Male ICR mice 5–7 weeks of age were used for all experiments (Harlan, Indianapolis, IN). Mice were housed on a 12 : 12 light–dark cycle (lights on at 06:00), with food and water available ad libitum. All studies were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals.
N-(piperidin-1-yl)-5-(4-iodophe-nyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide (AM251), and R-( + )-(2, 3-dihydro-5-methyl-3-[(4morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone mono-methanesulfonate (WIN55212-2), and (5Z,8Z,11Z,14Z)-5,8,11, 14-eicosatetraenyl-methyl ester phosphonofluoridic acid (MAFP) were obtained from Tocris (Ellisville, MO). Tetrahydrolipstatin (THL) and essentially fatty-acid free bovine serum albumin (BSA) were obtained from Sigma (St Louis, MO). 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphonovalerate (AP-5) were purchased from Ascent Scientific (Princeton, NJ). All cannabinoids were made up as stock solutions in DMSO; 0.5 g/l BSA was added to artificial cerebral spinal fluid (ACSF) to increase solubility and minimize nonspecific binding of lipophilic compounds. Equal amounts of DMSO and BSA were used in control experiments.
1-stearoyl-2-arachidonoylglycerol (SAG), 2-AG, AA, and their deuterated analogs were purchased from Cayman Chemicals (Ann Arbor, MI). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and 4-(dimethylamino)pyridine (DMAP) were purchased from Aldrich Chemical Company (Milwaukee, WI) and used without further purification. Solvents were HPLC grade and purchased from Fisher Scientific (Pittsburg, PA). 1-oleoyl-2-arachidonoylglycerol (OAG) was synthesized by a reaction of 2-AG and oleic acid using EDC and DMAP (catalyst) in heated toluene (60°C). The reaction was quenched with water. The organic compounds were extracted with ethyl acetate and the product was purified with preparative HPLC.
Mice were housed four per cage during all experiments. Mice were brought into the restraint room daily and subjected to 1 h of tube restraint in modified 50 ml conical tubes for 1 day or 10 consecutive days (between 9 and 11am). During this time mice were placed in ventilated animal housing cabinet. On termination of the stressor, mice were placed back in their home cage and returned to the animal care facility housing room. Mice were weighed daily before restraint stress. Control mice were left undisturbed in their home cages except for tail marking at the beginning of the experiment and as needed to maintain identifying marks throughout the 10-day protocol. After each stress episode plastic tubes were washed with soap and water, and then rinsed in 70% ethanol.
Tissue Preparation and Lipid Extraction
Control mice or stressed mice were killed by decapitation, the brain rapidly removed, and a 2 mm block containing the amygdala was made using a coronal brain matrix. Blocks were placed on a glass dish on top of dry ice-cooled metal block. Less than 90 s elapsed between decapitation and tissue freezing. Once frozen, 1 mm brain punches were made of the amygdala. Tissue was stored at −80° C until used for tissue extraction. Lipid extraction from amygdala micropunches was carried out exactly as described earlier (Patel et al, 2005b
), except that the homogenization volume was spiked with 50 pmol d8
-2-AG, and 500 pmol d8
-SAG, and 1000 pmol d8
-AA per sample. Immediately before LC-MS analysis, the samples were reconstituted in 200 µl of 9:1 methanol:water (v:v), vortexed, and transferred to autosampler vials.
Chromatographic separation of the analytes was achieved on a Phenomenex Synergi Polar-RP column (7.5 cm × 0.2 cm, 4 µm held at 40°C) using the following gradient: 85%B for 0.5 min, %B increased to 99% in 3 min and held at 99% for an additional 2 min. The column was re-equilibrated at initial conditions for 3 min before each injection. The flow rate was 0.5 ml/min. Component A was water and B was methanol and each component contained 80 µM silver acetate and 0.5% acetic acid (v:v).
The analytes were detected through single reaction monitoring (as [M + Ag] + complexes except AA, which is ionized as [(M—H)+2Ag]+) in the positive ion mode using the following reactions (the mass in parentheses represents the mass of the deuterated internal standard): AA (m/z 519(527)→409(417)); 2-AG (m/z 485(493)→411(419)); SAG (m/z 751(759)→411(419)) and OAG (m/z 759→411). Quantification was achieved through stable isotope dilution for AA, 2-AG, and SAG whereas OAG was quantified relative to SAG-d8. Levels of analytes are given in pmols of analyte per mg wet tissue weight.
Immnunohistochemistry and immunofluorescence were performed as described earlier (Patel and Hillard, 2003
). Briefly, mice were anesthetized with isoflourane and transcardially perfused with 20 ml of phosphate-buffered saline (PBS) followed by 10 ml of ice-cold 4% paraformadahyde. Brains were removed and post fixed for 24 h followed by a 48 h incubation in 30% sucrose solution. Frozen sections 30 µm thick were cut on a cryostat and stored in an ethylene glycol solution at −20°C until use.
