Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol

Key points center dot 2-Arachidonoylglycerol (2-AG), one of the best-characterized retrograde messengers at central synapses, has been thought to be produced on demand' through a diacylglycerol lipase (DGL)-dependent pathway upon activation of postsynaptic neurons (on-demand synthesis hypothesis). center dot However, recent studies propose an alternative hypothesis that 2-AG is pre-synthesized by DGL, stored in neurons, and released from such pre-formed pools' without the participation of DGL (pre-formed pool hypothesis). center dot To test these hypotheses, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde 2-AG signalling. center dot We found that 2-AG-mediated retrograde signalling was blocked after 1 h treatment with OMDM-188 in acute slices from the hippocampus, striatum and cerebellum, and was blocked several minutes after OMDM-188 application in cultured hippocampal neurons. center dot These results fit well with the on-demand synthesis hypothesis, rather than the pre-formed pool hypothesis. Abstract The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase (DGL) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGL and stored in neurons, and that 2-AG is released from such pre-formed pools' without the participation of DGL. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGL or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca2+ elevation, G(q/11) protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca2+-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre-Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca2+ elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGL rather than mobilization from presumptive pre-formed pools.