The protocols for animal care and use were approved by the Ethics and Research Committee at the Hospital Universitario Regional de Málaga and Universidad de Málaga. All experimental animal procedures were carried out in strict accordance with the European Communities directive 86/609/ECC (24 November 1986) and Spanish legislation (BOE 252/34367-91, 2005) regulating animal research. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Male Wistar rats (approximately 250 g, 10–12 weeks old; Charles Rivers, Barcelona, Spain) were housed individually in cages maintained in standard conditions (Servicio de Estabulario, Facultad de Medicina, Universidad de Málaga) at 20 ± 2°C room temperature, 40 ± 5% relative humidity and a 12-h light/dark cycle with dawn/dusk effect. Water and standard rodent chow (Prolab RMH 2500, 2.9 kcal/g) were available ad libitum, unless otherwise indicated for specific experimental procedures.

The animals were daily weighed, handled for 10 min and habituated to injection procedures (holding and pseudo-injection) during 1 week before the experimentation in order to minimize stress effects. To further habituate the animals to feeding procedures, 84 h before testing with drug, the animals were food deprived for 24 h with ad libitum access to water. Then, the rats were maintained under free-feeding period for 48 h. After this time, the animals were definitively food-deprived for 12 h (with free access to water) before the beginning of the food exposure and treatment. Finally, a can with a measured amount of food (usually 50–60 g per day) and a bottle containing 250 ml of fresh water were placed in the cage at time 0. Food pellets and rats were periodically weighed.

Fatty acid amide hydrolase (FAAH) inhibitor URB597 (cyclohexyl carbamic acid 3′-carbamoyl-biphenyl-3-yl ester; Cayman Chemical, cat. no. 10046, Ann Arbor, MI, USA) was dissolved in a vehicle containing 33% (v/v) DMSO in sterile 0.9% NaCl solution, just before each experiment. The vehicle or URB597 were intraperitoneally (i.p.) injected in a final volume of 1 ml/kg body weight. The optimal dose at which acute treatment would be more effective was selected for the repeated treatment experiment (see Supplementary Materials and Figure S1). Thus, URB597 was acutely or repeatedly administrated at a dose of 0.3 mg/kg body weight for 5 consecutive days at intervals of 24 h (08:00 a.m.). During the repeated administration of URB597, the cumulative food intake and body weight gain were daily measured for the 5 days of treatment. In a first batch, the animals were sacrificed 2 h after the last injection of vehicle or URB597. Finally, we generated three experimental groups (n = 16/group): (1) Vehicle administration for 5 days (vehicle group); (2) One URB597 administration (0.3 mg/kg/day) and repeated vehicle administration for the remaining 4 days (acute URB597 group); (3) Repeated URB597 administration (0.3 mg/kg/day) for 5 days (repeated URB597 group) (Figure S2). In a second batch, the animals were sacrificed 3 weeks after the last treatment day. We generated two experimental groups (n = 8/group): (1) Vehicle administration for 5 days (vehicle group); (2) Repeated URB597 administration (0.3 mg/kg/day) for 5 days (repeated URB597 group) (Figure S2).

5′;-bromo-2′-deoxyuridine (BrdU, cat. no. B5002, Sigma, St. Louis, MO, USA) was dissolved at 15 mg/mL in sterile 0.9% NaCl solution, and i.p. administrated at a dose of 50 mg/kg body weight twice per day at intervals of 10 h (08:00, 18:00 h), for 4 consecutive days (Cifuentes et al., 2011).

Previous to sacrifice, all animals were anaesthetized (sodium pentobarbital, 50 mg/kg body weight, i.p.) 2 h or 3 weeks after the last dose of treatment in a room separate from the other experimental animals. Most blood samples (n = 12/group) were briefly collected into tubes containing EDTA-2Na (1 mg/ml blood) and centrifuged (1600 g for 10 min, 4°C). Plasma samples were then frozen and stored at −80°C for biochemical, hormonal and liquid chromatography–mass spectrometry analyses. Half of the first batch of animals (n = 8/group) were sacrificed by decapitation and their brains were collected, quick frozen and stored at −80°C. These brains were then prepared on dry ice to obtain sections of 1 mm thick by using razor blades and a rat brain slicer matrix. The hippocampus, hypothalamus and striatum were precisely removed from −2.16 to −4.20 and from 2.28 to −0.24 Bregma levels (Paxinos and Watson, 2007) with fine surgical instruments. Brain samples were weighed and stored at −80°C until they were used for RT-qPCR and liquid chromatography–mass spectrometry analyses. Another half of the first batch of animals and the second batch of animales (n = 8/group) were transcardially perfused with 4% formaldehyde in 0.1 M phosphate buffer (PB) and the brains were dissected out and kept in the same fixative solution overnight at 4°C. The brains were then cut into 30-μm-thick coronal sections and were divided in eight parallel series by using a vibratome (Leica VT1000S). Sections were stored at 4°C in PB with 0.002% (w/v) sodium azide until they were used for immunostaining.

