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(< 0.01, MannCWhitney assessments). Picospritzer device and introduction of JWH-133 (5 M) into neurons via the patch pipette also depolarized neurons (= 38 4 mV and 22 6 mV; < 0.0001 and < 0.05). Sch.356036 (1 M), introduced into Rabbit Polyclonal to ALS2CR8 neurons via the patch pipette, largely prevented the depolarization by JWH-133 (1 M, = 4 2 mV; ***< 0.001). L-Valyl-L-phenylalanine (< 0.01, MannCWhitney assessments). Numbers of observations are indicated in bars (and and and < 0.01) and not in the presence of SR1 (a CB1R antagonist; 44 3 fmol/mg). Plasma membrane binding of [3H]CP55.940 (126 6 fmol/mg) was reduced after incubation with SR1 (36 1 fmol/mg; ***< 0.001) or SR2 (102 4 fmol/mg; *< 0.05). The reduction of plasma membrane binding was significantly larger in the presence of SR1 compared with SR2 (71 1% and 19 4%, respectively; < 0.001). Numbers of observations are indicated in bars; error bars represent SEM. CB2R Activation Opens Ca2+-Activated Cl? Channels via IP3R. The signaling cascade after CB2R activation can lead, through phospolipase C production, to Ca2+ release via IP3R (23) and, thus, to the potential activation of Ca2+-activated conductances (24). Introduction of the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-< 0.001) depolarizations (= 4 1 mV and 8 2 mV). The average response evoked with 5 M JWH-133 (in the absence of BAPTA) is the same as is usually depicted in Fig. 1< 0.05). (shows representative current traces. (< 0.05, = 12). These results indicate an endogenous tonus of eCBs and/or constitutive activity of the receptor and show that, when firing is usually evoked with an input that could resemble spontaneous background synaptic activity, further CB2R activation modulates the firing rate of mPFC neurons. Open in a separate window Fig. 4. CB2R activation decreases firing activity of rat mPFC layer II/III pyramidal neurons. (< 0.001). After preincubation (of at least 10 min before going whole-cell) with and continuous presence of 5 M Sch.356036, baseline firing frequency 0.83 0.15 Hz, normalized to 100 3%, JWH-133 (1 M) could not induce a response (92 6%, JWH-133 plus Sch.356036). (tests unless otherwise stated. In the figures, the significance is usually indicated with asterisks (*< 0.05, **< 0.01, and ***< 0.001). Drugs. For the radioactive binding assay, the synthetic cannabinoid CP55.940 5-(1,10-dimethyheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl)-cyclohexyl] phenol was purchased from Sigma Chemical. [3H]CP55.940 (126 Ci/mmol) was from PerkinElmer Life Sciences. SR141716 [N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-3-pyrazole-carboxamide], and SR144528 N-[(1S)-endo-1,3,3-trimethy-1-bicyclo[2.2.1]-heptan-2-yl]5-(4-choro-3-methyl-phenyl)-1-(4-methyl-benzyl)-pyrazole-3-carboxamide were kind gifts from Sanofi-Aventis Recherche (Paris, France). For the electrophysiological experiments, JWH-133, HU-308, and Sch.356036 were generous gifts from Abbott Laboratories (Weesp, the Netherlands). Cannabinoid receptor ligands were dissolved in DMSO to 50 mM and diluted in aCSF that never contained a final concentration of DMSO higher than 0.1%. BAPTA, 2-APB, and DIDS were all purchased from Sigma-Aldrich. TTX (0.5 M, Latoxan) was present during all recordings, with the exception of the experiments shown in Fig. 4. Supplementary Material Supporting Information: Click here to view. Acknowledgments We thank Dr. N. L. M. Cappaert and Dr. J. A. van Hooft for critical reading of the manuscript and Dr. T. Z. L-Valyl-L-phenylalanine Baram for critical suggestions in an early phase of the project. This study was supported L-Valyl-L-phenylalanine by Dutch Top Institute Pharma Grant T5-107-1. M.B., S.O., and M.M. were supported by Fondazione TERCAS Grant 2009-2012 and by Fondazione Italiana Sclerosi Multipla Grant 2011-2012. Footnotes The authors declare no conflict of interest. This article is usually a PNAS Direct Submission. This article contains supporting information online at