Supplementary Methods
Chemicals. Other chemicals were from the National Institute on Drug Abuse
(rimonabant, SR144528), Sigma-Aldrich (WIN55,212-2, morphine sulfate), Tocris
(VDM11, AM251), and Cayman ([2H8]-2-AG, URB597). Drugs were dissolved in a
vehicle of emulphor:ethanol:saline (1:1:8 by volume) or dimethylsulfoxide (DMSO).
Injection volumes were 1 ml-kg-1 for systemic administration, 10 μl for
intracerebroventricular administration, and 1 μl for PAG microinjection. Rimonabant,
SR144528, naltrexone and AM251 were administered by intraperitoneal (i.p.) injection
25 min before the tests or by microinjection 10 min before the tests. URB602, URB597,
VDM11 and MAFP were administered by i.p. (65 min before tests) or intracranial (35
min before tests) injection in the presence or absence of rimonabant.
Tolerance Induction. Sprague-Dawley rats received daily i.p. injections of vehicle or
WIN55212-2 for 2 weeks (10 mg-kg-1 once daily). Morphine antinociception (2.5 mg-kg1
s.c. on day 15) was assessed in separate groups treated chronically with WIN55212-2 or
vehicle. Separate groups received subcutaneous (s.c.) injections of vehicle or morphine
(10 mg-kg-1 once daily for 7 days). Post-injection tail-flick latencies were measured on
days 2, 7 and 14 (chronic WIN55212-2 study) or days 1 and 7 (chronic morphine study)
to confirm that the injection paradigm induced tolerance to the antinociceptive effects of
each agonist prior to administration of the stressor. 24 h after the last injection, rats were
subjected to foot shock, and stress antinociception was quantified. Ceiling tail-flick
latencies were 15 s for evaluation of SIA.
Biochemical Assays. We conducted CB1 and CB2 binding assays in rat cerebellar
membranes and CB2-overexpressing CHO cells (Receptor Biology-Perkin Elmer,
Wellesley, Massachusetts), respectively, using [3H]WIN-55212-2 (NEN-Dupont, Boston,
Massachusetts, 40-60 Ci/mmol) as a ligand. We measured phospholipase C and
phospholipase D activities at 37 °C for 15 min in 35 mM Tris–maleate buffer (0.5 ml, pH
7.3) containing calcium chloride (5 mM), fatty acid-free BSA (2 mg-ml-1, Sigma),
phospholipase C (B. cereus, 1 U; Sigma) or phospholipase D (S. chromofuscus, 10 U,
Sigma). Phosphatidylcholine [3H]methylcholine (8 mM, ARC, 60ci/mmol, 20,000 dpm)
was used as a substrate. Reactions were terminated by adding chloroform:methanol (1:1,
1 ml). Radioactivity was determined by liquid scintillation counting. We measured DGL
activity at 37oC for 30 min in 0.5 ml Tris buffer (50 mM, pH 7.5), rat brain protein (800
g, supernatant, 100 mg protein) and [3H]dioleoylglycerol (50 M, 75,000 dpm; ARC, St.
Louis, Missouri). After stopping the reactions with chloroform/methanol (1:1, 1 ml), we
collected 0.5 ml of organic layer and added 5 g of diolein, 5 g monoleoylglycerol and
12.5 g oleic acid and dried under a stream of nitrogen. Thin-layer chromatography
analyses were carried out on silica gel G plates, eluted with a solvent system consisting of
chloroform/methanol/ammonium hydroxide (85:15:0.1). Lipids were visualized by iodine
staining, and the bands scraped. Radioactivity was determined by liquid scintillation
counting. We performed cyclooxygenase (Cox) assays with a commercial kit using
purified enzymes (Cox-1 from ram seminal vesicles, Cox-2 human recombinant)
(Cayman Chemicals, Ann Harbor, Michigan).