The brain sections were divided in eight parallel series. Each parallel series consisted of one coronal section for each 240 μm. Free-floating coronal sections from −2.16 to −4.20 Bregma levels (hippocampus and hypothalamus), from Bregma 2.28 to -0.24 Bregma levels (striatum) and from 7.60 to 6.60 Begma levels (olfactory bulbs) of each parallel series were selected for each immunohistochemistry. Sections were incubated overnight in the following diluted primary antibody at 4°C: rabbit anti-FAAH (1:200, Cayman, cat no. 101600; Gulyas et al., 2004), rabbit anti-CB1 (1:200, Frontier Institute, cat. no. CB1-Rb-Af380-1; Uchigashima et al., 2007), rabbit anti-phospho-histone H3 (Ser10) (2 μg/ml, Upstate, cat. no. 06-570), mouse anti-BrdU (1:2000, Hybridoma Bank, Iowa City, IA, USA; ref. G3G4), rabbit anti-cleaved caspase-3 (1:500, Cell Signaling; cat. no. 9661), mouse anti-glial fibrillaric acidic protein (GFAP) (1:500, Sigma, cat. no. G3893) and rabbit anti-Iba-1 (1:1000, Wako, cat. no. 019-19741) (Cifuentes et al., 2011; Rivera et al., 2011, 2013). The following day the sections were incubated in the respective secondary antibody for 90 min: biotinylated goat anti-mouse IgG (1:500, Sigma; cat. no. B7264) or biotinylated donkey anti-rabbit IgG (1:500, Amersham, Little Chalfont, England; cat. no. RPN 1004). The sections were then incubated in ExtrAvidin peroxidase (Sigma, St. Louis, MO) diluted 1:2000 in darkness at room temperature for 1 h. Finally, immunolabeling was revealed with 0.05% diaminobenzidine (DAB; Sigma), 0.05% nickel ammonium sulfate and 0.03% H₂O₂ in PBS. Brain sections from the second batch of animals were only used to check cell survival by BrdU immunohistochemistry.

The following antibodies were used: rat anti-BrdU (1:2000; Accurate Chemical and Scientific, Westbury, NY, USA, cat. no. OBT0030G), mouse anti-NeuN (1:500; Millipore, cat. no. MAB377), mouse anti-β3 tubulin (1:5000; Promega, Madison, WI, USA) and mouse anti-GFAP (1:500; Sigma, cat. no. G3893). Sections were incubated overnight at 4°C with a cocktail of the primary antibodies. The rat anti-BrdU was detected with the donkey anti-rat IgG (H+L) labeled with Alexa Fluor® 488 (1:1000; Molecular Probes, Invitrogen, Paisley, UK, cat. no. A21208). The antibodies NeuN, β3-tubulin and GFAP were detected with the donkey anti-mouse IgG (H+L) labeled with Alexa Fluor® 594 (1:1000; Molecular Probes, cat. no. A21203). The sections labeled by double immunofluorescence (BrdU+NeuN, BrdU+β3-tubulin or BrdU+GFAP) were visualized with a confocal laser (spectral) scanning microscope (Leica TCS NT; Leica Microsystems) equipped with a 561 nm DPM laser (argon 30%) and a 40× objective (HCX PL APO CS 40.0x1.25 OIL UV). The emission filter settings were 500–550 nm for PMT2 (green) and 610–700 nm for PMT3 (red). Depending on the level of zoom used in each image, the XY voxel size was from 100 to 77 nm, approximately. Settings of light and brightness/contrast were adjusted by using the Leica LAS AF Lite imaging software.

