Animals

Male CD-1 mice (Charles River, Lyon, France) weighting 25–30 g were used. Mice were housed in cages of four and maintained at a controlled temperature (21±1 °C) and humidity (55±10%). Food and water were available ad libitum. Lighting was maintained at 12 h cycles (on at 8 am and off at 8 pm). All the experiments were performed during the light phase of the dark/light cycle. Animals were habituated to the experimental room and handled for one week before starting the experiments. All animal procedures were conducted in accordance with the standard ethical guidelines (European Communities Directive 2010/63/EU) and approved by the local ethical committee (Comitè Ètic d'Experimentació Animal, CEEA-PRBB). All behavioral analysis was performed blind to the experimental conditions.

Drugs and Treatments

Delta9-THC was purchased from THC-Pharm-GmbH (Frankfurt, Germany); rapamycin and chelerythrine (CHE) were purchased from Calbiochem (San Diego, CA, USA); cremophor-EL, MK801, PEAQX, NPC-15437 and anisomycin were purchased from Sigma–Aldrich (Madrid, Spain); temsirolimus was purchased from LC Laboratories (Woburn, MA); RO 25-6981 was purchased from Tocris Bioscience (Bristol, UK) and rimonabant was kindly provided by Sanofi-Aventis (Sanofi-Aventis Recherche, Montpellier, France).

MK801, NPC-15437, RO 25-6981, and PEAQX were dissolved in 0.9% sodium chloride (saline). THC and rimonabant were diluted in 5% ethanol, 5% cremophor-EL, and 90% saline. CHE was dissolved in dimethyl sulfoxide (DMSO) as a stock, 14.3 mM (equivalent to 50 mg/kg), and diluted in saline to the doses indicated (0.01, 0.1, 1, 3 mg/kg). NPC-15437 was dissolved in saline for intra-hippocampal microinjections and used at 0.3, 1, 2.5, and 5 μg/0.5 μl, bilaterally. Rapamycin was dissolved in DMSO. All drugs were administered intraperitoneally in a volume of 10 ml/kg, except rapamycin (2 ml/kg). Rimonabant (1 mg/kg), temsirolimus (1 mg/kg), anisomycin (18 mg/kg), MK801 (0.1 mg/kg), NPC-15437 dihydrochloride hydrate (1 mg/kg), RO 25-6981 maleate (6 mg/kg), PEAQX tetrasodium hydrate (1.2 mg/kg), and CHE (0.01 mg/kg) were administered 20 min before THC administration. Rapamycin (1 mg/kg) was administered once daily for 5 d, and mice were tested 3 h after the last rapamycin injection.

For the surgery, ketamine hydrochloride (Imalgène; Merial Laboratorios S.A., Barcelona, Spain) and medetomidine hydrochloride (Domtor; Esteve, Barcelona, Spain) were mixed and dissolved in sterile 0.9% physiological saline and administered intraperitoneally in a volume of 10 ml/kg of body weight. Atipamezole hydrochloride (Revertor; Virbac, Barcelona, Spain) and Meloxicam (Metacam; Boehringer Ingelheim, Rhein, Germany) were dissolved in sterile 0.9% physiological saline and administered subcutaneously in an injection volume of 10 ml/kg of body weight. Gentamicine (Genta-Gobens; Laboratorios Normon, S.A., Madrid, Spain) was dissolved in sterile 0.9% physiological saline and administered intraperitoneally in an injection volume of 10 ml/kg of body weight.

Immunoblot Analysis

Brain samples were obtained 30 min or 1 h after THC or vehicle treatment. Hippocampal tissues were dissected from brains, frozen on dry ice and stored at −80 °C until used as previously reported (Puighermanal et al, 2009). The antibodies used for immunoblot were: anti-phospho-Ser PKC (1:2,000), anti-phospho-PKC(pan)(βII Ser660) (1:500) (Cell Signaling, Beverly, MA, USA); anti-phospho-neurogranin (Ser36)/phospho-neuromodulin (Ser41) (1:1,000), (Millipore, Darmstadt, Germany), anti-neurogranin (1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:15,000), PKC alpha (1:500), PKCβI (1:500), PKCβII (1:500), PKCδ (1:500), PKCɛ (1:500), PKCη (1:500) and PKCθ (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The optical densities obtained with the phospho-specific antibody against pNeurogranin were normalized to the detection of total Neurogranin or GAPDH in the same sample and expressed as a percentage of control/vehicle treatment. The optical densities obtained for the rest of antibodies were normalized to the detection of GAPDH.

