Animal experiments accomplished the criteria of the European Communities Council Directive (86/609/EEC) and the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice (4–6 weeks old male, C57B1/6 strain) were housed, grouped and acclimatized to laboratory conditions (12 hr light/dark cycles) 1 week before the experiment and had ad libitum food and water access.
Slice preparation and electrophysiology
Whole cell patch-clamp and extracellular field recordings were made from visualized pyramidal cells in coronal slices of mouse prefrontal cortex. In brief, mouse were anesthetized with isoflurane and decapitated. The brain was sliced (300 µm) in the coronal plane using a vibratome (Integraslice, Campden Instruments, Loughborough, UK) and maintained in physiological saline at 4°C. Immediately after cutting, slices containing the PrPFC were stored for 30 min at 32–35°C in artificial cerebrospinal fluid (ACSF) that contained (in mM): 126 NaCl, 2.5 KCl, 2.4 MgCl2, 1.2 CaCl2, 18 NaHCO3, 1.2 NaH2PO4, and 11 Glucose, and was equilibrated with 95% O2/5% CO2. Slices were stored at room temperature until time of recording. For recording, slices were placed in the recording chamber and superfused (2 ml/min) with ACSF. All experiments were done at 32–35°C. The superfusion medium contained picrotoxin (100 µM) to block GABAA receptors. All drugs were added at the final concentration to the superfusion medium.
To evoke synaptic currents, stimuli (100–150 µsec duration) were delivered at 0.1 Hz through a glass electrode filled with ACSF and placed either in layer 2/3 or in layer 5/6 as previously described 
. No difference was observed between the two sites of stimulation and thus data were pooled together. Both the EPSC area and amplitude were measured (graphs depict amplitudes).
For extracellular field experiments, the recording pipette was filled with ACSF. Both the field excitatory postsynaptic potential (fEPSP) area and amplitude were measured (graphs depict area). The glutamatergic nature of the extracellular fEPSP was confirmed at the end of the experiments through the application of the non-NMDA ionotropic glutamate receptor antagonist DNQX (20 µM), which completely blocked the synaptic N2 component without altering the non-synaptic N1 component (not shown).
For whole cell patch-clamp experiments, layer 5/6 pyramidal neurons were visualized using an upright microscope with infrared illumination. Recordings were made with electrodes containing the following (mM): Cesium Methane-Sulfonate (CH3O3SCs) or K+Gluconate 128, NaCl 20, MgCl21, EGTA 1, CaCl2 0.3, Na2+-ATP 2, Na+-GTP 0.3, Glucose 10 buffered with Hepes 10, pH 7.3, osmolarity 290 mOsm. Electrode resistance was 4–6 MOhms. For the BAPTA experiments, 20 mM BAPTA was added to the intracellular medium.
A −2 mV hyperpolarizing pulse was applied before each evoked excitatory post-synaptic current (EPSC) in order to evaluate the access resistance, and those experiments in which this parameter changed >20% were rejected. Access resistance compensation was not used and acceptable access resistance was <25 MOhms. The potential reference of the amplifier was adjusted to zero prior to breaking into the cell. An Axopatch-1D (Molecular Device, Sunnyvale, USA) was used to record the data, which were filtered at 1–2 kHz, digitized at 5 kHz on a DigiData 1200 interface (Molecular Device, Sunnyvale, USA) and collected on a PC using Clampex 9.2 and analyzed using Clampfit 9.2 (Molecular Device, Sunnyvale, USA).
Spontaneous miniature excitatory postsynaptic currents (sEPSCs) were recorded in the whole cell voltage-clamp configuration using Axoscope 9.2 (Molecular Device, Sunnyvale, USA). sEPSC amplitude and inter-interval time were detected and analyzed using Clampfit 9.2 (Molecular Device, Sunnyvale, USA). For this analysis, a template of sEPSCs generated from averaging several typical synaptic events was slid along the data trace one point at a time. At each position, this template is optimally scaled and offset to fit the data and a detection criterion is calculated. The detection criterion is the template-scaling factor divided by the goodness-of-fit at each position. An event is detected when this criterion exceeds a threshold and reaches a sharp maximum.