Phospho-H3, BrdU, GFAP, Iba-1 and cleaved caspase 3-immunoreactive (-ir) nuclei and cells that came into focus were manually counted from Bregma −2.16 to −4.20 mm at hippocampal and hypothalamic levels, from Bregma 2.28 to −0.24 mm at striatal levels and from Begma 7.60 to 6.60 mm at olfactory bulb levels (Paxinos and Watson, 2007). Representative counting frames were evaluated using a standard optical microscope with the 40× objective (Nikon Instruments Europe B.V., Amstelveen, The Netherlands) coupled to the NIS-Elements Imaging Software 3.00 (Nikon). Focusing in the hippocampus, phospho-H3 and BrdU-ir nucleus counts were performed in the subgranular zone (SGZ) of the dentate gyrus (DG), while survived BrdU-ir nucleus counts and GFAP, Iba-1 and cleaved caspase 3-ir cell counts were carried out in the whole hippocampus (DG, CA3 and CA1 areas). Focusing in the hypothalamus, counting was performed in the paraventricular (PVH), ventromedial (VMH) and arcuate (ARC) nuclei and median eminence. Focusing in the striatum, phospho-H3 and BrdU-ir nucleus counts were performed in the subventricular zone (SVZ) of the lateral ventricles, while survived BrdU-ir nucleus counts and GFAP, Iba-1 and cleaved caspase 3-ir cell counts were carried out in the whole striatum. Survived BrdU-ir nucleus counts were also performed in the olfactory bulbs. Immunostained cells located in the uppermost side that came into focus while moving down through the thickness of the section were counted. Overall, quantification was expressed as the average number of cells per area (mm²) for each experimental group.

Densitometric analysis of the GFAP, FAAH, and CB1 immunoreactivity was evaluated in the three hippocampal areas (dentate gyrus, CA3 and CA1) and hypothalamus from Bregma −2.16 to −4.20 mm, and in the striatum from Bregma 2.28 to −0.24 mm (Paxinos and Watson, 2007). Digital high-resolution microphotographs of representative areas were taken with a 10× objective under the same conditions of light and brightness/contrast with an Olympus BX41 microscope equipped with an Olympus DP70 digital camera. Quantification was determined using the analysis software ImageJ 1.38X (NIH, USA). Focusing in CA1 and CA3 areas, we considered the following layers: stratum oriens (SO), stratum pyramidale (SP), stratum radiatum (SR), stratum lucidum (SL) and stratum lacunosum-moleculare (SL-M). For dentate gyrus, we considered the molecular layer (ml), the granular cell layer (gcl) and the polymorphic cell layer (pcl). Focusing in the hypothalamus, densitometry were performed in the paraventricular (PVH), ventromedial (VMH) and arcuate (ARC) nuclei and median eminence.

We performed real-time PCR (TaqMan, Applied Biosystem, Carlbad, CA, USA) as described previously (Crespillo et al., 2011; Decara et al., 2012) using specific sets of primer probes (Faah: Rn00577086_m1, Amplicon length: 63; Cnr1: Rn02758689_s1, Amplicon length: 92; Actb: Rn00667869_m1, Amplicon length: 91; Life Technologies). Briefly, brain regions were homogenized on ice and RNA was extracted following Trizol® method according to the manufacture's instruction (Gibco BRL Life Technologies, Baltimore, MD, USA). RNA samples were isolated with RNAeasy minelute cleanup-kit including digestion with DNase I column (Qiagen, Hilden, Germany). After reverse transcript reaction from 1 μg of mRNA, quantitative real-time reverse transcription polymerase chain reaction (qPCR) was performed in a CFX96TM Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and the FAM dye label format for the TaqMan® Gene Expression Assays (Applied Biosystem). Melting curve analysis was performed to ensure that only a single product was amplified. After analyzing several control genes, values obtained from the brain samples were normalized in relation to β-actin (Actb) levels.

The determination of AEA, PEA, OEA, and 2-arachidonoyl glycerol (2-AG) was done as previously described (Pastor et al., 2014). Briefly, aliquots of 175 μL of plasma were spiked with AEA-d4, PEA-d4, OEA-d4, and 2-AG-d5 (Cayman Chemical, Ann Harbor, MI), diluted up to 1 mL with 0.1M ammonium acetate pH 4.0 (Merck, Darmstadt, Germany), extracted with tert-butyl-methyl-ether (Merck) and analyzed by LC/MS-MS on a triple quadrupole mass spectrometer (Agilent 6410, Wilmington, DE) that operated with an electrospray ionization source (ESI) on the positive mode. Chromatographic separation was done with a Waters C18-CSH column (3.1 × 100 mm × 1.8 μm particle size), detection was done by selected reaction monitoring (SRM) and quantification was done by isotope dilution. 2-MGs were reported as the sum of isomer 1 and isomer 2 due to the instability of isomer 2 to isomerization.

Glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, creatinine, urea, uric acid and the hepatic enzymes aspartate transaminase (AST) and alanine transaminase (ALT) were analyzed using commercial kits according to the manufacturer's instructions in a Hitachi 737 Automatic Analyzer (Hitachi Ltd., Tokyo, Japan). Plasma levels of IGF-1, insulin, leptin and adiponectin were determined with three different enzyme-linked immunosorbent assay (ELISA) kits from BioVendor (Modrice, Czech Republic), Mercodia AB (Uppsala, Sweden) and B-Bridge International, Inc. (Mountain View, CA, USA), respectively. To perform the ELISA protocols in rat samples, we used 50, 25, 100, 100 μl of plasma to determine the concentrations of IGF-1, insulin, leptin, adiponectin, respectively. In all cases, a calibration curve and internal controls were included in each assay.

Statistical analysis of the results was performed using the computer program GraphPad Prism version 5.04 (GraphPad Software Inc., San Diego, CA, USA). Data were represented as the mean ± s.e.m. for at least six determinations per experimental group. Kolmogorov-Smirnov normality tests indicated that all data followed a Gaussian distribution (P > 0.1), so we selected a parametric statistical test. Statistical analysis was performed using one or Two-Way ANOVA and Bonferroni's test or Student's t-test when appropriate. P < 0.05 was considered to be significant.

We analyzed the effect of URB597 on food intake and body weight gain for 5 days (Figure 1). After Two-Way ANOVA analysis, a treatment effect on body weight gain was observed [F_{(2, 84)} = 5.89, P = 0.004]. Moreover, the effect of the repeated treatment on body weight gain showed a higher significance [F_{(1, 56)} = 10.7, P = 0.001] than that of the acute treatment [F_{(1, 56)} = 5.82, P = 0.019]. As a consequence, repeated URB597-treated rats showed a decrease in their body weight gain up to 48 and 72 h after the beginning of the repeated treatment when they were compared with vehicle-treated rats (^*P < 0.05) (Figure 1B). Moreover, a transitory decrease in the body weight gain was also observed in the second day after acute treatment of URB597.

To better understand the metabolic context associated to URB597 treatment, we evaluated the biochemical and hormonal profile in the plasma of these rats (Table 1). Biochemical parameters included basal glucose, triglycerides, total cholesterol, high-density lipoprotein (HDL)-cholesterol, creatinine, urea, uric acid and aspartate transaminase (AST)/alanine transaminase (ALT) ratio. Metabolic hormones included insulin, leptin, adiponectin and IGF-1. The plasma of rats acutely and repeatedly treated with URB597 showed lower levels of basal glucose [F_{(2, 15)} = 5.95, ^*P < 0.05], triglycerides [F_{(2, 15)} = 6.69, ^*P < 0.05], total cholesterol [F_{(2, 15)} = 6.29, ^*P < 0.05], HDL-cholesterol [F_{(2, 15)} = 5.21, ^**P < 0.01 and ^*P < 0.05, respectively] and urea [F_{(2, 15)} = 5.22, ^*P < 0.05] (Table 1). The AST/ALT ratio, used as diagnostic marker for liver damage, was lower in the plasma of repeated URB597-treated rats than that of vehicle or acute URB597-treated rats [F_{(2, 15)} = 7.13, ^**/##P < 0.01]. URB597 didn't induce any change in the plasma levels of uric acid. Regarding insulin, we observed a decrease in the plasma levels of the rats repeatedly treated with URB597 [F_{(2, 15)} = 5.03, ^**P < 0.01]. The decrease of both basal glucose and insulin levels in plasma resulted in reduced values of the Homeostatic Model Assessment (HOMA)-IR [F_{(2, 15)} = 8.44, ^**P < 0.01], defined as an index for insulin resistance, as well as increased values of HOMA-β [F_{(2, 15)} = 7.74, ^**P < 0.01], defined as an index for beta-cell function, in the rats acutely and repeatedly treated with URB597 (Table 1). In addition, leptin levels were significantly reduced only in the plasma of the repeated URB597-treated rats [F_{(2, 15)} = 25.22, ^***P < 0.001]. No effects were detected in the plasma levels of adiponectin and IGF-1 after URB597 treatment (Table 1).