Object-Recognition Task

Object-recognition memory was assayed in the V-maze paradigm 3 h (short-term memory assay) or 24 h (long-term memory assay) after the training session as described previously (Busquets-Garcia et al, 2011). On day 1, mice were habituated to the empty maze for 9 min. On the second day, mice were introduced in the maze for 9 min, where two identical objects were presented. For the memory test, mice were placed again in the V-maze for a period of 9 min, where one of the familiar objects was replaced by a novel object, and the total time spent exploring each of the two objects (novel and familiar) was recorded. Object exploration was defined as the orientation of the nose towards the object at a distance less than 2 cm. A discrimination index (DI) was calculated as the difference between the time spent exploring either the novel (Tn) or familiar (Tf) object divided by the total time exploring both objects (Tn+Tf) (DI=(Tn−Tf)/(Tn+Tf)). A positive DI reflects memory retention for the familiar object as mice explore the novel object for longer than the familiar object. This DI was different between control groups depending on animal batches or experimental conditions (eg, local vs systemic injections). For this reason, experimental groups to be compared were run in parallel. Drugs were injected immediately after training (rimonabant, temsirolimus, anisomycin, MK801, RO 25-6981, PEAQX, NPC-15437, and CHE) or 20 min after training (THC). Rapamycin was administered during 5 days previous to the task, and the fifth administration was performed immediately after training following the procedure described previously (Puighermanal et al, 2009). Given the variability in the absolute DI values, all experimental groups to be compared were run in parallel in continuous sessions where experimental groups were intermingled.

Surgery

Mice were deeply anaesthetized by intraperitoneal injection of a mixture of ketamine (75 mg/kg) and medetomidine (1 mg/kg). After surgery, anesthesia was reversed by subcutaneous injection of the synthetic α2 adrenergic receptor antagonist, atipamezole (2.5 mg/kg) indicated for the reversal of the sedative and analgesic effects of medetomidine (α2 adrenergic receptor agonist). In addition, mice received an intraperitoneal injection of gentamicine (1 mg/kg) along with subcutaneous administration of the analgesic meloxicam (2 mg/kg). During surgery, mice were placed in a stereotaxic frame (David Kopf, Tujunga, CA) under sterile conditions, and the upper incisor bar was set 1.0 mm below the interaural line so that the skull was horizontal between bregma and lambda. An incision was made over the skull, thus the skull was surgically exposed, the periosteum removed, and burr holes were subsequently drilled with the size of the guide cannula. A bilateral 26-gauge guide cannula (Plastics One, Roanoke, VA) was implanted into the dorsal hippocampus to be used to guide a bilateral injection cannula (33-gauge internal cannula, Plastics One). The bilateral guide cannula was held in place using dental cement (Dentalon plus, Heraeus Kulzer GmbH, Hanau, Germany) and two stainless-steel screws. The placement was set at 1 mm above the target injection site and the guide cannula was sealed with a dummy of stainless-steel wire with 0.5 mm of projection to prevent obstruction. The target injection site coordinates were as follows: anteroposterior, −1.80 mm; mediolateral, ±1.00 mm; dorsoventral, 2.00 mm (Paxinos and Franklin, 2001). Animals were kept on a 37 °C heating pad during the surgery, and until recovery from anesthesia. The behavioral experiments started 4 days after surgery.

Intra-Hippocampal Microinjections

Immediately after training, mice received a bilateral intra-hippocampal injection of 0.50 μl of NPC (0.3, 1, 2.5, or 5 μg) or vehicle at a constant rate of 0.25 μl/min by using a microinfusion pump (Harvard Apparatus, Holliston, MA) during 2 min. The 33-gauge bilateral injection cannula was connected to polyethylene tubing (PE-20, Plastics One) attached to a 10 μl Hamilton microsyringe (Hamilton, Reno, NV). The injection cannula projected 1.00 mm below the ventral tip of the implanted guide cannula. The displacement of an air bubble inside the length of the polyethylene tubing that connected the syringe to the injection needle was used to monitor the microinjections. After infusion, the injection cannula was left in place for an additional period of 2 min to allow the fluid to diffuse and to prevent reflux, then it was slowly withdrawn. In order to minimize surgical interventions, memory assessment was performed in the minimum number of subjects (n=3–6) to reveal a significant effect of pharmacological treatments.

