Animals

All experimental procedures were performed in accordance with the European Directive for the Protection of Vertebrate Animals used for experimental and Other Scientific Purposes (European Union Directive 2010/63/EU) and approved by the Ethic Committee for Animal Welfare of the University of the Basque Country (UPV/EHU) (CEBA 270M/2012, 189/2011). Male CD-1 mice (Charles Rivers, Wilmington, MA, USA) were housed (6–8 animals per cage) under controlled temperature (23 ± 1 °C), on a normal 12 h light/dark cycle, with free access to food and water. Only male mice were used in this study to avoid the effects of the female estrous cycles that can interfere with the pharmacological parameters.

Treatments

THC, rapamycin and vehicle were administered for 30 days starting at postnatal day 21. Drugs were dissolved in ethanol:cremophor:saline (0.9% NaCl) at 1:1:18 ratio. THC (THCPharm. GmbH. Frankfurt, Germany) (10 mg/kg ip) [15] was injected daily. Rapamycin (Shelleck Chemicals LLC, Houston, TX, USA) (5 mg/kg ip) was administered in a 1 day on/1 day off schedule [16], 30 min before THC injection. For prepulse inhibition (PPI) experiments, (±)-DOI (Sigma-Aldrich, St. Louis, MO, USA) (0.5 mg/kg ip) [17] was dissolved in saline and administered immediately before the behavioral assay. Experiments were carried out after a 5-day washout period to avoid the interference of acute effects of THC.

Experimental design

Experimental design of each assay was conducted as follows. A first batch of two groups of animals (vehicle and THC, n = 16 animals per group) was treated with THC or vehicle for 30 days. After a 5-day washout period, a number of animals (n = 8 per group, vehicle and THC) were killed, brains removed and THC concentrations measured. The rest of the animals (n = 8 per group, vehicle and THC) were used to perform basal PPI, 5-HT2AR density, radioligand binding and functional coupling experiments (n = 8 per group, pooled for in vitro assays). Briefly, after the 5-day washout period, PPI experiments were carried out and later, animals were killed, brains removed, and in vitro assays performed.

Another different batch of four groups of animals (vehicle-vehicle, vehicle-THC, rapamycin-vehicle and rapamycin-THC) (n = 8 per group) was stablished to carry out basal PPI, western blot experiments (Akt, p-Akt, rpS6, p-rpS6) and functional coupling experiments (n = 8 per group, pooled for in vitro assays) following these different pharmacological treatments.

Finally, another batch of four groups (vehicle-vehicle, vehicle-THC, rapamycin-vehicle and rapamycin-THC) was used to accomplish PPI experiments after acute DOI in a timely matched manner (n = 8 per group).

Quantification of THC concentration in mouse brain by liquid chromatography–tandem mass spectrometry

Pooled homogenized THC-free mouse brain samples (n = 6–8) were used for the development and validation. Experiments were carried out with a 1290 Infinity II ultrahigh performance liquid chromatography (UHPLC) system coupled to a 6495 iFunnel triple Quad mass spectrometer equipped with a JetStream electrospray ionization source (Agilent Technologies, Santa Clara, CA, USA). A Kinetex EVOC18 100A 3 × 100 mm (2.6 μm) column was selected. Sample analysis in positive ionization mode was performed using as mobile phase water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B) with elution gradient mode. The flow rate was 0.5 ml/min; injection volume was 5 μl, and the column temperature was maintained at 30 °C. The calibration curve was prepared by spiking 20 μl of standard working solution to obtain brain THC final concentrations of 8–2000 ng/g. Quality control samples (QCs) were prepared by spiking brain samples containing 60 and 1000 ng/g as the final THC concentration.

Prepulse inhibition of the startle reflex

Prepulse inhibition (PPI) test was performed in a startle chamber (PanLab, Barcelona, Spain) where a 60-dB background-noise was present for a 10 min acclimatization period, as well as throughout the entire experiment. Each session began with five startle pulse-alone trials to achieve a more stable response of the animals and were not included in the analysis. Mice were then subjected to a pseudo-randomized combination of: ten pulse-alone trials consisted of a white noise burst (120 dB, 40 ms); ten prepulse-alone trials for each prepulse intensity (10 ms at 77, 82, or 87 dB); ten prepulse-pulse trials for each prepulse intensity with an interval of 60 ms between both and ten no-stimulus trials, in which only the background noise was present. The inter-trial intervals were 10, 12, 15, 20, and 25 s. The maximum amplitude of the startle reaction was recorded for every trial, and the average startle for each trial subgroup was used for the analysis. No-stimulus trials and prepulse-alone trials did not exert any startle response and data were not included in the analysis. PPI was calculated as a percentage score: % PPI = 100—[(startle response to prepulse-pulse trial/startle response to pulse-alone trial) × 100]. Inhibition of the PPI response was induced by administration of the 5-HT2AR agonist (±)-DOI (0.5 mg/kg, i.p.) immediately before acclimation period. Each session lasted 30–33 min.