To investigate the impact of URB597 (0.3 mg/kg) on cell proliferation in relevant neurogenic zones of the adult brain, we evaluated the number of newborn cells in the SGZ, hypothalamus and SVZ by the analysis of the mitosis-related protein phospho-histone H3 (Figure 2) and after the administration of BrdU (50 mg/kg) for 4 days (Figure 3). The number of phospho-H3 and BrdU-ir cells was differentially detected depending on the dose and the neurogenic zone analyzed. The number of phospho-H3-ir cells was lower in the SGZ and hypothalamus after repeated URB597 administration [F_{(2, 19)} > 7.13, ^*P < 0.05] (Figures 2A,B,D–I), but higher in the SVZ after acute URB597 administration [F_{(2, 21)} = 4.24, ^*P < 0.05] (Figures 2C,J–L) compared with the vehicle group. Equally, the number of BrdU-ir cells was lower in the SGZ after repeated URB597 [F_{(2, 15)} = 5.1, ^*P < 0.05] (Figures 3A,D–F) and higher in the SVZ after acute URB597 [F_{(2, 15)} = 4.43, ^*P < 0.05] (Figures 3C,J–L). Contrary to phospho-H3, a significant increase in the number of BrdU-ir cells was detected in the hypothalami of acute URB597-treated rats [F_{(2, 15)} = 15.21, ^***P < 0.001] (Figures 3B,G–I). Moreover, no change in the number of BrdU-ir cells was observed in the hypothalami of repeated URB597-treated rats compared with the vehicle group, but it was significantly reduced compared with the acute URB597-treated rats [F_(2,15) = 15.21, ^##P < 0.01].

To determine the putative neuronal linage of these BrdU-ir cells, we also evaluated the presence of their co-expression with the neuron-specific β3-tubulin. As is shown in the representative images of Figures 3M–V, we can observe some co-localization of BrdU and neuron-specific β3-tubulin in a number of cells in the SGZ (Figures 3M–O), hypothalamus (Figures 3P–R) and SVZ (Figures 3S–V) of the three experimental groups. These data indicate a neuronal linage of at least part of the newborn cells after 5 days of BrdU treatment.

To investigate the impact of URB597 on cell survival, we evaluated the number of BrdU-ir cells in the hippocampus, hypothalamus, striatum and olfactory bulbs of rats sacrificed 3 weeks after administering BrdU (Figure 4). The hippocampus, striatum and olfactory bulbs of rats repeatedly treated with URB597 did not show any difference in the number of BrdU-ir cells compared with those of vehicle-treated rats (Figures 4A,C,D). However, we observed a significant decrease in the number of BrdU-ir cells specifically in the hypothalami of URB597-treated rats [F_{(1, 6)} = 11.43, ^**P < 0.01] (Figure 4B).

To determine whether these BrdU-ir cells are neurons, we analyzed their co-expression with the biomarker for mature neurons NeuN (Figures 4M–T). We observed co-localization of BrdU and NeuN in a number of cells in the hippocampus (mainly in the granular cell layer) (Figures 4M,Q), striatum (Figures 4O,S) and olfactory bulb (mainly in the internal plexiform and granule cell layers) (Figures 4P,T) of the two experimental groups. In contrast, we could not detect BrdU/NeuN⁺ cells in the hypothalamic areas analyzed (Figures 4N,R).

To investigate the impact of URB597 on astroglia, we evaluated the intensity of GFAP immunoreactivity and the number of astrocytes that express GFAP in the hippocampus, hypothalamus and striatum (Figure 5). The hippocampi and hypothalami of rats acutely or repeatedly treated with URB597 showed an overall decrease in the number of GFAP-ir cells compared with those of vehicle-treated rats [Hippocampus, one-dose: F_(2,21) = 12.89, ^**P < 0.01, 5-doses: ^***P < 0.001; Hypothalamus, one-dose: F_{(2, 21)} = 9.17, ^**P < 0.01, 5-doses: ^*P < 0.05] (Figures 5A,B). Regarding the hippocampus, the reduction in the number of cells expressing GFAP was significant in the dentate gyrus (^***P < 0.001), CA3 (^**P < 0.01) and CA1 (^*P < 0.05) fields. This decrease in the number of GFAP-ir cells agrees with a lower immunoreactivity of GFAP in both brain regions (Hippocampus, acute and repeated URB597: ^**P < 0.01; Hypothalamus, repeated URB597: ^**P < 0.01) (Figures 5D,E). It should be noted that the decrease of GFAP immunoreactivity in the hippocampus and hypothalamus after URB597 treatment can be also attributed as a consequence of a reduction in the density of branched processes that characterize the labeled astrocytes (Figures 5G–L, insets). This effect contrasted with the increase in the number of GFAP-ir cells and GFAP immunoreactivity observed in the striatum of acute URB597-treated rats [F_{(2, 18)} = 5.11,^*P < 0.05] (Figures 5C,F,M–O). However, when the treatment of URB597 was prolonged for 5 days, we could not observe any change in the number of GFAP-ir cells or GFAP immunoreactivity in the striatum.

