Depolarization and Ca(2+) down regulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons.
Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 G alpha(i/o)-coupled cannabinoid receptors (CB1) to modulate glutamate release. To study CB1 signaling investigators use primary cultures of granule neurons, typically grown in medium supplemented with elevated KCl to improve long-term survival. Herein, we demonstrate that CB1 expression and signaling are perturbed under these conditions. Specifically, immunochemical and RT-PCR assays indicate that depolarizing growth conditions decrease CB1 protein, mRNA and CB1-mediated inhibition of adenylyl cyclase compared to cultures grown in physiologic medium containing 5mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na(+)-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5mM versus 25 mM KCl confirms that depolarization reduces CB1 mRNA, but not mRNAs encoding several G-protein subunits or adenylyl cyclases. However, significant alterations in synaptic signaling proteins that likely lie downstream of CB1 are observed, including K(+) channels, alpha-neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca(2+)-dependent signaling pathway represses CB1 gene transcription.
Vallano
Mary Lou
ML
Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY 13210, USA. vallanom@upstate.edu
Beaman-Hall
Carol M
CM
Bui
Cuong J
CJ
Middleton
Frank A
FA
D000367:Age Factors; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D015152:Blotting, Northern / Q000379:methods; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003511:Cyclohexanols / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005455:Fluorescent Antibody Technique / Q000379:methods; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D017403:In Situ Hybridization / Q000379:methods; D008954:Models, Biological; D009474:Neurons / Q000502:physiology* / Q000528:radiation effects; D009543:Nifedipine / Q000494:pharmacology; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013778:Tetrazolium Salts; D013779:Tetrodotoxin / Q000494:pharmacology; D013844:Thiazoles; D013997:Time Factors
D002121:Calcium Channel Blockers; D003511:Cyclohexanols; D043884:Receptor, Cannabinoid, CB1; D013778:Tetrazolium Salts; D013844:Thiazoles; D013779:Tetrodotoxin; D011189:Potassium Chloride; C054649:3-(2-hydroxy-4-(1,1-dimethylheptyl)phenyl)-4-(3-hydroxypropyl)cyclohexanol; C022616:thiazolyl blue; D009543:Nifedipine; D002118:Calcium