Brain cortex membranes preparation

Animals were killed by cervical dislocation, brains removed, cortex dissected, and samples stored at −80 °C until use. Membrane-enriched fraction (P2) was prepared as described [18].

Radioligand binding

[3H]Ketanserin saturation binding assays were performed as previously reported [19] with minor modifications. [3H]Ketanserin binding (0.05–10 nM; eight concentrations) was used to calculate density (Bmax) and affinity (Kd) of 5-HT2AR. Non-specific binding was determined in the presence of the 5-HT2AR antagonist M100907 (1 μM). Competition curves of [3H]ketanserin binding (2 nM) with increasing concentrations of the agonist (±)-DOI (10−12–10−3 M) were also performed in order to delineate both the G-protein coupled and uncoupled 5-HT2AR conformations. Briefly, after incubation (60 min, 37 °C), free radioligand was discarded by rapid filtration under vacuum (1450 FilterMate Harvester, Perkin Elmer, Waltham, MA, USA). Filters were then rinsed, dried and bagged in Sample Bag with BetaPlate Scint scintillation cocktail. Radioactivity was detected by liquid scintillation spectrometry using a MicroBeta TriLux counter (Perkin Elmer, Waltham, MA, USA).

Antibody-capture [35S]GTPγS scintillation proximity assay (SPA)

Specific activation of different subtypes of Gα-proteins by (±)-DOI (10 µM) was determined using a previously described protocol [18]. The concentration of (±)-DOI (10 µM) was chosen for being the one which induces stimulation values around the Emax for any Gα subunit subtype under our experimental conditions [19]. [35S]GTPγS binding was performed in buffer containing 0.4 nM [35S]GTPγS, 15 μg of protein/well and different GDP concentrations depending on the Gα-subtype tested. Specific antibody for each Gα-subunit and polyvinyltoluene SPA beads coated with protein-A were added and incubated (3 h, room temperature). Plates were centrifuged and bound radioactivity detected on a MicroBeta TriLux scintillation counter. Non-specific binding was defined as the remaining [35S]GTPγS binding in the presence of 100 μM unlabeled GTPγS. The antagonist ketanserin (10 μM) was used to confirm the 5-HT2AR involvement in the observed stimulations (data not shown).

Western blot

Western blot experiments were performed with total homogenate fractions (S1) from mouse brain cortex tissue. Samples (30 μg) were heated (95 °C), loaded onto polyacrylamide gel (12%) and submitted to SDS-PAGE. Nitrocellulose membranes were blocked (5% non-fat dry-milk or/and 0.5% BSA) in TBS buffer followed by overnight incubation with primary antibodies (4 °C). Antibodies against 5-HT2AR, Akt, phospho-Akt(Ser473), rpS6, phospho-rpS6(Ser235/236) and β-actin were used. Incubation with fluorescent anti-IgG secondary antibodies was performed at room temperature (1 h). Immunoreactivity was quantified using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) (further details in Supplementary Table 1).

Data analysis and statistical procedures

Data were analyzed with GraphPad Prism™ 5.01, and InVivoStat software. Before the statistical analyses, the data were inspected for outliers (critical value, Z > 1.96) using Grubb’s test (GraphPad Software, www.graphpad.com/quickcalcs/grubbs1.cfm). In PPI experiments, data from animals (between 0 and 3 animals per group) showing an outlier value for a particular dB or for the startle amplitude to the pulse were discarded for further analysis. Two animals were also discarded for not showing any response to the startle stimulus. Two-group comparisons were made by unpaired Student’s t-test. Multiple groups’ comparisons were studied by one-way, two-way or three-way analysis of variance (ANOVA), followed by Bonferroni’s or Benjamini-Hochberg’s post hoc analyses. Statistical significance was set at p < 0.05. Displacement curves were analyzed using a nonlinear fit, and the selection between models was made by the extrasum-of-squares (F-test). Following the nonlinear curve fitting, Ki values for (±)-DOI were calculated from the corresponding IC50 values. Differences in the binding profiles were assessed by unpaired Student’s t-test of normalized (log) parameters. Specific binding values obtained from SPA assays were transformed to percentage of basal binding (binding values observed without agonist drug) obtained for each Gα-protein and analyzed by one-sample or two-sample Student’s t-test. The immunodensitometric values of the different target proteins were normalized to the intra-assay values obtained with anti-β-actin antibody and expressed as mean ± SEM of the percentages over an inter-assay normalization sample included in every experiment. Each sample was analyzed at least in two independent experiments.