Sections were thoroughly washed in tris-buffered saline (TBS), then incubated in 10% methanol, 2% hydrogen peroxide solution in TBS for 30 min followed by washing and incubation in 4% normal donkey serum and 0.2% Triton X-100 in TBS. Sections were incubated in an earlier characterized rabbit-anti DAGL
at 1:2000 dilution overnight at room temperature (Katona et al, 2006
). Sections were washed and then incubated in biotin-conjugated donkeyanti rabbit at 1:500 for 2 h, followed by HPR-conjugated streptavidin at 1:16000 for 90 min (Jackson ImmunoResearch, West Grove, PA). Sections were developed using diaminobenzadine substrate with nickel/cobalt heavy metal intensification as described earlier. For immunofluorescence, rabbit-anti DAGL
was used at 1:500, followed by 2 h incubation in Cy-3-conjugated donkey-anti rabbit at 1:250 for 2 h (Jackson ImmunoResearch).
For biocytin visualization of recorded neurons, 1% biocytin (Jackson ImmunoResearch) was included in the internal patch solution. After termination of the experiment (30–60 min), slices were placed in 4% paraformaldahyde for 24 h. Sections were then washed in PBS for 30 min and incubated in Cy-3-conjugated streptavidin (Jackson ImmunoResearch) at 1:500 dilution for 2 h at room temperature. Sections were washed and mounted on slides using Aquamount and visualized using confocal microscopy.
Brightfield photomicrographs were obtained using an upright Olympus SZX12 microscope coupled to an Olympus CMAD3 camera using Q-capture software. Confocal images of immunofluorescently stained sections were obtained using a Zeiss LSM510 confocal microscope. Small brightness and contrast adjustments, and figure arrangement were done using Adobe Photoshop CS.
Animals were retrieved from the colony and allowed to rest in sound attenuating boxes for a minimum of 1 h after which they were killed by decapitation. Stressed animals were killed by cervical dislocation and decapitated immediately following termination of the last 1 h restraint exposure. A 3 mm coronal block containing the amygdala was cut using a coronal brain matrix kept on ice; 300 µm coronal slices were made on a Leica VT1000S vibratome (Leica Microsystems, Bannockburn, IL) in a 1–4°C, oxygenated (95% O2, 5% CO2), 30% sucrose, low Na+ artificial cerebral spinal fluid containing in mM: 194 sucrose, 20 NaCl, 4.4 KCl, 2 CaCl2, 1 MgCl2, 1.2 NaH2PO4, 10 glucose, 26 NaHCO3. Once cut, sections were transferred to a holding chamber containing oxygenated ACSF in mM: 124 NaCl, 4.4 KCl, 2 CaCl2, 1.2 MgSO4, 1 NaH2PO4, 10 glucose, 26 NaHCO3 at 28°C. After a minimum of 1 h, sections were placed in the recording chamber superfused with oxygenated ACSF at a flow rate of 2 ml/min. To pharmacologically isolate inhibitory postsynaptic currents (IPSCs), ACSF was supplemented with a combination of 10 µM CNQX and 50 µM D/L AP-5. All experiments were carried out at 23–25°C.
Patch electrodes (3–6 ΩM) were pulled on a Flaming/ Brown microelectrode puller (Sutter Instruments) and filled with internal solution containing in mM: K-gluconate 70, KCl 80, EGTA 1, HEPES 10, Na-ATP 4, Na-GTP 0.3, QX-314 2, with osmolarity adjusted to 285–290 mOsm. Visually identified pyramidal neurons within the BLA were used for electrophysiological studies. Recordings were made using an Axopatch 1D amplifier (Axon instruments). Recordings were made at a holding potential of −70 mV. Evoked IPSCs (eIPSCs) were elicited by a bipolar stimulating electrode placed in the BLA with stimulation intensities varied from 3 to 40 V. eIPSP amplitudes were typically adjusted to 150–500 pA. Data were recorded using PClamp 9.2 (Molecular Devices).
For CB1 agonist and antagonist experiments, evoked simulations were elicited at 0.1 Hz with six stimulations being averaged to obtain one data point per minute. For depolarization-induced suppression of inhibition (DSI) studies, stimulations were made at 0.33 Hz. Access resistance (Ra) was monitored online using a 5 mV voltage step preceding each stimulation. Cells that showed increases in Ra of > 20% during an experiment or DSI trial or had an Ra >35 mΩ were excluded from the analysis.
Statistical analyses were performed using Prism Graphpad (San Diego, CA). Differences in 2-AG, AA, OAG, and SAG were evaluated by ANOVA followed by Dunnett’s test post hoc. For drug and stress DSI time-course experiments, data were analyzed by repeated measures ANOVA factoring time and treatment (drug or stress). For DSI summary data, we divided the postdepolarization period into a maximal DSI (average amplitude of the first three eIPSCs after the depolarizing pulse) and a late DSI (average amplitude of the last 10 eIPSCs of the experiment) component, which were compared with the average amplitude of the 10 baseline eIPSCs. Selected comparisons of summary DSI data were analyzed by ANOVA followed by Bonferroni’s test post hoc. Typically, data from two to three DSI trials per cell were averaged before analysis. p < 0.05 was considered significant throughout. Data are presented as mean ± SEM.