Focusing on the SGZ of the dentate gyrus, we observed the presence of some BrdU-ir cells that expressed GFAP (Figures 5P–R) in the three experimental groups. These data can suggest a glial linage of part of the newborn cells in the SGZ after 5 days of BrdU treatment. We could not find co-localization of BrdU and GFAP in cells of the hypothalamus and striatum.

To investigate the impact of URB597 on microglia, we evaluated the number of microglial cells that express Iba1 in the hippocampus, hypothalamus and striatum (Figure 6). The hippocampus of rats acutely and repeatedly treated with URB597 showed an overall decrease in the number of cells expressing Iba1 compared with that of vehicle-treated rats [F_{(2, 17)} = 7.12, ^*P < 0.05] (Figures 6A,D–F). Iba1-ir cells in the hippocampus of repeated URB597-treated rats also showed an apparent reduction in the density of branched processes that characterize the microglial cells (Figures 6D–F, insets). On the contrary, no effect was observed in the hypothalamus and striatum of the acutely and repeatedly treated rats (Figures 6B,C,G–L).

Apoptotic cells, as determined by cleaved caspase-3, were assessed in the hippocampus, hypothalamus and striatum of rats acutely and repeatedly treated with URB597 (Figure 7). Differences in the number of cleaved caspase-3-ir cells were detected depending on the dose and brais zone analyzed. The hippocampus of rats treated with repeated URB597 showed an overall decrease in the number of cleaved caspase-3-ir cells compared with that of vehicle-treated rats [F_{(2, 19)} = 21.31, ^***P < 0.001] (Figures 7A,D–F). The decrease in the number of apoptotic cells was significant in the dentate gyrus, CA3 (both at ^**P < 0.01) and CA1 (^*P < 0.05), being especially relevant in those rats acutely treated with URB597 (DG: ^**P < 0.01; CA3: ^*P < 0.05). No changes in the number of cells expressing cleaved caspase-3 were observed in the hypothalamus (Figures 7B,G–I). Opposed effects were observed in the striatum depending on the dose of URB597 used (Figures 7C,J–L). Thus, as was described above for BrdU and GFAP, we observed an increase in the number of cleaved caspase-3-ir cells in the striatum of acute URB597-treated rats compared with vehicle-treated rats [F_{(2, 20)} = 12.17, ^*P < 0.05]. On the contrary, the striatum of the rats repeatedly treated with URB597 showed a decrease in the number of cells expressing cleaved caspase-3 compared with that of the rats treated with vehicle (^*P < 0.05) or after 5 days of the acute URB597 (^###P < 0.001) (Figures 7C,J–L).

To determine the endocannabinoid tone after the URB597 treatment, we analyzed the levels of AEA, PEA, OEA, and 2-AG in the plasma (Figure 8). As we expected, increased levels of AEA, PEA, and OEA were exclusively observed in the plasma of repeated URB597-treated rats compared with vehicle [F_{(2, 33)} > 17.38, ^***P < 0.001] or acute URB597 (^###P < 0.001)-treated rats (Figure 8). Neither of the two administrations used for URB597 modified the plasma levels of 2-AG.

To assess whether the changes observed in the endocannabinoid tone are in concordance with the expression of pertinent components of the endocannabinoid system, we evaluated the gene and protein (immunohistochemical) expression of the NAEs-hydrolyzing enzyme FAAH and the CB1 cannabinoid receptor in the hippocampus, hypothalamus and striatum of rats after URB597 treatment (Figure 9). Rats repeatedly treated with URB597 showed decreases in both gene and immunohistochemical expressions of FAAH and CB1 in the hippocampus (Figures 9A,D,G–I,P–R), FAAH in the hypothalamus (Figures 9B,E,J–L) and CB1 in the striatum (Figures 9C,F,V–X). In addition, we observed additional changes in gene expression, which is not confirmed by the correspondent immunohistochemical analysis, such as increased levels of Faah mRNA in the striatum of acute and repeated URB597-treated rats as well as the decrease in the hypothalamic levels of Faah mRNA and the striatal levels of Cnr1 mRNA in acute URB597-treated rats (Figures 9B,C).

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.