Depolarization and Ca super(2+) downregulate CB1 receptors and CB1-mediated signaling in cerebellar granule neurons

• Published in: Neuropharmacology Vol. 50; no. 6; pp. 651 - 660
• Main Authors: Vallano, Mary Lou, Beaman-Hall, Carol M, Bui, Cuong J, Middleton, Frank A
• Format: Journal Article
• Language: English
• Published: 01.01.2006
• ISSN: 0028-3908
• DOI: 10.1016/j.neuropharm.2005.11.012

Presynaptic terminals of cerebellar granule neurons are primary targets of cannabinoids, which act through type 1 G alpha sub(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 5 mM KCl. Depolarization-dependent downregulation of CB1 mRNA, like survival, is attenuated by L-type VDCC antagonists but not the Na super(+)-channel antagonist, tetrodotoxin. Comparison of oligonucleotide microarrays from cultures grown in 5 mM 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 super(+) channels, alpha -neurexins, cAMP-GEFII, Munc13-3, secretogranin and synaptotagmin. These findings make a compelling argument to adopt cultures grown in 5 mM KCl for future study of CB1 signaling in granule neurons. Further, they suggest that a depolarization and Ca super(2+)-dependent signaling pathway represses CB1 gene transcription.