Depolarization and Ca2+ downregulate 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 Gαi/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+-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+ channels, α-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 Ca2+-dependent signaling pathway represses CB1 gene transcription.