The statistical analyses were performed using the Prism software (GraphPad Prism Software, San Diego, CA).