The coefficient of variation (CV) was calculated for as standard deviation / mean amplitude of individual evoked EPSCs and expressed as 1/CV2.
Data analysis and materials
All values are given as mean±S.E.M. For field recording/patch-clamp experiments, n corresponds to the number of individual cells/slices analyzed, with at least 4 animals included in each condition. Statistical significance between groups was tested using the Mann-Whitney U-test. Kolmogorov-Smirnov test was used for the statistical comparison of the cumulative distributions. All statistical tests were performed with Kyplotβ13 (Koichi Yoshioka) using a critical probability of p<0.05.
The fitting of concentration response curves were calculated according to y
)n}+ymin (where ymax
response in the absence of agonist, ymin
response remaining in presence of maximal agonist concentration, x
concentration of agonist producing 50% of the maximal response and n
slope) with Kaleidagraph 3.5 software (Synergy Software, Reading, PA, USA).
U73122, picrotoxin, CP55,940, THL and BAPTA from SIGMA (St. Quentin Fallavier, France) ; AM-251, DNQX, AM404, MPEP, and 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY341495) from Tocris (Bristol, UK). URB 597 and URB 754 were from Cayman (SPI-BIO, Montigny Le Bretonneux, France). SR141716A was a generous gift from Sanofi-Aventis Recherche (Montpellier, France). Other chemicals were from the highest commercial grade available.
4, 4–6 weeks old male, C57B1/6 strain) were deeply anesthetized with chloral hydrate (400 mg/kg body weight) and were transcardially perfused at room temperature (20–25°C) with PBS, pH 7.4, for 20 sec, followed by 500 ml of 4% formaldehyde (freshly depolymerized from paraformaldehyde) in 0.1 M phosphate buffer (PB), pH 7.4, for 10–15 min. Then, brains were removed from the skull and postfixed in 4% formaldehyde for up to 1 hr at room temperature. 50 µm-thick coronal sections cut from prPFC in a vibratome, were washed and blocked in 0.1 M PBS containing 3% newborn calf 
primary antibodies for 2 days at 4°C. We used polyclonal CB1R goat antibodies (3 µg/ml in 1.5% NCS/PBS) and polyclonal DGL-α guinea pig and rabbit antibodies (2 µg/ml in 1.5% NCS/PBS) generously provided by Dr. Masahiko Watanabe (Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan) 
. Polyclonal rabbit antibodies against mGluR5 (AB5675, Chemicon, CA, USA) were diluted at 1.36 µg/ml in 1.5% NCS/PBS. Slices were then washed in blocking solution and incubated with the following fluorochrome-conjugated secondary antibodies at working dilutions of 1:800 in 1.5% NCS/PBS: donkey anti-goat Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA); donkey anti-rabbit Cy3 (Jackson ImmunoResearch Inc.); goat anti-guinea pig Alexa Fluor-594 and goat anti-rabbit Alexa Fluor-488 (Molecular Probes, Eugene, OR, USA). Incubations were overnight at 4°C. Slices were washed again in 0.1 M PBS and then mounted in Vectashield medium (Vector laboratories, Burlingame, USA), coverslipped, and imaged on a laser-scanning confocal microscope (Olympus Fluoview FV500). Photomicrographs were taken and presented using Adobe Photoshop 7 (Adobe Systems, San Jose, CA, USA).
