Cannabinoids Enhance NMDA-Elicited Ca²⁺Signals in Cerebellar Granule Neurons in Culture

Abstract

A physiological role for cannabinoids in the CNS is indicated by the presence of endogenous cannabinoids and cannabinoid receptors. However, the cellular mechanisms of cannabinoid actions in the CNS have yet to be fully defined. In the current study, we identified a novel action of cannabinoids to enhance intracellular Ca2+ responses in CNS neurons. Acute application of the cannabinoid receptor agonists R(+)-methanandamide, R(+)-WIN, and HU-210 (1-50 nM) dose-dependently enhanced the peak amplitude of the Ca2+ response elicited by stimulation of the NMDA subtype of glutamate receptors (NMDARs) in cerebellar granule neurons. The cannabinoid effect was blocked by the cannabinoid receptor antagonist SR141716A and the Gi/Go protein inhibitor pertussis toxin but was not mimicked by the inactive cannabinoid analog S(-)-WIN, indicating the involvement of cannabinoid receptors. In current-clamp studies neither R(+)-WIN nor R(+)-methanandamide altered the membrane response to NMDA or passive membrane properties of granule neurons, suggesting that NMDARs are not the primary sites of cannabinoid action. Additional Ca2+ imaging studies showed that cannabinoid enhancement of the Ca2+ signal to NMDA did not involve N-, P-, or L-type Ca2+ channels but was dependent on Ca2+ release from intracellular stores. Moreover, the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate (IP3) receptor antagonist xestospongin C blocked the cannabinoid effect, suggesting that the cannabinoid enhancement of NMDA-evoked Ca2+ signals results from enhanced release from IP3-sensitive Ca2+ stores. These data suggest that the CNS cannabinoid system could serve a critical modulatory role in CNS neurons through the regulation of intracellular Ca2+ signaling.

Agonist‐dependent cannabinoid receptor signalling in human trabecular meshwork cells

“…Our (Howlett, 2005), the majority of these studies identified the involvement of G i/o proteins, which are capable of mobilizing Ca 2 þ via bg-subunit activation of PLC (Sugiura et al, 1996;Netzeband et al, 1999;Rubovitch et al, 2004 In hTM cells, WIN55,212-2 activation of endogenous CB 1 -G q/11 signalling involved activation of PLC and mobilization of Ca 2 þ from thapsigargin and cyclopiazonic acid-sensitive intracellular stores. The possibility of WIN55,212-2 increasing Ca 2 þ in hTM cells through direct inhibition of the sarcoplasmic/endoplasmic reticulum Ca 2 þ ATPase pumps is unlikely as hTM cells were still able to respond to 10 mM ATP with an increase in Ca 2 þ following exposure to WIN55,212-2 (data not shown), a process that is known to require intact Ca 2 þ stores (von Kügelgen and Wetter, 2000).…”

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

“…In primary hippocampal cells, CB1 receptor activation was capable of inhibiting the calcium release, in a cAMP/PKA-dependent manner, from ryanodine-sensitive intracellular store (Zhuang et al, 2005). In contrast, CB1 receptor activation increased NMDA-evoked Ca 2ϩ release from inositol triphosphate (IP 3 )-sensitive intracellular store in cerebellar granule neurons (Netzeband et al, 1999). Therefore, cannabinoids may enhance or inhibit the NMDA-induced increase in Ca 2ϩ levels depending on the cell type, the type of cannabinoid ligands, and the subsequent signaling pathways activated.…”

Cellular and Molecular Mechanisms Underlying Learning and Memory Impairments Produced by Cannabinoids: Figure 1.

“…Different and/or additional mechanisms may mediate cannabinoid effects in these brain regions and need to be studied. For example, in cultured cerebellar neurons, cannabinoid receptor activation enhances release from intracellular calcium stores (Netzeband et al 1999). Although this mechanism is unlikely to have a direct effect on transmitter release, it could affect profoundly synaptic transmission through second messenger pathways.…”