Technical Reference #1729

1729.

Signaling Pathways from Cannabinoid Receptor-1 Activation to Inhibition of N-Methyl-D-Aspartic Acid Mediated Calcium Influx and Neurotoxicity in Dorsal Root Ganglion Neurons Qing Liu; Manjunatha Bhat; Wayne D. Bowen; Jianguo Cheng, Anesthesiology Institute and Lerner Research Institute Brown University, Journal of Pharmacology and Experimental Therapeutics, 331(1729), (2009)
Link To Paper

Keywords:
cannabinoid receptor-1; NMDA; N-methyl-D-aspartic acid; PKA; Dose-Dependent Calcium Rise

Materials & Methods:
All procedures used in the animal experiments were approved by the Institutional Animal Care and Use Committee of Cleveland Clinic Foundation. DRG neurons were obtained as described earlier (Dedov et al. 2001). In brief DRG isolated from adult Sprague-Dawley rats (200–300 g) were incubated in Hanks’ balanced saline solution (Invitrogen Carlsbad CA) with 0.05% collagenase and 0.25% trypsin for 25 min at 37°C. Neurons were dissociated by trituration of ganglia with fire-polished Pasteur pipettes of decreasing diameter and afterward the cellular suspension was washed twice in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 2 mM L-glutamine. Freshly isolated neurons were plated onto collagen-coated coverslips and cultured in Dulbecco’s modified Eagle’s medium/F-12 Ham media containing 2 mM L-glutamine supplemented with 10% fetal bovine serum HAT supplement (100 M hypoxanthine 400 nM aminopterin 16 M thymidine) 50 ng/ml nerve growth factor 100 units/ml penicillin and 100 g/ml streptomycin. Cells were kept under 5% CO2 at 37°C and passed twice a week using nonenzymatic cell dissociation solution

Microscopic Technique
Leica DMIRB inverted microscope

Cell Type(s)
DRG neurons; F-11 cells