Double mGluR5/CB1R and DGL-α/CB1R immunocytochemistry for electron microscopy
8, 4–6 weeks old male, C57B1/6 strain) were deeply anesthetized with chloral hydrate (400 mg/kg body weight). The animals were transcardially perfused with phosphate-buffered saline (PBS 0.1M, pH 7.4) and then fixed by 500 ml of a fixative made up of 0.1% glutaraldehyde, 4% formaldehyde (freshly depolymerized from paraformaldehyde) and 0.2% picric acid in PBS. Perfusates were used at 4°C.Tissue blocks were extensively rinsed in 0.1M PBS (pH 7.4). Coronal prPFC vibrosections were cut at 50 µm and collected in 0.1 M PBS (pH 7.4) at room temperature. Sections were preincubated in 10% blocking NCS serum prepared in PBS for 1 h at room temperature and then incubated overnight at room temperature with rabbit polyclonal antibodies to mGluR5 (AB5675, Chemicon, CA, USA; 1.36 µg/ml diluted in 1.5% NCS/PBS), or with polyclonal guinea pig antibodies to DGL-α (2 µg/ml in 1.5% NCS/PBS) generously gifted by Dr. Masahiko Watanabe (Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan) for 2 days at 4°C. The localization of mGluR5 or DGL-α was carried out by means of a preembedding immunoperoxidase method. Briefly, prPFC sections were incubated sequentially at room temperature with a biotinylated secondary antibodies and avidin-biotin complex (ABC, Elite, Vector laboratories, Burlingame, CA, USA), each for 1 h. The immunoreaction was visualized with 0.05% 3,3′-diaminobenzidine (DAB)/ 0.01% hydrogen peroxide as chromogen.
The preembedding silver-intensified immunogold method described previously 
was used for the localization of CB1R in prPFC sections processed for the immunoperoxidase method with either mGluR5 or DGL-α antiserum. Following incubation with the primary polyclonal goat antibodies against CB1R (3 µg/ml in 1.5% NCS/PBS; generously gifted by Dr. Masahiko Watanabe, Department of Anatomy, Hokkaido University School of Medicine, Sapporo, Japan) for 2 days, prPFC sections were incubated with 1.4 nm gold-labeled rabbit anti-goat IgG (Fab' fragment, 1:100, Nanoprobes Inc., Stony Brook, NY, USA) diluted in 1% NCS/PBS for 4 hours at room temperature. In another set of experiments, mGluR5/DGL-α and DGL-α/mGluR5 were colocalized by the preembedding immunogold and immunoperoxidase methods, being the first protein of each combination revealed by silver-intensified gold particles (mGluR5: 1.4 nm gold-labeled goat anti-rabbit IgG, Fab' fragment, 1:100; DGL-α: 1.4 nm gold-labeled goat anti-guinea pig IgG, Fab' fragment, 1:100; Nanoprobes Inc., Stony Brook, NY, USA). PrPFC tissue was subsequently postfixed in 1% glutaraldehyde for 10 min, rinsed extensively in double-distilled water, and gold particles were silver intensified with an HQ Silver kit (Nanoprobes Inc.) for about 8 min.
Successfully double stained sections were osmicated (1% OsO4 in 0.1 M PB, pH 7.4, 20 min), dehydrated in graded alcohols to propylene oxide and plastic-embedded flat in Epon 812. Ultrathin sections were collected on mesh nickel grids, stained with uranyl acetate and lead citrate, and examined in a JEOL (Peabody, MA) X-100 electron microscope.
Preparations were photographed by using standard electron microscopy negative plates. Figure compositions were scanned at 300 dots per inch (dpi). Labeling and minor adjustments in contrast and brightness were made with Adobe Photoshop (7.0, Adobe Systems, San Jose, CA, USA).
PrPFC sections from 3 mice were analyzed. Electron micrographs were taken at a final magnification of x15.000 from 150 µm-grid squares showing good and reproducible DAB immunoreaction and silver-intensified gold particles for any condition studied. Image-J (version 1.36) was used to measure the membrane length. Positive labeling was considered if immunoparticles were in close proximity to the plasmalemma. Metal particles on membranes and positive immunoreactive profiles were visualized and counted manually. The total area studied for CB1R was
. Density of immunoparticles were averaged from different samples and presented as mean±SEM.