Identification of the endocannabinoid working as a retrograde messenger and its release mechanism

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Recent studies have revealed that endocannabinoids work as a retrograde messenger and play an important role in activity-dependent modulations of synaptic transmission in the brain. The endocannabinoid release is induced by postsynaptic depolarization or activation of group I metabotropic glutamate receptors (I-mGluRs). The released endocannabinoid then activates presynaptic CB1 cannabinoid receptors and suppresses the transmitter release. The mechanisms of endocannabinoid production, however, have not been fully determined. In this study, we used rat cultured hippocampal neurons and recorded cannabinoid-sensitive inhibitory postsynaptic currents (IPSCs), which can be used as biosensor of released endocannabinoids. We obtained the following results.1.Among three candidates of endocannabinoids (anandamide,2-AG, noladin ether),2-AG was the most effective and reversible in suppressing IPSCs.2.The endocannabinoid release was induced by postsynaptic activation, of M1/M3 muscarinic receptors … More as well as I-mGluRs. This receptor-driven endocannabinoid release was eliminated from the neurons prepared from PLCβ1-knockout mice.3.The depolarization-induced endocannabinoid release, which is dependent on Ca^<2+> elevation, was suppressed by the PLC inhibitor ET-18,but not eliminated from PLCβ1- or PLCδ-knockout mice.4.When weak depolarization and weak activation of I-mGluRs or M1/M3 muscarinic receptors, both of which can not induce endocannabinoid release, were simultaneously treated, endocannabinoids were released. This type of release was also eliminated from PLCβ1-knockout mice. Using TRPC6 channels as biosensor of PLC activity, we demonstrated that the synergistic effect of depolarization and receptor activation on endocannabinoid release can be explained by the Ca^<2+>-dependence of receptor-driven PLCβ activation.These results suggest that the postsynatpic activation (depolarization or receptor activation) stimulates PLCs and induces 2-AG production through the subsequent enzymatic activity by DAG lipase. The isozymes of PLCs involved might be different in different stimulation conditions. Less