Verification of Cannula Position

After behavioral experiment, mice were killed by cervical dislocation, and the brains were immediately removed, frozen and stored at -80 °C until sectioning. Coronal sections (30 μm) were cut on a cryostat and mounted on slides. The slides were stained with cresyl violet (Supplementary Figure S4A), and the injection sites were verified under a light microscope by an experimenter blind to the treatment. Mice with cannula location outside of the hippocampus were excluded from the results.

Tissue Preparation for Immunofluorescence

Mice were deeply anesthetized by i.p. injection (0.2 ml/10 g of body weight) of a mixture of ketamine (100 mg/kg) and xylazine (20 mg/kg) prior to intracardiac perfusion with 4% PFA in 0.1 M Na 2 HPO 4 / 0.1 M NaH 2 PO 4 buffer (PB), pH 7.5, delivered with a peristaltic pump at 19 ml per min for 3 min. Subsequently, brains were extracted and post-fixed with 4% PFA for 24 h and transferred to PB 0.1 M at 4 °C. Coronal frozen sections (30 μm) of the dorsal hippocampus (coordinates relative to bregma, –1.22 mm to –1.82 mm) were obtained on a vibratome and stored in the freezer with cryoprotectant solution until used.

Immunofluorescence

Free-floating brain slices were rinsed in PB 0.1M. Brain slices were blocked with 3% normal goat serum, 0.3% Triton X-100 in PB (NGS-T-PB) at room temperature for 2 h, and incubated overnight in the same solution with the primary antibody to anti-phospho-PKC(pan)(βII Ser660) (1:500 and 1:300) (Cell Signaling), anti-neurogranin (1:500), anti-PKCθ (1:500) (Santa Cruz Biotechnology), anti-CB1 (1:500) (Frontier Institute Co.Ltd, Japan), anti-neuronal nuclei (NeuN) (1:500) (Millipore) and anti-microtubule-associated protein 2 (MAP2) (1:500) (Millipore) at 4 °C. The next day, after three rinses in PB 0.1 M, sections were incubated at room temperature with the secondary antibody to rabbit or mouse conjugated to Alexa 555 (Cy3), Alexa 488 (Cy2), or Alexa 647 (IR) (1:500, Jackson ImmunoResearch, West Grove, USA) in NGS-T-PB for 2 h. After incubation, brain sections were rinsed and mounted immediately after onto glass slides coated with gelatin. Mowiol was used as mounting medium.

Confocal Imaging

Double immunofluorescence (pPKC (S660)/CB1) and triple immunofluorescence (neurogranin, pPKC (S660) and PKCθ/NeuN/MAP2) confocal images were obtained by using a Leica TCS SP8 confocal microscope adapted to an inverted Leica DMi8 microscope. General hippocampus images were obtained with a 10 × 0.3 NA dry Leica Semi-apochromatic objective and specific CA1 images with a × 63 1.3 NA glycerin immersion Leica Plan Apochromatic CS2. Alexa 488 (Cy2), Alexa 555 (Cy3), and Alexa 647 (IR) were excited with a 488 nm line of argon laser, a 561 nm diode-pumped solid state laser and a 633 nm helium neon laser. An acousto-optical beam splitter was used detecting on windows 501–566 nm (Alexa 488), 566–647 nm (Alexa 555) and 638–783 nm (Alexa 647). All images were 1024 × 1024 pixels, 8 bits.

Statistical Analysis

Comparisons between two groups were performed by Student’s t-test. One-way or two-way analysis of variance (ANOVA) were performed where appropriate for multiple-group comparisons as indicated in the text. Post hoc comparisons were performed by Student–Newman–Keuls test only when significant main effect of ANOVA (one-way ANOVA) or significant interaction between factors (two-way ANOVA) was revealed. All results are expressed as mean±SEM and differences were considered significant at P<0.05. Statistica Software was used for these statistical comparisons.