The objective of this study was to investigate the modulation of substance P release in the spinal cord by cannabinoid

receptors. We used neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo to measure substance P release in terms of the activation of its receptor (Mantyh et al., 1995; Abbadie et al., 1997; Allen et al., 1997; Marvizon et al., 2003a; Adelson et al., 2009). These data Epacadostat supplier were previously presented as a meeting abstract (Zhang et al., 2008). Animals used in this study were male Sprague–Dawley rats purchased from Harlan (Indianapolis, IN, USA). A total of 107 rats were used in the study. Spinal cord slices were prepared from 78 juvenile rats (3–5 weeks old). Intrathecal catheters were implanted in 29 adult rats (2–4 months old), of which 16 rats were used to induce NK1R internalization with noxious stimulation

and 13 rats were used to measure paw withdrawal responses to radiant heat. The anesthetic used and other procedural details are given below. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Veteran Affairs Greater Los Angeles Healthcare System, and conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to minimize the number of animals used. ACEA (arachidonyl-2-chloroethylamide), AM251 (N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide), AM281 (1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide),

Y-27632 purchase CGP-55845 ((2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl) phosphinic acid) and Tocrisolve (20% soya oil emulsified in water with Pluronic F68) were purchased from Tocris (Ellisville, MO, USA). Rimonabant (SR141716A) was from the National Institute of Drug Farnesyltransferase Abuse. Isoflurane was from Halocarbon Laboratories (River Edge, NJ, USA). Prolong Gold was from Invitrogen (Eugene, OR, USA). Capsaicin, CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2,) and other chemicals were from Sigma. Compounds were dissolved in water except for the following. Capsaicin and ACEA were dissolved in ethanol. For experiments in slices, AM251, AM281 and CGP-55845 were dissolved at 10 mM in dimethyl sulfoxide (DMSO) and then diluted to their desired concentrations. For the intrathecal injection of 1 nmol AM251 (in 10 μL), a stock solution of 10 mm AM251 was prepared in 100% DMSO and then diluted to 0.1 mm in saline. For the intrathecal injection of 10 nmol AM251 (in 10 μL), AM251 was diluted from 10 to 1 mm in 1% Tocrisolve in saline. Artificial cerebrospinal fluid (aCSF) contained (in mM): NaCl, 124; KCl, 1.9; NaHCO3, 26; KH2PO4, 1.2; MgSO4, 1.3; CaCl2, 2.4; and glucose, 10; K+-aCSF contained 5 mm of KCl, and sucrose-aCSF contained 5 mm KCl and 215 mm sucrose instead of NaCl (iso-osmotic replacement).